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

INSPECTORATE SECRETARY

Deputy for Deputy for Deputy for Deputy for Deputy for
Technology Technology Technology of Technology of Technology of
Policy of Natural Agroindustry & Energy, Industrial
Resources Biotechnology Materials & Design &
(Dr. Wahono S.) Environtment Engineering

§ Policy § Environment § Food § ICT § Security & Defence

§ Innovation § Earth Science § Health § Energy § Transportation
§ Marine Science § Bioenergy § Materials § Manufacturing
§ Disaster Mitigation § Environment
3
 Ten ranks of majority diseases in Indonesia

SKRT 1992 SKRT 1995 Suskesnas 2001

Diseases % 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, 2001
Health Profile of Indonesia 2003, Health Dept,2005
Multi pathways cancer development
DNA Mutation

1. Differentiation
2. Mitosis
3. Metastasis
4. Invasion Cancer cell & its roots

5. Angiogenesis
6. Apoptosis

Needs comprehensive
work to prevent
pathways
Anginogenesis
 Treatment of Cancer

— Surgery
— Chemotherapy
— Radiotherapy
— Hormonal therapy Natural
— Biologic therapy Product
— Gene therapy ???

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 Marine Diversity

• 30,000 sp Flowering Plants Algae: 782 spesies

spesies..
à 9,600 as Medicinal Plants
Seagrass : 13 spesies
spesies..
• 250 sp have been used Mangrove : 38 spesies
comercially as herbal
Sponge : 850 spesies
medicines
Coral : 910 spesies
• Hundreds of thousands of
microbes as endophyte or Thousands more…
soil microbes

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

8
 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
 “GAP” Ethnobotany
Good Agricultural • Empirical Claim
Practice
• Placebo effect
Traditional JAMU
knowledge S&T
Extraction, Pharmacological Validation

Medicinal “Modern” Standardized Phytomedicine

Plant Knowledge Herbal Med
Clinical
Pre-clinical On human
(on animal)
Efficacy & toxicity testing

S&T assessment: Pharmaco-Epidemologic

• Phytochemistry
• Cultivation GEP GCP GMP
• Processing
• Formulation
• Pharmacological GAP (Good Agricultural Practices)
validation GEP (Good Extracting Practices)
GCP (Good Clinical Practice)
GMP (Good Manufacturing Practices) 10
 Carcinogenesis and the Action of Phytochemicals &
other Chemopreventive Agents

Activation of Gene Mutation Angiogenesis

Procarcinogens by Proto-oncogene Program Cell Death
CYPs Tumor Suppressor

DNA Damage

X
Initiation
X
Promotion
X
Progression

Normal Mutant Benign Malignant

Cells Cells Tumors Tumors

Phytochemicals
Other Chemopreventive Agents

11
 PROCARCINOGEN
Chemopreventive
phytochemical based Metabolic activation
on Mechanism of
action
ULTIMATE
Blocking agents CARCINOGEN
Ellagic acid
Indole-3-carbinol
Sulforaphane Interaction with cellular DNA
flavonoids
DNA DAMAGE,
MUTAGENESIS
(Initiated cells)

Supressing agents Promotion, Progression

•-carotene
Curcumin
Gingerol MALIGNANT
EGCG TRANSFORMATION
Resveratrol (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 A cyclopenta[bc]benzopyran (2,5-methano-1-

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

14
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 16
 Modulation of Tumor Biomarkers by Curcumin

J.K. Lin and S.Y. Lin-Shiau, 2001 17

 Biochemical action of Curcumin
Biochemical action Reference
Scavenges 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) Huang et al. (1988)
mRNA and activity
Inhibits TPA-induced cellular 8- Shih and Lin (1993)
hydroxydeoxyguanosine
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 Huang et al. (1994)
tumorigenesis
Inhibits ENNG-induced duodenal tumorigenesis Huang et al. (1994)
Inhibits azoxymethane-induced colon tumorigenesis in Rao et al. (1995)
mice and rats

J.K. Lin and S.Y. Lin-Shiau, 2001 18

 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 FGF-
2 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)

19
R&D by BPPT

20
 Scope of works : Target

Phytochemistry : • Lead compound

Isolation and identification of active • Marker
compound

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

Animal model (In vivo) : • Preclinical data

• Inhibition of DMBA - induced mammary
tumorogenesis
• Immunostimulant assay

21
 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
Optimum Standardization,
Standardization and validation validation of extraction process

Ethnofarmacology

SELECTED SELECTED Formulation FORMULA

PLANTS PLANTS OBAT
In-vitro Screening KANKER
Cell proliferation (MTT Method)

Pre-clinical Test

STANDARDIZED
In vitro assay (BPPT & RSK Dharmais) Pre-clinical test HERBAL
(BPPT & RSK Dharmais) MEDICINE
Cell cycle (Flow cytometry)
Apoptosis (DNA fragmentation & Caspase)
Clinical Test
Molecular target (p53, Protein kinase, etc)

PHYTOMEDICINE
 in vitro anticancer evaluation
Sampel
100 µL 100 µL MTT 10 µL

Incubation, 24 h,
37oC, CO2 5% Incubation, 72 h,
37oC, CO2 5%

Cell
Culture microplate microplate microplate

Incubation 4 h,
100 µL 37oC, CO2 5%
Isopropanol-HCl

Absorb. 570 nm

Microplate reader microplate

N
Dehidrogenase
N
NH
S CH3
Data : prefent cell proliferation, LC50
N S CH3
(mitokondria) H
N
N+
N N
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 Sampel, single dose
(10 X, 1 week 2 X) (morning)

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

Inhibition of cell proliferation curve after Inhibition of cell proliferation after

treatment with ethanolic extract, n-hexsane treatment with ethyl acetate fraction on
fraction and water ethanolic fraction on T- T-47D cell
47D cell, respectively.

