APPENDICES

APPENDIX - 1 AUTHENTICATION LETTER

**r5T»r »f IKVDONMSIt > f OSJSTS

BOTANICAL SURVEY OP INDIA
' 4 THI ! 0 : M 0 fctCTO* vrwfta w^rwfi* wsFann
* ( STMJl CBCLE. J. K t * » G » Q s f C * p HWWlflBI
?^*? = ^ 0 * «T3ft5E ^ J ^ ?if?UWSi-
» . * t » f 8 £«. 53! «t»«<
•:• r«.-ii-"2i • •; 0,1!=- -ilfiOS W08

! I KTIFH A IK

!"his is ' o eerily that tha o\sri specimen b r o u g h ! by M i s s Yogini S Jaiswa:

from C . U . S h a h College of P h a r m a c y : S g n t a ; r u z M i i r r b a i - 4 9 is tdarAfied & authenticated a

Nams Family
Anaeardium ©ecidentale L. Anacardiaceae

u \ ^
IIM: W \ V * M R .
hi-

P a g e | 365
 APPENDIX - II LIST OF PUBLICATIONS

LIST OF PUBLICATIONS

> International Research Articles (02)

• Y.S. Jaiswal, P.A.Tatke, S.Y Gabhe , A. B. Vaidya. Antioxidant activity of
various extracts of leaves of Anacardium occidentale linn. (Cashew), Research
Journal of Pharmaceutical, Biological and Chemical Sciences, 1 (4), 112-119,
2010.

• Y.S. Jaiswal, P.A.Tatke, S.Y Gabhe , A. B. Vaidya. Isolation and quantitative

analysis of a bioactive polyphenol - Catechin in Anacardium occidentale Linn.
(Leaves and Testa) by HPLC analysis. Research Journal of Pharmacognosy and
Phytochemistry, 2 (5), 372-376, 2010.

> Research Articles in Indian Journals (02)

• Y.S. Jaiswal, P.A.Tatke, S.Y Gabhe , A. B. Vaidya. Application of Microwave

assisted extraction for fast Extraction of Catechin from Leaves of Anacardium
occidentale L. (Cashew), Journal Of Medicinal And Aromatic Plant Sciences.
32(4), 187-192,2010.

• Y.S. Jaiswal, P.A.Tatke, S.Y Gabhe , A. B. Vaidya. Antioxidant activity of leaves

of Anacardium occidentale Linn. Indian Journal of Natural Products., 26 (4),
17-22,2010.

> International Review Article (01)

• P.A.Tatke, Y.S. Jaiswal, An Overview of Microwave Assisted Extraction and its

Applications in Herbal Drug Research. Research Journal of Medicinal Plant,
5(1):21-31,2011.

Page | 366
 ISSN: 0975-8585

Research Journal of Pharmaceutical, Biological and Chemical

Sciences

Antioxidant Activity of Various Extracts of Leaves of Anacardium Occidentale

(Cashew)

YS JAISWAL 1 , PA TATKE1*, SATISH Y GABHE 1 , ASHOK VAIDYA 2

Department of Pharmaceutical Chemistry, C.U.Shah College of Pharmacy, S.N.D.T Women's University, Mumbai-
400049, India.
2
Medical Research Centre, Kasturba Health Society, Vile Parle-(W), Mumbai- 400 056, India.

ABSTRACT

The antioxidant properties and the effect on nitric oxide (NO) production of various extracts of leaves of
Anacardium occidentale were investigated. Radical-scavenging potential was evaluated using the l,l-diphenyl-2-
picrylhydrazyl (DPPH) radical. Griess assay was used to assess NO-inhibitory activity of the extracts. The antioxidant
activity of aqueous, ethanol, and petroleum ether (60-80 C) extracts of the leaves of Anacardium occidentale was
estimated. The order of the antioxidant potency of the plant extract is ethanol > aqueous > petroleum ether. The
results suggest that the leaves of A.occidentale are a potent source of natural antioxidants.
Key words: Antioxidant activity, Anacardium occidentale extracts, Total Polyphenol Content, l,l-Diphenyl-2-
picrylhydrazyl (DPPH) radical, Nitric oxide.

