THE LEAVES OF ARTOCARPUS ALTILIS CONSTITUENTS POTENTIAL FOR CARDIOVASCULAR DISEASE TREATMENTLeonardus B.S. Kardono, Tjandrawati Mozef, Nina Artanti, Puspa Dewi, M. Hanafi and Umar A. Jenie Research Center for Chemistry Indonesian Institute of Sciences

Screening of 42 plant species against cardiovascular system (endothel cells, monocyte cells, cardiomyocytes cells)

Selected plant: Artocarpus altilis (Parkinson) Fosberg. (syn. Artocarpus camansi Blanco, A. communis J.R. and G. Forster): family Moraceae (Mulberry family) known as bread fruit or sukun (Indonesian name). ETNOBOTANY/ETNOPHARMACOLOGY REVIEW Tropical plant (Indonesia, Thailand, Vietnam and Cambodia). Grows in Wallacea regions. In Indonesia the leaves used traditionally for treatment of liver diseases, inflammation, renal diseases, tooth ache and rash In Taiwan the roots and branch is used for treatment of liver disease, cirrhosis and hypertension.

Extraction of Sukun leaves

Further isolation20 fractions: Bioassays against cardiovascular diseases

Atherosclerosis: Induction: ox-LDLControl: SimvastatinAnalysis: cell proliferation using WST method (respiration activity)U937 cells -derived foam cellsEndothelial cells (HUVEC)Hypertension : Left ventricle hypertrophyInduction : Angiotensin IIControl: PropanololAnalysis: Cell viability by measuring the total protein (Bradford method)Cardiomyocytes cells: Neo natal rat left ventricle (1 day old)In vitro assay : Cell model

Effect of Sukun extract on proliferation of U937 cells induced by ox-LDL*: p

Pengaruh ekstrak sukun terhadap proliferasi sel endotel yang diinduksi ox-LDL

Effect of sukun extract on cardiomyocytes cells with hypertrophy (induced by Angiotensin II)*: p

Screening results ActiveNot activeActive fractions against the 3 kind of cell model: A1, A9, A20

A1A2A3A4A5A6A7A8A9A10Endothelial cellU937cardiac myocytesA11A12A13A14A15A16A17A18A19A20Endothelial cellU937cardiac myocytes

Further fractionation of the active fractions using chromatography method Structure elucidation using NMR, MS, IR and UV spectrum.

Active fractionType of compoundMain active constituentA1sterol-sitosterolA9flavonoid1-(2,4-Dihydroxyphenyl)-3-[8-hydroxy-2-methyl-2-(4-methyl-3-pentenyl)-2H-1-benzopyran-5-yl]-1-propanoneflavonoid8-geranyl-4',5,7- tirhydroxyflavoneflavonoid2-geranyl-2',3,4,4'-tetrahydroxychalcone

-sitosterol: 1-(2,4-Dihydroxyphenyl)-3-[8-hydroxy-2-methyl-2-(4-methyl-3-pentenyl)-2H-1-benzopyran-5-yl]-1-propanone8-geranyl-4',5,7- tirhydroxyflavone2-geranyl-2',3,4,4'-tetrahydroxychalcone Isolated chemical compounds from the ethyl acetate fraction2,4,5-trihydroxy-7-methoxy-8-prenylflavone2-geranyl-3,4,7-tirhydroxyflavone

IN VIVO STUDIES :Platelet agregationBlood viscosityThrombosisAcute ischemiaAtherosclerosis:Lipid accumulation on aortaBlood cholestherol

ANTI PLATELET AGGREGATION AND BLOOD VISCOSITY EXPERIMENTAIM:to determine which proportion of drugs giving the best effect on inhibition of platelet aggregation and on blood viscocity.

SAMPELS:Total flavonoids and sitosterol from Artocarpus altilis

ANIMAL:Sprague Dawley male rats (250g- 300g) 7 groups (6 rats/group)FEEDING:

once a day for 1 week: Group NaCl: 0.9% NaCl (2mL) Group Control 1: aspirin 312.5mg/kg BW Group Control 2: Gingko Biloba 5.76mg/kg BW Group Drugs 1: high total flavonoid 200mg/kg Group Drugs 2: total flavonoid (LD) 50 mg/kg BW and sitosterol 10 mg/kg BW (MD) Group Drugs 3: total flavonoid (MD) 100 mg/kg BW and sitostero (HD)l 20 mg/kg BW Group Drugs 4: total flavonoid 200 (HD)mg/kg BW and sitosterol (LD) 5 mg/kg BW

ABDOMINAL ARTERYCentrifugation 800rpm,10min PRP

Further centrifugation 2000rpm, 10min PPPIn a cuvette: 300 l of PRP or PPP10 l of Adenosine diphosphate (33.3M)

AGGREGOMETER% Inhibition of platelet aggregationFROM EYEBlood 3ml + 60l heparin (40 u/ml)

0.8ml injected into the viscometer.