Sample IC50 value (ppm)

Ethanol extract 265,26
n-hexane fraction 210,14
Ethylacetate fraction 22,11
Water ethanolic fraction 214,58
27
 Result of Proliferation Test of Aglaia elliptica Extract on MCF-7 Cell

Ethanol extract n-hexane fraction Ethyl acetate fraction

Hambatan proliferasi (%)

90.00
70.00 90.00
88.00
60.00 86.00 80.00
84.00
82.00
70.00
50.00
80.00 60.00
40.00 78.00
76.00 50.00
30.00 74.00
40.00
72.00
20.00 70.00 30.00
250 500 750 1000 68.00
50 100 250 500 750 1000 20.00
Konsentrasi (ppm) Konsentrasi (ppm) 10 20 40 80 160 320

80.00

60.00

40.00

20.00

0.00
250 500 750 1000
ExtractFraction IC50 value (ppm)
-20.00

-40.00 Butanol fraction Ethanol fraction 373,23

K o n s e n t r a s i (p p m)

n-hexane fraction ~
Hambatan proliferasi (%)

20.00
Ethylacetate fraction 18,51
10.00

0.00
Butanol fraction 1273,20
250 500 750 1000
-10.00 Water fraction 1478,67
-20.00
Water fraction
Konsentrasi (ppm)
28
 Preliminary Exploration Using MTT Test
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)
Demethoxy 12,07 (MCF-7)
andrographolide
29
 Preliminary Exploration Using MTT Test

PLANT ISOLATE IC 50 (ppm)

Temu lawak Curcumine 3,13 (MCF-7)
Demethoxy curcumine 0,21 (MCF-7)
Isolate 1 8,8 (MCF-7)
Isolate 2 0,03 (MCF-7)
Isolate 3 96,82 (MCF-7)
Isolate 4 25,49 (MCF-7)
Isolate 5 1,74 (MCF-7)
Isolate 6 0,01 (MCF-7)
Isolate 7 9,08 (MCF-7)
Isolate 8 2,87 (MCF-7)

30
 Preliminary Exploration Using MTT Test

PLANT ISOLATE IC 50 (ppm)

Rumput mutiara Isolate 1 10,28 (MCF-7)
Isolate 2 6,36 (MCF-7)
Isolate 3 2,61 (MCF-7)
Isolate 4 3,24 (MCF-7)
Isolate 5 66,22 (MCF-7)
Isolate 6 9,43 (MCF-7)

31
Spectrum of the Odorin (main compound of Aglaia extract)

3' O
4'
5
5' 1
N 2
N H
3
4
2"
O
9" 3"
8"
Posisi • H (ppm) 4"
2 2,19 (m) 7" 5"
3 A : 1,75 (m)
6"
B : 1,60 (m)
4 0,71 (t, 7,3)
5 1.10 (d, 6,7)
2’ 6,14 (d, 5,5 Hz) Odorin, C18H24N2O2
3’ A: 2,26 (m)
B: 2,02 (m)
MW : 300
4’ A: 2,09 (m)
B: 1,90 (m) MTT test on MCF-7.
5’ A: 3,73 (ddd, 3; 5;3,1 Hz)
B; 3,50 (ttm) LC50= 153,32 ppm
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)
32
Hasil uji in vivo ekstrak etanol daun Aglaia elliptica Blume
3 3 160

Hari ke (setelah DMBA)

2.6
2.5
2.2 140
Nodul/tikus

2 1.8
1.5 1.4 120

1
100
0.5

0 80
D1 D2 D3 KNG KPS 1 2 3 4 5
Perlakuan Nodul ke

D1 D2 D3 KNG KPS
Grafik multiplisitas tumor
Grafik waktu latensi
Rata-rata volume tumor (mm3)

90 83.5 Ket :
80
70 D1 : 250 mg/kg BB
60 55
61.6
51.5 55.2 D2 : 500 mg/kg BB
50
40 D3 : 1000 mg/kg BB
30 KNG : Kontrol negatif (DMBA)
20
10 KPS : positive control (Doxorubicin
0 0,5µg/200 g BB, iv weekly 3x, 1 day after
D1 D2 D3 KNG KPS
tumor appearance)
Perlakuan

Grafik rata-rata volume tumor

 In Vivo
Histopatology of mamae tissue
Group of Score of
treatment Carcinogenesis
D1 4,5
D2 4,5
D3 4,5
KNG 5 (4)
KPS 5
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)
34
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
35
 Aplication of herbal medicine for anticancer

Cancer Herbal medicine Remarks

Prelim. Stadium Late Stadium

36
36
 Application of herbal medicine for anticancer

Cancer Herbal medicine Remarks

Prelim. Stadium Late Stadium

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.

38
 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).

40
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
41
 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)

42
 Chemopreventive dietary compounds.

Jonathan T, 2005 43
 Molecular targets of chemopreventive compounds

Jonathan T, 2005 44