*Corresponding author
Email: patatke@gmail.com

October - December 2010 RJPBCS 1(4) Page No. 112

Research J. Pharmacognosy and Phytochemistry 2010: 2{5): 372-376
ISSN 0975-2331
Research Journal of Pharmacognosy
and Phytochemistry. 2(5): Sept.-Oct.
2010. 372-376
Research Article
fliEiimii.i.-M
www.anvpublication.org
* Corresponding Author:
Dr. Pratima Tatke.
Associate Professor in Pharm. Chem..
C. U. Shah College of Pharmacy,
S.N.D.T. Women's University, Juhu
Campus, Juhu Tara Road, Santacruz
West,
Mumbai-400 049, M.S.
E. Mail.- patatke@yahoo.com
Received on 29.05.2010
Accepted on 07.07.2010
© A&V Publication all right reserved
Pratima A. Tatke ei.al.
Isolation and Quantitative Analysis of a
Bioactive Polyphenol - Catechin in Anacardium
occidentale Linn. (Leaves and Testa) by HPLC
Analysis
Yogini S. Jaiswal1, Pratima A. Tatke 1 *, Satish Y.Gabhe1 and
Ashok B. Vaidya2
C.U.Shah College of Pharmacy, S.N.D.T Women's University. Mumbai-
400049, India.
TCMR Advanced Centre of Reverse Pharmacology in Traditional Medicine,
Kasturba. Health Society, Vile Parle-(W), Mumbai- 400 056, India.
ABSTRACT:
The present paper reports a method developed for isolation, characterization,
structural elucidation and quantitative analysis of bioactive polyphenol -
Catechin, from extracts of leaves and testa of Anacardium occidentale Linn
(Cashew). Isolation of Catechin was carried out by Preparative TLC. The
isolated Catechin was characterized by P-NMR, MS, IR spectroscopy and
various chemical and chromatographic analysis. The method was established
by using HPLC analysis and quantitation of isolated Catechin with marker
Catechin. The purity of isolated Catechin was found to be 99.30% based
upon the comparison of peak purity and peak areas of isolated Catechin and
marker Catechin.
The HPLC separation system consisted of a CI8 reversed-phase column, an
isocratic elution system of methanol-water (9:1 v/v), and a UV-Visible
detector with 254nm as the detection wavelength. The limit of detection
(LOD) and limit of quantitation (LOQ) were found to be 0.2 ug and 0.6 (jg
respectively with an Rt value of 2.5 min.
The maximum amount of Catechin was found in aqueous extract of leaves
(5.70%) and testa (13.65%).
Thus the developed HPLC method is rapid and simple technique for
separation and determination of Catechin from extracts of A.occidentale
leaves and testa. The method can be suitably applied for determination of
Catechin from various plant extracts.
KEYWORDS: Catechin: Anacardium occidentale Linn.; Polyphenol:
HPLC
1. INTRODUCTION:
The polyphenols like Catechin comprise of a range of substances that play a
role in protecting biological systems against the deleterious effects of
oxidative processes on macromolecules, such as proteins, lipids,
carbohydrates and DNA1. Malaysian population consumes traditional
vegetables and herbs, raw or cooked as accompaniments with their main
meal.
Many of these vegetables are claimed to possess medicinal properties
although there are no scientific evidences to support the claims. One of the
commonly consumed vegetables is the leaves of A. occidentale (commonly
known as Cashew).
372
Journal of Medicinal andAromatic Plant Sciences 32 (4) (2010) 187-192
Application of microwave assisted extraction for fast extraction of
catechin from leaves of Anacardium occidentale
YOGINI S. JAISWAL, I. S. R. NIDHIYA, PRATIMAA. TATKE', ASHOK B. VAIDYA**, SATISHY.
GABHE
C. U. Shah College of Pharmacy, S.N.D.T Women's University, Santacruz (W). Mumbai 400049, India.
Received 26th December, 2009
ABSTRACT
The present paper reports a novel technique for fast
extraction using microwave assisted extraction (MAE) and
high performance liquid chromatography (HPLC) method
for quantitative analysis of catechin in Anacardium
occidentale L. (cashew) leaf extracts. Water and methanol
were used as extracting solvents. Several influential
parameters of MAE namely, microwave power, irradiation
time, and amount of extracting solvent were studied for
optimization of extraction protocol. The maximum yield of
catechin with MAE was obtained by extraction with water at
low microwave power in 2 minutes. The optimal yield of
catechin with MAE, (88.40±0.04 mg/g) was about 2.5 times
the yield obtained by conventional Soxhlet extraction with
water for 18 hours (35.60 ±0.02 mg/g). The present work
reports a novel microwave-assisted extraction (MAE)
technique and high performance liquid chromatography
(HPLC) method for quantitative analysis of catechin in
Anacardium occidentale L. (cashew) leaf extracts.
Key words: Anacardium occidentale, catechin, HPLC,
Microwave-assisted extraction.
INTRODUCTION
Extraction and characterization of
phytoconstituentsfromplants have led to discoveries
of high activity profile drugs. Extraction can be done
by various methods based on the nature of the
constituents present. The use of traditional and
conventional extraction techniques for plant materials
are time and solvent consuming, and causing thermal
decomposition in case of soxhlet extraction. The newer
Author for correspondence : Email: patatke@gmail.com
** ICMR Advanced Centre of Reversed Pharmacology in
Traditional Medicine, Kasturba Health Society, Vile-Parle-(W),
Mumbai-400 056, India
l Pratimi_Tatke
extraction techniques such as Ultrasonic Assisted
Extraction (UAE), Microwave Assisted Extraction
(MAE) and Supercritical Fluid Extraction (SCF) have
been tried recently. These techniques enable automation
with shortened extraction time and reduced organic
solvent consumption. Microwave energy can be used
to heat the solvent and the sample to increase the mass
transfer rate ofthe solutesfromthe sample matrix into
the solvent and has shown tremendous research interest
and potential [7]. The microwave-assisted extraction
(MAE) technique is innovative and promising tool which
is highlighted by increased yield, decreased extraction
time and solvent consumption, moreover the
reproducibility is better [4]. Catechin is present as an
essential component of various herbal antioxidant,
antidiabetic and antiageing formulations available world
wide for treatment of diseases like aging, diabetes and
atherosclerosis [5,8]. Anacardium occidentale,
commonly known as cashew, is indigenous to Brazil,
and is a member of the family Anacardiaceae [3].
However, nowit is also widely grown in other tropical
places like India. The biological activities of this plant
are widely reported and it has been reported to possess
anti-viral, anti-bacterial, anti-fungal, anti diabetic and
anti-inflammatory activities [1,2,6,9-11]. Catechin is
present as one of the constituents in cashew and
probably responsible for its therapeutic benefits.
MATERIALS AND METHODS
Plant material
Plant samples were collectedfromTungareshwar
forests of Vasai Taluka, Dist. Thane in the state of
Maharashtra, India. The plant specimen was
 Indian J. Nat Prod.,2010, 26(4),17

