research in natural product for supporting cancer management

Research in Natural Productfor Supporting Cancer Management

Herbal Medicine Herbal Medicine Research GroupResearch GroupAgency for Assessment and Application of TechnologyAgency for Assessment and Application of Technology (BPPT)(BPPT)

Workshop on “Implementation Cancer Control Program : Accelerating Integrated System in Comprehensive Cancer Treatment”,

National Cancer Inst. RS. Dharmais,

Jakarta, 9-11 November 2009

Deputy for Technology

Policy

Deputy forTechnology of Natural Resources

Deputy forTechnology of Agroindustry & Biotechnology(Dr. Wahono S.)

Deputy for Technology of

Energy, Materials &

Environtment

Deputy forTechnology of

Industrial Design &

Engineering

CHAIRMANCHAIRMAN

SECRETARYINSPECTORATE

BPPTBPPTORGANIZATIONORGANIZATIONALAL

STRUCTURE STRUCTURE President of Republic President of Republic

of Indonesiaof Indonesia MinistryMinistryResearch & Research & TechnologyTechnology

§ Security & Defence§ Transportation§ Manufacturing

§ Food§ Health§ Bioenergy

§ Environment§ Earth Science§ Marine Science§ Disaster Mitigation

§ Policy§ Innovation

§ ICT § Energy§ Materials§ Environment

Ten ranks of majority diseases in Indonesia

SKRT 1992 SKRT 1995 Suskesnas 2001Diseases % Diseases % Diseases %

1. Circulation Syst 16.0 1. Circulation Syst 18,9 1. Circulation Syst 26,4

2. Tuberculosis 11,0 2. Respiration Syst 15,7 2. Respiration Syst 12,7

3. Kead.tidak jelas 9,8 3. Tuberculosis 9,6 3. Tuberculosis 9,4

4. Respiration Syst 9,5 4. Infection 7,9 4. Digestic Syst 7,0

5. Diarrhea 8,0 5. Diarrhea 7,4 5. Cancer 6,0

6. Infection 7,8 6. Digestic Syst 6,6 6. Accident 5,6

7. Bronchitis, asma 5,6 7. Perinatal Disorder 5,2 7. Perinatal Disorder 4,9

8. Trauma, Poisoning 5,3 8. Accident 5,2 8. Thyphus 4,3

9. Digestic Syst 5,3 9. Cancer 5,0 9. Diarrhea 3,8

10. Cancer 4,0 10. Neuro 2,5 10.Endokrin &Metabol. 2,7

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

DNA Mutation

1. Differentiation

2. Mitosis

3. Metastasis

4. Invasion

5. Angiogenesis

6. Apoptosis

Multi pathways cancer development Multi pathways cancer development

Needs comprehensive work to prevent pathways

Cancer cell & its roots

Anginogenesis

Treatment of Cancer

ó Surgery ó Chemotherapyó Radiotherapy ó Hormonal therapyó Biologic therapyó Gene therapy

6

Natural Product

???

7

India (60,000)

South America(100,000)

Africa (70,000)

China (40,000)

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

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

Indonesia (~ 80,000)

So: KRIBB KOREA, 2008

Korea (4,000)

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Algae: Algae: 782 782 spesiesspesies..SeagrassSeagrass : : 13 13 spesiesspesies..Mangrove : Mangrove : 38 38 spesiesspesiesSponge : Sponge : 850 850 spesiesspesiesCoral : Coral : 910 910 spesiesspesiesThousands more…Thousands more…

•• 30,000 sp Flowering Plants 30,000 sp Flowering Plants àà 9,600 as Medicinal Plants9,600 as Medicinal Plants

•• 250 sp have been used 250 sp have been used comerciallycomercially as herbal as herbal medicines medicines

•• Hundreds of thousands of Hundreds of thousands of microbes as microbes as endophyteendophyte or or soil microbessoil microbes

Terestrial DiversityTerestrial Diversity Marine DiversityMarine Diversity

So. BPOM, 2004 So. R. Dahuri, 1997

Official References :

1. National Policy on Traditional Medicine, ~ Decree of Ministry of Health No. 381/Menkes/SK/III/2007.

2. Complementary & Alternative Therapy Management in Health Service Facility. Regulation of Ministry of Health No. 1109/Menkes/Per/IX/2007.

