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Healing Power from Nature: Current Trends & Perspectives

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HEALING POWER FROM NATURE:
CURRENT TRENDS
AND
PERSPECTIVES

PROCEEDINGS OF THE

14th SEMINAR ON MEDICINAL & AROMATIC PLANTS

11 – 12 OCTOBER 2016
 FRIM Proceedings No. 8

HEALING POWER FROM NATURE:

CURRENT TRENDS AND PERSPECTIVES

PROCEEDINGS OF THE
14th SEMINAR ON MEDICINAL & AROMATIC PLANTS
11 – 12 OCTOBER 2016

Editors

B.J. Chee
M. Mastura
K. Getha
M.G.H. Khoo
J. Mailina
M.A. Adiana
M.S. Roshan Jahn

2016
© Institut Penyelidikan Perhutanan Malaysia 2016

Segala pertanyaan hendaklah dikemukakan kepada:

Ketua Pengarah
Institut Penyelidikan Perhutanan Malaysia
52109 Kepong
Selangor Darul Ehsan
Malaysia
Tel: 603-62797000
Faks: 603-62731314
http://www.frim.gov.my

Perpustakaan Negara Malaysia Cataloguing-in-Publication Data

HEALING POWER FROM NATURE : CURRENT TRENDS AND PERSPECTIVES :

PROCEEDINGS OF THE 14TH SEMINAR ON MEDICINAL & AROMATIC
PLANTS 11-12 OCTOBER 2016 / Editors B.J. Chee, M. Mastura, K.
Getha, M.G.H. Khoo, J. Mailina, M.A. Adiana, M.S. Roshan Jahn
(FRIM Proceedings ; No. 8)
ISBN 978-967-0622-66-8
1. Medicinal plants--Malaysia. 2. Herbs--Therapeutic use--Malaysia.
3. Herbs--Malaysia. I. B. J. Chee. II. M. Mastura. III. K. Getha.
IV. M. G. H. Khoo. V. J. Mailina. VI. M. A. Adiana VII. M. S. Roshan
Jahn. VIII. Series.
635.7

MS ISO 9001:2008
Diset dalam Calibri 11/12
Dihasilkan di Malaysia oleh Institut Penyelidikan Perhutanan Malaysia,
Kepong
Contents
Objectives xii

Sekapur Sirih xiii

Kata Alu-aluan xiv

Programme xv

Program Simbion Bioherba FRIM-MARA xix

Program Teknousahawan FRIM-MARA xx

PLENARY LECTURES

Integrated Approach in Natural Products Research to Discover 3

New Drug and Develop Herbal Medicines and Health Products.
Ibrahim, J.

Relevance of Traditional Knowledge in Herbal Research 5

Abdul Ghani, H.

ETHNOBOTANY, AGRONOMY & CONSERVATION

Industri Herba: Bahan Tanaman Berkualiti 9

Mohd Zaki, A.

Program Pemulihan Tanaman Kacip Fatimah oleh Jabatan Pertanian 10

Mohamed Redza, B.

Pembangunan Cameron Highlands Montane Park (CHiMP) bagi 11

Memperkasakan Program Konservasi Ex-situ Tumbuhan Ubatan
Tanah Tinggi di Semenanjung Malaysia.
Noorsiha, A., et al.

Kepentingan Tumbuhan Ubatan dan Beraroma di Kalangan 17

Masyarakat Melanau di Mukah, Sarawak.
Zahora, I.

v
Breeding Strategies of Eurycoma longifolia: Present and Future. 18
Nor Fadilah, W. et al.

Penghasilan Bahan Tanaman Tumbuhan Ubatan Menggunakan 24

Teknologi Kultur Tisu.
Nor Hasnida, H. et al.

Nilai Taksonomi Ciri Anatomi Daun Beberapa Spesies Tumbuhan 27

Ubatan Terpilih Daripada Famili Apocynaceae dan Dipterocarpaceae
Ummu Hani, B. et al.

Preliminary Observations on Soil Amendment Effects Using Biochar 32

on Labisia pumila var alata at Nursery Stage.
Jeyanny, V. et al.

Penubuhan Plot Ujian Progeni Limau Purut (Citrus hystrix) di 37

Empat Stesen Penyelidikan FRIM: Satu Kajian Awal.
Farah Fazwa, M.A. et al.

Effect of Fertilizer Application on the Growth of Eurycoma longifolia 42

(Tongkat Ali) Plantlets in Greenhouse Conditions.
Muhd Fuad, Y. et al.

In Vitro Germination of Synsepalum dulcificum (Pokok Ajaib) 46

from Seed Explants.
Mohd Saifuldullah, A.W. et al.

Establishment of Surface Sterilization Protocol for Tissue Culture 50

of Christia vespertilionis (Red Butterfly Wing).
Nazirah, A. et al.

Peranan Tapak Warisan FRIM Sebagai Bank Baka Bagi 54

Pemuliharaan Tumbuhan Ubatan dan Phytotourism.
Ainnur Amira, A.M. et al.

Ekosistem Tanah Tinggi Sebagai Santuari Tumbuhan Penawar di 59

Semenanjung Malaysia.
Nurliyana, A.L et al.

NATURAL PRODUCT DISCOVERY

Plant Extracts in Anticancer Treatment: Proof and Myths. 68

Aishah, A.

vi
The Powess of Oyster Mushroom (Pleurotus sajor-caju, PSC) as 69
an Alternative Nutritive Functional Ingredient.
Wan Rosli, W.I. et al.

Discovering the Anti-ovarian Cancer Potential of a Cardiac Glycoside 75

Derivative Using In Vitro, In Silico and Proteomics Approaches.
Nurhanan, M.Y. et al.

Purification of Flavonoid Methyl Ether from Tetracera indica Using 85

Automated Chromatographic Approach.
Fauziah, A. & Nor Hadiani, I.

Xanthine Oxidase Inhibitory Activity of Flavonoid Compound from 86

Hibiscus rosa-sinensis L. and Brassica oleracea L.
Hussin M. Z. et al.

Multidrug-Resistant Stahyplococcus aureus (MRSA) Activity of 92

Naringenin Related Compounds.
Adiana, M.A. et al.

Determination of Total Protein Content in Selected Plant Species 93

by Using Bicinchoninic Acid (BCA) Assay.
Norsuhaina, Z. & Abd Rashid, L.

Chemical Profiles of Four Selected Labisia pumila High Yielding 94

Accessions Based on Uterus Contraction Activity.
Farah Fazwa, M.A. et al.

Produk Hasilan Semula Jadi dari Tumbuhan Kesukaan Kucing. 101

Wan Mukhtar, W.R. et al.

Quantification of Volatine Organic Compound (Hexanoic Acid) in 108

Morinda citrifolia L. Juice Distillates by Gas Chromatography.
Nurul Raihana, A. et al.

Statistical Analysis of Agarwood Oil Chemical Compounds from 113

High Quality Using Boxplot.
Nurlaila, I. et al.

A Preliminary Study of Chemical Properties and In Vitro Anti-cancer 118

Activities of Christia sp.
Muhammad Haffiz, J. et al.

vii
Bioassay-Guided Isolation of Anti-trypanosomal Active Compound 123
from Actinomycete Strain TY035-025.
Muhammad Syamil, A. et al.

Chemical Composition of Litsea cubeba Essential Oils from 128

Cameron Highlands.
Nor Azah, M.A. et al.

Prismalayanoside from Prismatomeris terandra. 133

Nor Hayati, A. et al.

STANDARDISATION, QUALITY CONTROL & PROCESSING TECHNOLOGY

Status Pemiawaian dalam Industri Herba Tempatan 140

Zhari, I.

FRIM’s Commitment Towards Local Herbal Industry: Quality Control 141

of Herbal Product.
Ong, B.K. et al.

Herbal Product Deliverables: Seeds to Shelves. 145

Mohd. Shahidan, M.A. et al.

Effects of Temperature on Drying of Eurycoma longifolia Jack 147

Roots: Drying Time and Product Quality.
Hada Masayu, I. et al.

Perkhidmatan Ujian Kawalan Kualiti Pencemaran Mikroorganisma 153

yang Berakreditasi ISO/IEC 17025 untuk Produk dan Bahan Mentah
di Makmal NPQC, FRIM.
Amira Rina Nurdiana, M.S. et al.

Verification of Specified Microorganisms Test Method for 158

Capsule Matrice.
Norulaiman, Y. et al.

Validation of Heavy Metal Analysis (Pb, Cd, Hg, As) for Hard Gel Capsule 162
Using Microwave Digester and Atomic Absorption Spectrometry.
Nurhazwani, M.H. et al.

Optimisation of Supercritical Fluid Extraction Condition of Curcuma 167

domestica (Kunyit) Rhizome Using Response Surface Methodology.
Mohd Shafik Yuzman, T. et al.

viii
Screening of Selected Cinnamomum Spp. as Reducing Agent for 177
Nano Silver Synthesis.
Noor Rasyila, M.N. et al.

MARKETING, TRADE, BUSINESS POTENTIAL & LEGISLATION

Selling Our Story to the World: Branding with What We Have Best. 188
Mohamad Faisal, A.F.

Commercialisation Opportunity with BioAlpha 189

Faizzudin, S.

Kembara Penyelidikan, Pembangunan dan Pengkomersilan 190

Rangkaian Produk Disinfektan dan Antiseptik Mesra Alam.
Mastura, M.

PRECLINICAL, CLINICAL & PRODUCT DEVELOPMENT

Challenges and Experiences in Conducting Preclinical Studies 196

for Herbal Products: Pharmaniaga Perspective.
Vanessa Shalini, D.

Andrographolide Derivatives Target the Elusive Oncogenic K-Ras: 197

Promising Compounds to Combat K-Ras-Driven Malignancies
Johnson, S. et al.

Glucose Uptake Activity of Ficus deltoidea Extracts. 199

Nur Sumirah, M.D. et al.

Antioxidant Properties, Total Phenolic Content & Antiglycation 200

Activity in Four Varieties of Ficus deltiodea Extracts.
Nurshieren, Y. et al.

Natural Sweetener of Fresh Pineapples as Hepatoprotective Agent. 201

Nurul Hafizatul Syafiqah, M.A. et al.

Determination and Evaluation of Antioxidant Constituents of 202

Entada spiralis Ridl. Leaves.
Sharifah Nurul Akilah, S.M. et al.

Antiinflammatory Potential of Backhousia citriodora F. Muell. 203

Methanolic Extract by In Vitro Bioactivity Assessment.
Siti Nur Aisyah, M.H. et al.

ix
Larvicidal Properties of β-Carryophyllene of Major Component 204
Melaleuca cajuputi Essential Oils Against Dengue Vector Aedes
aegypti (L) and Aedes albopictus (Skuse).
Azlinda, A.B. & Hamdan, A.

Potential of Polygalacturonic Acid Vanadyl (IV) Comples as Oral 207

Insulin-mimetic Agent.
Nurnadiah, R. et al.

Comparison on Prediction and Experimental Lipophilicity 213

(Log P) of an Anticancer Compound 17- βH Neriifolin.
Asiah, O. et al.

The Effect of Tocotrienol Rich Fraction (TRF) Derived from Palm Oil 218
on Glutathione S-Transferases Protein Expression in Mice Liver.
Abdullah, A. et al.

Anti-Gout Properties from Baeckea frutescens via Xanthine 224

Oxidase Inhibition.
Fadzureena, J. et al.

Antioxidant Properties of Extracts from Different Part of Four 229

Malaysian Medicinal Plants.
Ihsan Safwan, K. et al.

Assessing Malaysian Forest Species for Lipogenase Inhibitory Activity. 236

Mazura, M.P. et al.

Evaluation of Antiinflammation and Antipyretic Activities of 240

Piper nigrum Fruit Extracts.
Ong, B.K. et al.

Potential Proteolytic Endophytes from Local Shiitake Mushroom. 246

Wan Nur Fatihah, W.M. & Zaidah, Z.A.

Assessment of Phytoconstituents, Antioxidants and Alpha- 251

Glucosidase Inhibitory Activities of Entada spiralis Ridl. Stem
Bark Extracts.
Fatimah, O.R. et al.

Antifungal Activity Against Phypathogenic Fungi Exhibited by 254

Antinobacteria from Root Soil of Selected Plant Species.
Getha, K. et al.

x
Phototoxicity Evaluation of Medicinal Plants and Products. 259
Khoo, M.G.H. et al.

Preliminary Observation of Proteomic Profiles of Cancer Cell Lines 266

Against Epigallocatechin Gallate (EGCG).
Nor Datiakma, M.A & Nurhanan Murni, Y.

In Vitro Anti-trypanosomal Activity of Selected Forest Plant Species. 269

Norhayati, I. et al.

Effect of pH and Initial Glucose Concentration on Bioactive 274

Metabolites Production from Ganoderma Sp. DSM 24013.
Roshan Jahn, M.S. et al.

Preliminary Study on Preparation of Gelatin Nanoparticles for 277

Plant Extract Towards Product Development.
Saidatul Husni, S. et al.

A Biological Approach: Intracellular Antioxidant Activity on 282

Camellia sinensis (Green Tea) Leaf Extract Using Human Red
Blood Cells
Shalini, M. et al.

Acknowledgement & Sponsors 288

Organising Committee 291

xi
 Objectives

To highlight and share scientific

and technological findings in research, development
and application related to natural products
and medicinal aromatic plants

To provide a forum for discussion

and exchange of ideas on the latest issues related
to natural products, medicinal and aromatic plants

To strengthen ties and facilitate networking among

entrepreneurs, researchers, academicians and policy
makers

xii
Sekapur Sirih
Industri berasaskan herba telah disenaraikan sebagai satu industri yang
mempunyai potensi dan daya saing untuk membantu meningkatkan ekonomi
negara bagi mencapai negara berpendapatan tinggi menjelang tahun 2020.
Pembangunan industri herba pula telah menjadi satu agenda penting yang telah
diketengahkan dalam sektor pertanian untuk Bidang Ekonomi Utama Negara
(NKEA) serta menjadi satu daripada cabang utama penggerak kepada Dasar
Agromakanan Negara.
Kementerian Sumber Asli dan Alam Sekitar (NRE) menganggarkan
pasaran herba tempatan dijangka meningkat sebanyak 15% setahun daripada
RM7 billion pada tahun 2010 ke RM29 billion pada tahun 2020. Dalam usaha
menembusi pasaran global dan meningkatkan ekonomi negara melalui
sumbangan industri berasaskan herba, produk sedia ada dan baharu perlu
ditingkatkan dari segi kualiti, keberkesanan dan keselamatan. Pada masa yang
sama, industri herba turut berhadapan dengan cabaran meningkatkan
pendapatan kumpulan sasar supaya jurang ekonomi antara kawasan bandar
dengan luar bandar dapat dirapatkan.
Faktor utama pertumbuhan positif nilai pasaran herba ini adalah
berdasarkan manfaat dan khasiat tersendiri herba yang diamalkan sejak dahulu
lagi yang secara tidak langsung mencetuskan kesedaran atas penggunaan herba
dalam kalangan rakyat Malaysia. Oleh yang demikian, penganjuran Seminar
Tumbuhan Ubatan dan Beraroma ke-14 yang bertema “Penawar daripada Alam
Semula Jadi: Trend dan Perspektif Semasa” amat tepat pada masanya. Saya
berharap agar semua peringkat perbincangan serta interaksi para pakar, wakil-
wakil industri, pengusaha tanaman herba dan penglibatan komuniti pemilik
pengetahuan tradisi dapat dilangsungkan dengan jayanya.
Ucapan penghargaan ikhlas saya terhadap usaha dan sokongan yang
berterusan daripada Institut Penyelidikan Perhutanan Malaysia (FRIM) dan
semua rakan agensi yang lain dalam menjayakan persidangan kali ini. Selamat
maju jaya.

Y.B. Datuk Ir. Haji Hamim bin Samuri

Timbalan Menteri Sumber Asli dan Alam Sekitar (NRE)

xiii
Kata Alu-Aluan
Terlebih dahulu saya ingin mengalu-alukan kehadiran tuan-puan ke Seminar
Tumbuhan Ubatan dan Beraroma ke-14 yang bertemakan, “Penawar daripada
Alam Semula Jadi: Trend dan Perspektif Semasa”. Ucapan terima kasih kepada
pembentang kertas kerja, wakil industri dan pengusaha-pengusaha herba, para
penyelidik, ahli akademik serta wakil komuniti yang sudi meluangkan masa
untuk berkongsi ilmu dan pengalaman dalam persidangan ini demi kebaikan
semua.
Seperti yang kita ketahui, permintaan produk berasaskan herba di
peringkat global semakin bertambah. Walau bagaimanapun, lambakan produk
herba di pasaran tempatan, khususnya produk yang tidak bersandarkan
penyelidikan serta dicemari dengan bahan beracun harus diberi penekanan.
Fokus juga perlu diberi untuk menghasilkan produk berasaskan herba yang
berkualiti tinggi, berkesan dan selamat bagi memastikan industri herba negara
mampu bersaing di pasaran global yang semakin terbuka.
Kini, tiba masanya untuk semua golongan yang terlibat dalam industri
herba untuk berganding bahu dan berkongsi ilmu pengetahuan. Persidangan ini
menyediakan platform untuk individu dan organisasi berkongsi pengetahuan,
pengalaman dan peluang baharu demi memperkasa industri herba negara ke
tahap yang lebih tinggi. Saya berharap melalui persidangan ini, semua pihak
yang terlibat akan menggunakan kekuatan negara termasuk sumber
kepelbagaian biologi serta pengetahuan kepelbagaian etnik untuk menghasilkan
produk-produk baharu yang bukan sahaja berdaya saing tinggi malah menepati
cita rasa pengguna.
Akhir kata, saya merakamkan setinggi-tinggi penghargaan kepada semua
pihak yang terlibat dalam menganjurkan persidangan kali ini dan berharap agar
kerjasama yang terjalin akan diteruskan demi kecemerlangan industri herba
negara. Selamat maju jaya.

Y.Bhg. Dato’ Dr Abdul Latif bin Mohmod

Ketua Pengarah Institut Penyelidikan Perhutanan Malaysia (FRIM)

xiv
 Programme
DAY 1: 11 October 2016

8.00 am Registration
8.45 am Arrival of VIP and Invited Guests
9.00 am Arrival of YB Deputy Minister of Natural Resources and
Environment (NRE)
9.05 am National Anthem : Negaraku & Doa Recital
9.15 am Director General of FRIM Opening Speech
9.25 am YB Deputy Minister of NRE Speech and Opening Ceremony
9.40 am ‘Gunung Ledang Malaysia’ Book Launch
9.50 am FRIM-MARA Product Launch
10.00 am Graduation Certificate Presentation –
Program Teknousahawan FRIM-MARA
10.15 am Video Montage
10.20 am Visit to Exhibition & Posters and Press Conference

PLENARY 1 Moderator: Dr Ismail Harun TKP(P) (FRIM)

11.00 am Prof Ibrahim Jantan (UKM)
Integrated Approach in Natural Products Research to Discover
New Drug and Develop Herbal Medicines and Health
Products.

SESSION 1 ETHNOBOTANY, AGRONOMY & CONSERVATION

Moderator: Dr Nor Azah Mohd Ali (FRIM)
Invited Papers
11.45 am Invited 1: Dr Mohd Zaki Abdullah (FRIM)
Industri Herba: Bahan Tanaman Berkualiti
12.15 pm Invited 2: En Mohammad Redza Baba (DOA)
Program Pemulihan Tanaman Kacip Fatimah oleh Jabatan
Pertanian
12.45 pm Q & A Session
1.00 pm Lunch and Poster Session

Oral Papers
2.00 pm Oral 1: Cik Noorsiha Ayop (FRIM)
Pembangunan Cameron Highlands Montane Park (CHiMP)
bagi Memperkasakan Program Konservasi Ex-situ Tumbuhan
Ubatan Tanah Tinggi di Semenanjung Malaysia.

xv
2.15 pm Oral 2: Pn Zahora Ismail (UPM, Bintulu)
Kepentingan Tumbuhan Ubatan dan Beraroma di Kalangan
Masyarakat Melanau di Mukah, Sarawak.
2.30 pm Oral 3: Pn Nor Fadilah Wook (FRIM)
Breeding Strategies of Eurycoma longifolia: Present and
Future.
2.45 pm Oral 4: Dr Nor Hasnida Hassan (FRIM)
Penghasilan Bahan Tanaman Tumbuhan Ubatan
Menggunakan Teknologi Kultur Tisu.
3.00 pm Q & A Session

SESSION 2 NATURAL PRODUCT DISCOVERY

Moderator: Prof Ibrahim Jantan (UKM)
Invited & Oral
Papers
3.15 pm Invited 1: Prof Aishah Adam (UiTM)
Plant Extracts in Anticancer Treatment: Proof and Myths
3.45 pm Invited 2: Prof Wan Rosli Wan Ishak (USM)
The Powess of Oyster Mushroom (Pleurotus sajor-caju, PSC)
as an Alternative Nutritive Functional Ingredient.
4.15 pm Invited 3: Dr Nurhanan Murni Yunos (FRIM)
Discovering the Anti-ovarian Cancer Potential of a Cardiac
Glycoside Derivative Using In Vitro, In Silico and Proteomics
Approaches.
4.45 pm Oral 1: Pn Fauziah Abdullah (FRIM)
Purification of Flavonoid Methyl Ether from Tetracera indica
Using Automated Chromatographic Approach.
5.00 pm Oral 2: En Mohamad Hussin Zain (UMT)
Xanthine Oxidase Inhibitory Activity of Flavonoid Compound
from Hibiscus rosa-sinensis L. and Brassica oleracea L.
5.15 pm Q & A Session
5.30 pm Afternoon Tea & End of Day 1

xvi
 DAY 2: 12 October 2016

SESSION 3 STANDARDISATION, QUALITY CONTROL &

PROCESSING TECHNOLOGY
Moderator: Dr Ling Sui Kiong (FRIM)
Invited & Oral
Papers
8.30 am Invited 1: Prof Zhari Ismail (USM)
Status Pemiawaian dalam Industri Herba Tempatan
9.00 am Invited 2: En Ong Boo Kean (FRIM)
FRIM’s Commitment Towards Local Herbal Industry: Quality
Control of Herbal Product
9.30 am Invited 3: En Mohd Shahidan Mohd Arshad (FRIM)
Herbal Product Deliverables: Seeds to Shelves
10.00 am Oral 1: Pn Hada Masayu Ismail (FRIM)
Effects of Temperature on Drying of Eurycoma longifolia
Jack
Roots: Drying Time and Product Quality.
10.15 am Q & A Session
10.30 am Tea Break & Poster Session

PLENARY 2 Moderator: Dr Rasadah Mat Ali (FRIM)

11.00 am Dr Abdul Ghani (Herbwalk Consultancy)
Relevance of Traditional Knowledge in Herbal Research

SESSION 4 MARKETING, TRADE, BUSINESS POTENTIAL &

LEGISLATION
Moderator: En Mohd Shahidan Mohd Arshad (FRIM)
Invited Papers
11.45 am Invited 1: Tn Hj Shahrizin Abdul Sarhadat (MARA)

12.15 pm Invited 2: En Mohamad Faisal Ahmad Fadzil (Tanamera)

Selling Our Story to the World: Branding with What We
Have Best.
12.45 pm Q & A Session
1.00 pm Lunch and Poster Session

Invited & Oral

Papers
2.00 pm Invited 3: En Faizzudin Serimasran (BioAlpha)
Commercialisation Opportunity with BioAlpha

xvii
2.30 pm Oral 1: Dr Mastura Mohtar (FRIM)
Kembara Penyelidikan, Pembangunan dan Pengkomersialan
Rangkaian Produk Disinfektan dan Antiseptik Mesra Alam
2.45 pm Q & A Session

SESSION 5 PRECLINICAL, CLINICAL & PRODUCT DEVELOPMENT

Moderator: Prof Zhari Ismail (USM)
Invited Papers
3.00 pm Invited 1: Prof Madya Dr Aman Shah Abdul Majid (QIUP)

3.30 pm Invited 2: Ms Vanessa Shalini Daniel (Pharmaniaga)

Challenges and Experiences in Conducting Preclinical Studies
for Herbal Products: Pharmaniaga Perspective
4.00 pm Invited 3: Prof Johnson Stanlas (UPM)
Andrographolide Derivatives Target the Elusive Oncogenic
K-Ras: Promising Compounds to Combat K-Ras-Driven
Malignancies
4.30 pm Q & A Session
4.45 pm Award Presentation & Closing Ceremony
5.15 pm Afternoon Tea & End of Seminar

xviii
 PROGRAM SIMBION BIOHERBA FRIM-MARA
Objektif:

Menjayakan aspirasi Program Transformasi Ekonomi Negara. Kerjasama

melibatkan penawaran pakej pengkomersilan komoditi bernilai tinggi bagi
mengupaya dan memperkasa usahawan teknopreneur glokal. MARA
menyediakan 90% dana keseluruhan manakala FRIM bertindak selaku penyedia
teknologi dan penyelia program.

Penyertaan & Output Projek:

 Produk Mandian `Head to Toe’ dan  Rangkaian Produk Herba untuk

Losyen Berantiseptik Penjagaan Ibu Bersalin
 Syarikat Nature Profusion Sdn. Bhd.  Syarikat Bionature Formula Sdn. Bhd

 Produk Biskut Serat Berkhasiat  Pengekstrakan Skala Komersil Bahan

 Syarikat Nourish Care Sdn. Bhd. Aktif Polisakarida
 Syarikat Poly-Xtract Sdn. Bhd.

xix
 PROGRAM TEKNOUSAHAWAN FRIM-MARA
Objektif:

 Memberikan pendedahan dan pengetahuan mengenai teknologi pemprosesan

& penghasilan produk terkini kepada usahawan industri berasaskan herba.

 Merealisasikan “Proof of Concept” (POC) daripada pihak usahawan kepada

produk-produk herba bermutu tinggi melalui pendekatan R&D.

 Membantu mengeluarkan produk-produk berkualiti mematuhi peraturan

pemasaran tempatan dan antarabangsa mengikut kategori produk

 Membantu menaiktaraf produk yang sedia ada dari usahawan menggunakan

pendekatan saintifik.

Penyertaan & Output Projek:

 Pembangunan Produk Kosmetik  Pembangunan Produk Minuman

Berasaskan labu Berasaskan bidara
 The Cucurbits Company Sdn. Bhd.  Syarikat TG Green Tech Industry Sdn.
Bhd. Bhd

 Produk ubatan tradisional  Produk ubatan tradisional

berasaskan tunjuk langit. berasaskan asam gelugor.
 Syarikat Tyzo Sdn. Bhd.  Syarikat Zulkifli Bamadhaj Sdn. Bhd.

xx
PLENARY LECTURES

1
INTEGRATED APPROACH IN NATURAL PRODUCTS RESEARCH TO
DISCOVER NEW DRUG LEADS AND DEVELOP HERBAL MEDICINES AND
HEALTH PRODUCTS

Ibrahim, J.

Drug and Herbal Research Center, Faculty of Pharmacy, Universiti Kebangsaan

Malaysia, Jalan Raja Muda Abdul Aziz, 50300 Kuala Lumpur, Malaysia

ABSTRACT

Drug discovery involves integration of enormous spectrum of research activities,

beginning from initial target identification and validation, through assay
development, high throughput screening (HTS), hit identification, lead
optimization and finally the selection of a candidate molecule for clinical
development. In the past, majority of new drugs have been discovered by direct
isolation from natural sources and molecular modification of natural products.
The single target or bullet-based approach has been the dominant paradigm to
discover natural small molecules from natural resources as new leads or models
for the development of synthetic molecules for the discovery of drug targets.
However, the reductionist approach in finding bioactive compounds from the
tropical rainforests at several major pharmaceutical companies had generally
declined due to the many major hurdles faced by them such as difficulties in
obtaining sufficient supply of high quality natural products screening libraries,
ownership issues and research in this field is lengthy, expensive, highly complex
and ineffective with low success rate. Natural products drug discovery has been
marginalized in favor of the rational design of synthetic compounds to target
specific molecules after the advent of HTS, combinatorial chemistry and
advancement in the knowledge of molecular mechanisms, cell biology and
genomics. However, there is a recent revival of interest in natural products
research due to disappointing results of combinatorial chemistry and HTS in
delivering potent chemical leads. The emergence of new technologies in mass
spectrometry, NMR and other spectroscopic techniques, and the advances in
screening methodologies, the development of molecular biology and
biotechnology, the use of computer technology in rational drug design are
already playing major roles in natural products research. Recently there is a
growing interest to use innovative approaches to drug discovery from natural
products by network pharmacology. The integrated multidisciplinary concept of
multiple targets, multiple effects and complex diseases in network
pharmacology have enriched our understanding of complicated pathogenesis
and multi-target pathologies of systemic diseases and reduced difficulty in
identifying relevant interventions to target such complexities. The ‘-omic’
technologies in system biology have now been widely used to correlate and

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elucidate multiple targets and network of human diseases and drug actions.
Herbal medicines may serve as valuable resources for network-based multi-
target drug discovery. Multi-target drugs could be developed from herbal
extracts by first evaluating the efficacy of the extracts, followed by identification
of their major bioactive components and redevelopment of a completely new
multi-component formulations composed of the major bioactive components in
order to reach a synergistic and optimal combination. In this paper, the use of
integrated approach to discover new drug leads, and development of herbal
medicines and health products will be discussed. A review of the past six
decades of scientific interests and advances in natural products research in
Malaysia will be presented and recent work on the phytochemistry, biochemical
and pharmacological activities of some medicinal plants carried out by the
author will be illustrated as examples.

Key words: New drug leads, natural product research, herbal medicine, health
products, medicinal plants

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FROM TRADITIONAL KNOWLEDGE TO THE LABORATORY

Abdul Ghani, H.

Herbwalk Consultancy,
Batu 8, Jalan Ayer Hangat,
Mukim Ayer Hangat, 07000 Langkawi, Kedah.

ABSTRACT

The tireless process of documenting, understanding, correctly interpreting

traditional knowledge on the art of making medicine is very essential in correctly
directing laboratory research in eliciting pharmacological activities of medicinal
plants. I have been frequently asked why results in herbal research is very
inconsistent. The reason to this is that many of the researches done did not take
into consideration many aspects of medicinal uses as per those done by the
custodian of traditional knowledge. In order to make effective medicines, the
traditional practitioners manipulate and formulate the plant materials in
manners that would bring out the effects they want. Only those who had learnt
this fine art during their training would be able to rightfully make consistent
medicine that is effective all the time. If only modern researchers take the pain
to study these methods, then many of their efforts would be fruitful and
successful. This presentation would be an attempt to share some of these
essentials as I understand it with the research community.

Key words: traditional knowledge, traditional practitioners, herbal research,

medicinal plants,

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ETHNOBOTANY, AGRONOMY
&
CONSERVATION

5
INDUSTRI HERBA: BAHAN TANAMAN BERKUALITI

Mohd Zaki, A.

Program Membiak-biak Tumbuhan,

Bahagian Bioteknologi Perhutanan,
Institut Penyelidikan Perhutanan Malaysia,
FRIM 52109 Kepong Selangor

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PROGRAM PEMULIHAN TANAMAN KACIP FATIMAH OLEH JABATAN
PERTANIAN

Mohamed Redza, B.

Seksyen Tanaman Industri, Aras 12,

Bahagian Padi, Tanaman Industri dan Florikultur,
Jabatan Pertanian, Wisma Tani,
No 30, Persiaran Perdana, Presint 4,
62624 Putrajaya

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PEMBANGUNAN CAMERON HIGHLANDS MONTANE PARK (CHiMP) BAGI
MEMPERKASAKAN PROGRAM KONSERVASI EX-SITU TUMBUHAN
UBATAN TANAH TINGGI DI SEMENANJUNG MALAYSIA

Noorsiha, A., Norliyana, A.L., Nuranis Suraya, B. & Ainnur Amira, A.M.

Bahagian Perhutanan dan Alam Sekitar, Institut Penyelidikan Perhutanan

Malaysia, 52109 Kepong, Selangor

Tel: 03-6279 7618 / 03-6279 7277 Emel: noorsiha@frim.gov.my

PENGENALAN

Ekosistem kawasan tanah tinggi mewakili kira-kira 5% keluasan tanah di

Semenanjung Malaysia. Rupabentuk mukabumi kawasan pergunungan ini
membentuk seakan-akan seperti tulang belakang yang memisahkan kawasan
pantai barat dan kawasan pantai timur. Antaranya Banjaran Titiwangsa,
Banjaran Bintang, Banjaran Keledang, Banjaran Nakawan di mana gugusan
puncak gunung tertinggi melakari banjaran-banjaran ini seperti Gunung Tahan,
Gunung Ulu Kali, Gunung Ledang, Gunung Brinchang, Gunung Jerai, Gunung
Korbu, Gunung Hijau dan sebagainya. Hutan tanah tinggi diklasifikasikan sebagai
sebahagian dari formasi hutan daratan utama dan terbahagi kepada tiga jenis
iaitu Hutan Gunung Rendah, Hutan Gunung Tinggi dan Vegetasi Sub-Alpine.
Hutan gunung rendah atau dikenali sebagai lower montane forest, hutan
gunung oak-laurel atau cloud forest menduduki kawasan beraltitud antara 800
meter hingga 1,500 meter dari paras laut. Hutan gunung tinggi, upper montane
forest atau hutan gunung ericaceous pula menduduki paras ketinggian melebihi
1,500 meter dari paras laut. Manakala vegetasi sub-alpine berada pada paras
ketinggian melebihi 13,000 kaki dari paras laut. Hutan tanah tinggi ini amat
berbeza jika dibandingkan dengan kawasan hutan tanah pamah. Ciri utama
hutan tanah tinggi ini ialah mempunyai purata ketinggian silara pokok antara 15
– 35 meter, mengandungi kumpulan tumbuhan berdaun kecil seperti jarum,
berbanir kecil serta kaya dengan pelbagai tumbuhan epifit atau penumpang.
Sebaliknya hutan tanah tinggi kurang mempunyai spesies berdaun majmuk,
pemanjat berkayu atau liana, tumbuhan kauliflori atau berbunga dan berbuah di
batang serta mengandungi komposisi spesies yang rendah berbanding hutan
tanah pamah. Namun, hutan tanah tinggi merupakan salah satu ekosistem
terpenting kerana mewakili pelbagai spesies terkhusus dan unik. Antara
kumpulan flora utama yang sering dijumpai di ekologi pergunungan ini adalah
kaum clusiaceae atau kaum kandis, fagaceae atau kaum berangan, lauraceae
atau kaum medang, pteridophytes atau kaum paku pakis, bryophytes atau kaum
lumut, kaum orchidaceae atau orkid gunung liar yang unik serta pelbagai spesies
palma, ginger, aroid yang tumbuh di kawasan lantai hutan. Berdasarkan laporan,

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kira-kira 3,300 spesies flora negara hanya ditemui di kawasan pergunungan
Semenanjung Malaysia dan mewakili 22% komposisi flora semula jadi di negara
ini. Bahkan status sebahagian besar dari komposisi flora gunung ini turut
diklasifikasikan sebagai tumbuhan endemik, lanka atau jarang-jarang ditemui,
terancam atau hampir pupus.

Umum mengetahui bahawa wujud pelbagai ancaman ke atas ekosistem

pergunungan kesan dari tekanan populasi, penggunaan tanah akibat aktiviti
pertanian dan hortikultur, industri pelancongan dan sebagainya. Ancaman ini
mengakibatkan impak dan kesan negatif yang teruk ke atas ekologi hutan tanah
tinggi yang sensitif ini. Natijahnya, perlu diwujudkan segera penubuhan sebuah
pusat konservasi tanah tinggi di Semenanjung Malaysia yang bertindak sebagai
one stop centre bagi program penyelidikan, pemuliharaan serta pendidikan dan
kesedaran alam sekitar berteraskan biodiversiti pergunungan. Ini penting agar
pelbagai isu ancaman dan kepupusan biodiversiti dan kemerosotan kualiti
ekosistem pergunungan negara yang sangat sensitif ini dapat ditangani dan
dikurangkan. Inisiatif sebegini perlu juga dilaksanakan segera sejajar dengan
komitmen kerajaan dalam menangani isu pemeliharaan dan pemuliharaan alam
sekitar bagi tujuan penghijauan kawasan tanah tinggi seperti Program Cameron
Highlands Dreams (CHD) di bawah pemantauan pihak Kementerian Sumber Asli
dan Alam Sekitar (NRE). Strategi ini juga penting dalam usaha untuk
mengembalikan dan mengukuhkan daerah Cameron Highlands sebagai destinasi
penting dan ikon pelancongan utama negara bagi kawasan tanah tinggi di
Malaysia. Juga perlu diwujudkan ialah satu platform bagi mendorong dan
memupuk nilai kesedaran dan kebertanggungjawaban masyarakat terhadap
mengekalkan kelestarian kawasan tanah tinggi di negara ini.

RANGKA KERJA

Keperluan membangunkan sebuah tapak konservasi ex-situ sebagai pusat

pengumpulan flora gunung negara berjaya direalisasikan menerusi penubuhan
projek kerjasama antara FRIM, Jabatan Perhutanan Semenanjung Malaysia
(JPSM) dan Jabatan Perhutanan Negeri Pahang (JPNP) yang dikenali sebagai
Projek Taman Gunung Cameron Highlands atau Cameron Highlands Montane
Park (CHiMP). Program kolaborasi penyelidikan dan pemuliharaan kawasan
tanah tinggi yang julung kali dijalankan di Semenanjung Malaysia ini telah
dimeterai menerusi dokumen Nota Kerjasama antara FRIM-JPN Pahang pada
Januari 2016 yang lalu. Kewujudan pusat rujukan biodiversiti pergunungan yang
berkonsepkan ”taman dalam persekitaran tanah tinggi” sebenarnya amat
relevan kepada keperluan para saintis dan pencinta kawasan tanah tinggi dalam
dan luar negara.

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 Lokasi CHiMP terletak antara 4o 28.324’ hingga 4o 28.487’ Utara dan 101o
22.946’ hingga 101o 23.081’ Timur di kawasan Hutan Lipur Parit Falls, Hutan
Simpan Hulu Bertam pada paras ketinggian antara 1,400 – 1,500 meter dari
paras laut. Terletak berhampiran Pejabat Hutan Daerah Cameron Highlands dan
boleh diakses oleh orang awam samada dengan menaiki kenderaan atau dengan
berjalan kaki dari pekan Tanah Rata ke pekan Brinchang dengan mengambil
laluan ke Taman Sedia. Satu program survei sempadan telah dijalankan bagi
menentusahkan kedudukan CHiMP secara terperinci bagi menghasilkan peta
kawasan seluas 10 ha. yang terdiri dari lima (5) zon utama iaitu zon konservasi
ex-situ, zon konservasi in-situ, zon rekreasi dan riadah, zon pendidikan alam
sekitar serta zon pengurusan dan fasiliti penyelidikan. Tapak lokasi ini memiliki
ciri-ciri yang sesuai untuk dimajukan berdasarkan kewujudan pelbagai flora
spesies gunung yang menarik, kemudahan infrastruktur seperti jalanraya,
tempat letak kereta, kawasan penempatan masyarakat, denai hutan, kawasan
riadah, kemudahan tandas awam serta merupakan lokasi tumpuan para
pencinta alam dan pelancong dari dalam dan luar negara. Pembangunan fizikal
tapak CHiMP telah menggunapakai dana RMK-10 sumbangan pihak SUK-Negeri
Pahang Darul Makmur yang disalurkan menerusi JPN Pahang terdiri dari
pembinaan pejabat operasi projek, pondok pengawal, tembok pintu masuk
utama dan pagaran, ruang taklimat pelawat, laluan pejalan kaki dan golongan
OKU, gazebo dan ruang rehat, trelis, pergola dan struktur rumah orkid serta
fasiliti tapak semaian. Fasa pembangunan Projek CHiMP melibatkan:

Fasa 1 (2006 – 2010) - Penubuhan dan Pembangunan Awal

Fasa 2 (2011 – 2015) - Pembangunan Lanjutan
Fasa 3 (2016 – 2020) - Kematangan Projek

Program pembangunan koleksi tumbuhan hidup bagi tujuan konservasi

ex-situ telah dijalankan dari masa ke semasa di sekitar kawasan tanah tinggi
utama di Semenanjung Malaysia melibatkan 30 kawasan hutan simpan di sekitar
negeri Johor, Kedah, Kelantan, Pahang, Perak, Perlis, Pulau Pinang dan
Terengganu. Secara umumnya, aktiviti pembangunan koleksi ex-situ ini
mengumpulkan sampel dari tumbuhan yang kerap menduduki kawasan lantai
hutan terdiri dari kumpulan monokot dan dikot seperti famili Araceae,
Begoniaceae, Gesneriaceae, Liliaceae, Nepenthaceae, Orchidaceae, Palmae,
Zingiberaceae dan sebagainya. Koleksi sampel-sampel hidup ini akan diproses
dan dibungkus dengan cermat dan berhati-hati semasa di lapangan bagi
memastikan kualiti kutipan terjamin. Satu standard operational procedure (SOP)
dalam mengendalikan koleksi bermula dari lapangan pengutipan sehingga ke
tapak CHiMP perlu dipatuhi dengan baik bagi mengurangkan kadar mortaliti.

Selepas aktiviti pengutipan, kesemua koleksi hidup ini perlu dibawa

segera ke tapak CHiMP untuk ditabung dan dirawat di tapak semaian
menggunapakai good nursery practices. Koleksi yang ditabung akan menjalani

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proses aclimatise dan hardening atau pengerasan selama 6-12 bulan di tapak
semaian sebelum bersedia sebagai bahan tanaman. Seterusnya, bahan tanaman
tersedia ini akan menjalani aktiviti penanaman di laman-laman pameran atau
showcase gardens CHiMP di bawah pembangunan landskap lembut atau
softscapes. Laman pameran ini disusunatur mengikut kumpulan floristik dan
diselenggara secara harian termasuk memantau aspek pest and disease (P&D).
Catatan data kutipan di lapangan dan laman pameran perlu didokumentasikan
dari masa ke semasa menerusi sistem pangkalan data Projek CHiMP. Aktiviti
pengecaman diperlukan bagi melabel tanaman dengan nama saintifik yang betul
demi manfaat pembangunan maklumat dan digunapakai bagi aktiviti pendidikan
botanikal CHiMP yang digemari di kalangan pelawat dan pelajar.

Program pembangunan koleksi ex-situ flora tanah tinggi ini turut

memfokuskan kumpulan tumbuhan ubatan termasuk spesies etnobotanikal
yang berguna kepada manusia. Pengumpulan sampel flora ini amat penting
untuk dijadikan asas rujukan bagi aktiviti pembangunan pelbagai produk
sebatian semula jadi berasaskan tumbuhan tanah tinggi di Semenanjung
Malaysia.

HASIL KERJA

Sehingga kini, projek CHiMP dianggarkan telah berjaya mengumpul dan

mendokumentasi sejumlah 5,595 nombor koleksi di kebanyakan banjaran
pergunungan utama Semenanjung Malaysia. Pembangunan landskap lembut
pula berjaya mempamerkan hampir 10,000 jumlah individu tanaman di 14 buah
showcase gardens CHiMP mewakili kaum ginger, aroid, asam batu,
rhododendron, orkid liar terrestrial, orkid epifitik, tumbuhan hiasan, buluh, paku
pakis, palma, tumbuhan herba, periuk kera dan pelbagai jenis pepanjat hutan.
Secara keseluruhan, dianggarkan jumlah spesies terkumpul adalah 550 taksa
mewakili kira-kira 17% jumlah keseluruhan flora kawasan tanah tinggi di
Semenanjung Malaysia.

Hasil pembangunan koleksi ex-situ ini juga berjaya mengenalpasti flora

tanah tinggi yang berguna sebagai sumber makanan tradisional, tumbuhan
hiasan, tumbuhan beraroma, untuk upacara ritual dan mengandungi nilai
ubatan yang berpotensi dalam pembangunan produk sebagai rempah-ratus
gunung. Dianggarkan lebih dari 50% flora etnobotanikal terkumpul di tapak
Projek CHiMP ini mampu dikategorikan sebagai tumbuhan berstatus ubatan dan
sebahagian besar turut berpotensi diketengahkan bagi tujuan pengkomersialan.
Antara genera utama tumbuhan ubatan yang dijumpai di tapak CHiMP ini adalah
Homalomena spp. (Kelemoyang gunung), Piper spp. (Sireh gunung), Begonia
spp. (Asam batu), Labisia spp. (Kacip fatimah gunung), Ficus deltoidea (Mas
cotek), Cheilocostus speciosus (Setawar hutan), Pentaphragma aurantiacum
(Salang serong), Dipteris conjugata (Payung ali), Phyllagathis hispida (Tapak

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gajah), Dianella javanica (Siak-siak), Mapania petiolata (Serapat), Rhododendron
javanicum (Rhododendron), Elettariopsis curtisii (Pijat-pijat) termasuk pelbagai
spesies halia gunung yang beraroma.

Manakala pembangunan penyelidikan tanah tinggi di CHiMP kini

menumpu kepada kajian dalam bidang taksonomi tumbuhan peringkat tinggi,
pembangunan koleksi biji benih, aspek hortikultur serta fitokimia bagi
pembangunan produk. Jaringan kerjasama penyelidikan melibatkan bidang
kajian biodiversiti tanah tinggi antara FRIM, pihak agensi kerajaan, NGO dan
pihak universiti merupakan inisiatif projek di masa hadapan. Penerbitan pelbagai
data saintifik turut dizahirkan dalam bentuk kertas kerja saintifik dan buku
seperti Preliminary Checklist Montane Flora of Peninsular Malaysia dan Colours
of CHiMP: Botanical Illustration termasuk cadangan penulisan Monograph of
Montane Medicinal Forest Species bagi kawasan tanah tinggi di Semenanjung
Malaysia.

RUMUSAN

Program pembangunan koleksi ex-situ flora tanah tinggi melibatkan proses

pengumpulan dan pendokumentasian penting bagi aspek penyelidikan dan
pemuliharaan ekosistem tanah tinggi di Semenanjung Malaysia. Inisiatif
penubuhan CHiMP sebagai sebuah pusat pameran dan rujukan flora kawasan
tanah tinggi pertama di Semenanjung Malaysia amat relevan bagi menampung
keperluan penyelidikan, pemuliharaan, pendidikan dan kesedaran alam sekitar
agar pertumbuhan hijau kawasan tanah tinggi serta pemerkasaan industri eko-
pelancongan Cameron Highlands dapat direalisasikan. Rangka kerja
pembangunan projek CHiMP ke arah memperkasakan pemuliharaan biodiversiti
dan eko-pelancongan Cameron Highlands berjaya direalisasikan di bawah dana
RMK-11 bagi tahun 2017-2020 menerusi Projek Pemuliharaan Biodiversiti Ke
Arah Pertumbuhan Hijau Kawasan Tanah Tinggi Bagi Memperkasakan Industri
Eko-Pelancongan Semenanjung Malaysia. Usaha pengumpulan koleksi ex-situ
yang dilaksanakan di tapak CHiMP ini juga penting dalam memenuhi keperluan
pembangunan koleksi tumbuhan ubatan negara agar dapat dijadikan asas bagi
pembangunan produk sebatian semula jadi berasaskan flora tanah tinggi yang
amat berpotensi diketengahkan bagi tujuan pengkomersialan.

RUJUKAN

Nuratikah, A. (2014), Laporan Projek CHiMP- Pembangunan Koleksi ex-situ dan

Landskap Lembut Taman.
Noorsiha, A., Ainnur Amira, A.M., Kamariah, M., Ng, S.C. & Ajeera, T. (2015).
Senarai Semak Awal Flora Gunung di Semenanjung Malaysia. Cameron

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 Highlands Montane Park (CHiMP). Institut Penyelidikan Perhutanan
Malaysia (FRIM).
Noorsiha, A., Nurliyana, A.L., Ng, S.C., Nuranis Suraya, B., Ainnur Amira, A.M.,
Kamariah, M., & Ajeera, T. (2016). Pangkalan Data Cameron Highlands
Montane Park (CHiMP). Institut Penyelidikan Perhutanan Malaysia (FRIM).
Padua, L.S., Bunyapraphatsara, N. & Lemmens, R. H. M. J. (Eds.). (1999). Plant
Resources of South-East Asia No 12(1). The Prosea Foundation, Bogor,
Indonesia. Pp. 705.
Van Valkenburg, J. L. C. H. & Bunyapraphatsara, N. (Eds.). (2001). Plant
Resources of South-East Asia No 12(2). Medicinal and Poisonous Plants.
The Prosea Foundation. Backhuys Publishers, Leiden. Pp. 776.
Lemmens, R. H. M. J. & Bunyapraphatsara, N. (Eds.). (2003). Plant Resources of
South-East Asia No 12(2). Medicinal and Poisonous Plants. Prosea
Foundation, Backhuys Publishers, Leiden. Pp. 776.

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KEPENTINGAN TUMBUHAN UBATAN DAN BERAROMA DI KALANGAN
MASYARAKAT MELANAU DI MUKAH, SARAWAK

Zahora, I.

Pusat Kecemerlangan Ekosistem Sains Boreneo (BORNEO EKOSAINS)

Fakulti Sains Pertanian dan Makanan, Universiti Putra Malaysia,
Kampus Bintulu Sarawak, 97008 Bintulu, Sarawak, Malaysia.

E-mel: zahora_i@upm.edu.my

ABSTRAK

Kajian ini dijalankan dengan tujuan untuk mendokumenkan penggunaan spesies-

spesies tumbuhan ubatan beraroma yang sering digunakan oleh masyarakat
Melanau dalam kehidupan seharian. Bidang kajian akan meliputi aspek
perubatan, makanan dan juga adat istiadat kaum Melanau. Maklumat diperolehi
daripada kaedah soal selidik, melalui perbualan dengan pengamal perubatan
tradisional dan tinjauan secara langsung. Melalui kajian ini, kita dapat melihat
kebergantungan suatu kominuti kepada tumbuh-tumbuhan dalam pelbagai cara.
Oleh yang demikian, mengenalpasti tumbuhan yang sering digunakan oleh
komuniti Melanau bukan sahaja membantu dalam pemeliharaan budaya
masyarakat Melanau malah ianya juga dapat memelihara khazanah semula jadi
dan pengetahuan tradisi bagi kaum tersebut.

Kata kunci: Tumbuhan ubatan, Melanau, Mukah, Sarawak

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BREEDING STRATEGIES OF EURYCOMA LONGIFOLIA: PRESENT & FUTURE

Nor Fadilah, W., Mohd Zaki, A. & Mohd Lokmal, N.

1
Plant Improvement Programme of Forest Research Institute Malaysia (FRIM),
52109, Kepong, Selangor, Malaysia.

Email: norfadilah@frim.gov.my

INTRODUCTION

Nowadays, most of the raw materials of E. longifolia needed for the herbs
industries, especially from the root parts, are obtained from natural forest and
very few are planted on commercial basis. The problem arises when the market
demands for raw materials of this species increases dramatically but the
plantation plots are nowhere to be seen. Consequently, it has resulted in over
exploitation of the plant species from its natural habitats which are the forest
reserve areas. This practice in the long run could endanger the plant species
diversity and also impractical in term of the economic aspect.

To conserve biodiversity in tropical rainforest, it is a need to not utilize E.

longifolia from the wild. Thus, it is important to provide a solution for the
problems of producing raw materials of E. longifolia and to ensure the genetic
diversity of this species is not in danger. Adapting from the success breeding
history of rubber and oil palm species in Malaysia, the first step to be taken is to
provide the necessary breeding information for the plant species in order to
produce high quality planting materials especially for commercialisation and in
breeding strategies, provenance trial is the first step.

For now, plant breeding information of this species is still in its early stages.
Currently, the raw materials obtained from forest reserve areas are varied in
their quality; growth and phytochemical contents. With the provenance trial
assessment, the information of high quality planting materials would be very
beneficial for the industries. A provenance performance trial is an experiment
established in a statistically valid design to compare any characteristics of
interest among provenances. Provenance trials are established to study the
growth variation between and within populations.

Thus, provenance trial of E. longifolia is conducted with the aims to cater the
needs for breeding information and conservation strategies. The main objective
of conducting this trial is to identify the most suitable provenance or
provenances for introduction to afforestation programmes. Among the
important characteristics to be considered in the selection of the best

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provenance are; growth performances, tolerance to pests and diseases,
physiological adaptation to the site (Hettasch 2002) and phytochemical contents
(Rasheed et al. 2012).

MATERIALS AND METHODS

Growth Performance of E. longifolia Provenances

Seeds and wildings of E. longifolia were collected from 27 locations of natural

forest reserve areas throughout Peninsular Malaysia and a total of 10
provenances [Pahang A, Pahang B, Pahang C, Pulau Pinang, Terengganu,
Selangor, Johor, Melaka, Perak and Pahang D] were grouped according to the
distances between each location.

The trial plots were established at four FRIM sub-stations, namely SPF Mata
Ayer, Perlis; SPF Jeli, Kelantan; SPF Maran, Pahang, and University Technology
Malaysia (UTM) Skudai, Johor. All these plots represent different regions in
terms of climatic and environmental conditions in Peninsular Malaysia.

Randomized Complete Block Design (RCBD) was adopted in each trial replicated
in three blocks. Each treatment plot was planted with 16 (4 x 4) plants at the
spacing of 2 m x 2 m. The area for each unit plot is 6 m x 6 m = 36 m 2. A total of
160 seedlings will be assigned in each block making a total of 480 seedlings per
trial plot. The trial plots are having a length of 38 m x 46 m. The total area of
each trial plot is approximately 0.2 hectares.

The parameters measured were quantitative data which includes growth in

height and diameter at collar region at 3 months interval with the first data
taken after a month planting. While qualitative data such as pest occurrence,
the presence of tiger moth and spider-mites were observed and recorded.

The data were statistically analysed by Analysis of Variance (ANOVA) to

determine the variation of growth performances (height and diameter at collar
region) among provenances by Duncan Multiple Range Test (DMRT) to
determine the significance differences of means among provenances. All
statistical analyses were carried out using SPSS statistical packaged.

Preliminary Phytochemical Screening of Eurycomanone

Plant materials of E. longifolia were harvested from natural forest reserve areas
of eight different provenances which were Johor, Melaka, Pahang B, Perak,
Pulau Pinang, Selangor and Terengganu. The size of plant samples taken was
standardised. The dried root, stem and leaves parts were extracted with water

16
19
extraction method. The extracts were analyzed by Ultra Performance Liquid
Chromatography (UPLC), Waters. The size of the column used was 5 cm long
with a diameter of 2.1 cm. As for the binary solvent manager, 0.05% phosphoric
acid and acetonitrile were used. The run time for each samples was about 8
minutes with 5 injections per sample (there were total of 3 replicates per
samples of each root, stem and leaves part). Eurycomanone compound is
detected at 244 nm wavelength with retention time (RT) approximately at 1.59
minutes. Quantity calculations were made according to the linear calibration
curves of standard (y = mx+c). Percentage of eurycomanone compounds were
calculated based on the peak area produced by UPLC profiles.

Preliminary Phytochemical Screening of Polysaccharides

Plant materials of E. longifolia were harvested from natural forest reserves of

nine different provenances namely Johor, Melaka, Pahang A, Pahang B, Pahang
D, Perak, Pulau Pinang, Selangor and Terengganu. The size of plant samples
taken was standardised. The harvested plants were washed and dried in an oven
for sample preparation. About 30 g dried root of E. longifolia were extracted by
double boiling procedure. The freeze-dried samples were prepared by Phenol-
Sulfuric acid method and light absorption at 493 nm was recorded by UV-VIS
detection. Quantity calculations were made according to the linear calibration
curves of standard (y = mx + c). The actual weight of polysaccharides content of
each provenance was calculated based on the peak area produced by UV-VIS
profiles.

RESULTS AND DISCUSSION

Growth Performance of E. longifolia Provenances

Data on field growth for the 12 months after planting in respect to growth traits
such as height and diameter at collar region pertaining to 4 sites and 10
provenances are summarized in Tables 1 and 2.

Table 1: Growth Performance of E. longifolia by site comparison at one year old

after planting
Rank Site Growth Traits
Height (cm) Collar diameter (mm)
1 SPF Mata Ayer, Perlis 28.50a ± 0.70 6.76a ± 0.12
2 UTM Skudai, Johor 28.43a ± 1.59 7.11a ± 0.28
3 SPF Jeli, Kelantan 24.30b ± 1.82 5.54b ± 0.32
4 SPF Maran, Pahang 22.66b ± 1.77 5.17b ± 0.45

17
20
Variation between the provenances was found significant with the Melaka
provenance performed the best and attained the maximum height, but second
best for the collar diameter (Table 2). Selangor provenance attained the
maximum collar diameter but showed no significant differences between
Melaka and Johor.

Table 2: Growth Performance of E. longifolia Provenances at one year old after

planting
Rank Provenances Growth Traits
Height (cm) Collar diameter
(mm)
1 Melaka 34.04a ± 3.48 7.44a ± 0.61
2 Johor 32.98ab ± 1.68 7.32a ± 0.29
3 Selangor 30.17abc ± 3.16 7.47a ± 0.55
4 Pahang B 28.98abc ± 2.63 5.77cd ± 0.46
5 Terengganu 28.10bcd ± 1.94 7.17a ± 0.34
6 Pahang D 25.35cde ± 1.58 6.91ab ± 0.28
7 Pulau Pinang 23.22def ± 3.63 6.27bc ± 0.64
8 Pahang C 23.03def ± 2.32 4.44e ± 0.28
9 Pahang A 22.71ef ± 2.04 5.71cd ± 0.36
10 Perak 19.50f ± 4.19 5.26de ± 0.28

Preliminary Phytochemical Screening of Eurycomanone

Figure 1 summarised the percentage of eurycomanone compounds found in

roots and stems of E. longifolia provenances. Terengganu provenance was found
with the highest content of eurycomanone in root part while there was none
detected in stems part, followed by Pulau Pinang and Johor provenance (Mohd
Zaki et al. 2015a).

Preliminary Phytochemical Screening of Polysaccharides

Table 3 summarised the percentage of polysaccharide content found in roots of

E. longifolia provenances (Mohd Zaki et al. 2015b). The data showed that
polysaccharides content in the roots of Terengganu was the highest with 1.06 %
actual weight of polysaccharides, followed by Pahang B and Johor.

18
21
 1.8
1.6 1.46
1.4
1.2
1
0.8
0.55 0.52
0.6 0.38 0.33 0.29 0.29 0.28
0.4 0.18 0.21 0.170.15
0.2 0.1
0
0
Pahang A Pulau Terengganu Selangor Johor Melaka Perak
Pinang

Mean (%) of eurycomanone in roots. Mean (%) of eurycomanone in stems.

Figure 1: Mean percentage (%) of eurycomanone of roots and stems from

different provenances

Table 3: Percentage and Actual Weight of Polysaccharide Content in E. longifolia

Provenances
Provenance Dry weight of Dry Polysaccharides Actual Weight
E. longifolia Polysaccharide Content (%) Polysaccharides in
(g) Fraction (g) E. longifolia (%)

Johor 30.00 0.318 41.5 0.44

Pahang B 30.00 0.633 35.8 0.76
Pahang D 30.00 0.302 40.8 0.41
Perak 30.00 0.265 39.1 0.34
Terengganu 30.00 0.939 34.0 1.06
Melaka 30.00 0.574 ND ND
Pahang A 30.00 0.820 ND ND
Pulau 30.00 0.130 ND ND
Pinang
Selangor 30.00 0.103 ND ND
ND = not detected

CONCLUSION

Based on the preliminary assessment, the top five rank according to the growth
performances analysis are; Melaka, Johor, Selangor, Pahang B and Terengganu.
While for the screening of eurycomanone compound, the top three of the
highest content recorded by Terengganu, Pulau Pinang and Johor. However, the
assessment of eurycomanone compound need to be taken cautiously as the
highest content recorded might not be as the best indicator. As for the
polysaccharides analysis, the top three provenances are recorded by
Terengganu, Pahang B and Johor. Additionally, pathogenicity test will be carried

19
22
out in the future for the assessment of provenances that are resistant to
selected pest and diseases.

ACKNOWLEDGEMENTS

Special thanks to Ministry of Agriculture and Agrobased industry, Malaysia for

providing the NKEA Research Grant Scheme (NRGS-MT-0512-02).

REFERENCES

Hettasch, M.H., Lunt, K.A., Pierce, B.T., Snedden, C.L., Steyn, D.J., Venter, H.M. &
Verryn, S.D. (2002). Tree Breeding Course Manual. Environmentek, CSIR.
Pretoria, South Africa.
Mohd Zaki, A., Nor Fadilah, W., Mohd Radzi, A., Sui Kiong, L., Ab. Rasip, A.G.,
Mohamad Lokmal, N., Abdul Rashid L., Ahmad Fauzi M.S., Farah Fazwa
M.A. & Regina Mariah, J. (2015a). Preliminary Phytochemical Screening
of Eurycomanone for Selection of High Quality Planting Materials:
Eurycoma longifolia. Malaysian Applied Biology 44(1):25–28.
Mohd Zaki, A., Nor Fadilah, W., Abdul Rashid, L., Nurnadiah, R., Mohd Radzi, A.,
Mohamad Lokmal, N., Ahmad Fauzi, M.S., Farah Fazwa, M.A., Ab.
Rasip, A.G., Regina Mariah, J. & Mohamad O. (2015b). Preliminary
Phytochemical Screening of Polysaccharides Content for Selection of
High Quality Planting Materials: Eurycoma longifolia. Jurnal Teknologi
(Sciences & Engineering) 77(24):101–105.
Rasheed, N.M.A., Nagaiah, K., Goud P.R. & Sharma, V.U.M. (2012). Chemical
Marker Compounds and Their Essential Role in Quality Control of Herbal
Medicines. Annals of Phytomedicine 1(1):1–8.

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23
PENGHASILAN BAHAN TANAMAN TUMBUHAN UBATAN
MENGGUNAKAN TEKNOLOGI KULTUR TISU

Nor Hasnida, H., Nazirah, A., Muhd Fuad, Y., Mohd Saifuldullah, A.W., Siti
Suhaila, A.R., Haliza, I., Fadhilah, Z., Rozidah, K., Rohani, A., Sabariah, R.,
Normah, B., Naemah, H., Rukiah, M. & Sharifah, M.

Tissue Culture Laboratory, Forest Biotechnology Division, Forest Research

Institute Malaysia (FRIM), 52109 Kepong, Selangor

Tel: 03-62797154 Fax: 03-62804614 E-mail: hasnida.frim@1govuc.gov.my

PENGENALAN

Perladangan hutan dan herba diperlukan untuk menangani masalah kekurangan

bekalan bahan mentah bagi memenuhi permintaan industri perkayuan, industri
herba dan farmaseutikal. Oleh itu, bahan tanaman yang berkualiti diperlukan
untuk menubuhkan ladang hutan dan herba. Bahan tanaman boleh dihasilkan
secara konvensional dan melalui kaedah bioteknologi iaitu kultur tisu.

Bidang kultur tisu menjadi semakin popular dalam penghasilan bahan

tanaman perladangan hutan disebabkan oleh ciri-cirinya yang unik. Diantaranya
boleh menggandakan anak pokok dengan cepat, boleh dihasilkan secara
berterusan sepanjang tahun, penghasilan anak pokok secara berjadual dan
penghasilan anak pokok spesies yang susah tumbuh. Selain dari itu, kultur tisu
juga boleh digunakan untuk strategi pemuliharaan iaitu untuk penyimpanan
germplasma secara jangka panjang (Akin-Idowu et al. 2009).

Kaedah kultur tisu yang biasa digunakan oleh makmal-makmal komersil

adalah penggandaan pucuk aksilari. Tunas aksilari dirawat dengan pengawalatur
tumbuhan untuk memecahkan kedormanan dan membentuk pucuk baru. Pucuk
baru yang terhasil seterusnya di subkultur ke dalam medium baru dan diakarkan
jika perlu untuk menghasilkan anak pokok atau plantlet (Philips &
Hubstenberger 1995).

BAHAN DAN KAEDAH

Kaedah kultur tisu yang biasa digunakan di Makmal Kultur Tisu FRIM adalah
penggandaan pucuk aksilari kerana anak pokok yang terhasil daripada kaedah ini
adalah serupa dengan induknya. Terdapat lima peringkat dalam teknik
penggandaan pucuk aksilari iaitu: pemilihan eksplan, penghasilan kultur bersih
(aseptik), penggandaan dan pemanjangan pucuk, pengakaran dan pengikliman

21
24
seterusnya dipindah tanam ke tapak semaian dan lapangan (Philips &
Hubstenberger 1995).

HASIL DAN PERBINCANGAN

Makmal Kultur Tisu FRIM sehingga kini telah berjaya membangunkan kaedah
kultur tisu bagi spesies-spesies tumbuhan ubatan seperti Tongkat Ali (Eurycoma
longifolia), Kacip Fatimah (Labisia pumila), Misai Kucing (Orthosiphon aristatus),
Mas Cotek (Ficus deltoidea), Halia (Zingiber officinale), Kunyit (Curcuma longa),
Kantan (Etlingera eliator), Spesies-spesies Temu (Curcuma sp.), Pegaga (Centella
asiatica), Periuk Kera (Nepenthes sp.), Karas (Aquilaria malaccensis) dan
Chandan (Aquilaria hirta).

Kultur tisu Tongkat Ali

Kultur tisu Kacip Fatimah Kultur tisu Misai Kuching

Kultur tisu Mas Cotek Kultur tisu Halia

Kultur tisu Kunyit Kultur tisu Kantan

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25
Kultur tisu spesies Temu Kultur tisu Pegaga

Kultur tisu Periuk Kera Kultur tisu Karas

Kultur Tisu Chandan

KESIMPULAN

Teknologi kultur tisu tumbuhan ubatan yang telah dibangunkan ini sedia untuk
diperlesenkan kepada syarikat atau individu yang berminat. Spesies-spesies
ubatan ini juga merupakan sebahagian produk jualan Makmal Kultur Tisu.

PENGHARGAAN

Penghargaan kepada kakitangan Bahagian Bioteknologi Perhutanan, FRIM dan

juga kepada pihak MOSTI, NKEA/MOA dan MFRDB yang menyumbangkan dana.

RUJUKAN

Akin-Idowu, P.E., Ibitoye, D.O. & Ademoyegun, O.T. (2009). Tissue Culture as a
Plant Production Technique for Horticultural Crops. African Journal of
Biotechnology 8(16):3782–3788.
Phillips, G.C. & Hubstenberger, J.F. (1995). Micropropagation by proliferation of
axillary buds. In. Plant Cell, Tissue and Organ Culture. Fundamental
Methods. Gamborg, O.L. & Phillips, G.C. Springer lab manual.

23
26
NILAI TAKSONOMI CIRI ANATOMI DAUN BEBERAPA SPESIES
TUMBUHAN UBATAN TERPILIH DARIPADA FAMILI APOCYNACEAE DAN
DIPTEROCARPACEAE

Ummu Hani, B.1, Nur Munirah, S.1 & Tan, A.L.1

1
Bioresources Programme, Natural Products Division, Forest Research Institute
Malaysia (FRIM), 52109 Kepong, Selangor

Tel: 03-62797602 Fax: 03-62729805 E-mail: ummuhani@frim.gov.my

PENGENALAN

Apocynaceae merupakan salah satu famili terbesar dalam kumpulan tumbuhan

angiosperma, mengandungi kira-kira 375 genus dan 5100 spesies di seluruh
dunia (Endress et al. 2007). Kebanyakan spesies terdiri daripada pelbagai habit
pertumbuhan i.e. liana, herba, syrub dan pokok (Endress & Bruyns 2000). Famili
ini mempunyai nilai perubatan seperti merawat menyakit kanser, penyakit kulit,
diabetes, cirit-birit dan penyakit malaria (Middleton 2007).