FREE RADICAL SCAVENGING ACTIVITY OF EXTRACTS OF

CASHEW TESTA
Yogini S. Jaiswal1, Pratima A. Tatke1*, Ashok Vaidya2and Satish Y.Gabhe1
C.U.Shah College of Pharmacy, S.N.D.T. Women's University, Santacruz,
Mumbai1- 400 049.
and
ICMR Centre of Reverse Pharmacology in Traditional Medicines, Kasturba Health
Society, Vile Parle (W), Mumbai2- 400 056.

ABSTRACT

Free radical scavenging activity of aqueous, ethanol and methanol extracts of

cashew (Anacardium occidentale Linn.) testa powder was evaluated employing 1,1-
diphenyl-2-picrylhydrazyl (DPPH) and nitric oxide radical methods. Results indicated the
ethanol extract of A. occidentale testa to be most potent reducing agent and radical-
scavenger among the tested extracts suggesting that cashew testa, a by-product of
cashew processing industry, can be used as an economical source of natural
antioxidants.

INTRODUCTION strong antioxidant activities, which is mainly

Reactive oxygen species (ROS) are
implicated in pathophysiological events such
as aging, cancer, atherosclerosis and
diabetes1. As natural antioxidants from herbal
source provide substantial protection that
slows down the process of oxidative damage
caused by ROS , there has been growing
interest in natural antioxidants of plant origin
which also find use as nutraceuticals due to
their impact on human health and disease
prevention3. As several fruits, nuts, leaves,
roots and barks have been exploited as
potential sources of natural antioxidants4
which yields considerably higher ratio of by-
products from them specially from seeds and
nuts5, and hence it would be befitting and
useful if these by-products are exploited as
potential sources of natural antioxidants. The
seed coat/testa/nut skin forming a protective
coat for cotyledons, is reported to have high
concentration of phenolic compounds6.
Recent studies have shown that extracts of
almond skins7"9, peanut skins10, hazelnut
testa and green leafy cover1112 possess
*For correspondence
due to the presence of large amounts of
phenolic compounds in these and
morphological parts specially the seed coat13"
15
. Cashew nut (Anacardium occidentale L) is
a major cash crop of the world, where India is
its one of the largest producer16. The kernel
of cashew nut is covered with a thin reddish-
brown skin or testa that has been reported as
a good source of hydrolysable tannins17 with
catechin and epicatechin as the major
polyphenols18. However, there are no reports
on antioxidant activity of the testa of cashew
nuts. The purpose of this study therefore was
to evaluate the in-vitro ability of the extracts
of testa of cashew nuts to scavenge the
reactive oxygen species.
]
MATERIAL AND METHODS
Plant Material
Testa samples were collected from
Sawantwadi region of Goa. The plant
specimen was authenticated at the Botanical
17
Research J o u r n a l of Medicinal P l a n t 5 (1): 21-31, 2011
ISSN 1819-3455 / DOI: 10.3923/rjmp.2011.21.31
© 2011 Academic J o u r n a l s Inc.

An Overview of Microwave Assisted Extraction and its Applications

in Herbal Drug Research

P. Tatke and Y. Jaiswal

C.U. Shah College of Pharmacy, S.N.D.T Womens University, Mumbai-400049, India

Corresponding Author: Pratima A. Tatke, Department of Pharmacology Chemistry, C.U. Shah College of Pharmacy,
S.N.D.T. Women's University, Santacruz, Mumbai- 400 049. M.S., India Tel: +91-022- 26603968, +91 9920685857

ABSTRACT
The application of microwave assisted extraction process for isolation a n d extraction of
phytoconstituents from plant m a t e r i a l has gained a n increasing importance. The techniques used
for extraction since decades have t h e limitations of requiring longer extraction times, large solvent
volumes a n d cause degradation of t h e r m o labile components. In t h e present review an a t t e m p t is
been made to emphasize upon t h e importance of Microwave Assisted Extraction (MAE) in herbal
d r u g research. The merits of MAE which help to prove it of being b e t t e r t h a n conventional
techniques are discussed. The technique requires less solvent volume, gives high and fast extraction
performance and offers protection to thermo-labile constituents. The theory a n d basic principle of
using microwave-energy for extraction, p a r a m e t e r s affecting t h e microwave-extraction efficiency,
its statistical optimization strategies and potential applications have also elucidated.

