Research in Natural Product for Supporting Cancer Management

Herbal Medicine Research Group Agency for Assessment and Application of Technology (BPPT)

Workshop on “Implementation Cancer Control Program : Accelerating Integrated System in Comprehensive Cancer Treatment”, National Cancer Inst. RS. Dharmais, Jakarta, 9-11 November 2009

BPPT
ORGANIZATIONAL ORGANIZATION AL STRUCTURE President of Republic of Indonesia Ministry Research & Technology CHAIRMAN
INSPECTORATE SECRETARY

Deputy for Technology Policy

Deputy for Technology of Natural Resources

Deputy for Technology of Agroindustry & Biotechnology (Dr. Wahono S.)

Deputy for Technology of Energy, Materials & Environtment

Deputy for Technology of Industrial Design & Engineering

§ Policy § Innovation

§ Environment § Food § Earth Science § Health § Marine Science § Bioenergy § Disaster Mitigation

§ ICT § Energy § Materials § Environment

§ Security & Defence § Transportation § Manufacturing

3

Ten ranks of majority diseases in Indonesia
SKRT 1992
Diseases
1. Circulation Syst 2. Tuberculosis 3. Kead.tidak jelas 4. Respiration Syst 5. Diarrhea 6. Infection 7. Bronchitis, asma 8. Trauma, Poisoning 9. Digestic Syst

SKRT 1995
%
16.0 11,0 9,8 9,5 8,0 7,8 5,6 5,3 5,3 4,0

Suskesnas 2001
%
18,9 15,7 9,6 7,9 7,4 6,6 5,2 5,2 5,0 2,5

Diseases
1. Circulation Syst 2. Respiration Syst 3. Tuberculosis 4. Infection 5. Diarrhea 6. Digestic Syst 7. Perinatal Disorder 8. Accident

Diseases
1. Circulation Syst 2. Respiration Syst 3. Tuberculosis 4. Digestic Syst

%
26,4 12,7 9,4 7,0 6,0 5,6 4,9 4,3 3,8 2,7

5. Cancer
6. Accident 7. Perinatal Disorder 8. Thyphus 9. Diarrhea 10.Endokrin &Metabol.

9. Cancer
10. Neuro

10. Cancer

Resource: Dept health, National Statistic Biro, 1992 dan 1995, 2001 Health Profile of Indonesia 2003, Health Dept,2005

Multi pathways cancer development
DNA Mutation

1. Differentiation 2. Mitosis 3. Metastasis 4. Invasion 5. Angiogenesis 6. Apoptosis
Cancer cell & its roots

Needs comprehensive work to prevent pathways
Anginogenesis

Treatment of Cancer
— — — — — —

Surgery Chemotherapy Radiotherapy Hormonal therapy Biologic therapy Gene therapy

Natural Product ???

6

Nature as Chemical Library
Distribution of 300,000 Plant Species in the World

Korea (4,000)

China (40,000)

India (60,000)

Indonesia (~ 80,000)
Africa (70,000) South America (100,000)

Only 2% of plants have their components and efficacy screened chemically 98% of plants yet to be thoroughly investigated Plant is a major source for drug materials

So: KRIBB KOREA, 2008
7

Terestrial Diversity
• 30,000 sp Flowering Plants à 9,600 as Medicinal Plants • 250 sp have been used comercially as herbal medicines • Hundreds of thousands of microbes as endophyte or soil microbes
So. BPOM, 2004

Marine Diversity
Algae: 782 spesies. spesies. Seagrass : 13 spesies. spesies. Mangrove : 38 spesies Sponge : 850 spesies Coral : 910 spesies Thousands more…

So. R. Dahuri, 1997
8

Official References :
1. 2.

3. 4. 5. 6.