Dipterocarpaceae adalah famili tumbuhan berkayu, mempunyai 17

genus dan kira-kira 510 hingga 680 spesies di seluruh dunia (Ashton 1982 &
Symington 2004). Terdapat 14 genus dan mengandungi kira-kira 160 spesies di
Semenanjung Malaysia (Symington 2004). Spesies dalam Dipterocarpaceae
adalah unik terutamanya pada ciri pembungaan. Oleh kerana musim
pembungaan yang tidak menentu, proses pengecaman spesies sukar
terutamanya apabila spesimen tidak mempunyai bunga atau buah.

Kebelakangan ini, kaedah anatomi sering digunakan terutamanya untuk

tujuan pengesahan identiti bagi spesimen herbarium (Culter et al. 2008).
Dickison (2000) menyatakan bahawa ciri anatomi tumbuhan seperti petiol,
tulang daun, trikom dll. mempunyai nilai sistematik. Oleh itu, objektif kajian ini
adalah untuk mengenalpasti variasi ciri unik anatomi daun yang boleh digunakan
untuk membezakan antara genus atau spesies dalam famili Apocynaceae dan
Dipterocarpaceae.

BAHAN DAN KAEDAH

Sampel daun spesies kajian iaitu A. angustifolia Miq., D. costulata (Miq.) Hook.f.,
D. aromatica C.F. Gaertn dan D. oblongifolia Dyer. diperoleh daripada kampus
Institut Penyelidikan Perhutanan Malaysia (FRIM), Kepong, Malaysia. Sampel
daun ditetapkan dalam larutan penetap AA (nisbah 1:3) selama 48 jam sebelum
hirisan dengan mikrotom gelongsor dapat dijalankan. Seterusnya proses

24
27
pewarnaan menggunakan Alcian Blue dan Safranin diikuti dengan penyahairan
menggunakan siri alkohol. Proses pelekapan dengan pelekap Euparal dan proses
pengeringan dalam ketuhar pada suhu 60 °C selama dua minggu. Pemerhatian
imej dilakukan dengan menggunakan mikroskop cahaya yang dihubungkan
dengan kamera video digital serta perisian CellSens. Teknik yang digunakan
dalam kajian ini adalah mengikut teknik yang telah diubahsuai daripada kaedah
yang dicadangkan oleh Johansen (1940) dan Sass (1958).

HASIL DAN PERBINCANGAN

Berdasarkan hasil kajian, terdapat beberapa variasi ciri unik anatomi dan ciri
sepunya (Rujuk Rajah 1 & 2; Jadual 1 & 2) yang boleh digunakan sebagai data
sokongan untuk membantu proses pengecaman genus atau spesies dalam famili
Apocynaceae dan Dipterocarpaceae.

Hasil kajian menunjukkan satu lapisan sel mesofil palisad dan kehadiran
trikom papila pada bahagian abaksial sel epidermis hanya dijumpai dalam famili
Apocynaceae sahaja. Kehadiran hablur pada sel palisad, dan sistem berkas
vaskular jenis terbuka pada petiol hanya hadir pada Alstonia angustifolia sahaja.

Kehadiran sel sklerenkima yang membentuk tiang pada lamina daun,

hablur pada bahagian adaksial sel epidermis lamina, salur resin pada petiol dan
tulang daun serta bentuk luaran petiol, tulang dan tepi daun dapat membezakan
Dipterocarpaceae dan Apocynaceae. Selain itu, Metcalfe dan Chalk (1979)
menyatakan bahawa struktur vaskular pada petiol dan tulang daun dalam
Dipterocarpaceae sentiasa kompleks dan mempunyai pelbagai struktur yang
boleh membuktikan nilai taksonomi terutamanya dalam pembezaan di
peringkat famili, genus atau spesies. Berdasarkan bentuk luaran petiol dan
tulang daun, serta kehadiran hablur pada sel epidermis lamina telah
membuktikan bahawa ciri anatomi daun sangat membantu untuk membezakan
spesies Dryobalanops oblongifolia dan Dryobalanops aromatica.

Berdasarkan kajian ini, terdapat beberapa ciri sepunya yang boleh

dijumpai pada ke empat-empat spesies kajian iaitu hablur dan lapisan sel
epikutikel pada petiol dan tulang daun. Kehadiran ciri unik dan sepunya pada
keratan rentas daun sangat membantu untuk pengecaman spesies terutamanya
apabila ciri spesimen tidak lengkap (tidak mempunyai buah atau bunga). Selain
itu, ciri anatomi keratan rentas juga boleh dijadikan sebagai sumber rujukan
mikroskopi apabila sampel dalam bentuk serbuk atau serpihan. Maklumat
mikroskopi ini sangat membantu untuk menentukan ketulenan suatu bahan
dalam penyediaan suatu produk atau ubatan.

25
28
KESIMPULAN

Kesimpulannya, variasi ciri anatomi seperti bentuk luaran petiol, tulang dan tepi
daun, kehadiran trikom dan hablur pada lamina, kehadiran sel sklerenkima, salur
resin dan struktur tisu vaskular yang kompleks boleh digunakan untuk
membezakan antara spesies dalam famili Apocynaceae dan Dipterocarpaceae.

PENGHARGAAN

Terima kasih kepada kakitangan Taman Etnobotani, FRIM yang memberi

bantuan dalam penyediaan spesimen dan pengecaman spesies kajian.

d
a b c

e f g h
Rajah 1: Keratan rentas lamina daun: (a) Alstonia angustifolia. (b) Dyera
costulata. (c) Dryobalanops aromatica. (d) Dryobalanops oblongifolia; Keratan
rentas tepi daun: (e) Alstonia angustifolia. (f) Dyera costulata. (g) Dryobalanops
aromatica. (h) Dryobalanops oblongifolia

a b c d

Salur resin
e g
f h
Rajah 2: Keratan petiol daun: (a) Alstonia angustifolia. (b) Dyera costulata. (c)
Dryobalanops aromatica. (d) Dryobalanops oblongifolia; Keratan tulang daun:
(e) Alstonia angustifolia. (f) Dyera costulata. (g) Dryobalanops aromatica.
(h)Dryobalanops oblongifolia

26
29
RUJUKAN

Ashton, P.S. (1982). Dipterocarpaceae. In Flora Malesiana, Series 1 -

Spermatophyta (Seed Plant), edited by van Steenis, C.G.G.J. 9(2):237–
552
Cutler, D.F., Botha, C.E.J. & Stevenson, D.W. (2008). Plant Anatomy: An Applied
Approach. New York: Blackwell Publishing.
Dickison, W.C. (2000). Integrative Plant Anatomy. San Diego: Harcourt Academic
Press. Pp. 530.
Endress, M.E., R.W. Van der Ham, S. Nilsson, L. Civeyrel, M.W. Chase, B.
Sennblad, K. Potgieter, J. Joseph, M. Powell, D. Lorence, Y.M.
Zimmerman, and V.A. Albert (2007). Aphylogenetic analysis of Alyxieae
(Apocynaceae) based on rbcL, matK, trnLintron, trnL-Fspacer sequences,
and morphological characters. Ann. Missouri. Bot. Gard. 94:1–35.
Endress, M. E., and P. V. Bruyns (2000). A revised classification of the
Apocynaceae. Botanic. Rev. 66:1–56.
Johansen, D.A. (1940). Plant Microtechnique. New York & London: McGraw-Hill.
Metcalfe, C.R. & Chalk, L. (1950). Anatomy of the Dicotyledons. Oxford: The
Clarendon Press. 2. Pp 223–262.
Middleton, D.J. (2007). Apocynaceae (subfamilies Rauvolfioideae and
Apocynoideae), Flora Malesiana, ser.I, Seed plants. The National
Herbarium of the Netherlands, Leiden, TheNetherlands. 18:474
Sass, J.E. (1958). Botanical Microtechnique 3rd ed. Ames: Iowa State University
Symington, C.F. (2004). Foresters’ Manual of Dipterocarps 2nd Ed. Puchong:
Academe Art & Printing Services Sdn. Bhd.

27
30
 Jadual 1. Variasi ciri anatomi keratan rentas lamina dan tepi daun spesies kajian
Sampel Lamina daun Bentuk luaran
tepi daun
Sel epidermis Sel klorenkima Sel Trikom Hablur
skelerenkima
adaksial abaksial Sel Sel mesofil
palisad span
Alstonia 1:1-1:2 1:2 1 8-10 Hadir pada Papila Tunggal dan drus Membulat-
angustifolia berkas vaskular hujung menirus;
lurus

Dyera 1:1 1:2 1 8-10 Hadir pada Papila Tidak hadir Membulat-
costulata berkas vaskular hujung
membulat
tumpul;
melengkung
40o arah
abaksial

Dryobalanops 3:1 1:1 2-3 7-8 Hadir; Tidak hadir Tidak hadir Membulat-
aromatica membentuk hujung menirus;
tiang sel lurus
sklerenkima
Dryobalanops 2:1 1:2 2-3 5-6 Hadir; Tidak hadir Tunggal dan drus Membulat-
oblongifolia membentuk hujung menirus;
tiang sel melengkung
sklerenkima 40o arah
abaksial

28
30
 Jadual 2. Variasi ciri anatomi keratan rentas petiol dan tulang daun spesies kajian
Sampel Bentuk luaran Bentuk luaran tulang Berkas vaskular (bv) Salur Hablur Trikom Lilin
petiol daun resin epikutikel
Petiol Tulang daun
Alstonia Adaksial- Adaksial-bonggol; Sistem terbuka Sistem Tidak Hadir Tidak Hadir
angustifolia cembung; Abaksial-bentuk arka tertutup hadir hadir
Abaksial-bentuk
arka

Dyera Adaksial- Adaksial-bonggol; Sistem tertutup; Sistem Tidak Hadir Tidak Hadir
costulata cembung; Abaksial- ¾ bulatan floem medulari tertutup; hadir hadir
Abaksial-bentuk hadir floem
‘U’ medulari hadir

Dryobalanops Adaksial-cekung; Adaksial-sedikit Sistem tertutup; Sistem Hadir Hadir Tidak Hadir
aromatica Abaksial- ¾ melengkung/cekung; bv medulari tertutup; bv hadir
bulatan Abaksial-bentuk ‘U’ hadir medulari hadir

Dryobalanops Adaksial-cekung; Adaksial- Sistem tertutup; Sistem Hadir Hadir Tidak Hadir
oblongifolia Abaksial- ½ melengkung/cekung; bv medulari tertutup; bv hadir
bulatan Abaksial- ¾ bulatan hadir medulari hadir

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31
PRELIMINARY OBSERVATIONS ON SOIL AMENDMENT EFFECTS USING
BIOCHAR ON LABISIA PUMILA VAR. ALATA PLANTS AT NURSERY STAGE

Jeyanny, V1., Farah Fazwa, M.A2., Syafiqah Nabilah S.B.2, Norhayati, S., Siti
Suhaila, A.R.2 & Fakhri, I1.

1
Forest Plantation Programme, Forest Biotechnology Division, 2Plant
Improvement Programme, Forest Research Institute Malaysia (FRIM), 52109
Kepong, Selangor.

Tel: 03-62797136 Fax: 03-62804614 E-mail: jeyanny@frim.gov.my

INTRODUCTION

Labisia pumila is an important herbal plant belonging to the Primulaceae family.

Traditionally, the water decoction of this plant was used for pre and post-
partum treatment by the Malay women (Jamia 2006). L. pumila has the ability to
regulate body weight (Fazliana et al. 2009), prevent photo-aging and has anti-
bacterial and anti-fungal effects (Karimi et al. 2011; Ali & Khan 2011). As natural
supplies are dwindling due to over exploitation, it is imperative that attempts be
made to cultivate this local popular herb. Thus, it is important to determine the
agronomy practices of this herb which may assist to increase the
phythochemical compounds and lead to increased productivity.

Fertilizer applications are essential in order to supply the important

nutrients that increase growth, vigour, yield and maintain the health of plants.
Biochar is a carbon-rich by product resulting from pyrolyzing biomass
(Biedermen & Harpole 2013). Addition of biochar to soil will help to ameliorate
the soil fertility and plant growth (Schulz & Glaser 2012). Besides optimum
growth, it is important to identify how plant nutrition can increase the
phytochemical compounds of L. pumila. Maintenance and/or enhancement of
phytochemicals present via fertilization may garner high quality plants for
production of phythochemical compounds. The objectives of this research were
to test the different effects on soil amendments on 6 months L. pumila plants at
nursery stage.

MATERIALS & METHODS

As preliminary observations, 12 plants at the age of 6 months derived from

tissue culture technique were transferred into 8’ x 10’ polybags (3 kg) in March
2016. Growing media used were topsoil, leaf compost and sand (2:3:1), whereby
6 treatments were represented by 2 replications for observation purposes. The
six treatments applied were:

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32
 1. Inorganic fertilizer (NPK Green 15:15: 15), 90 kg N/ha (T1) (control)
2. Inorganic fertilizer (NPK Green 15:15: 15), 90 kg N/ha + Biochar, 5% of media
(T2)
3. Controlled release fertilizer (AJIB NPK Granular 10:15: 17: 2: 5), 90 kg N/ha
(T3)
4. Controlled release fertilizer (AJIB NPK Granular 10:15: 17: 2: 5)+ Biochar, 5%
of media (T4)
5. Controlled release fertilizer + LM Plus Bio organic fertilizer 3:2:2:1 90 kg N/ha
(T5)
6. Controlled release fertilizer + LM Plus Bio organic fertilizer, 90 kg N/ha +
Biochar, 5% (T6)

Every month, the variables measured were collar diameter (mm), total
height (cm), and total leaf length (mm) using a digital calliper and a standard
ruler. At the end of the experiment the chlorophyll content of the leaves were
measured at 6 months after transplanting using a Minolta SPAD 502 chlorophyll
meter. The initial data measurement and the 6th month measurement were
summarized using the relative growth rate (RGR) equation below for collar
diameter and total height. The total phenolic content (TPC) of the plants were
also determined using Folin Ciocalteu method (Singleton & Rossi 1965).

RGR = [ln (Mo – Mt)]/ t

Mo = Data at initial
Mt = Data at final
t = time (months)

RESULTS & DISCUSSION

The results showed that the effects of T2 [NPK Green + Biochar] is 96% and 42%
higher compared to control for collar diameter and total height, respectively
(Figure 1). The height values for T3 and T4 whereas were 1.5 fold higher
compared to control. The values for chlorophyll content were also relatively
higher (>16%) for T2, T3, T4 and T6 compared to control (Figure 2). The highest
total phenolic content (Figure 3) was for T3 (AJIB NPK Granular) which was 545
mg/100g GAE (gallic acid equivalent). However, this value was in par with
control and T2. Values for T6 was the lowest (26%) compared to control.

From our initial observations, it was obvious that the addition of biochar
to normal fertilizer treatments increased plant productivity (Figures 1 and 2).
This is because we believe that biochar has the potential to improve water-
holding capacity of soils which typically result in increased plant growth with the
addition of fertilizers (Woolf et al. 2010). The effects of controlled release
fertilizer were also prominent compared to control as the nutrients are released
gradually due to the semi- permeable coating in line with plant growth.

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33
Furthermore, we believe that other nutrients incorporated in the controlled
release fertilizer such as magnesium and micronutrients also enhanced the TPC
values. Micronutrients are known to assist in the phenol metabolism of plants
(Marschner 1995)

Figure 1. Effects of different soil ammendments on the relative growth rate

of L. pumila var. alata

Figure 2. Effects of different soil ammendments on the chlorophll content

of L. pumila var. alata

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34
Figure 3: Effects of different soil ammendments on the total phenolic content
of L. pumila var. alata

CONCLUSION

This preliminary findings show that T2 (NPK Green + Biochar) promotes good
growth performances in L. pumila plants. The total phenolic content of L. pumila
plants were also enhanced with control (NPK Green), T2 and T3 (AJIB NPK
Granular). However, further evaluation should be conducted with more number
of samples to get more accurate results.

ACKNOWLEDGEMENTS

The authors are indebted to the staffs of Soil Management Branch and Tree
Improvement Branch for the assistance rendered throughout the experiment.
Financial sponsorship by the NRGS research grant (No NH1015A024) is greatly
acknowledged for this paper presentation.

REFERENCES

Ali, Z. & Khan, I. A. (2011). Alkyl Phenols and Saponins from the Roots of Labisia
pumila (Kacip Fatimah). Phytochemistry, (16):2075–2080.
Biederman, L. A., & Harpole, W. S. (2013). Biochar and its Effects on Plant
Productivity and Nutrient Cycling: A Meta‐analysis. GCB
Bioenergy. 5(2):202–214.
Fazliana, M., Wan Nazaimoon, W.M., Gu, H.F. & Ostenson, C.G. (2009). Labisia
pumila Ovariectomized rats. Maturitas. 62(1):91– 97.
Jamia, A.J. (2006). Malay Traditional Medicine. Tech Monitor (Special Feature:
Traditional Medicine: S & T Advancement), Pp. 37–49.

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Karimi, E., Jaafar, H. Z., & Ahmad, S. (2011). Phenolics and Flavonoids Profiling
and Antioxidant Activity of Three Varieties of Malaysian Indigenous
Medicinal Herb Labisia pumila Benth. J. Med. Plant Res. 5:1200–1206.
Marschner, H. (1995). Mineral Nutrition of Higher Plants. Pp. 889. London.
Academic Press
Schulz, H. & Glaser, B. (2012). Effects of Biochar Compared to Organic and
Inorganic Fertilizers on Soil Quality and Plant Growth in a Greenhouse
Experiment. Z. Pflanzenernähr. Bodenk. 175:410–422.
doi:10.1002/jpln.201100143
Singleton, V.L. & J.A. Rossi. (1965). Colorimetry of Total Phenolics with
Phosphomolybdic Phosphotungstic Acid Reagents. Am J Enol. Vitic. 16:144–
158
Woolf, D., Amonette, J. E., Street-Perrott, F. A., Lehmann, J. & Joseph, S. (2010).
Sustainable Biochar to Mitigate Global Climate Change. Nature
Communications.1:56.

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PENUBUHAN PLOT UJIAN PROGENI LIMAU PURUT (CITRUS HYSTRIX) DI
EMPAT STESEN PENYELIDIKAN FRIM: SATU KAJIAN AWAL

Farah Fazwa, M.A., Muhammad Asri, L., Norhayati, S., Mashitah, M.T., Mohd
Zaki, A., Samsuri, T.H. & Mohd Zaini, Z.

Program Membaikbiak Tumbuhan, Bahagian Bioteknologi Perhutanan, Institut

Penyelidikan Perhutanan Malaysia, 52109, Kepong, Selangor

Email: farah@frim.gov.my

PENGENALAN

Citrus hystrix lebih dikenali nama tempatannya sebagai limau purut adalah satu
daripada spesis liar di Malaysia. Ia juga dikenali dengan nama “Wild Lime” bagi
English, “Kaffir Lime” di Danish dan “Som Makrut” di Thailand. Tanaman saka ini
berasal dari rantau Asia Tenggara dan telah banyak ditanam di Indonesia,
Thailand dan Malaysia. Spesis ini tumbuh liar di tanah kering dan terdedah
kepada cahaya matahari. Pertumbuhan limau adalah sekitar 3-5 meter dan
mencapai sehingga ketinggian 30 meter, tetapi jika dibandingkan dengan spesis
limau lain, pertumbuhannya adalah perlahan. Daun limau purut mempunyai
kepanjangan antara 7.5–10.0 cm dan mempunyai tangkai bersayap yang
memberi gambaran dua helai daun yang dicantumkan hujung ke hujung (Yahaya
& Ahmad Puat 2005). Buahnya berbentuk ‘pear’ dan mencapai kepanjangan
sehingga 10 cm dan mempunyai diameter antara 5.0 cm hingga 7.5 cm. Buahnya
berwarna hijau dan bertukar kepada kekuningan apabila telah masak, berkedut
dan berpermukaaan kasar (Chin & Yong 1980).

Daun limau purut digunakan sebagai perasa dalam masakan Thailand

seperti tomyam. Bahagian daun limau purut juga digunakan sebagai salah satu
bahan untuk mandian wap. Kajian juga ada menunjukkan daun limau purut
sebagai sumber berkesan penggalak antitumor atau pencegah kanser. Buah
limau purut digunakan secara tradisi untuk mencuci rambut dan air dari
buahnya digunakan untuk membunuh pacat, mencuci mulut dan menguatkan
gusi. Kulit buahnya pula sering digunakan untuk membuat jamu, dan
memberikan aroma terhadap masakan berdaging. Minyak pati limau purut yang
diekstrak dari bahagian daun dan kulit buah mempunyai aroma khusus
digunakan untuk aromaterapi, nutraseutikal produk penjagaan diri. Minyak pati
ini juga mempunyai sifat-sifat antiseptik, astrigen dan antibakteria yang boleh
merawat luka dan lebam. (Yahaya et al. 2005). Ia juga baik untuk sakit sendi dan
sakit kepala (Khatijah 2006). Dalam kajian Nor Azah et al. (2004), bahagian buah
dan kulit buah limau purut telah diekstrak minyak patinya dan dirumus dengan

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37
beberapa surfaktan dan bahan aditif untuk menghasilkan produk pembersih
tangan dan produk penjagaan diri.

Melihat kepada kepentingan spesies ini di dalam industri herba negara,

satu kajian bagi mendapatkan baka-baka berkualiti dari segi hasil minyak pati
dan kandungan sitronellalnya telah dijalankan oleh Program Membaikbiak
Tumbuhan dari Institut Penyelidikan Perhutanan Malaysia (FRIM) melalui
kaedah penyaringan dan pemilihan (Farah Fazwa et al. 2015). Pemilihan induk
atau baka yang baik merupakan langkah penting dalam mana-mana aktiviti
pembiakbaikan tumbuhan sebelum dibuat perambatan tampang dan
sebagainya. Oleh itu sebagai langkah seterusnya dalam aktiviti pembiakbakaan
spesies ini, satu kajian yang dinamakan sebagai ujian progeni telah dijalankan
bagi memilih pokok ibu atau progeni yang unggul untuk dijadikan baka.

BAHAN DAN KAEDAH

Sebanyak 15 progeni limau purut yang telah dipilih dari kajian lepas dijadikan
sebagai bahan tanaman. Daripada 15 progeni ini sebanyak 900 anak progeni
telah dihasilkan melalui pembiakan biji benih (Jadual 1). Lokasi kajian bagi 4 plot
ujian progeni ialah di Stesen Penyelidikan FRIM (SPF); SPF Jeli (Kelantan), SPF
Selandar (Melaka), SPF Mata Ayer (Perlis) dan SPF Maran (Pahang). Rekabentuk
kajian adalah berdasarkan kepada rekabentuk RCBD dengan 3 blok dan setiap
blok mempunyai 75 sampel pokok. Keluasan kawasan bagi setiap plot ujian
progeni ialah seluas 0.2 hektar dengan jarak tanaman 3 meter x 3 meter. Di
antara parameter kajian yang diukur ialah ketinggian pokok (cm), diameter
(mm), silara pokok. Kesemua anak pokok yang ditanam di plot kajian
mempunyai umur yang seragam iaitu (9 bulan).

KEPUTUSAN DAN PERBINCANGAN

Plot ujian progeni limau purut telah ditubuhkan di 4 kawasan SPF bermula dari
bulan Februari 2016. Didapati semua kawasan ini adalah berbeza dari segi
penerimaan taburan hujan, suhu dan jenis tanah. Jadual 2 menunjukkan
kedudukan koordinat bagi setiap SPF dan maklumat persekitaran. Ujian progeni
dijalankan adalah bagi melihat keupayaan induk-induk hidup dalam pelbagai
keadaan persekitaran yang berbeza-beza dan dapat mengeluarkan hasil yang
tinggi (Zobel & Talbert 1984). Rekabentuk plot kajian bagi setiap kawasan adalah
seperti dalam Gambarajah 1 di bawah. Adalah dijangkakan penubuhan plot ujian
progeni ini (Gambarajah 2) akan memberikan keputusan hasil minyak pati dan
kompaun kimia oleh setiap progeny. Keputusan ini akan membawa kepada
pemilihan baka limau purut yang paling unggul/elit.

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38
KESIMPULAN

Di akhir kajian ini adalah dijangkakan keputusan hasil minyak pati dan kompaun
kimia bagi setiap progeni limau purut yang diuji di 4 lokasi SPF akan dapat
diperolehi setelah pokok mencapai usia matang (3-5 tahun). Ini adalah kerana
spesies limau purut mempunyai pertumbuhan yang agak perlahan di peringkat
awal penanamannya iaitu sekitar satu hingga tiga tahun. Namun begitu, prestasi
pertumbuhan setiap progeni terus dinilai sehingga pokok benar-benar mencapai
usia matang. Sekurang-kurangnya 1 daripada 15 progeni yang diuji akan
memberi keputusan hasil minyak pati (sitronellal) yang tinggi dan akan dipilih
sebagai baka elit. Seterusnya bahan tanaman ini akan dikomersialkan bagi
memenuhi permintaan bekalan bahan mentah dan pasaran industri herba
tempatan.

Jadual 1: Senarai progeni, bilangan ramets dan peratus kompaun sitronellal

yang digunakan dalam ujian ini

No progeni KOD RAMETS % Sitronelal

PA13 1 60 80.52
PA18 2 60 84.09
PA8 3 60 81.95
PC4 4 60 81.82
PR12 5 60 84.06
PP14 6 60 80.61
PR14 7 60 79.34
PR9 8 60 75.43
PS14 9 60 77.16
PS8 10 60 74.18
PC18 11 60 54.59
PC19 12 60 48.33
PR5 13 60 68.19
PS10 14 60 63.62
PK10 15 60 48.96

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39
Jadual 2: Maklumat kawasan ujian progeny yang dijalankan di 4 Stesen
Penyelidikan FRIM

Bil. Lokasi SPF Koordinat Siri tanah Taburan Purata suhu

hujan Jan – dis 2016
Jan – dis
2016
1. Mata Ayer, N 6, 64 70.25 - 200mm – 24°C - 33°C
Perlis E 100, 24 33. 47 300mm
2. Jeli, N 5, 39 21.8 Renggam 200mm – 29°C - 30°C
Kelantan E101,41 12.7 300mm
3. Maran, N 3,63 97.65 Bungor 160mm – 24°C - 33°C
Pahang E102,80 46.91 240mm
4. Selandar, N 2,25 27.0 TaiTak/ 160mm – 24°C - 33°C
Melaka E 102,23 36.8 Bungor 240mm

Gambarajah 1: Rekabentuk plot kajian bagi setiap plot ujian progeni di 4 Stesen
Penyelidikan FRIM

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Gambarajah 2: Plot ujian progeni limau purut yang ditubuhkan di SPF Mata
Ayer, Perlis

RUJUKAN

Adam, R.P. (2005). Identification of Essential oil Components by Gas

Chromatography/Mass spectrometry, 4th Edition. Allured Business Media:
Carol Stream, Illinois.
Chin, H.F. & Yong, H.S. (1980). Malaysian Fruits in Color. Tropical Press Sdn. Bhd,
Kuala Lumpur, Malaysia. Pp 52.
Farah Fazwa, M.A., Mailina, J., Nor Azah, M.A., Nur Nazihah, M., Mohd. Zaki, A.,
Syafiqah Nabilah, S.B., Norhayati, S.,& Mohd Asri, L. (2015). Pemilihan dan
Penghasilan Baka Limau Purut (Citrus hystrix) Bermutu Tinggi. Pp. 106–112
in Asiah, O. et al. (Eds). Prosiding Persidangan Industri Herba 2015.
Khatijah H. (2006). Anatomical atlas of Malaysian Medicinal Plants Volume 1.
Universiti Kebangsaan Malaysia, Bangi. Pp. 58–61.
Nor Azah, M.A., Mastura, M., Zaridah, M.Z., Mailina, J., Mohamad Shahidan.,
Norseha, A., Zainon, A.S., Norhanan, M.Y., Abdul Majid, J. & Abu Said, A.
(2004). Potential application of Citrus hystrix essential oils for personal care
products. Prosiding Hasil Kajian IRPA RMK8 2004 FRIM. Pp. 273–277.
Yahaya H. & Ahmad Puat N. (2005). Limau purut. Penanaman Tumbuhan Ubatan
dan Beraoroma. Cetakan pertama. Eds. Musa Yaacob, Muhamad.
Zobel, B.J. & Talbert, J. (1984). Applied Forest Tree Improvement. John Willey &
Son. New York.

39
41
EFFECT OF FERTILIZER APPLICATION ON THE GROWTH OF EURYCOMA
LONGIFOLIA (TONGKAT ALI) PLANTLETS IN GREENHOUSE CONDITIONS

Muhd Fuad, Y.1, Nor Hasnida, H.1, Nazirah, A.1, Siti Suhaila, A.R.1, Haliza, I.1,
Rozidah, K.1 , Rohani, A.1 , Sabariah, R.1 ,& Muhd Shazrizal Shahmy, M.S.2

1
Forest Research Institute Malaysia, 52109 Kepong, Selangor,Malaysia
2
Universiti Perguruan Sultan Idris, 35900 Tanjung Malim, Perak, Malaysia

Tel: 03-62797353 Fax: 03- 62804614 E-mail: muhdfuad@frim.gov.my

INTRODUCTION

Eurycoma longifolia or tongkat ali is naturally distributed in the tropical forest of

Southeast Asia. Tongkat ali is well known for an energy booster and treating
erectile dysfunction. As the demand for tongkat ali planting material is always
on the rise. Tongkat ali roots took around 7 years before it can be harvested.
Therefore, tissue culture technology is the best alternative to avoid the
depletion of the plant natural habitat and to meet industrial demand. In green
house environment, the growth rate of tissue culture plantlets were slow.
Therefore organic or chemical fertilizers were applied to keep plants healthy
and as growth booster. This was done to achieve the average height for normal
plantlets, suitable for outside planting at 30 cm tall. Fertilizer application
practices and cropping systems play a key role in the regulation of C and N
contents in agricultural soils (Tong et al. 2009). While the use of mineral
fertilizers is the fastest way in providing nutrients to plant (Mando et al. 2005).
However several studies have shown that excessive use of chemical fertilizers,
could reduce crop yield due to soil acidification, loss of soil biological activity,
loss of soil physical properties and soil nutrient imbalance (Adediran et al 2004).

MATERIALS AND METHODS

Acclimatized tongkat ali tissue culture plantlets were potted into mixture of
baked soil and peat moss medium. All plantlet were placed in greenhouse
environment with average temperature of 28°C. The plantlets were watered
twice daily. All plantlets were divided into 5 groups (1 control and 4 treatments)
with 11 replicates. After 1 week potting, the plantlets were fertilized with NPK
Green, EFB compost, goat manure fertilizer and complehumus respectively. The
growths of plantlets were observed weekly. The height and stem diameter of
each plantlets were recorded.

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42
RESULT AND DISCUSSION

Mean height increment (cm) 0.7 0.6182

0.6 0.5364
0.5
0.4
0.2818
0.3 0.254

0.2 0.1091
0.1
0

Treatment

Figure 1: Effect of different fertilizer on growth of plantlets (height)

0.6
0.5055
Mean Stem Diameter Increment

0.5 0.4491
0.4
0.4
0.3
0.2055
0.1755
(mm)

0.2
0.1
0

Treatment

Figure 2: Effect of different fertilizer on growth of plantlets (stem diameter)

After I month observation, It was observed that Tongkat Ali tissue

culture plantlets fertilized with Complehumus showed the highest increase in
height followed by goat manure which are 0.62 cm and 0.54 cm respectively
(Figure 1). Plantlets fertilized with NPK Green showed the least increment even
compared with control. In term of stem diameter, plantlets fertilized with EFB
compost showing the highest increment which is 0.5 mm followed by NPK green
0.45 mm (Figure 2).

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43
 (a (b) ( (d (e
) c ) )
)

(e (d (c) (b (a
) ) ) )
Figure 1: (a) Control (b) NPK Green (c) EFB compost (d) Goat manure
(e) Complehumus
Polybags potted plantlets have limited source of nutrient. Overtime, it
will cause the plantlets to be stunted and easily attacked by pest and disease.
Scheduled fertilization will be the best way to increase the growth rate and keep
the plantlets healthy.

Different fertilizer regime gave different effects. Although plantlets

fertilized with EFB compost showed the highest stem diameter increment, but
did not show much increment in height. The same condition was also observed
in plantlets fertilized with NPK Green fertilizer. Only plantlets fertilized with
Complehumus showed good results in both height and stem diameter
increment. The mean height increment was double compared to control.

Among the fertilizer used in this experiment, only NPK Green was
categorized as a chemical fertilizer. Based on overall observation, organic
fertilizers provided better results in the greenhouse environment. Furthermore,
Nejatzadeh et al (2011) mentioned that the mineral nutrients of non-chemical
fertilizers were released at a slowly rate, making it more permanent and stayed
longer in the soil. Organic manure also reduced soil pH and increased the
electrical conductivity and the ability of absorbing soil nutrients. Non-chemical
fertilizers are more favored nowadays as they are not harmful to the
environment and less hazardous to health and cost less than chemical fertilizers.

CONCLUSION

Based on the study, complehumus fertilizer gave the best results for the growth
of tongkat ali tissue culture plantlets in the greenhouse condition.

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44
ACKNOWLEDGEMENT

We could like to thank the staff of Tissue Culture Laboratory, FRIM and Mohd
Shahrizal Shahmy from UPSI for their assistance and support.

REFERENCES
Adediran, J.A., Taiwa, L.B., Akande, M.O., Sobulo, R.A. & Idown, O.J. (2004).
Application of organic and inorganic fertilizer for sustainable maize and
coupea yield in Nigeria. Journal of Plant Nutrition, 27:1163–1181.
Nejatzadeh, F., Tahmasebi Enferadi, S., Naghavi, M., Hasani, A., Mostoufi, Y. &
Mousavi, A. (2011) Iranian Journal of Medical and Aromatic Plants, 5
(18):4537–4544
Mando, A., Ouattara, B., Sedago, M., Stroosnijder, L., Ouattara, K., Brussaard, L.
& Vanlauwe, B. (2005). Long-term effects of tillage and manure
application on soil organic fractions and crop performance under Sudano-
sahelian conditions. Soil and Tillage Research 80: 95–101.
Tong, C., Xiao, H., Tang, G., Wang, H., Huang, T., et al. (2009) Long-term fertilizer
effects on organic carbon and total nitrogen and coupling relationships of
C and N in paddy soils in subtropical China. Soil and Tillage Research 106:
8–14. doi: 10.1016/j.still.2009.09.003

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45
IN VITRO GERMINATION OF SYNSEPALUM DULCIFICUM (POKOK AJAIB)
FROM SEED EXPLANTS

Mohd Saifuldullah, AW.1, Nor Hasnida, H.1, Nazirah, A.1 Muhd Fuad, Y.1,
Rozidah, K.1, Rohani, A.1, Sabariah, R.1, Ayu Fazlina, K.2, Amirah Hanun, A.2, Siti
Suhaila, A. R.1 & Haliza, I.1

1
Forestry Biotechnology Division, Forest Research Institute Malaysia, 52109
Kepong, Selangor Darul Ehsan. 2Universiti Malaysia Kelantan, Kampus Kota,
Karung Berkunci 36, Pengkalan Chepa, 16100 Kota Bharu, Kelantan.

Tel: 03-62797160 Fax: 03-62804614 e-mail: saifuldullah@frim.gov.my

INTRODUCTION

Synsepalum dulcificum known as miracle fruit or “Buah Ajaib” is a medicinal

plant (Figure 1). It is a slow growing shrub from Tropical West Africa. It is a
unique plant as it can alter the taste of all sour or bitter foods into sweet taste
due to the presence of Miraculin, a type of protein that binds to the sweet
receptors and changes its configuration in the tongue (Ogunsola & Ilori 2008). It
can grow up to 5 to 6 feet but it also can reach up to 18 feet if resembles to
their like natural habitat. It also produces fruits (Figure 2) the entire year. It has
little white of flower and also berry shaped fruit green in color when young and
will turn bright red when ripening (Hiday 2014). According to Zuraida et al.
(2014), plant tissue culture is a science of growing plant cells, tissues or organs
isolated from the mother plant, and also a well established technology. It is also
called micropropagation. This technique was practiced under controlled
environment.

Figure 1 (left): S. dulcificum in FRIM's Botanical Garden, Kepong

Figure 2 (right): Fruits of S. dulcificum

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46
MATERIALS AND METHODS

Plant material

In this study, S. dulcificum fruits were collected from FRIM’s Botanical Garden,
Kepong. The seeds were used as explants in this experiment.

Surface sterilization method

Surface sterilization is the most crucial method in the development of tissue

culture protocol. The fruits of S. dulcificum were washed under running water to
remove the dirt or debris. The fruits were rinsed with a few drops of Tween 20
for 10 minutes. 0.1% Thiram (fungicide) was applied and constantly shook for 30
minutes. Then washed with 70% Clorox® plus 1 drop of Tween 20 for 20 minutes
(Figure 3). The fruits were rinsed using sterilized distilled water and excised. The
seeds were washed with 20% Clorox® plus 1 drop of Tween 20 and shook for 10
minutes. Lastly, they were rinsed with sterilized distilled water and cultured in
Murashige & Skoog (MS) (Murashige & Skoog 1962) media supplemented with
0.5 mg/L BAP. Observation of clean cultures and germination rates were
recorded weekly.

(A) (B)

Figure 3 A & B: Surface sterilization of S. dulcificum fruits using Clorox®

RESULTS AND DISCUSSION

Commercial bleach or Clorox® was used in tissue culture surface sterilization

technique. Bleach was the agent used to remove debris and contaminants from
the explants surface. However, the disadvantage of using bleach is it can destroy
plant cell if exposed under high concentration and in longer exposure duration
(Akin-Idowu 2009). But in the study, the seeds of S. dulcificum can withstand the

45
47
high concentration or percentage of Clorox®. In order to minimize the risk of
contamination during surface sterilization, Thiram was also used as the fungicide
reagent.

After 4 weeks in culture, observation showed that 94.4% clean culture

was obtained (Figure 4) and but the germination of seeds (Figure 5 & 6) was
only at 41.7% after 4 weeks observation. Even though the high percentage of
clean culture was obtained, there was no guarantee that the clean culture will
have higher germination rate. It depends on the plant species and seeds
characteristic (mature or young when it is cultured).

6% 42%

Clean Cultures
Germinate

94% Contamination 58%

Not Germinate

Figure 4: Percentage of clean culture Figure 5: Germination rate

Figure 6: Germination of seeds and produced complete plantlet in MS media

supplemented with 0.5 mg/L BAP

CONCLUSION

The protocol of surface sterilization for S. dulcificum or Miracle Fruit using seed
explants has been established. Clean cultures and complete plantlets from
germinated seeds were obtained from this study. Shoot cultures from in vitro
germinated seedlings were transferred to the shoot multiplication media for
further study.

46
48
ACKNOWLEDGEMENTS

Our sincere appreciation to Forest Research Institute Malaysia, FRIM for funding
and all the staff in FRIM Tissue Culture Laboratory for support and help.

REFERENCES

Akin-Idowu, P.E., Ibitoye, D.O. & Ademoyegun, O.T. (2009). Tissue Culture as a
Plant Production Technique for Horticultural Crops. African Journal
of Biotechnology 8(16):3782–3788.
Hiday, M. 2014. Manfaat Miracle Fruit Buah Ajaib. (http://tanaman--
herbal.blogspot.my/2014/5/manfaat-miracle-fruit-buah-ajaib.html)
Murashige, T. & Skoog, F. (1962). A Revised Medium For Rapid Growth And
Bioassays With Tobacco Tissue Cultures. Physiol. Plant 15:473–497
Ogunsola, K.E. & Ilori, C.O. (2008). In Vitro Propagation Of Miracle Berry
(Synsepalum Dulcificum Daniel) Through Embryo And Nodal
Cultures. African Journal Of Biotechnology 7(3):244–248
Zuraida , A.R., Fatin, L.I.K. & Ayu, N.O. (2014). In Vitro Plant Propagation For
Rapid Multiplication Of Melicope Lunu-Ankenda: A Plant Species Of
High Medicinal Value. Inter Jour Of Phar And Bio Scie. 5(1B):1148–
1156

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49
ESTABLISHMENT OF SURFACE STERILIZATION PROTOCOL FOR TISSUE
CULTURE OF CHRISTIA VESPERTILIONIS (RED BUTTERFLY WING)

Nazirah, A., Nor Hasnida, H., Muhd. Fuad, Y., Mohd Saifuldullah, A.W., Siti
Suhaila, A.R., Haliza, I., Rozidah, K., Sabariah, R. & Rohani, A.

Forestry Biotechnology Division, Forest Research Institute Malaysia (FRIM),

52109, Kepong, Selangor.

Tel: 03-62797646 Fax: 03-62804614 Email: nazirah.frim@1govuc.gov.my

INTRODUCTION

Christia vespertilionis or Rerama, belongs to Fabaceae family and originally

found in South East Asia region. Rerama can grow up to 1.5 to 2 feet height after
4 months germinated and comes in various colour (Figure 1). The green leaves
Rerama were widely used as traditional medicine and has been proven by
scientific study. In vivo studies on this plant extract showed anti-cancer, anti-
inflammatory and anti-plasmodial property (Hofer et al. 2013; Upadhyay et al.
2013).

Figure 1: C. vespertilionis or Rerama

MATERIALS AND METHODS

Sample Source

The plant was cultivated until 3 – 4 feet in height and produced multiple
branches, free from disease and in healthy greenhouse condition.

Surface Sterilization

3 different surface sterilization methods were tested to determine the

percentage of clean culture and viability rate. Pre-surface sterilization steps
were similar for all three methods as shown in Figure 2. All the leaves foliage
was trimmed and removed (2B). The nodal segments were washed under
running tap water to remove the debris and macro contaminant (2C). 0.1%

48
50
fungicide (Thiram) was used to soak the nodal segments of Rerama for 30
minutes (2D).

A C

B D

Figure 2: Pre-surface sterilization of Rerama nodal segments

The nodal segments were transferred into new sterilized bottled for
surface sterilization steps performed in laminar airflow. The differences
between the surface sterilization methods were the concentration of the
commercial bleach used and the time of samples exposed to the bleach as
stated in Table 1. Figure 3 showed the surface sterilization performed in laminar
airflow; A) Explants soaked in Ethanol, B) Explants soaked in Clorox® with Tween
20, C) Explants were cut into 2 cm in length, D) Explants cultured in new media
and E) New shoot emerged from clean culture.

Table 1: Surface sterilization method of Rerama nodal segments

Surface Sterilization Method

1. The explants were washed with sterilized distilled water to remove dirt and
macro contaminant
2. The explants were soaked in 50% Ethanol for 2 minutes
3. The explants were soaked in Clorox® with a drop of Tween 20 (Method 1:
50% Clorox® for 15 min; Method 2: 50% Clorox® for 15 min; Method 3:
60% Clorox® for 10 min)
4. The explants were rinsed with sterilized distilled water and cut into 2 cm
long
5. The 2 cm explants were soaked again in Clorox® with a drop of Tween 20
(Method 1: 10% Clorox® for 2 min; Method 2: 10% Clorox® for 5 min;
Method 3: 10% Clorox® for 10 min)
6. The explants were rinsed with sterilized distilled water and cultured in
Murashige & Skoog (MS) media for shoot induction

49
51
 A B
F

D E

Figure 3: Surface sterilization method of Rerama nodal segments.

RESULT AND DISCUSSION

Rerama is an ornamental herb with thin and soft stem. It is generally more
sensitive and fragile compared to woody plant. Double surface sterilization
method was used in this study where 2 steps of soaking in Clorox® were
performed. Figure 4 showed that lower Clorox® concentration (50%) produced
higher percentage of clean culture and explants viability rate in producing new
shoots compared to 60% Clorox®.

The success of surface sterilization protocol was measured from the

percentage of clean cultures obtained as well as the viability of the explants in
vitro. Commercial bleach or Clorox® was widely used in tissue culture. The
bleach function is to remove the contaminant from the explants surface with
the aid of surfactant Tween 20. When dissolved in water, hypochlorous acid
produced from Clorox® has an anti-microbial activity due to its ability to
breaking down bacterial cell membranes and react with proteins and DNA of the
bacteria. Beside their importance to remove the contaminants, the bleach has
also side effect as this chemical will destroy plant cell if exposed under high
concentration and in longer exposure duration (Mihaljević et al. 2013). The
destruction on the plant cell can be seen with the discoloration of green
explants to whitish. The whitish explants eventually started to die. From this
study, method 2 was more suitable in establishing Rerama clean cultures and
shoot induction.

CONCLUSION

The protocol for surface sterilization of Rerama explants has been established
and clean cultures have been obtained. The clean cultures were transferred into
shoot multiplication media for future study.

50
52
 100 Percentage

Percentage (%)
80 of clean
60 culture (%)
40 Viability
rate (%)
20
0
Method 1 Method 2 Method 3
Surface sterilization methods of Rerama explants

Figure 4: Percentage of clean culture and explants responsive rate in 3 different

surface sterilization methods

AKNOWLEDGEMENT

The authors would like to thanks FRIM for providing fund and staff from Tissue
Culture Laboratory for their assistances in completing this research.

REFERENCES

Hofer, D., Schwach, G., Tabrizi-Wizsy, N.G., Sadjak, A., Sturm, S., Stuppner, H. &
Pfragner, R. (2013). Christia vespertilionis Plant Extracts As Novel
Antiproliferative Agent Against Human Neuroendocrine Tumor Cells.
Oncology Reports. 29:2219–2226
Mihaljević, I., Dugalić, K., Tomaš, V., Viljevac, M., Pranjić, A., Čmelik, Z., PuškarB.,
& Jurković, Z. (2013). In Vitro Sterilization Procedures For
Micropropagation Of ‘Oblačinska’ Sour Cherry. Journal of Agricultural
Sciences. 58(2):117–126
Upadhyay, H.C., Sisodia, B.S., Cheema, H.S., Agrawal, J., Pal, A., Darokar, M.P. &
Srivastava, S.K. (2013). Novel Antiplasmodial Agents from Christia
vespertilionis. Natural Product Communication. 8(11):1591–1594

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PERANAN TAPAK WARISAN FRIM SEBAGAI BANK BAKA BAGI
PEMULIHARAAN TUMBUHAN UBATAN DAN PHYTOTOURISM

Ainnur Amira, A.M., Abu Huzaifah, A.M., Ajeera T., Mohd Nazrin, A. Mohamad
Alif, R. & Noorsiha A.

Bahagian Perhutanan dan Alam Sekitar, Institut Penyelidikan Perhutanan

Malaysia, 52109 Kepong, Selangor

Tel: 03-6279 7277/7619 Emel: ainnuramira@frim.gov.my

PENGENALAN

Kampus FRIM diwartakan sebagai Tapak Warisan Semula Jadi Negara pada 2009
dan seterusnya diangkat sebagai Tapak Warisan Kebangsaan pada 2012.
Kampus FRIM berkeluasan 544.3 ha merupakan satu-satunya institusi
penyelidikan di dunia diiktiraf sebagai tapak warisan. Justeru, pelbagai usaha
sedang dilaksanakan bagi menggerakkan Kampus FRIM sebagai World Heritage
Site UNESCO (WHS-UNESCO) menjelang tahun 2020. Pencalonan sebagai WHS-
UNESCO bakal mengukuhkan lagi usaha penjenamaan Tapak Warisan FRIM
sebagai tapak warisan bertaraf dunia seiring dengan status FRIM sebagai sebuah
institusi penyelidikan perhutanan terulung dunia.

Enam (6) tapak konservasi dikenalpasti bagi menjaga kepentingan

koleksi tumbuhan hidup iaitu Taman Etnobotani sebagai pusat domestikasi
tumbuhan ubatan dan beraroma; Taman Botani Kepong sebagai pusat
kecemerlangan penyelidikan dan pendidikan dalam bidang botani, hortikultur,
landskap dan pendidikan alam sekitar; Denai Alam sebagai kawasan rekreasi;
Arboretum sebagai pusat pengumpulan tumbuhan endemik dan terancam;
Rumah Tradisional Melayu Melaka sebagai pusat tumbuhan etnobotanikal dan
plot penyelidikan FRIM sebagai kawasan hutan yang ditanam sejak 1920-an.

Antara program terawal bagi pembangunan bank baka tumbuhan

ubatan ialah menerusi Projek WWF-FRIM sekitar tahun 1990an melibatkan
pengutipan koleksi buah-buahan nadir, koleksi tumbuhan ubatan dan beraroma
di bawah Bahagian Tumbuhan Ubatan, program konservasi ex-situ koleksi buluh
menerusi program IDRC, Projek Flora Malesiana Center (FMC-3) iaitu Plant
Introduction for Kepong Botanic Gardens termasuk program pertukaran bahan
tanaman dengan Taman Botani Pulau Pinang di bawah projek (FMC-10)

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54
KAEDAH

Pembangunan Sistem Pangkalan Data Flora Kampus FRIM

Pembangunan sistem pengkalan data biodiversiti Kampus FRIM berjaya

mengkompilasikan pelbagai rekod tumbuhan yang pernah ditanam seawal
tahun 1923. Sehingga kini sejumlah 21,743 rekod (Rajah 1) telah terkumpul
menerusi pangkalan data myBOTANI yang diperoleh berdasarkan 12 sumber
maklumat botanikal tersedia termasuk daripada Bahagian Perhutanan dan Alam
Sekitar, Bahagian Biodiversiti Hutan dan Bahagian Hasilan Semula Jadi.
Manakala analisa rekod dan penyediaan senarai semak awal menggupakai buku
Plant Resources of South-East Asia (PROSEA) Jilid 1-3: Medicinal and Poisonous
Plants

A Checklist of Plantation
Trials in Peninsular Malaysia
FRIM Species (BRAHMS)

FRIM A Sanctuary for

Threatened Trees
99 Spesies Buah di FRIM

Survey Flora In FRIM (2014)

Rajah 1. Sumber maklumat botanikal Kampus FRIM

Program Inventori Pokok di Plot-Plot Penyelidikan

Kampus FRIM dibahagikan kepada 55 FIELD atau plot penyelidikan yang

dikategorikan kepada sepuluh (10) jenis dirian hutan. Inventori pokok atau
pengumpulan maklumat pokok telah dijalankan di lapan (8) FIELD terpilih yang
merangkumi 20% keluasan sebenar kampus FRIM. Tumpuan diberikan ke atas
individu pokok dengan saiz minimum 10 cm ke atas dan sehingga kini sejumlah
19,752 rekod telah terkumpul menerusi sistem pangkalan data
myTREEINVENTORI.

Pelaksanaan Kajian Tapak Warisan FRIM

Bagi memperkasa bidang penyelidikan di Tapak Warisan FRIM, beberapa kajian

yang melibatkan pendokumentasian tumbuhan peringkat tinggi (herbasus) dan
juga peringkat rendah (paku-pakis) telah dijalankan yang melibatkan para
pegawai penyelidik FRIM menerusi Jawatankuasa Penilaian Institut (JPI).

53
55
Platform penyelidikan kajian kampus FRIM perlu diwujudkan dengan
penglibatan para penyelidik FRIM termasuk institusi penyelidikan, jabatan dan
agensi luar khususnya bagi aktiviti penyelidikan tumbuhan ubatan berteraskan
pembangunan produk sebatian semula jadi Tapak Warisan FRIM.

KEPUTUSAN DAN PERBINCANGAN

Perbandingan telah dibuat antara tumbuhan ubatan di kawasan Asia Tenggara

berdasarkan buku PROSEA dan tumbuhan ubatan di Tapak Warisan FRIM.
Sejumlah 906 taksa tumbuhan ubatan direkodkan di kawasan Asia Tenggara dan
daripada keseluruhan jumlah tersebut, 510 taksa (56%) diwakili oleh spesies
tumbuhan ubatan di Semenanjung Malaysia.

Hasil penemuan daripada 2,300 spesies yang telah disenaraikan dari

sistem pangkalan data flora, dianggarkan terdapat 274 taksa (11.91%) mewakili
198 genera dari 102 famili tumbuhan ubatan telah direkodkan. Berdasarkan
rekod ini, didapati Tapak Warisan FRIM mewakili 53.74% (Rajah 2) daripada
keseluruhan spesies tumbuhan ubatan di Semenanjung Malaysia.

906
1000
BILANGAN TAKSA

800 510
274 (53.74%)
600
400
200
0
Asia Tenggara Semenanjung Tapak Warisan FRIM
Malaysia

Rajah 2. Perbandingan taksa tumbuhan ubatan mengikut lokasi

Sepuluh (10) Famili utama tumbuhan ubatan Tapak Warisan FRIM

disenaraikan seperti Jadual 1. Famili Eurphorbiacea mencatatkan bilangan
spesies tumbuhan ubatan tertinggi dengan jumlah 24 spesies (Rajah 3). Bagi
meningkatkan potensi pyhtotourism di Tapak Warisan FRIM, pembangunan
strategik dan terancang menerusi konsep pembangunan “Heart of Heritage”
FRIM (HOH-FRIM) mampu menjadi contoh terbaik bagi usaha melestarikan alam
sekitar di negara ini berlandaskan konsep konservasi inter-situ tumbuhan
ubatan yang akan melibatkan tiga (3) tapak utama iaitu Pusat Interpretif
Biodiversiti (PIB), Crown Shyness Circle (CSC) dan Budaya Warisan Alam (BWA).
Dijangkakan tapak HOH-FRIM ini akan bertindak sebagai daya penarik Kampus
FRIM yang terbaharu dan mampu menarik tumpuan masyarakat dari dalam dan

54
56
luar negara untuk berkunjung ke Kampus FRIM sekaligus meningkatkan potensi
sebagai pyhtotourism.

Jadual 1. Sepuluh (10) Famili utama tumbuhan ubatan Tapak Warisan FRIM
NO. FAMILI GENERA SPESIES
1. Annonaceae 5 9
2. Apocynaceae 5 9
3. Dipterocarpaceae 4 6
4. Euphorbiaceae 12 24
5. Lamiaceae 6 7
6. Leguminosae 12 14
7. Moraceae 5 12
8. Myrtaceae 5 7
9. Rubiaceae 13 20
10. Zingiberaceae 6 7
TOTAL 73 115

Zingiberaceae 7
Rubiaceae 20
Myrtaceae 7
Moraceae 12
Leguminosae 14
Lamiaceae 7
Euphorbiaceae 24
Dipterocarpaceae 6
Apocynaceae 9
Annonaceae 9

0 5 10 15 20 25
Bilangan Spesies

Rajah 3. Bilangan spesies tumbuhan ubatan berdasarkan sepuluh (10) Famili

utama Tapak Warisan FRIM

RUMUSAN

Tumbuhan ubatan Tapak Warisan FRIM berjumlah 274 spesies daripada 198
genera dari 102 famili mewakili 53.74% daripada keseluruhan spesies tumbuhan
ubatan di Semenanjung Malaysia. Jesteru, pembangunan Arboretum Tumbuhan
Ubatan dilihat sebagai satu usaha murni bagi memperkasa nilai tumbuhan
ubatan dan beraroma di Kampus FRIM selain bertindak sebagai bank baka bagi
pemuliharaan tumbuhan ubatan yang berpotensi sebagai phytotourism dan
mampu memperkukuhkan industri pelancongan selaras dengan Bidang Ekonomi

55
57
Utama Negara (NKEA) yang mensasarkan Malaysia sebagai hab biodiversiti
global yang cemerlang sekaligus memelihara keunikkan khazanah alam hutan
tropika regenerasi di Kampus FRIM.

RUJUKAN

Abd. Latif, M., Shamsudin, I. & Nik Zanariah, N.M. (2013). FRIM Warisan
Kebangsaan. Forest Research Institute Malaysia, Kepong. Pp 42.
Padua, L.S., Bunyapraphatsara, N. & Lemmens, R. H. M. J. (Eds.), (1999). Plant
Resources of South-EastAsia No 12(1). The Prosea Foundation, Bogor,
Indonesia. Pp 705.
Van Valkenburg, J.L.C.H. & Bunyapraphatsara, N. (Eds.). (2001). Plant Resources
of South-East Asia No 12(2). Medicinal and Poisonous Plants. The Prosea
Foundation. Backhuys Publishers, Leiden. Pp 776.
Lemmens, R.H.M. J. & Bunyapraphatsara, N. (Eds.). (2003). Plant Resources of
South-East Asia No 12(2). Medicinal and Poisonous Plants. Prosea
Foundation, Backhuys Publishers, Leiden. Pp 776.

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58
EKOSISTEM TANAH TINGGI SEBAGAI SANTUARI TUMBUHAN PENAWAR
DI SEMENANJUNG MALAYSIA.

Nurliyana, A.L., Nuranis Suraya, B., Ainnur Amira, A.M., Abu Huzaifah, A.M. &
Noorsiha, A.

Bahagian Perhutanan dan Alam Sekitar, Institut Penyelidikan Perhutanan

Malaysia (FRIM), 52109 Kepong, Selangor

Tel: 03-62797277/03-62797619 Email: nurliyana@frim.gov.my

PENGENALAN

Malaysia merupakan negara ke-12 yang kaya dengan kepelbagaian biodiversiti.

Hutan gunung merupakan salah satu ekosistem utama dan jenis hutan ini
berbeza daripada hutan dipterokarpa bukit melalui ciri-ciri struktur dan floristik
tumbuhan serta komposisi spesiesnya. Secara semula jadinya, semakin tinggi
altitut, semakin rendah kanopi flora gunung tersebut. Hutan gunung mempunyai
kegunaan yang bernilai dari segi ekonomi. Antara sumber kegunaan yang boleh
didapati dari hutan gunung ialah sebagai ubatan, hiasan, makanan dan minyak
pati (Ruth Kiew 1992). Pelbagai ancaman ke atas ekosistem pergunungan
mengakibatkan impak negatif ke atas ekosistem pergunungan yang sensitif ini.
Antara ancaman yang berlaku ke atas hutan pergunungan ialah penebangan
hutan dan penukaran tanah untuk pembangunan dan pertanian serta
pengutipan hasil hutan secara haram.

Dianggarkan 25 peratus flora Semenanjung Malaysia berasal dari

kawasan pergunungan yang kaya dengan kepelbagaian spesies kerana taburan
kawasan pergunungan adalah kecil, yang meliputi kawasan tulang belakang
utama Semenanjung Malaysia iaitu banjaran Titiwangsa dan banjaran-banjaran
lain. Antara kawasan pergunungan utama adalah Gunung Jerai (Kedah), Gunung
Tahan (Pahang), Gunung Stong (Kelantan), Gunung Ledang (Johor) dan lain-lain
pergunungan utama yang digazetkan sebagai hutan simpan kekal negara.
Kepelbagaian dan keunikan flora yang terdapat di kawasan pergunungan,
seharusnya dilaksanakan pemuliharaan in-situ dengan mewujudkan santuari
bagi memelihara dan menjamin khazanah semulajadi negara ini.

Flora Pergunungan Semenanjung Malaysia

Di Semenanjung Malaysia, dianggarkan 8,500 merupakan spesies flora dan kira-

kira 40 peratus iaitu 3,323 spesies flora boleh ditemui di hutan gunung atau
montane forest dan hanya 11 peratus sahaja daripada jumlah tersebut, iaitu 353

57
59
spesies mempunyai kualiti materia medica iaitu memiliki unsur-unsur yang
boleh merawat dan menyembuhkan penyakit.

KAEDAH

Data flora pergunungan Semenanjung Malaysia diekstrak melalui Senarai Semak

Awal Flora Pergunungan Semenanjung Malaysia (Preliminary Checklist of
Montane Flora of Peninsular Malaysia) dan senarai ekstrakan tersebut yang
mempunyai nilai perubatan dirujuk menggunakan Plant Resources of South-East
Asia 12 (1-3) (PROSEA) dan beberapa buah buku rujukan utama yang lain. Data
flora pergunungan yang diperoleh dikelaskan mengikut kumpulan floristik iaitu
dikot, monokot, gimnosperma serta paku pakis dan kerabat. Senarai tersebut
diringkaskan lagi kepada famili dominan dan beberapa spesies flora
pergunungan unik yang mempunyai nilai perubatan, julat altitut yang berbeza
seperti hutan dipterokarp bukit atas (upper hill dipterocarp forest), hutan
gunung rendah (lower montane forest) dan hutan gunung tinggi (upper montane
forest) serta pengasingan data flora pergunungan ubatan kepada habit yang
berbeza iaitu epifit, herbasus, pepanjat dan pokok.

KEPUTUSAN DAN PERBINCANGAN

Berdasarkan analisis data yang dijalankan ke atas preliminary checklist, daripada

3,323 spesies flora pergunungan dan hanya 11 peratus sahaja daripada jumlah
tersebut, iaitu 353 spesies mempunyai nilai ubatan dan hanya kumpulan
monokot, dikot, paku pakis dan kerabat sahaja yang mempunyai nilai ubatan
atau penawar.

Hasil analisis data mendapati daripada 138 famili kumpulan dikot, 91

famili terdiri daripada tumbuhan ubatan dan mencatatkan kumpulan floristik
yang paling tinggi mempunyai flora ubatan. Manakala hanya sembilan (9)
daripada 30 famili dibawah kumpulan monokot yang mempunyai nilai ubatan
dan bagi kumpulan paku-pakis dan kerabat hanya 13 famili sahaja yang
mempunyai nilai ubatan daripada 41 famili. Namun, daripada tiga famili yang
mewakili kumpulan gimnosperma, tiada famili yang mempunyai nilai perubatan
direkodkan.

Daripada 212 famili flora pergunungan Semenanjung Malaysia, hanya

113 famili yang mempunyai nilai perubatan. Perbandingan jumlah famili utama
flora pergunungan yang mempunyai nilai ubatan ditunjukkan dalam Rajah 1.

Flora ubatan tanah tinggi boleh dijumpai pada julat altitut yang berbeza
mengikut tiga (3) jenis julat altitut habitat iaitu hutan dipterokap bukit atas
(upper hill dipterocarp forest), hutan gunung rendah (lower montane forest) dan

58
60
hutan gunung tinggi (upper montane forest) seperti Rajah 3. Flora penawar
banyak direkodkan di habitat hutan gunung rendah sebanyak 55 peratus dan
kebanyakkannya tabiat/habit yang direkodkan adalah pokok (Rajah 4).

150 138
130
110 91 FAMILI FLORA
90 PERGUNUNGAN
70 SEMENANJUNG
50 41
30 MALAYSIA
30 9 13
3 0 FAMILI FLORA
10
-10 PERGUNUNGAN
UBATAN

Rajah 1. Graf perbandingan jumlah keseluruhan famili flora pergunungan

Semenanjung Malaysia dan jumlah famili yang mempunyai nilai
perubatan

APOCYNACEAE 5 10
ORCHIDACEAE 6 9
LAURACEAE 6 11
ANNONACEAE 9
7 SPESIES
MELIACEAE 7 10
GENERA
MELASTOMATACEAE 7 13
FABACEAE 8 14
ARACEAE 1011
COMPOSITAE 15
15
RUBIACEAE 17 22

0 5 10 15 20 25
Rajah 2. Senarai 10 famili utama flora pergunungan Semenanjung Malaysia yang
mempunyai nilai ubatan

Antara kegunaan flora ubatan pergunungan adalah sebagai tonik

kesihatan bagi kaum lelaki dan perempuan, ubat periuk selepas bersalin,
penyakit umum seperti batuk, lesu badan dan lain-lain. Ringkasan spesies
ubatan gunung direkodkan dalam Jadual 4.

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61
 Hutan Dipterokarp
9% Bukit Atas (800-1,000
36% m)
Hutan Gunung Rendah
(1,000-1,500m)
55%

Hutan Gunung Tinggi

(1,500m)

Rajah 3. Taburan julat altitut bagi flora ubatan tanah tinggi di Semenanjung
Malaysia

EPIFIT
RENEK
4%
24% HERBASUS
21%

POKOK
31%
PEPANJAT
20%

Rajah 4. Tabiat spesies flora ubatan tanah tinggi

Berdasarkan taburan flora ubatan gunung yang lebih tertumpu di

banjaran utama Semenanjung Malaysia, adalah sesuai sekiranya sebuah santuari
flora penawar ini diwujudkan di kawasan hutan gunung rendah di banjaran
Titiwangsa selain memelihara ekosistem pergunungan yang sensitif dan
terdedah kepada pelbagai ancaman.

RUMUSAN

Taburan kepelbagaian flora pergunungan yang berpotensi sebagai tumbuhan

penawar ini seharusnya diperkemaskan lagi dengan usaha inventori di lapangan
seperti penggunaan teknologi geospatial agar rekod kepelbagaian flora ubatan
di Semenanjung Malaysia sentiasa dikemaskini. Pembinaan santuari khas
pemuliharaan dan pemeliharaan flora ubatan gunung dapat diperluaskan di
Semenanjung Malaysia dan usaha murni ini mampu membantu pihak berwajib
memangkinkan usaha pemuliharaan dan pemeliharaan khazanah alam negara.

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62
Jadual 4. Senarai 12 spesies ubatan gunung serta kegunaannya sebagai
tumbuhan penawar

Nama
Famili Spesies Taburan Kegunaan
Tempatan
Araceae Homalomena Keladi Semenanjung Tonik
griffithii kelemoyang Malaysia wanita
Pentaphrag Pentaphragma Salang serong; Perlis Tonik wanita
-mataceae aurantiacum P.Pinang
Pahang
Terengganu
Kelantan
Melastoma Phyllagathis Tapak gajah Semenanjung Batuk, mual,
-taceae hispida Malaysia tonik lelaki
Xanthor Dianella Siak-siak Perak Tonik wanita,
-rhoeaceae javanica Pahang buasir,
senggugut
Myrsinaceae Labisia pumila Kacip fatimah Semenanjung Tonik wanita
Malaysia
Ericaceae Rhododendron Rhododendron Pahang Badan lesu
javanicum Kelantan
Taccaceae Tacca Basung baning Semenanjung Darah tinggi,
integrifolia Malaysia kencing
manis,
radang,
bengkak
Araceae Epipremnum Giant pothos Semenanjung Rheumatism
pinnatum vine Malaysia dan kanser,
lebam,
batuk,
paralisis
Piperaceae Piper porphyro Belimbing Pahang Kurap susu
-phyllum tanah; Sireh
murai
Cyperaceae Mapania Akar serapat Semenanjung Ubat periuk
petiolata Malaysia selepas
bersalin;
antidote
racun
Dipteridaceae Dipteris Payung ali Pahang Tonik lelaki
conjugata
Hypoxidaceae Molineria Lemba Semenanjung Senggugut,
latifolia Malaysia keputihan

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63
RUJUKAN

de Padua, L.S., Bunyapraphatsara, N. & Lemmens, R.H.M.J. (1999). Plant

Resources of South-East Asia No 12(1): Medicinal andPoisonous
Plants 1. Prosea Foundation. Bogor, Indonesia. Pp 711.
Fatan Hamamah, Y. (2000). Potensi Tumbuh-Tumbuhan Sebagai Ubat-
Ubatan. Journal e-Bangi Fakulti Sains dan Sosial Kemanusiaan
(FSSK). Universiti Kebangsaan Malaysia, Bangi, Selangor.
Kiew, R. (1992). The Montane Flora of Peninsular Malaysia: Threats
and Conservation. Background Paper on Malaysian National
Conservation Strategy. Economic Planning Unit, Kuala Lumpur.
Lemmens, R.H.M.J. & Bunyapraphatsara, N. (2003). Plant Resources
of South-East Asia No 12(3): Medicinal and Poisonous Plants 3.
Prosea Foundation. Bogor, Indonesia. Pp 644.
Noorsiha, A., Ainnur Amira, A.M., Kamariah, M., Ng, S.C. & Ajeera, T.
(2015). Senarai Semak Awal Flora Gunung di Semenanjung
Malaysia. Cameron Highlands Montane Park (CHiMP). Institut
Penyelidikan Perhutanan Malaysia (FRIM).
Noorsiha, A., Ainnur Amira, A.M. & Kamariah, M. (2013). Pangkalan
Data Cameron Highlands Montane Park (CHiMP). Institut
Penyelidikan Perhutanan Malaysia (FRIM).
Noorsiha, A., Ainnur Amira, A.M., Kamariah, M. & Ajeera, T. (2014).
Pangkalan Data Cameron Highlands Montane Park (CHiMP).
Institut Penyelidikan Perhutanan Malaysia (FRIM).
Noorsiha, A., Ng, S.C., Nurliyana, A.L., Ainnur Amira, A.M., Kamariah,
M. & Ajeera, T. (2015). Pangkalan Data Cameron Highlands
Montane Park (CHiMP). Institut Penyelidikan Perhutanan
Malaysia (FRIM).
Noorsiha, A., Nurliyana, A.L., Ng, S.C., Nuranis Suraya, B., Ainnur Amira,
A.M., Kamariah, M., & Ajeera, T. (2016). Pangkalan Data Cameron
Highlands Montane Park (CHiMP). Institut Penyelidikan
Perhutanan Malaysia (FRIM).
Shamsul, K. & Tajuddin, A.M. (2011). Tinjauan Tumbuhan Ekonomi di Cameron
Highlands. Siri Kepelbagaian Biologi Hutan. Jabatan Perhutanan
Semenanjung Malaysia (JPSM).
van Valkenburg, J.L.C.H. & Bunyapraphatsara, N. (2002). Plant
Resources of South-East Asia No 12(2): Medicinal and
Poisonous Plants 2. Prosea Foundation. Bogor, Indonesia. Pp 782.