3. Standardized Health Service Using Herbal Medicine-Decree of Ministry of Health No. 121/Menkes/SK/II/2008

4. WHO General Guidelines for Methodologues on Research and Evaluation of Traditional Medicine, 2000

5. WHO Guidelines for the Regulation of Herbal Medicines in the South-East Asia Region, 2003

6. WHO Guidelines on Safety Monitoring of Herbal Medicines in Pharmacovigilance System, 2004

9

1010

JAMUJAMU

• Empirical Claim Empirical Claim •• Placebo effectPlacebo effect

GEPGEPGEPGEP GMPGMPGMPGMPGCPGCPGCPGCP

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

ClinicalOn human

Standardized Standardized Herbal Med Herbal Med

PhytomedicinePhytomedicine

Extraction, Pharmacological ValidationExtraction, Pharmacological ValidationS&TS&T

Pre-clinical(on animal)

MedicinalMedicinalPlantPlant

TraditionalTraditionalknowledgeknowledge

“GAP”“GAP”Good AgriculturalGood Agricultural

PracticePractice

“Modern”“Modern”KnowledgeKnowledge

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

Ethnobotany

Efficacy & toxicity testing

Pharmaco-Epidemologic

Carcinogenesis and the Action of Phytochemicals & other Chemopreventive Agents

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Activation of Activation of ProcarcinogensProcarcinogens by by CYPsCYPs

Activation of Activation of ProcarcinogensProcarcinogens by by CYPsCYPs

Gene Mutation Proto-oncogene Tumor Suppressor

Gene Mutation Proto-oncogene Tumor Suppressor

AngiogenesisProgram Cell DeathAngiogenesisProgram Cell Death

DNA DamageDNA Damage

NormalCellsNormalCells

PromotionPromotion

MutantCellsMutantCells

ProgressionProgression

BenignTumorsBenignTumors

MalignantTumorsMalignantTumors

PhytochemicalsOther Chemopreventive AgentsPhytochemicalsOther Chemopreventive Agents

InitiationInitiationXX XXXX

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PROCARCINOGEN

ULTIMATE CARCINOGEN

DNA DAMAGE, MUTAGENESIS (Initiated cells)

MALIGNANT TRANSFORMATION

Blocking agentsEllagic acid Indole-3-carbinol Sulforaphane flavonoids

Supressing agents•-carotene Curcumin Gingerol EGCG Resveratrol

Metabolic activation

Interaction with cellular DNA

Promotion, Progression

Chemopreventive phytochemical based on Mechanism of action

(Surh et al., 2000)

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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

Rocaglamide (Agalaia Sp. = Pacar China)

So. Bohnenstengel, 2000

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

Aglain Derivatives Rocaglamid Derivatives

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

Active compound of Aglaia sp for example :

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Forbagline derivativesAglaforbesin Derivatives

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Mechanisms of Cancer Chemoprevention by Curcumin

1. Suppression of c-jun and c-fos Expression2. Inhibition of Protein Kinase C (PKC) and EGFR Tyrosine Kinase3. Suppression of Colonic Aberrant Crypt Foci through Inhibiting

iNOS4. Inhibition of COX-2 by Curcumin in Bile Acid and PMA-Treated

Cells5. Inhibition of Xanthine Oxidase6. Modulation of Ca+2 and Cellular p53 Protein7. Reduction of ER(+)PgR(+) Mammary Tumor8. Curcumin acts as Inducer of Phase-2 Detoxification Enzymes9. Suppression of Hepatocellualr Carcinoma Invasion by Inhibiting

MMP-9J.K. Lin and S.Y. Lin-Shiau, 2001

Curcumin

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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 ReferenceScavenges superoxide anion and hydroxyl radical Kunchandy and Rao (1990)Scavenges singlet oxygen Subramanian et al. (1994)Inhibits lipid peroxidation Sreejayan (1994)Inhibits TPA-induced ornithine decarboxylase (ODC) mRNA and activity

Huang et al. (1988)

Inhibits TPA-induced cellular 8-hydroxydeoxyguanosine

Shih and Lin (1993)

Inhibits TPA-induced skin inflammation Huang et al. (1997)Inhibits lipoxygenase and cyclooxygenase activities Huang et al. (1991)Inhibits arachidonic acid metabolism Conney et al. (1991)Inhibits the formation of carcinogen-DNA adducts Conney et al. (1991)Inhibits skin tumor initiation and promotion Huang et al. (1992)Inhibits BaP induced forestomach and lung tumorigenesis

Huang et al. (1994)

Inhibits ENNG-induced duodenal tumorigenesis Huang et al. (1994)Inhibits azoxymethane-induced colon tumorigenesis inmice and rats

Rao et al. (1995)

Biochemical action of Curcumin

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

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Angiostatic Activity of Curcuminoids

Localized delivery of curcuminoids inhibits the angioproliferativeresponse to FGF-2 stimulation in rabbit corneas.Photograph of rabbit eyes showing the cornea-scleral region ofneovascularized corneas 12 days post-surgery. The slow release 80-ng FGF-2 pellet in B and C are indicated by the asterisks, and the 2-mg curcuminoidpellet in A and C appear as yellow circular discs.