National Policy on Traditional Medicine, ~ Decree of Ministry of Health No. 381/Menkes/SK/III/2007. Complementary & Alternative Therapy Management in Health Service Facility. Regulation of Ministry of Health No. 1109/Menkes/Per/IX/2007. Standardized Health Service Using Herbal MedicineDecree of Ministry of Health No. 121/Menkes/SK/II/2008 WHO General Guidelines for Methodologues on Research and Evaluation of Traditional Medicine, 2000 WHO Guidelines for the Regulation of Herbal Medicines in the South-East Asia Region, 2003 WHO Guidelines on Safety Monitoring of Herbal Medicines in Pharmacovigilance System, 2004

9

Good Agricultural Practice

“GAP”

Ethnobotany
• Empirical Claim • Placebo effect Traditional knowledge JAMU S&T

Extraction, Pharmacological Validation

Medicinal Plant

“Modern” Knowledge
Pre-clinical (on animal)

Standardized Herbal Med

Phytomedicine
Clinical On human

Efficacy & toxicity testing

S&T assessment: • Phytochemistry • Cultivation • Processing • Formulation • Pharmacological validation

Pharmaco-Epidemologic

GEP

GCP

GMP

GAP (Good Agricultural Practices) GEP (Good Extracting Practices) GCP (Good Clinical Practice) GMP (Good Manufacturing Practices)

10

Carcinogenesis and the Action of Phytochemicals & other Chemopreventive Agents
Activation of Procarcinogens by CYPs DNA Damage Gene Mutation Proto-oncogene Tumor Suppressor Angiogenesis Program Cell Death

X Initiation
Normal Cells Mutant Cells

X Promotion
Benign Tumors

X Progression
Malignant Tumors

Phytochemicals Other Chemopreventive Agents

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PROCARCINOGEN
Chemopreventive phytochemical based on Mechanism of action
Blocking agents Ellagic acid Indole-3-carbinol Sulforaphane flavonoids Metabolic activation

ULTIMATE CARCINOGEN
Interaction with cellular DNA
DNA DAMAGE, MUTAGENESIS (Initiated cells)

Supressing agents •-carotene Curcumin Gingerol EGCG Resveratrol

Promotion, Progression

MALIGNANT TRANSFORMATION
(Surh et al., 2000)
12

Rocaglamide (Agalaia Sp. = Pacar China)
Anticancer and Activity of Rocaglamide & Derivatives (27 compounds)

• Antiproliferation Activity (MTT assay) • Inhibition DNA-synthesis (Thymidine upatake) • Inhibition RNA-synthesis (uridine uptake) • Inhibition protein-synthesis (Leucine uptake) • Cellular viability test • Cell cycle • Proteine kinase • Inhibition transcription NF-kB
So. Bohnenstengel, 2000
13

Active compound of Aglaia sp for example :
Rocaglamid Derivatives Aglain Derivatives

rocaglamid (1H-2,3,3a,8b-tetrahydrocyclopenta[b] benzofuran)

A cyclopenta[bc]benzopyran (2,5-methano-1benzoxepin)
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Aglaforbesin Derivatives

Forbagline derivatives

15

Curcumin
Mechanisms of Cancer Chemoprevention by Curcumin
1. Suppression of c-jun and c-fos Expression 2. Inhibition of Protein Kinase C (PKC) and EGFR Tyrosine Kinase 3. Suppression of Colonic Aberrant Crypt Foci through Inhibiting iNOS 4. Inhibition of COX-2 by Curcumin in Bile Acid and PMA-Treated Cells 5. Inhibition of Xanthine Oxidase 6. Modulation of Ca+2 and Cellular p53 Protein 7. Reduction of ER(+)PgR(+) Mammary Tumor 8. Curcumin acts as Inducer of Phase-2 Detoxification Enzymes 9. Suppression of Hepatocellualr Carcinoma Invasion by Inhibiting MMP-9
J.K. Lin and S.Y. Lin-Shiau, 2001
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Modulation of Tumor Biomarkers by Curcumin