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 NATURAL
PRODUCT
DISCOVERY

63
PLANTS EXTRACTS IN ANTICANCER TREATMENT: PROOF AND MYTHS

Aishah, A.

Faculty of Pharmacy, Universiti Teknologi MARA,

UiTM Puncak Alam Campus,
42300 Bandar Puncak Alam, Selangor

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THE PROWESS OF OYSTER MUSHROOM (PLEUROTUS SAJOR-CAJU) AS
AN ALTERNATIVE NUTRITIVE FUNCTIONAL INGREDIENT

Wan Rosli, W.I.1, Sze Han, N.1 & Wan Amir Nizam, W. A.2

1
Nutrition Programme; 2Biomedicine Programme, School of Health Sciences,
Universiti Sains Malaysia, 16150 Kota Bharu, Kelantan

Tel: 09-7677783 Fax: 09-7677505 E-mail: wrosli@usm.my

INTRODUCTION

Pleurotus sajor-caju (PSC), one of the species from Pleurotus spp., is an edible
oyster mushroom firstly discovered in India and characterized by a white spore
print and gills attachment as well as eccentric strip occasionally (Miles & Chang
1997). It grows on trunks and stumps of deciduous trees in tropical and
subtropical rainforests and could be artificially cultivated on various agricultural
residues. It ranks second most popular cultivated mushrooms after button
mushroom and accounted for 14.2%of the total world mushroom production
(Mohamed Imran et al. 2011). For centuries, it has been widely used for culinary
and medicinal purposes due to its pleasant taste and pharmacological
properties. This mushroom is claimed to possess considerable importance in
human diet as it shows favourable dietetic properties attributed to its high
protein, minerals (calcium, phosphorus, and iron), B vitamins (thiamin,
riboflavin, and folic acid), and dietary fibre contents Manzi et al. (1999). On the
other hand, it is low in fat content and calorific value (kcal/g) and completely
devoid of starch; hence it could be an excellent inclusion in the diet of
individuals with hyperlipidemia and diabetes (Agrawal et al. 2010). Therefore, it
is considered to be advantageous in the prevention and management of
diabetes mellitus (DM). Previously, attention has been focused primarily on its
immune modulating, hypotensive, hypocholesterolemic, and antitumor
properties (Shah et al. 2007). Nevertheless, there is a lack of scientific
information on its antidiabetic properties. Thus, it is worthwhile to study the
mushroom for its utilization in diabetes management. This study is aimed at
investigating the hypoglycemic and antidiabetic effects of PSC aqueous extract
by evaluating glucose tolerance and certain important serum profiles in normal
and STZ-induced diabetic rats. The oyster mushroom powder was used in the
preparation of butter cookies, meat-based patties and herbal seasoning.

MATERIALS AND METHODS

The PSC aqueous extract was screened for hypoglycaemic potential by Blood
Glucose (BG) study. Variable doses of 500, 750 and 1000 mg/kg of extract were

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given orally by gavage to normal rats. Oral glucose tolerance test (OGTT) was
also performed in normal and diabetic rats with the same doses of extract.
Using the most effective dose (750 mg/kg) of PSC aqueous extract identified in
previous experiments, the antidiabetic effects of PSC aqueous extract were
assessed for 21 days in diabetic rats. The experimental design used was based
on studies by Jaiswal et al. (2009) with some modifications. The GI values were
determined according to the methods of FAO/WHO (1998) in which
measurements of BG in 11 healthy human subjects who were given various
types of carbohydrate from butter cookies formulated with PSC powder. For
morphological investigation, the sample was processed by critical point drying,
coated with gold and viewed in a Quanta FEG 450 Scanning Electron Microscope
(SEM) using XTm Product Version 4.1.7.2095 viewer software.

RESULTS AND DISCUSSION

Hypoglycaemic Effect of Oyster Mushroom Aqueous Extract on OGTT in

Diabetic Rats

Figure 1 illustrated the results of glucose tolerance test up to 3 h of glucose

loading. At the 500 mg/kg lower dose of PSC extract, BG level showed no
significant difference compared to control after 1 h of glucose administration,
indicating no hypoglycaemic effect. However, diabetic rats treated with 750 and
1000 mg/kg of the PSC extract significantly (P<0.05) exhibited lower BG level
compared to control group, but no significant difference compared to
metformin group. Besides this, the increment in BG level at 750 (74.7%) and
1000 mg/kg (73.1%) doses were lower compared to the 500 mg/kg dose
(97.7%). After 3 h of glucose loading, BG level of rats treated with 750 and 1000
mg/kg doses of PSC extract exhibited significant reduction (P<0.05) compared to
control but not significant compared to metformin. At 750 mg/kg dose, a
maximum fall of 36.5% in BG level was observed, whereas a 33.5% and 34.8%
reduction were observed at PSC extract doses of 500 and 1000 mg/kg,
respectively.

Morphological Characterisation of Butter Cookies Added with PSC

Powder

The diameter of wheat starch granules in PSC-based cookies and control cookies
ranged from 15.91 to 19.25 µm and 25.95 to 36.42 µm, respectively. The
present observation indicated that the addition of PSC in butter cookies resulted
in reduction of starch granules diameter. This may be due to the presence of
natural insoluble dietary fibre (33.0 g/100g) and β-glucan (25.83%) from PSC
powder (Aishah & Wan Rosli 2013) which competed with wheat starch to absorb
limited amount of available moisture in the cookies’ ingredients. The low

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70
surface area of starch granules resulted in reduced rate of starch hydrolysis and
thus, slowly raised blood glucose (Figure 2).

Figure 1: Hypoglycaemic effect of graded doses of PSCE (Pleurotus sajor-caju

aqueous extract) on glucose tolerance in diabetic rats. ap < 0.05 as
compared to diabetic control group (distilled water); bp < 0.05 as
compared to diabetic treated group (metformin 150 mg/kg).

Figure 2: Photomicrograph of butter cookies added with PSC powder (right) and
control without PSC powder (left) viewed at 1000 X magnification.

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Morphological Characterisation of Meat Patty Added with PSC

At 500 X magnification, the fibrous pileus cap of oyster mushroom carpophore

dried in oven dryer (Figure 3a) was more condensed and thick compared to
those dried using the bio-dehydration method (Figure 3b). Both treatments
showed a multi-layered spongy and rubbery looking network of sheath-like
structure of the dried oyster mushroom. This explained its ability to enhance
juiciness and tenderness of prepared food products when added with this dried
fungi. Fibrous longitudinal shrivel forming irregular form of structure was
observed as a result of drying. Majority of the fibrous materials are joined to
each other in a ‘Y’ form giving a substance with spongy multi layers of rubbery
network. This feature facilitates in the enhancement of both water and oil
retention in meat-based products especially to sustain tenderness and juiciness
of finished emulsion-type of processed meat patties and sausages.

“Y” shape
shape

3a 3b
Figure 3. The fibrous cut of pileus cap of carphopore of the oyster mushrooms
fruiting body were dried in a laboratory oven (3a) and Bio-dehydration
low temperature drying system (3b).

Mean Incremental Area Under Curve (iAUC) and Glycemic Index (GI)
Values of Butter Cookies Added with PSC Powder

Mean incremental area under curve (iAUC) of butter cookies added with PSC
powder was lower (82.3 mmol x min/l) than both control and glucose (96.1 and
168.0 mmol x min/l). The cookies added with PSC power had low GI (49) while
control and glucose recorded intermediate GI (57.2) and high GI (100) values
(Table 1). It was reported that PSC had 33.0% dietary fibre (Aishah & Wan Rosli
2013), which may play a crucial role in lowering the peak glycemic responses
when incorporated in butter biscuits. Presence of dietary fibre can delay gastric

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72
emptying and retards digestion and absorption rates of available carbohydrates
in small intestines (Benini et al. 1995).

Wolever (1986) demonstrated that cellulose and uronic acid component of

insoluble fibre contents had closer relationship with GI than soluble fibre. On
the contrary, other findings showed that soluble fibre had more effect on
glycemia. Soluble dietary fibre can slow gastric emptying and macronutrient
absorption from the gut while insoluble fibre can increase insulin sensitivity
whereby both will control elevation of postprandial glycemic response
(Chandalia et al. 2000).

Table 1: Mean incremental area under curve (iAUC) and glycemic index (GI)
value of butter cookies added with PSC powder

Test food AUC mmol x min/l GI

0% PSC 96.1 ± 8.1 57.2 ± 4.8
8% PSC 82.3 ± 11.0a 49.0 ± 6.5a
Glucose (Ref food) 168.0 ± 4.3 100

CONCLUSION

The present study concludes that aqueous extract has demonstrated significant
effects on BG level and apparent improvement on glucose tolerance in single
administration study. In addition, PSC powder has shown significant effect in
lowering GI values in butter cookies. Dietary fibres and natural β-glucan
occurred in PSC powder have shown significant action in suppressing the starch
granules diameter which delay gastric emptying and slows digestion and
absorption rate of available carbohydrates. This means that the PSC extract
could give immediate hypoglycemic effect such as reduced postprandial
glycemic response. These results evidently express the possible benefits of PSC
aqueous extract in controlling diabetes and preventing its complications.
Further biochemical, pharmacological, and clinical investigations should be
undertaken to elucidate the possible mechanism of the hypoglycemic and
antidiabetic activities of PSC extracts.

ACKNOWLEDGEMENTS

The authors acknowledge Research University Grant no: 1001/PPSK/813057 for

providing financial assistance. The authors are also thankful to contributions of
the staff members from Animal Research and Service Centre and nutrition lab in
Universiti Sains Malaysia.

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REFERENCES

Agrawal, R.P., Chopra, A. & Lavekar, G.S. (2010). Effect of Oyster Mushroom on
Glycemia, Lipid Profile and Quality of Life in Type 2 Diabetic Patients.
Australian Journal of Medical Herbalism 22(2):50–54.
Aishah, M.S. & Wan Rosli, W.I. (2013). Effect of Different Drying Techniques on
the Nutritional Values of Oyster Mushroom (Pleurotus sajor-caju). Sains
Malaysiana 42(7):937–941.
Benini, L., Castellani, G., Brighenti, F., Heaton, K.W., Brentegani, M.T., Casiraghi,
M.C., Sembenini, C., Pellegrini, N., Fioretta, A. & Minniti, G. (1995).
Gastric Emptying of a Solid Meal Is Accelerated by the Removal of
Dietary Fibre Naturally Present in Food. Gut 36: 825-830.
http://dx.doi.org/10.1136/gut.36.6.825.
Chandalia, M., Garg, A., Lutjohann, D., von Bergmann, K., Grundy, S.M. &
Brinkley, L.J. (2000). Beneficial Effects of High Dietary Fiber Intake in
Patients with Type 2 Diabetes Mellitus. New England Journal of
Medicine 342: 1392-1398. http://dx.doi.org/10.1056/
NEJM200005113421903.
FAO/WHO. (1998). Carbohydrates in human nutrition. (FAO Food and Nutrition
Paper - 66). Report of a Joint FAO/WHO Expert Consultation Rome, 14-
18 April 1998.
Jaiswal, D., Kumar Rai, P., Kumar, A., Mehta, S. & Watal, G. (2009). Effect of
Moringa oleifera Lam. Leaves Aqueous Extract Therapy on
Hyperglycemic Rats. Journal of Ethnopharmacology 123(3):392–396.
Manzi, P., Gambelli, L., Marconi, S., Vivanti, V. & Pizzoferrato, L. (1999).
Nutrients in Edible Mushrooms: an Inter-Species Comparative Study.
Food Chemistry 65(4):477–482.
Miles P.G. & Chang, S.T. (1997). Mushroom Biology: Concise Basics and Current
Development, World Scientific, New York, NY, USA.
Mohamed Imran, M., Mohamed Mahroop M. R., Abdul Basith, J. & Asarudeen,
A. (2011). Determination of Total Phenol, Flavonoid and Antioxidant
Activity of Edible Mushrooms Pleurotus florida and Pleurotus eous.
International Food Research Journal 18(2):574–577.
Shah, S., Ghosh, D. & Mallick, S.K. (2007). Immunomodulatory and Antitumor
Activities of Water-Soluble Proteoglycan Isolated from the Fruiting
Bodies of Culinary-Medicinal Oyster Mushroom Pleurotus ostreatus.
International Journal of Medicinal Mushrooms 9(2):23–138.
Wolever, T.M.S., Jenkins, D.J.A., Kalmusky, J., Giordano, C., Giudici, S., Jenkins,
A.L., Thompson, L.U., Wong, G.S. & Josse, R.G. (1986). Glycemic
Response to Pasta: Effect of Surface Area, Degree of Cooking, and
Protein Enrichment. Diabetes Care 9: 401-404.
http://dx.doi.org/10.2337/diacare.9.4.401.

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DISCOVERING THE ANTI-OVARIAN CANCER POTENTIAL OF A CARDIAC
GLYCOSIDE DERIVATIVE USING IN VITRO, IN SILICO AND PROTEOMICS
APPROACHES

Nurhanan, M.Y.1, Siti Syarifah, M.M.1, Muhammad Haffiz, J.1, Asiah, O.1, Nor
Datiakma, M.A.1, Puteri Syafinaz Akma, A.R. 2, Mohd Ilham, A.3

1
Natural Products Division, Forest Research Institute Malaysia, 52109 Kepong,
Selangor; 2University of Malaya Centre for Proteomic Research, University of
Malaya, 50603 Kuala Lumpur; 3Atta-ur-Rahman Institute for Natural Product
Discovery, Universiti Teknologi MARA, UiTM Puncak Alam, 42300 Bandar Baru
Puncak Alam, Selangor.

Tel: 03-62797659 Fax: 03-62804623 E-mail: hanan@frim.gov.my

INTRODUCTION

Ovarian cancer is fourth most common cancer among Malaysian women (Zainal
Ariffin & Nor Saleha 2011) and fifth most common cancer among American
women in which there are approximately 190,000 ovarian cancer cases reported
worldwide (Siegel et al. 2016). The search for new anti-cancer drug candidate is
in dire needs since the survival rate of patients with ovarian cancer is still poor.
In fact, ovarian cancer is known to cause the highest mortality rate among
gynaecology type of cancer (Gallion et al. 1995). Among the reasons include
poor in ovarian cancer detection method and the anti-cancer drugs used for the
treatments may become ineffective once drug resistance and toxic side effects
occur. One of the reasons of the drug resistance occurrence is due to the drug
not able to reach its target. Cancer arises due to prolonged uncorrected DNA
mutations that lead to the production of dysfunctional proteins which are
responsible for abnormal cell divisions, growth and proliferations. Thus, active
compound that capable in targeting these proteins may increase the chance to
restore the normal functions of the cells. For example, apoptosis is a
programmed cell death that involved a series of signalling pathways is one of
important mechanism to discard cells that have abnormal growth in order to
retain healthy cells. However, many cancer cells experienced malfunction in
apoptosis which explains on why the cancer cells still proliferate and grow.
Hence, any compound that capable to restore the proteins that involve in
regulating apoptotic pathway(s) may have an anti-cancer potential.

To date, we have screened at least 500 extracts against breast and

ovarian cancer cell lines and the most potent extract had been used to isolate
the active compound which was then characterised as 17H-neriifolin; a cardiac
glycoside. Cardiac glycosides comprise of a five membered lactone ring

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(cardenolides or aglycone) and sugar moieties (glycone). These compounds can
be found in animals as well as plants. Popularly plant derived cardiac glycosides
such as digoxin and digitoxin have long being used for treating congestive heart
failure alongside with other drugs. The investigations of cardiac glycoside
compounds as anti-cancer agents have started to gain attentions among the
cure hunters due to its high potency, lower recurrence rate and unique
mechanisms of action (Stenkvist et al. 1980; Newman et al. 2008). Due to these
promising scientific claims on anti-cancer property of cardiac glycosides, 17H-
neriifolin was further evaluated on its anti-ovarian cancer potential by
elucidating its mechanisms of action using proteomics and in silico approaches.

MATERIALS AND METHODS

In vitro Anti-cancer Studies

The ovarian cancer cell lines (SKOV-3) was purchased from American Type
Culture Collections (ATCC), USA and A2780 and A2780cisR were purchased from
ECACC, UK. The cells were seeded in 96-well plate at a density of 4000-6000
cells/ml and incubated at 37C and 5 % carbon dioxide in air. The cells were
treated with 17βH-neriifolin at different concentrations (0.001, 0.002, 0.004,
0.008, 0.016 µg/mL). The reactions were stopped using Sulphorhodamine B
(SRB) assay (Skehan et al. 1990) after 72 hr and the results were measured at
OD of 492nm with Magellan V.4 microtiter plate reader. The percentage of cells
viability will be calculated as follows: (OD492nm of treated cells/ OD492nm of
non-treated cells) x 100. The IC50 values were analysed based on the dose-
responsed curves from at least three independent experiments. Chemotherapy
drugs namely cisplatin and paclitaxel and also cardiac glycosides compounds
(digoxin and ouabain) were used as positive controls.

Molecular Docking of 17H-neriifolin on Na+K+ATPase

The crystal structure of N+K+-ATPase (PDB: 3A3Y) was used as a docking target.
The protein molecule was prepared by deleting ligand, extra chain, and metal
ions using ViewerLite 5.0 program (accessed from http://www.accelrys.com).
The ligand was prepared using UCSF Chimera 1.7 (Pettersen et al. 2004).
Protein-ligand docking is conducted using AutoDock 4.2 with AutoDockTools
(ADT) (Morris et al. 2009). Input structures were prepared using Gasteiger
charges for both the ligand and the protein. Grid parameter files were built
using Autogrid 4 and docking simulations were performed using the Lamarckian
Genetic Algorithm.

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Proteome Expressions with Two Dimensional Gel Electrophoresis (2-DE)

Briefly, 17βH-neriifolin-treated and non-treated SKOV-3 protein samples were

separated by its charges (pI) and MW via Iso-electric Focusing (IEF) using 13 cm
IPG strip and 2-DE, respectively. 2-DE profiles of 17βH-neriifolin treated- and
non-treated samples were stained using silver staining method (Siti Syarifah et
al. 2014). The differential analysis was then performed using Progenesis
SameSpot software (Nonlinear Dynamics Ltd., Newcastle, UK). The proteins that
were differentially expressed were then identified using Matrix-assisted laser
desorption/ionization-Mass spectrometries (MALDI-MS/MS) and Mascot
database search.

Protein Interactions Analysis

The interaction of Na+ATPases and protein identified from the proteomics

studies was performed using Ingenuity Pathway Analysis software (Qiagen,
USA). In the IPA analysis, the right-tailed Fisher Exact test is used to calculate p-
value and score (p-score=–log10 p-value) (Ingenuity Systems,
http://www.ingenuity.com/). A score of 2 was established by the IPA to increase
confidence that a particular protein was not randomly assembled into a
network.

RESULTS AND DISCUSSION

In vitro Anti-cancer Evaluation

17H-neriifolin was found to be more active than the cardiac glycosides and
chemotherapy drugs listed in Table 1. Cisplatin was found to be less active in
A2780cisR cell line (cells that was resistance to cisplatin) compared to A2780. It
had been reported that cisplatin had caused drug resistance effect in many
ovarian cancer patient. This might due to the drug failed to reach its target that
is DNA (Lippard 1995). Another drug namely paclitaxel that was initially derived
from Taxus brevifolia has also been used to treat ovarian cancer. The results in
Table 1 showed that paclitaxel was much more active than cisplatin and the
drug had been used currently as single or in combination with cisplatin to treat
the ovarian cancer patient (Daud et al. 2001). However, less than 50% cancer
patients did not benefited from these treatment regimens due to intrinsic or
extrinsic drugs resistance effects (Utsunomiya et al. 2006). Due to the anti-
cancer activity exerted by 17H-neriifolin is higher than both drugs. Other
cardiac glycosides (digoxin and ouabain) were also found to be highly active in
inhibiting the proliferations of selected cancer cell lines. The use of cardiac
glycosides in the field of cancer therapy has been reported by Stenkvist et al.
(1980) in which the breast cancer cells obtained from women on digitalis

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therapy were characterized by a series of more benign features compared with
cancer cells from control patients. In fact, digoxin has been reported to enter
clinical trial in cancer drug development phase (Shim and Liu 2014).

Table 1. The IC50 values (M) of 17H-neriifolin, selected cardiac glycosides (CG)
and anti-cancer drugs (AC) in ovarian cancer cell lines
Cancer cell lines
Compound/Drug
SKOV-3 A2780 A2780cisR
17H-neriifolin 0.015 ± 0.001 0.023 ± 0.001 0.029 ± 0.0011
Digoxin (CG) 0.080 ± 0.005 0.082 ± 0.014 0.082 ± 0.006
Ouabain (CG) 0.023 ± 0.0006 0.044 ± 0.006 0.041 ± 0.012
Paclitaxel (AC) 0.033 ± 0.002 0.043 ± 0.001 0.049 ± 0.0012
Cisplatin (AC) 9.60 ± 0.27 5.83 ± 0.70 45.46 ± 1.87

Molecular Docking of 17H-neriifolin on Na+K+ATPase

Na+K+-ATPase was discovered by Skou (1957) as an energy transducing pump. It

consists of α and β subunits and was found to be the main target for cardiac
glycoside compounds (digitoxin, digoxin, ouabain, etc.). The proteins have
started to be studied on its role in cell adhesion, inducing cancer cell death and
a versatile signal transducer since a decade ago (Chen et al. 2006).
Molecular docking was performed using AutoDock 4.2 program to
determine the target protein for 17H-neriifolin. The optimization of docking
procedure has been performed using ouabain, a cardiac glycoside to Na+K+-
ATPase. From the docking analysis, 17H-neriifolin bound to the same binding
site as ouabain at α subunit. The best pose was chosen based on the closest
conformation geometries to the crystal structure of the complex of ouabain
with Na+K+-ATPase (pdb: 3a3y). It was found that the binding affinity of 17H-
neriifolin to Na+K+-ATPase is comparable with ouabain with the inhibition
constant (Ki) of 3.4 µM and 1.3 µM respectively (Figure 1).

Ouabain 17H-neriifolin

BE = -8.03 BE = -7.46
IC = 1.3 µM IC = 3.4 µM

Figure 1. 3D picture of binding sites of ouabain and 17H-neriifolin analysed

using AutoDock 4.2 program.

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The Effect of 17H-neriifolin on Proteins Expressions and Interactions

At least 1300 proteins were found to be separated using the mentioned 2-DE
protocol. 24 proteins were found to be differentially expressed based on the
proteome profiling analysis between the 2-DE protein profiles of 17H-
neriifolin-treated and non-treated SKOV-3 cells. The proteins had been
identified using MALDI-MS/MS and the results had been published in Siti
Syarifah et al. 2014. Based on Ingenuity Software Analysis (IPA), four key
proteins were found to be significantly involved in cell death and survival,
haematological system development and function, cell-to-cell signalling and
interactions pathways were PKM, HNRNPA1, SMN and TAGLN (Figure 2).

Figure 2. The protein-protein interaction network of the differentially expressed

proteins in SKOV-3 ovarian cancer cells treated with 17H-neriifolin.

The functions of these proteins namely PKM, HNRNPA1, SMN and

TAGLN were described in Table 2. The proteins were found to cross talk with
other including P38 MAPK, TNF and JUN proteins. These proteins are known to
be that affect proliferation, differentiation, survival and migration and apoptosis
(Wagner and Nebreda 2009).

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Table 2. Key proteins that involved in the cell death pathways affected by 17H-
neriifolin.

Proteins Identified MW pI Protein Functions References

(Da) Expressions
PKM 66459 7.96 Down- An isoenzyme of Goldberg et
Pyruvate kinase, regulated the glycolytic al. 2012;
muscle enzyme pyruvate Spoden et
kinase which plays al. 2009.
as key regulator
for glycolysis. The
knock-down of
PKM2 was
reported to
decreased viability
and induce
apoptosis in
multiple cancer
cell lines (e.g.
HepG2, SKOV-3)
but not in normal
fibroblast or
endothelial cells.
Dysregulation of
glycolysis that
leads to starvation
of cells and cell
death.
HNRNPA1 29482 9.19 Down- HNRPA 1 is an Carpenter
Heterogeneous regulated oncogene. et al. 2006;
nuclear Involve in Patry et al.
ribonucleoprotein inhibition of 2003.
A1 apotosis,
angionesis and cell
invasion. This
finding was
supported by
other studies eg.
The knock-down
of HNRPA1 elicits
programmed cell
death in ovarian,
cervix, colon,
breast and brain
cancer cell lines,
thus HNRPA1
suggested to have
potential to be

76
80
 developed as
biomarker in the
therapy of cancer.
SMN 2696 4.68 Down- Human SMN Vyas et al.
Survival of motor regulated delays the onset of 2002.
neuron 1, apoptosis by
telomeric acting on
mechanism that
mediate cell death
through the
mitochondrial
release of
cytochrome C.
Down regulation
of SMN will
prevent the
process of
apoptosis being
delayed, instead,
promotes the
induction in
apoptosis. This
finding was
supported by an in
vivo studies.
TAGLN 3834 10.29 Up- Transgelin was Assinder et
Transgelin regulated reported to be al. 2009;
involved in a Chunhua et
multiple signaling al. 2013;
pathway to induce Zhe-Wei et
apoptosis in al. 2010.
cancer cells.
Transgelin over-
expression
suppressed the
expression of
metallo-matrix
proteinase) MMP-
9. MMP-9
indirectly interact
with Akt. Akt will
then act directly
on Caspase 3 to
cause DNA
fragmentation.

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81
 New generation of anti-cancer drugs were developed by targeting
various protein targets that were not restricted to the classical type of
classification such as anti-metabolites, topoisomerase inhibitor, alkylating
agents, to name a few. Post human genome project since 2001 had leads to the
discoveries of 21,000 distinct human protein-coding genes which some may be
the potential of being the new and novel targets for future anti-cancer drugs
(Frazer 2012). Examples of new drugs approved by FDA recently for cancer
treatments that targeted protein molecules include palbociclib (targeted
tyrosine kinase), panobinostat (targeted histone deacetylase), ramucirumab
(targeted VEGF receptor 2), to name a few (Buffery 2015). Thus, future studies
in need are to perform validation studies on these identified key proteins
affected by 17βH-neriifolin at in vitro and in vivo levels.

CONCLUSION

In conclusion, it was found that the binding affinity of 17βH-neriifolin targeted

and binds to Na+K+-ATPase. PKM, HNRNPA1, SMN and TAGLN proteins identified
were found to be significantly involved in cell death and survival, haematological
system development and function, cell-to-cell signalling and interactions
pathways that eventually converged and killed the cancer cells via apoptosis.
This compound may serve as potential anti-cancer bioactive hit-to-lead
compound candidate.This new discovery on anti-ovarian cancer potential of
17βH-neriifolin had been granted a patent numbered of MY 153745 A.

ACKNOWLEDGEMENTS

We are grateful to MOSTI for providing e-Science fund to conduct these studies
and Ms. Ruzana Rabuzin for her assistance during cell culture works.

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Buffery, D. (2015). The 2015 Oncology Drug Pipeline: Innovation Drives the Race
to Cure Cancer. American Health and Drug Benefits 8: 216–222.
Carpenter, B., MacKay, C., Alnabulsi, A., MacKay, M., Telfer, C., Melvin, W. T., &
Murray, G.I. (2006). The roles of heterogeneous nuclear
ribonucleoproteins in tumour development and progression. Biochimica
et Biophysica Acta 1765: 85-100.
Chen, J-Ql., Contreras, R.G., Wang, R., Fernandez, S.V., Shoshani, L., Russo,
I.H., Cereijido, M. & Russo J. (2006). Sodium/potassium ATPase (Na+, K+-
ATPase) and ouabain/related cardiac glycosides: A new paradigm

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and Treatment 96: 1-15.
Chunhua, L ., Donglan., L , Xiuqiong, F., Lihua, Z., Qin, F., Yawei, L., Liang, Z., Ge,
W., Linlin, J., Ping, Z., Kun, L. & Xuegang, S. (2013). Apigenin up-
regulates transgelin and inhibits invasion and migration of colorectal
cancer through decreased phosphorylation of AKT. Journal of Nutritional
Biochemistry 24: 1766-1775
Daud, A., Munster, P. and Spriggs, D. R. 2001. New drugs in gynecologic cancer.
Current Treatment Options in Oncology 2: 119-28.
Frazer, K.A. (2012). Decoding the human genome. Genome Research 22: 1599–
1601.
Gallion, H. H., Pieretti, M., DePriest, P. D. & van Nagell, J. R., Jr. (1995). The
molecular basis of ovarian cancer. Cancer 76(10 Suppl): 1992-1997.
Goldberg, M.S., & Sharp, P.A. (2012). Pyruvate kinase M2-specific siRNA induces
apoptosis and tumor regression. Journal of Experimental Medicine 209:
217-224
Lippard, S. J. (1995). Progress in inorganic chemistry, Bioinorganic chemistry.
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Morris,G. M., Huey, R., Lindstrom, W., Sanner, M. F., Belew, R. K., Goodsell, D. S.
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Chemistry: 30(16):2785-91.
Newman, R.A., Yang, P., Pawlus, A.D. & Block, K.I. (2008). Cardiac glycosides as
novel cancer therapeutic agents. Molecular Interventions 8:36-49
Nurhanan, M. Y., Ling, S.K., Zunoliza, A., Siti Syarifah M.M. & Nor Afiedatul
Akmal M.Y. (2015). Anti-proliferative effect of Clinacanthus nutans on
ovarian, breast and colorectal cancer cell lines. Pp. 200-208 in Asiah et
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Tani, Putrajaya.
Nurhanan, M.Y., Asiah, O., Mohd Ilham, M.A., Siti Syarifah, M.M., Norhayati, I. &
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20(2): 77-81
Patry, C., Bouchard, L., Labrecque, P., Gendron, D., Lemieux, B., Toutant, J. &
Chabot, B. (2003). Small interfering RNA-mediated reduction in
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apoptosis in human cancer cells but not in normal mortal cell lines.
Cancer Research 63: 7679-7688.
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neriifolin on its anticancer effect in skov-3 ovarian cancer cell line.
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p53. Asian Journal of Andrology 12: 186-195.

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PURIFICATION OF FLAVONOID METHYL ETHER FROM TETRACERA
INDICA USING AUTOMATED CHROMATOGRAPHIC APPROACH

Fauziah, A.1,2,3 & Nor Hadiani, I.1,2

1
Faculty of Applied Sciences, University Teknologi Mara, Shah Alam, 40450 Shah Alam,
Selangor. 2Atta-Ur-Rahman Institute of Natural Product for Drug Discovery (RiND),
University Teknologi Mara, Puncak Alam, 42300 Kuala Selangor, Selangor.
3
Phytochemistry Programme, Natural Products Division, Forest Research Institute
Malaysia (FRIM) 52109 Kepong, Selangor

E-mail: fauziahabdullah@frim.gov.my

ABSTRACT

Tetracera indica belongs to the Dilleniaceae family. Locally known as mempelas.

The plant is small woody about 2 m in height or liana up to 5 m long. This plant
grows in the forest fringes of Thailand and throughout Peninsular Malaysia. In
traditional medicine practice, various part of T. indica roots are used for the
treatment several diseases such as hypertension, fever, skin inflammation, put
on poisonous snake bites and festering finger, pulmonary hemorrhages and also
used as gargle for the treatment of aphthae. It is also believed that the plant is
used to treat diabetes. T. indica reported previously to contain common
flavonoids such as quercetin and kaempferol, methyl ether flavonoids and
terpenes group of compound such as betulinic acid and betulin. Despite of the
reported traditional uses and variety types of compound isolated, therefore
isolation and purification of compound from stem of T. indica has been done.
This paper is focusing on purification of flavonoid methyl ether from semi-
purified fractions obtained from Yamazen Dual Channel Automated Flash
Chromatography and further purification using preparative recycling HPLC
technique. Compounds purification was performed using Recycling Preparative
HPLC LC-9130 II NEXT. Separation and purification was achieved using a JAIGEL-
ODS-AP30, SP 120-15 using isocratic elution of 100% methanol, 10 ml of
injection volume with 5ml/min flow rate. The detection was performed using
UV-370 NE at 214, 254, 270 and 285 nm. The structures of the purified flavonoid
methyl ether were confirmed by a combination spectroscopic ultraviolet,
infrared, mass and nuclear magnetic resonance as well as by comparison with
literature values or standard samples.

Key words: Tetracera indica, Automated Flash Chromatography

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XANTHINE OXIDASE INHIBITORY ACTIVITY OF FLAVONOID COMPOUND
FROM HIBISCUS ROSA-SINENSIS L. AND BRASSICA OLERACEA L.

Hussin, M.Z.1, Farah, S.2 & Nurliyana, A.2

1
Center for Fundamental and Liberal Education, 2School of Fundamental Science,
Universiti Malaysia Terengganu, 21030, Kuala Terengganu.

Tel: 09-6683554 Fax: 09-6683434 E-mail: mhuzain@umt.edu.my

INTRODUCTION

Flavonoids are phytochemical compounds present in plants and they contribute

to the colour of fruits and vegetables (Umamaheswari et al. 2007). They can be
found in any parts of plant since one of their function is to attract pollinators
Flavonoid compound is one of the active ingredient in Hibiscus rosa-sinesis L. as
it gives a red colour to flowers (Consolacion et al. 2011) and a purple colour in
Brassica species (Bridle et al. 2008 and Lin et al, 2008). There are many studies
on flavonoid compounds from plant sources since they have versatile health
benefits. Flavonoids in plants have biological activities such as antioxidant
(Vanden-Berghe et al. 1993; Kumar et al. 2013) by inhibition of xanthine oxidase
activity (Paul et al. 1998; Mo et al. 2007; Hendriani et al. 2014). The anti-
oxidative effect found in these plant extracts has led researchers to investigate
their inhibitory activity on xanthine oxidase during uric acid production to help
patients with gout.

Hibiscus species have been found to have potential bioactive compounds as

antioxidant and cardio-protective agents (Ghaffar & El-Elaimy 2012). The red
flower of Hibiscus species has been reported to be used against paralysis and to
regulate menstruation, although sometimes it can cause abortion (Ragasa &
Rufino 2011). On the other hand, Brassica species (red cabbage) has been
widely known as a vegetable which also have health benefit effects (Lucier et al.
2013). A study on the influence of certain flavonoids in Brassica species,
including myricetin, quercetin, kaemferol and luteolin, on human health verified
their capacity to reduce the risk of coronary heart disease (Franke at al. 2004).

Gout, a disease related to deposition of uric acid crystals within the joint tissues,
is currently being cured by the urate-lowering drug known as allopurinol.
However, this drug is known to cause Stevens-Johnson syndrome and toxic
epidermal necrolysis (Mockenhaupt et al, 2008). Therefore, both plant extracts
could be promising alternatives as remedy for gout by decreasing uric acid level
in human tissue.

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MATERIALS AND METHODS

Chemicals and plant materials

Chemicals and reagents used were allopurinol (Fluka), xanthine and xanthine
oxidase (Merck), monobasic sodium phosphate (NaH2PO4), dibasic sodium
phosphate (Na2HPO4), uric acid (Sigma-Aldrich), hydrochloric acid, sodium
hydroxide, methanol, deionized water and absolute ethanol. The samples of
Hibiscus rosa-sinensis L. were collected in Kuala Terengganu and Brassica
oleracea L. was purchased from a local supermarket.

Preparation of crude extracts

The raw plant material of Hibiscus rosa-sinensis L. (5.0 g) and Brassica oleracea
L. (50.0 g) were ground before subjected to solvent extraction process. The
grounded sample was soaked and homogenized in methanol for 1, 5 and 10
days. Three different batches of solution mixture was filtered and concentrated
to give a sample extract for enzymatic inhibitory assay.

Inhibitory assay

The inhibitory effect on xanthine oxidase was measured spectrophotometrically

at 290 nm under aerobic condition. A commercial xanthine oxidase inhibitor,
allopurinol (100 µg/ml) was used as a positive control for inhibitory activity
assay. The reaction mixture consist of 1500μl (50 mM sodium phosphate buffer;
pH 7.5), 300 μl of sample solution (0.1mg/ml) in deionized water, 300 μl of
freshly prepared enzyme solution (0.2 units/ml of xanthine oxidase in phosphate
buffer) and 300 μl of deionized water. The assay mixture was pre-incubated at
25°C for 15 min. Then, 600 μl of substrate solution (0.15 mM of xanthine) was
added into the mixture. The absorbance was measured at intervals of 2 minutes
for 20 minutes using UV/VIS spectrophotometer against a blank prepared in the
same way but the enzyme solution was replaced with the phosphate buffer.
Another reaction mixture was prepared (control) consist of 300 μl of deionized
water instead of test samples in order to have maximum uric acid formation.
The Inhibition activity of xanthine oxidase was calculated as (1- ΔAbsBlank /
ΔAbsExp) x 100.

RESULTS AND DISCUSSSIONS

Optimization of extraction process.

All different batches of samples were filtered and concentrated before

evaluating the production rate of uric acid in enzymatic assay. The production

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87
rate of uric acid catalysed by xanthine oxidase without treatment with extract
samples was 0.915 µM/min. The result shows that activity of xanthine oxidase
reduced when the extract of H. rosa-sinensis L. and B. oleracea L. were
introduced to the reaction mixture. The lowest production rate of uric acid for
H. rosa-sinensis L. (0.424 µM/min) and B. oleracea L. (0.512 µM/min) was at 5
days (Figure 1). The findings showed that the optimum and effective soaking
period to extract active flavonoid compounds was 5 days. However, the
production rate of uric acid is still higher than the positive control (allopurinol).
This result may be due to the low concentration of active flavonoid compound in
the extracts if compared with allopurinol.

1
Production rate of ric acid

0.9
0.8
(µmolar/min)

0.7
0.6
0.5
0.4
0.3 Red Cabbage
0.2
0.1 Hibiscus rosa-sinensis
0

Samples

Figure 1: Production rate of uric acid for different batches of extract

Xanthine oxidase inhibitory activity

Extract at 5 days soaking was selected as the sample because of its effective
activity in xanthine oxidase (XO) inhibitory assay. The inhibition percentages of
sample were tabulated in Table 1. The comparison was also made between the
optimised plant extracts and positive control (allopurinol). The results showed
that inhibition percentage of samples fluctuated due to the nature of interaction
between the enzyme (xanthine oxidase) and sample. The data showed that
extract of H. rosa-sinensis L. was much effective in inhibiting the activity of
xanthine oxidase compared with B. oleracea L. The inhibition percentage of H.
rosa-sinensis L. (54.66%) was higher than B. oleracea L. (30.00%) at effectiveness
concentration of 0.08 mg/ml and 0.06 mg/ml, respectively. However, the
inhibition percentages of both plant extracts were much lower than allopurinol
due to the low concentration of active flavonoid compounds in the extracts. The
lowest difference of inhibition percentage between extract of H. rosa-sinensis L.
and allopurinol was 37.16% at 0.08 mg/ml.

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Table 1: Inhibitory percentage of difference concentration of samples
Concentration Inhibition Inhibition Inhibition
(mg/ml) percentage of percentage of percentage of
Hibiscus rosa- Brassica oleracea L. allopurinol (%)
sinensis L. (%) (%)
0.04 43.96 25.31 92.09
0.06 43.47 30.00 92.35
0.08 54.66 26.72 91.82
0.10 48.09 23.44 90.68

IC50 evaluation of the optimized extract on xanthine oxidase inhibitory

activity.

IC50 of xanthine oxidase activity was used to represent the concentration of

optimised extract that gives 50% inhibition under experimental conditions. The
graph showed that the lowest IC50 value was for allopurinol (0.009 mg/ml)
followed by H. rosa-sinensis L. (0.083 mg/ml) and B. oleracea L. (higher than 0.1
mg/ml). The results showed that only 0.083mg/ml of extract (H. rosa-sinensis L.)
was needed to give the 50% inhibition activity of allopurinol. Therefore, it could
be a potential plant extract to be used in treating gout disease.

100
Percentage of XO inhibition (%)

y = 21.128ln(x) + 148.47
80 R² = 0.9633
y = 11.234ln(x) + 77.957 Hibiscus rosa-sinensis
60 R² = 0.9709

40
Red cabbage
20 y = 5.9653ln(x) + 42.087
R² = 0.92
0 Allopurinol
0 0.05 0.1
Concentration (mg/ml)

Figure 2: IC50 curve for inhibition activity of samples.

CONCLUSION

Screening of phytochemical compounds in tropical plants or herbs for

pharmacological activity promises alternative treatment for various illnesses
such as gout. The extracts of flavonoid compounds from petal of H. rosa-
sinensis L. and B. oleracea L. showed the ability to inhibit xanthine oxidase

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activity. The efficiency of the extract to inhibit the activity depends on the
content of active flavonoid compounds in the extract. The data showed that
both samples should be soaked in methanol for 5 days to extract the optimum
amounts of active compounds before carrying out the inhibitory assay. The
extract of H. rosa-sinensis L. (54.66%) is more effective in inhibiting the xanthine
oxidase activity compared to the extract of B. oleracea L. (30.00%). The lowest
percentage difference of inhibition between optimised extract of H. rosa-
sinensis L. and commercial anti-gout (allopurinol) was 37.16%. However, the
percentage difference is quite high and these findings encouraged further
studies to be conducted on the extract for anti-gout product. Nevertheless, the
concentration of H. rosa-sinensis L. extract that gave 50% inhibition activity as
that of allopurinol was only 0.083mg/ml.

ACKNOWLEGEMENTS

We are grateful to University Malaysia Terengganu for the financial grant (GGP
Fasa 2/2013).

REFERENCES

Bridle, P. & Timberlake, C.F. (2008). Anthocyanins as natural food colours-

selected aspects. Food Chem 58:103–109.
Consolacion, Y.R. & Leslie-Ann, A.R. (2011). Antimicrobial Flavonoid from
Hibiscus rosa-sinensis Linn. The Manila Journal of Science 7:12–18.
Franke, A.A., Custer, L.J., Arakaki, C. & Murphy, F.P. (2004). Vitamin C and
Flavonoid Levels of Fruits and Vegetables Consumed in Hawaii. Food
Compos Anal. 17:1–35
Ghaffar, F.R.A. & El-Elaimy, I.E. (2012). In vitro, antioxidant and scavenging
activities of Hibiscus rosa-sinensis crude extract. Journal of Applied
Pharmaceutical Science 2(1):51–58.
Hendriani, R., Sukandar, E.Y., Kusnandaranggadiredja & Sukrasno. (2014). In
Vitro Evaluation Of Xanthine Oxidase Inhibitory Activity Of Sonchus
Arvensis Leaves. Int J. Pharm. Pharm. Sci. 6(2):501–503
Kumar, S. & Pandey, A.K. (2013). Review Article: Chemistry and biological
activities of flavonoids: an overview. The Scientific World Journal
Volume 2013.
Lin, J.Y., Li, C. & Hwang, I. (2008). Characterization of the Pigment Components
in Red Cabbage and their Anti-inflammatory Effects. Food Chemistry
109:771–781.
Lucier, G. & Plummer, C. (2013). Vegetables and Melon Outlook. Economic
Research Service 297.

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Mo, S.F., Zhou, F., Lu, Y.Z., Hu, G.H., Zang, D.M. & Kong, L.D. (2007).
Hypouricemicaction of selected flavonoids in mice: structure-activity
relationships. Biological & Pharmaceutical Bulletin 30:1551–1556.
Mockenhaupt, M., Viboud, C., Dunent, A., Naldi, L., Halevy, S., Bouwes-Bavinck,
J.N., Sidoroff, A., Schneck, J., Roujeau, J.C. & Flahault, A. (2008). Stevens-
Johnson syndrome and toxic epidermal necrolysis: assessment of
medication risks with emphasis on recently marketed drug. The
EuroSCAR-study. J. Invest Dermatol. 128(1):35–44.
Paul, C., Li, Y., Mario,C., Jia,P.H., Kanyanga,C., Bart, V.P., Luc, P., Arnold, J.V. &
Dirk, V.B. (1998). Structure-Activity relationship and Classification of
Flavonoids as Inhibitors of Xanthine Oxidase and Superoxide Scavengers.
J. Nat. Prod. 61:71–76.
Ragasa, C.Y. & Rufino, A.L. (2011). Antimicrobiol Flavonoid from Hibiscus rosa-
sinensis Linn. The Manila Journal of Sciences 7(1):12–18.
Umamaheswari M., Asokkumar K., Somasundaram A., Sivashanmugam T.,
Subhadradevi V. & Ravi T.K. (2007). Xanthine activity of some Indian
medical plants. J. Ethnopharmacol 109:247–551
Vanden-Berghe, D.A.R., Haemers, A. & Vlietinck, A.J. (1993). In Bioactive Natural
Products: Detection, Isolation and Structural Determination. CRC Press
London. Chapter 17, pp 405–440.

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MULTIDRUG-RESISTANT STAPHYLOCOCCUS AUREUS (MRSA) ACTIVITY
OF NARINGENIN RELATED COMPOUNDS

Adiana, M.A.1, Saiful Azmi, J.1, Mastura, M.1, Ling, S.K1 & Farediah, A.2
1.
Natural Products Division, Forest Research Institute Malaysia (FRIM), 52109,
Kepong, Selangor, Malaysia. 2.Department of Chemistry, Faculty of Science,
Universiti Teknologi Malaysia, 81310, Skudai, Johor.

Tel: 03-62797366 Fax: 03-62729805 E-mail: adiana@frim.gov.my

ABSTRACT

Increase prevalence of Methicillin-resistant Staphylococcus aureus (MRSA), a

clinically important pathogen, remains a major threat to healthcare worldwide
as the existing antibiotics become less in numbers and powers. The need to
search for alternative antibiotics becomes even more crucial when Vancomycin
resistant isolates was reported from various parts across the globe. Previously,
we have reported the isolation of naringenin-4’-methyl ether from a local
medicinal plant, Chromolaena odorata. However, this flavanone compound
displayed no inhibitory activity with minimum inhibitory concentration (MIC)
value of > 500 µg/ml against a panel of MRSA isolates. In this study, 13
synthetically-produced naringenin-related compounds were evaluated for their
potential inhibitory activity against clinical MRSA isolates. The analysed
flavonoids belong to three well-differentiated structural patterns; chalcones,
flavanones and flavones. A prenylated naringenin, namely 4’,5,7-trihydroxy-3’-
prenylflavanone showed good inhibitory activity against all three MRSA isolates
with MIC value of 31.3 µg/ml. All of the inhibitory-active flavonoids are from the
flavanone sub-group.

Key words: Flavanones, Methicillin-resistant Staphylococcus aureus (MRSA),

flavonoids

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DETERMINATION OF TOTAL PROTEIN CONTENT IN SELECTED PLANT
SPECIES BY USING BICINCHONINIC ACID (BCA) ASSAY

Norsuhaina, Z. & Abd Rashid, L.

Natural Product Division, Forest Research Institute Malaysia, 52109 Kepong,

Selangor.

Tel: 0362797337 Fax: 0362729805 E-mail: norsuhaina@frim.gov.my

ABSTRACT

This paper reports the determination of total plant protein content of six
selected plant species, including pineapple stem and fruit, noni fruit, petai
belalang seeds, roselle calyx, soybean, soursop fruit and tongkat ali roots by
using bicinchoninic acid assay. Plant proteins have reduced content of essential
amino acids in comparison to animal proteins. A significant reduction of limiting
amino acids (methionine, lysine, tryptophan) denotes lower protein synthesis.
The bicinchoninic acid assay for the determination of total protein content was
first introduced by Smith et al. It was developed for protein quantification for
UV – absorbance method. This assay counts on the reduction of Cu2+ ions by
protein. The Cu+ formed is detected by conversion into a violet-colored
substance by reaction with bicinchoninate. The color produced from this
reaction increases in a proportional line over a broad range of increasing protein
concentration. The plant protein extracts from each sample were extracted
previously by using 0.1 M phosphate buffer. The protein content in each plant
protein extracts were as follows; pineapple stem (84.6%) and fruit (70.65%),
noni fruit (38.07%), petai belalang seeds (68.8%), roselle (16.96%), soybean
(83.76%), soursop (65.2%) and tongkat ali (1.13%). We concluded that pineapple
stem has the highest total protein content compared to other plant species,
thus it could be considered as protein food supplement helping to improve
nutrient intake and has health benefits.

Key words: plant protein, total protein content, bicinchoninic acid assay (BCA),
UV-absorbance method, pineapple stem

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CHEMICAL PROFILES OF FOUR SELECTED LABISIA PUMILA HIGH
YIELDING ACCESSIONS BASED ON UTERUS CONTRACTION ACTIVITY

Farah Fazwa, M. A., Zunoliza, A.1, Nur Nazihah, M., Syafiqah Nabilah, S.B.,
Norhayati, S. & Mohd Zaki, A.

Forest Biotechnology Division, 1Natural Products Division, Forest Research

Institute Malaysia, 52109 Kepong, Selangor, Malaysia

Email: farah@frim.gov.my

INTRODUCTION

Herbal plants are widely used as a medicinal herbs and are important part of
health care since the ancient times. In certain countries such as Africa, more
than 90% of the populations consumed herbal plants as their major source of
uterotonic agents. Labisia pumila or locally known as “Kacip fatimah” is one of
the herbs being widely used by Malay women to induce and facilitate childbirth
as well as to improve their post partum health (Ibrahim et al. 2011). There are
three verieties of L. pumila, namely L. pumila var. alata, L. pumila var. pumila
and L. pumila var. lanceolata (Sunarno 2005). These three varieties can be
characterised through the size of petiol. L. pumila var alata and L. pumila var.
pumila have a short petiole within 2 cm to 8 cm, but L. pumila var. alata has a
wide wing. While L. pumila var. pumila have long petiole within 5 cm to 13 cm
without wing (Jamia Azdina et al. 1999).

Several research have been done focusing on the uses of L. pumila.

There is a preliminary study on traditional medicines containing L. pumila var.
alata for pre-natal and L. pumila var. pumila for post-natal (Jamia Azdina et al.
1999). Parts used for traditional preparation includes leaves, roots or the entire
herb. Water decoction of L. pumila helps to reduce illness during labor, to
facilitate childbirth, to tone abdominal muscles and to regain body strength
(Wan Ezumi et al. 2007). The uses of L. pumila in medicine are not restricted to
women as it can also be consumed by men to treat dysentery, flatulence and
gonorrhea (Muhammad & Mustafa 1994).

Increasing request for high yielding material of this plant from the
herbal industry serve as catalyst for researchers to establish high quality
planting materials to fulfill the industrial demand. In order to produce high
quality raw materials to support Malaysian herbal industries, it is important to
select genotype which either exhibiting notable bioactivity and/or contained
high quality active ingredients (Ibrahim et al. 1990) before it can be
domesticated. Therefore, a study conducted in 2015, identified four accessions

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of L. pumila for having high uterus contraction activity (Farah Fazwa et al. 2015).
In subsequent, this study was carried out to identify the chemical profiles of the
four selected accessions using high performance liquid chromatography (HPLC),
ultraviolet-visible (UV-Vis), fourier transform infrared (FTIR) and high
performance thin layer chromatography (HPTLC).

MATERIAL AND METHODS

Chemical fingerprinting by High Performance Liquid Chromatography

(HPLC)

A total of 20 mg of extracted L. pumila was dissolved in 50% MeOH/water and

sonicated for 15 minutes. Each of resulting solution were filtered through
membrane filter (pore size 0.45 µm) prior to analysis. The samples were
analyzed by means of HPLC system (Waters Delta 600 with 600 controllers) with
Photodiode array detector (Waters 996). A Phenomenex-Luna (5 mm x 4.6 i.d. x
250 mm) was used and for elution of constituents, two solvents denoted as A
and B was employed. A was 0.1% aqueous Formic acid and B was acetonitrile.
Initial conditions were 85% A and 15% B with a linear gradient reaching 60% B at
30 minutes. The flow rate used was 1.0ml/min and the injection volume was 10
l.

Chemical fingerprinting by UV-Vis Spectroscopy (UV-VIS)

A total of 10 mg of L. pumila extracts was diluted with 50 mL distilled water. The

dilution was scanned at the range of 200–400 nm using UV-Vis
spectrophotometer.

Chemical fingerprinting by Fourier Transform Infrared Spectroscopy

(FTIR)

A total of 2 mg of dried leaf was ground into powder and then blended with KBr
powder, ground again and pressed into a tablet. FTIR analysis was performed
using Spectrum 100 FTIR system (Perkin Elmer), equipped with a DTGS detector.
Infra-red (IR) spectra were recorded form an accumulation of 16 scans in 4000
cm-1 – 450 cm-1 range with resolution of 4 cm-1. Each sample was analysed in
triplicate.

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RESULTS AND DISCUSSIONS

Chemical fingerprinting by High Performance Liquid Chromatography

(HPLC)

The HPLC profile shows similar pattern of peaks between LP15 and LP30 (L.
pumila var. alata) and LP26 and LP28 (L. pumila var. pumila) (Figure 1). In this
study determination of the compounds present were carried out by comparing
the retention time of standard compounds and extracts. HPLC profile of LP15 (L.
pumila var. alata) showed the presence of caffeic acid and myricetin at the
retention time of 16.596 and 22.560 minutes, respectively. Whereas for LP26
representing L. pumila var. pumila, indicated the presence of gallic acid and
rutin at retention time of 8.215 and 17.550 minutes, respectively. This finding is
comparable to the previous study by Hawa et al. (2012) where presence of gallic
acid, caffeic acid, pyrogallol, myricetin, quercetin and rutin in L. pumila were
reported. The identified compounds can be used as a reference to identify the
relationship between the chemical compounds and uterus contraction activity
of these four accessions.

Figure 1: HPLC profile of two varieties of L. pumila with higher uterus

contraction activity; (a) L. pumila var. alata, (b) L. pumila var. pumila
with seven marker compounds

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Chemical fingerprinting by UV-Vis Spectroscopy (UV-VIS)

Figure 2 showed the UV profile of LP15, LP30, LP26 and LP28. This UV spectrum
shows UV maxima at wavelength of 260 to 280 nm, which the presence of
phenolic compound groups is indicated. Previous study by Ehsan et al. (2011)
has reported that higher accumulation of phenolics such as gallic acid and
pyrogallol were found in L. pumila var. alata, whereas the higher accumulation
of flavonoids such as rutin, quercetin and kaempferol were found in L. pumila
var. pumila. Therefore, in this study, the UV profile of the four accessions
probably represented the phenolic group such as gallic acid and derivatives of
gallic acid such as pyrogallol.

a) LP15

b) LP30

c) LP26

d) LP28

Figure 2: Uv-Vis profile of two varieties of L. pumila with higher uterus

contraction activity; (a) & (b) L. pumila var. alata, (b) &(c) L. pumila
var. pumila

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 Fourier Transform Infrared (FTIR) fingerprint

The FTIR spectra of four accessions of L. pumila are shown in Figure 3.

Comparison of the FTIR spectrum between the four accessions indicated the
differences of the main constituent for the four accessions seems to be similar.
Table 1 showed the presence of strong peak at 3340, 3339 and 3262 which
indicated the presence of hydroxyl (O-H) group. Another peaks observed at
2920 – 2923 cm-1 for the four accessions assigned as symmetrical vibration of C-
H group indicates as asymmetrical vibration of C-H. Spectra also exhibited peaks
in the range from 1635 to 1640, which usually a characteristic for
the presence of C=C. While, peaks observed at 1750-1735cm-1 being assigned as
carbonyl groups (C=O). Thus, all the observed peaks can suggest the presence of
phenolic compounds in all extracts.

Table 1: Frequencies of four accessions of L. pumila

Frequencies [cm-1] Functional group

3262 – 3340 v(O-H, N-H)

2920 – 2923 vas(C-H)

1635 – 1640 v(C=O) main, v(C=C), δas(N-H)

1445 – 1448 δas(C-H)

1369 – 1371 δas(C-H)

1236 δs(C-CO), v(C-O),vllas(=C-O-C), vf(C-C)

1155 – 1159 v(C-H)

1033 – 1035 vll(=C-O-C), vf(C-C)

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Figure 3: FTIR spectra of four accessions of L. pumila

CONCLUSION

Findings of this study will be used as a reference for identification of

phytochemical constituents in L. pumila and as criteria in selecting of high
yielding plants in plant breeding programme.

REFERENCES

Ehsan, K., Hawa, Z.E.J., & Sahida, A. (2011) Phytochemical Analysis and
Antimicrobial Activities of Methanolic Extracts of Leaf, Stem and Root
from Different Varieties of Labisa pumila Benth Molecules. 16:4438–
4450.
Hawa, Z.E.J., Mohd Hafiz, I. & Ehsan, K. (2012). Phenolics and Flavonoids
Compounds, Phenylanine Ammonia Lyase and Antioxidant Activity

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 Responses to Elevated CO2 in Labisia pumila (Myrisinaceae). Molecules.
17:6331– 6347.
Ibrahim, J. & Abdul Razak, M.A. (1990). A still for distillation of essential oils in
the field. FRIM Technical Information No. 17 April 1990.
Ibrahim, M.H. & Hawa, Z.E.J. (2011). Photosynthetic Capacity, Photochemical
Efficiency and Chlorophyll Content of Three Varieties of Labisia pumila
Benth Exposed to Open Field and Greenhouse Growing Conditions. Acta
Physiol. Plant. 33:2179–85.
Jamia Azdina, J., Houghton, P.J. & Milligan, S.R. (1999). Kacip Fatimah: A Malay
Traditional Herb for Pregnant Women. In Abdul Manaf, A., Khozirah, S.
& Zuriati, Z. (Eds.). Phytochemicals and Biopharmaceutins From The
Malaysiam Rain Forest, (Pg. 166–176). Kepong: Institut Penyelidikan
Perhutanan Malaysia.
Muhamad, Z. & Mustafa, A. M. (1994). Traditional Malay Medicine Plants.
Penerbit Fajar Bakti Sdn. Bhd. Kuala Lumpur. Pp 26
Sunarno, B. (2005). Revision of the Genus Labisia (Myrsinaceae). Blumea -
Biodiversity, Evolution and Biogeography of Plants. National Herbarium
Nederland. 50:579–597
Wan Ezumi M.F, Siti Amrah, S., Suhaimi, A.W.M, & Mohsin, S.S.J. (2007).
Evaluation of Female Reproductive Toxicity of Aqueous Extract of
Labisia pumila var. alata in rats. Indian Journal of Pharmacology.
39(1):30–32.

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PRODUK HASILAN SEMULA JADI DARI TUMBUHAN KESUKAAN KUCING

Wan Mukhtar, W.R., Abdul Wahab, I. & Mohsin, H.F.

Jabatan Farmakologi & Kimia, Fakulti Farmasi, Universiti Teknologi MARA,

42300 Bandar Puncak Alam, Selangor Darul Ehsan, Malaysia.

E-mail: ibtisam@puncakalam.uitm.edu.my

PENGENALAN

Masyarakat kini kian tertarik dengan kucing sebagai personaliti di dalam media
elektronik dan sosial (Oh My Jep, Maru The Cat, Cole and Marmalade). Sebelum
era teknologi, kucing merupakan ikon dalam kalangan pembaca buku kanak-
kanak (Seuss 1985). Sasterawan Negara juga merupakan penulis yang mendapat
ilham dengan memerhati perilaku kucing (Zain 2015). Para novelis turut
mengikuti kaedah yang sama di dalam penghasilan penulisan kreatif (Tehrani
2007 & Saad 2013). Terdapat juga kucing yang menarik perhatian tanpa
menggunakan agen atau produk semula jadi. (Rajah 1).

Aktiviti seharian manusia boleh terjejas melalui gelagat kucing seperti

berguling di atas papan seterika. Tindakan yang lebih dramatik dari seseekor
kucing untuk mengalihkan perhatian manusia, boleh juga dilihat apabila
seseorang pemilik kucing berada di depan komputer. Ini seterusnya menghalang
seseorang dari menekan papan kekunci. Pelbagai aktiviti dan sikap kucing boleh
diperhatikan serta direkod. Pemerhatian ini seterusnya, boleh dijadikan
kompilasi yang bersifat lucu dan humor (Canfield et al. 2012). Interaksi kucing
dengan manusia juga boleh bersifat terapeutik, seperti yang dilaporkan di dalam
wad klinikal oleh Dosa (2011).

Rajah 1: Aksi kucing yang boleh mengganggu aktiviti manusia.

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 Maklumat berkaitan sikap haiwan peliharaan ini boleh diperolehi dari
pelbagai media komunikasi, namun, kebanyakannya menyasarkan golongan
pembaca spesifik serta penyelidik. Misalnya, data sebatian kimia yang
bertanggungjawab untuk menarik perhatian kucing, diakses dari jurnal saintifik
tertentu, penerbitan konferens dan artikel berwasit. Ini adalah berkaitan dengan
kepenggunaan tumbuhan sebagai agen yang menarik perhatian kucing. Spesis
pokok berubat dan herba mampu berfungsi sebagai agen untuk mengawal
perangai kucing agar menjadi lebih baik, jinak serta lebih disayangi. Kertas kerja
ini akan mengulas spesies Nepeta dan Actinidia (masing-masing dari keluarga
pokok Lamiaceae dan Actinidiaceae). Informasi mengenai molekul sebatian
semula jadi tersebut juga akan diperjelaskan.

KAJIAN LITERATUR MENGENAI SPESIS NEPETA DAN ACTINIDIA

Melalui literatur, pokok genus Nepeta berasal dari Eropah, Asia dan Afrika, di
mana kepelbagaian spesies ini lebih tinggi di Mediterranean dan timur Cina.
Sementara itu, spesies Actinidia atau silver vine (matatabi di dalam Bahasa
Jepun), tumbuh di Asia dan Jepun. Satu spesis Actinidia, iaitu Actinidia
polygama, mengandungi asid askorbik, lakton monoterpen dari jenis iridan dan
triterpenoid (Sakai et al. 1980; Matsuzawa et al. 1986, Sashida et al. 1992).
Kepentingan tumbuhan ini mendapat perhatian penyelidik untuk pemfailan
paten berkaitan metodologi yang meningkatkan rangsangan kepada kucing
(Loew 2010). Ekstrak Actinidia menunjukkan aktiviti anti-radang (Kim et al.
2003) dan anti-obesiti (Sung et al. 2013). Komposisi kimia yang aktif terhadap
kesan tersebut merupakan monoterpen iridoid, masing-masing dirujuk sebagai
nepetalakton (Ciaccio et al. 2013) dan matatabilakton (Loew 2010) (Rajah 2).

Nepetalakton Matatabilakton

Rajah 2: Struktur kimia diastereomer untuk nepetalakton dari Nepeta (kiri) dan
matatabilakton (kanan), iaitu biomolekul dari spesis Actinidia.

Iridoid ialah satu kelas sebatian semula jadi yang diterbitkan daripada 1-
isopropil-2,3-dimetilsiklopentana (Rajah 3). Penomboran di dalam gelang lima
ahli atau pentana siklik tersebut memberi pemahaman terhadap kedudukan
kumpulan isopropil dan metil pada atom karbon 1, 2 dan 3. Di dalam biosintesis

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tumbuhan, monoterpen 8-oksogeranial atau (2E,6E)-2,6-dimetil-2,6-
oktadiendial, mengalami pensiklikan berenzim, untuk membentuk kumpulan
lakton siklik, yang menjadi sebahagian struktur utama di dalam sebatian ini.
Berdasarkan metodologi, sebatian ini dijangka terhasil dari proses
pengekstrakan yang menggunakan pelarut organik.

1-isopropil-2,3-dimetilsiklopentana 8-oksogeranial

Rajah 3: Struktur kimia asas untuk iridoid (kiri) dan 8-oksogeranial (kanan).

Melalui pemerhatian, rangka kimia untuk 8-oksogeranial melibatkan E-

isomer pada dua buah atom karbon, yang dilabelkan sebagai nombor 2 dan 6
(Rajah 3). Keadaan ini akan menghasilkan pusat stereoisomer kepada lakton
tersebut, sebaik sahaja pensiklikan berlaku. Orientasi atom hidrogen di antara
lakton dan gelang siklopentana di dalam nepetalakton (Rajah 2) mendatangkan
kewujudan dua struktur kimia yang mungkin. Cabaran di dalam analisis kimia
untuk molekul ini juga berlaku semasa proses penulenan secara kromatografi
serta interpretasi data spektroskopi. Kaedah yang lebih kompleks diperlukan
untuk mengenal pasti isomer yang memberi rangsangan lebih tinggi kepada
kucing. Tambahan itu, pemahaman rangka molekul ini juga turut menjadi satu
perkara yang penting. Melalui teknik kimia sintesis, stereokimia sebatian ini
boleh dikenal pasti.

BAHAN DAN KAEDAH

Sampel Nepeta diperolehi dari kedai haiwan tempatan. Pengekstrakan melalui

kaedah maserasi dilakukan pada suhu bilik. Kromatografi lapisan nipis secara
preparatif digunakan untuk mengasingkan komponen kimia di dalam herba
tersebut. Kaedah spektroskopi (500 MHz, CDCl3) dilakukan untuk mencirikan
struktur kimia sebatian tersebut.

PENEMUAN DAN PERBINCANGAN

Ciaccio dan sekumpulan penyelidik (2013) menulis tentang pengasingan dan

pencirian struktur isomer nepetalakton daripada herba yang dikomersialkan
untuk kucing. Pengetahuan ini menjadi satu pengalaman yang mendidik ahli

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kimia muda di dalam latihan makmal mereka. Informasi ini juga dirujuk di dalam
satu kursus penyelidikan untuk pelajar farmasi di UiTM (Wan Mukhtar 2016).
Melalui eksperimen ini, teknik kimia yang serupa diperkenalkan kepada pelajar
diploma sains (Sarwan et al. 2013). Melalui kromatografi, satu sebatian (dilabel
sebagai sebatian A) berjaya diasingkan.

