THE LEAVES OF ARTOCARPUS ALTILIS

CONSTITUENTS POTENTIAL FOR

CARDIOVASCULAR DISEASE TREATMENT
Leonardus B.S. Kardono, Tjandrawati Mozef, Nina Artanti,
Puspa Dewi, M. Hanafi and Umar A. Jenie
Research Center for Chemistry
Indonesian Institute of Sciences
(1) Achyranthes aspera L. (Amaranthaceae)
(2) Ageratum conyzoides L. (Asteraceae)
(3) Allamanda cathartica L. (Apocynaceae)
(4) Allium cepa L. (Lilyaceae)
(5) Allium sativum L. (Lilyaceae)
(6~7) Aloe vera L. (Lilyaceae)
(8) Andrographis paniculata Ness (Acanthaceae)
(9) Artocarpus altilis Lamk (Moraceae)
(10~11) Averrhoa bilimbi L. (Oxalidaceae)
(12~13) Averrhoa carambola L. (Oxalidaceae)
(14) Bixa orellana (Bixaceae)
(15) Blumea balsamifera (L.) D.C. (Asteraceae
(16~17) Bougainvillea glabra Choisy (Nyctaginaceae)
(18) Caesalpinia pulcherrima (Fabaceae)
(19) Carica papaya (Caricaceae)
(20) Cassytha filiformis L. (Lauraceae)
(21) Catharanthus roseus (L.) G. Don (Apocynaceae)
(22) Celosia argentea L. (Amaranthaceae)
(23~24) Cicca acida (L.) Merr. (Euphorbiaceae)
(25) Cucumis sativus L. (Cucurbitaceae)
(26) Cyperus rotundus L. (Cyperaceae)
(27) Elephantopus scaber L. (Asteraceae)
(28) Euphorbia hirta L. (Euphorbiaceae)
(29) Excocaria bicolor Hassk (Euphorbiaceae)
(30) Gynandropsis ginandra (L.) Briq.
(Capparidaceae)
(31~32) Hibiscus rosasinensis (Malvaceae)
(33) Impatiens balsamina L. (Balsaminaceae)
(34~35) Ipomoea batatas (Convolulaceae)
(36~37) Jasminum sambac (Oleaceae)
(38) Kalanchoe pinnata (Lom) Pers. (Crassulaceae)
(39) Leuceuna leucocephala Bth. (Mimosaceae)
(40) Lophatherum gracile Brongn (Poaceae)
(41) Melia azedarach L. (Meliaceae)
(42) Micromelum pubescens Bl. (Rutaceae)
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
t t f t l t i l i
T h e P E T f r a c t i o n
6 2 g
p a r t i t i o n e d w i t h P E T
T h e w a t e r f r a c t i o n
p a r t i t i o n e d w i t h E A
T h e E A f r a c t i o n
1 3 1 g
T h e w a t e r f r a c t i o n
p a r t i t i o n e d w i t h n - B u O H
T h e n - B u O H f r a c t i o n
3 8 g
T h e w a t e r f r a c t i o n
c o n d e n s e d
c o n d e n s e d
c o n d e n s e d c o n d e n s e d
Ethanol extract of Artocarpus altilis
Further
isolation
PET fraction
column chromatography
A1 A2 A3
A4 A5
A6 A7
PE:EA
15:1
PE:EA
12:1
PE:EA
10:1
PE:EA
6:1
PE:EA
4:1
PE:EA
2:1
PE:EA
1:1
EA fraction
column chromatography
PE:EA
5:1
PE:EA
3:1
PE:EA
1:1
PE:EA
1:2
PE:EA
1:5
PE:EA
1:8
PE:EA
1:10
EA
A8
A9 A10 A11 A12 A13 A14 A15
n-BuOH fraction
absorptive resin
25%ethanol
50%ethanol 75%ethanol
100%ethanol
A16 A17
A18
A19
Water fraction
A20
20 fractions: Bioassays against cardiovascular diseases
Atherosclerosis:
Induction: ox-LDL
Control: Simvastatin
Analysis: cell proliferation using WST method (respiration activity)
U937 cells -derived foam cells
Endothelial cells (HUVEC)
Hypertension : Left ventricle hypertrophy
Induction : Angiotensin II
Control: Propanolol
Analysis: 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<0.05 vs model, **: p<0.01 vs control;
#: p<0.05 vs simvastatin, ##: p<0.01 vs simvastatin
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<0.05 **: p<0.01
Screening results
Active
Not active
A1 A2 A3 A4 A5 A6 A7 A8 A9 A10
Endothelial cell
U937
cardiac myocytes
A11 A1
2
A1
3
A1
4
A1
5
A1
6
A1
7
A1
8
A19 A20
Endothelial cell
U937
cardiac myocytes
Active fractions against the 3 kind of cell model: A1, A9, A20
Further fractionation of the active fractions using chromatography method
Structure elucidation using NMR, MS, IR and UV spectrum.
Active
fraction
Type of
compound
Main active constituent
A1 sterol -sitosterol
A9 flavonoid 1-(2,4-Dihydroxyphenyl)-3-[8-hydroxy-
2-methyl-2-
(4-methyl-3-pentenyl)-2H-1-
benzopyran-5-yl]-1-propanone
flavonoid 8-geranyl-4',5,7- tirhydroxyflavone
flavonoid 2-geranyl-2',3,4,4'-
tetrahydroxychalcone
HO
H H
H
O OH
HO
OH
O
O
OH
OH
HO
O
HO
OH
OH
O
OH
-sitosterol:
1-(2,4-Dihydroxyphenyl)-3-[8-hydroxy-2-methyl-2-
(4-methyl-3-pentenyl)-2H-1-benzopyran-5-yl]-1-propanone
8-geranyl-4',5,7- tirhydroxyflavone
2-geranyl-2',3,4,4'-
tetrahydroxychalcone
Isolated chemical compounds from the ethyl acetate fraction
2,4,5-trihydroxy-7-methoxy-8-prenylflavone
2-geranyl-3,4,7-
tirhydroxyflavone
IN VIVO STUDIES :
Platelet agregation
Blood viscosity
Thrombosis
Acute ischemia
Atherosclerosis:
Lipid accumulation on
aorta
Blood cholestherol
ANTI PLATELET AGGREGATION AND BLOOD VISCOSITY
EXPERIMENT
AIM:
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 ARTERY