(Royce Mohan et al, 2000)

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R&D by BPPT

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Scope of works :

Phytochemistry :Isolation and identification of active compound

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

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

• Immunostimulant assay

Target

• Lead compound• Marker

• Efficacy data (in vitro)

• Mechanism

• Preclinical data

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)

DATABASE Med-Plants

SELECTED PLANTS

SELECTED PLANTS

Ethnofarmacology

In-vitro Screening Cell proliferation (MTT Method)

FORMULA OBAT

KANKER

Formulation

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)

Phytochemical AnalisysStandardization and validation

EXTRACTIONOptimum Standardization,

validation of extraction process

Pre-clinical Test

Clinical Test

STANDARDIZED HERBAL

MEDICINE

PHYTOMEDICINE

PROCESS SCHEMEPROCESS SCHEME

in vitro anticancer evaluation

N

N+

NN

N

S CH3

Cell Culture microplate

100 µL

Incubation, 24 h, 37oC, CO2 5%

microplate

Sampel 100 µL

Incubation, 72 h, 37oC, CO2 5%

microplate

MTT 10 µL

microplate

100 µL Isopropanol-HCl

Incubation 4 h, 37oC, CO2 5%

Microplate reader

Absorb. 570 nm

N

HN

NHN

N

S CH3

MTT

Dehidrogenase (mitokondria)

MTT-formazan

Data : prefent cell proliferation, LC50

Animal : female rats sp. Sprague DawleyMetode : induction with carcinogen dimetil benz(a)antracen (DMBA)

DMBA (oral)20 mg/Kg BW

(10 X, 1 week 2 X)Sampel, single dose

(morning)

5 weeks 4 weeks 12 weeks

Evaluation5 week old

Data :• Tumor Incident• Tumor Multiplicity• Histopatology (proliferation,

nekrosis, apoptosis)

in vivoin vivo anticancer evaluationanticancer evaluation

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Herbal Medicine

IC50(ppm) on cell MCF-7

IC50 (ppm) oncell T-47D

MeOH EtOH MeOH EtOH

Herba Bandotan(Ageratum conyzoides) - - 207.5 62

Daun Binahong(Anredera cordifolia) 67 88 15.8, 87,5

Umbi Keladi tikus(Typhonium divaricatum) 12 51 15.2 20

Biji Klabet (Trigonela foenum graecum) 215 285 140 129

Rimpang Kunyit putih(Curcuma zedoaria) 546 - 116.8 1248

Daun Laban abang(Aglaia elliptica) 26.9 372 282 286.26

Buah Mahkota dewa(Phaleria macrocarpa) - 55780 14000 -

Rumput mutiara(Hedyotis corimbosa) 142 119 -

Herba Sambiloto(Andrographis paniculata) 122 275 70 112

In Vitro Preliminary Test

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

Inhibition of cell proliferation curve aftertreatment with ethanolic extract, n-hexsanefraction and water ethanolic fraction on T-47D cell, respectively.

Inhibition Test of Aglaia elliptica Extract onCellular Inhibition of Cell T-47D

Sample IC50 value (ppm) Ethanol extract 265,26n-hexane fraction 210,14Ethylacetate fraction 22,11Water ethanolic fraction 214,58

-20.00

-10.00

0.00

10.00

20.00

250 500 750 1000

Konsentrasi (ppm)

Ham

bat

an p

rolif

eras

i (%

)

28

Result of Proliferation Test of Aglaia elliptica Extract on MCF-7 Cell

20.00

30.00

40.00

50.00

60.00

70.00

250 500 750 1000

Konsentrasi (ppm)

Ham

bata

n pr

olife

rasi

(%)

Ethanol extract

68.0070.0072.0074.0076.0078.0080.0082.0084.0086.0088.0090.00

50 100 250 500 750 1000

Konsentrasi (ppm)

n-hexane fraction

Water fraction

Ethyl acetate fraction

20.00

30.00

40.00

50.00

60.00

70.00

80.00

90.00

10 20 40 80 160 320

-40.00

-20.00

0.00

20.00

40.00

60.00

80.00

250 500 750 1000

Konsentrasi (ppm)