VISCOMETERMaximum value of the blood viscosity.BLOOD COLLECTION

ANTI-PLATELET AGGREGATION EXPERIMENTCONCLUSIONMTF (100mg/kg)+HBS (20mg/kg) and LTF (50mg/kg)+MBS (10mg/kg) active as anti platelet agregation (comparison: Aspirin and Gingko)

BLOOD VISCOSITY EXPERIMENTCONCLUSIONMTF+HBS and LTF+MBS have activity to reduce blood viscosity compare to normal, and similar to the positive control (gingko and aspirin) especially on high shear rate.

Effect of anti-blood viscosity

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THROMBOSIS EXPERIMENTFEEDING:Group NaCl: 0.9% NaCl (2mL)Group Control: Ginkgo biloba (5.76mg/kg)Group Drugs 1: LTF (total flavonoid 50mg/kg) and MBS ( sitosterol 10mg/kg)Group Drugs 2: MTF (total flavonoid 100 mg/kg) and HBS ( sitosterol 20 mg/kg)Conclusion:MTF+ HBS and LTF+MBS active as anti thromosis compare to positive control (Gingko biloba)

Catheter at jugular vein injected with 1ml of 1% heparin. Micro vein (diameter: 20 40m) of the mesenteryFluorescein Sodium injected through the catheter. Light of 490nmRecord of thrombus formationThrombosis.mpg

ACUTE CARDIAC ISCHEMIA EXPERIMENTAIM:To assess effect of drugs on expression of 4 keys enzymes (LDH, CK, SOD and MDA) involved in myocardial infarction caused by ischemia

SAMPEL:Total flavonoids and sitosterol from Artocarpus altilis

ANIMAL:Sprague Dawley male rats (250g- 300g) 5 groups (10 rats/group),

FEEDING:once a day for 1 week: Group NaCl: 0.9% NaCl (2mL)Sham operation Group Model: 0.9% NaCl (2mL) Group Control: DI AO (Chengdu Pharmaceutical) 0.06g/kg BWGroup Drugs 1: total flavonoid (LD) 50 mg/kg BW and sitosterol 10 mg/kg BW (MD) Group Drugs 2: total flavonoid (MD) 100 mg/kg BW and sitostero (HD)l 20 mg/kg BW

Catheter in jugular vein injected with 1ml of 1% heparin 1 ml of pituitrin (0.6 u/kg BW) to induce ischemia ECG monitored for 10 minutes. BLOOD COLLECTIONFrom abdominal arteryASSAY OF 4 KEYS ENZYMESLactate dehydrogenaseCreatine KinaseSuperoxide DismutaseMaleic dialdehydeCentrifugation at 1200g for 10 minute to get the serum

EKSPERIMEN ANTI ISCHEMIACONCLUSIONThe mixture of total flavonoid and b-sitosterol (MTF+HBS) shows anti-ischemia activity compare to the model (positive control DiAO) especially at key enzyme LDH (Lactate dehydrogenase) and CK (Creatin Kinase).

Experiment on lipid accumulation at aorta vesselAim:To study the effect of flavonoids on lipid accumulation at aorta vesselSample:Flavonoids of A. altilis Animal model: Wistar male rat, weight 180-200 g, 2months old. 3 groups (5 rats /group) and negative control group (2 rats) Feeding:High cholesterol diet for 30 days: Group I (Positive control): high cholesterol dietGroup II: high cholesterol diet+ sukun fraction 150 mg/kg BWGroup III: high cholesterol diet+ sukun fraction 300 mg/kg BW Group IV (Negative control): standard dietOn Day 31:Blood sample taken from the tailLaparatomi an histology of aorta blood vessel to observe the lipid accumulation on every group