K e y w o r d s : Microwave-assisted extraction, conventional extraction, optimization strategies, plant

research, h e r b a l

INTRODUCTION
P l a n t s h a v e been t h e source of potential t h e r a p e u t i c agents ever since m a n k i n d h a s evolved.
Although several active phytoconstituents and high activity profile drugs have been discovered
from plants b u t t h e quality and safety related problems of herbal drugs h a v e still been a challenge
for researchers. The major reasons for t h e s e drawbacks are t h e lack of high performance, reliable
extraction techniques and methodologies for establishing t h e purity and s t a n d a r d for herbal
medicines (Huie, 2002). It is due to these factors t h a t t h e h e r b a l medicines have still to find their
w a y in order to be accepted in global m a r k e t . In research related to discovery of new active
phytoconstituents, extraction is one of t h e i m p o r t a n t steps as it is t h e s t a r t i n g point for t h e
isolation and purification procedures. An individual p l a n t may consist of several active
phytoconstituents existing in a b u n d a n c e along with certain constituents of low activity profile.
T h u s , there arises a need for t h e development of extraction a n d analysis techniques with high
performance (Smith, 2003). There h a s been a need for b e t t e r a n d newer extraction techniques, in
t h e h e r b a l d r u g i n d u s t r y so t h a t t h e extraction t i m e a n d t h e cost of solvent consumption is
decreased (Nyiredy, 2004).
Amongst the various traditional and conventional extraction techniques, Soxhlet extraction has
b e e n t h e most widely used. Soxhlet extraction serves not only as a technique for extraction of
phytoconstituents b u t also as a reference to compare newer extraction techniques. Soxhlet

21
 APPENDIX - III LIST OF PRESENTATIONS

LIST OF PRESENTATIONS

> Presentations in International Conferences (04)

• Y.S. Jaiswal, P.A.Tatke, S.Y Gabhe , A. B. Vaidya. Application of Microwave

assisted extraction for fast Extraction of Catechin from Leaves of Anacardium
occidentale L. (Cashew). International symposium on current status and
opportunities in Aromatic & Medicinal plants, held in Lucknow in Feb. 2010.

• Y.S. Jaiswal, P.A.Tatke, S.Y Gabhe , A. B. Vaidya. A simple method for isolation
of Catechin - a potential Biomarker from medicinal plants and its evaluation,
presented at 21st International Symposium on Pharmaceutical and Biomedical
Analysis, held at Orlando, USA on 11-14 October 2009.

• Y.S. Jaiswal, P.A.Tatke, S.Y Gabhe , A. B. Vaidya. Rapid densitometric

quantitative screening of various extracts of Anacardium occidentale Linn, for
Catechin content, presented at 21st International Symposium on Pharmaceutical
and Biomedical Analysis, held at Orlando, USA on 11-14 October 2009.

• Y.S. Jaiswal, P.A.Tatke, S.Y Gabhe , A. B. Vaidya. Antioxidant activity of

various extracts of leaves of Anacardium occidentale (Cashew), presented at
International Conference on New Developments in Drug Discovery from
Natural Products and Traditional Medicines, held at NIPER- Chandigarh, on 16-
20, November 2008.

Page | 367
 APPENDIX-IV LIST OF AWARDS

LIST OF AWARDS RECEIVED

• Selected for "Short stay fellowship programme for students from India and
China" by the Executive board of Utrecht University Region Committee Asia, for
a short term stay and fellowship at the Department of Nephrology and
Hypertension at Utrecht University Medical Centre, Utrecht; Netherlands, Europe
for a project under the guidance of Prof. M.C.Verhaar and Dr. Joost Fledderus.

• Received second prize at ANVESHAN-2009- ZONAL Research Festival, held

at Bhopal, on 21-29^ , January 2009 for the project entitled, Isolation And
Chemical Characterization Of Catechin - A Potential Biomarker With
Antioxidant Activity, and was selected as one of the best research projects.

• Received second prize at the ANVESHAN-2009 - 2 nd National Level Research

Convention, held by Association of Indian Universities -AIU, at Calcutta, on
23-25111, February 2009 for the paper entitled, Isolation And Chemical
Characterization Of Catechin - A Potential Biomarker With Antioxidant Activity.