ExtractFraction IC50 value (ppm)

Ethanol fraction 373,23

n-hexane fraction ~

Ethylacetate fraction 18,51

Butanol fraction 1273,20

Water fraction 1478,67

Butanol fraction

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PLANT ISOLATE IC 50 (ppm)

Binahong (leaves) Isolate 1 60,47 (MCF-7)

Isolate 2 7,14 (T47-D)

Binahong (tuber) Isolate 1 -

Isolate 2 -

Isolate 3 -

Isolate 4 -

Keladi tikus (tuber) Hexadecanoic acid -

Mahkota dewa Phalerin 111,13 (MCF-7)

Sambiloto Andrographolide 20,84 (MCF-7)

Demethoxyandrographolide

12,07 (MCF-7)

Preliminary Exploration Using MTT Test

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Temu lawak Curcumine 3,13 (MCF-7)

Demethoxy curcumine 0,21 (MCF-7)

Isolate 1 8,8 (MCF-7)









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Rumput mutiara Isolate 1 10,28 (MCF-7)







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Spectrum of the Odorin (main compound of Aglaia extract)

NNH

O

O

4"

3"

2"

5'

4' 3'

1 2

5

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5"7"

8"9"

6"

Odorin, C18H24N2O2

MW : 300

MTT test on MCF-7.LC50= 153,32 ppm

Posisi • H (ppm)2 2,19 (m)3 A : 1,75 (m)

B : 1,60 (m)4 0,71 (t, 7,3)5 1.10 (d, 6,7)2’ 6,14 (d, 5,5 Hz)3’ A: 2,26 (m)

B: 2,02 (m)4’ A: 2,09 (m)

B: 1,90 (m)5’ A: 3,73 (ddd, 3; 5;3,1 Hz)

B; 3,50 (ttm)2” 6,61 (d, 15,9 Hz)3” 7,09 (d, 15,2 Hz)

5”/9” 7,59 (m)6”/8” 7,58 (m)

7” 7,54 (m)

Hasil uji in vivo ekstrak etanol daun Aglaia elliptica Blume

Grafik multiplisitas tumor

Grafik rata-rata volume tumor

80

100

120

140

160

1 2 3 4 5

Ha

ri k

e (s

etel

ah

DM

BA

)

Nodul ke

D1 D2 D3 KNG KPS

Grafik waktu latensi

Ket :D1 : 250 mg/kg BBD2 : 500 mg/kg BBD3 : 1000 mg/kg BBKNG : Kontrol negatif (DMBA)KPS : positive control (Doxorubicin 0,5µg/200 g BB, iv weekly 3x, 1 day after tumor appearance)

0

0.5

1

1.5

2

2.5

3

D1 D2 D3 KNG KPS

2.6

2.21.8

1.4

3N

odul

/tik

us

Perlakuan

0

10

20

30

40

50

60

70

80

90

D1 D2 D3 KNG KPS

5561.6

51.5

83.5

55.2

Ra

ta-r

ata

vol

ume

tum

or (

mm

3)

Perlakuan

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Histopatology of mamae tissue

Group of treatment

Score of Carcinogenesis

D1 4,5

D2 4,5

D3 4,5

KNG 5

KPS 5

SCORE0= normal1= mild ductal hyperplasia2= severe ductal hyperplaisa3= hyperplasia with atipia4= ductal carcinoma in situ5= ductal carcinoma invasive (5)

(4)

In Vivo

Sample : leaves extract of A. elliptica

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Result of In Vivo Test Using Sambiloto-Extract

Note : PREV : preventive, KUR : curativeD1: 250 mg/kg BW, D2 ; 500 mg/ kg BW, D3 ; 1000 mg/kg BW

Treated Group

3636

Aplication of herbal medicine for anticancer

Cancer Herbal medicinePrelim. Stadium Late Stadium

Remarks

3737

Cancer Herbal medicinePrelim. Stadium Late Stadium

Remarks

Application of herbal medicine for anticancer

Source : Suprapto Maat, 2001

ó Chemo-preventive

ó Palliative treatment, if chemotherapy radiotherapy, or surgery no more effective

ó Adjuvant in addition of conventional therapy

ó Alternative medicine for those facing health cost constraint using conventional therapy.

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Conclusion

Herbal Medicine could be applied for Supporting Cancer Management as :

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

Thank you

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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).

BINAHONG

41Ga

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)

(a) (c)(b)

(e) (f)(d)

BINAHONG

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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

research in natural product for supporting cancer management

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