Spektrum 1H-NMR (500 MHz, CDCl3) untuk sebatian A ditunjukkan di

dalam Rajah 4. Dirumuskan bahawa A merupakan sebatian hidrokarbon bersifat
alifatik kerana anjakan kimia yang terhad di antara δH 0.7–2.0 ppm sahaja.
Ketiadaan signal pada julat 3 ppm dan ke atas, menunjukkan sebatian ini tidak
mempunyai kumpulan olefinik, sebagaimana yang dapat dilihat pada struktur
kimia untuk nepetalakton di dalam Rajah 2. Namun, terdapat empat unit
kumpulan metil (-CH3) yang hadir di dalam A, seperti mana yang ditunjukkan
secara jelas pada δH 0.78 (triplet), 0.99 (singlet), 1.00 (singlet) dan 1.05 (triplet)
ppm. Kewujudan signal metil yang mempunyai dua jenis multiplisiti ini, adalah
disebabkan keadaannya yang berada di dalam atau di luar gelang siklik. Metil
yang berada di dalam kumpulan isopropil mungkin kelihatan singlet, berbanding
dengan metil yang terikat secara terus dengan gelang hidrokarbon. Terdapat
satu signal pada δH 1.91 ppm (multiplet) yang menunjukkan intergrasi untuk
satu proton, serta boleh dirujuk kepada proton metin (-CH) di dalam kumpulan
isopropil. Multiplet pada δH 1.76 ppm (2H) pula ditujukan kepada dua proton
metin di dalam gelang. Signal pada δH 1.63 dan δH 1.55 ppm (masing-masing dua
proton), diinterpretasikan sebagai proton metilen (-CH2) yang berada di dalam
gelang. Justeru satu rangka karbon gelang lima ahli, atau siklopentana
dicadangkan untuk sebatian A, yang memiliki struktur kimia asas untuk satu
iridoid, iaitu 1-isopropil-2,3-dimetilsiklopentana (Rajah 3). Dengan ini, prekursor
untuk nepetalakton telah diasingkan dari sampel dalam ujikaji ini. Namun,
sebarang kewujudan sebatian lakton hanya akan dapat disahkan dengan lebih
lanjut setelah spektrum karbon dapat diperolehi.

Rajah 4: Spektrum 1H-NMR (500 MHz, CDCl3) sebatian A dari Nepeta.

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 Masyarakat tempatan boleh mengaitkan rangsangan tumbuhan Nepeta
dan Actinidia dengan kesan daripada pokok kucing galak (Acalypha indica) (Jajuli
2015) dari keluarga Euphorbiaceae apabila membandingkan kandungan iridoid
yang seumpama daripada spesies tersebut. Pengesanan fitokimia daripada
pelbagai bahagian tumbuhan dijalankan dan laporan mengenai metabolit
sekunder telah pun dilakukan (Mohd Nazri et al. 2016). Namun, informasi
molekul daripada pokok ini baharu sahaja didedahkan (Scaffidi et al. 2016)
(Rajah 5). Sekali lagi tumpuan perlu diberi kepada rangka karbon 8-oksogeranial,
yang hanya melibatkan E-isomer pada karbon 2 dan 6 (Rajah 3), lakton dari
spesis Acalypha mempunyai orientasi kumpulan metil yang sama (Rajah 4). Kini,
kewujudan struktur isomer yang lain adalah satu hipotesis (Rajah 5). Oleh yang
demikian, lebih banyak kajian boleh dilakukan ke atas bahan semula jadi ini.

Isodihidronepetalakton Isoiridomirmecin

Rajah 5: Struktur kimia untuk dua sebatian iridoid dari A. indica.

Struktur satu dimensi Struktur dua dimensi

Rajah 6: Cadangan struktur molekul satu lakton yang terkini. Orientasi kumpulan
metil (–CH3) yang bertentangan (dilukis sebagai ikatan tebal dan
terputus) (kiri) dan model komputer, di mana kesemua atom hidrogen
tidak dipamerkan (kanan).

RUMUSAN

Aplikasi bahan aktif daripada tumbuhan Nepeta dan Actinidia menjadikan kucing
lebih aktif, jinak dan riang. Kajian juga boleh meluaskan pengetahuan di dalam

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bidang farmasi veterinari dan zoologi (Abraham 2011; Abramson et al. 2012;
Dhaliwal et al. 2014) kerana pokok ini boleh merawat kucing yang liar, sakit dan
pasif. Usaha dan tenaga perlu digembeling bagi pengumpulan data kimia serta
mencadangkan perusahaan dan perladangan pokok kucing galak tempatan
untuk peningkatan bioekonomi negara. Akhir sekali, terdapat kemungkinan satu
sebatian prekursor iridoid untuk nepetalakton telah diperolehi. Produk semula
jadi ini, iaitu 1-isopropil-2,3-dimetilsiklopentana sebagaimana yang ditemui di
dalam kajian ini belum pernah dilaporkan.

PENGHARGAAN

Penulis mengucapkan terima kasih kepada pihak fakulti di atas sokongan yang
diberi. Penghargaan juga ditujukan kepada Encik Mohd Syukri Baharudin, Atta-
ur-Rahman Institute for Natural Products Discovery (AuRIns), Universiti
Teknologi MARA, Puncak Alam, di atas bantuan teknikal.

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Sarwan, H., Abd Rahim, H.S., Mohd Amin, N.H., Mohsin, H.F. & Abdul Wahab, I.
(2013). Designing Chromatographic Protocol of the Plant Extracts for
Diploma of Science Students. 2nd Students Innovation & Design
Competition, 5th December 2013, Faculty of Health Sciences, UiTM Puncak
Alam.
Sashida, Y., Ogawa, K., Mori, N. & Yamanouchi, T. (1992). Triterpenoids from the
fruit galls of Actinidia polygama. Phytochemistry. 31(8):2801–2804.
Scaffidi, A., Algar, D., Bohman, B., Ghisalberti, E. & Flematti, G. (2016).
Identification of the Cat Attractants Isodihydronepetalactone and
Isoiridomyrmecin from Acalypha indica. Aust. J. Chem. 69(2):169–173.
Sung, Y.Y., Yoon, T., Yang, W.K., Moon, B.C. & Kim, H.K. (2013). Anti-obesity
effects of Actinidia polygama extract in mice with high-fat diet-induced
obesity. Mol. Med. Rep. 7(2):396–400.
Tehrani, F. (2007). Manikam Kalbu, Dewan Bahasa dan Pustaka, Malaysia.
Wan Mukhtar, W.R. (2016). The Extraction of Nepeta and Actinidia Species.
Bachelor of Pharmacy Dissertation, Faculty of Pharmacy, UiTM
Zain, B. (2015). Berguru Pada Binatang (Edisi Khas Sasterawan Negara), Dewan
Bahasa dan Pustaka, Malaysia.

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QUANTIFICATION OF VOLATILE ORGANIC COMPOUND (HEXANOIC ACID)
IN MORINDA CITRIFOLIA L. JUICE DISTILLATES BY GAS
CHROMATOGRAPHY

Nurul Raihana, A.1, Muhd Fauzi, S.1,3, Zaidah, Z.A.2,3

1
School of Chemistry, 2School of Biology, Faculty of Applied Sciences, Universiti
Teknologi MARA, 40450 Shah Alam, Selangor; 3Atta-ur-Rahman Institute for
Natural Product Discovery, Universiti Teknologi MARA, Puncak Alam Campus,
42300 Puncak Alam, Selangor

Tel: 03-55437852 Fax: 03-55444562 Email: mohdf956@salam.uitm.edu.my

INTRODUCTION

This study was done to quantify the amount of hexanoic acid in commercial and
pure Morinda citrifolia L. fruit juice distillates by gas chromatography-flame
ionization detector (GC-FID). This species has various vernacular names that are
commonly used such as “Indian mulberry”, “nuna”, or “ach” in India, “nhau” in
South East Asia, “noni” in Hawai and in Malaysia, M. citrifolia L. is known as
mengkudu. M. citrifolia L. is one of the members in family Rubiaceae and all
parts of the tree have multi functions. M. citrifolia L. plant has been used for
food, medicinal and cosmetic purposes for centuries (Wei et al. 2011). The plant
is a small evergreen tree with large bright green elliptical leaves (Pino et al.
2010). The colour of the fruit reflects their ripeness and it has two ripening
stages. The fruit will give very pungent smell and sour taste when it matures.
The immature fruit will has green colour meanwhile the fruit will turns to whiter
colour as it ripens. Most M. citrifolia L. is consumed as juice, although leaves,
flowers, bark and roots can also be used (Dixon et al. 1999). Plant essential oils
have been used for many purposes such as ornamental (cosmetic), dietary
(spices and aromas) or medicinal. Chemical compositions of essential oils in the
plant will change to adapt with the internal and external environment.
According to Pino et al. (2010), the composition of some esters increased due to
the ripening of the M. citrifolia L. fruit. Wei et al. (2011) reported a total of 51
volatile compounds were found from the ripe M. citrifolia L. fruit. Octanoic acid
and the hexanoic acid are the most abundance compounds found in the volatile
profile (Wei et al. 2011). The benefits of consuming this plant attract the
attention of researchers. M. citrifolia L. is reputed to have antibacterial,
antiviral, antifungal, antitumor, antihelminthic, analgesic, hypotensive, anti-
inflammatory and immune enhancing effects (Usha et al. 2010).

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METHODOLOGY

Plant Material

The fresh fruits of M. citrifolia L. were collected from Puchong, Selangor were
used for the extraction of volatile compounds. While, the commercial M.
citrifolia L. fruit juice was bought at superstore in Shah Alam.

Extraction of Volatile Compound

The extraction of volatile compounds of the investigated samples was done by

steam distillation-extraction (SDE). 300 g of fresh fruits were blended for five
minutes and the juice obtained was mixed with 300 mL water and 150 g of
sodium chloride in a 1000 mL round bottom flask. 40 ml of hexane in a 100 ml of
another round bottomed flask was placed in a 70C water bath. The volatile
compounds were extracted from the samples by steam distillation extraction for
2 hours. For commercial M. citrifolia L. juice, the extraction process was
performed similar as the fresh fruit. The collected hexane was dried over
anhydrous sodium sulphate. The sample then filtered and the filtrate was
subjected to GC analysis.

Gas Chromatography-Mass Spectrometry (GC-MS) Analysis

GC-MS analysis was performed on an Agilent GC-MSD system equipped with a

fused silica capillary column (30 m x 0.25 mm; 0.25 µm film thickness). Helium
gas (He) was used as a carrier gas with a flow rate of 1 ml/min. The injector
temperature was set at 275C. The GC oven temperature was set using
temperature programme 40C for 10 min, increased to 240C at a rate of
4C/min, and held at this final temperature for 10 min. The mass analyser
temperature and ion source temperature were 150C and 230C, respectively.
The injection port was operated in splitless mode. 1.0 µL of samples were
injected throughout this experiment. Mass spectra were obtained by electron
ionization (EI) at 70 eV and the mass scan was from 40 to 451 amu.

Gas Chromatography-Flame Ionisation Detector (GC-FID) Analysis

GC-FID analysis was performed on an Agilent GC-FID system. Nitrogen gas (N2)
was used as a carrier gas with a flow rate of 15 ml/min. The injector
temperature was set at 200C. Temperature programme was employed as
follows: 80C for 10 min, increased to 160C at a rate of 4C /min, and held at
this final temperature for 2 min. The FID detector was set at 280C. The
injection was operated in splitless mode. 0.1 µL of samples were injected
throughout this experiment.

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RESULTS AND DISCUSSION

The presence of two main peaks in the GC-MS chromatograms from the two
distillates were confirmed as hexanoic acid and octanoic acid. GC-FID was used
for the quantification of hexanoic acid in both distillates. Hexanoic acid peak in
the samples were identified by comparing its retention time to the retention
time of standard solution. Quantifications of the hexanoic acid were performed
using external standard and internal standard method calibration curves. n-
Hexane was used as a solvent in the analysis. The retention times for the solvent
and external standard (hexanoic acid) were 0.3 min and 21.6 min respectively.
Internal standard (IS) used in the analysis was pentanoic acid and its retention
time was 18.3 min as shown in Figure 1.

Figure 1. GC-FID chromatogram of internal standard (pentanoic acid)

and external standard (hexanoic acid)

Calibration curve for external standard method of hexanoic acid was found to be
linear in the range of 4640 mg/L to 23200 mg/L with correlation coefficient (R2)
of 0.9888. The linearity of this method provides measurable quantification
analysis (Figure 2). Internal standard calibration curve was plotted based on the
ratios of hexanoic acid peak areas over pentanoic acid peak areas against a
series of ratio of hexanoic acid concentrations over constant pentanoic acid
concentration. The graph for the internal standard was linear in the ranges of
0.494 to 2.470 with the R2 of 0.9988 (Figure 3).

Figure 2. External standard calibration curve of hexanoic acid

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 Figure 3. Internal standard calibration curve of hexanoic acid

The concentrations of hexanoic acid in M. citrifolia L. pure juice and commercial

juice filtrates were determined and calculated using both calibration methods as
tabulated in Table 1. The analysis showed that the concentration of hexanoic
acid in the M. citrifolia L. pure juice distillate was higher than the commercial
juice distillate for both methods. The concentration of the hexanoic acid in the
pure juice for external standard and internal standard were 238830.0 mg/L and
264328.4 mg/L respectively. Meanwhile, the concentrations of hexanoic acid in
the commercial juice for external standard was 20662.0 mg/L and for internal
standard was 29502.1 mg/L. Internal standard method give slightly higher result
compared to external standard method. Further detail method validation is
required to confirm the analysis.

Table 1. Analysis of M. citrifolia L.

Average concentration of
Type of method hexanoic acid (mg/L)
Pure juice Commercial juice
External standard 238830.0 20662.0
Internal standard 264328.4 29502.1

CONCLUSION

Determination of M. citrifolia L. hexanoic acid juice distillates have been

successfully analysed by GC-MS and GC-FID. The analysis of internal standard
method showed slightly higher content of hexanoic acid compared to external
standard method. However, further method validation is required to give
precise analysis results.

ACKNOWLEDGEMENT

The authors would like to thank Universiti Teknologi MARA (UiTM) and Ministry
of Higher Education Malaysia for financial support under Fundamental Research
Grant Scheme FRGS/1/2013/ST01/UiTM/02/4. The authors are thankful to

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Madam Noor Haida Kamalull Khudzri, Madam Shaheda Ismail and Mr. Adnan
Ismail from UiTM for their technical assistance.

REFERENCES

Wei, G.J., Ho, C.T. & Huang, A.S. (2011). Analysis of Volatile Compounds in Noni
Fruit (Morinda citrifolia L.) Juice by Steam Distillation-Extraction and Solid
Phase Microextraction Coupled With GC/AED And GC/MS. Journal of Food
and Drug Analysis 19(1):33–39.
Pino, J.A., Márquez, E., Quijano, C.E. & Castro, D. (2010). Volatile Compounds in
Noni (Morinda citrifolia L.) At Two Ripening Stages. Ciência e Tecnologia
de Alimentos 30(1): 183-187. doi:10.1590/s0101-20612010000100028.
Dixon, A.R., Mcmillen, H. & Etkin, N.L. (1999). Ferment this: The Transformation
of Noni, A Traditional Polynesian Medicine (Morinda citrifolia, Rubiaceae).
Ecological Botany 53(1): 51-68. doi:10.1007/ bf02860792.
Usha, R., Sashidaran, S. & Palaniswamy, M. (2010). Antimicrobial Activity of A
Rarely Known Species, Morinda citrifolia L. Ethnobotanical Leaflets 14:
306–311.

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STATISTICAL ANALYSIS OF AGARWOOD OIL CHEMICAL COMPOUNDS
FROM HIGH QUALITY USING BOXPLOT

Nurlaila, I.1, Nor Azah, M.A.2, Mailina, J.2, Abd Majid, J.2, Mohd Hezri, F.R.,1 &
Mohd Nasir, T.1

1
Faculty of Electrical Engineering, Universiti Teknologi MARA, 40450 Shah Alam;
2
Natural Products Division, Forest Research Institute Malaysia, 52109 Kepong,
Selangor

Tel: 03-55436065 Fax: 03- 55435007 Email: nrk_my@yahoo.com

INTRODUCTION

Agarwood oil is the oil extracted from agarwood trees. Agarwood or gaharu is
the resin impregnated heartwood of the Aquilaria species, a genus which
belongs taxonomically to the family Thymelaeceae. The agarwood oil is highly
demanded due to its special usage; an incense for religious ceremony, in
perfume and traditional medicine preparations (Naef 2011). In the Middle East,
it is a symbol of wealth and widely used during the wedding ceremony (Pravina
2008). The agarwood oil is traded according to its quality where high quality oil
is sold expensively and vice versa. Currently, the grading of the agarwood oil to
the high and low quality is done manually using human trained grader which
refers to human experience and perception to the oil colour, odour and long
lasting aroma. The high grade normally refers to strong odour and dark colour
(black). The brownish and yellowish colour refers to medium and low grade,
respectively (Naef 2011). This method has limitation such as human nose easily
get fatigue to deals with repeatability experiment (oil odour), hence the result
obtained by trained grader usually is not consistent and time consuming.
Researchers have found that there is no standard yet available in grading the
agarwood oil and the oil quality result is questionable (Pravina 2008).
Therefore, the objective of this study is to propose scientific method in grading
the agarwood oil using its chemical properties so that an accurate result can be
achieved. In specific, a statistical analysis focusing on boxplot has been
performed in analysing the agarwood oil compounds especially from high
quality samples.

MATERIALS AND METHODS

In this study, seven samples from high quality agarwood oils are obtained from
Forest Research Institute Malaysia (FRIM). The samples are coded as T, MA1, LA,
KB, JBD, MA2 and MA. GC-MS is performed in extracting the chemical
compounds of the oil using a standard procedure as in previous work (Nor Azah

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et al. 2008). After that, all the compounds were tabulated and the major
compounds that exist in all samples were identified. These compounds is
analysed their distribution using bar graph and boxplot. All the analysis works
are done via dedicated Matlab software version R2010a.

RESULTS AND DISCUSSION

The chemical compounds and their abundances (%) in high quality agarwood oil
of all selected samples are listed in Table 1. Generally, it can be seen that the
agarwood oil samples consist of sesquiterpene hydrocarbon, oxygenated
sesquiterpenes and their chromone derivatives which are from terpenes group.
At least 43 compounds are found and only four compounds exist in all samples.
The compounds are β-agarofuran, α-agarofuran, 10-epi--eudesmol, and
dihydrocolumellarin. The variation of chemical compounds with different
abundances notified that the chemical profile as one factor that influence the
quality of agarwood oil and has the same agreement by the other researchers
(Ishihara et al. 1993; Adam et al. 2012).

Figure 1 shows the major compounds; β-agarofuran, α-agarofuran, 10-epi--

eudesmol, and dihydrocolumellarin with their abundances in all samples It is
noticed that, 10-epi--eudesmol contributed the most by having the highest
abundances among all with the total abundances of 72.41%. In specific, the
abundances of 10-epi--eudesmol are 9.76%, 10.91%, 7.65%, 7.07%, 8.14%,
8.28% and 20.60% in T, MA1, LA, KB, JBD, MA2 and MA, respectively. For β-
agarofuran, α-agaroufran, and dihydrocolumellarin, their total abundances in all
samples are 15.55%, 14.06% and 14.86%, accordingly. The finding in this study
revealed that 10-epi--eudesmol is the most important in high quality samples in
this study.

Table 1. Chemical compounds and their abundances (%) in high quality

agarwood oils of T, MA1, LA, KB, JBD, MA2 and MA
Abundances, %
Chemical Compounds
T MA1 LA KB JBD MA2 MA
2-Phenyl propanal 0.00 0.00 0.71 0.00 0.00 0.00 0.00
Benzaldehyde 1.74 1.74 0.69 0.00 0.00 0.00 0.00
Benzeneacetonitrile 1.38 1.76 0.00 0.00 0.00 0.00 0.00
4-Phenyl-2-butanone 0.00 0.00 0.68 2.66 0.85 1.34 1.21
α-Funebrene 0.00 0.28 0.00 0.00 0.00 0.00 0.00
α-Gurjunene 0.00 0.00 0.00 0.00 0.00 2.83 1.21
β-Copaene 0.00 0.93 0.00 0.00 0.00 0.00 0.00
α-Guaiene 0.00 0.45 0.33 0.58 0.14 0.82 0.00
-Elemene 0.00 0.00 0.96 0.00 0.00 0.00 0.00

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 Aromadendrane 25.92 1.30 9.80 0.00 0.00 0.98 0.00
Valencene 0.00 1.42 0.00 0.00 0.00 0.00 0.00
-Gurjunene 0.00 0.45 0.00 0.00 0.25 0.00 0.44
β-Agarofuran* 4.33 1.73 2.04 1.02 1.02 1.42 5.01
ar-Curcumene 0.00 0.57 0.00 0.59 1.20 0.83 0.61
α-Muurolene 0.00 0.00 0.00 0.00 1.19 2.43 0.00
-Guaiene 0.00 0.00 0.71 0.00 0.00 0.00 0.00
β-Dihydroagarofuran 0.00 2.72 0.55 1.31 1.05 2.90 0.04
α-Agarofuran* 3.53 1.72 1.37 1.47 1.76 1.48 2.73
Elemol 0.00 0.00 0.46 1.86 0.00 0.00 1.02
Dodecanoic acid 0.00 0.00 0.55 0.00 0.00 0.00 0.00
β-Vetivene 0.00 0.00 0.37 0.00 0.00 0.00 0.00
-Vetivenene 0.00 0.00 0.36 0.87 0.00 0.00 0.00
Spathulenol 0.00 0.00 0.40 0.00 0.00 1.63 0.00
β-Gurjunene 0.00 0.00 1.07 0.00 0.00 0.46 0.00
10-epi--Eudesmol* 9.76 10.91 7.65 7.07 8.14 8.28 20.60
-Eudesmol 0.00 8.16 0.00 30.36 14.85 12.93 3.11
Agarospirol 9.31 0.00 24.18 2.15 0.00 0.00 0.00
Alloaromadendrene
0.00 1.21 0.00 0.00 0.00 1.70 0.00
epoxide
Guaia-3,9-dien-11-ol 0.00 4.92 0.00 0.00 0.00 0.00 0.00
β-Eudesmol 0.00 4.15 8.42 0.00 0.00 0.00 0.00
α-Eudesmol 17.24 0.00 0.00 0.00 0.00 0.00 0.00
Valerianol 19.38 0.00 14.14 2.29 0.00 0.00 1.73
β-Costol 1.98 0.00 0.00 0.00 0.00 0.00 0.00
α-Bisabolol 0.00 2.87 0.00 0.00 0.00 1.23 0.00
Selina-3,11-diene-9-one 0.00 0.00 0.99 0.42 0.00 0.34 0.00
Cyperotundone 0.00 0.00 1.69 0.00 0.00 0.00 0.00
10-nor-Calamenene 2.81 0.00 0.00 0.00 0.00 0.00 0.00
Selina 3,11-dien-14-ol 0.00 0.00 1.84 0.00 7.16 0.00 0.00
Aristolone 0.00 0.00 0.63 0.00 0.00 0.00 0.00
Hexadecanoic acid 0.00 7.03 5.70 4.46 2.71 0.00 2.65
Thujopsenal 2.02 0.00 0.00 0.00 1.03 0.00 0.00
Cyclohexadecanolide 0.00 1.09 0.00 0.00 0.00 0.00 0.00
Dihydrocolumellarin* 2.84 3.13 0.37 1.86 1.79 2.27 2.60
Note: *identified as significant by exist in all samples

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 Figure 1. Major compound with their abundances in MA,
MA2, JBD, KB, LA, MA1 and T

Figure 2 demonstrates boxplot of major compounds identified exist in all

samples represents data distribution for those compounds. As expected, 10-epi-
-eudesmol has the highest mean, which is above 5%. One outlier has been
detected which is originally from sample MA, with abundances of 20.60%. The
mean for β-agarofuran, α-agarofuran, and dihydrocolumellarin are closed to
each other within 0 to 5%. These three compounds have abundances within the
same range for all samples since no outliers are detected.

CONCLUSION

Statistical analysis of agarwood oil chemical compounds for high quality using
box plot has been presented in this study. The study found that β-agarofuran, α-
agarofuran, 10-epi--eudesmol, and dihydrocolumellarin as major compounds as
they exist in all samples. 10-epi--eudesmol is identified as an important
compound and contributed to high quality of agarwood oil. The finding in this
study is significant as it will benefit for future work especially in agarwood oil
quality grading.

Figure 2. Boxplot of major compounds identified exist in all samples

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ACKNOWLEDGEMENTS

The authors would like to thank all staff involved from Faculty of Electrical
Engineering, UiTM and Natural Products Division, FRIM, as well as research
grant vote no. 50-41-02-01-001.

REFERENCES

Adam, F., Tajuddin, S.N., & Johari, J. (2012) Rahsia dan Keunikan Gaharu, 1st ed.
Pahang: Universiti Malaysia Pahang.
Ishihara, M., Tsuneya, T., & Uneyama, K. (1993). Fragrant sesquiterpenes from
agarwood, Phytochemistry 33:1147–1155 .
Naef, R. (2011). The volatile and semi-volatile constituents of agarwood, the
infected heartwood of Aquilaria species: a review. Flavour and
Fragrance Journal 26:73–87 .
Nor Azah, M.A., Chang, Y.S., Mailina, J., Abu Said, A., Abd Majid, J, Saidatul
Husni, S., Nor Hasnida, H. & Nik Yasmin, N.Y. (2008). Comparison of
Chemical Profiles of Selected Gaharu Oils from Peninsular Malaysia. The
Malaysian Journal of Analytical Sciences 12: 340.
Pravina, A.K. (2008). Application of solid phase microextraction in gaharu
essential oil analysis. Bachelor of Chemical Engineering, Faculty of
Chemical Engineering and Natural Resources, Univerisiti Malaysia
Pahang (UMP), Pahang.

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A PRELIMINARY STUDY OF CHEMICAL PROPERTIES AND IN VITRO ANTI-
CANCER ACTIVITIES OF CHRISTIA SP.

Muhammad Haffiz, J., Nurhanan Murni, Y. and Nordati Akma, M.A.

Biomolecule Research Laboratory, Natural Product Division, Forest Research

Institute Malaysia, Kepong, 52109, Selangor

Tel: +60-362797651 Fax: +60-362804623 Author: haffiz@frim.gov.my

INTRODUCTION

Christia sp. known as ‘pokok rerama, is a non climbing perennial herb used as
ornamental plant cultivated in Southeast Asia region. This species has a unique
trifoliate shape leaves and grow to a height of about 60-120 cm in open
grassland, thickets, seaside and roadsides. This species is reported to be used to
treat bronchitis and inflamed tonsils, muscle weakness and poor blood
circulation (Whiting 2007). A few classes of compounds were reported in this
Christia sp. including alkaloids, triterpenes, phenols and fatty acids. Palmitine
and corynoxidine were reported presence in aerial part of the plant and
isoquinoline alkaloids, usually typical for Papaveraceae or closely related
families, were found in Christia plant extracts. The cyclohexane extract of this
plant showed potential anti plasmodial activity against chloroquine resistant
FcBI/Colombia starain of Plasmodium falciparum with IC50 value 10.8 μg/mL
(Nguyen-Pouplin et al. 2007). The objective of this study is to determine possible
anti-proliferative effects of water and methanol extracts from Christia sp.
against cell lines of breast cancer (MCF-7), ovarian cancer (SKOV-3) and
colorectal (HT-29). The results of this study may provide evidence on the claims
for their use in herbal practice.

MATERIAL AND METHODS

Plant materials

Fresh sample was obtained from commercial nursery located in Sg. Buloh. It was
identified by Mr Kamaruddin Salleh and voucher specimen deposited at FRIM.

Water extract

The fresh sample were oven dried at 50-60 °C for three days, ground and
subjected to methanol and water extract. The dried sample (2g) was refluxed in
methanol (200 mL) in 3 hours at 55-60°C. The crude extract was then filtered
and evaporated using rotary evaporator to yield an extract (0.4 g, 20 %).

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Ethanol extract

The dried sample (2 g) was soaked in water (200mL) using water bath at 95°C for
3 hours. The solution was then filtered and concentrated using evaporator and
kept in -20°C freezer. After 24 hours the concentrated crude was freeze dried at
-80°C for 3-4 days to afford water extract (0.31 g, 17.5 %).

Anti-proliferative Assay

The breast (MCF-7), ovarian (SKOV-3) and colorectal (HT29) cancer cell lines
were cultured in Dubelcco’s Modified Eagle’s Medium (DMEM), supplemented
with 5% Foetal Calf Serum, 1% fungizone, 1% penicillin streptomycin and 0.125%
gentamycin sulphate until it reached sub-confluent state. Cell plating was
performed in 96 well plate at a density of 7000-10000 cells/ well and left
overnight in the incubator set at 37oC and 5% carbon dioxide in air. The medium
was renewed the next day and the cells in different wells were treated with a
series of plant extracts (1% v/v) prepared at five different concentrations (1,5,
25,125 and 625 µg ml-1) in triplicate. The control wells contained cells and
medium only. Another set of wells on the plate contained cells that were not
treated with any plant extracts but were treated with ethanol (1% v/v) as a
vehicle control. The cells were incubated for 72 hours. SRB assay (Skehan et al.
1990) was then performed and the OD values were read at 492 nm using
Magellan V.4 Elisa reader and software. The percentage of living cells were
calculated based on the OD values as below:

OD of plant-treated cells- OD of ethanolic-treated cells

OD of control-OD of ethanolic-treated cells

IC50 values of each plant extracts treated in MCF-7, SKOV-3 and HT-29 were
obtained from the dose-response curve (percentage of living cells versus the
concentrations of plant extracts used). The experiment was repeated at least
three times.

Sample preparation and HPLC profiling

About 5 mg of each sample was weighed and placed into 2 mL eppendorf tube.
1 mL methanol and water were added for respective methanol and water
extract, and the solutions were sonicated for 10 min. The solutions were filtered
using MiliporeMillex-HN, Nylon 0.45 μm. The analyses of all samples were
carried out on a synergi 4μm fusion RP 80 Å; 150mm x 4.6 mm. The
chromatographic system Agilent 1160 Infinity was used for the analyses. The
column was set at 30C and UV detection was performed at 254 and 366 nm. An

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injection volume of 50uL of samples flow rate 1mL/min was used in the
analyses. The 0.1% formic acid in acetonitrile and water in gradient were
employed as mobile phase.

RESULTS AND DISCUSSION

The HPLC analysis of water and methanol extracts of this plant showed the
presence of unidentified four major peaks P1a-P4a and P1b-P4b for respective
spectra for both extracts as shown in Figure 1. As comparative study, P1a, P3a
and P4a showed similar profile to P1b, P3b and P4b (Figure 2) .Meanwhile peak
P2a at 9.1 min showed different profile to P2b at 9.09 min (Figure 3).
Differences UV spectra on peaks P2a and P2b in both extracts may give different
activities on the cell lines.

P1a P2a Water extract P4a

P3a

P1b
Methanol extract
P3b

P2b
P4b

Figure 1: HPLC profiles of water and methanol extract of Christia sp. at 280 nm

From this study, all the extracts and drug recorded IC50 in μg/mL range
attributed to the anti-proliferative effects in the respective cancer cell lines
(Table 1). The results (Table 1) also showed cisplatin as control drug exert very
high effect against colorectal (HT29), breast (MCF7) and ovarian (SKOV3) cell
lines.

Water extract of this plant showed better anti-proliferative effects as compared

to the methanol extract. However, very high concentration is needed to inhibit
the proliferation of respective cancer cell lines as compared to cisplatin as

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standard drug. It may be noted that cisplatin is one of the anti-cancer drugs that
has and still being used to treat various type of cancers.

CONCLUSION

The anticancer activities of methanol and water extracts of Christia sp. Showed
very low i anti-proliferative effects against colorectal (HT29), breast (MCF7) and
ovarian (SKOV3) cancer cell lines as compared to cisplatin drug.

P3b
P1a & P1b

P3a

P4a & P4b

Figure 2: Spectra of peaks P1a-4a and P1b-4b

P2b
P3b P2a

Figure 3: Comparison spectra for peaks P2a and P2b

Table 1: IC50 of the both methanol and water extracts of Christia sp against colorectal
(HT29), breast (MCF7) and ovarian (SKOV3) cancer cell lines.
HT29 MCF7 SKOV3
Cell Lines / Extracts
(IC50, μg/mL) (IC50, μg/mL) (IC50, μg/mL)
Methanol extract 365.40 ± 7.60 349.92 ± 13.44 353.86 ± 2.51
Water extract 254.94 ± 5.13 119.22 ± 9.35 217.24 ± 3.13
Drug: Cisplatin 0.71 ± 0.03 0.73 ± 0.09 0.56 0± .06

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REFERENCES

Bunawan, H., Bunawan, S.N. & Baharum, S.N. (2015). The Red butterfly wing
(Christia vespertilionis): A promising cancer cure in Malaysia. Int J Pharm
Pharm Sci 7(8):1.
Hofer, D., Schwach, G., Tabrizi-Wizsy, N.G., Sadjak, A., Sturm, S., Stuppner, H. &
Pfragner, R. (2013). Christia vespertilionis plant extracts as a novel
antiproliferative agent against human neuroendocrine tumor cells. Oncol
Rep 29(6):2219–2226.
Gouri Kumar, D. & Bakh. F (2016). An Appraisal of Christia vespertilionis (L. F.). A
Promising Medicinal Plant, International Journal of Pharmacognosy and
Phytochemical Research 8(6):1037–1039.
Nguyen-Pouplin, J., Tran, H., Phan, T.A., Dolecek, C, Farra, J., Tran, T.H., Caron,
P., Bodo, B. & Grellier, P. (2007). Antimalarial and cytotoxic activities of
ethnopharmacologically selected medicinal plants from south Vietnam. J
Ethnopharmacol 109:417–427
Skehan, P., Storeng, R., Scudiero, D., Monks, A., McMahon, J., Vistica, D.,
Warren, J.T., Bokesch, H., Kenney, S. & Boyd, M.R. (1990). New
Colorimetric assay for anticancer-drug screening. Journal of the national
cancer institute 82:1107–1112
Upadhyay H.C., Sisodia B.S. , Cheema H.S. , Agrawal J., Pal A. , Darokar M.P. &
Srivastava S.K. (2013). Novel antiplasmodial agents from Christia
vespertilionis. Natural Product Communications 8(11):1591–1594
Van Meeuwen, M.S., van Steenis, C.G.G.J. & Stemmerik, J. (1961). Preliminary
revisions of some genera of Malaysian Papilionaceae II. Vol.6, Part 1,
Reinwardtia: Herbarium Bogoriense.
Whiting, P.A. (2007). Commercial production of Christia subcordata Moench by
establishing cultural practices and by applying plant growth regulators
[dissertation]; The University of Georgia
(http://athenaeum.libs.uga.edu/handle/10724/17094)

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BIOASSAY-GUIDED ISOLATION OF ANTI-TRYPANOSOMAL ACTIVE
COMPOUND FROM ACTINOMYCETE STRAIN TY035-025

Muhammad Syamil, A. 1, Getha, K.1, Lili Sahira, H.1, Norhayati, I.1, & Roshan, J.1

1
Biomolecule Research Laboratory, Natural Products Division, Forest Research
Institute Malaysia, 52109 Kepong, Selangor

Tel: 03-62797658 E-mail: syamil@frim.gov.my

INTRODUCTION.

Human African trypanosomiasis (HAT) or sleeping sickness, is an illness endemic

to sub-Saharan Africa in human and animal. It is caused by the flagellate
protozoan Trypanosoma brucei. Current treatment is to use pentamidine and
melarsoprol. However, adverse side effect and treatment failure in the second
stage of the diseases encouraged continuous effort for development of new
drug or treatment (Keiser et al. 2001 and Lejon et al. 2003).

Actinobacteria are gram-positive, often filamentous, microorganism

known for their unsurpassed capacity for the production of secondary
metabolites, particularly from genus Streptomyces (Hemashenpagam 2011).
These molecules present original and unexpected structure and are selective
inhibitors of their molecular targets. At FRIM, we have established FRIM
Microbial Culture Collection (FRIM-MCC) to provide microbial isolates for
screening activities on extracts using anti-trypanasomal assay. Actinobacteria
strain TY035-025 which showed strong activity with IC50 value of 0.96 ug/mL was
selected for large scale fermentation. The aim of the present study was to
isolate the anti-trypanosomal active compound.

MATERIALS AND METHODS

Fermentation of Strain Ty035-025 and Crude Extract Preparation

Mycelia agar plugs were taken from fresh culture of strain TY035-025 and
transferred to 20 mL of media M2. After 3 days of submerged cultivation at 28 ±
2°C and 200 rpm, 10 mL of aliquot of the culture broth was inoculated into 200
mL of media M2 in 1000 mL flask. A total of 10 flasks were inoculated and
incubated at 28 ± 2°C and 200 rpm. After 8 days of fermentation, culture broth
was dispensed into 8 freeze dry flasks, stored at -80 °C for 24 hour and freeze-
dried. After the drying was completed, the culture broth powder was immersed
in mixture of dichloromethane and methanol (1:1, v/v), sonicated for 20

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minutes and filtered. Solvent layer was dried in vacuo to yield the crude extract
(23-124-C).

Anti-trypanosomal assay

Bloodstream form trypanosomes were inoculated into 96-well plate at a density

of 2000-2500 trypanosomes/100 uL medium. The trypanosomes were incubated
for 72 hours in the presence of a standard drug or extracts. About 10 uL of
fluorescent dye Alamar Blue was added into each well after 6 hours. Background
fluorescence and absorbance of the drug containing medium were determined
after 72 hours for each drug dilution. Wells without the drugs served as
controls. The plates were read at excitation wavelength 528/20 nm and
emission wavelength 590-35 nm with optics position on the bottom to
determine the IC50 (Hitomi & Kazuhiko 2005).

Bioassay-guided isolation of active compound

Crude extract was dissolved in chloroform. The solution was passed through a
preconditioned silica gel (230-400 mesh) in chloroform. Gradient elution was
carried out with chloroform and methanol. Fractions collected were tested using
TLC (solvent system, chloroform-methanol, 4:1 v/v) and subsequently pooled
together according to their Rf value and sent for anti-trypanosomal assay. All
fractions which showed high anti-trypanosomal activity, was pooled together
and proceed to purification using C18 column followed by TLC preparative.

RESULTS AND DISCUSSION

Evaporation in vacuo for solvent layer yielded 3.0 g of crude extract (23-124-C).
A total of 17 fractions were collected when 23-124-C was eluted. Fractions 124-
7 to 124-10 showed high anti-trypanosomal activity (Table 1). These fractions
was then pooled together and proceed to second column chromatography.
From this process, 7 fractions were collected. Fraction 130-3 which showed
highest activity was further purified with TLC preparative. These isolation
processes were illustrated in Figure 1. Bioassay-guided isolation identified one
active compound, MS02 (12.1 mg, IC50 0.01 ug/mL) from the crude extract
column fractionation.

Table 1: Active fractions from isolation of 23-124-C.

Fraction No Weight (mg) IC50 (ug/mL)
124-7 660.3 1.11
124-8 764.6 1.09
124-9 1015.2 0.14
124-10 25.7 0.30

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 Figure 1: Isolation and purification of MS02.

Compound MS02 has a mass of m/z 456 [M+H] (Figure 2). Further investigation
with one dimensional NMR, showed that MS02 consist of 56 protons and 25
carbons (Figure 3 and Figure 4).

Figure 2: The ESI/MS spectra of compound MS02.

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 Figure 3: 1H NMR analysis of compound MS02.

Figure 4: 13C NMR analysis of compound MS02.

CONCLUSION

One active anti-trypanosomal compound (MS02) has been successfully isolated

from crude extract of actinobacteria strain TY035-025. This compound showed
an activity of IC50 0.01 ug/ml. Further investigation is needed to elucidate the
structure of MS02 to determine the novelty of the compound.

REFERENCES

Eperon, G., Schmid, C., Loutan, L. & Chappuis, F. (2007). Clinical presentation
and treatment outcome of sleeping sickness in Sudanese pre-school
children. Acta Tropica. 101:31–39.

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Getha, K., Hatsu, M., Wong. H.J. & Lee, S.S. (2009). Submerged Cultivation of
Basidiomycete Fungi Associated with Root Diseases for Production of
Valuable Bioactive metabolites. Jour of Tropical Forest Science. 21:1–7
Hemashenpagam, N. (2011). Purification of Secondary Metabolites from Soil
Actinomycetes. Inter Journal of Microbiology Research. 3:148–156.
Hitomi, S. & Kazuhiko, O. (2005). Trypanasoma brucei in vitro screening model.
Standard Operating Procedure. Version 1.5:1–11.
Lejon, V., Legros, D., Savignoni, A., Etchegorry, M. G., Mbulamberi, D. & Büscher,
P. (2003). Neuro-inflammatory risk factors for treatment failure in ‘‘early
second stage’’ sleeping sickness patients treated with Pentamidine.
Journal of Neuroimmunology. 144:132–138.
Keiser, J., Stich, A. & Burri, C. (2001). New drugs for the treatment of human
African trypanosomiasis: Research and Development. Trends in
Parasitology. 17:42–49.

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CHEMICAL COMPOSITION OF LITSEA CUBEBA ESSENTIAL OILS FROM
CAMERON HIGHLANDS

Nor Azah, M.A.1, Mailina, J.1, Abd Majid, J.1, Saidatul Husni, S. 1, Sahrim, L1.,
Noorsiha, A.1, Mohammad Faridz, Z.P.1, Chung, R.C.K.1, Azrina, A.2,
Kamaruddin, S.1, Christine, S.A.B.3 & Nurliyana, A.L. 1

1
Forest Research Institute Malaysia, 52109 Kepong, Selangor, Forest Research Institute
Malaysia, 52109 Kepong, Selangor, 2School of Chemical Engineering, Universiti Sains
Malaysia , 14300 Nibong Tebal, Pulau Pinang, 3Faculty of Applied Sciences, University
Teknologi Mara, 40450 Shah Alam.

Tel: 03-62797344 Fax: 03-62729805 Email: norazah@frim.gov.my

INTRODUCTION

Litsea cubeba (Lour.) Pers belongs to the genus of Litsea and family of
Lauraceae. The genus Litsea is mainly distributed in the tropical and subtropical
regions (Kong et al. 2015). L. cubeba is also known as May Chang or Mountain
pepper. L. cubeba was introduced in the early 1950s as material source of citral
which competing with lemon grass. Essential oils from the fruit are rich with
citral components. Due to its pleasant citrus-like smell with spicy peppery
aroma, it is often used as a flavouring modifier, enhancer in food and cosmetics
(Nor Azah & Susiarti 1999). L. cubeba oils have been extensively studied for its
anti-oxidant, anti–microbial, anti-inflammatory and anti-asthma properties
(Chen et al. 2013; Wang et al. 2013; Liu & Yang, 2012; Tajkrimi et al. 2010; Gogoi
et al. 1997). Besides that, the essential oils were also found to have good
pesticidal effect such as nematicidal activity against pine wood nematode (Park
et al. 2007) as well toxic towards third-instar Trichoplusia ni larvae (Jiang et al.
2009) and fungicidal effect against Sclerotinia sclerotiorum, Thanatephorus
cucmeris, Pseudocer-cospora musae and Colletotrichum gloeosporioides (Yang
et al. 2010).

The chemical composition of the oils from the various parts may differ
due to different in locality and geographical locations (Bighelli et al. 2011; Siakia
et al. 2013).The differences in chemotype components may play a role in the
chemistry and biological application of the L. cubeba. As part of our study on the
wild montane flora of Cameron Highlands, the essential constituents of the leaf
and fruit oils will be reported in this paper.

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MATERIALS AND METHODS

Plant Material

The leaves and fruity parts of Litsea cubeba were collected from Cameron
Highlands Montane Park (CHiMP) in September 2015. A voucher specimen of
this plant (KAMP-CH-0009) has been deposited in FRIM’s herbarium for
authentication and reference.

Distillation of Essential Oils

The fresh leaf, twig and fruit of L. cubeba were cut into small pieces and
immediately extracted by water-distillation for 6 hours. The oily layers obtained
were separated and dried over anhydrous sodium sulphate. The yields were
calculated on a dry weight basis of the plant material (v/w %).

Chemical Analysis

Analyses of the essential oils were carried out using Shimadzu GC2010Plus and
Agilent Technologies GCMS 7890A/5975C Series MSD apparatus. Both systems
were equipped with fused silica capillary columns HP-5MS (30m x 0.25mm,
0.25mm film thickness). The gas chromatograph (GC) was equipped with FID
using split mode injection technique and the operating parameters were helium
gas as carrier gas at a flow rate of 1ml/min, injector temperature 250°C and
detector temperature 250°C. The gas chromatography was programmed initially
at 60°C for 10 min, then to 230°C for 1 min at 3°C/min. For gas
chromatography/mass spectrometry (GCMS) analysis, the temperature
programme was set similarly to GC programme. The chemical constituents were
confirmed by comparison of the samples’ spectrum to mass spectral library
(HPCH2205.L; Wiley7Nist05.L). The results of the peak areas were expressed as
peak area counts.

RESULTS AND DISCUSSION

Chemical Composition of Essential Oils

The yield of leaf, twig and fruit essential oil of L. cubeba were calculated based
on dry weight materials which gave 2.59, 0.02 and 0.27%v/w respectively. All
volatile chemical compositions detected in the oils were shown in Table 1. 1, 8-
cineole was identified as the major constituents of leaf (48.8%) and twig (57.7%)
oils respectively. Meanwhile, the major constituents of the fruit oil were made
up of citral; geranial (44.7%) followed by neral (28.7%). 1,8-Cineole was not

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detected in the fruit oil. Geranial and neral were present only in low amounts
for leaf and twig oil (below 2%).

The main components of L. cubeba leaf oil were sabinene (14.9%), α-

terpinyl acetate (8.9%), α-pinene (5.4%), α-terpineol (5.0%), terpinen-4-ol
(4.6%), β-caryophyllene (2.3%), myrcene (1.8%), -terpinene (1.5%) and
terpinolene (1.1%). The remaining components detected were below than 1%
or in trace amounts. For twig oil, the main components detected included α-
terpinyl acetate (11.4%), terpinen-4-ol (4.1%), sabinene (4.0%), β-pinene (3.2%),
α-pinene (2.8%), -terpinene (2.0%), linalool (2.0%), α-terpineol (2.0%), myrcene
(1.4%), geranial (1.4%) and β-caryophyllene (1.4%).

Apart from citral compounds, other components found present in the

fruit oils were in percentage of below than 1% except for limonene (1.2%). Kai et
al. (2014) reported the fruit oil of L. cubeba from Guangxi, China contained
geranial (27.5%), neral (23.6%) and also limonene at 18.8%. The essential oil of
L. cubeba from southern area of China gave neral (63.8%) and limonene (7.4%)
as the major constituents (Wang & Liu 2010). Ko et al. (2009) reported the
presence of neral (41.3%) and followed by geranial (30.1%) as the major
component of the fruit oil.

CONCLUSION

The differences in chemotype components may play a role in the chemistry and
biological application of the L. cubeba. The essential oil contents showed the
potential of this species to be incorporated as an active ingredient in personal
care products as well as for aromatherapy purpose.

ACKNOWLEDGEMENTS

We are grateful to all staff of CHiMP and from Natural Product Division for their
technical assistance and encouragements during the work. This research was
supported by the FRIM funding (RPP Project).

REFERENCES

Bighelli, A., Muselli ,A., Casanova, J. Nguyen, T.T., Vu Van, A. & Bessiere, J.M.
(2011). Chemical variability of Litsea cubeba leaf oil from Vietnam.
Journal of Essential Oil Research 17:86–88
Gogoi, P., Baruah, P. & Nath, S.C. (2011). Antifungal activity of the essential oil of
Litsea cubeba. Journal Of Essential Oil Research 9(2):213–215

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Chen, Y., Wang, Y., Han, X., Si L. Q. & Liyuan, L. (2013). Biology and chemistry of
Litsea cubeba, a promising industrial tree in China. Journal of Essential
Oil Research 25(2):103–111.
Jiang, Z., Yasmin, A., Bradbury, R., Zhang, X. & Isman, M.B. (2009). Comparative
Toxicity Of Essential Oils of Litsea pungens and Litsea cubeba and Blends
of Their Major Constituents against the Cabbage Looper, Trichoplusia ni.
Journal of Agriculture and Food Chemistry. 4833–4837.
Kai, Y., Cheng, F.W., Chun, X.Y., Zhu, F.G., Rui, Q.S., Shan, S.G., Shu, S.D., Zhi, L.L.
& Zhi, W.D. (2014). Bioactivity of Essential Oil of Litsea cubeba From
China and Its Main Compounds Against Two Stored Product Insects.
Journal of Asia-Pacific Entomology 17:459–466.
Ko, K., Waraporn, J. & Angsumarn, C. (2009). Repellency, fumigant and Contact
Toxicities of Litsea cubeba (Lour.) Persoon Against Sitophilus zeamais
Motschulsky and Tribolium castaneum (Herbst). Kasetsart J. (Nat. Sci.)
43:56–63.
Kong, D.G., Zhao, Y., Li, G.H., Chen, B.J., Wang, X.N., Zhou, H.L., Lou, H.X., Ren, D.
M. & Shen, T. (2015). The Genus Litsea in Traditional Chinese Medicine:
An Ethnomedical, Phytochemical and Pharmacological Review. Journal
of Ethnopharmacology. 256–264.
Liu, T.T & Yang, T.S. (2012). Antimicrobial Impact of the Components of Essential
Oils of Litsea cubeba from Taiwan and Antimicrobial Activity of the Oil in
Food Systems. International Journal of Food Microbiology 68–75.
Nor Azah, M.A. & Susiarti, S. (1999). Litsea Cubeba (Lour.) Parson. Pp. 123–126
in Essential oil plants. Plant Resources of South-East Asia 19. Oyen L.P.A.
& Nguyen Xuan Dung (Eds). Backhuys Publishers., Leiden
Park, I.K., Kim, J., Lee, S.G. & Shin, S.C. (2007). Nematicidal Activity of Plant
Essential Oils and Components from Ajowan (Trachyspermum ammi),
Allspice (Pimenta dioica) And Litsea (Litsea cubeba) Essential Oils against
Pine Wood Nematode (Bursaphelenchus Xylophilus). The Journal of
Nematology. 275–279.
Saikia, A.K., Chetia, D., D’Arrigo, M., Smerigloi, A., Strano, T. & Ruberto G.
(2013). Screening of fruit and leaf essential oils of Litsea cubeba Pers.
from north- east India-chemical composition and antimicrobial activity.
Journal of Essential Oil Research 25(4):330–338
Tajkrimi, M.M., Ibrahim, S.A. & Cliver, D.O. (2010). Antimicrobial herb and spice
compounds in food. Food control. 1199–1218.
Wang, H.W. & Liu, Y.Q. (2010). Chemical Composition and Antibacterial Activity
of Essential Oils From Different Parts of Litsea cubeba. Chem. Bodivers.
7:229–235.
Yang, Y., Jiang, J., Qimei, L., Yan, X., Zhao, J., Yuan, H., Qin, Z. & Wang, M. (2010).
The Fungicidal Terpenoids and Essential Oils from Litsea cubeba in Tibet.
Molecules. 7075–7082.

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Table 1. Chemical composition of the leaves, twigs and fruits of L. cubeba oils
Constituents RI Leaf Twig Fruits Mode of
identification
α-Thujene 924 0.5 0.3 t MS, KI
α-Pinene 932 5.4 2.8 0.2 MS, KI
Camphene 946 0.1 0.1 t MS, KI
Sabinene 969 14.9 4.0 t MS, KI
β-Pinene 974 0.3 3.2 t MS, KI
Myrcene 988 1.8 1.4 t MS, KI
6-Methyl-5-hepten-2-one - - - 0.7 MS
-2 Carene 1001 t 0.9 t MS, KI
limonene 1024 - - 1.2 MS, KI
1,8-Cineole 1026 48.8 57.7 - MS, KI
-Terpinene 1054 1.5 2.0 - MS, KI
cis-Sabinene hydrate 1065 0.1 - - MS, KI
cis-Linalool oxide 1067 - t t MS, KI
Trans-linalool oxide 1084 - t t MS, KI
Terpinolene 1086 1.1 0.8 - MS, KI
Linalool 1095 0.3 2.0 0.7 MS, KI
cis-p-Menth-2-en-1-ol - 0.2 0.2 - MS
trans-p-Menth-2-en-1-ol - 0.1 0.1 - MS
Rosefuran epoxide 1173 - - 0.1 MS, KI
Terpinen-4-ol 1174 4.6 4.1 - MS, KI
α-Terpineol 1186 5.0 2.0 - MS, KI
Neral 1235 0.2 1.1 28.7 MS, KI
Geranial 1264 0.1 1.4 44.7 MS, KI
Bornyl acetate 1287 0.1 0.1 0.5 MS, KI
-Terpinyl acetate 1316 0.7 0.8 - MS, KI
α-Terpinyl acetate 1346 8.9 11.4 - MS, KI
Longicyclene - t - - MS
α-Copaene 1374 0.2 0.3 - MS, KI
β-Elemene 1389 t t - MS, KI
cis-α-Bergamotene 1411 0.2 0.6 - MS, KI
β-Caryophyllene 1417 2.3 1.4 - MS, KI
epi-β-Santalene 1445 t - - MS, KI
α-Humulene 1452 0.2 0.1 - MS, KI
β-Santalene 1457 0.3 0.3 - MS, KI
Bicyclogermacrene 1500 0.2 0.3 - MS, KI
(E,E)-α-Farnesene 1505 t 0.2 - MS, KI
7-epi-α-Selinene 1520 t t - MS, KI
-Cadinene 1522 t t - MS, KI
Caryophyllene oxide 1582 0.1 0.4 t MS, KI
Note: t=trace; MS=mass spectrum; KI=Kovat index.

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PRISMALAYANOSIDE FROM PRISMATOMERIS TERANDRA

Nor Hayati, A.1, Khalijah, A.2, Yasodha, S.2, Khalit, M.2, Ling, S.K.1, Ong, B.K.1,
Norul Aiman, Y.1, Amira Rina Nurdiana, M.S.1 & Nurhazwani, M.H.1

1
Natural Product Division, Forest research Institute Malaysia, 52109 Kepong,
Selangor. 2Department of Chemistry, Faculty of Science, Universiti Malaya, Kuala
Lumpur, Malaysia

Tel: 03-62797357 Fax: 03-62729805 E-mail: norhayatiab@frim.gov.my

INTRODUCTION

Prismatomeris tetrandra (Roxb.) K. Schum is also synonym to P. malayana Ridley

and P. albidiflora King (Lemmens & Bunyapraphatsara 2003). Locally it is known
as tongkat aji samat. This species can be found in East Asia and the Malesian
region. It grows mainly in lowlands regions up to 500 m altitude, but sometimes
in montane forest up to 1700 m altitude, in humid evergreen forest as well as
dry forest, scrub vegetation, on dunes, also on shale and limestone.
In the traditional medicinal system, every part of Prismatomeris
tetrandra is very useful. In Peninsular Malaysia and Thailand traditional
medicinal system, the leaves have been applied as a poultice to fresh wounds
(Burkill, 1966). In Cambodia, a decoction of the leaves, made by extended
boiling with coconut palm and lawsonia roots, is used to treat bronchitis. In
Thailand, the macerated roots in water are used to treat snakebites whereas in
Indochina, the decoction has been used in a mixture with coconut and henna to
treat bronchitis (Lemmens & Bunyapraphatsara, 2003).
Past reports have illustrated that there has been a few biological activity
studies on Prismatomeris sp. An extract of P. tetrandra was found to be
cytotoxic in brine shrimp lethality bioassay and on human tumor cell line (Dey et
al. 2003). Kartsen et al. (2007) reported on the isolation and determination of
absolute configuration of the new cytotoxic iridoids from Prismatomeris
tetrandra namely as prismatomerine. The root methanol extracts of
Prismatomeris sessiflora Pierre ex Pitard showed significant anti-malarial
potential against T9/94 Plasmodium falciparum (Kittisak et al. 1999). However,
the isolated rubiadin and rubiadin-1-methyl ether (anthraquinones) revealed
weak activity on the anti-malarial assay with IC50 range 13,000 ng/mL and 1,560
ng/mL respectively. Examination of the literature has demonstrated that
Prismatomeris species produces mainly anthraquinones, iridoids and
triterpenoids (Lee 1968; Kittisak et al. 1999; ZiMing et al. 2005; Kwanjai et al.
2005; Kartsen et al. 2007).

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MATERIALS AND METHODS

Chemicals and instruments

All chemicals were obtained from commercial sources (Aldrich, Merk, and
Sigma) and used without further purification. Solvents were used without
further purification or drying, unless stated otherwise. Reactions and isolations
were monitored using thin layer chromatography (TLC) (aluminum supported
silica gel 60 F254 plates were used for TLC). TLC spots were visualized under
ultra-violet light (254 nm and 365 nm). The plates were then sprayed with 10%
sulphuric acid followed by heating using a hot plate to detect the presence of
phenolics and terpenes which were indicated by the presence of colourful spots.
Several packing materials were used for column chromatography i.e MCI gel
CHP 20P, Sephadex LH-20, Chromatorex ODS, silica gel 60 (70-230 Mesh ASTM
or equivalent to silica gel of size 0.063-0.200 mm). The infrared spectra were
recorded on a Perkin Elmer Spectrum 100 Fourier Transform Infrared (FT-IR)
spectrometer equipped with a mid-infrared deuterated triglycine sulphate
(DTGS) detector. NMR analyses were carried out on a Bruker DRX 300 NMR
spectrometer (300 MHz for 1H NMR and 75 MHz for 13C NMR) system with
deuterated pyridine (C5D5N). The mass spectra were obtained using a LTQ
Orbitrap Mass spectrometer equipped with an electrospray ionization probe by
employing either a negative or positive ion mode, which ever could afford the
best limits of detection for the compounds.

Plant material

P. tetrandra was collected from Setiu, Terengganu, identified and deposited in

the herbarium (FRI 50080) of the Forest Research Institute Malaysia (FRIM). The
plant material was dried in an oven at 40 oC, divided into different parts and
ground. Oven-dried leaves (2.041 kg) were ground and extracted with MeOH by
soaking for three times at room temperature. The concentrated extract (20.1 g)
was suspended in H2O and partitioned with petroleum ether, followed by CHCl3
and finally with EtOAc. The EtOAc layer was chromatographed over Sephadex
LH-20 column eluted with 100% MeOH. The resulting fraction obtained were
further purified by a combination of column chromatography employing silica
gel with CM system, MCI gel with water- methanol (WM) system and MCI gel
CHP20P to finally afforded one iridoids 1.

RESULTS AND DISCUSSION

Prismalayanoside 1 was isolated as yellowish amorphous solid. It produced

pseudo-molecular ion peak [M+Na]+ at m/z 685.17523 (calcd. 685.16944) with
ESI-MS which is consistent with the molecular formula of C31H33O16.

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1
CO2CH3

O
O
OGlu
O
H
HO
OH
2 3

The NMR spectra reveal that this compound has the characteristics of
iridoid skeleton. The signals for the glucose protons (H-1’, H-2’, H-3’, H-4’, H-5’
and H-6’) were resonated between 5.86-3.89 ppm. Combined analysis of the
13
CNMR and the DEPT spectra showed thirty one carbon signals which consisted
of one methyl, three methylene, eighteen methine and nine quartenary
carbons. The analysis on COSY and HMQC spectra showed the correlations
between H-6, H-7, H-5, H-9 and H-1, thus supported the cis-fused
cylopentanopyran ring system found in iridoid skeletons which particularly the
plumieride type iridoids (Kuigoua et al. 2010). The correlations between H-18
and H-16a, H-16b and H-17, H-17b suggested the presence of the five
membered ring. The unusual seven-membered unsaturated spiro lactone ring
attached to C-8 was constructed by the long range correlations in the HMBC
spectrum.

CONCLUSION

Combined analysis using NMR techniques such as 1HNMR, 13CNMR, HMQC,

HMBC, COSY, NOESY and ROESY and other spectroscopic techniques including
IR, UV and mass on compound 1 finally suggest the structure of the compound
namely as prismalayanoside. It has basic skeleton as the reported
prismatomerin 2 and gaertneroside 3. The structure of 1 was however novel
because it was having unprecedented spiro-hepta-lactone ring as compared to
the other two compounds. The ring was built up of carbons and protons at the
position 19, 15, 14, 12, 10 and 8.

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ACKNOWLEDGEMENTS

This work was supported financially by the Ministry of Science and Technology
Malaysia (MOSTI) under project number 02-03-10-SF0110.

REFERENCES

Burkill, I.H. (1966). A dictionary of the economic products of the Malay

Peninsula. Kuala Lumpur, Malaysia: Ministry of Agriculture and co-
operatives.
Dey, S.K., Islam, S., Mostafa, M., Nahar, N. & Mosihuzzaman, M. (2003). Some
secondary metabolites from cytotoxic extract of Prismatomeris tetrandra.
J. Bangladesh Chem. Soc., 6:22–27.
Kartsen, K., Dietmar, G., Sujit, K.D., Nilufar, N., Mohammad, M., Nasim, S.M.,
Hossain, S, Philip, J.S. & JianJung, P.F.S. (2007). Prismatomerin, a new
iridoid from Prismatomeris terandra. Structure elucidation, determination
of absolute configuration and cytotoxicity. J. Nat. Prod., 70(8):1339–1343
Kittisak, L., Sukanya, D.A., Vichien, J. & Jerapan, K. (1999). Anti-malarials from
Stephania venosa, Prismatomeris sessiliflora, Diospyros Montana and
Murraya siamensis. Planta Medica 65:754–756
Kuigoua, G.M., Kouam, S.F., Ngadjui, B. t., Schulz, B., Green, I.R., Choudhary,
M.I., Krohn, K. (2010). Secondary metabolic products from the stem bark
of Plumeria rubra Linn. displaying anti-microbial activities. Planta Med,
76: 620-625.
Kwanjai, K., Somdej, K. & Ruchanee, P. (2005). Biological activity of
anthraquinones and triterpenoids from Prismatomeris fragrans. Journal of
Ethnopharmacology 100:284–288
Lee, H.H. (1968). Colouring matters from Prismatomeris malayana. Phytochem.,
8:501–503.
Lemmens, R.H.M.J. & Bunyapraphatsara, N. (2003). Plant resources of South-
East Asia, Medicinal and Poisonous Plants 3 12(3). Bogor, Indonesia:
Prosea Foundation.
ZiMing, F., JianShuang, J., YingHong, W. & PeiCheng, Z. (2005). Anthraquinones
from the roots of Prismatomeris tetrandra. Chem. Pharm. Bull.,
53(10):1330–1332

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 STANDAREDISATION,
QUALITY CONTROL
& PROCESSING TECHNOLOGY

133
STATUS PEMIAWAIAN DALAM INDUSTRI HERBA TEMPATAN

Zhari, I

School of Pharmaceutical Sciences,

Universiti Sains Malaysia,
11800 Pulau Pinang

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FRIM’S COMMITMENT TOWARDS LOCAL HERBAL INDUSTRY: QUALITY
CONTROL OF HERBAL PRODUCT

Ong, B.K., Norulaiman, Y., Nor Hayati, A., Nurhazwani, M.H. Amira Rina
Nurdiana, M.S. & Nor Azah, M.A.

Natural Products Division, Forest Research Institute Malaysia, FRIM, 52109

Kepong, Selangor, Malaysia.

Tel : 03-62797370 Fax: 03-62729805 Email : ongbk@frim.gov.my

INTRODUCTION
Herbal Products and Quality Control

Malaysians are more health conscious nowadays. Such awareness and the back
to the nature trend have increased the popularity of herbal based product
usage among Malaysians. Currently, the Malaysia market is flooded with all
types of herbal-based products. More than 40 % of those products are
imported from neighbouring countries such as Indonesia, Thailand, and some
are originated from China and India. The remaining products are locally
manufactured. Quite a number of these herbal products are lack of scientific
study and safety evaluation. The quality of these products have not been given
sufficient due attentions by the manufacturer. Most of the time, these herbal
products are produced at small scale and processed in an uncontrolled
environment. The quality, effectiveness and safety of the produced product are
not guaranteed. Incident of poisoning after consuming certain herbal products
and cases of products tested with harmful chemicals and adulterant happened
nowadays. These incidents will definitely reduce the level of consumer trust
and confidence in using the herbal products especially those produced by local
manufacturers.

Quality control is a process employed to ensure a certain level of

quality in product or service. The basic goal of quality control is to ensure the
products meet specific requirement and are dependable and satisfactory.
Herbal product quality control is an important criterion that should be
emphasized before the products being delivered to the consumer. Consumer’s
concerns on safety, quality and effectiveness of herbal products can be
addressed and their confidence being restored if the procedures of product
quality control were carried out. The Ministry of Health has set some
guidelines that must be complied by manufacturers prior to market their herbal
products.

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Natural Products Quality Control Laboratory (NPQC)

Natural Products Quality Control Laboratory (NPQC) has been established under
Natural Products Division (formerly known as Medicinal Plants Division) in FRIM.
Currently, NPQC provides quality control technical services to herbal industries.
These technical services testing scope include a) microorganism contamination
evaluation, b) heavy metal analyses, c) tablet and capsule weight uniformity
test, d) tablet and capsule disintegration test. These tests are developed and
carried out in accordance with the guidelines outlined under the drug
Registration Guidance Document and Control of Drugs and Cosmetics
Regulations 1984.

MS ISO/IEC 17025:2005 Accreditation

NPQC has been accredited by Department of Standards Malaysia under the

‘Skim Akreditasi Makmal Malaysia’ (SAMM) for the scope area of microbiology
and chemical (Figure 1). The above scheme meets the requirements of MS
ISO/IEC 17025:2005 ’General requirement for competence of testing and
calibration laboratories’. This Malaysian Standards (MS) is identical with ISO/IEC
17025:2005 published by the International Organization for Standardization
(ISO). The accredited testing scopes are as following:

A) Microbial Enumeration Tests:

i. Total aerobic microbial count (TAMC)
ii. Total combined yeasts/moulds
(TYMC)
B) Test for Specified Micro-organism:
i. Test for absence of Escherichia coli
ii. Test for absence of Salmonella
iii. Semi-quantitative test for Bile-
Tolerent Gram-Negative Bacteria
iv. Semi-quantitative test for Escherichia
coli
v. Test for absence of Pseudomonas
aeruginosa
vi. Test for absence of Staphylococcus
aureus
vii. Test for absence for Candida albicans
C) Determination of Heavy Metal: Figure 1 : Accreditation by Department
i. Mercury (Hg) of Standards Malaysia under
SAMM
ii. Arsenic (As)
iii. Cadmium (Cd)
iv. Lead (Pb)
D) Determination of moisture

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Quality Control Technical Services

Microorganism Contamination

Plant and herbal raw material, semi-finished or finished products received from
the industry will firstly undergo microorganism contamination determination.
Total aerobic microbial count (TAMC) and total combined yeasts/moulds count
(TYMC) will be determine to evaluate the level of microorganism contamination
in the received sample. In addition, specific microorganism detection test will
be carried out to determine the presence of specific microorganisms such as
Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, Salmonella
spp, the bile tolerant gram negative bacteria and Candida albicans.

Heavy Metal Analyses

Heavy metal limit test is one of the mandatory quality control testing required
by the Ministry of Health for any herbal products registration in Malaysia.
Heavy metal analyses of lead (Pb), cadmium (Cd), mercury (Hg) and arsenic (As)
in samples were carried out using atomic absorption spectrometry. Sample
digestion was performed using nitric acid and hydrogen peroxide in microwave
digester prior to heavy metal analysis.

Results obtained from the above technical services will assist herbal
product manufacturer in formulating corrective action plan to ensure the
quality and safety of their final product before being marketed to the
consumer. Losses as a result of producing large quantities of microorganism
and/or heavy metal contaminated herbal product that will not comply with the
safety guidelines of Ministry of Health will be prevented.

Weight Uniformity Test and Disintegration Test for Tablet and Capsule

Product weight uniformity test is one of the quality control requirements for
herbal tablet and capsule product. The weight uniformity test provide limits for
the permissible variations in the weights of individual tablets or capsules,
expressed in terms of the allowable deviation from the average weights of a
sample. Whereas, disintegration test is another quality control testing
mandatory for herbal and capsule product. The above test is provided to
determine whether tablets or capsules disintegrate within the prescribe time
when placed in a liquid medium under the experimental conditions presented.
Both the above quality control tests are currently provided by NPQC, FRIM.
Both the above test for tablet and capsule are based on guidelines from British
Pharmacopoeia and/or United States Pharmacopoeia. NPQC is expected to
receive the MS ISO/IEC 17025:2005 accreditation for the above mention test
scope from Department of Standards Malaysia before end of 2016.