Page I 369
APPENDIX-V LIST OF ABBREVIATIONS

LIST OF ABBREVIATIONS

ABTS : 2,2'-azino-bis (3-ethylbenzthiazoline-6-sulphonic acid)

AChE : Acetylcholinesterase
ADA : American Diabetes Association
ADP : Adenosine-5-diphosphate
ADPrib Polyadenosine Diphosphoribose
ALP Anti-lipid peroxidation
ALT Alanine aminotranferase
ARE Antioxidant-response element
ANOVA : One-Way Analysis Of Variance
BCS : Bovine Calf Serum
BPT . Bronchial Provocation Tests
CAM Complementary And Alternative Medicine
CG Cashew gum
CHD Coronary Heart Disease
CMCG Carboxymethylated Cashew gum
CNS Central Nervous System
CNSL Cashew nut shell liquid
CRP C-Reactive Protein
CSBME Cashew Stem Bark Methanolic Extract
CSE Cashew Nut Skin Extract
DAP Dihydroxyacetone Phosphate
DCP Dibasic Calcium Phosphate
DPE Dry Powder Extract
DSHEA Dietary Supplement Health and Education Act
E Epichlorohydrin
EASD European Association for the Study of Diabetes
EC Endothelial cells
ECGS Endothelial Cell Growth Supplement
EDHF Endothelium Derived Hyperpolarizing Factor

Page | 370
APPENDIX-V LIST OF ABBREVIATIONS

EMEA : European Agency for the Evaluation of Medicinal

products
ESRD : End Stage Renal Disease
FAA Formalin-Acetic Acid-Alcohol
FIP International Pharmaceutical Federation
GAE Gallic Acid Equivalence
GCLC Glutamate-Cysteine Ligase, Catalytic Subunit
GCLM Glutamate-Cysteine Ligase, Regulatory Subunit
GC-MS Gas Chromatography-Mass Spectrometry
GLDH Glutamate Dehydrogenase
GLP Good Laboratory Practices
GLP-1 Glucagon-Like Peptide -1
GLUT-4 Glucose Transporters
G3P Glycerol-3- phosphate
GMP Good Manufacturing Practices
GOD Glucose oxidase
GOT Glutamate Oxaloacetate
GPO Glycerol Phosphate Dehydrogenase
GPT Glutamate pyruvate transaminase
GSH Glutathione
HbAlc Glycohemoglobin
H2DCFDA : 2',7'-dichlorodihydrofluoresceindiacetate
HDL High density lipoprotein
HLA Human Leucocyte Antigen
HMPS Hexose Monophosphate Shunt
HO-1 Heme oxygenase-1
HPLC High Performance Liquid Chromatography
HPTLC High Performance Thin Layer Chromatography
ICD-10 International Classification of Diseases
IND International Nomenclature of Disease
IRS-1 Insulin Receptor Substrate-1

Page | 371
APPENDIX - VI SYNOPSIS

Synopsis submitted for the dissertation entitled

PHYTOCHEMICAL AND PHARMACOLOGICAL

INVESTIGATION OF
ANACARDIUM OCCIDENTALE (LINN.) FOR
ANTIDIABETIC ACTIVITY"

Page | 373
PHYTOCHEM1CAL AND PHARMACOLOGICAL INVESTIGATION

OF Anacardium occidental (Linn.) FOR ANTIDIABETIC ACTIVITY

A Synopsis submitted to

S.N.D.T. Women's University, Mumbai

For the degree of

Doctor of Philosophy in Pharmacy
(Faculty of Technology)

In the subject of
Pharmaceutical Chemistry

By
Miss. Yogini S. Jaiswal
M. Pharm

C. U. Shah College of Pharmacy,

SNDT Women's University,
Juhu Campus, Santacruz (W),
Mumbai - 400 049

March-2011
 SYNOPSIS

The term diabetes is a complex and multifarious group of disorders characterized by

hyperglycemia that has reached epidemic proportions in the present century. Infection is
a leading cause of morbidity and mortality among the diabetic population. There are two
types of diabetes: Type-1 diabetes mellitus formerly known as insulin dependent diabetes
mellitus (IDDM) and Type-2 diabetes mellitus formerly known as non-insulin dependent
diabetes (NIDDM). The vast majority of diabetic patients are Type-2 diabetes mellitus.
The World Health Organization (WHO) has predicted that the global prevalence of Type-2
diabetes will be from 135 million in 1995 to 300 million in 2025 and that this increase will
affect both industrialized and developing countries expecting the greatest increase in India,
from 19.4 to 57.2 million.'"2 Treatment of diabetes mellitus involves changes in the lifestyle
and pharmacological intervention with insulin or oral hypoglycemic drugs. For most patients
with Type-2 diabetes, changes in lifestyle are the cornerstone of treatment, particularly in the
early stages of the disease. Pharmacologic intervention is a secondary treatment.
Ethnobotanical information indicates that more than 100 plants have been used in
traditional remedies for the treatment of diabetes. The hypoglycemic activity of large
number of these plants have been evaluated and confirmed in different animal models.
Reactive oxygen metabolites (ROMs) which include super oxide anion (O2), hydrogen
peroxide (H2O2), hydroxyl radicals (OH), have been implicated in several degenerative
diseases including atherosclerosis, cataractogenesis, insulin dependent diabetes mellitus
and ischemia. Free radicals and other reactive oxygen species are formed constantly in the
human body and are removed by the enzymatic and non enzymatic antioxidant defense
system. In spite of the presence of number of marketed oral synthetic antidiabetic drugs,
researchers have now diverted their attention 10 different herbs and medicinal plants in
order to find out new active principles with better anti diabetic activity.
Traditional anti diabetic plants might provide new oral hypoglycemic compounds, which
can counter the high cost and poor availability of the current medicines / present day
drugs for many rural populations in developing countries.3'4

i ' < L> i- 1 1

 SYNOPSIS

AIMS AND OBJECTIVES:

The present project aims at systematic phytochemical and pharmacological investigation
of A. occidentale (Linn.) with antidiabetic activity with specific objectives given below:
1. Screening of extract/s and fraction- s for antioxidant activity.
2. Evaluation of bioactive extract s and fraction's for antidiabetic activity and
assessment of metabolic parameters like glucose and lipid profile.
3. Acute oral safety studies of extract's and fractions by OECD guidelines.
4. Detailed evaluation of bioactive extract s and or fraction-s on type 2 diabetes
animal models.