J.K. Lin and S.Y. Lin-Shiau, 2001

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Biochemical action of Curcumin
Biochemical action Scavenges superoxide anion and hydroxyl radical Scavenges singlet oxygen Inhibits lipid peroxidation Inhibits TPA-induced ornithine decarboxylase (ODC) mRNA and activity Inhibits TPA-induced cellular 8hydroxydeoxyguanosine Inhibits TPA-induced skin inflammation Inhibits lipoxygenase and cyclooxygenase activities Inhibits arachidonic acid metabolism Inhibits the formation of carcinogen-DNA adducts Inhibits skin tumor initiation and promotion Inhibits BaP induced forestomach and lung tumorigenesis Inhibits ENNG-induced duodenal tumorigenesis Inhibits azoxymethane-induced colon tumorigenesis in mice and rats Reference Kunchandy and Rao (1990) Subramanian et al. (1994) Sreejayan (1994) Huang et al. (1988) Shih and Lin (1993) Huang et al. (1997) Huang et al. (1991) Conney et al. (1991) Conney et al. (1991) Huang et al. (1992) Huang et al. (1994) Huang et al. (1994) Rao et al. (1995)

J.K. Lin and S.Y. Lin-Shiau, 2001

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Angiostatic Activity of Curcuminoids

Localized delivery of curcuminoids inhibits the angioproliferative response to FGF-2 stimulation in rabbit corneas. Photograph of rabbit eyes showing the cornea-scleral region of neovascularized corneas 12 days post-surgery. The slow release 80-ng FGF2 pellet in B and C are indicated by the asterisks, and the 2-mg curcuminoid pellet in A and C appear as yellow circular discs. (Royce Mohan et al, 2000)
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R&D by BPPT

20

Scope of works :
Phytochemistry : Isolation and identification of active compound

Target
• Lead compound • Marker

Bioassay (in vitro) : • MTT ploripheration assay • Cell cycle assay (in process)

• Efficacy data (in vitro) • Mechanism

Animal model (In vivo) : • Inhibition of DMBA - induced mammary tumorogenesis • Immunostimulant assay

• Preclinical data

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Selected Medicinal Plants
1. Sambiloto (Andrographis paniculata) 2. Laban abang (Aglaia elliptica Blume) 3. Biji klabet (Trigonella foenum graecum) 4. Binahong (Anredera cordifolia) 5. Keladi tikus (Typhonium divaricatum) 6. Rumput mutiara (Hedyotis corimbosa) 7. Mahkota dewa (Phaleria macrocarpa) 8. Kunyit putih (Curcuma zedoaria) 9. Herba Bandotan (Ageratum conyzoides) 10.Temu lawak (Curcuma xantorrhiza Roxb)

PROCESS SCHEME
DATABASE Med-Plants EXTRACTION Phytochemical Analisys
Standardization and validation Optimum Standardization, validation of extraction process

Ethnofarmacology

SELECTED PLANTS

In-vitro Screening
Cell proliferation (MTT Method)

SELECTED PLANTS

Formulation

FORMULA OBAT KANKER

Pre-clinical Test

In vitro assay (BPPT & RSK Dharmais)
Cell cycle (Flow cytometry) Apoptosis (DNA fragmentation & Caspase) Molecular target (p53, Protein kinase, etc)

Pre-clinical test (BPPT & RSK Dharmais)

STANDARDIZED HERBAL MEDICINE Clinical Test PHYTOMEDICINE

in vitro anticancer evaluation
100 µL Incubation, 24 h, 37oC, CO2 5% Sampel 100 µL Incubation, 72 h, 37oC, CO2 5% MTT 10 µL

Cell Culture

microplate

microplate

microplate
Incubation 4 h, 37oC, CO2 5%

100 µL Isopropanol-HCl

Absorb. 570 nm

Microplate reader

microplate

N

N N+ N

S

CH3

Dehidrogenase (mitokondria)

N

NH H N N

S

CH3

Data : prefent cell proliferation, LC50

N

N

MTT MTT-formazan

in vivo anticancer evaluation
Animal : female rats sp. Sprague Dawley
Metode : induction with carcinogen dimetil benz(a)antracen (DMBA)
5 weeks 4 weeks 12 weeks

DMBA (oral) 20 mg/Kg BW (10 X, 1 week 2 X)

Sampel, single dose (morning)

5 week old

Evaluation Data : • Tumor Incident • Tumor Multiplicity • Histopatology (proliferation, nekrosis, apoptosis)