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Recognition from National Pharmaceutical Regulatory Agency (NPRA)

Since the early 2016, NPQC receive recognition and endorsement from National
Pharmaceutical Regulatory Agency (NPRA), Ministry of Health as a testing
laboratory for traditional product analyses services. NPRA is formerly known as
National Pharmaceutical Control Bureau (NPCB). The quality control testing
report issued by NPQC, FRIM is recognised and accepted by NPRA. Thus, herbal
product manufacturer able to utilise the NPQC issued quality control report as
partial fulfilment for herbal product registration requirement.

CONCLUSION

Natural Products Quality Control Laboratory (NPQC), FRIM which

accredited by Department of Standards Malaysia under the ‘Skim Akreditasi
Makmal Malaysia’ (SAMM; MS ISO/IEC 17025:2005), is competent to provide
quality control technical services; a) microorganism contamination evaluation,
b) heavy metal analyses, c) tablet and capsule weight uniformity test, d) tablet
and capsule disintegration test. Currently, NPQC is ready to provide the testing
for plant and herbal raw material, semi-finished or finished products to herbal
industry. NPQC is recognised as a testing laboratory for traditional product
analyses services by National Pharmaceutical Regulatory Agency (NPRA),
Ministry of Health Malaysia.

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HERBAL PRODUCT DELIVERABLES: SEEDS TO SHELVES

Mohd. Shahidan, M.A1, Khairul Kamilah, A.K.1, Munirah, M.F1, Hada Masayu,
I.2, Zaki, A.3, Zamree, M.S.1, Syed Othman, S.O.1, Fadhilah, Z.1, Nur Fairus, Y.1 &
Mazdiana, Z.1

1
Innovation and Commercialization Division, Forest Research Institute Malaysia
(FRIM), Kepong Selangor; 2Natural Products Division, Forest Research Institute
Malaysia (FRIM), Kepong Selangor. 3Forest Biotechnology Division, Forest
Research Institute Malaysia (FRIM), Kepong Selangor.

Tel: 03-62797509 Fax: 03-62729805 E-mail: shahidan@frim.gov.my

ABSTRACT

Market for herbal products has evolved steadily with the demand and related
quality management, globally. The quality management has progressed via the
adaptation and embracement of technological applications and market mutual
agreements. Looking at the current market shapes of the herbal products, easily
the herbal products can be seen as Food and Beverages (F&B), Traditional
Products, Health Supplements, Cosmetics Products and also as botanical drugs
and Phytomedicine products. Each individual product type being regulated by
respective authorities and market requirements/legislation that includes
cascade of quality management system and procedures. Further understanding
of the supply chain of the herbal products, the raw materials are coming from
cultivated of wild crafted sources, followed by primary and secondary
processing activities that usually referred to the activity related to Pre and Post
Harvest Handling, Fresh Cut Technologies and Farm Gate Technologies. Certain
products need further refinement/isolation processes such as Aqueous/Solvent
Extraction, Fractionation, Fermentation and other physical mechanical
maneuvers. The semi-finished products, active ingredients (AI) or the extracts
will be further manipulated to form final of finished products as mentioned
earlier. Based on this supply chain, this paper will further discuss the issues by
clustering the products by few market deliverables or herbal product
deliverables such as planting materials, raw materials, the extracts and the
finished products. In addition to that, the term Quality Planting Materials
(QPM), Quality Raw Materials (QRM), Quality Extracts (QE) and Quality Finished
Products (QFP) will be used for the purpose of discussion in this paper. The
objective of the this paper is to discuss and elaborate the attribution and the
importance of quality management to all herbal product deliverables by
focusing on the technological application and specific activities that mandatorily
needed prior to maintain the quality of the deliverables to the market. For

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instance, the discussion on production of the quality planting material (QPM)
will mainly refers to the international accepted guidelines such as Quality
Declared Planting Materials (QDPM) by FAO. The successive steps of
methodologies that involved in the quality management will be discussed by
giving the suitable examples. The quality raw materials will be further discussed
by quoting the pertinent elements in Pre and Post Harvest Technologies. The
specific activities will be discussed by giving the suitable example. The extraction
technologies that responsible in transforming the raw materials into more
refined extracts will involve the cascades of processing procedures and also the
standardization activities. The Quality Finished Products (QPM) attributions will
differ based on the market segmentation and requirements.

Key words: Herbal products deliverables, standardization process

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EFFECTS OF TEMPERATURE ON DRYING OF EURYCOMA LONGIFOLIA
JACK ROOTS: DRYING TIME AND PRODUCT QUALITY

Hada Masayu, I.1,2, Mohd. Nordin, I1, Pin, K.Y.2, Rabitah, Z.1 & Mohd. Radzi, A.2

1
Process and Food Engineering Department, Universiti Putra Malaysia (UPM),
Serdang Selangor; 2Natural Products Division, Forest Research Institute Malaysia
(FRIM), Kepong Selangor.

Tel: 03-62797781 Fax: 03-62729805 E-mail: hada@frim.gov.my

INTRODUCTION

Eurycoma longifolia (EL) is a medium-sized tree growing to a height of about 10

m. The plant is mostly found as an under-storey growth of lowland forests in
Peninsula Malaysia and other Southeast Asian Regions (Ang et al. 1995). In
Vietnam it is the tree “that cures a hundred diseases” but in Malaysia it is better
known for its aphrodisiac properties (Chuah et al. 2006). EL can be harvested
starting 5 years after cultivation (Mohd Radzi et al. 2005). The plant is pulled out
from the soil and the taproot is harvested which is yellowish with a bitter taste.

Eurycomanone (EUR) is a plant quassinoids derived from EL roots. It is

selected as one of the commercial potential chemicals other than eurycomanol
and eurycomalactone (Indu bala and Lean Teik, 2000). Biological studies have
shown that EUR possess antimalarial activity against Plasmodium falciparum.
Encouraging results found where study was done on the effects of daily
replacement of culture media containing EL root extracts (rich in quassinoids)
against six Malaysian isolates (Ang et al. 1995). Four quassinoids were separated
namely pasak bumin-A, -B, -C and -D from E. longifolia and found that
pasakbumin-A (=eurycomanone) and pasakbumin-B to exhibit potent antiulcer
activity (Tada et al. 1991).

Drying, also known as dehydration is a process of removing moisture

from products. This will cause reduction in weight and volume of products that
will facilitate savings in the transportation cost, storage space and minimize
packaging. Other than that dried products have the advantages to prevent
contamination from microbes due to its stability during storage. Convection
oven drying is a well-known and widely used drying method by practitioners in
the local herbal industry. In drying process, the quality of the dried product will
depend on the drying parameters such as velocity of drying air, humidity of
drying air and product characteristics. The effect of drying temperature is the
most widely studied parameter. Extreme temperature applied during drying, will

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bring about undesirable changes in the quality of the output products. Previous
studies revealed that the use of high temperature is often led to the
degradation or loss of quality of the product (Alean et al. 2016). The major
consideration in drying of herbs like EL roots is the preservation of the chemical
compounds, which are mostly heat sensitive.

Many studies have reported on the bioactivity of EL but the effect of

drying on the drying kinetics and quality of EL roots in order to determine the
optimum temperature for drying of this herb yet to be studied. In this study,
EUR as an active compound was selected as quality indicator for EL roots. The
content of this EUR in the dried roots was determined using ultra-performance
liquid chromatography (UPLC). The concentration change of EUR subjected to
different drying temperatures was obtained from the UPLC results.

MATERIALS AND METHODS

Materials

EL was collected from cultivation plot at Bukit Hari in FRIM. The selected part of
the plant was the root to be used in the experiment. The collected samples were
cleaned from dirt using tap water, rinsed, chopped and then kept in polystyrene
container while being transported to the laboratory. Initial moisture content of
the roots was 63.0 ± 2.9% (wet basis) measured before each experiment using
calibrated Halogen Moisture Analyser.

Experimental design

The convection oven dryer used in this study consists of three main components
namely heating elements, drying trays and temperature control button. The
drying process was operated at three selected temperatures of 50, 60 and 70°C.

Initial moisture content of the sample was determined before each experiment.
Approximate weight of 0.5 g fresh sample was distributed uniformly as a thin layer on
aluminum tray with square openings of 14 x 8 cm. Water loss from the samples was
measured by weighing the sample outside the drying chamber periodically using
electronic balance. Weights were recorded every 10 minutes until the equilibrium
moisture content was reached. Each drying experiment was triplicated. Final product
moisture content (wb) was determined using Halogen moisture analyser by drying the
sample at 100°C.

Chemical analysis

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Quantification analysis of EUR from EL roots was performed using Ultra
Performance Liquid Chromatography (UPLC) equipped with an Acquity ® BEH
C18 column (2.1 x 150.0 mm, 1.7 µm particle size). The elution was done using
the gradient solvent system with a flow rate as in the table at room
temperature. Mobile phase consist of eluted with 0.1% (v/v) formic acid in H2O
and acetonitrile. Table 1 shows the elution profile of the analysis. The
wavelength was set at 244.4 nm and sample injection volume was 10 µL.

Table 1: The mobile phase change in UPLC analysis

Time Flow rate 0.1% Formic Acid, 100% Acetonitrile,
(min) (ml/ min) (%) (%)
0 0.20 90 10
2 0.25 85 15
5 0.30 75 25
10 0.25 60 40
12 0.20 90 10

The dried materials were ground using pulverising machine and

extracted using distilled water in 1:10 ratio (Radzi et al. 2005). The mixture was
sonicated for 60 minutes at 37°C to optimize the extraction of the
phytochemicals. Then the solution was filtered using 0.45µm prior to UPLC
analysis for the chemical profiles and concentrations of EUR.

Identification of selected peak indicating targeted compound was done

by comparing their retention time with the standard. The concentration of the
compounds was calculated from their peak area by using the calibration curves
developed from the standard.

Analysis of drying data

Drying Curve

The free moisture versus drying time graphs was plotted for each of the
experiments. Moisture content (dry basis) of the sample was described by the
percentage equivalent of the ratio of the weight of water to the total weight of
the dry material. It was calculated by using equation 1 (Ramaswamy & Marcotte
2006):

𝑀𝑜𝑖𝑠𝑡𝑢𝑟𝑒 𝑐𝑜𝑛𝑡𝑒𝑛𝑡 = (𝑀⁄𝑆) × 100

Where M is the content of water and S is the content of solid.

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Drying Rate Curve

The drying rate versus free moisture graph were plotted for each of
experiments. The drying rate of the sample was calculated using equation 2
(Kavak Akpinar et al., 2003):

Drying rate = M(t+dt) - Mt

dt

Where M(t + dt) and Mt are moisture content at t+dt (g water / g dry solid) and
moisture content at time t, respectively and t is drying time (min).

RESULTS AND DISCUSSION

Drying Kinetics

Figure 3 shows the changes in free moisture against time at 50, 60 and 70°C of
convection oven drying process. It was found that the drying time for 50, 60 and
70°C was 60, 40 and 30 minutes respectively. This indicates that higher drying
temperature resulted in shorter drying times. This was because of a larger
driving force for heat transfer at temperature of 70°C as compared with at lower
temperature.
2.0
(g water / g dry solid)

1.5
Free moisture

5050°C
C
1.0 60 C
60°C
0.5 70 C

0.0
0 20 40 60 80
Time (min)
Figure 3: Drying curves of EL roots dried at 50, 60 and 70°C using
convection oven

Effect of convection oven drying on EUR content

Figure 4 shows the effect of convection oven drying temperature from 50 to

70°C on the quality of EL roots measured by EUR concentration. The control
sample was EL roots dried at room temperature.

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 15

Eurycomanone
concentration
(mg / L)
10

0
control 50 60 70
Temperature (C)

Figure 4: EUR concentration in EL roots subjected to convection oven

temperature at 50, 60 and 70°C

It can be seen that EUR was stable when drying was conducted at 50
and 60°C. However, there was a drop about 30% of EUR concentration in the
sample dried at 70°C. The degradation was significant (p<0.05) as affected by
that temperature. Based on Davidson et al. (2004), undesirable quality of
ginseng root occurred if the air temperature was increased to 50°C when the
moisture content exceeded 1.22 g H2O/g dry solid. On contrary, the quality of
ginseng root was maintained when temperature above 38° was applied at lower
values of moisture content. From the current study, EL root was found to be
stable when dried at temperature 50 and 60°C, hence it was suggested that the
roots to be dried at these temperature for optimum quality of product.

CONCLUSIONS

The effects of temperature on EL roots using convection oven was successfully

investigated. Based on the findings on drying times and EUR concentration, the
optimum drying temperature for EL roots was at 60°C.

ACKNOWLEDGEMENTS

The authors wish to acknowledge the support and help given by the staff of
Natural Products Division and Maran Research Station, FRIM.

REFERENCES

Alean, J., Chejne, F. & Rojano, B. (2016). Degradation of polyphenols during the
cocoa drying process. Journal of Food Engineering 189:99–105
Ang, H.H., Chan, K.L. & Mak, J.W. (1995). Effect of 7-day daily replacement of
culture medium containing Eurycoma longifolia Jack constituents on the
Malaysian P. falciparum isolates. J Ethnopharmacol 49:17–15.

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Chuah, C.H., Mok, J.S.L., Liew, S.L. & Ong, H.C. (2006). 101 Plants to fight cancer.
Sarawak Biodiversity Centre.
Davidson, V.J., Li, X. & Brown Ralph, B. (2004). Forced-air drying of ginseng root:
Effects of air temperature on quality. Journal of Food Engineering 63:
361–367.
Guarte, R.C. (1996). Modeling the drying behavior of copra and development of
a natural convection dryer for production of high quality copra in the
Philippines. PhD dissertation. Stuttgart, Germany: Hohenhaim
University.
Indubala, J. & Ng, L.T. (2000). Herbs: The green pharmacy of Malaysia. Vinpress
Sdn. Bhd.
Kavak Akpinar, E., Bicer, Y. & Cetinkaya, F. (2006). Modelling of thin layer drying
of parsley leaves in a convective dryer and under open sun. Journal of
Food Engineering 75:308–315.
Midili A., Kucuk H. & Yapar, Z. (2002). A new model for single layer drying.
Drying technology, 20(7):1503–513.
Mohd Radzi, A., Mohd Ilham, A., Norijas, A.A., Mohd Noh, J. & Mohammad
Ghawas. (2005). Penentuan kualiti berdasarkan kandungan kimia dan
usia penanaman tongkat ali (Secara perladangan). Pp. 279–285 in
Chang, Y.S., Vimala S., Mazura M.P. and Ong B.K. (Eds). Current trends
and perspectives. Proceedings of the Seminar on Medicinal and Aromatic
Plants (MAPS 2004). 20–21 July 2004, Kuala Lumpur.
Pin, K.Y., Chuah, T.G., Abdull Rashih, A., Rasadah, M.A., Law, C L. & Choong, T.S.
Y. (2009). Drying of betel leaves (Piper betle L.): Quality and drying
kinetics. In: Proceedings of the Seminar on MAPS 2008, In Chang, Y.S.,
Saiful Azmi, J. & Hada Masayu, I. (Eds), 21–22 Oct 2008, Kuala Lumpur.
Rajeev, B. & Karim, A.A. (2010). Tongkat ali (Eurycoma longifolia Jack): A review
on its ethnobotany and pharmacological importance. Fitoterapia 81:
669–679.
Ramaswamy, H. & Marcotte, M. (2006). Food processing: Principles &
applications. CRC Press.
Tada, H., Yasuda, F., Otani, K., Doteuchi, M., Ishihara, Y. & Shiro, M. (1991). New
untiulcer quassinoids from E.longifolia. Eur J Med Chem. 26:345–9.

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PERKHIDMATAN UJIAN KAWALAN KUALITI PENCEMARAN
MIKROORGANISMA YANG BERAKREDITASI ISO/IEC 17025 UNTUK
PRODUK DAN BAHAN MENTAH HERBA DI MAKMAL NPQC, FRIM.

Amira Rina Nurdiana, M.S., Ong, B.K., Norulaiman, Y., Nurhazwan,i M.H., Nor
Hayat,i A. & Nor Azah, M.A.

Makmal Kawalan Kualiti Produk Hasilan Semula Jadi (NPQC), Bahagian Hasilan
Semula Jadi, Institut Penyelidikan Perhutanan Malaysia (FRIM), 52109 Kepong,
Selangor. Malaysia

Tel : 03-62797785 Fax: 03-6272980 Email : amirarina@frim.gov.my

PENGENALAN

Tumbuhan ubatan ialah flora yang dijadikan ubat-ubatan atau produk herba
bagi merawat penyakit secara tradisional. Kini, produk herba berasaskan
tumbuhan ubatan dihasilkan secara meluas tidak kira dalam bentuk produk siap
mahupun separa siap. Walaupun produk herba bermanfaat untuk kesihatan, ia
juga boleh memudaratkan jika diproses tanpa mengikut piawai yang dibenarkan
atau mengandungi bahan-bahan yang memudaratkan kesihatan. Maka, Makmal
Kawalan Kualiti Hasilan Semula Jadi (NPQC) menawarkan perkhidmatan ujian
kawalan kualiti terhadap sampel herba bermula dari peringkat pemprosesan
bahan mentah sehingga ke produk siap untuk industri herba tempatan. Makmal
NPQC juga berfungsi membangunkan prasarana dan kaedah ujian kawalan
kualiti berpandukan kaedah piawaian antarabangsa. Makmal NPQC telah
berjaya memperolehi akreditasi MS ISO/IEC 17025:2005 oleh Skim Akreditasi
Makmal Malaysia (SAMM) semenjak tahun 2011. Makmal NPQC juga
merupakan sebuah makmal di bawah program Pembangunan Produk Herba
(PPH) telah diberi status BioNexus Partner (BNP) oleh Malaysian Biotechnology
Corporation. Makmal NPQC telah mendapat pengiktirafan sebagai Makmal
Panel Biro Pengawalan Farmaseutikal Kebangsaan (BPFK), Kementerian
Kesihatan Malaysia untuk Analisis Produk Tradisional.

KAEDAH UJIAN

Ujian pencemaran mikroorganisma terbahagi kepada Ujian Bebanan Mikrob

(Microbial Enumeration Test) dan juga Ujian Mikroorganisma Spesifik.

Ujian Bebanan Mikrob

Untuk ujian ini, sampel yang diperlukan adalah sebanyak 10 gram atau ml. Bagi
sampel separa siap dan produk siap, sebanyak 5 pencairan bersiri manakala

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untuk sampel bahan mentah pencairan bersiri sampel disediakan sehingga 7
pencairan. Pencairan bersiri sampel disediakan di dalam larutan Sodium klorida
pepton (Naclpp). Setiap pencairan dipiringkan sekurang-kurangnya dengan 2
replikasi untuk jumlah pengiraan mikroorganisma aerobik (TAMC) dan jumlah
pengiraan yis dan kulat (TYMC). TAMC akan menggunakan Tryptic soy agar (TSA)
dan dieramkan pada suhu 32.52.5 °C selama 3-5 hari. TYMC pula menggunakan
media Sabouraud dextrose agar (SDA) dan dieramkan pada suhu 22.52.5 °C
bagi TYMC untuk selama 5-7 hari. Purata kiraan koloni dikira dan unit
penghasilan koloni (CFU) bagi setiap gram atau ml sampel ditentukan. Bacaan
(CFU/g@ml) pada pencairan di dalam julat kiraan yang mempunyai nilai
perbezaan (SD) yang kecil antara plat/replikasi akan diambil untuk dianalisis.
(BP2016 Appendix XVI B)

Ujian Mikroorganisma Spesifik

Pengesanan mikroorganisma spesifik dijalankan mengikut kaedah standard

British Pharmacopoiea (2016) untuk kesemua spesies mikroorganisma yang
terlibat. Ujian pengesahan spesies dijalankan dengan menggunakan Sistem
Pengenalpastian ID (Biolog).

Ujian Pengesanan dan Semi-kuantitatif Escherichia coli

1 gram @ ml sampel yang diterima akan di masukkan kedalam 9 ml kaldu Tryptic

soy (TSB) dan pencairan bersiri di dalam TSB sehingga 3 pencairan akan
disediakan untuk ujian semi-kuantitatif. Campuran sampel dan kaldu dieramkan
pada suhu 32.5  2.5 °C selama 18-24 jam. 1 ml kultur kemudian di pindahkan ke
dalam 100ml kaldu MacConkey (McB) dan dieramkan lagi selama 24-48 jam
pada suhu 431 °C. Selepas pengeraman, subkultur ke atas media MacConkey
agar (McA) dijalankan untuk mendapatkan koloni tunggal dan dieramkan selama
18-72 jam pada suhu 32.5  2.5 °C. Koloni yang dijangkakan positif akan
dijalankan ujian pengesahan.

Ujian Pengesanan Salmonella spp.

25 gram sampel ditimbang dan dimasukkan ke dalam 225 ml Air penimbal

pepton (BPW). Campuran kemudiannya dieramkan pada suhu 32.5  2.5 °C
selama 24 jam. 0.1ml kultur dipindahkan ke dalam 10 ml kaldu Rappaport
Vasilidis Salmonella (RVS) dan dieramkan pada suhu 32.5  2.5 °C selama 24
jam. Selepas pengeraman, subkultur ke atas media Xylose lysine deoxycholate
(XLD) dijalankan untuk mendapatkan koloni tunggal dan dieramkan selama 18-
72 jam pada suhu 32.52.5 °C. Koloni yang dijangkakan positif akan dijalankan
ujian pengesahan.

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Ujian Semi-kuantitatif Bile tolerant bakteria Gram negatif

1 gram @ ml sampel yang diterima akan di masukkan kedalam 9 ml kaldu Tryptic

soy (TSB). Campuran sampel dan kaldu dieramkan pada suhu 22.5  2.5 °C
selama 2-3 jam. 1 ml kultur kemudian di pindahkan ke dalam 9 ml kaldu Mossel
dan sebanyak 4 pencairan bersiri dilakukan sebelum dieramkan lagi selama 24-
48 jam pada suhu 32.5  2.5 °C. Selepas pengeraman, subkultur ke atas media
Violet Red Bile Dextrose (VRBD) dijalankan untuk mendapatkan koloni tunggal
dan dieramkan selama 18-24 jam pada suhu 32.5  2.5 °C. Koloni yang hidup
atas media VRBD dianggap positif .

Ujian Pengesanan Staphylococcus aureus

1 gram @ ml sampel yang diterima akan di masukkan kedalam 9 ml kaldu Tryptic

soy (TSB). Campuran sampel dan kaldu dieramkan pada suhu 32.5  2.5 °C
selama 18-24 jam. Selepas pengeraman, subkultur ke atas media Mannitol Salt
dijalankan untuk mendapatkan koloni tunggal dan dieramkan selama 18-72 jam
pada suhu 32.5  2.5 °C. Koloni yang dijangkakan positif akan dijalankan ujian
pengesahan.

Ujian Pengesanan Pseudomonas aeruginosa

1 gram @ ml sampel yang diterima akan di masukkan kedalam 9 ml kaldu Tryptic

soy (TSB). Campuran sampel dan kaldu dieramkan pada suhu 32.5  2.5 °C
selama 18-24 jam. Selepas pengeraman, subkultur ke atas media Cetrimide
dijalankan untuk mendapatkan koloni tunggal dan dieramkan selama 18-72 jam
pada suhu 32.5  2.5 °C. Koloni yang dijangkakan positif akan dijalankan ujian
pengesahan.

Ujian Pengesanan Candida albicans

1 gram @ ml sampel ditimbang dan dimasukkan ke dalam 100 ml Kaldu

Sabouraud dextrose (SDB) . Campuran kemudiannya dieramkan pada suhu 32.5
 2.5 °C selama 3-5 hari. Selepas pengeraman, subkultur ke atas media
Sabouraud dextrose agar (SDA) dijalankan untuk mendapatkan koloni tunggal
dan dieramkan selama 24-48 jam pada suhu 32.5  2.5 °C. Koloni yang
dijangkakan positif akan dijalankan ujian pengesahan.

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KEPUTUSAN DAN PERBINCANGAN

Ujian Bebanan Mikrob

Setiap satu koloni yang dikira pada media agar akan direkodkan dalam unit CFU.
Nilai CFU/g@ml untuk setiap sampel yang diuji akan memberikan tahap
kandungan mikroorganisma di dalam sesuatu produk tersebut. Kriteria
penerimaan yang digariskan di dalam British pharmacopoeia bagi produk ubatan
tradisional untuk penggunaan oral juga dapat digunakan sebagai garis panduan
dalam menilai kualiti produk herba yang dihasilkan.

Ujian Mikroorganisma Spesifik

(a) (b) (c) (d) (e) (f)

Gambarajah 1: Spesies mikroorganisma di atas media agar a) E.coli, b)
Salmonella typhii, c) Bile tolerant bakteria Gram negatif,
d) S. aureus, e) P. aeruginosa, dan f) C. albicans

Ujian Pengesanan dan Semi-kuantitatif Escherichia coli

Koloni yang dijangkakan positif di atas media McA adalah kelihatan merah bata,
dengan bentuk yang tidak sekata dan warna mendakan merah disekeliling koloni
[Gambarajah 1(a)]. Kehadiran spesies Escherichia coli hanya akan disahkan
selepas keputusan ujian pengesahan adalah positif.

Ujian Pengesanan Salmonella spp.

Koloni yang dijangkakan positif di atas media XLD adalah kelihatan berwarna
merah dengan pusat - pusat hitam [Gambarajah 1(b)]. Kehadiran spesies
Salmonella spp. hanya akan disahkan selepas keputusan ujian pengesahan
adalah positif.

Ujian Semi-kuantitatif Bile tolerant bakteria Gram negatif

Koloni yang hidup atas media VRBD dikategorikan sebagai positif untuk Bile
tolerant bakteria Gram negatif [Gambarajah 1(c)]. Julat keberangkalian bilangan
(PN/g@ml) Bile tolerant bakteria Gram negatif bakteria untuk sampel adalah
merujuk kepada keputusan positif pada pencairan bersiri.

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Ujian Pengesanan Staphylococcus aureus

Koloni yang dijangkakan positif di atas media Mannitol Salt adalah kelihatan
berwarna kuning dengan zon kuning [Gambarajah 1(d)]. Kehadiran spesies
Staphylococcus aureus hanya akan disahkan selepas keputusan ujian
pengesahan adalah positif.

Ujian Pengesanan Pseudomonas aeruginosa

Koloni yang dijangkakan positif di atas media Cetrimide adalah kelihatan

berwarna fluorescein kuning hijau ke biru hijau [Gambarajah 1(e)]. Kehadiran
spesies Pseudomonas aeruginosa hanya akan disahkan selepas keputusan ujian
pengesahan adalah positif.

Ujian Pengesanan Candida albicans

Koloni yang dijangkakan positif di atas media SDA adalah kelihatan berwarna
putih dan berbentuk convex [Gambarajah 1(f)]. Kehadiran spesies Candida
albicans hanya akan disahkan selepas keputusan ujian pengesahan adalah
positif.

RUMUSAN

Kesimpulannya, Ujian Kawalan Kualiti Pencemaran Mikroorganisma yang

Berakreditasi ISO/IEC 17025 untuk produk dan bahan mentah herba di makmal
NPQC, FRIM dapat membantu dalam khidmat perundingan, nasihat dan
perkhidmatan teknikal ujian kawalan kualiti dalam membangunkan produk
herba yang lebih berkualiti bermula dari peringkat pemprosesan bahan mentah
sehingga ke produk siap untuk industri herba tempatan.

RUJUKAN

British Pharmacopoiea. (2016). Microbial Enumeration Tests. British

Pharmacopoiea , Volume V. British Pharmacopoiea Commission.
London. A491-A495
British Pharmacopoiea. (2016). Test for Specified Micro-Organism. British
Pharmacopoiea , Volume V. British Pharmacopoiea Commission.
London. A486-A489

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VERIFICATION OF SPECIFIED MICRO-ORGANISMS TEST METHOD FOR
CAPSULE MATRICE

Norulaiman, Y.1, Ong, B.K.1, Nurhazwani, M.H.1, Nor Hayati, A.1, Amira Rina
Nurdiana, M.S.1, Adib Zubaidi, R.2 & Nor Azah, M.A. 1

1
Herbal Product Development Programme, Natural Products Division, 2Herbal
Technology Centre, Innovation and Commercialisation Division, Forest Research
Institute Malaysia (FRIM), 52109 Kepong, Selangor.

Tel: 03-62797783 Fax: 03-62729805 E-mail: norulaiman@frim.gov.my

INTRODUCTION

Nowadays, various types of herbal product are produce and these products
consist of different type of matrices such as capsule, tablet, liquid, powder, oil,
cream, and ointment. Due to matrices effect in test result, verification of test
method has to be performed to estimate the potential influence of matrices on
the test. Verification of test method is essential requirements for accreditation
of ISO/IEC 17025. In ISO/IEC17025: 2005 clause 5.4.2 did mention about
selection of test method by the laboratory; Methods published in international,
regional or national standards shall preferably be used. The laboratory shall
confirm that it can properly operate standard methods before introducing the
tests. If the standard method changes, the confirmation shall be repeated
(Malaysian Standard, 2005). Confirmation or verification in microbiology test
method involves several parameters such as Limit of detection (LOD),
repeatability, reproducibility, accuracy, sensitivity and selectivity. These
parameters were evaluated using different types of matrices to verify the test
method (NATA 2012). In herbal industries, herbal products registration requires
quality control testing. One of the quality control tests is a test for specified
micro-organism. The current specified micro-organisms test methods used are
based on British Pharmacopoeia standard method (BP2016 Appendix XVI).The
above mention test will determine for a) the absence or limited occurrence of
five selected microbial species; namely Escherichia coli, Salmonella spp.,
Staphylococcus aureus, Pseudomonas aeruginosa and Candida albicans, b) semi-
quantitative bile tolerant gram negative bacteria.

MATERIALS AND METHODS

For the verification parameters: limit of detection (LOD), selectivity and

sensitivity were evaluated using capsule matrices. Herbal capsule used in this
verification were formulated and produced in FRIM Herbal Technology Centre
(HTC). Specified micro-organism species used are traceable to ATCC culture.

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Standard Inoculum of Target Micro-organism

Standard inoculum of (E. coli, Salmonella spp., S. aureus, P. aeruginosa and C.

albicans) were prepared in normal saline (0.58% Sodium chloride (NaCl) from
overnight pure culture and standardised at absorbance of 0.5A in 600 nm
wavelength using spectrophotometer. Serial dilutions up to 10-10 of inoculum
were plate in Trypticase Soy Agar (TSA) for E. coli, Salmonella spp., S. aureus,
and P. aeruginosa while for C. albicans were plated in Sabouraud dextrose Agar
(SDA) and incubated at 32.5°C. Colony forming Unit (CFU) of serial dilution was
counted and recorded for each micro-organisms species.

Limit of Detection

For the LOD determination of the test for absence of specified micro-organism
(E. coli, Salmonella spp., S. aureus, P. aeruginosa and C. albicans), sterile
capsules were spiked with less than 10 CFU of the target micro-organism which
is 1, 4, 3, 9 & 5 CFU of E. coli, Salmonella spp., S. aureus, P. aeruginosa and C.
albicans respectively in six replicates. The spiked capsules were then being
subjected to standard test method for each species (British Pharmacopoeia
2016).

Selectivity

In the selectivity determination, low counts of target micro-organisms and high

counts of non-target micro-organisms with 102 and 104 CFU respectively were
spiked together into sterile capsules. The spiked capsules then proceed with
standard test method for each species in 6 replicates (British Pharmacopoeia
2016).

Sensitivity

For the sensitivity determination in semi-quantitative test; bile tolerant gram

negative bacteria where E. coli were used as target micro-organism. <10, 10,
102, 103, and 104 CFU of E. coli was spike into sterile capsule in six replicates. The
spiked capsules then proceeded with standard test method (British
Pharmacopoeia 2016). The sensitivity determination for test for absent of E. coli,
Salmonella spp., S. aureus, P. aeruginosa and C. albicans; sterile capsule in 6
replicates were spiked with not more than 100 CFU of target micro-organisms.
The spiked capsules were then being subjected to standard test method for
each species (British Pharmacopoeia 2016).

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RESULTS AND DISCUSSION

Limit of Detection

Verification on LOD of test for absent of specified micro-organism (E. coli,

Salmonella spp., S. aureus, P. aeruginosa and C. albicans) showed spiked herbal
capsule with below 10 CFU of target micro-organism were able to detect the
specified micro-organism (Table 1). The species identification of target micro-
organism were confirmed with >90% probability.

Table1. Verification method performance of test for absence of specified

micro-organism.
Species LOD Selectivity Sensitivity
Bile tolerant gram negative bacteria Positive NA 100%
Escherichia coli Positive 100% 100%
Salmonella paratyphi Positive 100% 100%
Staphylococcus aureus Positive 100% 100%
Pseudomonas aeruginosa Positive 100% 100%
Candida albicans Positive 100% 100%
NA: not applicable; LOD: Limit of detection

Selectivity

In the selectivity determination, there is zero false positive and false negative
result even though high counts of non-target micro-organism were spiked
together into the capsule matrices. The selectivity rate of the test for absent of
method specified micro-organism (E. coli, Salmonella spp., S. aureus, P.
aeruginosa and C. albicans) is 100% for capsule matrices. It showed that the
tests are highly specific and selective to the target micro-organism. In test for
absent of E. coli and S. aureus, non-target micro-organism spiked was S. aureus
and E. coli vice versa. Bile salts and crystal violet in MacConkey media were
responsible to inhibit the growth of S. aureus. The colony of S. aureus on
mannitol salt agar appeared yellow in colour and surrounded with yellow zone
due to degradation of mannitol into acid. In test for absent of Salmonella spp.
and P. aeruginosa, the species was spiked vice versa into capsule matrices as
non-target micro-organism. Spiked of P. aeruginosa was inhibited in RVS broth
and subcultures of spiked sample on Xylose lysine deoxycholate (XLD) agar were
only revived Salmonella paratyphi. Production of hydrogen sulphite of
Salmonella paratyphi on XLD agar as the colonies appeared in red translucent
with black centre. While P. aeruginosa on cetrimide agar were differentiated
from Salmonella spp with big, raised green colonies. Lastly for test for absent of
C. albicans, E. coli was spiked as non-target micro-organism. Result showed that
C. albicans grew dominantly on SDA agar with white convex colonies.

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Sensitivity

In sensitivity of semi-quantitative test for bile tolerant gram negative, showed

present of E. coli on Violet Red Bile Dextrose (VRBD) agar for all spiked capsule
of 10, 102, 103, and 104 CFU. The sensitivity of bile tolerant gram negative
bacteria semi-quantitative test was verified for capsule matrices. Test for
absence of specified micro-organism (E. coli, Salmonella spp., S. aureus, P.
aeruginosa and C. albicans) were 100 % sensitive in matrices of hard gel capsule.
All replicates of spiked capsule were able to detect the present of target micro-
organism. It shows the test methods were sensitive to the target micro-
organism spiked in capsule matrices.

CONCLUSION

Based on these method verification results obtained for the capsules matric, the
above specified micro-organisms test methods are suitable and ‘fit to be used’
for capsule products in NPQC laboratory.

ACKNOWLEDGEMENTS

Author would like to thank all NPQC laboratory and Herbal Product
Development Programme members for their technical assistance.

REFERENCES

Department of Standards Malaysia (2005). General Requirements for the

Competence of Testing and Calibration Laboratories. Malaysian
Standard MS ISO/IEC 17025:2005. Malaysian Standard Committee,
Selangor. 13 pp.
NATA (2012). Guidelines for the validation and verification of quantitative and
qualitative test methods. NATA Technical Note 17-June 2012 National
Association of Testing Authorities, Australia.
British Pharmacopoeia (2016). Test for Specified Micro-organisms (S. aureus, P.
aeruginosa & C. albicans). British Pharmacopoeia, Volume V. Appendix
XVI B: Microbiological Examination of Non-sterile Products. British
Pharmacopoeia Commission, London. A486-A489.
British Pharmacopoeia (2016). Test for Specified Micro-organisms (E.coli,
Salmonella spp & Bile tolerant gram negative bacteria). British
Pharmacopoeia, Volume V. Appendix XVI F: Microbiological Examination
of Herbal Medicinal Products for Oral Use. British Pharmacopoeia
Commission, London. A505-A507.

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VALIDATION OF HEAVY METAL ANALYSIS (PB, CD, HG, AS) FOR HARD
GEL CAPSULE USING MICROWAVE DIGESTER AND ATOMIC ABSORPTION
SPECTROMETRY

Nurhazwani, M.H.1, Nor Hayati, A.1, Adib Zubaidi, R.2, Ong, B.K.1, Norulaiman,
Y.1, Amira Rina Nurdiana, M.S.1 & Nor Azah, M.A.1

1
Natural Products Division, 2Herbal Technology Centre, Innovation and
Commercialization Division, Forest Research Institute Malaysia (FRIM), 52109
Kepong, Selangor

Tel : 03-62797785 Fax: 03-62729805 Email: nurhazwani@frim.gov.my

INTRODUCTION

Validation applies to a defined protocol, for the determination of a specified

analyte and range of concentrations in a particular type of test material, used
for a specified purpose. In general, validation should check that the method
performs adequately for the purpose throughout the range of analyte
concentrations and test materials to which it is applied (Thompson et.al 2002).
Method validation is an essential component that can signify the reliability of
analytical methods in all areas of analysis. It is appropriate in ensuring the
laboratory capability and demonstrates it can repeat the method performance
by using the chosen parameters such as specificity, linearity, precision
(repeatability and intermediate precision), accuracy, recovery, limit of detection
and limit of quantification. In addition, it can certify the laboratory is compliance
with regulation and product quality, safety and consistency is guaranteed.

MATERIALS AND METHODS

Instrumentation

An Atomic Absorption Spectrometry (AAS) Model Analyst 600 by Perkin Elmer

was used to determine the concentrations of heavy metals. THGA graphite
(transversely-heated graphite) with pyrolytic coating and L’vov platform as
atomizer was used to measure lead, cadmium and arsenic metals, while Flow
Injection for Atomic Spectroscopy System (FIAS 100) was used to determine
mercury metals. This AAS system is equipped with hollow cathode lamp (HCL)
for cadmium and electrode less discharge lamp (EDL) for lead, mercury and
arsenic as the source of the energy. 99.999% of purified argon was used as a
protective gas. For the digestion of the samples, a Multiwave 3000 Microwave
Oven 50 Hz by AntonPaar was used.

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Reagents and Samples

Standard solutions of Pb, Cd, Hg, As (traceable to SRM from NIST 1000 mg/l) was
used as stock solutions with appropriate dilution. Hydrogen peroxide 30% and
nitric acid 65% were added into samples to undergo microwave digestion
process. Atomic absorption modifier solution (Mg), atomic absorption modifier
solution (Pd) and matrix modifier 10% NH4H2PO4 as modifier while hydrochloric
acid (fuming 37%) as carrier in FIAS analysis. Ultra pure (Milli-Q) water and blank
matrix sample; hard gel encapsulation with vegetable capsule that contains
maltodextrin as excipient was used throughout the experiments.

Sample Preparation and Analysis Procedure

Based on BS EN 14084:2003, about 0.5 g of blank matrix sample (hard gel

capsule) was accurately weighed into PTFE digestion vessel. 5 mL of
concentrated HNO3 and 2 mL of concentrated H2O2 were added to the vessel
and then it was spike with known amount and concentration of standard
solutions Pb, Cd, Hg, and As. Decomposition of the samples was carried out in a
microwave digestion system. The solution then was transferred into a
volumetric flask and made up to 50 mL with ultra-pure (Milli-Q) water. The
calibration curve was prepared in the range of 0-5 µg/l for Cd, 0-30 µg/l for Hg,
0-100 µg/l for Pb and As elements. All calibration curves showed good linear
regression (r2 > 0.995) within test ranges.

RESULTS AND DISCUSSION

Specificity

Specificity is the ability to assess unequivocally the analyte in the presence of

components which may be expected to be present. Typically these might
include impurities, degradants, matrix, etc. (ICH Harmonised Tripartite Guideline
2005). Blank matrix sample that was spike simultaneously with known amount
and concentration of Pb, Cd, Hg, As elements shows that method and
instrument used is able to select and analyze the concentration of heavy metals
even in the presence of other elements and analytes. The obtained mean
values; 61.35, 2.99, 21.07, 96.73 µg/l were compared with the actual value;
60.00, 3.00, 20.00, 100.00 µg/l for Pb, Cd, Hg, As, respectively. It showed a
difference of less than 10% from the actual value.

Linearity

The linearity of an analytical procedure is its ability (within a given range) to

obtain test results which are directly proportional to the concentration

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(amount) of analyte in the sample. (ICH Harmonized Tripartite Guideline 2005).
Linearity was determined by calculating the regression plots by the least squares
method and expressed as the correlation coefficient (r2) (S.Gupta et.al 2010).
The regression equations and correlation coefficients (r2) of Pb was y = 1.03985X
+ 4.1403 (r2 = 0.9999), for Cd y = 0.9776X + 0.03847(r2 = 0.9955), for Hg y =
0.9533X + 0.3464 (r2 = 0.9987) and for As it was y = 0.9938X + 0.5263 (r2 =
0.9924).

Precision (Repeatability & Intermediate precision)

Repeatability expresses the precision under the same operating conditions over
a short interval of time. Repeatability is also termed intra-assay precision while
intermediate precision expresses within-laboratories variations: different days,
different analysts, different equipment, etc. (ICH Harmonized Tripartite
Guideline 2005). The results were expressed through SD and RSD%, calculated
on the concentration for each metal for each validation day. The intra-day and
inter-day AAS analysis of Pb, Cd, Hg and As showed good precision. The
precision of the analysis, expressed in relative standard deviations (RSD), ranged
between 0.84% and 8.23% for intra-day analysis (n = 10) and 3.72-7.65% for
inter-day analysis (n = 2).

Accuracy

The accuracy of an analytical procedure expresses the closeness of agreement

between the value which is accepted either as a conventional true value or an
accepted reference value and the value found. (ICH Harmonized Tripartite
Guideline 2005). The obtained mean values; 92.52, 4.59, 27.86, 101.96 µg/l
were compared with the real value; 100.00, 5.00, 30.00, 100.00 µg/l for Pb, Cd,
Hg, As, respectively, were in the tolerance of 15% from the real value.

Recovery

The fraction of analyte added to a test sample (fortified or spiked sample) prior
to analysis, the unfortified and fortified samples, percentage recovery (%R) is
calculated as follows:

%R = [(CF-CU)/CA] x 100

Where CF is the concentration of analyte measured in the fortified sample; CU is

the concentration of analyte measured in the unfortified sample; CA is the
concentration of analyte added (measured value, not determined by method) in
fortified sample (EURACHEM Guide 1998). The total amount of each compound
was calculated from the corresponding calibration curve and the recovery of

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each compound was obtained. It ranged from 85.61% to 116.83% for Pb, Cd, Hg
and As in all the cases. The percentage of recovery value is in the range of 80-
120.

Limit of Detection

The detection limit of an individual analytical procedure is the lowest amount of

analyte in a sample which can be detected but not necessarily quantitated as an
exact value. (ICH Harmonised Tripartite Guideline 2005). The threshold
concentration at which the test becomes unreliable were determined and
showed positive/negative results; 10/0 for Pb, Cd, Hg and As in all the cases.

Limit of Quantification

The quantitation limit of an individual analytical procedure is the lowest amount

of analyte in a sample which can be quantitatively determined with suitable
precision and accuracy. The quantitation limit is a parameter of quantitative
assays for low levels of compounds in sample matrices, and is used particularly
for the determination of impurities and/or degradation products. (ICH
Harmonized Tripartite Guideline 2005). The limits of quantification for Pb, Cd,
Hg and As was 20.00, 1.00, 10.00 and 20.00 µg/l, compared with the mean
values 23.28, 0.86, 10.07, 19.71 µg/l, respectively. From the results, it shows
that the coefficient of variation, CV less than 20% and quantitative value within
the acceptance criteria in tolerance of 20% from target concentration.

CONCLUSION

Our method validation results indicated the established heavy metal analyses
testing for capsule matrix meets the acceptance criteria and therefore is capable
of giving accurate and reliable readings. We can conclude that the above test
method is ‘fit for intended use’ for capsule products in NPQC laboratory.

REFERENCES

Thompson, M., Stephen L., Ellison, R., & Wood, R. (2002). Harmonized
Guidelines for Single-Laboratory Validation of Methods of Analysis.
(IUPAC Technical Report). International Union of Pure and Applied
Chemistry: 74(5):835–855.
BS EN 14084:2003, (2003). Foodstuffs – Determination of Trace Elements –
Determination of Lead, Cadmium, Zinc, Copper and Iron by Atomic
Absorption Spectrometry (AAS) After Microwave Digestion.
ICH Harmonised Tripartite Guideline (2005). Validation of Analytical Procedures:
Text and Methodology Q2 (R1).

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Gupta, S., Pandotra, P., Gupta, A.P., Dhar, J.K., Sharma, G., Ram, G., Husain, M.K.
& Bedi, Y.S. (2010). Volatile (As and Hg) and Non-volatile (Pb and Cd) Toxic
Heavy Metals Analysis In Rhizome of Zingiber officinale Collected From
Different Locations of North Western Himalayas by Atomic Absorption
Spectroscopy. Journal of Food and Chemical Toxicology 2966–2971.
EURACHEM Guide (1998). The Fitness for Purpose of Analytical Methods: A
Laboratory Guide to Method Validation and Related Topics.

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OPTIMISATION OF SUPERCRITICAL FLUID EXTRACTION CONDITION OF
CURCUMA DOMESTICA (KUNYIT) RHIZOME USING RESPONSE SURFACE
METHODOLOGY.

Mohd Shafik Yuzman, T., Saidatul Husni, S, Hada Masayu, I., Nor Azah, M.A.,
Mohd Asyraf, O., Nurul Nabilah, M.H. & Mohammad Faridz, Z.P.

Natural Product Division, Forest Research Institute Malaysia, 52109 Kepong,

Selangor, Forest Research Institute Malaysia, 52109 Kepong, Selangor

Tel: 03-62797349 Fax: 03-62729805 Email: saidatul@frim.gov.my

INTRODUCTION

Curcuma domestica or kunyit is a rhizomatous perennial herb of the

Zingiberaceae family. Kunyit is traditionally used to treat liver and gall bladder
problems, stop bleeding, relief chest congestion and menstrual problem
(Awasthi and Dixit 2009). C. domestica is also popular as spice and colourings in
cookings. C. domestica oil have unique antioxidant, antitumorigenic,
anticarcinogenic, antimutagenic, antiarthritic and antimicrobial activities (Naz et
al. 2010). Ar-turmerone was reported as the major compound in C. domestica
oil by 41.4% and 59.7% (Naz et al. 2010). In recent study, ar-tumerone also
induces neural stem cell proliferation and promoted neurogenesis
(Hucklenbroich et al. 2014). Supercritical fluid extraction (SFE) has been proven
as better alternative technique for extraction of oils compare to other
conventional method due to cleaner extract, non-toxic by-products and shorter
duration time of extraction (Herrero et al. 2006; Huang et al. 2012; Reverchon et
al. 2006). Percentage yield and total chemical composition of extract can be
affected by the parameter of the machine. Therefore, different operating
parameter can yield different extract output (Siti Hafsah et al. 2016).

MATERIALS AND METHODS

Plant Material

Dried ground rhizome of C. domestica were purchase from Ethnoresources Sdn

Bhd. The moisture content was recorded at 5.8% using halogen analyser.

Supercritical carbon dioxide (SC-CO2) extraction

Nine extractions were carried out at pressures of 100-300 bar and temperature
of 40-60°C using constant flow of 35g/min of carbon dioxide (CO2). 150 g of
sample was loaded into the extractor for each extraction. Sample was soaked in

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SC-CO2 for 30 minutes and was extracted for 120 minutes. The extract was
recovered at the end of the experiment from the collection container due to the
difference of pressure.

Chemical analysis

Quantitative analysis of the oil was carried out using Shimadzu GC 2010 and
Agilent Technologies GCMS 7890A/5975C Series MSD. The chemical
constituents were identified by mass spectral library (HPCH2205.L;
Wiley7Nist05.L; NIST05a.L). The results of the peak areas were expressed as
peak area counts.

Response surface methodology (RSM)

The study is to investigate the effect of two independent variables (pressure and
temperature) towards on the oil yield and ar-tumerone concentration. The
relationship between independent variables and both responses mention above
was examined by employed the Response Surface Response (RSM). 13
experiments included 4 factorial points, 4 central points and 5 central points
were assigned based on the Central Composite Design (CCD). The contrast
coefficients among the obtained experimental data were well tuned with the
suggested mathematical model by using Expert Design version 7.0 software and
were statistically analyzed using Analysis of Variance (Manickam & Tan 2012).
The mathematical models for each responses were predicted by using multiple
regression model and were fitted into second order polynomial equation 1 as
shown below.
𝑌 = 𝛽𝑜 + ∑ 𝛽𝑖 𝑋𝑖 + ∑ 𝛽𝑖𝑖 𝑋𝑖 2 + ∑ ∑ 𝛽𝑖𝑗 𝑋𝑖 𝑋𝑗 Eq (1)
Where Y is the response variable, 𝛽𝑜 is a constant, and 𝛽𝑖 , 𝛽𝑖𝑖 ,
and 𝛽𝑖𝑗 represents the linear, quadratic and interactive coefficients respectively.
𝑋𝑖 and 𝑋𝑗 are the independent variables. The coefficient of determination, R2
were determined and the F-test of significance of the equation parameters for
each response variable was analyzed. Previous study found only the factors with
significance higher than or equal to 5% (p ≤ 0.05) were considered. Lastly, an
optimization of both responses were performed by kept all independent
variables within range while the responses were maximized.

RESULTS AND DISCUSSION

Supercritical carbon dioxide (SC-CO2) extraction

Highest oil yield was obtained from extraction condition 5 (200 bar and 50°C).
While the lowest yield was produced from extraction condition 7, (300 bar and
40°C). For the highest concentration of ar-tumerone (%area) was obtained from

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extraction condition 7. On other hand the lowest concentration of ar-tumerone
(%area) was produced from extraction condition 5.

Table 1. Yield (%) and ar- tumerone (% area) of C. domestica oils from 9
conditions
Condition Pressure Temperature Weight of Yield (%) ar-
(bar) (oC) extract (g) tumerone
(% area)
1 100 40 1.03 0.6843 55.96
2 100 50 0.81 0.5328 55.47
3 100 60 0.8 0.5226 41.87
4 200 40 0.8 0.5228 53.85
5 200 50 1.4 0.9276 41.75
6 200 60 1.27 0.8354 47.77
7 300 40 0.3 0.1986 57.73
8 300 50 1 0.6579 49.56
9 300 60 0.7 0.4635 50.04

Chemical composition of oils

A total of 18 compounds were identified from the oil of C. domestica extracted

using supercritical fluid extraction (SFE). From the condition stated in the table
below, condition 2 (pressure 100 bar and temperature 50°C) yield the highest
amount compounds while condition 5 (pressure 200 bar and temperature 50°C)
and condition 9 (pressure 300 bar and temperature 60°C) yield the lowest
number of compounds. All the compound were identified and confirmed using
mass spectrometer library and Kovat’s index.

Analysis of RSM model

The Response Surface Methodology (RSM) was employed to determine the best
combinations of variables for obtaining high extraction yield in supercritical CO2
process (Azmir et al. 2014). In this case, oil yield and ar-tumerone concentration
are the responses while pressure and temperature are two independent
variables. The relationship between two independent variables and response
has been analyzed by RSM so that the predicted of both responses can be
obtained. Table 3 showed the Central Composite Design (CCD) matrix of
experimental and predicted of oil yield and ar-tumerone concentration based on
the independent variables (pressure and temperature). A quadratic model is
suggested since CCD can fit a full quadratic model.

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 Table 2. Chemical composition of C. domestica oils
Condition
Constituents KI 1 2 3 4 5 6 7 8 9 Identification
Z-caryophyllene 1373 - 0.34 - - - - - 0.40 - MS, KI
ar-curcumene 1441 2.21 2.42 1.03 2.40 1.54 2.2 1.80 1.72 1.66 MS, KI
α-zingerberene 1453 - 0.94 - - 0.65 0.64 0.35 0.78 - MS, KI
β-bisabolene 1466 0.29 0.41 0.15 0.38 - - 0.28 0.32 - MS, KI
β-sesquiphellandrene 1482 - 2.52 0.72 2.34 1.56 2.06 1.87 1.91 1.69 MS, KI
Geranyl butyrate 1537 0.18 0.25 0.16 - - - - 0.11 - MS, KI
Cis - sesquisabinene 1544 1.12 1.01 1.26 0.88 - - 0.86 0.22 - MS, KI
hydrate
(E)-nerolidol 1554 0.31 0.34 0.35 - - - - 0.97 10.5 MS, KI
3
ar - turmerol 1567 0.49 0.64 0.71 - - - - 0.36 - MS, KI
cis-β-elemene 1576 - 0.34 0.41 - - - - 0.65 - MS, KI
ar-turmerone 1642 55.9 55.4 41.8 53.8 41.7 47.7 57.7 49.5 50.0 MS, KI
6 7 7 5 5 7 3 6 4
α-turmerone 1661 - 0.71 - - - - - - - MS, KI
6S,7R-bisabolone 1674 21.4 22.8 19.7 23.9 16.9 22.1 24.6 - - MS, KI
0 8 8 9 7 4 5
Germacrone 1681 0.17 0.21 0.31 - - 7.77 - 22.8 - MS, KI
1
1-bisabolene 1711 0.97 0.90 0.62 0.86 - - 0.89 0.97 0.80 MS, KI
Geranyl hexanoate 1722 0.60 0.28 - - - 0.45 - - - MS, KI
(E)-α-atlantone 1743 3.79 3.18 4.94 5.19 2.40 5.08 5.55 - 4.91 MS, KI
Furanodienone 1752 - 0.29 0.49 - - - - - MS, KI

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Table 3: Independent variables for extraction C. domestica with experimental
and predicted values of oil yield and ar-tumerone concentration

Factors Oil Yield % Ar-tumerone

concentration %
Run A= B= Experiment Predicted Experimental Predicted
Pressure Tempt
(Bar) (OC)
1 200.00 40.00 0.5228 0.6513 53.85 51.9447
2 200.00 50.00 0.9276 0.9101 41.75 43.1534
3 200.00 50.00 0.9276 0.9101 41.75 43.1534
4 300.00 40.00 0.1986 0.2006 57.73 56.8693
5 100.00 50.00 0.5328 0.7068 55.47 48.3347
6 300.00 60.00 0.4635 0.5525 50.04 50.7826
7 200.00 50.00 0.9276 0.9101 41.75 43.1534
8 200.00 50.00 0.9276 0.9101 41.75 43.1534
9 100.00 40.00 0.6843 0.5538 55.96 58.726
10 300.00 50.00 0.6579 0.5669 49.56 49.678
11 200.00 60.00 0.8354 0.7899 47.77 42.658
12 200.00 50.00 0.9276 0.9101 41.75 43.1534
13 100.00 60.00 0.5226 0.4791 41.87 46.2393

The Analysis of Variance (ANOVA) was performed so that the goodness of the fit
can be evaluate. The ANOVA of the model predict the values for oil yield and ar-
tumerone concentration calculated using regression model and compared with
experimental values obtained from the experiment. Figure 1 and 2 showed the
correlation between experimental and predicted values of oil yield and ar-
tumerone concentration. Both response variables were fitted into a second
order polynomial equation presented in Eq (2) and (3) respectively. Based on the
figure 1 and 2, the experimental responses are nearer to the predicted
responses as most of the point close to the straight line.
2 2
Oil yield (%) = 0.91- 0.07A + 0.069B + 0.11AB - 0.27A - 0.19B Eq (2)

Ar-tumerone conc. (%) = 43.15 +0 .67A - 4.64B + 1.60AB + 5.85A2 + 4.15B2 Eq (3)

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 Figure 1: Relationship between experimental and predicted values of oil yield
based on equation

Figure 2: Relationship between experimental and predicted values of ar-

tumerone concentration based on equation

According to Azmir et al. (2014), the coefficient of determination (R2) of the

model of 0.99 indicates a good agreement between the experimental and
predicted of both response variables. Meanwhile, Siti Hafsah et al.(2015), stated
that the R2 value for all response variables that were higher than 0.75 shows a
regression model explained the response well. The R2 value of oil yield and ar-
tumerone concentration were 0.8740 and 0.7565 respectively. Thus, it can be
conclude that the regression model explained the response well and a good fit.

Based on table 4, it showed that the quadratic term of pressure (A2) and
quadratic term of temperature (B2) effects were highly significant (p ≤ 0.05).
However, both linear pressure and temperature effects give value (p ≥ 0.05)
which is non-significant. The interaction between temperature and pressure was
non-significant (p ≥ 0.05) within the experimental ranges. The ANOVA regression
model for ar-tumerone concentration was shown in table 5. The linear and
square effects of pressure both gives a non significant effects. For the
temperature effects, both linear and quadratic term expressed a significant and

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non-significant respectively. The interaction between pressure and temperature
displayed a non-significant effects within the experimental ranges.

Table 4: The ANOVA for regression model and respective model term for oil
yield

Source Sum of df Mean F Prob < Remarks

Squares Square value F
Model 0.59 5 0.12 9.71 0.0047 Significant
A-Pressure 0.029 1 0.029 2.41 0.1647 Non significant
B-Temperature 0.029 1 0.029 2.36 0.1682 Non significant
AB 0.045 1 0.045 3.73 0.0947 Non Significant
A2 0.21 1 0.21 17.02 0.0044 Significant
B2 0.10 1 0.10 8.21 0.0242 Significant
Residual 0.085 7 0.012
Lack of Fit 0.085 3 0.028
Pure Error 0.000 4 0.000

Table 5: The ANOVA for regression model and respective model term for ar-
tumerone concentration

Source Sum of df Mean F Prob < Remarks

Squares Square value F
Model 368.33 5 73.67 4.35 0.0404 Significant
A-Pressure 2.71 1 2.71 0.16 0.7013 Non significant
B-Temperature 129.36 1 129.36 7.64 0.0280 Significant
AB 10.24 1 10.24 0.60 0.4623 Non significant
A2 94.61 1 94.61 5.59 0.0501 Non significant
B2 47.52 1 47.52 2.81 0.1379 Non significant
Residual 118.57 7 16.94
Lack of Fit 118.57 3 39.52
Pure Error 0.000 4 0.000

Figure 3 and 4 illustrates the interaction between independent variables and

response represented in three dimensional response surface. Based on the
figure 3, the oil yield is increasing as the pressure and temperature increasing.
However, it only increasing up to the 200 bar for all range temperature. Above
200 bar, there is fluctuation in oil yield percentage even though the pressure is
high. It is obviously shown that increasing pressure at constant 40oC did not
exhibit a good oil yield and thus, make it became least percentage of oil yield

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produced when comparing to 50 oC and 60 oC. Figure 4 indicated that the highest
ar-tumerone concentration was founded at low temperature which is 40 oC for
each point of pressure. The highest amount of concentration obtained were
55.96%, 53.85% and 57.73% for 100 bar, 200 bar and 300 bar respectively. In
contrast, increasing the temperature makes the ar-tumerone concentration
degraded down to 41.87 %.
Design-Expert® Software

Oil yield
Design points above predicted value
Design points below predicted value
0.9276
0.93
0.1986

X1 = A: Pressure 0.745
X2 = B: Temperature
0.56
Oil yield

0.375

0.19

60.00 300.00
55.00 250.00
50.00 200.00

B: Temperature45.00 150.00
A: Pressure
40.00 100.00

Figure 3: Response surface for oil yield percentage as a function of temperature

and pressure
Design-Expert® Software

ar-Tumerone concentration
Design points above predicted value
Design points below predicted value
57.73
59
41.75

X1 = A: Pressure 54.5
ar-Tumerone concentration

X2 = B: Temperature

50

45.5

41

60.00 300.00
55.00 250.00
50.00 200.00
45.00 150.00
B: Temperature A: Pressure
40.00 100.00

Figure 4: Response surface for oil yield percentage as a function of temperature

and pressure

Optimization using RSM-CCD

An optimization technique is vital in order to improve performance of the

systems and increase the yield of the processes. Optimization can be defined as

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the process of finding the most effective or favourable value or condition (Kelley
2010). In making optimal design decision, engineers are often combine
judgement, modelling, opinions and experience together. The decision is made
by considering other factors such as cost, energy consumption, power
consumption and more. In this case, the optimal design decision has been made
by modelling the experiment. The optimum pressure and temperature in
producing highest yield of oil and ar-tumerone concentration were 115 bar and
40 C respectively. While the prediction for oil yield percentage and ar-tumerone
concentration are 0.60 % and 56.97 %, respectively. Logically, these optimum
values are reasonable due to no high power needed to generate in CO2 pump,
and the used smallest heat exchanger that accomplishes the desired heat
transfer.

CONCLUSION

Based on optimisation by RSM, the optimum condition for extracting oil with
high ar-tumerone concentration from C. domestica rhizome is 115 bar and 40°C.
At this extraction condition, the oil yield and ar-tumerone concentration are
0.60 % and 56.97 %, respectively. The percentage of oil yield is significantly
influenced by pressure and temperature but ar-tumerone concentration is
significantly influenced by temperature.

ACKNOWLEDGEMENTS

The authors would like to thank all staff of PPH and Natural Products Division for
their advice and technical assistance in this study.

REFERENCES

Awasthi, P.K, & Dixit, S.C. (2009). Chemical Composition of Curcuma Longa
Leaves and Rhizome Oil from the Plains of Northern India. .J Young
Pharm. 1(4):312–316.
Azmir, J., Zaidul, I.S.M, Sharif, K.M., Uddin, M.S., Jahurul, M.H.A, Jinap, S., Hajeb,
P. & Mohamed, A. (2014). Supercritical Carbon Dioxide Extraction of
Highly Unsaturated Oil from Phaleria Macrocarpa Seed. Food research
International 65:394–400.
Herrero, M., Cifuentes, A. & Iban˜ez, E. 2006. Sub- and supercritical fluid
extraction of functional ingredients from different natural sources: Plants,
food-by-products, algae and microalgae: A review. Food Chemistry
98:136–148.
Huang, Z., Shi, X.H. & Jiang, W.J. (2012). Theoretical models for supercritical fluid
extraction. Journal of Chromatography 1250:2–26.

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Hucklenbroich, J., Klein, R., Neumaier, B., Graf, R., Fink, G.R., Schroeter &
Rueger, M. (2014). Aromatic-turmerone induces neural stem cell
proliferation in vitro and in vivo. Stem Cell Research and Therapy 5:100.
Kelley, T.R. (2010). Optimization, an important stage of Engineering Design. The
Technology Teacher 69(5):18–23.
Manickam, S. & Tan, K.W. (2012). Response Surface Methodology, an Effective
Strategy in the Optimization of the generation of Curcumin-loaded
micelles. Asia Pacific Journal of Chemical Engineering.
Paulicci, V.P., Couto, R.O., Teixeira, C.C.C. & Freitas, L.A.P. (2012). Optimization
of the extraction of curcumin from Curcuma Longa rhizomes. Brazillian
Journal of Pharmacognosy 23(1):94–100.
Reverchon, E. & Iolanda, D.M. (2006). Supercritical fluid extraction fractionation
of natural matter. The Jour of Supercritical Fluids 38:146–166.
Shagufta Naz, Saiqa Ilyas, Zahida Parveen and Sumera Javed. (2010). Chemical
Analysis of Essential Oils from Turmeric (Curcuma longa) Rhizome
Through GC-MS. Asian Journal of Chemistry 22(4):3153–3158.
Siti, H.M.S., Zaibunnisa, A.H., Khudzir, I., Nooraain, H. & Wan, I.W.I. (2015).
Optimization of Curcuma Loga L. Rhizome supercritical carbon dioxide
extraction (SC-CO2) by Response Surface Methodology (RSM). Jurnal
Teknologi (Sciences and Engineering) 78(6):87–92.

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SCREENING OF SELECTED CINNAMOMUM SPP. AS REDUCING AGENT
FOR NANO SILVER SYNTHESIS

Noor Rasyila, M.N.1, Nor Azah, M.A.1, Saidatul Husni, S.1, Vimal Angela, T.2,
Mohd. Faridz, Z.F.1.

1
Herbal Products Development Programme, Natural Products Division, Forest
Research Institute Malaysia, Kepong, Selangor. 2Universiti Malaysia Terengganu,
21230 Kuala Terengganu, Terengganu

Tel: 03-62797782 Fax: 03-62729805 Email: rasyila@frim.gov.my

INTRODUCTION

Nanoparticles green or eco-friendly synthesis has become an important branch

of nanotechnology and the commercial demand for nanoparticles are increasing
due to their wide applications. According to Shakeel et al. (2016), various
literatures had reported on the biological synthesis of silver nanoparticles using
microorganisms including bacteria, fungi and plants. It was due to their
antioxidant or reducing properties responsible for the reduction of metal
compounds in their respective nanoparticles. It was relatively simple, cost-
effective, nontoxic and environmental friendly. Silver nanoparticles produced by
plants are more stable and faster in comparison with those produced by other
organisms. However, better experimental procedures are needed for synthesis
of well-characterized nanoparticles (Hassan et al. 2012). Silver nanoparticles are
known to be a universal antimicrobial substances in biology and medicine, which
has been used in prevention and to treat various diseases and infections (Yuet
et al. 2012). Oei et al. (2012), also reported that silver nanoparticles possess
anti-fungal, anti-inflammatory, anti-viral, anti-angiogenesis and anti-platelet
activity (Kim et al. 2009; Nadwomy et al. 2010; Lara et al. 2010; Kalishwaralal et
al. 2009; Shirvastava et al. 2009).

Most of the traditional herbal drugs and herbal extracts despite of their
impressive in-vitro findings demonstrates less or negligible in-vivo activity due
to their poor lipid solubility or improper molecular size, resulting in poor
absorption and hence poor bioavailability. Poor bioavailability increases system
clearance requiring repeated administration or higher dose, which makes the
drug as a poor candidate for therapeutic use. One way to overcome poor
bioavailability is to improve the characteristic of micro-structured bioactive
compound in the herbal drugs such as water solubility, bioavailability, increasing
absorbency to organism as well as antioxidant properties; by enhancing the
reduction particle size of herbal drugs which facilitate the active ingredients to
disperse and dissolve stably and homogeneously. It is also reported that

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antioxidant components with a wide variety of free radical scavenging
compounds have low absorption activities in the body system and in food due to
their larger particle size, complex chemical structure and poor water solubility of
the constituent compounds. Nano particles measure between one and about
100 nanometres with a very high surface-to-volume ratio, which essentially
allows them to be more active than larger particles. Nano herbal drugs usually
has a few million times larger surface area than traditional herbal drugs. It
enhances bioavailability by improving the component solubility and
permeability, increasing absorbency of the organism, reducing herb doses and
achieving steady-state therapeutic levels of drugs over an extended period (Ace
Nano 2012).