PLAN OF WORK:
PART- I

1. Literature survey: Relevant literature search regarding the plant Anacardium

occidentale Linn. (Cashew) was carried out.
2. Procurement and Authentication of plant material: Plants were authenticated by
botanist from Botanical Survey of India. Pune and the samples were deposited for
future reference. •
3. Extraction of the plant material: Different extracts were prepared by conventional
soxhlet extraction and microwave assisted extraction technique.
4. Phytochemical investigation of the extracts: Qualitative phytochemical evaluation
of the extracts to investigate the presence/absence of various phytoconstituents was
carried out.
5. TLC and HPLC studies of the extract/s and fraction/s: TLC and HPLC profiles
and HPTLC fingerprints of the extracts of cashew leaves and testa prepared by
conventional extraction process were developed.
6. In vitro screening of extract/s and fraction/s for its free radical scavenging
activity: In vitro antioxidant activity of extract/s was assessed by DPPH, Greiss and
TBARS assays.
7. Isolation of catechin: Biomarker Catechin was isolated from extracts of Cashew
leaves and testa by preparative Thin Layer Chromatography (TLC) technique and its
identity and purity was confirmed by comparing with standard through
chromatographic techniques.
P age | 2
 SYNOPSIS

PART - II

1. Quantitation of extracts by HPTLC and HPLC: The extracts of leaves and testa
of cashew were quantified for the content of Catechin through an optimized HPTLC
and HPLC method.
2. Acute oral toxicity studies: Acute Oral Toxicity Study was carried out in Albino
mice following OECD 423 for various extracts of leaves and testa and polyphenol
fraction of cashew.
3. Anti lipid peroxidation activity of extracts (in vitro):A\\ the extracts of cashew
leaves and testa were evaluated for ferrous sulphate induced lipid peroxidation using
mice liver homogenate (TBARS Assay).
4. Evaluation of Antioxidant potential of cashew extracts by various assays on cell
lines: The evaluation of cashew extracts, polyphenol fractions and Catechin, was
carried out by ROS Assay, Angiogenesis and cell viability assay.

PART-I1]

1. Streptozotocin Induced Nicotinamide model for antidiabetic activity:

The antidiabetic activity of bioactive extracts of cashew leaves, and testa and
polyphenol fraction were evaluated by STZ induced nicotinamide model. This model
was used for preliminary screening of bioactive extracts. The effect of various
extracts of cashew testa, fractions of Cashew Testa and Ethanol Extract Of Cashew
leaves was evaluated in rats with STZ-Nicotinamide induced type 2 diabetes mellitus.
2. Formulation of tablets from bioactive extract of cashew testa: Tablets of bioactive
extract of testa were prepared and evaluated for various parameters like friability,
hardness, content uniformity testing, dissolution, disintegration and stability studies
etc.

P a ji < | 3
 SYNOPSIS

Literature survey reveals few reports for the anti-hyperglycemic, hypoglycemic and anti-
oxidant potential of various extracts of the leaves and testa of A.occidentale. Hence the
plant was selected for a systematic and detailed study to explore the anti-diabetic activity
of phytoconstituents of leaves and testa using various in vitro and in vivo parameters.5"8

Phytochemica) Investigation of Extracts

O Procurement and Authentication of plant material:

Plant samples of Leaves were collected from Tungareshwar forests of Vasai Taluka.
Dist. Thane and the Testa were collected from Sawantwadi region. Goa in the state of
Maharashtra. India. The plant specimen was authenticated at the Botanical Survey of
India. Pune: (MS). A herbarium of the plant specimen (specimen voucher number no.
YOGA 1/No.BSl/WC Tech/2008/69) was submitted at the Botany Department of BSL
Pune.

O Extraction ojplant material by various solvents and extraction parameters:

The samples of fully matured leaves and Testa of A.occidentale were dried, ground to
coarse powder form and extracted by Soxhlet extractor. An attempt was made to optimize
and design a procedure for extraction of dry powder of dried leaves of A. occidentale by
microwave assisted process which is. considered as a potential alternative to traditional
extraction methods. Extraction of leaves (subjected to various drying conditions )was
carried out to evaluate the effect of varying drying temperatures on the phytoconstituents.

TLC and HPTLC studies of Various Extracts of Testa and Leaves

In order to establish a chromatographic profile for the phytoconstituents of various
extracts, a suitable mobile phase was designed based upon solubility and phytochemical
characteristics of various constituents.