In Vitro Preliminary Test
Herbal Medicine
Herba Bandotan (Ageratum conyzoides) Daun Binahong (Anredera cordifolia) Umbi Keladi tikus (Typhonium divaricatum) Biji Klabet (Trigonela foenum graecum) Rimpang Kunyit putih (Curcuma zedoaria) Daun Laban abang (Aglaia elliptica) Buah Mahkota dewa (Phaleria macrocarpa) Rumput mutiara (Hedyotis corimbosa) Herba Sambiloto (Andrographis paniculata) 67 12 215 546 26.9 IC50(ppm) on cell MCF-7 MeOH EtOH 88 51 285 372 55780 142 122 275 IC50 (ppm) on cell T-47D MeOH 207.5 15.8, 15.2 140 116.8 282 14000 119 70 EtOH 62 87,5 20 129 1248 286.26 112
26

Inhibition Test of Aglaia elliptica Extract on Cellular Inhibition of Cell T-47D

Inhibition of cell proliferation curve after treatment with ethanolic extract, n-hexsane fraction and water ethanolic fraction on T47D cell, respectively.

Inhibition of cell proliferation after treatment with ethyl acetate fraction on T-47D cell

Sample Ethanol extract n-hexane fraction Ethylacetate fraction Water ethanolic fraction

IC50 value (ppm) 265,26 210,14 22,11 214,58
27

Result of Proliferation Test of Aglaia elliptica Extract on MCF-7 Cell
Ethanol extract
Hambatan proliferasi (%) 70.00 60.00 50.00 40.00 30.00 20.00 250 500 750 1000 Konsentrasi (ppm)

n-hexane fraction
90.00 88.00 86.00 84.00 82.00 80.00 78.00 76.00 74.00 72.00 70.00 68.00 50 100 250 500 750 1000

Ethyl acetate fraction
90.00 80.00 70.00 60.00 50.00 40.00 30.00 20.00 10 20 40 80 160 320

Konsentrasi (ppm)

80.00 60.00 40.00 20.00 0.00 250 -20.00 -40.00 K o n s e n t r a s i (p p m) 500 750 1 000

ExtractFraction Butanol fraction Ethanol fraction n-hexane fraction

IC50 value (ppm) 373,23 ~ 18,51 1273,20 1478,67
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Hambatan proliferasi (%)

20.00 10.00 0.00 250 -10.00 -20.00 Konsentrasi (ppm) 500 750 1000

Ethylacetate fraction Butanol fraction Water fraction Water fraction

Preliminary Exploration Using MTT Test PLANT Binahong (leaves) Binahong (tuber) ISOLATE Isolate 1 Isolate 2 Isolate 1 Isolate 2 Isolate 3 Isolate 4 Hexadecanoic acid Phalerin Andrographolide Demethoxy andrographolide IC 50 (ppm) 60,47 (MCF-7) 7,14 (T47-D) 111,13 (MCF-7) 20,84 (MCF-7) 12,07 (MCF-7)
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Keladi tikus (tuber) Mahkota dewa Sambiloto

Preliminary Exploration Using MTT Test PLANT Temu lawak ISOLATE Curcumine Demethoxy curcumine Isolate 1 Isolate 2 Isolate 3 Isolate 4 Isolate 5 Isolate 6 Isolate 7 Isolate 8 IC 50 (ppm) 3,13 (MCF-7) 0,21 (MCF-7) 8,8 (MCF-7) 0,03 (MCF-7) 96,82 (MCF-7) 25,49 (MCF-7) 1,74 (MCF-7) 0,01 (MCF-7) 9,08 (MCF-7) 2,87 (MCF-7)
30

Preliminary Exploration Using MTT Test PLANT Rumput mutiara ISOLATE Isolate 1 Isolate 2 Isolate 3 Isolate 4 Isolate 5 Isolate 6 IC 50 (ppm) 10,28 (MCF-7) 6,36 (MCF-7) 2,61 (MCF-7) 3,24 (MCF-7) 66,22 (MCF-7) 9,43 (MCF-7)

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Spectrum of the Odorin (main compound of Aglaia extract)

4' 5' N 2"

3'

O 5 N H O 1 2 3 4

9" 8"

3" 4"

Posisi 2 3 4 5 2’ 3’ 4’ 5’ 2” 3” 5”/9” 6”/8” 7”