The plants or plants extract, which act as reducing and capping agents
for nanoparticles synthesis, are more advantageous over other biological
processes. Huang et al. 2007, reported that sun-dried biomass of Cinnamomum
camphora leaf extract when treated with aqueous silver or gold precursors at
ambient temperature, produced silver and gold nanoparticles in the range of
55–80 nm. The polyol component and the water-soluble heterocyclic
components present in the C. camphora were mainly responsible for the
reduction and stabilization of silver or chloroaurate in nanoparticles. The
reduction of silver ions and stabilization of the AgNPs were thought to occur
through the involvement of proteins. It is the best platform for synthesis of
nanoparticles; being free from toxic chemicals as well as providing natural
capping agents for the stabilization of silver nanoparticles. Cinnamomum spp.
plant parts are important ingredient in Traditional Chinese Medicine; as
household remedy against various human ailments and also added to improve
taste and aroma of food (Singh et al. 2007). According to Natural Standard
Monograph (2008), extracts of Cinnamomum spp., as well as the major
components cinnamaldehyde and eugenol, have antibacterial and antimicrobial
properties which demonstrated activity against Campylobacter jejuni,
Escherichia coli, Listeria monocytogenes and Salmonella enterica in vitro. It also
reduces discomfort from heartburn as well as carminative that relieves bloating
and gas (Joe et al. 2004). Some of the species from the genus of Cinnamomum
are C. camphora, C. cassia, C. burmannii, C. iners, C. javanicum, C. sintoc, C.
tamala and C. zeylanicum. The objective of this study is to screen the potential
Cinnamomum spp. water extract that is capable to synthesis silver nitrate to
silver nanoparticles as reducing agent as well as capping agent and determine
the average size for the silver nanoparticles.

MATERIALS AND METHODS

Plant Material

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Samples of Cinnamomum spp. (C. zeylanicum, C. iners, C. sintoc and C.
mollisimum) fresh leaves were collected from the Taman Ethnobotani, Forest
Research Institute Malaysia. The samples were washed, cut and oven dried at
60°C for two days. Dried samples were ground and stored double layered pack.

Water extraction

Water extraction method by Veerasamy R. et al. (2011), with slight modification

on the weight of sample and technique of reflux was used. Each dried ground
Cinnamomum spp. leaves were weighed for 10g and 100ml of distilled water
were added into the boiling flask. The flask was then heated for 25 minute using
reflux technique. Then, the boiled sample was filter through Filter Paper
Whatsman No.1 and aliquot was kept at 4°C for further synthesis of silver
nanoparticles.

Synthesis of Silver Nanoparticles Using Water Extract

The synthesis of silver nanoparticles was prepared using aqueous solution (1

mM) of silver nitrate (AgNO3). 5ml water extract of Cinnamomum spp. were
added into 95ml of aqueous solution of 1 mM silver nitrate for reduction into
Ag+ ions in 250-ml Erlenmeyer flask and heated in water bath with different
temperatures (75°C and 90°C) for 60 min respectively. The reductions of silver
nitrate to silver ions were confirmed by the changing of the solution colour. The
fully reduced solution was centrifuged at 5000 rpm for 30 min. The supernatant
liquids were discarded and the pellet redispersed in deionized water.
Centrifugation was repeated twice to trice to wash off any absorb substances on
the surface of the silver nanoparticles (Veerasamy et al. 2011).

Control Samples

Control samples were prepared to ensure the formation of nanoparticles by the

biosynthesis of the extracts with the aqueous silver nitrate. The control samples
were untreated samples of Cinnamomum spp. water extract, 1mM of aqueous
silver nitrate and the distilled water.

Characterization of Silver Nanoparticles

UV-Visible Spectral Analysis

The UV-Vis spectroscopy analysis was used to examine the presence of silver
nanoparticles in the aqueous solution. UV-Vis absorption spectrophotometer
with resolution of 1 nm between 200 and 600 nm possessing a scanning speed
of 240 nm/min. The AgNPs solution was put in quartz cuvette for the analysis.

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Before analyzing, the silver nanoparticles solution was sonicated and vortex in
order to produce uniform dispersion of nanoparticles.

Zetasizer Analyzer

The size of the nanoparticles was determined by using Zetasizer Analyzer. The
Zetasizer Analyzer provided the average diameter of the nanoparticles in
nanometer (nm).

RESULTS AND DISCUSSION

Synthesis of silver nanoparticles using water extract

Silver nitrate was synthesized into silver nanoparticles using water extract of C.
zeylanicum, C. iners, C. sintoc and C. mollisimum with temperature variation of
75°C and 90°C. The formation of silver nanoparticles was monitored via color
changes and UV-Vis spectroscopy. Changes in colour of the reaction mixtures
from yellowish to slightly brownish grey respectively (Figure 1), suggesting the
rapid formation of silver nanoparticles which indicates the biosynthesis of silver
nanoparticles. The brown colour in the solution is due to the Surface Plasmon
Resonance Phenomenon (Sasikala et al. 2012). The same colour change of silver
nanoparticles from colourless and yellow to dark brown or deep brown in the
previous study showed nanoparticles formed. (Basavegowda et al. 2014; Khalil
et al. 2013).

i) ii)
Figure 1: Biosynthesis of silver nanoparticle shows the colour changes i) before
and ii) after heating

UV-Vis study of silver nanoparticles

Besides the colour changes, the synthesis of silver nanoparticles was confirmed
by measuring the UV-Vis spectrum of the reaction mixtures. The control samples
of both 1mM aqueous silver nitrate and Cinnamomum spp. water extract were
analyzed. As shown in Figure 2 and 3 there were no peaks appeared at both
spectra in range of 400 nm to 500 nm wavelength.

After the synthesis process, the maximum peak of wavelengths for each
Cinnamomum spp. samples are reported for both temperatures as in Table 1.

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Figure 4-5 showed the UV-Vis spectrum of colloidal solutions of silver
nanoparticles synthesized from leaf extract of Cinnamomum spp. heated at 75°C
and 90°C.

0
0 100 200 300 400 500
-2

AgNO3 75 °C AgNO3 90 °C

Figure 2: Control samples of AgNO3 heated at 75°C and 90°C

0
0 200 400 600 800
-2

C. iners C. sintoc
C. zeylanicum C. mollisimum

Figure 3 : Control samples of Cinnamomum spp. extracts

Therefore, the peaks from the analysis were actually indicating the formation of
silver nanoparticles after reaction between aqueous silver nitrate and water
extract.

Table 1: The UV-VIS spectrum silver nanoparticles heated at 75°C and 90°C for
60 minutes
Sample Maximum peak (nm) : Absorbance (A)
75°C 90°C
AgNO3 C. zeylanicum 422 : 0.978 432 : 0.850
AgNO3 C. iners 424 : 2.084 420 : 1.996
AgNO3 C. sintoc 423 :1.333 413 : 1.082
AgNO3 C. mollisimum 274 : 0.709 271 : 0.752

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Similar to many other cases, silver nanoparticles synthesized by using leaf
extracts of Eucalyptus hybrid, Acalypha indica, Nelumbo nucifera, Solanum
torvum, Helianthus annus and Cassia auriculata the absorbance peaks were
between 400 and 450 nm (Sasikala et al. 2012).

0
0 100 200 300 400 500 600
C. iners C. sintoc C. zeylanicum C. mollisimum

Figure 4: The UV-VIS spectrum silver nanoparticles heated at 75°C

3
2
1
0
0 100 200 300 400 500 600
-1
C. iners C. sintoc C. zeylanicum C. mollisimum

Figure 5: The UV-VIS spectrum silver nanoparticles heated at 90°C

Only silver nanoparticles synthesized from leaf extract of C. mollisimum showed

absorbance peaks at wavelength of 274 nm (75°C) and 271 nm (90°C) which are
considered active at relatively lower wavelength. This indicates that different
plant samples gives different results although from the same genus.

Particle size Analysis

The size of silver nanoparticles produced from the various extracts were
observed by using Zetasizer Analyser. The overall particle size of control AgNO3
and biosynthesis using Cinnamomum spp. are shown in Table 2. The method
formed particles with a disperse size range of 140 to 175 d.nm when heated at
90°C compared to silver nanoparticles heated at 75°C. C. sintoc displayed
highest size reduction (140.2 d.nm with smaller polydispersity index 0.230)
during synthesis process at 90°C. The higher the temperature reaction, the

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182
better the homogenous of Ag+ and the smaller the particles produced but on
the other hand, C. mollesimum created larger particle size( Khalil et al. 2013).
Figure 6 showed the size distribution graph on the consistency peak for C. sintoc
at 90°C.

Table 2: Diameter and polydispersity index of control AgNO3 and biosynthesis

using Cinnamomum spp. water extract at 75°C and 90°C
Sample Particle size (d.nm)
75°C PDI 90°C PDI
AgNO3 Control 356.3 0.446 297.7 0.627
AgNO3 C. zeylanicum 190.4 0.302 174.3 0.315
AgNO3 C. iners 250.0 0.424 168.6 0.420
AgNO3 C. sintoc 156.8 0.251 140.2 0.230
AgNO3 C. mollisimum 1149 0.683 1097 0.553

Figure 6: Size distribution of biosynthesis using C. sintoc water extract at 90°C

CONCLUSION

This study demonstrated that the C. zeylanicum, C. iners and C. sintoc are good
sources as reducing agent in synthesis of silver nanoparticles in the wavelength
ranging 410 – 430 nm with dispersible particles and uniform average size of 140
– 175 d.nm at temperature of 90°C. Different plant extracts produces different
results of silver nanoparticles. C. mollisimum was active at relatively lower
wavelength and can be studied further with different variables because it has
potential to synthesis the silver nanoparticles.

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http://acenano.aceenvironment.com/home Ace Nano (2012).

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Huang, J., Li, Q & Sun D. (2007). Biosynthesis of silver and gold nanoparticles by
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Khalil, M.M.H., Ismail, E.H., El-Baghdady, K.Z. & Mohamed, D. (2013). Green
synthesis of silver nanoparticles using olive leaf extract and its
antibacterial activity. Arabian Journal of Chemistry: 7:1131–1139.
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Lara, H., Ayala, N.N., Ixtepan, T.L. & Rodriguez, P.C. (2010). Mode of antiviral
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Nadworny, P., Wang, J., Tredget, E. & Burrell, R. (2010). Anti-inflammatory
activity of nanocrystalline silver-derived solutions in porcine contact
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Nanoparticles from Cochlospremum Religiosum and their Antibacterial
Efficacy. J. Pharm. Sci. & Res.: 4(6):1836–1839.
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(2009). Characterization of Antiplatelet Properties of Silver
Nanoparticles. ACS Nano: 3(6):1357–1364.
Singh, G., Maurya, S., DeLampasona, M.P. & Catalan C.A (2007). A comparison of
chemical, antioxidant & antimicrobial studies of cinnamon leaf & bark
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Veerasamy, R., Xin, T.Z., Gunasagaran, S., Wei Xiang, T.F., Chou Yang, E.F.,
Jeyakumar, N., & Dhanaraj, S.A. (2010). Biosynthesis of silver
nanoparticles using mangosteen leaf extract and evaluation of their
antimicrobial activities. Journal of Saudi Chemical Society: 15:113–120.
Yuet, Y.L., Buong, W.C., Mitsuaki, N. & Son, R. (2012). Synthesis of silver
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 MARKETING, TRADE,
BUSINESS POTENTIAL &
LEGISLATION

179
SELLING OUR STORY TO THE WORLD: BRANDING WITH WHAT WE HAVE
BEST

Mohamad Faisal, A.F

F.A. Herbs Sdn Bhd / Tanamera Sdn Bhd

Lot 3726, Jalan Tembusu,
Kg Melayu Subang,
40150 Shah Alam
Selangor

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COMMERCIALISATION OPPORTUNITY WITH BIOAPLHA

Faizzudin, S.

Bioalpha International Sdn Bhd

No 10, jalan P/9A, Seksyen 13,
43650 Bandar Baru Bangi,
Selangor

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KEMBARA PENYELIDIKAN, PEMBANGUNAN DAN PENGKOMERSILAN
RANGKAIAN PRODUK DISINFEKTAN DAN ANTISEPTIK MESRA ALAM.

Mastura, M.

Program Bioaktiviti, Bahagian Hasilan Semula Jadi, Institut Penyelidikan

Perhutanan Malaysia (FRIM), 52109, Kepong, Selangor.

Tel: 03-62797351 Fax: 03-62729805 E-mail: mastura@frim.gov.my

PENGENALAN

Penggunaan produk-produk penjagaan diri berantiseptik menjadi tren yang

melazimi masyarakat. Selain membersih produk-produk sedemikian
mengandungi bahan aktif mikrobiosid yang berfungsi untuk merencat mikrob
sekaligus memberikan persepsi `melindungi’ penggunanya daripada jangkitan
kuman. Namun, penggunaan meluas antibiotik seperti sikloheksamida dan
mikrobiosid triklosan / triklokarban dalam produk-produk penjagaan diri
berantiseptik boleh menggugat kesihatan pengguna selain memburukkan lagi
fenomena keresistanan mikrob terhadap agen kawalan. Penggunaan triklosan
iaitu sejenis bahan kimia sintetik mencetus kontroversi kerana kaitan
bioakumulasinya dengan gangguan hormon selain berpotensi menjadi pencetus
kanser (karsinogenik). Justeru inisiatif untuk menemukan agen-agen yang boleh
dibangunkan sebagai mikrobiosid yang berkesan dan selamat menjadi suatu
keperluan yang semakin mendesak.

FASA PENYELIDIKAN DAN PEMBANGUNAN

PDM3 iaitu salah satu daripada formulasi sebatian hasilan semula jadi yang
berpotensi untuk menggantikan triklosan sebagai mikrobiosid telah dikenalpasti
menerusi kajian FRIM terdahulu. Pemilihannya sebagai mikrobiosid untuk
dibangunkan sebagai produk penjagaan diri bernilai tinggi (jenama Ciera®)
adalah berdasarkan profil kualiti, keselamatan dan keserasiannya dengan lain-
lain ramuan utama produk cecair mandian dan cucian tangan. Keberkesanan
rangkaian produk Ciera® membersih dan melindungi penggunanya dari
jangkitan kuman telah dibuktikan menerusi ujian perencatan (Jadual 1).

Gambar 1: Kempen kesedaran terhadap Gambar 2: PDM3

bahaya triklosan

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Jadual 1: Ujian perencatan Mikrob

*Masa
Mikrob tindakan % perencatan
(saat)
Methicillin resistant Staphylococcus
5 99.9%
aureus (ATCC 33591)
Community acquired MRSA (BAA
5 99.9%
1556)
Pseudomonas aeruginosa (ATCC
5 99.9%
27853)
Escherichia coli (ATCC 8739) 5 99.9%
Bacillus cereus (ATCC 11778) 5 99.9%
Salmonella typhimurium (ATCC
10 99.9%
14028)

FASA AKREDITASI

Inisiatif untuk memperoleh akreditasi dari pihak berautoriti turut mewarnai

kembara panjang produk terbitan teknologi FRIM ini. Berbeza dengan
formulasi pencuci dan pembasmi kuman yang lain, ciri mesra-pengguna
diterjemahkan menerusi sifatnya yang lembut pada kulit (tidak merengsa
kulit -ujian dermal irritection), tanpa bau yang menusuk serta tidak mengkakis
atau mengeringkan kulit justeru selamat untuk digunakan oleh pengguna
berkulit sensitif. Produk mandian dan cucian tangan Ciera® ini juga bebas dari
bahan-bahan kimia berisiko seperti SLS/SLES dan paraben serta terbitannya.
Nombor notifikasi (NOT) daripada Kementerian Kesihatan Malaysia turut
diperoleh kerana produk mematuhi garis panduan kualiti dan keselamatan
yang ditetapkan oleh Biro Pengawalan Farmaseutikal Kebangsaan (BPFK).

Nilai pH produk, kandungan organik meruap yang rendah dan

keupayaannya untuk mengurai secara semula jadi di persekitaran (biorosot)
melayakkan formulasi ini untuk pensijilan eko daripada Jabatan Standard
Malaysia (SIRIM). Tahap biorosot yang tinggi di persekitaran, mengelakkan
kejadian akumulasi yang boleh mencetuskan kesan buruk dalam jangka masa
panjang. Perakuan HALAL daripada pihak Jabatan Kemajuan Islam Malaysia
(JAKIM) lambang kepatuhan terhadap konsep `halalan-toyiba’ turut
membanggakan. Dua produk terbaru yang mengkhusus untuk kegunaan bayi
berjaya memperoleh pensijilan “Organic Certified” daripada badan akreditasi
tersohor bagi produk organik iaitu EcoCert Organic Cosmetic yang
berpangkalan di Perancis.

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191
 Gambar 3: Pengiktirafan dan akreditasi yang berjaya diperoleh

FASA PENGKOMERSILAN
Rangkaian produk kini dihasilkan pada skala komersil untuk meneroka pasaran
pengguna khalayak. Aspirasi untuk menghasilkan produk disinfektan dan
antiseptik berinspirasikan alam semula jadi akhirnya berjaya direalisasikan
menerusi kerjasama bersinergi dengan syarikat Nature Profusion Sdn. Bhd.
Syarikat yang lahir menerusi program FMBiosis iaitu kerjasama pintar di antara
Institut Penyelidikan Perhutanan (FRIM) dan Malaysian Technology
Development Corporation (MTDC). Syarikat Nature Profusion Sdn Bhd.
menggalas misi untuk memartabatkan produk antiseptik dan disinfektan
terbitan R&D tempatan di bawah jenama utamanya iaitu Ciera® dan Ciera
Junior™.

Gambar 4: Logo syarikat dan jenama rangkaian produk premium

Berdepan dan bersaing dengan produk keluaran syarikat-syarikat

gergasi seperti Johnson & Johnson, Colgate Palmolive dengan percaturan arena
komersil yang sangat asing perlu cepat dipelajari. Data-data saintifik yang
menyokong setiap ciri unik masih sukar difahami apa lagi dihargai oleh majoriti.
Idealisma penyelidikan perlu terselaras bagi mengimbangi tabiat kepenggunaan
khalayak bakal pembeli.

Bermula dengan tiga varian produk disinfektan sediaan pekat, kini

rangkaian produk telah diperluas kepada disinfektan semburan pelbagai guna,
cecair mandian berantiseptik, cecair cucian tangan berantiseptik, busa cecair
cucian tangan, cecair mandian bayi `head to toe’ dan losyen berantiseptik.
Sebahagian dari rangkaian produk jenama Ciera® kini boleh ditemui di lebih 100
outlet pasaraya dan farmasi terpilih.

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192
 Gambar 5: Sebahagian daripada rangkaian produk jenama Ciera®

Gambar 6: Sebahagian outlet edaran produk disinfektan jenama Ciera®

RUMUSAN
Rangkaian produk disinfektan dan antiseptik jenama Ciera® dan Ciera Junior™
terbitan FRIM Technologies® ini merupakan salah satu inisiatif Institut
Penyelidikan Perhutanan Malaysia (FRIM) merakyatkan hasil R&Dnya, selaras
aspirasi kerajaan. Semoga rangkaian produk inspirasi semula jadi berteras
kepakaran tempatan ini akan berkembang maju dan terus lestari menyebarkan
manfaatnya ke seluruh pelusuk dunia.

PENGHARGAAN
Projek telah diupayakan oleh pelbagai dana penyelidikan dan pengkomersilan
daripada FRIM, MTDC, MARA dan MOSTI. Bantuan teknikal daripada Encik Saiful
Azmi Johari, Puan Mazurah Mohd. Isa, Puan Hannan Abdul Wahab, lain-lain
rakan penyelidik akan selalu dikenang. Sokongan tidak berbelah bahagi dari
pihak pengurusan FRIM dan khalayak pengguna produk Ciera®juga amat
dihargai.

185
193
 PRECLINICAL, CLINICAL
&
PRODUCT DEVELOPMENT

186
CHALLENGES AND EXPERIENCES IN CONDUCTING PRECLINICAL STUDIES
FOR HERBAL PRODUCTS: PHARMANIAGA PERSPECTIVE

Vanessa Shalini, D.

Clinical Affairs Department,

Regulatory Affairs Division,
Pharmaniaga Bhd,
No 7, Lorong Keluli 1B, Kawasan Perinduatrian Bukit Raja Selatan,
Seksyen 7, 40000 Shal Alam,
Selangor

187
196
ANDROGRAPHOLIDE DERIVATIVES TARGET THE ELUSIVE ONCOGENIC K-
RAS: PROMISING COMPOUNDS TO COMBAT K-RAS-DRIVEN
MALIGNANCIES

Johnson, S1,2, Quah, S.Y.1, Michelle Tan, S.,1, Wong, C.C.1, Pran, K.D.3,
Sreenivasa, R.S.3 & Khozirah, S.2

1
Pharmacotherapeutic Unit, Department of Medicine Faculty of Medicine and Health
Sciences, 2Laboratory of Natural Products, Institute of Bioscience; Universiti Putra
Malaysia, Serdang, Selangor; 3Department of Pharmaceutical Chemistry, International
Medical University, Bukit Jalil, 57000 Kuala Lumpur.

ABSTRACT

Our concerted effort in the discovery of drugs from natural products led to
identification of andrographolide as a potential anticancer agent. It is a major
bioactive constituent found in Andrographis paniculata (“Hempedu Bumi”) that
contributes to a variety of pharmacological activities. Chemical modification of
this compound via a semisynthetic approach yielded diverse analogues. These
compounds were tested for anticancer properties through in vitro, in vivo and in
silico studies. We were able to identify a few lead semisynthetic compounds
that exerted inhibitory effects on cancer cells harbouring the most notorious
and elusive cancer causing oncoprotein K-Ras (K-RasG12V, K-RasG12C, and K-
RasG12D). The mutation of K-Ras gene is prevalent in pancreatic (>90%), colon
(40–50%) and lung (30–50%) cancers but also found in cancers of the biliary
tract, endometrium, cervix, bladder, liver, breast and myeloid leukaemia. Our
previous computational molecular docking studies had identified an
andrographolide analogue termed SRJ23 to target K-Ras oncoprotein at a
specific binding pocket located between two switches of the protein (Hocker et
al, 2013, PNAS, 110:10201–10206). A subsequent analysis found the compound
anchored in the binding pocket via hydrogen bond interaction with hydroxyl and
carbonyl groups of the lactone ring at carbon positions 14 and 16, respectively.
In addition, molecular dynamics simulation showed the lactone ring portion was
held stably via 14-OH group in the binding pocket, while other parts, principally
the three-ring system also interacts via hydrophobic contact within the binding
site (Fig. 1). To confirm the in silico findings, we performed saturation transfer
difference-nuclear magnetic resonance (STD-NMR) study to investigate the
potential interaction between SRJ23 and K-RasG12V protein, on the basis of
proton resonances. The 14-OH and two -CH2 moieties on the three-ring system
were determined as the chemical moieties responsible for molecular
recognition in the binding site of K-Ras. These results support the compound’s
strong and prolonged inhibition on the oncogenic K-Ras protein. Further
chemical modifications of this compound yielded analogues with stronger

188
197
binding affinity and selectivity to the oncogenic K-Ras. A preliminary in vivo
study revealed one such compound named Raspholide to suppress the growth
of human pancreatic (PANC-1, K-RasG12D) cancer xenografts in mice better than
SRJ23.

Asp 54
Fig. 1. Interaction of SRJ23 with KRasG12V protein (PDB ID: 4EPX) showing hydrogen
bond between the 14-OH of the lactone ring and the side chain of aspartate at
amino acid position 54.

Key words: Andrographis paniculata, Rasopholide, computational molecular

docking studies, anticancer agent, oncoprotein K-Ras

189
198
GLUCOSE UPTAKE ACTIVITY OF FICUS DELTOIDEA EXTRACTS

Nur Sumirah, M. D.1, Nurshieren, Y.1, Zainah, A.2, Muhajir, H.1

1
Department of Microbiology, Faculty of Biotechnology and Biomolecular
Sciences, Universiti Putra Malaysia, 43400 Serdang, Selangor, Malaysia.
2
Malaysian Nuclear Agency, Bangi 43000, Kajang, Selangor, Malaysia

Tel: 0132125350 E-mail: nursumirah@gmail.com

ABSTRACT

Diabetes mellitus is a metabolic disorder characterized by chronic

hyperglycemia. Glucose uptakes into peripheral cells (muscle and adipocytes)
have been widely accepted as one of the mechanisms by which hyperglycemia is
reduced. Ficus deltoidea or locally known as mas cotek and belongs to the
Moraceae family and is native to Southeast Asia. It is traditionally user in herbal
remedies to treat diabetes. In order to investigate the underlying mechanism in
antihyperglycemic action of different varieties of F. deltoidea, we evaluate the
effect of the plant extracts F. deltoidea (intermedia) and F. deltoidea (kunstlery)
on glucose uptake activity into L6 myotubes cells. The effect of the extracts (10 –
1000 µg/ml) on glucose uptake activity was evaluated either in the absence
(basal) or presence (insulin-mediated) of 100 nM insulin. The results showed
that there is enhancement of insulin-mediated glucose uptake of F. deltoidea
(intermedia) extracts at concentration of 10, 50 and 100 µg/ml. Meanwhile, in F.
deltoidea (kunstlery) extract was observed to have insulin mimetic and insulin
sensitizing activity at 10 and 50 µg/ml. All concentrations of F. deltoidea
(kunstlery) extract showed significant enhancement of insulin-mediated glucose
uptake activity. Metformin was used as positive control and showed significant
enhancement of insulin-mediated glucose uptake. It is suggested that F.
deltoidea extracts have potential to be developed into a new oral antidiabetic
agent.

Key words: Ficus deltoidea, glucose uptake activity, insulin mimetic,

metformin, antidiabetic agent

190
199
ANTIOXIDANT PROPERTIES, TOTAL PHENOLIC CONTENT AND
ANTIGLYCATION ACTIVITY IN FOUR VARIETIES OF FICUS DELTOIDEA
EXTRACTS

Nurshieren, Y.1, Nur Sumirah, M.D.1, Zainah, A.2 & Muhajir, H.1

1
Department of Microbiology, Faculty of Biotechnology and Biomolecular
Sciences, Universiti Putra Malaysia, 43400 Serdang, Selangor, Malaysia
2
Malaysian Nuclear Agency, Bangi 43000, Kajang, Selangor, Malaysia

Tel: 012-5737345 E-mail : nurshieren.yahaya@gmail.com

ABSTRACT

Diabetes Mellitus is a chronic metabolic disorder which has been listed as one of
the seven top killer diseases in Malaysia. Extensive research is needed to reduce
the fatality effect of the disease. One of the most researched medicinal plants
include Ficus deltoidea and it was reportedly to have antidiabetic properties.
The four varieties of F. deltoidea namely variety Deltoidea, Angustifolia, Bilobata
and Motleyana was used in this research. The objective of this study is to
evaluate the amount of antioxidant present in the extracts, to examine the
phenolic content in the extracts and to evaluate the antiglycation activity in the
extracts. The antioxidant properties were measured through DPPH radical
scavenging activity. The total phenolic content was determined by the Folin–
Ciocalteu method whereas the total phenolic content was calculated based on
the gallic acid calibration curve. While the in-vitro glycation of Bovine Serum
Albumin (24 hours) was done by incubating 500 µl of 1 mg/ml of Bovine Serum
Albumin (BSA) with 400 µl of 500 mM fructose (final concentration) and 100 µl
of F. deltoidea standardized extract (100-5000 µg/ml) at 60 °C for 24 hours. F.
deltoidea Bilobata has the highest IC50 for DPPH radical scavenging activity
which is 66.81 mg/ml and has the highest phenolic content which is 241.58
GAE/g dry weight. In addition, F. deltoidea Bilobata has the highest IC50 for 24
hours antiglycation which is 1.22 mg/ml. Among the four varieties, F. deltoidea
Bilobata showed the highest properties of antioxidant, total phenolic content
and 24 hour antiglycation activity. This suggests that F. deltoidea Bilobata has
the potential developed into effective andiabetic agent in future antidiabetic
research.

Key words: Ficus deltoidea, antioxidant properties, phenolic content, in-vitro

glycation

191
200
NATURAL SWEETENER OF FRESH PINEAPPLE AS HEPATOPROTECTIVE
AGENT

Nurul Hafizatul Syafiqah, M.A., Mohd Kamal, N.H. & Abd Rashid, L.

Natural Product Division, Forest Research Institute Malaysia, 52109 Kepong

Selangor

E-mail: hafizatulazlan@yahoo.com.my

ABSTRACT

Natural sweetener of Ananas comosus aqueous extract has been investigated as

hepatoprotective agent in rats administered with paracetamol to induce hepatic
toxicity. The results revealed that the hepatotoxic-induced rats showed a
significant increase in levels of aspartate aminotransferase (AST) and alanine
aminotransferase (ALT) compared to the normal and extract treated group of
rats. Moreover, serum malondialdehyde (MDA) concentration of paracetamol
group had elevated significantly. The liver weight of paracetamol group is also
higher than normal and treated groups. On the other hand, the hepatotoxicity-
induced rats treated with different concentrations (100-450 mg/kg b.w.) of A.
comosus natural sweetener showed a significant decrease of AST and ALT,
serum MDA concentration and liver weight. The rats administered with
paracetamol showed significant increase in liver enzymes and liver weight
resulting in hepatic toxicity in rats however treatment of A. comosus natural
sweetener significantly alleviated the toxicity effects and provides hepatic
protection through its antioxidant properties.

Key words: Ananas comosus, natural sweetener, hepatic toxicity, paracetamol

192
201
DETERMINATION AND EVALUATION OF ANTIOXIDANT CONSTITUENTS
OF ENTADA SPIRALIS RIDL. LEAVES

Sharifah Nurul Akilah, S.M1, Siti Zaiton, M.S1, Norazian, M.H.1, Nik Mohd. Idris,
N.Y.1 & Aiza, H.2

1
Kulliyyah of Pharmacy, Pharmaceutical Chemistry Dept, International Islamic University,
Malaysia. 2Faculty of Applied Sciences, Chemistry Dept, Universiti Teknologi Mara
Pahang, Malaysia.

Tel: 09-570 4921 Fax: 09-571 6775 E-mail: dszaiton@iium.edu.my

ABSTRACT

In the modern times, great attention has been given to natural antioxidant
because the use of synthetic antioxidant caused harm to the human body.
Entada spiralis Ridl. is from the Leguminoceae family. The study aims to
determine and evaluate the potential of antioxidant activity of the E. spiralis
leaves. Petroleum ether, chloroform, and methanol crude extract were used for
the experiment. Antioxidant activity assay was performed in-vitro by 2,2-
Diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity, ferric reducing
antioxidant power (FRAP) and ABTS radical cation scavenging activity. Total
phenolic content was measured by Folin-Ciocalteu’s reagent method and
flavonoid was evaluated by calorimetric method. Determination of antioxidant
activity through the bioautographic method was carried out by dot-blot assay.
The results were compared with standards. All crude extracts were capable to
scavenge the radicals in a concentration dependent manner. Based on the
results obtained, the methanolic leaf extract exhibited good scavenging activity.
The inhibition concentration (IC50) values of methanolic extract on DPPH radical,
FRAP, and ABTS radical were found to be 40.23, 14.50 and 5.09 ug/ml,
respectively. As expected, phenolic and flavonoid content was found to be the
highest in methanol extract. The results indicated that the E. spiralis Ridl. leaves
possessed radical scavenging activity and is fit for further scientific investigation.

Key words: Entada spiralis, natural antioxidant, radical scavenging activity,

inhibition concentration (IC50)

193
202
ANTI-INFLAMMATORY POTENTIAL OF BACKHOUSIA CITRIODORA F.
MUELL. METHANOLIC EXTRACT BY IN-VITRO BIOACTIVITY ASSESSMENT

Siti Nur Aisyah, M.H., Shalini, M., Khoo, M.G.H., Rohana, S. & Mazura, P.

Natural Products Division, Forest Research Institute Malaysia, Kepong, 52109

Selangor, Malaysia

Tel: 03-6279 7608 Fax: 03-6272 9805 Email: sitinuraisyah@frim.gov.my

ABSTRACT

Backhousia citriodora F. Muell is an Australian woody species from the

Myrtaceae family with the crushed leaves have lemony scent. In-vitro anti-
inflammatory property of the stem and leaf extracts were investigated together
with antioxidant and safety profile. Anti-inflammatory potential of the methanol
extracts were evaluated by lipoxygenase inhibition assay. The stem extract
showed high anti-inflammatory activity with IC50 value of 12.78 µg/ml compared
to the positive control, nordihydroguaiaretic acid (NDGA) (1.00 µg/ml).
Meanwhile, the leaf extract showed mild inhibition activity with IC50 value of
23.48 µg/ml. Antioxidant activity was determined with 2,2-diphenyl-1-(2,4,6-
trinitrophenyl)hydrazyl (DPPH) free radical scavenging assay. Total phenolic (TP)
and flavonoid content was also assessed. The stem extract showed high activity
on DPPH free radical scavenging and TP content (2328 mg GAE/100 g extract)
compared to the leaf extract (679 mg GAE/100 g extract). For safety aspect,
cytotoxicity testing indicated that at 100 µg/ml, the stem extract was less toxic
than the leaf extract against normal kidney and liver cell lines at 100 µg/ml.
Anti-inflammatory potential of the extract might be due to the presence of
phenolic compounds (PCs) such as flavonoids, in which PCs are known to
possess anti-inflammatory properties. Thus, B. citriodora extracts have the
potential to be developed into anti-inflammatory agent, besides being a natural
source of phenolic and flavonoid compounds.

Key words: Backhousia citriodora F. Muell., anti-inflammatory, antioxidant,

cytotoxicity, phenolics

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LARVICIDAL PROPERTIES OF β-CARRYOPHYLLENE OF MAJOR
COMPONENT MELALEUCA CAJUPUTI ESSENTIAL OILS AGAINST DENGUE
VECTOR AEDES AEGYPTI (L) AND AEDES ALBOPICTUS (SKUSE).

Azlinda, A.B.1 & Hamdan, A.2

1
Department of Medical Microbiology & Parasitology, School of Medical
Sciences, Universiti Sains Malaysia, 16150 Kubang Kerian, Kelantan Malaysia
2
Vector Control Research Unit, School of Biological Sciences, Universiti Sains
Malaysia, 11900 Pulau Pinang Malaysia

Tel: 09–7676253 Fax: 09-767 6289 E-mail: azlindaab@usm.my

INTRODUCTION

Mosquitoes are not only the most important vector for the transmission of
malaria, filariasis, and viral diseases (James, 1992) such as yellow fever and
dengue (Prajapat et al. 2005). For instance, Aedes aegypti is main vector for the
transmission of both dengue fever and dengue haemorrhagic fever.
Organophosphates such as temephos and fenthion and insect growth regulators
such as diflubenzuron and methopreneare generally used for the control of
mosquito larvae. Although effective, their repeated use has disrupt natural
biological control systems and has led to outbreaks of insect species,
widespread development of resistance, with undesirable effects on non-target
organisms, and human health concerns (Yang et al. 2002). One of the earliest
reports on the use of plant extracts against mosquito larvae is credited to
Campbell et al. (1933), who found that plant alkaloids such as nicotine,
anabasine, methylanabasine and lupinine, extracted from the Russian weed
Anabasis aphylla, killed larvae of Culex pipiens, Culex territans and Culex
quinquefasciatus. The chemicals derived from plants have been projected as
weapons in future mosquito control programme as they are shown to function
as general toxicant, growth and reproductive inhibitors, repellents and
oviposition-deterrent (Sukumar et al. 1991).

MATERIALS AND METHODS

Extraction of essential oil

The extraction of essential oil from leaf specimens are carried out using a steam
distillation. Dried fresh leaves which were allowed to dry at room temperature
were grounded to a small particles. Grounded leaves were transferred into the
distillation flask (500 ml) and filled with distilled water (60–70°C), left to boil
slowly until distillation process completed. The mixture of oil and water are

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allowed to settle for 24 hours. The water layer is slowly drawn off and the
remaining oil were collected into a glass beaker and dried with anhydrous
sodium sulfate (kept at 4–5°C). The extraction process of essential oil were
repeated to produce stock. Stock of essential oil were kept in amber bottle and
stored at 4–5°C.

Analysis of essential oil

Qualitative analysis of the chemical constituents will be carried out under the
following conditions: Alltech 15897 AT-1MS capillary column (30 m×0.25 mm
ID×0.25 μm, film thickness); held at 60 °C for 1 min, raised to 150 °C at a rate of
6 °C/min, raised to 240°C at a rate of 10°C/min, and held for 6 min; 250°C
injector temperature; carrier helium gas at a flow rate of 1.0 ml/min; 300:1 split
ratio. Diluted oil (0.1 μl, 1:10, v/v, in dichloromethane) will be automatically
injected into the system using a splitless mode. The oil components were
identified by using GCMS libraries (NIST107.LIB and WILEY229.LIB). The
percentage of the identified compound will be computed from a total ion
chromatogram (TIC). The identified major constituents of the essential oil
standards Testing Materials (TM) will be purchased from Sigma-Aldrich / Fischer
– Scientific for larvicidal bioassay testing.

Mosquito and larval preparations

Field sampling

Larva used for the bioassay were collected from Hospital Universiti Sains
Malaysia (HUSM) and Kubang Kerian, Kelantan sampling area. Ovitrap are used
to attract wild strain mosquito to lay eggs. One unit ovitrap consisting of a
cylindrical container (black), filled with 250ml tap water and a paddle to provide
a suitable substrate for oviposition (hardboard, 2.0cmx 12.5cm x 0.3cm and is
placed vertically in the ovitrap). The ovitrap will then be placed in the field for
five days. On the fifth day, paddle and their water contents will be collected and
replaced with fresh ones. All collected paddles and its contents from the field
will brought back to the laboratory and transfer into enamel trays for
processing. Newly hatched larva are used for bioassay study.

Bioassay

Third-instar larvae of mosquitoes (n=20) were used for the larvicidal bioassay for
each testing solutions. Five replicates of identified TM were prepared by
dissolving the suitable amount of seasoned tap water (dechlorinated) to
produce a total of 800.0 ml of a desired (mg/L) testing solution (200.0 ml for
each cup). The control solutions were prepared by dissolving 2.0ml of acetone

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and 198 ml of seasoned tap water. The number of dead larvae in each cup were
counted after one (1) and 24 hour respectively. The larvae were considered
dead if they were immobile and do not respond to any mechanical touches after
24 hr (Macedo et al. 1997). Selected TM are assayed with a serial concentrations
with ranges ≤1.0 ppm (mg/L) to produce a range of mortality from 10 to 100 %
along with control.

Data Analysis

LC50 and LC90 value are determined by using Log Probit Analysis as followed:

% mortality = % test mortality - % control mortality X 100

100 - control mortality

RESULTS AND DISCUSSION

From the GCMS analysis, β-carryophyllene was identified as second largest

component of the total major constituents (20.04%). Standard testing materials
of β-carryophyllene was selected due to its affordable price and availability in
the market within short period of time. From the results obtained it was clearly
observed that A. aegypti exhibit higher mortality than A. albopictus in response
to the bioassay testing. Table 1.0 and 2.0 shows the highest of 76.0 % and 55.0
% larval mortality were noted at 0.009 ppm concentration of β-carryophyllene
from HUSM and Kubang Kerian respectively. Whereas the lowest larval mortality
of 1.0 % and 3.0 % were recorded at 0.0005 ppm concentration of A. albopictus.

Table 1.0 Percentage mortality of A. aegypti larvae from HUSM and Kubang
Kerian, Kelantan
A. aegypti (N=100)
Dose HUSM Kubang Kerian
ppm Percentage Mean Percentage Mean
(mg/L) Mortality Mortality ± SD Mortality Mortality ±
(%) (%) SD
0.0005 2 0.40 ± 0.55 3 0.60 ± 0.55
0.001 7 1.40 ± 1.14 9 3.20 ± 3.27
0.003 25 5.00 ± 2.83 30 6.00 ± 1.58
0.007 65 13.00 ± 2.74 40 8.00 ± 2.35
0.009 76 15.20 ± 1.64 55 11.00 ± 1.00
*control 0 0
*control (=aseton 0.1%)

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Table 2.0 Percentage mortality of A. albopictus larvae from HUSM and Kubang
Kerian, Kelantan
A. albopictus (N=100)
Dose HUSM Kubang Kerian
ppm(mg/L) Percentage Mean Percentage Mean
Mortality (%) Mortality ± SD Mortality Mortality ± SD
(%)
0.0005 3 0.60 ± 0.55 1 0.20 ± 0.45
0.001 7 2.80 ± 2.49 4 0.80 ± 0.45
0.003 17 3.40 ± 0.55 14 2.80 ± 0.84
0.007 25 5.00 ± 0.71 18 3.60 ± 0.89
0.009 30 6.00 ± 0.71 23 4.60 ± 1.14
*control 0 0
*control (=aseton 0.1%)

Table 3.0 indicate mean and percentage mortality of Aedes sp larval against
control positive solution, temephos. It was observed that highest concentration
gives the highest mortality. A. aegypti displayed similar pattern of mortality rate
as β-carryophyllene by producing highest mortality percentage compared to A.
albopictus

Table 3.0 Mean and mortality percentage of temephos (positive control)

A. aegypti (N=100) A. albopictus (N=100)
Dose Mean Percentage of Mean Percentage of
mg/L Mortality ± SD mortality (%) Mortality ± mortality (%)
SD
0.0005 6.0 ± 0.71 30 4.0 ± 0.00 20
0.001 11.0 ± 1.58 55 9.4 ± 2.30 47
0.002 14.0 ± 1.58 70 13.8 ± 0.84 69
0.003 16.6 ± 0.55 83 15.4 ± 1.14 77
0.005 18.0 ± 0.71 90 16.6 ± 0.55 83
Control* 0 0 0 0
*control (=aseton 0.1%)

Table 4.0 LC50 and LC90 values of β-carryophyllene against Aedes sp

A. aegypti A. albopictus
(N=100) (N=100)
Area LC50 ppm LC90 ppm LC50 ppm LC90
(CI95%) (CI95%) (CI95%) (CI95%)
0.572 0.777 0.907 1.424
HUSM (0.546-0.598) (0.735-0.838) (0.789-1.393) (1.115-2.872)

0.668 1.020 0.997 1.548

Kubang (0.623-0.730) (0.907-1.246) (0.846-1.614) (1.193-3.104)
Kerian

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Table 4.0 briefly, describe LC50 and LC90 values of β-carryophyllene produce by
Aedes sp from both sampling area HUSM and Kubang Kerian. Aedes sp from
HUSM gives the lowest LC50 and LC90 values respectively

CONCLUSION

This study findings concluded β-carryophyllene possess some lethal effect

against Aedes sp from HUSM sampling area. It is also indicated A. aegypti is
more susceptible compared to A. albopictus.

ACKNOWLEDGEMENTS

We wish to thank the School of Medical Sciences, Universiti Sains Malaysia,

Health Campus, Kelantan and Vector Control Research Unit, School of Biological
Sciences, Universiti Sains Malaysia, Pulau Pinang for supporting this study. The
study was supported by the short term grant, 304/PPSP/61313037

REFERENCES

Campbell, F.L, Sullivan, W.W. & Smith, L.N. (1933). The relative toxicity of
nicotine, anabasine, methylanabasine and lupinine for culicine
mosquito larvae. J Econ Entomol 26:500–509
James, A.A. (1992). Mosquito molecular genetics: the hands that feed
bite back. Science 257(5066):37–38
Macedo, M.E., Consoli, R.A., Grandi, T.S., dos anjos. A.M., de Oliveira, A.B.,
Mendes, N.M., Queiroz, R.O. & Zani, C.L. (1997). Screening of
Asteraceae (Compositae) plant extracts for larvicidal activity against
Aedes fluviatilis (Diptera: Culicidae). Mem. Inst. Oswaldo Cruz. 92:565
Prajapati V, Tripathi AK, Aggarwal KK & Khanuja SPS. (2005). Insecticidal,
repellent and oviposition-deterrent activity of selected essential
oils against Anopheles stephensi, Aedes aegypti and Culex
quinquefasciatus. Bioresource Technology 96(2005):1749–1757
Sukumar, K., Perich, M.J. & Boobar, L.R. (1991). Botanical derivatives in
mosquito control; a review. J. Am. Mosq. Control. Assoc. 72:210–
237.
Yang, Y.C., Lee, S.G., Lee, H.K., Kim, M.K., Lee, S.H. & Lee, H.S. (2002). A
piperidine amide extracted from Piper longum L. fruitshows
activity against Aedes aegypti mosquito larvae. J. Agric. Food
Chem. 50(13):3765–3767
WHO (1981). Instruction for determining the susceptibility of resistance of
mosquito larvae to insecticides. World Health Organisations
Mimeograph. WHO/VBC/81.807.

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POTENTIAL OF POLYGALACTURONIC ACID VANADYL (IV) COMPLEX AS
ORAL INSULIN-MIMETIC AGENT.

Abd Rashid, L., Nurnadiah, R., Mohd Kamal, N.H., Norsuhaina, Z., Muhammad
Khair, M.A. & Mohd Radzi, A.

Natural Product Division, Forest Research Institute Malaysia, 52109 Kepong,

Selangor.

Tel: 0362797337 Fax: 0362729805 E-mail: nurnadiah@frim.gov.my

INTRODUCTION

Polygalacturonic acid is the structural backbone of plant protein. It is a

carbohydrate type substances which consist of linear polymeric galacturonic
acid linked together via glycosidic linkages. It appears as linear type of
polysaccharide , thus making the chemical characterizations processes easier as
compared to the heterogeneous type polysaccharides such as pectin, chitosan,
hemicellulose etc. In addition, polysaccharide polymer is biodegradable and
biocompatible and would be effective in enhancing drug bioavailability through
mechanism of delaying release.

There were many research literatures highlighting vanadium compound ability

to improve metabolic disorder of diabetic patient due to its insulin mimetic/
enhancing effects both in vitro and in vivo (Lucy & John 2003, Hongyu et al
2011). Vanadium is a transition metal a wide range of oxidation states. Although
it was reported as an oral therapy for diabetes mellitus, vanadium absorption
from the gastrointestinal tract is poor due to its chemical nature and solubility.
Unabsorbed vanadium is excreted via feces, whereas absorbed vanadium
homeostasis is regulated by the kidney and excretion occurs in the urine.
Absorbed vanadium can also be stored in the bone, kidney, and liver prior to
excretion (Yuen et al 1992). In order to overcome systemic drug toxicity, to
improve treatment absorption rates and to avoid undesirable side effect of
vanadium compounds, vanadium complex with polygalacturonic acid were
synthesized and investigated on its potential as insulin alternative for diabetes
treatment.

MATERIALS AND METHODS

Synthesis of PGA-vanadyl (IV) complex (PGA-V).

The PGA was suspended in vanadium sulfate solution and adjusted to pH 4.0
with diluted NaOH. After stirring for 1 h, the suspension (coordination complex)

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was filtered and washed with water and air dried. The PGA and coordination
complex was compared and analyzed by FTIR.

Oral Glucose Tolerance Test (OGTT)

For OGTT analysis, the tested rats were divided into 4 groups. For the first
group, 5 rats were treated with distilled water as control group, second group
(n=5) rats were treated with 50 mg/kg PGA-V complex, third group (n=5) rats
were treated with vanadium sulfate (VOSO4) at 50 mg/kg and the fourth group
(n=5) rats were treated with insulin at 10 U/kg. Prior the treatment process,
blood samples of each group were taken. Then after 30 minutes of the
treatments, blood samples were taken and glucose was loaded to each rats. The
blood samples were taken interval until it reached 90 minutes of experiment.

RESULTS AND DISCUSSION

Synthesis of PGA-vanadyl (IV) complex (PGA-V).

The axial positions of hydroxyl functional group at C-3 and carboxylic acid group
at C-5 of the galacturonic acid residue of PGA play the important role for the
complexation with vanadyl (IV)(VO2+) ion as shown in Figure 1. The vanadyl (IV)
ions also functioning as the cross-linker of the complex. At pH 4 all PGA was
converted to polygalacturonate form that have COO- functional groups and
hydroxylate form which contained CO- groups. Those functional groups tend to
form ionic bonding with vanadyl (IV) ions which have dissociated from its salt
form. The vanadyl (IV) ions are expected to give the insulin mimetic property of
the complex.

COO-
COO-

* O O O
OH OH

O *
OH
OH OH
n
O
polygalacturonic acid (PGA) COO OH
pH 4.0, 2+
O O
O O
stir 1 h * O V O
+ O
OOC
O
COO
2+
V O
O
O
O O *
O O OOC
OH
2+
S *
O V
O HO n
O

polygalacturonate vanadyl (IV) complex (PGA-V)

vanadium (IV) oxide sulfate

Figure 1: Synthesis of PGA-Vanadyl (IV) complex

The binding of vanadyl (IV) to PGA was observed via FT-IR spectrum as shown in
Figure 2. The existence of strong peak at 1744 cm-1 in PGA-V sample indicate the

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present of carbonyl carbon of ester groups which suggested the carboxylic acids
have been converted to COO-VO2+.

Figure 2: FT-IR spectra of PGA-V complex and PGA

Oral Glucose Tolerance Test (OGTT)

The results of the OGTT from the graph plotted (Figure 3) indicated that
impaired glucose tolerance was improved after treatment with PGA-V complex
rats as compared to the untreated groups (control). PGA-V able to reduce blood
glucose levels in safe manner. These results indicated that the corporation of
essential trace element vanadium and polysaccharide could enhance their
biological activity, inter coordination and bioavailability. The insulin-enhancing
properties effect of PGA-V complexes was also similar to the literature reported
by Hongyu et al (2011) that use polysaccharide from chitosan and carrageenan.
From the experiment, the blood glucose levels of the treated rats reduced
drastically when treated with insulin and vanadium sulfate salt. Direct
application of vanadyl sulphate and uncontrolled dose of insulin can lead to
undesired effect such as hypoglycemic condition which can cause chronic
fatigue and dizziness. The finding is consistent with findings of past studies by
Karmaker et al (2010).

CONCLUSION

PGA-V complex was successfully synthesized and was able to reduce blood
glucose levels.

ACKNOWLEDGEMENTS

This project was funded by Research, Pre-Commercialization and Publication

Grants (RPP)-0514-FK-01.

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 In vivo studies - Effect of PGA-V on oral glucose
loading in mice
20
Blood glucose level (mmol/l)

15
Control
10
Insulin
5
VOSO4
0 PGA-V
-30 0 30 60 90
Time (min)

Figure 3: OGTT profiles of rats treated with insulin, vanadyl sulfate, PGA-V
complex and water

REFERENCES

Lucy, M., & John, H.M. (2003). Insulin-Like Actions Of Vanadium: Potential As A
Therapeutic Agent. The Journal Of Trace Elements In Experimental
Medicine. 16, 253-267.
Hongyu, Z., Yuetao, Y., Dawei F., Yipeng, Wang. & Song, Q. (2011). Hypoglycemic
Properties Of Oxavanadium (IV) Coordination Compounds with
Carboxymethyl-Carrageenan And Carboxymethyl-Chitosan in Alloxan-
Induced Diabetic Mice. Evidence Based Complementary and Alternative
Medicine.
Yuen, V.G., Orvig, C. & McNeill, J.H. (1993). Glucose-Lowering Effects Of A New
Organic Vanadium Complex, Bis (Maltolato) Oxovanadium (IV). Can. J.
Physiol. Pharmacol. 71:263–269.
Karmaker, S., Saha, K.T., Yoshikawa Y. & Sakurai, H. (2010). Vanadyl- poly (ϒ-
glutamic acid) Complexes as Oral Therapeutic Agents for the Treatment
of Type 1 like Diabetic Mice. African Journal Of Pharmacy And
Pharmacology. 4(5):235–243.

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COMPARISON ON PREDICTION AND EXPERIMENTAL LIPOPHILICITY (LOG
P) OF AN ANTICANCER COMPOUND 17-βH NERIIFOLIN

Asiah, O., Mohd Haffiz, J., Nurhanan Murni, Y. & Siti Syarifah, M.M.

Natural Products Division, Forest Research Institute Malaysia (FRIM), Kepong,

52109 Selangor Darul Ehsan.

Tel: 03-62797667 Fax: 03-62729805 Email: asiah@frim.gov.my

INTRODUCTION

Drug discovery and development is a lengthy and costly process. In general, 90%
or more of the budget is spent on clinical trials, mainly in Phase III
(http://www.manhattan-institute.org/html/fda_05.htm). From surveys done in
USA indicated that a new lead compound usually takes more than 13 years on
average to reach the market with cost estimated approximately US$1.8 billion
and the average success rate (from pre-clinical stage to launch) of only 8% (Paul
et al. 2010). A study done by the Biotechnology Industry Organization (BIO)
Industry Analysis and BioMed Tracker revealed that the overall success rate for
drugs moving from clinical trials to United State Food and Drug Administration
(FDA) approval from late 2003 to the end of 2011 is only one in 10
(https://www.bio.org/media/press-release/). Nevertheless, selection of good
candidates in the early stage of drug discovery or drug likeness filters is also
important to avoid permeability and solubility problems.

The lipophilicity or log P is one of the Lipinski rule of five (ROF) parameters that
are widely applied in drug discovery to understand the absorption and
permeability of compounds. Determination of lipophilicity in the early stage of
drug development can significantly limit the problem with poor absorption,
distribution, metabolism and excretion (ADME) properties of drug candidates
and improve its efficacy hence could reduce the overall development cost.

The experimental method for determination of log P is based on the partitioning

of a molecule in a system of two immiscible phases (aqueous and lipophilic). The
traditional method by using shake-flask procedure often time-consuming,
labour-intensive, it requires a high purity substances and also suffers from
solubility and stability problems. Hence, HPLC is the most frequently used
chromatographic method for determination of lipophilicity due to rapid,
accurate and highly reproducibility analysis of relatively large sets of sample
(Mrkvicková et al. 2008).

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Besides the experimental methods, lipophilicity can be estimated through
computational approaches. Various programmes/softwares have been
developed using various methods including fragment-based, atom-based and
knowledge based which rely solely on knowledge of the structure of the
chemical. This study was carried out to compare and validate the prediction and
experimental log P values of 17-βH neriifolin using computational and HPLC
methods respectively.

MATERIALS AND METHODS

Prediction of log P

Various free online computer programmes were used to predict the log P of 17-
βH neriifolin including Molinspiration (http://www.molinspiration.com/cgi-
bin/properties), OSIRIS property explorer (http://www.organic-
chemistry.org/prog/peo/), ACD/Labs, and ChemDraw.

Experimental log P

Log P of 17-βH neriifolin was measured using RP-HPLC following the procedure
from Mrkvicková et al. (2008) with minor modification. Briefly, the analyses of
all compounds were carried out on a monolithic chromatographic column
Chromolith RP18e; 100 mm x 4.6 mm (Merck, Darmstadt, Germany). The
chromatographic system Agilent 1160 Infinity (Agilent Technologies, CA, USA)
was used for the analyses. The column was set at 30oC and UV detection was
performed at 254 nm. An injection volume of 50 µL of samples was used in the
analyses. The methanol and 0.05M phosphate buffer (pH 7.4) in ratio 42:58 (v/v)
were employed as mobile phase. The flow rate was 2 ml/min. The log P of all
standard compounds is expressed as a logarithm of retention factor (log k),
which is calculated according to equation 1 and 2. The experimental log P for 17-
βH neriifolin was calculated from the regression analysis (Table 1, Fig. 1, Eq. 3).

log k = log [(tr-t0)/t0)] ......... Eq. 1;

where tr is retention time of sample and t0 is the dead retention time.

log Pcal= 5.08+2.86 (log k) ......... Eq. 2

log Pest = 2.8573(rt) + 5.0818 ......... Eq. 3,

where rt is retention time of 17-βH neriifolin

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Table 1. Log k values obtained by HPLC and calculated log P values of standard
compounds, t0=0.981.

Compound tr (min) Log k Log P

9MC 1.36 -0.95 2.4
DGX 1.031 -1.29 1.3
EURY 1.37 -0.91 2.3
ANDR 1.4 -0.85 2.7
EPI 1.21 -1.45 1.5
EPG 1.28 -1.2 1.8
JTA 0.98 -0.7 2.6

Figure 1. Linear regression derived from log P and retention time of seven
standard compounds used for estimation of log P of 17-βH neriifolin

RESULTS AND DISCUSSION

Lipophilicity of 17-βH Neriifolin

The lipophilicity for 17-βH neriifolin obtained from prediction and experimental
approaches is listed in Table 2. The predicted log P ranged from 2.00 to 3.72.
The lowest value is obtained from ALOGPS 2.1 program and the highest from
ACD/Chemsketch Freeware version. The former program was developed with
12,908 molecules from PHYSPROP database using 75 E-state indices, while the
later was based on an experimental data set over 18,000 reliable log P
measurements. Basically the computer software prediction is based on the
different mathematical models that either used the substructure or whole
molecular approaches (Mrkvicková et al. 2008). In this study, most of the
programmes showed almost the same log P value for 17-βH neriifolin except for
one outlier which is clogP predicted using ACD/Chemsketch from ACD/labs
although the programme used the same fragment/compound method as OSIRIS
and Datawarrior.

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Besides prediction, log P for 17-βH neriifolin was determined using RP-HPLC
method modified from Mrkvicková et al. (2008). In this experiment, log P for 17-
βH neriifolin was calculated from equation 3 derived from regression analysis of
seven standard compounds as shown in Table 1 and Figure 1. The retention time
for 17-βH neriifolin from the HPLC experiment was 1.07, hence the log P
calculated from Eq. 3 was 2.19. This value is very close to the prediction values.
Thus, the log P value obtained from the HPLC experiment validated the
prediction values of available freeware programmes as listed in Table 2 except
for ACD/Chemsketch.

Table 2. The comparison of prediction and experimental lipophilicity of 17-βH

neriifolin
Log P Program/Software
Prediction
clogP 3.72 ACD/Chemsketch
clogP 2.39 OSIRIS property explorer
clogP 2.44 Datawarrior
clogP 2.59 ChemDraw Ultra 11.0
alogP 2.00 ALOGPS 2.1 (vcclab)
milogP 2.17 Molinspiration
xlogP3 2.1 PubChem
Experimental
Log P 2.19 RHPLC

Drug likeness prediction of 17-βH Neriifolin

The prediction of drug likeness property of the compound performed by

Molinspiration programme is presented in Table 3. The compound 17-βH
neriifolin complied with the Lipinski ROF, with only one violation i.e. molecular
weight (mwt) more than 500 g/mol. According to ROF, an orally active drug
candidate should show no more than one violation of the following four criteria:
it should not have more than five hydrogen bond donors; it should not have
more than 10 hydrogen bond acceptors; it should not have mwt greater than
500 g/mol; and it should not have an octanol-water partition coefficient (log P)
greater than five (Lipinski, 2000).

CONCLUSION

In this study, the lipophilicity of 17-βH neriifolin determined using both

computational and experimental approaches showed almost similar values,
indicating the accuracy of the prediction programme regardless of the
mathematical method applied for this compound. The molecular properties also

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showed that 17-βH neriifolin has fulfilled the criteria of Lipisnki ROF for orally
active drug, hence has a great potential to be developed as an anticancer agent.

ACKNOWLEDGEMENTS

We are grateful to Ministry of Science, Technology and Innovation Fund grant

and Ministry of Finance for providing the financial support.

Table 3. Molecular properties prediction for 17-βH neriifolin using

Molinspiration program
17-βH neriifolin
miLogP 2.173
TPSA 114.69
No. of atoms 38.0
Mwt (g/mol) 534.69
No. of hydrogen acceptor 8
No. of hydrogen donor 3
No. of violations 1
No. of rotatable bonds 4
Volume (Å3) 506.03
TPSA (total polar surface area)

REFERENCES

ACD/LogP Freeware, Advanced Chemistry Development, Inc., Toronto, ON,

Canada, www.acdlabs.com, 2015.
Lipinski, C.A. (2000). Drug-like properties and the causes of poor solubility and
poor permeability. Journal of Pharmacoll & Toxicoll Methods 44:235–
249.
Mrkvičková, Z., Kovařikova, P., Baliková, S. & Klimeŝ, J. (2008). Determination of
lipophilicity of novel potential antituberculotic agents using HPLC on
monolithic stationary phase and theoretical calculations. Journal of
Pharmaceutical and Biomedical Analysis 48:310–314.
Paul, S.M., Mytelka, D.S, Dunwiddie, C.T., Persinger, C.C., Munos, B.H., Lindborg,
S.R. & Schacht, A.L. (2010). How to improve R&D productivity: the
pharmaceutical industry’s grand challenge. Nature Reviews Drug
Discovery 9:203–214.
VCCLAB, Virtual Computational Chemistry Laboratory, http://www.vcclab.org,
2005.

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THE EFFECT OF TOCOTRIENOL RICH FRACTION (TRF) DERIVED FROM
PALM OIL ON GLUTATHIONE S-TRANSFERASES PROTEIN EXPRESSION IN
MICE LIVER

Abdullah, A.1, Atia, A.1,2 & Alrawaiq, N.1

1
Department of Pharmacology, Faculty of Medicine, Universiti Kebangsaan
Malaysia Medical Centre (UKMMC), Jalan Yaacob Latif, Bandar Tun Razak, 56000
Cheras, Kuala Lumpur.2Department of Anesthesia and Intensive Care, Faculty of
Medical Technology, Tripoli University, Tripoli, Libya

Tel: 03-91459569 Fax: 03-91459547 E-mail: manlah1969@yahoo.com

INTRODUCTION

Vitamin E refers to a family of eight different isomers that belong to two classes
i.e. α, β, γ, and δ tocopherols and α, β, γ, and δ tocotrienols (Sylvester & Theriault
2003). All forms of vitamin E possess antioxidant activity, with, tocotrienols
shown to have stronger antioxidant potential than tocopherols (Pruthi et al.
2001). Tocopherols are abundantly found in oils extracted from leaves and
seeds of most plants e.g. corn, olive, soybean, sesame, peanut and sunflower.
Tocotrienols are less abundant and found only in the oil fractions of some cereal
grains such as barley, rice, annatto, wheat, and most abundantly, in palm fruit
(Sen et al. 2010). A standardised tocotrienol-rich fraction (TRF) consist mainly of
68% of a α, γ, δ-tocotrienols and 32% α-tocopherols mixture can be obtained
from palm oil after esterification and following distillation, crystallization and
chromatography (Sundram & Gapor 1992).

Glutathione S-transferases (GSTs) are family of phase II detoxification enzymes

that catalyse the conjugation of several endogenous and exogenous
electrophilic compounds. In mammals, GSTs are divided into numerous
cytosolic, mitochondrial and microsomal GST isoenzymes. The cytosolic enzymes
are encoded by five related gene families (known as; alpha, mu, pi, sigma, and
theta GST), while the membrane-bound enzymes and microsomal GST are
encoded by single genes and both have originated separately from the soluble
GST (Hayes et al. 2005). GST up-regulation is substantial for the redox state of
the cell because GST catalyses the conjugation of the reduced glutathione,
which is a major intracellular antioxidant (Hayes et al. 2005). In this study, the
ability of three different doses of TRF to modulate GSTs at the protein level in
the livers of ICR male white mice was investigated.

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MATERIALS AND METHODS

In this study, TRF (Gold Tri.E70) was purchased from Sime Darby Bioorganic
(Kuala Lumpur, Malaysia) and contains α-tocopherol at 159.5 mg/g, α-
tocotrienol at 205.1 mg/g, β-tocotrienol at 32.9 mg/g, γ-tocotrienol at 249.8
mg/g and δ-tocotrienol at 119 mg/g. RIPA buffer and Anti-rabbit IgG peroxidase
secondary antibody were purchased from Santa Cruz Biotechnology (Dallas,
USA). Chemiluminescence Western blotting detection reagents were purchased
from Amersham (Uppsala, Sweden). Nitrocellulose membrane was purchased
from Sigma-Aldrich (Seelze, Germany). GSTA1 mouse polyclonal primary
antibody, GSTM polyclonal primary antibody, GSTP polyclonal primary antibody
and β-actin rabbit polyclonal antibody were purchased from Abcam
Biotechnology (Cambridge, UK).

Animal handling and treatment

Male ICR white mice (25–30 g) were used in this study. Mice were kept in clean
polypropylene cages in a ventilated room with 12 hour light-dark cycle, with
food and water available ad libitum. Animals were treated with three different
doses of TRF (dissolved in corn oil). The mice were divided into 5 groups. Mice in
the first group (n = 6) was designated as the control group and were given only
the vehicle i.e. corn oil. Mice in the second group (n = 6) were treated with 200
mg/kg TRF. Mice in the third group (n = 6) were treated with 500 mg/kg TRF.
Mice in the fourth group (n = 6) were treated with 1000 mg/kg TRF. Mice in the
fifth group (n = 6) i.e. the positive control group were treated with 100 mg/kg
butylated hydroxyanisole (BHA). All treatments were administered via oral
gavage for 14 consecutive days. At day 15, mice were sacrificed via cervical
dislocation. Their livers were subsequently isolated, snapped frozen in liquid
nitrogen and stored at -80oC until further use. All experimental procedures were
approved by the Universiti Kebangsaan Malaysia Animal Ethics Committee
(UKMAEC).

Preparation of cytosolic protein fraction

Liver tissue samples were homogenized in RIPA lysis buffer (which contained 10
μl PMSF, 10 μl sodium orthovanadate and 10 μl protease inhibitor cocktail
solution per 1 ml of 1X RIPA lysis buffer). After centrifugation, the supernatants
(cytosolic fractions) were collected and their protein concentrations were
determined.

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Western blotting

Standard Western blotting procedure was used for immunodetection of

proteins. Briefly, 100 μg of liver protein was separated using 15% sodium
dodecyl sulfate–polyacrylamide gel (SDS–PAGE) electrophoresis. The proteins in
the gel were then transferred to nitrocellulose membrane. The membrane was
then incubated for 20 min at room temperature in blocking solution [150 mM
NaCl, 3 mM KCl, 25 mM Tris, 0.1% (v/v) tween-20 and 10% non-fat milk powder
(pH 7.4)]. After blocking, the membrane were incubated with the following
antibodies: primary polyclonal rabbit anti-mouse GSTA, primary polyclonal
rabbit anti-mouse GSTM1, primary polyclonal rabbit anti-mouse GSTP and
primary polyclonal rabbit anti-mouse actin for 1 hour at room temperature.
Subsequently, incubation with a peroxidase-conjugated goat anti-rabbit IgG
secondary antibody was carried out for another 1 h at room temperature.
Protein bands were visualized using enhanced chemiluminescence method
according to the manufacturer’s instructions (Amersham, Uppsala, Sweden). The
intensity of the protein bands were quantified, relative to the signals obtained
for actin, using ImageJ software.

Statistical analysis

Data are presented as mean ± standard error of the mean (SEM). Significant
differences between mean values of multiple groups were determined using
one-way ANOVA and Student’s t-test. Statistical analysis was conducted using
the SPSS software version 22. The result was considered statistically significant
when P < 0.05.

RESULTS AND DISCUSSION

Mice treated with TRF at concentrations of 200, 500 and 1000 mg/kg were
found to have significantly increased liver GSTA protein level by 1.8-, 2.3- and
3.3-fold (p < 0.05) respectively, as compared to controls (Figure 1). Mice treated
with TRF at concentrations of 200, 500 and 1000 mg/kg were found to have
significantly increased liver GSTM1 protein level by 2.8-, 2.9- and 3.9-fold (p <
0.05) respectively, as compared to controls (Figure 2). And mice treated with
TRF at concentrations of 200, 500 and 1000 mg/kg were found to have
significantly increased liver GSTP protein level by 1.6-, 2.3- and 2.9-fold (p <
0.05) respectively, as compared to controls (Figure 3).

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 9
8 *
normalized to actin 7
Relative density

6
5
4
*
* *
3
2
1
0
C T200 T500 T1000 BHA
GSTA
β-Actin

Figure 1. Effect of different doses of TRF on GSTA liver protein level. Mice were treated
with 200, 500 and 1000 mg/kg TRF for 14 days. Livers were then harvested and GSTA
protein was determined by Western blotting. The intensity of protein bands were
quantified relative to the signals obtained for actin using ImageJ software. The graph
represents the average optical density (± S.E.M.) of bands from three different
experiments (*) P < 0.05 compared to the control.