Evaluation Of Antioxidant Activity

O Quantification of total phenolics - by Folin-Ciocalteu method

The amount of total phenolic compounds in the extracts and fractions was determined
colorimetrically with the Folin-Ciocalteu (FC) reagent. The reaction mixture contained

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the sample, FC reagent, sodium carbonate solution.The volume was made stated quantity
with water and was incubated in dark under ambient conditions for 2 hrs to complete the
reaction. The absorbance of the resulting solution was measured at 760nm in a UV Vis
spectrophotometer. The gallic acid was used a standard. The concentration of total
phenolic compounds was expressed as mg of gallic acid equivalents (GAE) per g of dried
extract, using a standard curve of gallic acid. All the measurements were carried out in
triplicates.9

O DDPH (J, 1, diphenyl 2-piayl hydrazyl) assay I0

The antioxidant activity using the DPPH assay was assessed by the method of Ansari et.
al (2005). The hydrogen atoms or electron-donating ability of the extracts was
determined from the bleaching of purple-colored methanol solution of DPPH. Briefly, to
various concentrations of the extract 0.1 mM of DPPH was added. After an incubation of
30 min in dark at room temperature, absorbance was recorded at 517 nm. This activity is
given as percent DPPH radical scavenging, which is calculated with the equation: %
DPPH radical scavenging = [(control absorbance - sample absorbance)] / control
absorbance] * 100.

O FeS04 induced lipid peroxidation (TBARS Assay) in mice liver homogenate ]'12

The incubation mixture contained potassium chloride (150mM), 10% brain homogenate
as lipid source and various concentrations of test compound. Peroxidation was initiated
by adding ferrous sulphate. After incubating for 20 minutes of 37°C, reaction was
stopped by adding TBA/TCA/BHT solution, followed by heating at 80°C for 15 minutes,
cooled, centrifuged for 10 minutes and absorbance_of the supernatant liquid was recorded
at 535 nm.

Acute oral toxicity study '

Acute Oral Toxicity Studies were carried out in Albino mice following OECD 423
Guidelines, for extracts which showed a better antioxidant activity in wtro.
A single dose oral study was conducted to determine the acute toxic category of the
extracts used in the study. The acute oral toxicity study was conducted as per
Organization for Economic Co-operation and Development (OECD) 423 guidelines.
Each animal was fasted 4 h prior to dosing with free access to water. Drug was suspended
in suitable vehicle and administered to the animals. If mortality was observed in two out
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 SYNOPSIS

of three animals then the dose administered was considered as toxic dose. However if
mortality was observed in any one animal out of the three then the same dose was
repeated again to confirm the toxic effect. If no mortality was observed then the other
dose levels were employed for further toxicity studies. The animals were observed for
clinical signs (tremors, convulsions, lethargy and coma), gross behavioural changes and
mortality after 30 min, 1 st. 2nd, 3rd and 24th hr. These daily observations were continued
for a period of 14 days. The mean group body weight of the control and test group
animals were recorded on 0th, 7th and 14th day respectively.

STZ-NA induced Type 2 diabetes mellitus in adult rats: Intervention study 14'15
O Induction of Experimental diabetes (NIDDM)
A rat model of type 2 diabetes mellitus (non-insulin dependent diabetes mellitus.
NIDDM) was induced in overnight-fasted rats by a single intraperitoneal injection of
streptozotocin 15 min after the intraperitoneal administration of nicotinamide. The rats
were supplied with 5% glucose water and ad libitum basal diet during the next 24 hours
to avoid sudden hypoglycemia post-injection. On day 2. water was replaced with drinking
water. Blood samples were obtained from the retroorbital plexus in both streptozotocin-
injected and control animals at 72 hours and on day 7 after an overnight fast. Rats with
stable elevated fasting blood glucose levels on day 7 and above 200 mg/dL were
considered diabetic and chosen for the interventional study. Fasting blood glucose levels
were determined by glucose oxidase method. The animals were grouped randomly based
on their blood glucose levels, each having six animals. The control group received 0.05
% suspension of CMC and in the treatment group the drug/extracts were suspended in
0.05% CMC administered orally for 15 days. At the end of the experimental period, the
rats were fasted overnight and blood samples were withdrawn from the retro orbital
plexus. Serum samples were used for the various biochemical estimations.

O Neonatal streptozotocin-induced rat model of Type 2 diabetes mellitus^

The pups were weaned after 30 days, and the animals were checked for fasting glucose
levels after 3 months of STZ injection. When animals were 12 weeks old. an oral glucose
tolerance test was performed. Glucose was administered after 0 min reading. All the
animals were fasted overnight before experiments. Animals which were intolerant to
glucose by OGTT are chosen for the experiments.