• H (ppm) 2,19 (m) A : 1,75 (m) B : 1,60 (m) 0,71 (t, 7,3) 1.10 (d, 6,7) 6,14 (d, 5,5 Hz) A: 2,26 (m) B: 2,02 (m) A: 2,09 (m) B: 1,90 (m) A: 3,73 (ddd, 3; 5;3,1 Hz) B; 3,50 (ttm) 6,61 (d, 15,9 Hz) 7,09 (d, 15,2 Hz) 7,59 (m) 7,58 (m) 7,54 (m)
7" 6"

5"

Odorin, C18H24N2O2 MW : 300 MTT test on MCF-7. LC50= 153,32 ppm

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Hasil uji in vivo ekstrak etanol daun Aglaia elliptica Blume
2.5 Nodul/tikus 2 1.5 1 0.5 0 D1 D2 D3 Perlakuan D1 D2 KNG KPS 2.6 2.2 1.8 1.4 Hari ke (setelah DMBA) 3 3 160 140 120 100 80 1 2 3 Nodul ke D3 KNG KPS 4 5

Grafik multiplisitas tumor
Rata-rata volume tumor (mm3) 90 80 70 60 50 40 30 20 10 0 D1 83.5

Grafik waktu latensi
Ket : D1 : 250 mg/kg BB D2 : 500 mg/kg BB D3 : 1000 mg/kg BB KNG : Kontrol negatif (DMBA) KPS : positive control (Doxorubicin 0,5µg/200 g BB, iv weekly 3x, 1 day after tumor appearance)

55

61.6

51.5

55.2

D2

D3 Perlakuan

KNG

KPS

Grafik rata-rata volume tumor

In Vivo

Histopatology of mamae tissue
Group of treatment D1 D2 D3 KNG KPS Score of Carcinogenesis 4,5 4,5 4,5 5 5

(4)

Sample : leaves extract of A. elliptica
SCORE 0= normal 1= mild ductal hyperplasia 2= severe ductal hyperplaisa 3= hyperplasia with atipia 4= ductal carcinoma in situ 5= ductal carcinoma invasive

(5)
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Result of In Vivo Test Using Sambiloto-Extract

Treated Group

Note : PREV : preventive, KUR : curative D1: 250 mg/kg BW, D2 ; 500 mg/ kg BW, D3 ; 1000 mg/kg BW
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Aplication of herbal medicine for anticancer
Cancer
Herbal medicine Prelim. Stadium Late Stadium Remarks

36 36

Application of herbal medicine for anticancer
Cancer
Herbal medicine Late Stadium Prelim. Stadium Remarks

Source : Suprapto Maat, 2001

37 37

Conclusion
Herbal Medicine could be applied for Supporting Cancer Management as :
— Chemo-preventive — Palliative treatment, if chemotherapy

radiotherapy, or surgery no more effective
— Adjuvant in addition of conventional therapy — Alternative medicine for those facing health cost

constraint using conventional therapy.
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Thank you

Herbal Medicine Research Group Agency for Assessment and Application of Technology (BPPT)
39

BINAHONG A B C

D

E

F

Effect of treatment on proliferation activity of Rat’s Mamae Cell (AgNOR straining). Control corn oil (a), control without (b), extract 1500 mg/kgBW (c), DMBA (d), DMBA-extract 750 mg/kgBW (e), DMBA-extract 1500 mg/kgBW (f).
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BINAHONG

(a)

(b)

(c)

(d)

(e)

(f)

DMBA (a), control corn oil (b), control without treatment (c), DMBA-extract 750 mg/kgBW (d), DMBA-extract 1500 mg/kgBW (e), extract 1500 mg/kgBW. There is no significant histological different among group (400x enlargement)

Ga

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NATURAL PRODUCT FOR SUPPORTING CANCER MANAGEMENT
Exploring the efficacy, safety, and prospect for drug development Through ~ Secondary information ~ Primary information (BPPT’s, R&D for Herbal Medicine Development)

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Chemopreventive dietary compounds.

Jonathan T, 2005

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Molecular targets of chemopreventive compounds

Jonathan T, 2005

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