6 *
normalized to actin
Relative density

5
4 *
3 * *
2
1
0

GSTM1
β-Actin

Figure 2. Effect of different doses of TRF on GSTM1 protein level. Mice were treated
with 200, 500 and 1000 mg/kg TRF for 14 days. Livers were then harvested and GSTM1
protein was determined by Western blotting. The intensity of protein bands were
quantified relative to the signals obtained for actin using ImageJ software. The graph
represents the average optical density (± S.E.M.) of bands from three different
experiments. (*) P < 0.05 compared to the control.

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 *
normalized to control
5

Relative density
4
*
3
* *
2
1
0
C T200 T500 T1000 BHA

GSTP
β-Actin
Figure 3. Effect of different doses of TRF on GSTP protein level. Mice were treated with
200, 500 and 1000 mg/kg TRF for 14 days. Livers were then harvested and GSTM1
protein was determined by Western blotting. The intensity of protein bands were
quantified relative to the signals obtained for actin using ImageJ software. The graph
represents the average optical density (± S.E.M.) of bands from three different
experiments. (*) P < 0.05 compared to the control.

The results demonstrated that TRF increased liver GSTs protein levels in a dose-
dependent manner, with the highest level observed in animals administered
1000 mg/kg TRF, followed by 500 mg/kg and 200 mg/kg respectively. However,
the highest observed increase in GSTs protein levels by TRF was still below the
expression level induced by BHA (positive control) treatment. These results
suggest that the antioxidant activity of tocotrienols might also partly be
mediated through the induction of phase II enzymes (such as the GSTs).

The antioxidant activities of tocotrienols have been linked to Nrf2 activation in

an in vitro study (Hsieh et al. 2010). However, the exact mechanism by which
tocotrienol induces GSTs protein expression in mice liver is still not fully
understood. Since GSTs are phase II enzymes and phase II enzymes are
regulated by Nrf2 (Aleksunes & Manautou 2007), it is suggested that
tocotrienols are able to dissociate the Nrf2/Keap1 complex, allowing Nrf2 to
translocate to the nucleus and increase the expression of phase II enzymes
(including the GSTs) in the liver cells. Further studies are needed to confirm this
mechanism.

CONCLUSION

Oral administration of TRF for 14 days in mice significantly increased liver GSTs
protein expression, with the highest expression seen with mice treated with

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1000 mg/kg TRF, followed by 500 and 200 mg/kg respectively. It is suggested
that TRF regulate GSTs expression through the Nrf2 pathway.

ACKNOWLEDGEMENTS

The authors would like to thank Universiti Kebangsaan Malaysia (UKM) through
the receipt of research grants UKM-GGPM-TKP-051-2010 and FF-176-2013.

REFERENCES

Aleksunes, L.M. & Manautou, J.E. (2007). Emerging Role of Nrf2 in Protecting
Against Hepatic and Gatrointestinal Disease. Toxicologic Pathology 35:
459–473.
Hayes, J.D., Flanagan, J.U. & Jowsey, I.R. (2005). Glutathione transferases.
Annual Review of Pharmacology and Toxicology 45:51–88.
Hsieh, T.C., Elangovan, S. & Wu, J.M. (2010). Differential suppression of
proliferation in MCF-7 and MDA-MB-231 breast cancer cells exposed to
alpha-, gamma- and delta-tocotrienols is accompanied by altered
expression of oxidative stress modulatory enzymes. Anticancer Research
30:4169–4176.
Pruthi, S., Allison, T.G. & Hensrud, D.D. (2001). Vitamin E Supplementation in the
Prevention of Coronary Heart disease. Mayo Clinic Proceedings
76(11):1131–6.
Sen C.K., Rink, C., Khanna, S. (2010). Palm Oil–Derived Natural Vitamin E a-
Tocotrienol in Brain Health and Disease. Journal of the American College
of Nutrition 29:314S–323S.
Sundram, K. & Gapor, A. (1992). Vitamin E from Palm Oil. Its Extraction and
Nutritional Properties. Lipid Technology 4:137–141.
Sylvester, P.W. & Theriault, A. (2003). Role of Tocotrienols in the Prevention of
Cardiovascular Disease and Breast Cancer. Current
Topics in Nutraceutical Research 1:121–136.

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ANTI GOUT PROPERTIES FROM BAECKEA FRUTESCENS VIA XANTHINE
OXIDASE INHIBITION

Fadzureena, J.1, Siti Nur Aisyah, M.H.2, Mazura, M.P.2, Fauziah, A.3, Zunoliza,
A.3, Nuziah, H.3, & Hani, I.B.2

1 2 3
Bioresources Programme, Bioactivity Programme, Phytochemistry
Programme, Natural Products Division, Forest Research Institute Malaysia,
52109 Kepong, Selangor.

Email: fadzureena@frim.gov.my

INTRODUCTION

Baeckea frutescens is a shrub or small tree of the Myrtaceae family that can be
found in Sumatra, the Malay Peninsula and the coasts of southern China to
Australia (Burkill 1935). Phytochemical investigation on the aerial parts of this
plant has led to the isolation of unusual endoperoxide together with compounds
linked to a flavanone nucleus (Tsui & Brown 1996) as well as compunds from the
chromones and chromanones group (Tsui & Brown 1996). Chemical study on the
leaves of B. frutescens yielded phloroglucinols (Fujimoto et al. 1996), Flavonones
(Makino & Fujimoto 1999), Chromone C-glycoside (Satake et al. 1999) and
flavonol glycoside (Lu et al. 2008). More information on the identity of the
bioactive constituents and their mechanisms of action is needed in order to
provide a stronger scientific rational for the medicinal uses of the plants. This
indeed is the basis of the current study, the aim being to obtain more scientific
data mainly on anti-inflammatory properties with regards to validating the
claims mention earlier.

MATERIALS AND METHODS

Plant material

The stem of B. frutescens were collected in January 2010 from FRIM Research
Station at Setiu, Terengganu. The plant was identified by Ms. Tan Ai Lee and
voucher specimen was deposited at Natural Products Division Reference
Collection, FRIM, Kepong, Selangor.

Preparation of extract and fractions

First 2.0 kg of B. frutescens stem were oven dried, grounded and macerated 4
times in methanol at room temperature (20–30 oC). Methanol was dried under
reduced temperature and pressure using a rotary evaporator. The solid dark

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green residue designated as “extract” was kept in refrigerator at 4 oC. A total of
250 g of the crude extract was subjected to liquid-liquid partition between 50%
aqueous methanol and hexane to give hexane, dichloromethane, ethyl acetate
and water fractions.

Antiinflammatory Evaluation: Assay for inhibition of xanthine oxidase

activity

Xanthine oxidase inhibiting activity was conveniently measured by slightly

modifying the spectrometric method developed by Noro et al. (1983). Xanthine
oxidase was purchased from Roche, USA and xanthine (substrate) was
purchased from Sigma (St. Louis, MO, U.S.A.). All other chemicals were of
analytical grade. In assay protocol, 130 µl of 0.05 M potassium phosphate buffer
(pH 7.5), 10 µl of test-compound solution and 10 µl of xanthine oxidase enzyme
solution were mixed and incubated for 10 min at 25 °C. The reaction was then
initiated by the addition of 100 µl xanthine (substrate) solution. The enzymatic
conversion of xanthine to form uric acid and hydrogen peroxides measured at
absorbance of 295 nm. Test compounds and reference standards were dissolved
in DMSO. All reactions were performed in triplicates in a 96-well UV microplate.
The IC50 values were then calculated using the Probit Analysis program.

Isolation and Purification

A total of 250g stem crude MeOH extract were subjected to liquid-liquid

partitioning. Four fractions namely hexane, chloroform, ethyl acetate and water
fraction were obtained and were sent for bioassay. From the 4 fractions, ethyl
acetate fraction showed high activity and were selected to further
phytochemical work up. Isolation of ethyl acetate fraction yielded 4 compounds
namely BF 33, BF 6322, BF 71 and BF 84. All compounds were subjected to NMR
for identification and HPLC for fingerprinting evaluations.

HPLC Procedures

Chromatographic separations were carried out on a C18 analytical column

(Phenomenex ®-Luna 5u C18 100A, 250 x 4.60mm 5micron). The mobile phase
consisted of water-formic acid (A; 100:0.1 v/v)-acetonitrile (B) ; A:B was as
follows : 0min 85:15; 12 min, 75:25; 22 min, 75:25; 25min, 85:15; 35 min, 85:15;
flow-rate was 1.0ml/min and the column temperature was maintained at 30OC.
All solvents were HPLC grade.

RESULTS AND DISCUSSIONS

In vitro xanthine oxidase inhibitory activity

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The anti-inflammatory potential of B. frutescens stem MeOH extracts together
with four fractions obtained were screen for using in-vitro xanthine oxidase
inhibitory assay. Results were as shown in Table 1. Ethyl acetate fraction
exhibited high inhibition towards xanthine oxidase enzyme activities.
Allopurinol, a known inhibitor of xanthine oxidase, was adopted as positive
control in the studies and has an IC50 of 0.4388 µg/ml. The inhibitory activity of
the isolated compounds of B. frutescens were as shown in Table 2

Table 1: In-vitro xanthine oxidase inhibitory activity of stem extract and fractions

No Sample Xanthine Oxidase (%)a

1 MeOH extract (stem) 80.83 ± 4.66
2 Hexane fraction (stem) 25.76 ± 4.09

3 CHCl3 fraction (stem) 46.96 ± 3.91

4 EtOAc fraction (stem) 79.33 ± 2.66

5 H2O fraction (stem) 42.46 ± 6.52

Table 2: In-vitro xanthine oxidase inhibitory activity and IC50 values of

compounds isolated

No Sample Xanthine Oxidase (%)a IC50(µg/ml)

1 BF 33 43.13 ± 7.75 ND
2 BF 6322 99.47 ± 0.53 3.584
3 BF 71 40.63 ± 7.88 ND
4 BF 84 NA ND
5 Allopurinol 98.36 ± 0.98 0.4388
(Positive Control)

Final concentration of tested sample in reaction mixture was fixed at

100g/ml Note: H-high (71-100%) M-moderate (41-70%) L-low (0-
40%) NA - not active ND- not determine

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Structure elucidation
OH CH3

OH O OH

HO O
O

H3C OH O CH3

BF 6322 H3C
E 71
OH O

OH
OH
OH
OH

HO O
HO O
OH
Rhamnoside
Rhamnoside O
O
OH O
OH O

E 33 E 84
Myricitrin Quercitrin

HPLC Fingerprinting

Figure 1 and 2 showed the HPLC profile and chromatogram of the active extract
and active compound (BF 6322), respectively. Based on this profile, we
quantified the presence of BF 6322 in the active extract through standard
calibration curves. Results of the study showed the active extract contained 10%
of BF 6322.

1.50

BF 33 BF
1.00
AU

6322
0.50

0.00
2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00 32.00 34.00
Minutes

Figure 1: HPLC profile of active extract

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 0.10

0.08 BF
AU 0.06 6322
0.04

0.02

0.00

2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00 32.00 34.00
Minutes

Figure 2: HPLC profile of active compound, BF 6322

CONCLUSION

From this study, stem extract of B. frutescens showed potential xanthine oxidase
activity. Fractionation of the stem MeOH extract was conducted to get the
hexane, chloroform, ethyl acetate and water fractions. From 4 fractions tested,
ethyl acetate fraction showed high activity against xanthine oxidase inhibitory
assay (anti-gout properties). Further phytochemical work up led to the isolation
of 4 compounds, BF 6322, E 71, E 33 and E 84. Compound BF 6322 showed
potent anti-gout activity through xanthine oxidase inhibitory assay with IC50
value of 3.584 µg/ml.

ACKNOWLEDGEMENTS

We thank the Ministry of Agriculture and Agrobased Industry (MOA) for the
research funding

REFERENCES

Burkill, I.H., (1935). A Dictionary of the Economic Products of the Malay

Peninsula. Vol 1. Crown Agents for the Colonies, London.
Fujimoto, Y., Usui, S., Makino, M. & Sumatra, M. (1996). Phloroglucinols from
Baeckea frutescens. Phytochemistry. 41(3):923–925.
Lu, W.J., Ya, Q.K., Chen, J.Y. & Liu, B.M. (2008). A New Flavonol Glycoside from
Baeckea frutescens L. Yaoxue Xuebao. 43(10):1032–1035.
Makino, M. & Fujimoto, Y. (1999). Flavanones from Baeckea frutescens.
Phytochemistry. 50(2):273–277.
Noro, T., Oda, Y., Miyase, T., Ueno, A. & Fukushima, S. (1983). Inhibitors of
Xanthine Oxidase from the Flowers and Buds of Daphne genkwa. Chem.
Pharm. Bull. 31:3984–3987.
Tsui, W.Y. & Brown, G.D. (1996). Chromones and Chromanones from Baeckea
frutescens. Phytochemistry. 43(4):871–876.
Tsui, W.Y. & Brown, G.D. (1996). Unusual Metabolites of Baeckea frutescens.
Tetrahedron. 52(29):9735–9742.

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ANTIOXIDANT PROPERTIES OF EXTRACTS FROM DIFFERENT PART OF
FOUR MALAYSIAN MEDICINAL PLANTS

Ihsan Safwan K., Mohd Kamal N.H. & Nurul Hafizatul Syafiqah M.A.

1
Natural Products Division, Forest Research Institute Malaysia (FRIM), 52109
Kepong, Selangor

Tel: 03-6279 7727 Fax: 03- 6272 9805 E-mail: ihsan@frim.gov.my

INTRODUCTION

Antioxidant is any substances that have the ability to scavenge or reduce the
detrimental effect of free radicals on biomolecules such as protein, lipid and
DNA causing several undesired health condition including aging, atherosclerosis,
cancer and several other diseases (Jin et al. 2016; Gangopadhyay et al. 2016). In
human body, free radicals were produced from oxygen which is fundamental to
cellular metabolism and energy production (Wickens 2001). Synthetic
antioxidant such as butylated hydroxyanisole (BHA), butylated hydroxytoluene
(BHT) and tert-butyhydro-quinone (TBHQ) were used in food industry to stop
lipid peroxidation. However, previous studies showed that BHA and BHT
accumulate in the body and result in liver damage and carcinogenesis (Ito et al.
1986; Whysner et al. 1994). In order to reduce synthetic antioxidants
dependency, there were great interest to study antioxidant capacities and total
phenolics content in vegetables, fruits, spices and medicinal plants (Jin et al.
2016). In this study, antioxidant capacity of different part of four medicinal
plants have been evaluated by means of DPPH scavenging activities and ferric
reducing antioxidant power while extracts ability to quench oxidative damage to
membrane lipids was analysed by anti-lipid peroxidation assay. Phenolics
content of all extracts were also analysed in order to find the extract with high
phenolics content which were generally attributed to good antioxdant property.

MATERIALS & METHODS

Plant Material.

The (leaf (L), stem (S) and root (R), of four medicinal plants namely Aquilaria
malaccensis (AM), Brucea javanica (BJ), Averhoa carambola (AC) and Tetracera
indica (TI) were used in this study and coded as such for this communication.

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DPPH Radical Scevenging

Determination of antioxidant activity by DPPH radical scavenging method was

carried out using a method described by Li & Shah (2013) with slight
modification. The decrease of absorbance was monitored after 30 min
incubation period at the wavelength 517 nm at room temperature. All tests
were conducted in triplicate. The antioxidant activity was expressed using
following equation:

DPPH Scavenging effect (%) = (ODcontrol – ODsample)/ ODcontrol × 100

Ferric Reducing Antioxidant Power

The FRAP assay was performed according to the method of Benzie & Strain
(1996).

Total Phenolics Content

The determination of TPC was carried out using Folin-Ciocalteu method as

described by Velioglu et al. (1998). The absorbance was recorded at 725 nm
using 80 % (v/v) methanol as a blank. Total phenolic content was expressed as
milligram of gallic acid equivalents (GAE) per 100 gram of samples.

TBARS Assay

The effect of plant extracts on FeCl3-egg yolk induced lipid peroxidation was
determined as previously described (Pandey et al. 2007).

Statistical Analyses

Statistical analysis for DPPH, FRAP and TPC was performed by one-way ANOVA
with Turkey’s posthoc multiple group comparison. Statistical analysis for MDA
was performed by one-way ANOVA with Dunnet’s posthoc multiple comparison
test. The statistical analysis were performed using GraphPad Prism software
(Version 5). P<0.01 was considered significant for all tests.

RESULTS & DISCUSSION

DPPH Radical Scavenging Properties

DPPH is a radical which is commonly used for determination of antioxidant

properties of plant extracts. Plant with antioxidant properties scavenge DPPH

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radical by donating their free electron to the stable radical DPPH to the yellow-
coloured diphenylpicrylhydrazine. DPPH has an intense violet colour but turns to
a light yellow color as unpaired electrons are scavenged by antioxidants. Results
of this study showed great variance on DPPH radical scavenging activity
between species tested (Figure 1). Among those species, Aquilaria malaccensis
and Tetracera indica exhibit highest DPPH scavenging activities with > 80%
inhibition properties. AML, AMS and TIL DPPH scavenging activities were
88.81±0.63, 92.67±1.19 and 91.74±0.38%, respectively. While root part of
Averhoa carambola (ACR) showed substantially good DPPH scavenging activity
with 93.58% of DPPH inhibition, leaf part (ACL) showed lower activity with
77.40±0.54% of DPPH inhibition.

Figure 1: DPPH radical scavenging activities of A. malaccensis leaf and stem, B. javanica
leaf, stem and root, A. carambola leaf and root and T. indica leaf. Data was
expressed as % of DPPH inhibition ± SD Values with different letter are
significantly different (p<0.05).

Ferric Reducing Antioxidant Power (FRAP)

Result of FRAP (Figure 2) also showed almost similar result to DPPH radical
scavenging activity except that FRAP value of AMS was substantially higher
compared to other extracts. FRAP value of AMS was 343.62±14.75 mM of FeSO4
eqivalent per 100 mg of samples while FRAP value closest to that was ACL with
FRAP value of 205.29±1.82 mM of FeSO4 eqivalent per 100 mg of samples. These
results indicate very good antioxidant properties of A. malaccensis and T. indica
while A. carambola showed variation between leaf and root extrats in both
assays. FRAP is one of most commonly used method to determine the redox
potential of antioxidants (Müller et al., 2011). The principles of FRAP assay is to
determine the ability of tested samples to reduce ferric (Fe3+) to ferrous (Fe2+).
The FRAP and DPPH assay shared a similar principles in evaluating antioxidant
properties which were rely on either single eletron transfer or hydrogen atom
transfer reaction kinetics (Huang et al. 2005). Both DPPH and FRAP methods

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were widely used due to its simple, cost effective and easy to interprete nature
(Wootton-Beard et al. 2011).

Figure 2: Ferric reducing antioxidant power of A. malaccensis leaf and stem, B. javanica
leaf, stem and root, A. carambola leaf and root and T. indica leaf. Data was
expressed as mM FeSO4 equivalent in 100mg of extracts ± SD. Values with
different letter are significantly different (p<0.05).

Total phenolics content (TPC)

Antioxidants activity of plant extracts were commonly attributed to its phenolics

content. Phenolics are plant secondary metabolites which consists of about
8000 naturally occuring compounds and can be abundantly found in plants (Ai et
al. 2016). Epedemiological studies have provided evidences that long term
consumption of diets rich in plant polyphenols offered some protection against
development of cancers, cardiovascular diseases, diabetes, osteoporosis and
neurodegenerative diseases (Pandey & Rizvi 2009).

Total phenolics content were analyzed by Folin-ciocalteu method and

the results were shown in Figure 3. Phenolics content of each species observed
was found significantly different (p>0.05) to each other. Highest phenolics
content was observed in TIL with 22.36±1.94 mg GAE/g extract followed by
AMS, AML, BJS, ACR, BJL, ACL and BJR with 17.60±2.49, 10.86±1.13, 10.71±0.74,
10.00±0.80, 7.88±0.40, 5.48±0.87, 2.18±0.49 mg GAE/g extract, respectively.
Correlation between DPPH and TPC has been made (data not shown) and the
result showed moderate correlation (R2 = 0.6039). This result is similar to that of
previously published report (Bajalan et al. 2016) which indicate R2= 0.602 in
their correlation analysis between DPPH and TPC. Eventhough correlation value
should be higher than that of result we obtained, it could sometimes be lower.
That could result from another types of bioactive constitutes apart from

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phenolics which present those plants with high antioxidant properties which is
not yet explored in this study.

Figure 3: Total phenolics content of A. malaccensis leaf and stem, B. javanica leaf, stem
and root, A. carambola leaf and root and T. indica leaf. Data was expressed as
mg GAE equivalent in 1 g extracts ± SD.. Values with different letter are
significantly different (p<0.05).

Anti-lipid peroxidation properties

TBARS assay is the assay which measure the level of lipid peroxidation in certain
sample such as plasma, organ, tissue and cells. Lipid peroxidation is implicated
to the development of several diseases like atheosclerosis, diabetes and ageing.
In this study, lipid peroxidation were evaluated in vitro using egg yolk as a lipid
rich medium. Oxidative degeneration of lipid or lipid peroxidation produces
MDA and 4-hydroxynonenal (HNE) as bi-products and MDA can reacts with TBA
to produces pink colour MDA-TBA abducts which can be detected by
spectrophotometer at 593 nm. MDA value has become an indicative value for
the severity of lipid peroxidation. As been shown in Figure 4, lipid peroxidation
has been induced when egg yolk homogenate were incubated with 4 mM FeCl3.
This result is in agreement with previous study (Zeng et al. 2013) which indicate
increased level of MDA when it is induced with FeCl3 . Results of this study
demonstrated good anti-lipid peroxidation properties of several extracts such as
AML, ACL, ACR and TIL compared to that of FeCl3-induced negative control (NC)
(p>0.05) with 37.51±4.11, 30.47±5.55, 29.42±0.21 and 39.07±3.63% of
inhibition, respectively. However, those extracts inhibition properties were not
comparable to that of Vitamin C.

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Figure 4: MDA value of different part of A. malaccensis leaf and stem, B. javanica leaf,
stem and root, A. carambola leaf and root and T. indica leaf.. Data was
expressed as µM MDA value ± SD. AML; Values with asteriks (*) are significantly
different (p<0.05) compared to FeCl3-induced negative control (NC).

CONCLUSION

The antioxidant properties of different part from A. malaccensis, B. javanica, A.

carambola and T. indica have been evaluated and the result has showed varied
antioxidant property of those samples. However, some samples like AML, AMS,
BCR and TIL exhibited a very good antioxidant properties.

REFERENCES

Ai, G., Huang, Z.M., Liu, Q.C., Han, Y.Q. & Chen, X. (2016). The Protective Effect
of Total Phenolics from Oenanthe javanica on Acute Liver Failure
Induced by D-galactosamine. Journal of Ethnopharmacology. 186:53–60.
Bajalan, I, Mohammadi, M., Alaei, M. & Pirbalouti, A.G. (2016). Total Phenolic
and Flavonoid Contents and Antioxidant Activity of Extracts from
Different Populations of Lavandin. Industrial Crops and Products,
87:255–260.
Benzie, I.F. & Strain, J.J. (1996). The Ferric Reducing Ability of Plasma (FRAP) as a
Measure of ”Antioxidant Power”: The FRAP assay. Analatycal
Biochemistry. 239:70–76.
Gangopadhyay, N., Rai D.K., Brunton N.P., Gallagher E. & Hossain, M.B. (2016).
Antioxidant-guided Isolation and Mass Spectrometric Identification of
the Major Polyphenols in Barley (Hordeum vulgare) Grain. Food
Chemistry. 210:212–220

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Huang, D., Ou, B., & Prior, R.L. (2005). The Chemistry Behind Antioxidant
Capacity Assays. Journal of Agricultural and Food Chemistry.
53(6):1841−1856.
Ito, N., Hirose, M., Fukushima, S., Tsuda, H., Shirai, T. & Tatematsu, M. (1986).
Studies on Antioxidants: Their Carcinogenic and Modifying Effects on
Chemical Carcinogenesis. Food Chemistry and Toxicology, 24:1099–
1102.
Jin, L., Li, X.B., Tian, D.Q., Fang, X.P., Yu, Y.M., Zhu, H.Q., Ge, Y.Y., Ma, G.Y.,
Wang, W.Y., Xiao, W.F. & Li, M. (2016). Antioxidant Properties and Color
Parameters of Herbal Teas in China. Industrial Crops and Products,
87:198–209.
Li, S. & Shah, N.P. (2013). Effect of Various Heat Treatments on Phenolic Profiles
and Antioxidant Activities of Pleurotus eryngii Extracts. Journal of Food
Science. 78(8):1122–1129.
Müller, L., Fröhlich, K. & Böhm, V. (2011). Comparative Antioxidant Activities of
Carotenoids Measured by Ferric Reducing Antioxidant Power (FRAP),
ABTS Bleaching Assay (aTEAC), DPPH Assay and Peroxyl Radical
Scavenging Assay. Food Chemistry. 129:139–148.
Pandey, N., Chaurasia, J.K., Tiwari, A.P., Tripathi, Y.B. (2007). Antioxidant
Properties of Different Fractions of Tubers from Pueraria tuberosa Linn.
Food Chemistry, 105:219–222.
Pandey, K.B. & Rizvi, S.I. (2009). Plant Polyphenols as Dietary Antioxidants in
Human Health and Disease. Oxidative Medicine and Cellular Longevity.
2(5):270–278.
Velioglu, Y.S., Mazza, G, Gao, L. & Oomah, B.D. (1998). Antioxidant Activity and
Total Phenolics in Selected Fruits, Vegetables, and Grain Products.
Journal of Agricultural and Food Chemistry. 46:4113–4117.
Whysner, L., Wang, C. X., Zang, E., Iatropoulos, M. J., & Williams, G. M. (1994).
Dose Response of Promotion by Butylated Hydroxyanisole in Chemically
Initiated Tumours of the Rat Forestomach. Food and Chemical
Toxicology. 32:215–222.
Wickens, A.P (2001) Ageing and the Free Radical Theory. Respiration Physiology.
128:379–391.
Wootton-Beard, P.C., Moran, A. & Ryan L. (2011). Stability of the Total
Antioxidant Capacity and Total Polyphenol Content of 23 Commercially
Available Vegetable Juices Before and After in vitro Digestion Measured
by FRAP, DPPH, ABTS and Folin–Ciocalteu Methods. Food Research
International. 44:217–224.
Zeng, W.C., Zhang, W.C., Zhang W.H., He, Q. & Shi, B. (2013). The Antioxidant
Activity and Active Component of Gnaphalium affine Extract. Food and
Chemical Toxicology. 58:311–317.

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ASSESSING MALAYSIAN FOREST SPECIES FOR LIPOXYGENASE
INHIBITORY ACTIVITY

Mazura, M.P., Siti Nur Aisyah, M.H. & Hani Idayu, B.

Bioactivity Programme, Natural Products Division, Forest Research Institute

Malaysia, 52109, Kepong, Selangor.

Tel: 03-62797359 Fax: 03-62729805 E-mail: mazura@frim.gov.my

INTRODUCTION

Natural products, including those derived from higher plants have over the
years contributed greatly to the development of modern therapeutic drugs.
Most plant derived secondary metabolites are known to interfere directly or
indirectly with various inflammatory mediators that include arachidonic acid
metabolites. Lipoxygenases (LOXs) catalyse the first step of the arachidonic
pathway leading to a wide variety of bioactive lipids including leukotrienes (LTs).
It has been reported that LTs produced by the activities of LOXs play a major
role in numerous inflammatory and immune responses related to several
diseases including asthma, atherosclerosis and cancer (Steele et al. 1999; Porta
& Rocha-Sosa 2002.). It is of the view that the production of LTs can be
prevented via inhibition of the lipoxygenase pathway, therefore inhibition of the
biosynthesis of inflammatory mediators by blocking the activities of LOXs has
been proposed as an effective therapeutic strategy for attenuating and
protecting against inflammatory and allergic responses (Samuelsson & Funk
1989).
Our tropical forest contains an immensely flora, and this enormous
biodiversity generates a variety of natural resources which help to sustain the
livelihood, economy and the socio-cultural life of local communities. Forest trees
have been found to have potential therapeutic values that could meet the
medicinal needs of people in the rural area. Several of its plant species have
very large applications in the traditional folk medicine; various parts of these
plants are used by the local people as cure for various ailments. Despite such
enormous potential, remarkably few reports are available on these forest
species regarding their biological activities and the active principles responsible
for such activities. Thus, the aim of this study was to evaluate the anti-
inflammatory activity of the selected forest species found in the campus area of
FRIM using the anti-15 LOX model of inhibition.

MATERIALS AND METHODS

Plant Collection and Preparation of Extracts

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Plant materials were collected from the campus of Forest Research Institute
Malaysia and identified by Ms. Tan Ai Lee, a botanist at FRIM. The voucher
specimens were deposited in herbarium for future references. The plant
materials were air dried and ground to mesh size 40-60 using a grinding
machine. The dried pulverized materials were extracted in methanol (soaking in
methanol: 10 times the amount of plant materials) for 72h. The methanol
extracts were filtered and the solvent removed by distillation under reduced
pressure using rotary evaporator.

In vitro 15-Lipoxygenase Inhibitory Assay

Lipoxygenase inhibiting activity was conveniently measured by slightly

modifying the spectrometric method developed by Azhar-Ul-Haq et al. (2004).
Soybean lipoxygenase (1.13.11.12) type I-B and linoleic acid were purchased
from Sigma (St. Louis, MO, U.S.A.). All other chemicals were of analytical grade.
In assay protocol, 160 µl of 100mM sodium phosphate buffer (pH 8.0), 10 µl of
test-compound solution and 20 µl of lipoxidase enzyme solution were mixed and
incubated for 10 min at 25 °C. The reaction was then initiated by the addition of
10 µl linoleic acid (substrate) solution, with the formation of (9Z,11E)-(13S)-13-
hydroperoxyoctadeca-9,11-dienoate, the change in absorbance at 234 nm. Test
compounds and the positive control were dissolved in DMSO. All the reactions
were performed in triplicates in 96-well micro-plate and the absorbance was
read at 234 nm in a microplate reader (FLUOStar Omega: BMG Labtech,
Germany). NDGA was used as reference standard. The percent inhibition was
calculated from the following equation:

% inhibition= (Absorbance of control – Absorbance of test sample)/Absorbance

of control x 100.

RESULTS AND DISCUSSION

In the search for potential anti-inflammatory agent, particularly lipoxygenase

inhibitor from forest species, 45 methanolic extracts of different parts from 15
species were screened for their inhibitory action against lipoxygenase activity
using soybean lipoxygenase. For many in vitro studies, soybean lipoxygenase is
used due to difficulties in obtaining human lipoxygenase for bioassay. Results
for LOX inhibitory activity (%) are shown in Table 1. The chosen test
concentration of 100µg/ml for all the extracts in this bioassay shows varying
degrees of activity. We considered an inhibition below 40% to be insignificant at
the concentration tested. An inhibition between 40% and 70% is considered to
have moderate activity and above 70% as high. NDGA (Nordihydroguaiaretic
acid), a known lipoxygenase inhibitor was used as a positive control. The

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extracts from C. iners (twig), D. dao (all parts), D. aromatica (all parts), D.
oblongifolia (all parts), E. tapos (all parts), G. umbrosus (all parts), M. ferrea
(twig & bark), P. gutta (leaf & bark) and S. koetjape (twig & bark) displayed high
anti-lipoxygenase activity.

The potential of these medicinal plants could be credited to their

secondary metabolites which have the capacity to retard catalysis process of
arachidonic acid pathway. Natural products have been used as medicinal agents
for many years, and at the same time, they served as the starting points for
many semisynthetic analogues. Numerous plants and plant constituents have
already demonstrated anti-inflammatory properties illustrating that there is still
potential for novel innovative anti-inflammatory agents to be identified from
uncharacterized natural plant resources. Moreover, the traditional folklore and
ethno-pharmacological knowledge is helpful to identify plants with presumable
health-beneficial effects or potential anti-inflammatory activities. Therefore, the
results encourage further investigation of in vitro and in vivo studies along with
detailed phytochemical investigations to provide new leads and therapies for
inflammatory related diseases.

CONCLUSION

This preliminary study provides the biochemical evidence that the anti-
inflammatory activity of C. iners, D. dao, D. aromatica, D. oblongifolia, E. tapos,
G. umbrosus, M. ferrea, P. gutta and S. koetjape may act via inhibition of the
enzyme lipoxygenase in the arachidonic acid pathway. Further bioassay-guided
fractionation approaches is needed to identify and/or purify the major active
constituents in the various extracts discussed.

REFERENCES

Azhar-Ul-Haq, Malik, A., Anis, I., Khan, S.B., Ahmed, E., Ahmed, Z., Nawaz, S.A. &
Choudhary, M.I. (2004). Enzymes Inhibiting Lignans from Vitex negundo.
Chem. Pharm. Bull. 52(11):1269–1272.
Porta, H. & Rocha-Sosa, M. (2002). Plant Lipoxygenases, Physiological and
Molecular Features. Plant Physiol. 130:15–21.
Samuelsson, B. & Funk, C.D. (1989). Enzymes Involved in the Biosynthesis of
Leukotriene B4. J. Biol. Chem. 264: 19469–19472.
Steele, V.E., Holmes, C.A., Hawk, E.T., Kopelovich, L., Lubet, R.A., Crowell, J.A.,
Sigman, C.C. & Kelloff, G.J. (1999). Lipoxygenase Inhibitors as Potential
Cancer Chemopreventives. Cancer Epidemiol. Biomarkers Prevent..8:
467–483.
Table 1. Lipoxygenase inhibitory activities of selected Malaysian forest species

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Species Local name Part Lipoxygenase inhibition (%)a
Alstonia angustiloba Miq. Pulai Leaf 15.19 ± 3.47
Twig 9.17 ± 1.23
Bark 19.74 ± 3.37
Carallia suffruticosa Ridl. Meransi Leaf 29.97 ± 4.47
Twig 65.40 ± 2.45
Bark 43.94 ± 1.24
Cinnamomum iners Reinw. Medang Leaf 19.38 ± 1.50
teja Twig 93.92 ± 3.25
Bark 54.27 ± 2.45
Dracontomelon dao (Blanco) Merr. & Sengkuang Leaf 98.83 ± 1.17
Rolfe Twig 93.31 ± 3.61
Bark 90.06 ± 5.05
Dryobalanops aromatica C.F. Gaertn. Kapur Leaf 94.62 ± 1.33
Twig 95.35 ± 2.72
Bark 94.92 ± 3.01
Dryobalanops oblongifolia Dyer Keladan Leaf 84.93 ± 2.14
Twig 96.21 ± 2.52
Bark 94.74 ± 2.98
Dyera costulata (Miq.) Hook.f. Jelutong Leaf 57.74 ± 1.98
Twig 50.66 ± 1.73
Bark 12.44 ± 2.67
Elateriospermum tapos Blume Perah Leaf 78.85 ± 1.24
Twig 97.62 ± 1.25
Bark 96.61 ± 3.39
Fagraea fragrans Roxb. Tembusu Leaf 22.10 ± 1.26
Twig 59.66 ± 4.10
Bark 17.93 ± 0.19
Goniothalamus umbrosus J. Sinclair Kenerak Leaf 95.20 ± 2.43
Twig 98.14 ± 1.86
Bark 96.23 ± 2.00
Mesua ferrea L. Penaga lilin Leaf 66.68 ± 4.54
Twig 94.12 ± 3.29
Bark 80.94 ± 1.77
Palaquium gutta (Hook.f.) Baill. Nyatoh Leaf 71.51 ± 1.33
taban Twig 68.00 ± 1.88
merah Bark 93.09 ± 3.46
Sandoricum koetjape (Burm.f.) Merr. Sentul Leaf 44.41 ± 1.94
Twig 82.68 ± 2.86
Bark 85.63 ± 4.18
Scorodocarpus borneensis (Baill.) Becc. Kulim Leaf 19.08 ± 0.12
Tectona grandis Jati Leaf 19.31 ± 0.22
Twig 13.89 ± 0.81
Bark 25.57 ± 1.63
aLOX inhibition is based on triplicate measurements from at least 3 independent experiments.
Values are mean ± SEM of three parallel measurements.

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EVALUATION OF ANTI-INFLAMMATION AND ANTIPYRETIC ACTIVITIES
OF PIPER NIGRUM FRUITS EXTRACT

Ong, B.K., Lau, M.F., Norulaiman, Y., Nor Hayati, A., Nurhazwani, M.H. & Amira
Rina Nurdiana, M.S.

Natural Products Division, Forest Research Institute Malaysia (FRIM), 52109

Kepong, Selangor.

Tel : 03-62797370 Fax: 03-62729805 Email : ongbk@frim.gov.my

INTRODUCTION

Piper nigrum is part of the larger family of peppers called Piperaceae. There
have been reported with pharmacology actions such as anthelmintic,
antiperiodic, carmination and antipyretic. These species have been traditionally
used for the treatments of inflammation, anti-malarial treatment, weight loss
treatment, treatment for snake venom poisoning and anti-epileptic treatment
(Lindsey 2000). Inflammation is a protective response intended to eliminate the
initial cause of cell injury as well as the necrotic cells and tissue resulting from
the original insult. The most important mechanism of anti-inflammatory action
is the inhibition of prostaglandin (PG) synthesis at the injury site. The anti-
inflammatory potency of different compounds corresponds with their potency
to inhibit cyclooxygenase (COX) (Chakraborty 2004). Pyrexia is a physiological
condition of abnormally high body temperature that occurs due to the resetting
of the hypothalamic thermostat. This condition will manifest during the
occurrence of infection and inflammation. The most important mechanism of
anti-inflammatory action is the inhibition of prostaglandin (PG) synthesis at the
injury site. The anti-inflammatory potency of different compounds corresponds
with their potency to inhibit COX. Nonsteroidal anti-inflammatory drugs
(NSAIDs) are frequently used as antipyretic agents that mostly exert their
antifever effect by inhibiting COX. Thus, COX-2-selective drugs exhibited the
biological properties of reducing or preventing fever (Simmons et al. 2000).

MATERIALS AND METHODS

Preparation of Alcoholic Extract

100 g of dried fruit powder of P. nigrum was stirred overnight in 70% ethanol and
followed by centrifugation at 500 rpm for 10 minutes. The obtained supernatant
was being collected before subjected to concentration using evaporator. Ethanol
was removed. The obtained sample extract was kept in -20oC freezer.

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Animals

Swiss albino mice (20-40 g) and albino rats (100-150 g) of either sex were obtained
from the Animal House unit of Institute Medical Research. These animals were
maintained under suitable nutritional and environmental condition throughout the
experiment at Animal House of Forest Research Institute Malaysia (FRIM).

Antipyretic Activity in Rats

Mice were given 20 ml/kg (20%) suspension of brewer’s yeast subcutaneously.

Initial rectal temperature was recorded. After 18 hours animals that showed an
increase of 0.5°C in rectal temperature were selected. The treated group received
100, 200 and 400 mg/kg of extracted sample orally; control group received 0.3 ml
normal saline. Paracetamol was used as reference drug. Rectal temperature was
determined by digital thermometer at 1, 2, 3, 4, 5, 6 and 7 hours.

Anti-Inflammatory Activity

Carrageenan Induced Rat Paw Oedema Inhibitory Assay

Inflammation was induced by a modification of the method of Sugishita (Antonio

Navarro et al. 2001). 0.1 ml of 1% carrageenan suspension was injected into sub-
plantar surface of the on the left paw of each rat. The control group received 0.3
ml normal saline, the treated and positive control groups received 100, 200 and
400 mg/kg of extracted samples and 100 mg/kg of Indomethacine respectively via
subcutaneous route, 30 min before carrageenan injection. The paw volume is
measured by using plethysmometer at 0, 1, 2, 3, 4 and 5 hours.

TPA Induced Mouse Ear Oedema Inhibitory Assay

12-O-tetradecanoylphorbol-13-acetate, TPA (1 ug) dissolved in acetone (20 ul) was

applied to the ear of male ICR albino mice (25-30 g) by means of a micropipette.
The plant sample was dissolved in acetone and applied topically to the inner
surface of the right ear at 0.5 mg/ear, 1 mg/ear and 2 mg/ear about 30 min before
each TPA treatment. The other left ear which acted as a control was applied with
acetone, the sample vehicle. After 8 hours, the animals were killed by cervical
dislocation. A 6 mm biopsy tissue disc was obtained from both ears. The weights
for both the tissue disc were immediately measured. The increase in the weight of
the right ear punches over that of the left indicating the occurrence of oedema.
The results were expressed as percentage inhibition (IE%). Indomethacin was used
as a positive control for this study.

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Statistical Analysis

Data analysis in this study were performed using one-way analysis of variance
(ANOVA) test between two mean groups; control and test groups, follow by
application of student’s t-test.

RESULTS AND DISCUSSION

Results obtained for the rat paw oedema inhibitory assay, indicating PNE at all
different dosages exhibited anti-inflammatory activity (Figure 1). The paw oedema
volume exhibited reduction after the third hour of administration. The 100 mg/kg of
PNE treatment group showed the least of rat paw oedema between first to the third
hours when compared with other groups. The 200 mg/kg of PNE treatment group
exhibited anti-inflammatory response which is similarly with the Indomethacin,
positive group.

Volume of Oedema Carrageenan-Induced Rat Paw Oedema Inhibitoy Assay

1.60
1.40
1.20
Paw Oedema (ul)

Pa
w 1.00
Oe 0.80
de
m 0.60
a 0.40
(m
0.20
l)
0.00
1 2 3 4 5 6
Hours

negative Indomethacin 100mg/kg 200mg/kg 400mg/kg

Figure 1. Volume of Rats Paw Oedema

Results from rat paw oedema inhibitory assay also indicated that, 100 mg/kg PNE
treatment group gave the highest inhibitory effect (61.83%) at first hour after
carrageenan induction (Figure 2). The group demonstrated the most significant
(p<0.01) percentage inhibition. The 200 mg/kg PNE treatment group has
demonstrated low inhibition percentage during early stage of carrageenan
induction, however the effect increased gradually. The highest dose, 400 mg/kg of
PNE treatment group exhibited higher inhibition percentage during at the later
stage (5 hours) of the experiment, demonstrated significant (p<0.05) percentage of
anti-inflammatory activity.

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 Percentage Inhibition of PNE Using Carrageenan-induced Rat Paw
oedema Inhibitory Assay

70

Percentage Inhibitory(%)
60
50
40
30
20
10
0
1 2 3 4 5 6
Hours

100mg/kg 200mg/kg 400mg/kg Indomethacin

Figure 2. Percentage Inhibition of PNE in Carrageenan Assay

Group with treatment of 0.5 mg/ear/20 μl of PNE exhibited significant effect

(p=0.0001) when compares with the positive control, Indomethacin group. Both 1
mg/ear/20 μl and 2 mg/ear/20 μl of PNEs also demonstrated the availability to
inhibit inflammation significantly (p=0.0012 and p=0.01). The maximum effect of
anti-inflammation of PNE (64.23%) is at concentration of 1 mg/ear/20 μl. In Figure
3, plotted graph indicates that IC50 PNE is 0.5 mg/ear/20μl while it is found that
50% IC50 of Indomethacin is 0.76 mg/ear/20 μl. Thus, concentration of PNE
required to produce 50% of maximum effect is lower than Indomethacin needed.

% Inhibition of PNE Using TPA-Induced Mouse Ear Oedema

Inhibitory Assay 2
y = -20.539x + 117.49x - 103.62
80.0
R2 = 1
70.0
Percentage of Inhibitory (%)

60.0

50.0

40.0

30.0

20.0

10.0

0.0
0 0.5 1 2
-10.0
Concentration (mg/ear/20ul)

Figure 3. Percentage Inhibitions of PNE in TPA Assay

All dosages of PNE produced anti-inflammatory activity in non-concentration

dependent manner because the effect will not usually be linearly proportional
to the concentration (Katzung 2004). In TPA induced mouse ear oedema
inhibitory assay, IC50 of Indomethacin is 0.76 mg/ear/20 μl and IC50 of PNE is 0.50
mg/ear/20 μl. From this observation, PNE is more potent than Indomethacin
because the less concentration of PNE required producing 50% of maximum
effect. The anti-inflammation effect might due to its topical anti-inflammatory

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activity which was associated with reduction in neutrophils infiltration into
inflamed tissues (Antonio et al. 2001). Topical application exhibited higher anti-
inflammatory activities for treatment group with low concentration of PNE. This
observation might due to the effect of bioavailability which caused by the
factors of incomplete absorption and first-pass elimination (Katzung 2004).
Antipyretic Activity
40

39

38
Rectal Temperature ( C)

37

36

35

34
0 1 2 3 4 5 6 7
Hours
negative positive 100mg/kg 200mg/kg 400mg/kg

Figure 4. Rectal Temperatures of All Groups

The antipyretic activity experiment (Figure 4) indicated, the rectal temperature

for all treated groups had decreased significantly (p<0.01) as compared with the
untreated group. Animal group treated with low dosage of PNE (100 mg/kg)
exhibited antipyretic action at first hour after oral treatment. Rectal
temperature from this treatment group decreased significantly (P<0.001) from
38.0°C to 37.1°C within the first hour of treatment. Animal group treated with
highest dose of PNE (400 mg/kg) demonstrated most significantly (p<0.01)
antipyretic effect as compared with Paracetamol. The high dosage treatment
manifests a decreasing of 3°C in rectal temperature at the first hour.

All treated groups exhibited antipyretic activity in concentration dependent

manner. PNE at the highest dose, (400 mg/kg) demonstrated most significantly
effect. This highest dose of PNE also caused occurrence of hypothermia.
Hypothermia is defines as the reducing of core temperature to 35°C or lower.
COX-2 is the primary COX isoform implicated in the febrile response (Netea et al.
2000). Fever induction is accompanied by an increase in COX-2 expression
throughout the vasculature of the brain (Simmons et al. 2000). Endogenous
pyrogens (EP) activity in the plasma is responsible by cytokines with
proinflammatory properties. These proinflammatory cytokines such as IL-1, TNF,
lymphotoxin, IL-6, and IFNs once reach the CNS, via induction of central
mediators such as prostaglandins (PGs), would able to increase the temperature
set point and cause fever (Netea et al. 2000).

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CONCLUSION

Through this study, it can be concluded that Piper nigrum extract (PNE) exerts
significant anti-inflammatory and antipyretic effects. PNE extract exhibited the
antipyretic property in concentration dependent manner, however a non-
concentration dependent manner was observed during the anti-inflammation
evaluation.

ACKNOWLEDGEMENTS

We would like to thank all Natural Products Division staffs, especially the Herbal
Product Development Programme member for their technical assistance.

REFERENCES

Antonio, N., Beatriz, D.L.H. & Angel, V. (2001). Anti-Inflammatory and

Immunomodulating Properties of A Sterol Fraction from Sideritis Foetens
Chem. Biol. Pharm. Bul 24(5): 470–473.
Katzang, B.G.M., (2004). PhD. Basic & Clinical Pharmacology. Pp. 1–49.
Chakraborty, T.H.Y. & Tian-Shang, W. (2004). Anti-inflammatory and Analgesic
Activities from Root of Angelica pubescens. Plant Medical 61:2–8
Daniel, L.S., David, W. & Kenneth, W. (2000). Nonsteroidal Anti-Inflammatory
Drugs, Acetaminophen, Cyclooxygenase 2, and Fever. Clinical Infectious
Diseases 31:S211–S218
Lindsey, W. (2000). Uses of Piperine. The Cary Academy. (Online)
Http://Web1.Caryacademy.Org/Chemistry....Piperin_Home_Page.Htm.
Netea, M.G., Bart, J.K. & Jos, W.M.V.D.M. (2000). Circulating Cytokines as
Mediators of Fever. Clinical Infectious Diseases 31: S178-S184.

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POTENTIAL PROTEOLYTIC ENDOPHYTES FROM LOCAL SHIITAKE
MUSHROOM

Wan Nur Fatihah, W.M.1 & Zaidah, Z.A.1,2

1
School of Biology, Faculty of Applied Sciences, Universiti Teknologi MARA,
40450, Shah Alam; 2Atta-ur-rahman Institute for Natural Products Discovery,
Universiti Teknologi MARA, Puncak Alam Campus, 42300 Puncak Alam, Selangor.

Tel: 0355437851 Fax: 0355444562 Email: drzaidah@salam.uitm.edu.my

INTRODUCTION

Endophytic fungi are fungi that colonize living plant tissues without causing any
immediate negative effects (Hirsh & Braun 1992) and diseases (Petrini 1991)
which involve continual metabolic interaction between fungus and host.
Moreover, numerous promising and structural secondary metabolites have
been isolated (Schulz et al. 2002) and they range from various types of phenolics
to hormone-like compounds. Reports also have shown that the various classes
of endophytic fungi produce auxin (indole acetic acid) and gibberellins (GA3, GA
4 and GA7) (Khan et al. 2015). Other than that, endophytes have also been
found to produce various types of extracellular enzymes, such as phosphatase,
zylanase and cellulases (Corréa et al. 2014). Furthermore, microbial enzymes are
known to play a crucial role as metabolic catalysts, leading to their use in various
industries and applications (Gopinath et al. 2013; Gurung et al. 2013). Besides,
enzyme demands in industries are still mounting as the industries are growing
(Inacio et al. 2015). The study is timely because enzymes from animal and plant
sources have failed in meeting the demands from industries. Therefore,
screening for fungal protease is a crucial need for industrial biotechnology.

MATERIAL AND METHODS

Isolation of Endophytic Fungi from Local Shiitake Mushrooms

200 L of the mushroom solution were pipetted onto PDA and incubated at
30oC for five days. Then, the single and pure colony of endophytic fungi was sub-
cultured into a new PDA by using the streak plate method and incubated at 30oC
for three days.

Screening for Proteolytic Activity

Skimmed milk agar was used to detect the proteolytic activity by measuring the
clear zone of protein hydrolysis on agar plates containing protein substrates.

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The formation of clear halo around the colony was measured which represented
protease activity against protein in the agar.

Identification of Fungi Isolates

Macroscopic and microscopic observations were made based on their

morphological forms. Isolated fungi with positive proteolytic activity were
characterized by using 18S rRNA gene and internal transcriber spacer region
sequence analysis (Anderson et al. 2003). Moreover, DNA extraction was carried
out by using Cetyltrimetylammonium bromide (CTAB) with some modifications.
Primers used were ITS1 (5’-TCCGTAGGTGAACCTGCG-3’) and ITS4 (5’-
TCCTCCGCTTATTGATATGC-3’). A sequence similarity search was performed by
employing the National Centre for Biotechnology (NCBI) database and the Basic
Local Alignment Search Tool (BLAST) (URL:http://www.ncbi.nih.gov/BLAST/).
Sequences with identity match of 97% and above were retrieved.

RESULTS AND DISCUSSION

Proteolytic activity

Moderate proteolytic activities were observed in two out of four isolated

endophytic fungi. The activity was projected through the formation of clear
halos around the samples cultured on Skimmed Milk Agar. The diameters of
these clear halos were tabulated in Table 1.

Endophytic Isolates with Macroscopic and Microscopic Observations

The three potential protease sources were identified based on their

morphological characteristics, macroscopic and microscopic identification. The
top view of isolate H1 (Figure 1) shows the white colony and the color turned to
yellowish-greenish as the fungi colony aged. The yellowish-greenish shade
formed a ring-like zone. From the reverse view, the colour of the colony was
green-whitish. The microscopic observation, as shown in Figure 1, shows
branching conidiophores and irregular verticillates of isolate H1. Based on these
morphological characteristics and that claimed by Samuels (2006), the
endophytic fungi isolate H1 was tentatively Trichoderma sp. The top view of
endophytic fungi isolate H2 (Figure 2) shows white colony, but the color turned
brownish as the fungi colony aged. Meanwhile, from reverse view, the color of
the colony was brown-whitish. Furthermore, the microscopic observation, as
shown in Figure 2 shows the conidia and the conidiophore of isolate H2. The
endophytic fungi phialides grew in-group, forming brush-like structure. Based on
these morphological characteristics (Qin et al. 2015), the endophytic fungi
isolate H2 had been tentatively Penicillium sp.

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Table 1. Diameters of clear halo retrieved from isolated endophytic fungi on
skimmed milk agar
Samples Diameter SD (mm)
Positive (Fusarium sp) 1.000 ± 0.000
Negative (sdH2O) 0.000 ± 0.000
H1 0.633 ± 0.058
H2 0.767 ± 0.058
H3 0.150 ± 0.050
S 0.000 ± 0.000

Figure 1. Macroscopic observation of isolate H1, left is top view, while

right is reverse view on PDA, and bottom is microscopic
observation of isolate H1 under 100 x magnifications.

Figure 2. Macroscopic observation of isolate H2, left is top view, while

right is reverse view on PDA, and bottom is microscopic
observation of isolate H1 under 100 x magnifications.

Endophytic Isolates with 18S rRNA Sequence Analysis

The results of sequences were analyzed by using DNA Base software program,
while identification was performed by conducting a homology search of the ITS
region sequence via BLASTN on DNA database GenBank. Based on the sequence
alignment, two potential proteolytic endophytes had been identified, which were
Trichoderma harzianum (H1) and Penicillium aculeatum strain H23 (H2). The
BLASTN showed that the sequence of the fungal isolate H1 had 99% homology
with T. harzianum strain RCT5 and 97% homology with P. aculeatum strain H23
(H2). These results are summarized in Table 2. The findings of Trichoderma and

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Penicillium species in the current work, in fact, are consistent with the findings
obtained by Kim et al. (2012) and Qin et al. (2015)

Table 2. Endophytic isolate confirmation based on 18S rRNA Sequence

Analysis
Isolate Isolate Name Homology
Isolate H1 Trichoderma harzianum strain RCT5 99%
Isolate H2 Penicillium aculeatum strain H23 97%

CONCLUSION

In conclusion, T. harzianum strain RCT5 and P. aculeatum strain H23 have been
proven to be promising strains to produce proteolytic enzymes. Nonetheless,
purification and further investigation on strain improvement studies would
provide better yield of this enzyme.

ACKNOWLEDGEMENT

The authors would like to acknowledge Universiti Teknologi MARA (UiTM)

Shah Alam Selangor and Ministry of Education Malaysia (MOE) for financing
this project (FRGS/2/2014/STWN10/UITM/02/1 grant).

REFERENCES

Hirsh, Z.M. & Braun, L.H., (1992). Genetically shapping morphology of the
filamentous fungi Aspergillus glaucus for production of antitumor
polyketide aspergiolide A. Microbial Cell Factories 13:73.
Petrini, I.M. & Wilson, N.M. (1995). The unseen majority: endophytes as drivers
of plant biodiversity and productivity in terrestrial ecosystems. Ecology
Letters 11:296–310.
Schulz, B., Boyle, C., Draeger, S., Römmert, A.K. & Krohn, K. (2002). Endophytic
fungi: a source of novel biologically active secondary metabolites.
Mycological Research: 106:996–1004.
Khan, A.L., Hussain, J., Al-Harrasi A., Al-Rawahi A. & LeeI, J. (2015). Endophytic
Fungi: A source of Gibberellins and Crop Resistance to A Biotic Stress.
Critical Rev Biotech 35(1):62–74.
Corrêa, R.C.G., Rhoden, S.A., Mota, T.R., Azevedo, J.L., Pamphile, J.A., de Souza,
C.G.M., Polizell, M.L.T.M., Bracht, A. & Peralta, R.M. (2014) Endophytic
Fungi: Expanding The Arsenal of Industrial Enzyme Producers. J Indus
Microb Biotech. 41(10):1467–1478.
Gopinath, K.O., William, S., Hiroshi, J.N., & Robert, J. (2013). Endophytic fungi.
Importances of endophytic fungi in industries 26 (July):191–205.

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Li, J.H., Kjove, A.Z., Youshino, H., Song, H.B., Gurung, N, Ray, S., Bose, S. & Rai, V.
(2013). A Broader View: Microbial Enzymes and Their Relevance in
Industries, Medicine and Beyond. BioMed Res Intern. 2013:1–18.
Inacio, F.D., Ferreira, R.O., Aparecida, C. & Araujo, V.D.B. (2015). Proteases of
Wood for Fungi with emphasis on the Genus Pleurotus. BioMed Research
International: Pp 1-10.
Anderson, I.C., Campbell, C.D., & J.I. (2003). Potential Bias Of Fungal 18s rdna
And Internal Transcribed Spacer Polymerase Chain Reaction Primers For
Estimating Fungal Biodiversity. Environl Microbiology 5:36–47.
Samuels, G.J. (2006). Trichoderma: Systematics, The Sexual State, And Ecology.
Phytopathology 96:195–206.
Qin, P., Yang, Q., Huang, F., Liu, B. & Li, J. (2015). Effects of Penicillium spp. and
Trichoderma spp. on Pleurotus ostreatus Growth and screening of
Effective Disinfectants. Agric. Science & Tech. 16(3):435–438.
Kim, C.S., Shirouzu, T., & Nakagiri, A. (2012). Trichoderma mienum sp. nov.,
isolated from mushroom farms in Japan. Antonie van Leeuwenhoek
102(4):629–641.

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ASSESSMENT OF PHYTOCONSTITUENTS, ANTIOXIDANTS, AND ALPHA
GLUCOSIDASE INHIBITORY ACTIVITIES OF ENTADA SPIRALIS RIDL. STEM
BARK EXTRACTS

Fatimah, O.R., Siti Zaiton, M.S. & Qamar, U.A.

Pharmaceutical Chemistry Dept., International Islamic University Malaysia,

Bandar Indera Makota, 25200 Kuantan, Pahang, Malaysia

Tel: 01-1331-43623 E-mail: bukolami_fatty@yahoo.com

INTRODUCTION

There are about 29 to 30 different species of the genus ‘Entada ‘out of which
only 6 of them are found in Asia. In Malaysia, the most common specie of
entada is Entada spiralis ridl (family fabaceae synonym: Entada scheffleri) locally
known as Beluru or Sintok. It is a woody climber that can grow up to 25m tall
(Figure 1 (A) & (B))

A B

Figure 1: (A) E. spiralis stem bark (B) E.spiralis leaves.

Sintok possess a wide range of ethnomedicinal uses. The root decoction

is used to treat venereal diseases and hemorrhoid (Aiza et al. 2015) while the
stem bark is used for hair treatment, insect bite and washing. The leave paste is
used to cure postpartum headache. Despite the vast medicinal potential of this
plant, very few studies have been conducted to proof its biological activity. The
aim of this study is to determine the phytochemical component of different
extracts of E. spiralis ridl. stem bark, the antioxidant and α-glucosidase
inhibitory activities.

METHODOLOGY

Air- dried stem bark of E. spiralis was grounded into fine powder, pulverized and
macerated successively using petroleum ether, chloroform and methanol. Each
extract was concentrated in vacuo at 400C. Preliminary phytochemical screening
was conducted according to (Yadav et al. 2011), (Masih & Singh 2012) and

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(Wintola & Anthony 2011). Total phenolic and flavonoid contents were
estimated spectrophotometric and Folin-Ciocalteu’s methods adapted from
Sulaiman et al. (2011) and Ahmed et al. (2015), respectively. The antioxidant
activity was determined using 2,2 diphenyl-1-picrylhydrazyl (DPPH) and 2,2′-
Azino-bis 3-ethylbenzthiazoline-6-sulfonic acid (ABTS), while α–glucosidase
inhibitory activity was measured using modified method of (Telagari 2015).

RESULTS AND DISCUSSION:

Preliminary phytochemical screening of MeOH extract of E. spiralis indicates the

presence of saponin, phenols, flavonoids, terpenoids, tanins, and glycosides
while CHCl3 and petroleum ether extracts were observed to contain only
terpenoids and tannins. Alkaloids were completely absent in all the extracts.
Total phenolics (TPC) and total flavonoids (TFC) contents of these extracts were
observed in decreasing order from MeOH > CHCl3 >Et2O (with TPC values of 42.5
± 8.59, 28.3 ±1.38 and 2.6 ± 0.95 respectively). Overall, TFC were observed to be
much lower than TPC. All the extracts showed radical scavenging activity in a
concentration dependent manner as shown in Table 1.

Table 1: Free radical scavenging activity of various extracts of E. spiralis Ridl.

Sample % inhibition at 1000µg/ml IC50(µg/ml)
EM 77.83 ± 0.64 42.67 ± 4.10
EC 48.76 ± 1.24 472.83 ± 11.20
EP 20.27 ± 4.05 1050 ± 23.21
QC 74.0 ± 2.56 29.82 ± 3.73
AC 90.89 ± 0.05 24.67 ± 0.45
Values were expressed as mean ± SEM of triplicate measurement.
Samples were analyzed using one way ANOVA.

EM (E. spiralis methanol extract), EC (E.spiralis chloroform extract), EP (E

. spiralis petroleum ether extract), QC (quercetin) and AC (ascorbic acid). IC50
(concentration of a sample required to scavenge 50% of the free radicals).
Alpha-glucosidase inhibitory activity of MeOH and CHCl3 extracts were relatively
higher than Et2O with IC50 of 20.67 ±4.62 and 27.21 ± 2.14 respectively.

CONCLUSION

The result of this study suggests that the presence of secondary metabolites
responsible for the biological activities in plant sample is strongly dependent on
the extracting solvent. However, further research is ongoing to validate the
biological activities of this plant and also to isolate the active principles.

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ACKNOWLEDGMENT

We would like to acknowledge the support provided by the ministry of science,

innovation and technology, Malaysia (e-science) Grant No. F13-005-0055 and
also the technical support provided by the Kulliyah of Pharmacy, IIUM.

REFERENCES

Yadav, R.N.S. & Agarwala, M. (2011). Phytochemical analysis of some medicinal

plants. Journal of phytology, 3(12).
Masih, N.G., & Singh, B.S. (2012). Phytochemical screening of some plants used
in herbal based cosmetic preparations. Pp. 111–112 In Chemistry of
Phytopotentials: Health, Energy and Environmental Perspectives.
Springer. Berlin Heidelberg.
Wintola, O.A., & Afolayan, A.J. (2011). Phytochemical constituents and
antioxidant activities of the whole leaf extract of Aloe ferox Mill.
Pharmacognosy magazine, 7(28):325.
Sulaiman, S.F., Sajak, A.A.B., Ooi, K.L., & Seow, E.M. (2011). Effect of solvents in
extracting polyphenols and antioxidants of selected raw vegetables.
Journal of Food Composition and Analysis, 24(4):506–515.
Ahmed, I.A., Mikail, M.A., Bin Ibrahim, M., Bin Hazali, N., Rasad, M.S.B.A., Ghani,
R.A. & Yahya, M.N.A. (2015). Antioxidant activity and phenolic profile of
various morphological parts of underutilized Baccaurea angulata fruit.
Food chemistry, 172:778–787.
Telagari, M., & Hullatti, K. (2015). In-vitro α-amylase and α-glucosidase
inhibitory activity of Adiantum caudatum Linn. & Celosia argentea Linn.
extracts and fractions. Indian journal of pharmacology, 47(4):425.
Harun, A., Siti Zaiton M.S. & Norazian M.H. (2015). Bioassay guided isolation of
an antidermatophytic active constituent from the stem bark of Entada
spiralis ridl. Malaysian Journal of Analytical Sciences, 19(4):752–759

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ANTIFUNGAL ACTIVITY AGAINST PHYTOPATHOGENIC FUNGI EXHIBITED
BY ACTINOBACTERIA FROM ROOT SOIL OF SELECTED PLANT SPECIES

Getha, K., Lili Sahira, H. & Hema Thopla, G.

Bioactivity Programme, Natural Products Division, Forest Research Institute

Malaysia (FRIM), 52109 Kepong, Selangor

Tel: 03 6279 7653 E-mail: getha@frim.gov.my

INTRODUCTION

Over the years, use of chemical fungicides has been restricted due to increasing
concerns about the environmental risks and effects on sensitive non-target
microorganisms. Biological control using microbes has been considered as an
alternative safe method for controlling soil-borne fungal pathogens (Wu et al.
2015). Among the biocontrol agents, filamentous actinobacteria, especially
species belonging to genus Streptomyces have received considerable attention
(Shen et al. 2016). These include biocontrol potential of actinobacteria against
fungal pathogens such as Rhizoctonia and Phytium spp. (Yuan & Crawford 1995),
and Fusarium sp. (Getha et al. 2005).

Many studies reported that the rhizosphere of medicinal plants showed

high abundance of actinobacteria with fungal pathogen-suppressing activity.
Besides protecting roots against invasion by phytopathogenic fungi, these
actinobacteria also played important roles in promoting plant growth (Gangwar
et al. 2011). Taking these factors into consideration, the current study aims to
isolate actinobacteria from soil samples collected from the root region of
various medicinal and forest plant species, and to evaluate their in vitro
antifungal activity against important soil-borne phytopathogenic fungi.

MATERIALS AND METHODS

Soil sample collection and isolation of actinobacteria

A total of 10 soil samples were collected from around roots of medicinal and
forest plant species at Penang National Park, and air-dried at room temperature.
Samples were treated using chemical and physical pretreatment methods, and
suspension spread onto four different selective media according to in-house
protocols. After 4 weeks of incubation at 28 ± 2oC, actinobacteria colonies were
picked and transferred onto fresh ISP2 agar. Isolates were tentatively classified
into five groups based on morphological differences on ISP2 (Numata & Nimura
2003). Spore suspension of isolates was stored in 20% glycerol at -80oC.

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In vitro antifungal assay

A modified cross-plug method was used to screen the actinobacteria isolates for
antifungal properties against phytopathogenic fungi and percent inhibition of
fungal radial growth (PIFRG) was calculated (Getha et al. 2005). Test fungi used
were Fusarium oxysporum f.sp. cubense race 4 and Ganoderma boninense
(obtained from University Malaya), and Rigidoporus vinctus, Cylindrocladium sp.
and Phellinus noxius (obtained from Plant Pathology Lab, FRIM). All cultures
were grown on potato dextrose agar (PDA) at 272oC.

Micromorphological characterisation of active actinobacteria cultures

Selected isolates were streaked on ISP2 agar and sterile 12 mm diameter cover
slips were inserted at an angle of about 45o into the culture surface. After
incubation at 282oC for 14 days, cover slips with attached mycelial growth
were carefully removed from the agar plates, mounted in sterile saline on glass
slide, and examined for spore chain-type under light microscope at 600X
magnification.

RESULTS AND DISCUSSION

Isolation and macromorphological characterisation of actinobacteria

A total of 100 actinobacteria isolates were obtained from the soil samples
collected from root region of different medicinal and forest plant species at
Penang National Park. Fifty percent of the total isolates obtained were from soil
collected around the roots of Dipterocarpus grandiflorus, Alstonia angustiloba
and Aquilaria malacensis, showing a rhizospheric region rich in microbes such as
actinobacteria in these plants (Figure 1). Rhizosphere is a unique biological niche
that supports abundant diversity of microorganisms, and the diversity is
governed by physical and chemical characteristics of the plant species and soil
(Zhao et al. 2012). For example, soil microbes in the rhizosphere of medicinal
plants Matricaria chamomilla, Calendula officinalis and Solanum distichum
grown in desert ecosystem showed a high abundance of gram positive bacteria
with strong pathogen-suppressing abilities (Koeberl et al. 2013).

All isolates were categorised into different macromorphology groups

such as Streptomyces-type (35%), Oligosporic-type (27%), Micromonospora-type
(12%), Nocardioform-type (9%) and Actinoplanetes-type (3%). About 14%
isolates, not falling into these groups, were categorised as others (Figure 2). In
this study, Streptomyces-type isolates were the dominant group obtained from
root soil of most plant species and this finding supported results from other
studies on rhizospheric actinobacterial diversity in medicinal plants (Zhao et al.