Aorta HistologyPositive ControlNegative controlFlavonoids Total 150 mg/kgFlavonoids Total 300 mg/kg

Cholesterol content assayp

Toxicity studiesAcute toxicity: using ICR miceSubcronic toxicity: using Wistar rat

ACUTE TOXICITY EXPERIMENTFEEDING sitosterol 2.5g/kg BW or equivalent to 125x of human dose (2mg/kg BW)total flavonoid 4.5g/kg BW or equivalent to 400x of human dose (10mg/kg BW)OBSERVATION: after 3,7 and 14 day Body weighDeath rateBehaviour: walking, drinking and eating (total water and food taken was recorded), brightness of eyes and fur/hairsCONCLUSION:HighTF and BS is not toxic to animal model (no weight reduction and show normal behaviour )ICR mice: 10 males 10 females

SUBCRONIC TOXICITY STUDYDosage: flavonoid= 50 mg/kg bb/hari. Total flavonoid content in A. Altilis fraction=30%. Route: oral, 1xdayly for 90 days

GroupTreatmentNo. male rats (n)Numbe of female rats(n)1Given Flavonoids Total 83,33 mg/kg bw/ day10102Given sukun fraction 166,67 mg/kg bw/ day10103Given Flavonoids Total 333,33 mg/kg bw/ day10104Normal control, given 1% CMC solution1010

HEARTACTIVITY OF ASPARTATE AMINO TRANSFERASEACTIVITY OF CREATINE KINASEHEART HISTOLOGY ON MALE AND FEMALE RATOne way ANAVA analysis show that AAT and CK activities of the male and female rats in groups treated with flavonoids (Group I,II,III) did not show significant differences with the control (Group IV)

GroupRepetitionpolymorpho nuclear infiltration Lipid formation on chamber between FibrosisI6---II6---III6---IV6---

JANTUNG (HISTOLOGI) (A)nucleus, (B) fibroblast GROUP 1GROUP 2GROUP 4GROUP 3Comparison between control and treated groups showed that flavonoids treatment did not show any effect on heart of male and female rats.

KIDNEYOne way ANAVA analysis show that urea and creatine plasma contents of the male and female rats in groups treated with flavonoids (Group I,II,III) did not show significant differences with the control (Group IV)

KIDNEY (HISTOLOGY)One way ANAVA analysis show that diameter of kapsula bowman and the distance between glomerulus chamber and kapsula bowman of the male and female rats in groups treated with flavonoids (Group I,II,III) did not show significant differences with the control (Group IV)

LIVEROne way ANAVA analysis show that ALT and AP activities of the male and female rats in groups treated with flavonoids (Group I,II,III) did not show significant differences with the control (Group IV)

LIVER HISTOLOGYAverage number of damage cells on male and female rats after 90 days of treamentDiameter of Vena Centralis Liver histology shows that there is no damage cells after 90 days feeding with flavonoids total One way ANAVA analysis show that diameter of vena centralis of the male and female rats in groups treated with flavonoids total (Group I,II,III) did not show significant differences with the control (Group IV)

GroupDegree of Damage Cell0%(without damage)20% - 40% (medium necrosis)> 40% (heavy necrosis)IIIIIIIV100%100%100%100%0%0%0%0%0%0%0%0%

GroupMale RatFemale RatIIIIIIIV62,86 1,2963,39 1,0564,32 1,2563,21 1,5663,50 2,6463,73 2,5164,32 1,9363,05 2,29

HaematologyANAVA analysis show that the numbers of erythrocyte, leucocyte, thrombocyte and haemoglobin content on male and female rats in groups treated with flavonoids total (Group I,II,III) did not show significant differences with the control (Group IV)

CONCLUSIONTotal Flavonoids (50mg/kg) dan -Sitosterol (10 mg/kg) from showed activities against cardiovascular diseases :Atherosclerosis (in vitro, postive control: Simvastatin) in vivo: reduced lipid accumulation on aorta vessel and reduced cholesterol content in bloodHyperthrophy (in vitro, postive control : Propanolol)Thrombosis (in vivo, postive control : Aspirin, gingko biloba)Platelet agregation and reduced blood viscosity (in vivo, postive control : Aspirin, gingko biloba)Ischemia (in vivo, postive control : Diao)Acute and subcronic toxicity studies showed no toxic effect on the animal tested

THANK YOU

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