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RESULTS AND CONCLUSION:

Phytochemical and pharmacological investigation of Cashew testa and leaves was carried
out for evaluation of Antidiabetic activity.JThe phytochemical tests of extracts of leaves
reveal the presence of tannins and phenolics. saponins, flavonoids, alkaloids, steroids and
sugars. Extracts of testa also reveal the presence of tannins and phenolics, saponins,
flavonoids, alkaloids, steroids and sugars. Quantitation of various phytoconstituents in
the extracts is to be carried out. Presence of Catechin in the extracts was ascertained
based upon the peak of a similar Rf value found in the extracts of testa and leaves.
Out of six extracts and one fraction, two extracts and one polyphenol fraction was found
to exhibit significant antidiabetic activity in the selected in vivo animal models. The
extracts of cashew also exhibited significant antioxidant effect as evaluated through
various in vitro radical scavenging assays. A microwave assisted extraction technique
was designed to increase the yield of phytoconstituents from cashew leaves. A bioactive
constituent was successfully isolated and characterized from extracts of cashew. An
attempt is been been made to develop an antidiabetic formulation, which may overcome
the drawbacks of current commercial antidiabetic formulations.

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REFERENCES:

1. Ajgaonkar S.S. (1979). Herbal drugs in the treatment of diabetes. International

Diabetes federation Bulletin. 24, 10-17.
2. Arulmozhi D.K. Veeranjaneyulu A. Bodhankar SL. Neonatal streptozotocin-induced
rat model of Type 2 diabetes mellitus: A glance. Indian Journal of Pharmacology ,
2004;36:217-221.
3. Chattopadhyay. R.R.. 1999. A comparative evaluation of some blood sugar
lowering agents of plant origin. Journal of Ethnopharmacology 67, 367-372.
4. Beigh, S.Y., Nawchoo. LA. and IqbaL M. 2002. Herbal Drugs in India: Past and
Present Uses. Journal of Tropical Medicinal Plants 3: 197-204.
5. Abas F., Lajis N.. lsraf D.. Khozirah S.. and Umi Kalsom Y.. Antioxidant and nitric
oxide inhibition activities of selected Malay traditional vegetables. Food Chemistry,
95(4), 2006, 566-573.
6. Goncalves J., Lopes S., Oliveira D., Costa S., Miranda M.. Romanos M., In vitro anti-
rotavirus activity of some medicinal plants used in Brazil against diarrhea, Journal of
Ethnopharmacology, 99(3), 2005,403-407.
7. Kamtchouing P.. Sokeng S., Moundipa P., Watcho P., Jatsa H., and Lontsi D.,
Protective Role of Anacardium occidentale extract against streptozotocin induced
diabetes in rats. Journal of Ethnopharmacology, 62(2), 1998, 95-99.
8. Kubo L, Masuoka N., Ha T., and Tsujimoto K., Antioxidant activity of anacardic
acids.Food Chemistry, 99(3), 2006, 555-562.
9. Srinivasan R.,Chandrasekar M.J.N., Nanjan MJ,Suresh B.Antioxidant activity of
Caesalpinia digyna root. Journal of Ethnopharmacology 2007:113:284-291.
10. Ansari N.M., Houlihan L., Hussain B., Pieroni A. Antioxidant Activity of Five
Vegetables Traditionally Consumed by South-Asian Migrants in Bradford, Yorkshire,
UK Phytotherapy research.2005; 19: 907-911.
11. Narla R.S., Rao M.N .A., Scavenging of free radicals and inhibition of lipid
peroxidation by 3-phenylsydnone. Journal of Pharmacy and Pharmacology, 1995;
47:623-625.

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12. Sreejayan, Rao M.N .A.. Curcuminoids as potent inhibitors of lipid peroxidation.
Journal of Pharmacy and Pharmacology. 1994: 46:1013-1016.
13. Barik R., Jain S.. Qwtra D., Joshi A.. Tripathi G.S., Goyal R.Antidiabetic activity of
aqueous root extract of Ichnocarpus frutescens in streptozotocin-nicotinamide
induced type-11 diabetes in rats. Indian journal of pharmacology 2008; 40:19-22.
14. Shirwaikar A., Rajendran K., Kumar. Bodla R., Antidiabetic activity of aqueous leaf
extract of Annona squamosa in streptozotocin-nicotinamide type 2 diabetic rats.
Journal of Ethnopharmacology 2004; 91:171-175.
15. Gokhale M.S., Shah D.H.; Hakim Z.; Santani D.D., Goyal R.K., Effect of chronic
treatment with amlodipine in Non-insulin-dependent diabetic rats. Pharmacological
Research. 1998: 37:455-459.
16. Murali B.. Upadhyaya U.M.. Goyal R.K.,Effect of chronic treatment with
Enicostemma littorale in non-insulindependent diabetic (NIDDM) rats. Journal of
Ethnopharmacology 2002;81:199- 204.

Candidate Research Guide:

• o ^

flogini S. Jaiswal Dr. Pratima A. Tatke,

Research Student Associate Professor,
C. U. Shah College of Pharmacy, C. U. Shah College of Pharmacy
SNDT Women's University, SNDT Women's University,
Juhu Campus, Santacruz (W), Juhu Campus, Santacruz (W),
Mumbai - 400 049 Mumbai - 400 049

Date: j [ 5 \ O v 3 - o ^ O I )

Place: Mumbai

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 My
r~\
Research Guide

Date: 0^-0^'fl-0'1
(Dr. (pratimaA. Tat^e
Associate Professor in Pharm.Chem.
C.U.Shah College of Pharmacy,
S.N.D.T Women's University,
Mumbai.

• si-.