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2012). The Streptomyces morpho-type was distinguished from other
actinobacterial colonies by their formation of substrate mycelium and abundant
aerial mycelium with powdery spore mass (Numata & Nimura 2003).
No. of isolates

Figure 1. Total number of representative isolates of actinobacteria from root

region soil of different plant species at Penang National Park

Figure 2. Distribution of actinobacteria morpho-types isolated from root region

soil of different medicinal and forest plants at Penang National Park

In vitro antifungal activity of actinobacteria

The actinobacteria isolates were evaluated for in vitro antifungal activity against
banana wilt pathogen (F. oxysporum f.sp. cubense), oil palm basal stem rot
pathogen (G. boninense), and forest plant root pathogens (R. vinctus,
Cylindrocladium sp. and P. noxius). A total of 11 isolates exhibited strong
inhibitory activity with PIFRG values of more than 70% against one or more test
fungi (Table 1). Interestingly, a high number of these antifungal active
actinobacteria originated from the root region of A. malacensis. It is known that
the antagonistic profile of bacteria is strongly dependent on the different
conditions in the rhizosphere. Berg & Smalla (2009) showed that plant species
was a determinant factor in shaping actinobacteria communities in strawberry

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rhizosphere from the different bulk soil communities. Thus, the current results
supported the possible potential of A. malacensis root ecosystem in hosting a
rich pool of actinobacteria group (Figure 1), and also such actinobacteria
communities with biocontrol potentials. Factors such as root exudation of A.
malacensis could have affected the distribution, diversity and bioactivity of
actinobacteria as indicated in previous study on the diversity of arbuscular
mycorrhizal fungi in the rhizosphere of this plant species (Tabin et al. 2014).

Table 1: Inhibition of in vitro radial growth of different phytopathogenic fungi by

actinobacteria isolates assayed using the cross-plug method

Soil source % Inhibition of fungal radial growth (PIFRG)

Isolate
(plant F. oxysporum G. Cylindrocladium P.
code R. vinctus
species) f.sp. cubense boninense sp. noxius
A001 E. longifolia 69% 72% ± 70% 82%
S.
A005 54% 100% ± ± 80%
calophylla
A012 97% 92% 53% 93% 95%
A013 P. pinnata 82% 77% - ± 88%
A014 69% 94% - 57% 62%
A.
A023 67% 55% ± 65% 88%
angustiloba
A026 51% 86% - ± 99%
A027 65% 100% ± ± 94%
A.
A028 ± 95% ± ± 84%
malacensis
A033 - 73% - ± 71%
A034 - 72% - ± 65%
PIFRG: Getha et al. (2005); ± : 20% < PIFRG ≤ 50%; - : PIFRG ≤ 20%.

Isolate A012 displayed the strongest and widest antifungal activity spectra with
PIFRG > 90% against all test fungi except R. vinctus. It was the only isolate that
showed a PIFRG > 50% against R. vinctus. Isolate A027 also showed potential
activity against G. boninense and P. noxius. Micromorphological characterisation
showed both isolates to have spiral spore chain.

The influence of plant species on the abundance of root-associated

actinobacteria was clearly shown in this study. Rhizospheric soil of A.
malaccensis, a species important as a source of Agarwood products, exhibited
potential in hosting actinobacteria communities and those with antifungal
potential. Bioactive metabolites from the Streptomyces spp. identified in this
study could play a promising role in biological control of phytopathogenic fungi.

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ACKNOWLEDGEMENTS

Authors would like to thank FRIM for the research grant (40-30-04-04-004), and
University Malaya and FRIM Plant Pathology Lab for the phytopathogenic fungal
cultures.

REFERENCES

Berg, G. & Smalla, K. (2009). Plant species and soil type cooperatively shape the
structure and function of microbial communities in the rhizosphere.
FEMS Microbiology Ecology. 68:1–13.
Gangwar, M., Dogra, S. & Sharma, N. (2011). Antagonistic bioactivity of
endophytic actinomycetes isolated from medicinal plants. Journal of
Advanced Laboratory Research in Biology 2:154–157.
Getha, K., Vikineswary, S., Wong, W.H., Seki, T., Ward, A. and Goodfellow, M.
(2005). Evaluation of Streptomyces sp. strain g10 for suppression of
Fusarium wilt and rhizosphere colonization in pot-grown banana
plantlets. Journal of Industrial Microbiology and Biotechnology 32:24–32
Koeberl, M., Schmidt, R., Ramadan, E.M., Bauer, R. & Berg, G. (2013). The
microbiome of medicinal plants: diversity and importance for plant
growth, quality and health. Frontiers in Microbiology 4:400–410
Numata, K. & Nimura, S. (2003). Access to soil actinomycetes in Malaysian
tropical rain forests. Actinomycetologica 17:54–56.
Shen, T., Wang, C., Yang, H, Deng, Z., Wang, S., Shen, B. & Shen, Q. (2016).
Identification, solid-state fermentation and biocontrol effects of
Streptomyces hygroscopicus B04 on strawberry root rot. Applied Soil
Ecology 103:36–43
Tabin, T., Shrivastava, K. & Shukla, A.K. (2014). Distribution and diversity of AM
fungi in the rhizospheric soils of naturally and artificially growing
Aquilaria malaccensis Lamk. trees in Arunachal Pradesh and Assam
states of North East India. Indian Journal of Hill Farming 27(2):41–48.
Wu, Y., Yuan, J., Yaoyao, E., Raza, W., Shen, Q. & Huang, Q. (2015). Effects of
volatile organic compounds from Streptomyces albulus NJZJSA2 on
growth of two fungal pathogens. Journal of Basic Microbiology 54:1–14.
Yuan, W.M. & Crawford, D.L. (1995). Characterisation of Streptomyces lydicus
WYEC108 as a potential biocontrol agent against fungal root and seed
rots. Applied and Environmental Microbiology 61:3119–3128
Zhao, K., Penttinen, P., Chen, Q., Guan, T., Lindstrom, K., Ao, X., Zhang, L. &
Zhang, X. (2012). The rhizospheres of traditional medicinal plants in
Panxi, China, host a diverse selection of actinobacteria with
antimicrobial properties. Applied Microbiology and Biotechnology 94:
1321– 335.

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PHOTOTOXICITY EVALUATION OF MEDICINAL PLANTS AND PRODUCTS

Khoo, M.G.H., Rohana, S. & Mailina, J.

Natural Products Division, Forest Research Institute Malaysia, 52109 Kepong,

Selangor.

Tel: 03-6279 7342 Fax: 03-6272 9805 Email: mary@frim.gov.my

INTRODUCTION

Phototoxicity refers to abnormal skin reactions as a result of skin exposure to

solar irradiation, enhanced by endogenous or exogenous photoreactive
chemicals. Phototoxicity is a form of photosensitivity which includes
photoallergic reaction. Phototoxicity is non-immunologically mediated and is
more common than photoallergy. Exposure to sun is the most common stimulus
to phototoxicity, but artificial light source could also be the trigger. UV radiation
from the solar spectrum that reaches the earth, particularly UVA, which ranges
from 320-400 nm is responsible for most cases of phototoxicity. The UVB (290-
320 nm) is more energetic but UVA penetrates the skin more deeply and it is the
most important spectrum for inducing photodermatosis. Well-documented
reports due to exogenous photosensitivity of UVB are still rare (Goncalo 2010).
Phototoxic compounds enter the skin by topical administration or reach the skin
via systemic circulation by ingestion or parenteral administration. They must be
activated to become phototoxic by absorbing light. The absorbed energy
produces molecular changes, causing toxicity. Drugs such as tetracyclines or
fluoroquinolones are known to cause phototoxic effect (Kumar 2013).
Regulatory agencies such as U.S. Food and Drugs Administration (FDA) and
European Medicines Agency (EMA) have issued guidelines for photosafety
testing, especially on compounds that absorb UVB, UVA or visible light in the
range of 290-700 nm and can reach the skin or eyes (ICH 2015; EMA/CHMP/ICH
2012). Although this regulation is not mandatory in Malaysia, but Malaysia’s
weather may increase the chances of phototoxic event. Therefore, this study is
to examine the phototoxic effect of Alpinia malaccensis var. nobilis, Citrus
hystrix, and Aquilaria malaccensis extracts/essential oils, which are used to
formulate cosmeceutical products.

MATERIALS AND METHODS

Cell culture

BALB/c 3T3 clone A31 cells purchased from ATCC were used for the
phototoxicity assay. Cells were cultured in Dulbecco’s Modified Eagle’s Medium

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(DMEM) supplemented with 10% new-born calf serum, penicillin (100 IU),
streptomycin (100 g/ml); and incubated at 37C, 5% CO2.

Phototoxicity assay

Phototoxicity assay was conducted based on OECD (2004) but with modification.
MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay was
used to quantify cell viability instead of Neutral Red Uptake assay. For each
phototoxicity assay, two 96-well plates were seeded with cells at the density of
1 × 104 cells/well; 100 l/well of complete medium – one plate for irradiation
(+Irr) and the other as the non-irradiated (-Irr) plate. The cells were incubated
overnight at 37C, 5% CO2 to allow attachment to the wells. On the next day,
spent culture medium was removed and cells were washed with Earle’s
Balanced Salt Solution (EBSS) (150 l/well). Then, EBSS containing appropriate
concentrations of test samples were added to each well (150 l/well). Eight
different concentrations were used for each test sample. Chloropromazine and
L-histidine were used as the phototoxic and non-phototoxic reference
compounds, respectively. Control cells were treated with 0.5% DMSO. +Irr plate
was exposed to radiation emitted by a solar simulator with mercury-metal
halide lamp at room temperature for approximately 50 minutes. The energy
density (dose) was set at 5 J/cm2 (measured in the UVA range). This is because
at 5 J/cm2, it has been determined to be non-cytotoxic to BALB/c 3T3 clone A31
cells, yet sufficiently potent to excite chemicals to elicit phototoxic reactions
(OECD 2004). In order to obtain
5 J/cm2 within approximately 50 minutes, irradiance was adjusted to
1.7-2.0 mW/cm2. Meanwhile, -Irr plate was kept in dark at room temperature
for the same duration of irradiation time. After irradiation, solution containing
test sample was removed and cells washed with EBSS (150 l/well). Serum- and
phenol red-free culture medium was added into each well (100 l/well) and cells
were incubated at 37C, 5% CO2 for another 18-24 hours. Viability was assessed
by MTT assay (Mosmann 1983).

Data analysis and interpretation

Percentage of viability relative to control was calculated and to plot dose-

response curves. 50% inhibitory concentration (IC50) was determined using non-
linear regression. Photo-irritation-factor (PIF) and mean photo effect (MPE)
were calculated using Phototox Software version 2.0 developed by ZEBET.

Interpretation of phototoxic effect was based on guidelines by OECD (2004):

No phototoxicity: PIF < 2 or MPE < 0.1.
Probably phototoxicity: 2 < PIF > 5 or 0.1 < MPE > 0.15
Phototoxicity: PIF > 5 or MPE > 0.15

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260
RESULTS AND DISCUSSION
A total of nine test samples comprised of extracts, essential oils, active
ingredients, and formulated products from A. malaccensis var. nobilis, C. hystrix,
and A. malaccensis were tested for their phototoxic effect. Figure 1 shows the
dose-response curves of irradiated (+Irr) and non-irradiated (-Irr) reference
compounds – chloropromazine and L-histidine. Chloropromazine is a phototoxic
compound. The curves show that increasing concentration of chloropromazine
reduced the viability of BALB/c 3T3 clone A31 cells. Irradiation decreased cell
viability even more as compared to its non-irradiated counterpart (Figure 1a).
On the other hand, L-histidine is a non-phototoxic and non-cytotoxic compound.
Whether irradiated or non-irradiated, the compound did not affect cell viability
(Figure 1b). Based on OECD (2004) guidelines, the PIF and MPE values of
chloropromazine (PIF: 10.73 ± 2.26; MPE: 0.46 ± 0.03) clearly places it under the
category of phototoxic and L-histidine (PIF: 0.43 ± 0.23; MPE: 0.10 ± 0.03) as
non-phototoxic (Table 1).
The leaf extract of A. malaccensis var. nobilis (with removed chlorophyll)
exhibited phototoxic effect (Figure 2a). Increasing concentration of the extract
decreased cell viability and with irradiation, the reduction was greater. The
rhizome extract of A. malaccensis var. nobilis also affected cell viability in a
dose-dependent manner but the cytotoxic effect is less severe as compared to
the leaf extract. When irradiated, there was a slight increment of cell death at
high concentrations but not at low concentrations (Figure 2b). Figure 2c shows
the dose-response curve of a product formulated from A. malaccensis var.
nobilis. The product affected cell viability but no phototoxic effect was
observed.
(a) Chloropromazine
% Viability (relative to control)

% Viability (relative to control)

(b) L-histidine
120 140
-Irr -Irr
100 +Irr 120 +Irr
100
80
80
60
60
40
40
20 20
0 0
-1.0 -0.5 0.0 0.5 1.0 1.5 0 1 2 3
Log [Chloropromazine] (g/ml) Log [L-histidine] (g/ml)

Figure 1: Dose-response curves of irradiated (+Irr) and non-irradiated (-Irr)

reference compounds, (a) chloropromazine and (b) L-histidine.

The PIF and MPE values for A. malaccensis var. nobilis leaf extract (PIF:
59.53 ± 14.02; MPE: 0.59 ± 0.12) places it under the category of phototoxic
(Table 1), which is in agreement with the dose-response curves seen in Figure
2a. The PIF value of the rhizome extract (2.32 ± 0.88) shows that it is probably
phototoxic while the MPE value (0.04 ± 0.06) indicates that it is non-phototoxic

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(Table 1). Based on the dose-response curves (Figure 2b), UVA irradiation
caused the extract to become more toxic towards the cells, especially at high
concentration. Therefore, it is probably wise to categorise the rhizome extract
as “probably phototoxic” as a precautionary measures. For the product, the PIF
and MPE (PIF: 1.38 ± 0.15; MPE: -0.05 ± 0.03) values falls under the category of
non-phototoxic (Table 1). This result is similar to the observation based upon
the dose-response curves in Figure 2c.

Table 1. PIF and MPE values of reference compounds and test samples
PIF MPE Classes
Chloropromazine 10.73 ± 2.26 0.46 ± 0.03 Phototoxic

L-histidine 0.43 ± 0.23 0.10 ± 0.03 Not phototoxic

A. malaccensis var. nobilis 59.53 ± 14.02 0.59 ± 0.12 Phototoxic

leaf extract

A. malaccensis var. nobilis 2.32 ± 0.88 0.04 ± 0.06 Probably

rhizome extract phototoxic

Serum formulated from A. 1.38 ± 0.15 -0.05 ± 0.03 Not phototoxic

malaccensis var. nobilis

C. hystrix essential oil 24.05 ± 18.16 0.28 ± 0.14 Phototoxic

Handwash formulated from 1.73 ± 0.46 0.01 ± 0.10 Not phototoxic

C. hystrix

A. malaccensis essential oil 1.12 ± 0.34 0.04 ± 0.04 Not phototoxic

A. malaccensis active 1.33 ± 0.33 0.08 ± 0.07 Not phototoxic

ingredient

Serum formulated from A. 1.11 ± 0.22 0.08 ± 0.04 Not phototoxic

malaccensis

Cream formulated from A. 1.00 ± 0.00 0.02 ± 0.01 Not phototoxic

malaccensis

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262
 % Viability (relative to control)

% Viability (relative to control)

(a) A. malaccensis leaf A. malaccensis rhizome
(b)
140 140
-Irr -Irr
+Irr +Irr
120 120
100 100
80 80
60 60
40 40
20 20
0 0
-1 0 1 2 3 0 1 2 3
Log [A. malaccensis leaf] (g/ml) Log [A. malaccensis rhizome] (g/ml)

% Viability (relative to control)

(c) A. malaccensis - serum
140
-Irr
120 +Irr

100
80
60
40
20
0
0 1 2 3
Log [A. malaccensis - serum] (g/ml)

Figure 2: Dose-response curves of irradiated (+Irr) and non-irradiated (-Irr) A.

malaccensis var. nobilis (a) leaf extract, (b) rhizome extract and (c)
product (serum).

The dose-response curves of C. hystrix essential oil show that it is

phototoxic (Figure 3a) but not the product (handwash) (Figure 3b). The PIF and
MPE values also indicate that the essential oil falls under the category of
phototoxic (PIF: 24.05 ± 18.16 and MPE: 0.28 ± 0.14) while the product is non-
phototoxic (PIF: 1.73 ± 0.46 and MPE: 0.01 ± 0.10) (Table 1). Many Citrus spp.
has been reported to be phototoxic (Marcos & Kahler 2015; van Zoelen & van
Thiel 2014; Quaak et al. 2012; Pomeranz & Karen 2007; Kaddu et al. 2001).
Meanwhile, A. malaccensis essential oil and all the formulated products did not
show sign of phototoxicity effect (Figure 3c-f and Table 1).

CONCLUSIONS
The extracts of Alpinia malaccensis var. nobilis and essential oil of Citrus hystrix
have the potential to become phototoxic, and therefore must be use with
caution. Fortunately, none of the formulated products from these two plant
species were phototoxic. On the other hand, neither Aquilaria malaccensis
essential oil nor products formulated from it were phototoxic. Our findings
provide persuasive evidence that safety evaluation on herbal medicine must not
be neglected.

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 % Viability (relative to control)
% Viability (relative to control)
(a) Citrus hystrix oil (b) Citrus hystrix - hand wash
140 140
-Irr -Irr
+Irr +Irr
120 120
100 100
80 80
60 60
40 40
20 20
0 0
-1 0 1 2 3 0 1 2 3
Log [Citrus hystrix oil] (g/ml) Log [Citrus hystrix - hand wash] (g/ml)
% Viability (relative to control)

(c) A. malaccensis oil

% Viability (relative to control)

(d) A. malaccensis - active ingredient
140 140
-Irr
-Irr +Irr
120 +Irr 120
100 100
80 80
60 60
40 40
20 20
0 0
0 1 2 3 0 1 2 3
Log [A. malaccensis oil] (g/ml) Log [A. malaccensis - active ingredient] (g/ml)
% Viability (relative to control)

% Viability (relative to control)

(e) A. malaccensis - serum (f) A. malaccensis - cream

140 140
-Irr -Irr
+Irr +Irr
120 120
100 100
80 80
60 60
40 40
20 20
0 0
0 1 2 3 0 1 2 3
Log [A. malaccensis - serum] (g/ml) Log [A. malaccensis - cream] (g/ml)

Figure 2: Dose-response curves of irradiated (+Irr) and non-irradiated (-Irr) (a) C.

hystrix essential oil, (b) C. hystrix product (handwash), (c) A. malaccensis
essential oil and A. malaccensis products (d) active ingredient, (e) serum
and (f) cream.

ACKNOWLEDGEMENTS
This work was supported by FRIM’s internal grant – Grant for Research and Pre-
commercialisation, GPP (RPP-0613-BA-02). The author would like to thank
numerous persons especially Mr. Chee Beng Jin and Mr. Azman Mohamed for
their assistance.

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REFERENCES

European Medicines Agency/Committee for Medicinal Products for Human

use/International Conference on Harmonisation (EMA/CHMP/ICH).
(2012). S10 Photosafety evaluation of pharmaceuticals. ICH guidelines
S10. Guidance on photosafety evaluation of pharmaceuticals. Pp 15.
Goncalo, M. (2010). Phototoxic and photoallergic reactions. Contact Dermatitis.
Johansen J.D. et al. (eds.). Pp. 361–376.
ICH. (2015). S10 photosafety evaluation of pharmaceuticals. Guidance for
industry. Pp. 18.
Kaddu, S., Kerl, H. & Wolf, P. (2001). Accidental bullous phototoxic reactions to
bergamot aromatherapy oil. J. Am. Acad. Dermatol. 45(3):458–461.
Kumar, S.B. (2013). Phototoxicity of drugs. J. Pharm. Sci. & Res. 5(12):275–276.
Marcos, L.A. & Kahler, R. (2015). Phytophotodermatitis. Int. J. Infect. Dis. 38:7–8.
Mosmann, T. (1983). Rapid colourimetric assay for cellular growth and survival
application to proliferation and cytotoxicity assays. J. Immuno. Methods:
54:55–63.
OECD. (2004). Test no. 432: In vitro 3T3 NRU phototoxicity test, OECD guideline
for testing of chemicals. 15 pp.
Pomeranz, M.K. & Karen, J.K. (2007). Images in clinical medicine.
Phytophotodermatitis and limes. N. Engl. J. Med. 357(1):e1.
Quaak, M.S.W., Martens, H., Hassing, R.J., van Beek-Nieuwland, Y. & van
Genderen, P.J.J. (2012). The sunny side of lime. J. Travel Med. 19(5):327–
328.
van Zoelen, M.A.D & van Thiel, P.P.A.M. (2014). A sudden rash and blisters on
the left leg in Bali. “Lime disease” or phytophotodermatities as a result of
exposure of lime juice to her left leg. Neth. J. Med. 72(4):230, 234.

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PRELIMINARY OBSERVATION OF PROTEOME PROFILES OF CANCER CELL
LINES AGAINST EPIGALLOCATECHIN GALLATE (EGCG)

Nor Datiakma, M.A & Nurhanan Murni, Y.

Natural Products Division, Forest Research Institute Malaysia, 52109, Kepong,

Selangor

Tel: 03-62797658 Fax: 03-628046658 e-mail: nordatiakma@frim.gov.my

INTRODUCTION

Colorectal cancer is ranked as top three most common cancers among

Malaysians and leading cause of death due to cancer worldwide. Ministry of
Health Malaysia (Malaysian Cancer Statistics - Data And Figure Peninsular
Malaysia, 2006) via National Cancer Registry (NCR) reported that 13.2 % or a
total of 2866 new cases of colorectal cancer reported in 2006. In cancer
research, identification of biomarkers for early detection, prognosis, and
response to treatment; determination of novel targets for drug discovery; and
therapeutic intervention are important goals.
Proteomics is one of the most successful areas of modern biotechnology
research. The term proteome describes the protein complement expressed by a
genome, while proteomics is the study of the whole of proteins determined by a
genome for their expression, localization, interaction, and post translational
modifications (Chevalier 2010)
This paper reports the preliminary study on mode of action of EGCG
compound on colorectal cancer cell lines. 2-DE profiling work of non-treated and
treated Colorectal cancer cell lines with EGCG were performed to observe
protein expressions which may lead to new drug candidate target discoveries.

MATERIALS AND METHODS

Protein Sample Preparation

The colorectal cancer cell line (HT29) was seeded at 10 million cells/ T75 cm2
cell culture flask in DMEM supplemented with 5% FBS for 24 h, and kept at
37 oC, pH 7, 5% carbon dioxide in air. The cells were then treated with
Epigallocatechin Gallate (EGCG) using its IC50 value and incubated at 37 oC, pH 7
with 5% CO2 in air. Cells were sedimented by centrifugation and the pellet was
lysed in lysis buffer containing 6 M urea, 2M Thiourea, 4% CHAPS, 65mM
dithiothreitol (DTT) and protease inhibitor. Protein concentration was
determined using the 2-D Quant Kit (Amersham Biosciences, Uppsala, Sweden).
The quantification protocol was performed as described by the manufacturer

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using Bovine Serum Albumin (BSA) as protein standard. A standard calibration
curve of absorbance values over several protein concentrations was
constructed.

Two Dimensional - Polyacrylamide Gel Electrophoresis (2D PAGE)

Protein samples (70 μg) was diluted in rehydrating buffer containing 6 M urea,
2M Thiourea, 2% CHAPS, 65 mM dithiothreitol (DTT), 2% Pharmalyte of pH 3–10
and 0.0007% bromophenol blue and applied to 11 cm IPG strips of pH 3–10. The
IPGStrips were passive rehydrated at ambient temperature for overnight. The
proteins were then separated by isoelectric focusing (IEF) using the following
parameters with current limit of 50 μA/strip: 300 V for 30 minutes and 3500 V
for 12 000 V/hour. Then, the strips were subjected to two-step equilibration in
buffers containing 6 M urea, 375 mM Tris-HCl, pH 8.8, 2% sodium dodecyl
sulfate (SDS) and 25% glycerol with 65 mM DTT and followed by 260 mM
iodoacetamide and applied to the 12 % SDS-PAGE gels. The second dimension
electrophoresis was performed by SDS-PAGE with an SE 600 Ryby (GE
Healthcare, Sweden) electrophoresis unit. The gels were stained with silver
stain according to Berkelman and Stenstedt (1998). Digital images of the
analytical gels were acquired and analysed quantitatively for differentially
expressed proteins using ImageMaster 2D Platinium 7.0 analysis software (GE
Healthcare, Sweden).

RESULTS AND DISCUSSION

Sample preparation in proteomics is one of the most crucial, yet

problematic, steps for high – quality resolution of proteins in 2D-PAGE.
Most problems can be traced to coextraction of nonprotein cellular
components that can affect protein migrations (Gȍrg et al. 2000).
In this study, total protein extracted from both treated and non-
treated colorectal cancer cells with EGCG obtained yield range 6.8-7.3
µg/µL. Proteins from both samples were undergo 2D-PAGE protein
profiles using immobilized pH-gradient (IPG) strips pH 3–10, 11 cm. from
the profiling work done, at least 690 protein spots were detected in the
silver stained 2D-PAGE gels.
Gel image analysis was performed by comparing the occurrence of
every spot among the two sets of protein profiles from treated and non-
treated colorectal cancer cells with compound from EGCG cell proteins.
Figure 1 below shows the representative gel images of colorectal cell with
compound EGCG. Marked protein spots were the differentially expressed
proteins detected by using ImageMaster 2D Platinium 7.0 analysis
software.

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 From the analysis, 93 differentially expressed protein spots were
detected (selection criteria: >1.5 folding value & p<0.05) where; 42
protein were down-regulated and 51 were up-regulated.

CONCLUSION

Further work on identification of the 91 differentially expressed proteins

in 2D-PAGE protein profiles will be performed. The protein identification
data obtained will be useful to understand the mode of action of the
EGCG compound on colorectal cancer cells HT29.

Figure 1. Representative gel images of colorectal cancer cells with compound EGCG.
Marked protein spots were differentially expressed proteins detected by using
ImageMaster 2D Platinium 7.0 analysis software.

REFERENCE

Chevalier, F. (2010). Highlights On The Capacities of "Gel-based" Proteomics.

Proteome Science :8–23
Gorg, A., Obermaier, C., Boguth, G., Harder, A., Scheibe, B., Wildgruber, R. &
Weiss, W. (2000). The current state of two – dimensional
electrophoresis with immobilized pH gradients. Electrophoresis. 21:
1037–53.
Malaysian Cancer Statistics - Data And Figure Peninsular Malaysia. 2006.
http://www.moh.gov.my/images/gallery/Report/Cancer/Malaysia
CancerStatistics_2006.pdf (Retrieved August15, 2016).

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IN VITRO ANTI-TRYPANOSOMAL ACTIVITY OF SELECTED FOREST PLANT
SPECIES

Norhayati, I., Getha, K., Nurhanan Murni, Y., Ling, S.K., Lili Sahira, H., Asiah,
O., Mohd Hafidz Hadi, A. & Nor Azlianie, A.

Forest Research Institute Malaysia (FRIM), 52109 Kepong, Selangor

Email: norhayati@frim.gov.my

INTRODUCTION

African trypanosomes are protozoan parasites responsible for human African

trypanosomiasis or sleeping sickness are transmitted by the bite of an infected
tsetse fly. The causative agents of this disease are from the Trypanosoma spp.
The few registered trypanocides are frequently toxic, require lengthy parenteral
administration, lack efficacy and are unaffordable for most of the patients
(Legros et al. 2002).

Natural products remain an unmatched source of drugs lead with diverse and
novel chemo types. About 70% of the currently available drugs have origin from
natural product, mainly from plants (Rollinger et al. 2006). It is estimated that
two-third of the world population rely on traditional medical remedies due to
the limited availability and affordability of pharmaceutical products (Tagboto &
Townson 2001). The aim of the present study was to investigate the in vitro anti-
trypanosomal potential effects of 30 extracts derived from 10 forest plant
species.

MATERIALS & METHODS

Plant materials and preparation of extracts

Ten plants species (Table 1) were collected from various locations in FRIM. Plant
parts such as leaves, twig and bark were collected, dried and ground into
powder (40-60 mesh). A total of 200g of each samples was extracted with 2.5 L
methanol (3 x extraction), and dried in vacou. The methanolic extracts obtained
were kept in -20 °C until used.

Trypanosome culture

The experiments was performed with bloodstream form of trypanosomes

Trypanosoma brucei strain BS221. The parasites were cultured in 24 well plates
with Balz Minimal Essential Medium (BMEM), containing Minimal Essential

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Medium (MEM), Mercaptoethanol dilution with 10% heat inactivated fetal
bovine serum (FBS) and supplemented with Balz supplement and incubated in
CO2 incubator.

Antitrypanosomal assay

Five microliter of diluted extract samples in different concentrations were added

into the each wells of plates. A parasite suspension was then added to each well
(95 µl) to give final density of 20, 000 parasites/ ml (total culture volume 100 µl).
Control wells without plant extract were included, as well as control wells with
solvent. After incubation at 37 °C for 72 hours in humidified incubator
containing 5% CO2, the IC50 values were determined using the Alamar blue assay
(Raz et al. 1997). The IC50 was calculated according to Otuguro et al. (2008) and
Pentamidine was included as a standard drug.

RESULTS AND DISCUSSION

Antitrypanosomal activity of 30 methanolic extracts from 10 plant forest species

(nine plant families) was investigated against trypanosome parasite T. brucei
using Alamar blue assay. Results in Table 1 showed that, 33 % of extracts
showed potential antitrypanosomal activity. These active extracts were from D.
oblongifolia, D. aromatic, G. umbrosus, A. angustiloba, S. borneensis and D. dao.
Extracts were calssified as ‘active’ when they showed IC50 value <10 µg/ml
according (Freiburghaus et al. 1996). The highest activity was seen in the bark
extract of D. oblongifolia (IC50: 2.99 ± 0.08 µg/ml), the twig extract of this plant
also showed activity with IC50 value of 4.97 ± 0.28 µg/ml. However, their leaves
were not active. Interestingly, all parts of D. aromatica showed
antitrypanosomal activity (IC50: 4.40 ± 0.82 - 4.83 ± 1.36,µg/ml).

D. oblongifolia and D. aromatica, both species belong to the

Dipterocarpaceae family and are known for their chemical diversity such as
terpenoids, flavanoids, arylpropanoids and oligomer resveratrol (Wan Zuraida et
al. 2010). Resveratol isolated from D. aromatica was reported to show
antiprotozoal (Leiro et al. 2004), anticancer (Aggarwa et al. 2004; Igura et al.
2001), antioxidant (Ito et al. 2003) and anti-inflammation (Luo et al. 2006)
activities. Therefore, the antitrypanosomal activity showed by extracts of D.
oblongifolia and D. aromatic in this study, may contributed by the presence of
resveratrol in these plants.

Goniothalamus spp from Annonaceae family have been studied for

various bioactivities. Compounds acetogenins and styryl lactones have reported
to inhibit broad array of cancer cells including breast, colon, kidney and
pancreatic carcinoma cells (Wiart 2007), antimalarial, and antiprotozoal

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activities (Aali et al. 1999). Besides that, leaf extract of G. umbrosus showed
antioxidant, antibacterial and antiviral properties (Noor-Zarina et al. 2011).

CONCLUSION

This preliminary screening revealed potential plant extracts that showed

antitrypanosomal activity against T. brucei. Both, D. oblongifolia and D. aromatic
showed potential to be further studied to isolate their active compound as
antitrypanosomal agents.

ACKNOWLEDGEMENTS

Authors would like to thank FRIM for the funding used in this study. Thanks are
also due to Mr Mohd Faizul Zaki Mohd Yatim for technical assistance.

REFERENCES

Aggarwal, B.B., A. Bhardwaj, R.S. Aggarwal, N.P. Seeram, S. Shishodia & Y.

Takada. (2004). Role of resrovetrol in prevention and therapy cancer:
preclinical and clinical studies. Anticancer Res., 24(5A):2783–2840.
Freiburghaus, F., Kaminsky, R., Nkunya, M.H.H. & Brun. R. (1996). Evaluation of
African medicinal plants for their in vitro trypanocidal activity. J.
Ethnopharmacol, 55(1):1–11.
Igura, K., T. Ohta, Y. Kuroda & K. Kaji. (2001). Resveratrol and quercetin inhibit
angiogenesis. Cancer Lett. 171:11–16.
Legros, D., Ollivier, G., Gastellu-Etchegorry, M., Paquet, C., Burri, C., Jannin, J. &
Buscher, P. (2002). Treatment of human African trypanomiasis- present
situation and needs for research and development. The Lancet
Infectious Diseases 2:437–440.
Luo, L. & Huang, Y.M. (2006). Effects of resveratrol on the cognitive ability of
Alzheimeros mice.[article in Chinese] Zhong Nan Da Xue Xue Bao Yi Xue
Ban. 31(4):566–568.
Otuguro, K., Ishiyama, A., Namatame, M., Nishihara,A., Furusawa, T., Masuma,
R., Shiomi, K., Takashashi, Y., Yamada, H & Omuro, S. (2008). Selective
and potent in vitro antitrypanosomal activities of ten microbial
metabolites. Journal Antibiotics 61(6):372–378.
Tagboto, S. & Townson, S. (2001). Antiparasitic properties of medicinal plants
and other naturally occurring products. Advances in Parasitology. 50:
199–295.
Wan Zuraidah, W.M.Z., Norizan, A., Liliwirianis, N and Kamaruzaman, J. (2010).
Chemical prospecting of Malaysian dipterocarpaceae from H.S. UiTM,
Pahang Malaysia. World Apply Science Journal 8(9):1050–1055.

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Table 1: Antitrypanosomal activity of 30 methanol extracts from 10 forest plant species
Antitryp
Species Family Local name Plant part activity
(µg/ml)
Carallia suffruticosa Rhizophoraceae Meransi Leaf >10.0
Ridl.
Twig >10.0
Bark >10.0
Dryobalanops Dipterocarpaceae Keladan Leaf >10.0
oblongifolia Dyer 4.97 ±
Twig
0.28
2.99 ±
Bark
0.08
Cinnamomum iners Lauraceae Medang teja Leaf >10.0
Reinw.
Twig >10.0
Bark >10.0
Goniothalamus Annonaceae Kenerak / Leaf >10.0
umbrosus J. Sinclair Mentua binasa 4.31 ±
Twig
0.33
4.85 ±
Bark
0.09
Dracontomelon dao Anacardiaceae Sengkuang Leaf >10.0
(Blanco) Merr. & Rolfe 6.01 ±
Twig
0.30
Bark >10.0
Alstonia angustiloba Apocynaceae Pulai Leaf >10.0
Miq. 4.11 ±
Twig
0.33
Bark >10.0
Scorodocarpus Olacaceae Kulim 5.01 ±
Leaf
borneensis (Baill.) 0.83
Becc. Twig >10.0
Bark >10.0
Dryobalanops Dipterocarpaceae Kapur 4.4 ±
Leaf
aromatica C.F. 0.82
Gaertn. 4.4 ±
Twig
0.81
4.83 ±
Bark
1.36
Elateriospermum Euphorbiaceae Perah Leaf >10.0
tapos Blume
Twig >10.0
Bark >10.0
Palaquium gutta Sapotaceae Nyatoh taban Leaf >10.0
(Hook.f.) Baill. merah
Twig >10.0
Bark >10.0
Note: IC50 Pentamidine; 2.91 ng/ml; Values = IC50 ± SD

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EFFECT OF PH AND INITIAL GLUCOSE CONCENTRATION ON BIOACTIVE
METABOLITES PRODUCTION FROM GANODERMA SP. DSM 24013

Roshan Jahn, M.S.1, Getha, K.1, Mohamad Nazrin, C.S.2, Muhammd Syamil, A.2
& Hema Thopla, G.1

1
Bioactivity Programme, Natural Products Division, Forest Research Institute
Malaysia, 52109 Kepong, Selangor, 2Phytochemistry Programme, Natural
Products Division, Forest Research Institute Malaysia, 52109 Kepong, Selangor

Tel: 03 6279 7653 Fax: 03 6280 4623 E-mail: getha@frim.gov.my

INTRODUCTION

Basidiomycetes fungi have natural anti-bacteria and anti-fungal compounds to

survive their natural environment setting (Hawksworth 2001). Generally, it is
expected for the basidiomycetes to produce these anti-microbial substances
which can be exploited to provide beneficial effects towards the betterment of
human health.

Ganoderma spp. belonging to the basidiomycetes group have been recognized

for its potential to treat a range of diseases such as cancer, hepatopathy,
arthritis, hypertension, neurasthenia and chronic hepatitis (Liu et al. 2011).
Among the species, G. lucidum and G. sinense have been well studied.
Polysaccharides and triterpenoids have been identified as key contributors to
the biological activity and therapeutic effect observed in these species (Qiao et
al. 2007).

Submerged fermentation has been used as an alternative method to produce

bioactive compounds from basidiomycetes. Fermentation media pH and
substrate concentration play a significant role in obtaining substantial yield and
quality products during growth of basidiomycetes (Tang & Zhong 2002). Effects
of glucose concentration and media pH were studied for the production of
microbial metabolites from mycelia culture of Ganoderma sp. DSM 24013 in 5L-
Stirred Tank Bioreactor (5L-STB).

MATERIAL AND METHODS

Seed cultures of Ganoderma sp. DSM 24013

Ganoderma sp. DSM 24013 was obtained from FRIM Microbial Culture
Collection (FRIM-MCC). The fungal strain was grown on potato dextrose agar
(PDA) for 8 days.

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Fungal mycelia plugs were transferred from PDA agar into 150 mL seed medium
(YP5G medium) in 1L Erlenmeyer flask. Cultures were incubated at 282oC and
agitated at 200 rpm for 4 days.

Production culture in Stirred-tank bioreactor (STB)

A 5% (v/v) seed culture of strain DSM 24013 was inoculated into 3L Y5PG
medium in a 5-L STB (Minifors, Switzerland). Fermentation was performed
under the following conditions: agitation speed 150 rpm provided by two six-
bladed Rushton impellers and cascaded maximum at 300 rpm, initial pH 5.5,
aeration 0.5-1.5 vvm, dissolved oxygen (DO) 25±5%, temperature 26°C.

Effects of glucose concentration and media pH

Strain DSM 24013 was cultured for 9 days in 5-L STB using different glucose
concentrations (10, 15, 20, 25 g/L) at two different pH conditions: pH 5.5 (pH
not controlled) and pH 5.5 (pH controlled throughout bioreactor run).

Analytical determination

Fermentation broth samples were collected every 24 hours and subjected to

residual sugar determination according to methods of Gusakov et al. (2011).

Broth samples were extracted with butyl acetate and the solvent layer was dried
in vacuo to determine culture filtrate extract (CfE) yield and anti-MRSA activity.

Anti-MRSA assay

Anti-MRSA activity of CfE was tested against Staphylococcus aureus (MRSA)

ATCC 33591 using paper disk assay method (Bauer et al. 1966).

RESULTS AND DISCUSSION

This study looked at the effects of crucial fermentation parameters as identified

from our previous shake flask studies on the production of bioactive metabolites
from strain DSM 24013. These parameters are pH (controlled at 5.5 and not
controlled in which initial pH was set at 5.5) and effect of carbon source
(glucose) concentrations which was set at 10, 15, 20, 25 g/L.

Results showed when the pH is not controlled in which it was set at 5.5
initially, CfE production was favourable. The maximum CfE production yield

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(g/L), glucose consumption rate (mg/L d-1) and anti-MRSA activity (DIZ, mm)
after 6 days of fermentation were 1.01 ± 0.00, 1.216 ± 0.037 and 14 ± 0.35,
respectively for glucose concentration at 10 g/L (Table 1). On the other hand, in
a pH controlled environment (pH 5.5) CfE yield was very low (0.04 ± 0.01 g/L)
after 6 days of fermentation (Table 1). Growth rate of strain DSM 24013 was low
under this condition and therefore synthesis of the bioactive metabolites in a
culture medium with a controlled pH setting was also low. Effect of pH is
considered important to the development of a large-scale fermentation process
for efficient production of valuable metabolites (Fang & Zhong 2002).

Table 1: CfE production and bioactivity for DSM 24013 at 10 g/L glucose
concentration
Anti-MRSA activity (DIZ,
pH condition CfE yield (g/L)
mm)
pH 5.5 1.01 ± 0.00 14 ± 0.35
(not controlled)
pH 5.5 0.04 ± 0.01 ND
(controlled)
ND: Not determined

Table 2 summarizes the effect of initial glucose concentrations on

substrate consumption rate (by the maximum bioactivity obtained) and CfE
production. The extract yield had shown an increase in parallel with an increase
of initial glucose concentrations. At different initial glucose concentrations, the
maximum CfE yield (g/L) and glucose consumption rate (mg/L d-1) were 1.14 ±
0.00 and 1.71 ± 0.01, 1.35 ± 0.01 and 3.58 ± 0.07, and 2.35 ± 0.03 and 13.88 ±
2.9 at 15, 20 and 25 g/L glucose, respectively. Carbohydrates are important
carbon and energy sources for cultured cells (Mao et al. 2005). From our
previous shake flask studies (data not reported) we found glucose has a
significant effect in the targeted CfE metabolites synthesis.

Table 2. CfE yield and substrate consumption rate at pH 5.5 (not controlled)
Glucose Glucose consumption rate* CfE production yield
concentration (mg/L.d-1) (g/L)
(g/L)
10 1.216 ± 0.04 1.01 ± 0.00
15 1.71 ± 0.01 1.14 ± 0.00
20 3.58 ± 0.07 1.35 ± 0.01
25 13.88 ± 2.9 2.35 ± 0.03
* Glucose consumption rate (g/(L.d) ) = (initial glucose concentration - residual glucose
concentration when maximum bioactivity was achieved) / (cultivation time when
maximum bioactivity reached) (Mao et al. 2004);

With an increase in substrate concentration, strain DSM 24013 is

efficiently utilising the available carbon source, converting the carbon into cell

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proliferation resulting in high yield of CfE production. About two fold increase in
the CfE yield was seen when 25 g/L glucose was used in the fermentation,
compared to when the carbon source level was at 10 g/L glucose.

CONCLUSION

As a conclusion, an initial media pH 5.5 (pH not controlled) and 25 g/L glucose as
the carbon source were favorable for STB fermentation of Ganoderma sp. DSM
24013. At these parameters, a significant 2.3 fold higher in production of CfE
was achieved compared to the yield obtained when 10 g/L glucose was used as
the carbon source.

REFERENCES

Bauer, A.W., Kirby, W.M., Sherris, J.C. & Tuck, M. (1966). Antibiotic susceptibility
testing by a standardized single disk method. Am J Clin Pathol. 45:493–
496.
Fang, Q.H. & Zhong, J.J. (2002). Effect of initial pH on production of ganoderic
acid and polysaccharide by submerged fermentation of Ganoderma
lucidum. Process Biochemistry. 37(7):769–774.
Gusakov, A.V., Kondratyeva, E.G. & Sinitsyn, A.P. (2011). Comparison of two
methods for assaying reducing sugars in the determination of
carbohydrase activities. Int. J. Anal. Chem. 2011:283658.
10.1155/2011/283658
Hawksworth, DL. (2001). Mushrooms: the extent of the unexplored potential.
Int J Med Mushrooms. 3:333–337.
Liu, G.Q., Ren, G.P., Wang, X.L. & Zhao, Y. (2011). Statistical optimization of the
key medium components by response surface methodology to promote
ganoderic acid formation by medicinal mushroom Ganoderma sinense in
submerged culture. Journal of Medicinal Plants Research. 5(3):425–431.
Tang, Y.J. & Zhong, J.J. (2002). Fed batch fermentation of Ganoderma lucidum
for hyperproduction of polysaccharide and ganoderic acid. Enzyme and
Microbial Technology. 30(1):20–28.
Qiao, Y., Zhang, X.M. & Qiu, M.H. (2007). Two novel lanostane triterpenoids
from Ganoderma sinense. Molecules. 12:2038–2046.

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PRELIMINARY STUDY ON PREPARATION OF GELATIN NANOPARTICLES
FOR PLANT EXTRACT TOWARDS PRODUCT DEVELOPMENT

Saidatul Husni, S., Nor Azah, M.A., Nur Nadiah, R. & Nurul Insyirah, M.A.

Natural Product Division, Forest Research Institute Malaysia, 52109

Kepong, Selangor, Forest Research Institute Malaysia, 52109 Kepong,
Selangor

INTRODUCTION

Nanoparticles (NPs) had received growing attention in these recent years

especially as delivery system for bioactive molecules by entrap, encapsulate and
absorb them. Size of NPs are ranging from one to one thousand nm (Bahareh et
al. 2014). Some advantages using nanotechnology in developing nanometric
delivery system are bioactive compounds that been encapsulated can be
protected from unwanted reactions during the delivery process, impact on the
final product’s organoleptic properties can be minimised and the bioactivity of
encapsulated product can be enhance due to its high mass transfer rate on the
site of action (Spigno et al. 2013).

In nanopharmaceutical and medical application, gelatin has been widely

use because of its properties such as natural non-toxic biopolymer,
biocompatible and biodegradable (Habashi, 2002). Furthermore, gelatin
nanoparticles have been used as a carrier for delivery drug. This is because
gelatin molecules contain repeating sequences of glycine, proline and alanine
amino acid triplets, which offer possibilities for chemical modification and drug
attachment (Weber et al., 2000). Other benefits of gelatin nanoparticle (NPs)
are simple, low cost and reproducible production which make gelatin NPs has
potential to be upscaling and commercially attractive (Zwiorek et al. 2004).

According to Bahareh et al. (2014), quality of gelatin NPs from one-step

desolvation method is unsatisfying. Reproducibility result of this technique is
inadequate due to the broad size distributions and the formulation tends to be
unstable (Zwiorek et al. 2005). In order to overcome the problem, two-step
desolvation method is applied and found suitable in preparing gelatin
nanoparticles. The first step in the technique is discard low molecular weights
components of gelatin which would make the production of stable nanoparticles
with a uni-modal size distribution impossible. After sediment is resolved and pH-
adjustment is been done. Then, the in-situ particles are prepared by a second
desolvation step (Zwiorek et al. 2004).

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MATERIALS AND METHODS

Preparation of Gelatin Nanoparticles

Gelatin nanoparticles (NPs) were prepared by a two-step desolvation method,

modified from El-Houssiny et al. (2015). Gelatin was dissolved in distilled water
at 60 ℃ water bath. After fully dissolved, the gelatin solution is added dropwise
in acetone at ratio of 1:1 (gelatin solution:acetone) and left for 24 hours. The
precipitate produced high molecular weight gelatin. The solid gelatin particles
were separated from solution and were dissolved in distilled water. The pH of
solution was adjusted to 8.76 using triethanolamine. In order to produce gelatin
NPs and plant extract loaded gelatin NPs, the solution was separated into two
different beakers. Selected plant extract was dissolved in gelatin NPs at 4% (w/v)
to produce plant extract loaded gelatin NPs. Then, both solutions were drop-
wise into acetone at ratio gelatin NPs or plant extract loaded gelatin NPs to
acetone is 1:3 and left for overnight. 1M calcium chloride was added to form
cross link. The acetone was removed by using rotary evaporator and all NPs
were freeze-dried, weight and stored as powder prior to particle size
determination.

For comparison, all NPs samples were dissolved in distilled water at

0.05% (w/v) and were placed in dialysis tubing cellulose membrane (average flat
width 25 mm, 3500 Dalton pore size) and dialysed in running water. During this
process, particles with 35 kD molecular weight were eliminated. The solution
samples in dialysis tube were measured for particle size.

Particle size analysis

Particle size measurement was carried by dynamic light scattering (DLS) using
Zetasizer ZS (Malvern Instruments, UK). 1.5ml of 0.05% (w/v) of all NPs solutions
were injected into 3 ml disposable cuvette and measured in triplicate at 25C.
The result were described in form of average size in diameter (nm) and
polydispersity index (PDI).

RESULTS AND DISCUSSION

Formation of GNPs by two step desolvation also known as nanoprecipitation as

the NPs are formed during the precipitation process. This is done by adding
desolvating agent into gelatin solution to reduce the water availability and
resulted low hydration moreover the protein chain precipitated as nanoparticle
(El-Houssiny et al. 2015). In the second step of desolvation, NPs were formed by
adjusting the pH value of gelatin solution at 8.76 for calcium chloride (CaCl2) to
make the carbonyl group (-COOH) predominant for crosslinking with Ca+2. In this

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study, three different concentration of gelatin NPs were prepared; 9.0%, 5.0%
and 1.25% (w/v) and labelled as GNPs-1, GNPs-2 and GNPs-3 respectively. For
each concentration, 4% plant extract were loaded and namely as P-GNPs-1, P-
GNPs-2 and P-GNPs-3.

The particle size measurement were carried out for both GNPs and P-
GNPs, before and after dialysis. Based on Table 1, the particle size of GNPs is
smaller when higher concentration of gelatin was used. For example, the
particle size for GNPs-3 before dialysis is 68.3 nm compared to GNPs-1, 97.7 nm
and after dialysis process, the particle size of GNPs-3 is 87.6 nm compared with
GNPs-1, 143.6 nm. The plant extract were successfully loaded in gelatin NPs as
all sample were larger than gelatin NPs at the same gelatin concentration. For
instance, the particle size for P-GNPs3 after dialysis process is 136.5 nm
compared to GNPs-3 87.6nm. However, after dialysis process, particle size of
GNPs-1 is larger than P-GNP1, this condition may due to instability of NPs and
likely to agglomerate. From the result, P-GNPs2 after dialysis process has the
smallest particle size, 96.6nm, while P-GNPs-2 before dialysis process has the
largest particle size. Based on this result, P-GNPs may be transmitted through
the intracellular space in skin epidermis and stratum corneum as the gap
between skin cells in epidermis layer is 75 nm and the gaps between cells in
stratum corneum are 100-200nm (Choi et, al. 2014).

Polydispersity index (PDI) value of the samples after dialysis is smaller

than the value of samples before dialysis. From the result, higher concentration
of gelatin in NPs formulation gives lower PDI value. This is due to the smaller
particles had been removed through dialysis process. However, measurement
by DLS was reported to gives bigger size compared to size observed with
transmission electron microscopy (TEM) (El-Houssiny et a., 2015). According to
Prabha et al, (2002), particle size measurement by DLS gives hydrodynamic
diameter rather than the actual diameter of hydrophilic nanoparticle.

CONCLUSION

Gelatin concentration of 9.0% was found suitable to be used for producing

gelatin NPs and encapsulate 4.0% of plant extract. From the result, P-GNPs3
formulation was chosen as the best among other formulation for plant extract
loaded GNPs as it gives particles size below than 200 nm and low PDI value.
However, further study must be conducted to determine the particle size using
other technique such as TEM or SEM and carry on formulation study in
developing final product with P-GNPs 3 as the bioactive ingredient and finally to
evaluate the efficacy and safety.

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ACKNOWLEDGEMENT

Authors would like to express our gratitude to all Natural Product Division staff,
Dr. Zunoliza Abdullah and Mrs. Norulaiman Yusoff for their time, help and
advice.

Table 1: Particle size (average diameter, nm) and PDI value of GNPs and P-GNPs

Sampel Gelatin Type sample Before dialysis After dialysis

concentration Z-Ave Z-Ave
PDI PDI
(%) (nm) (nm)
GNPs 1 Gelatin NPs 97.7 1.0 143.6 0.6
Plant extract
1.25
P- GNPs1 loaded gelatin 109.1 0.6 97.3 0.5
NPs
GNPs 2 Gelatin NPs 86.0 0.7 83.8 0.6
Plant extract
5.0
P- GNPs2 loaded gelatin 153.8 0.6 96.6 0.5
NPs
GNPs 3 Gelatin NPs 68.3 0.5 87.6 0.7
9.0 Plant extract
P- GNPs3 loaded gelatin 135.1 0.4 136.5 0.3
NPs

REFERENCE

Baherah, A., Nourpanah, P., Mohammad, R. & Shahram, A. (2014). Producing

Gelatin Nanoparticle as Delivery System for Bovine Serum Albumin.
Iranian Biomedical Journal 18:34–40.
Choi, W., No, R. H., Kwon, H.S. & Lee, Y.H. (2014). Enhancement of skin anti-
inflammatory activities of Scutellaria baicalensis extract using a
nonoencapsulation process. Journal of Cosmetics and Laser Therapy 16:
271–278.
El-Houssiny, A.S., Ward, A.A., Mostafa, D.M., Ad-El-Messieh, S.L., Abdel-Nour,
A.L., Darwish, M.M. & Khalil, A.A. (2015). A Newly Developed Transdermal
Treatment of Osteoarthritis Using Gelation Nanoparticles. International
Journal of Biological & Pharmaceutical Research 6:264–272.
Habashi, A.A. (2002). HPLC determination of glucosamine in rat plasma. Journal
of Pharm. Pharmaceut Sci. 5:176–180.
Prabha, S., Zhou, W.Z., Panyam, J. & Labhasetwar, V. (2002) Size-dependency of
nanoparticle- mediated gene transfection: studies with fractioned
nanoparticles. International Journal of Pharmaceutics 244:105–115

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Spigno, G., Donsi, F., Amendola, D., Sessa, M., Ferrari, G., & De Faver, D.M.
(2013). Nanoencapsulation systems to improve solubility and antioxidant
efficiency of a grape marc extract into hazelnut paste. Journal of Food
Engineering 114:207–217.
Weber, C., Coester, C., Kreuter, J. & Langer, K. (2000). Desolvation process and
surface characterization of protein nanoparticles. Int J Pharm. 7:91–102.
Zwiorek, K., Kloeckner, J., Wagner, E. & Coester, C. (2004). Gelatin Nanoparticles
as a New and Simple Gene Delivery System. Journal of Pharmaceutical
Science 7:22–28

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A BIOLOGICAL APPROACH: INTRACELLULAR ANTIOXIDANT ACTIVITY ON
CAMELLIA SINENSIS (GREEN TEA) LEAF EXTRACT USING HUMAN RED
BLOOD CELLS

Shalini, M., Vimala, S. & Juliza, M.

Bioactivity Programme, Natural Products Division, 2Publication Branch, Technical

Service Division, Forest Research Institute Malaysia, 52109 Kepong, Selangor.

Tel: 03-6279 7355 Fax: 03-62729805 E-mail: shalini@frim.gov.my

INTRODUCTION

Recently, the evaluation of antioxidant bioavailability has gained interest among

researchers globally. A diet rich in antioxidant may not be biologically active. It is
important to assess the antioxidant efficacy by taking into consideration the
cellular antioxidant bioavailability, cellular uptake, distribution and metabolism.
Intracellular antioxidant activity in red blood cells (IAA-RBC) is a more
biologically relevant method than the in-vitro antioxidant assays. The IAA-RBC
method was designed comparable to oxygen radical absorbance capacity
(ORAC) which tested the antioxidant availability in living cells in-vitro (Honzel et
al. 2008) and antioxidant uptake in-vivo (Jensen et al., 2008). The choice of RBC
as the model for intracellular antioxidant protection is important (Buehler &
Alayash 2005) because mature RBC prevent oxidation and inflammation by
scavenging harmful ROS. IAA-RBC bioassay evaluates antioxidant protection that
shows the capability to absorb and protect living cells from oxidative damage.

Tea is a popular drink in the world, today and throughout history (Dattner &
Boussabba 2003). Traditional practitioners in China used green tea (GT) as a
stimulant, a diuretic and an astringent. Epidemiology studies have shown GT to
have health promoting benefits (Brown 1999; Friedman 2007). Polyphenols in
GT neutralize free radicals and reduce oxidative damage that contributes to
various health problems including cancer and heart disease (Kim et al., 2003). In
recent years, green tea extract has been included as an ingredient in
nutraceuticals and functional food products. GTE has been used in a variety of
food products including bread (Wang & Zhou 2004), biscuits, dehydrated apple
(Lavelli et al. 2010) and various meat products (Mitsumoto et al. 2005). GTE in
general has been identified as a source of antioxidants, its uptake and
bioavailability in human RBC against oxidative stress have been poorly
described. The aim of this study is to investigate effects on intracellular
antioxidant activity of GTE in human red blood cells. In-vitro antioxidant
activities of GTE were determined using ORAC and DPPH assay. The total
phenolic content (TPC) was also analyzed.

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MATERIALS AND METHODS

Sample Collection

Green Tea Leaf Extract (extract reference) was purchased from ChromaDex

IAA-RBC Analysis

The IAA-RBC assay was performed as described by Honzel et al. 2008, with some
modifications. The packed RBCs were diluted 2:100 in PBS before the incubation
with DCFH-DA and antioxidants. A stock solution, 20 mM DCFH-DA, was
prepared in DMSO and diluted in PBS to obtain a 76 µM working solution. The
RBC suspension was incubated at 37 0C with 25 µM DCFH-DA and GTE solutions
at the 200ug/ml concentrations. At the end of incubation, the RBCs were
washed twice in PBS to remove the antioxidants remaining in the extracellular
medium, resuspended in cold PBS and 260 µl transferred in a 96-well microplate
that was placed in the BMG Omega Fluostar Fluorescent Spectrophotometer.
Then 40 µl of 4.5 mM AAPH was applied to the cell suspension and the
fluorescence was read at 485 nm ex. and 520 nm em. each minute. In each
experiment quercetin was used as a standard. The fluorescence was then
monitored kinetically with data taken every 5 minute. The plate reader was
controlled by MARS data analysis software. IAA-RBC values were calculated
using MARS Data Analysis Software

ORAC Analysis

a. Lipophilic ORAC Assay

For the lipophilic antioxidant assay, the 2 mg of GTE was dissolved in 2ml of
acetone and then diluted with 8 ml of a 1.4% RMCD solution (50% acetone/50%
water, v/v). Any further dilution was with the 1.4% RMCD solution. The 7%
RMCD solution was used as a blank and to dissolve the Trolox standards for the
lipophilic assay. 25 µl of this solution was added to the 96 well solid black
microplate. The wells received 150 µl of fluorescein solution in the microplate.
The plate was then allowed to equilibrate by incubating for 10 minutes at 370C.

b. Hydrophilic ORAC Assay

Further dilution of the hydrophilic fraction was made with phosphate buffer. A
25 µl portion of the diluted GTE was added to a well in a 96 well solid black
microplate. The fluorescein solution and AAPH were added in the same manner
as that for the lipophilic assay. To the interior experimental wells, 150 ul of
working sodium fluorescein solution was added. The blank wells received 25 ul

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of Trolox dilution. The sample wells received 25 ul of samples. The plate was
then allowed to equilibrate by incubating for 10 minutes at 370C.

c. ORAC Instrument procedure

The both lipophilic and hydrophilic ORAC assay was performed in BMG Omega
Fluostar Fluorescent Spectrophotometer with injector was used with an
excitation filter of 485 nm bandpass and emission filter of 528 nm bandpass. The
plate reader was controlled by MARS data analysis software. Reactions were
initiated by the addition of 25 ul of AAPH solution (240mM) using the microplate
reader’s injector for a final reaction volume of 200 ul. The fluorescence was then
monitored kinetically with data taken every minute. The fluorescence of each
well was measured by top reading every 60 seconds. ORAC values were
calculated using MARS Data Analysis Reduction Software.

DPPH Free Radical Scavenging Activity

Antioxidant reducing activity on DPPH radical is estimated according to the

method of Blois (1958) with modification into a high-throughput microplate
system. GTE (50µl of 1.0 mg/ml) is added to 50µl of DPPH (FG: 394.32) (1mM in
ethanolic solution) and 150µl of ethanol (absolute, AR Grade) in a 96 well
microtiter plate, in triplicates. The plate is shaken (15s, 500rpm) and left to
stand at room temperature for 30 minutes. The absorbance of the resulting
solution is measured spetrophotometrically at 520 nm.

Determination of Total Phenolic Content

Determination of TPC on GTE was performed using Folin-Ciocalteu reagent

according to the method of Singleton and Rossi, 1965, with modifications into
high-throughput microplate system. Distilled water 0.1 ml and 0.1 ml diluted
Folin-Ciocalteu reagents (0.1 ml/0.9 ml) were added to 50 μl GTE. The sample
(GTE with Folin-Ciocalteu reagent) were set aside for 5 min before 0.1 ml 7.5%
sodium carbonate (w/v) was added. After 2 hours, the absorbance was
measured at 765 nm wavelength using a spectrophotometer. The calibration
curve of gallic acid (GA) was used for the estimation of sample activity capacity.

RESULTS AND DISCUSSION

Free radicals attack biological molecules, causing damage to cells and tissues
which lead to aging symptoms and age-dependent conditions. RBC are living
cells that carry oxygen to various organs in the human body. RBC also plays an
important role in antioxidant protection against oxidative stress (Buehler &
Alayash 2005).

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Table 1: Antioxidant activity of green tea extract (GTE) in intracellular
antioxidant assay

Test sample IAA-RBC

µmol Quercetin equivalent (QE) per 100 g
Green tea leaf extract 5,423 ± 150
(GTE)
IAA-RBC value is based on triplicate measurements from at least 3 independent experiments.
Values presented is averages ± SEM of three replicates.

In the current study, the GTE cellular protection and bioavailaility were assessed
in human RBC using IAA-RBC method. The GTE showed IAA-RBC value of 5,423 ±
150 µmol Quercetin equivalent (QE) per 100 g (Table 1). The results
demonstrated that GTE has biologically active antioxidants that were able to
cross plasma the membrane into the intracellular matrix of RBC. Antioxidant
uptake from GTE could protect living cells from radical induced oxidative stress
within the cells. The intracellular antioxidant activity within RBC could prevent
further damage to cells.

Table 2: Antioxidant activity of green tea extract (GTE) in in-vitro antioxidant

Assays
Test In-vitro Antioxidant Bioassay
sample Hydrophilic ORAC Lipophilic DPPH TPC
ORAC
µmol Trolox equivalent (TE) per (%) IC50 (mg
100 g (µg/ml) GAE/g)
Green tea 410,758 1,850 98.42 ± 6.97 ± 0.02 1,757 ±
leaf 0.66 17.96
extract
(GTE)
Results above is based on triplicate measurements from at least 3 independent experiments.
Values presented are averages ± SEM of three replicates.

The values of ORAC assay, both lipophilic and hydrophilic in similar conditions
for GTE are presented in Table 2. In GTE, both lipophilic and hydrophilic ORAC
showed total antioxidant capacity of 412,608 ± 3,280 µmol Trolox equivalent
(TE) per 100 g which is higher than the common plant extracts (e.g grape seed
extract, 112,470 µmol Trolox equivalent (TE) per 100 g) as reported by
Brunswick Laboratories. GTE was further measured for in-vitro DPPH radical
scavenging assay and inhibition concentration (IC50) showed 98.42 ± 0.66 % and 6.97 ±
0.02 µg/ml, respectively. The results indicated that the tested GTE possessed
high radical quenching properties which were able to donate electrons to free
radicals and stabilize them in the biological and food system. Furthermore, TPC,
1,757 ± 17.96 mg GAE/g of extract, corresponded to the high in-vitro antioxidant

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activities. In-vitro antioxidant assays example DPPH and ORAC, evaluated the
potential of natural products in counteracting ROS and act as an important tool
for screening purposes. However, cellular models better reflect the antioxidant
activity in physiological conditions for potential bioactivity. Thus, it is important
to combine both cellular and in-vitro antioxidant assays in natural product
development, formulation and screening for complete evaluation of the
antioxidant activity.

CONCLUSION

The antioxidants present in GTE has potential biological antioxidant activity in

protecting living cells and contribute towards health promoting effect at cellular
level.

ACKNOWLEDGEMENT

This research work was financially supported by Forest Research Institute of

Malaysia (FRIM) under the Research, Pre-Commercialisation & Publication
Grants (RPP) (41-31-10-03-002). National Blood Bank Malaysia for the human
blood supply.

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 using a multichannel liquid handling system coupled with a microplate
fluorescence reader in 96-well format. Jourl of agril and food chemistry,
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crossover study. Jourl of agril and food chemistry, 56(18):8326 – 8333.
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Mitsumoto, M., O’Grady, M.N., Kerry, J.P. and Buckley, D.J. (2005). Addition of
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stability during chilled storage in cooked or raw beef and chicken patties.
Meat Science 773–779.
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process. Jourl of agril and food chemistry :8224–8229.

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Acknowledgement & Sponsors
The organizer wishes to thank:

Kementerian Sumber Asli dan Alam Sekitar (NRE)

Malaysian Bioeconomy Development Corporation Sdn Bhd
Majlis Amanah Rakyat (MARA)
CIMB Bank
Biotek Abadi Sdn Bhd
Medigene Sdn Bhd
Pusat Penyelidikan Perubatan Herba, Institut Penyelidikan Perubatan Malaysia
Hydroscience Engineering
Active Advance Technology Sdn Bhd
Nourish Care Sdn. Bhd.
Nature Profusion Sdn. Bhd
Bionature Formula Sdn. Bhd
Poly-Xtract Sdn. Bhd.
Tyzo Sdn Bhd
TG (Tok Guru) Green Tech Industry Sdn Bhd
The Cucurbits Company Sdn Bhd
Syarikat Zulkufli Bamadhaj Sdn Bhd
MSI Technologies (Malaysia) Sdn Bhd
Avanticell Asia Pacific Sdn Bhd
Straits Scientific (M) Sdn Bhd
Ika Works (Asia) Sdn Bhd

Sincere appreciation also to the speakers, participants and individuals who have
directly or indirectly contributed to the successful organization of this seminar.

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Organising Committee
Sub Committee & Members
Patron:
Asmar Hj Hassan
Y.Bhg Dato’ Dr Abdul Latif Mohmod
Muhamad Jefri Govel
Kamal Ariffin Ismail
Chairperson: Maria Arlene Jackan Siba
Dr Rasadah Mat Ali Mohamad Nazrin Che Saad
Dr Asiah Osman Mohd Sharul Nizam Usof
Zaridah Mohd Zaki
Secretary: Roshan Jahn Mohd Salim
Dr Mazura Md. Pisar Ummu Hani Badron
Lili Sahira Husin Rohana Sahdan
Nurul Haslinda Makhtar
Treasurer: Nor Azlianie Abllah@Awang
Ong Boo Kean Siti Nur Aisyah Mohd Hashim
Mohd Radzi Ahmad Dionysia Modingin
Shalini Markandan
Scientific: Hani Idayu Bani
Chee Beng Jin Juliza Mohamed
Dr Mastura Mohtar Hanan Abdul Wahab
Dr Getha Krishnasamy Mazurah Mohamed Isa
Dr Mary Khoo Gaik Hong Nur Fasehah Zulkifli
Mailina Jamil Nur Munirah Sabki
Roshan Jahn Mohd Salim Muhammad Khair Mohd Ayob
Adiana Mohd Adib Norhayati Ismail
Nor Datiakma Mat Amin
Tan Ai Lee
Promotion:
Fauziah Abdullah
Dr Fadzureena Jamaludin
Dr Nor Hayati Abdullah
Dr Nor Azah Mohd Ali
Nurul Hafizatul Shafiqah Mohd Azlan
Dr Ling Sui Kiong
Nurnadiah Rahim
Noor Rasyila Mohamed Noor
Saiful Azwan Mohd Basari
Mohd Faizulzaki Mohd Yatim
Social: Mohd Nizal Salehin
Dr Nurhanan Murni Yunos Mohd Shafik Yuzman Tolmanan
Dr Nik Musa’adah Mustapha Mohd Hafidz Hadi Abdullah
Saidatul Husni Saidin Muhamman Syamil Azahar
Siti Syarifah Mohd. Mutalip Dr Zunoliza Abdullah
Muhammad Haffiz Jauri
Logistic: Azman Mohamed
Ihsan Safwan Kamarazaman Abdul Halim Talha
Abdul Rashid Li Mohd Faizal Sharuddin
Mohd Kamal Nik Hasan Selva Kumar Raman
Adiana Mohd Adib Hairol Nizam Haron
Abdul Majid Jalil Mohd Kafi Jaapar
Sahrim Lias All Staffs of Natural Product Division

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