Research in Natural Product for Supporting Cancer Management
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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
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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
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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
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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
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Treatment of Cancer
ó Surgery ó Chemotherapyó Radiotherapy ó Hormonal therapyó Biologic therapyó Gene therapy
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Natural Product
???
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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
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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
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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
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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
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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)
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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
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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
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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
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-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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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)
Preliminary Exploration Using MTT Test
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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)
Preliminary Exploration Using MTT Test
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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
34
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)
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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
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3636
Aplication of herbal medicine for anticancer
Cancer Herbal medicinePrelim. Stadium Late Stadium
Remarks
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Cancer Herbal medicinePrelim. Stadium Late Stadium
Remarks
Application of herbal medicine for anticancer
Source : Suprapto Maat, 2001
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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
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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