Centrifugation 800rpm,10min

PRP

Further centrifugation 2000rpm, 10min

PPP
In a cuvette:
300 l of PRP or PPP
10 l of Adenosine diphosphate (33.3M)


AGGREGOMETER
% Inhibition of platelet aggregation

FROM EYE
Blood 3ml + 60l
heparin (40 u/ml)

0.8ml injected into the
viscometer.

VISCOMETER
Maximum value of the
blood viscosity.
BLOOD COLLECTION
ANTI-PLATELET AGGREGATION EXPERIMENT
CONCLUSION
MTF (100mg/kg)+HBS (20mg/kg) and LTF (50mg/kg)+MBS (10mg/kg)
active as anti platelet agregation (comparison: Aspirin and Gingko)
BLOOD VISCOSITY EXPERIMENT

Effect of anti-blood viscosity
0
5
10
15
20
25
1 5 30 200
Shear rate(l/s)
Blood viscosity
normal
tf
high-tf+low-
mid-tf+low-

low-tf+mid-

gingko
aspirin
*
*
*
*
*
*
**
**
**
**
**
** **
**
**
CONCLUSION
MTF+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.
THROMBOSIS EXPERIMENT
FEEDING:
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 mesentery
Fluorescein Sodium injected
through the catheter.
Light of 490nm
Record of thrombus formation
Thrombosis.mpg
ACUTE CARDIAC ISCHEMIA EXPERIMENT
AIM:
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 BW
Group 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 COLLECTION
From abdominal artery
ASSAY OF 4 KEYS ENZYMES
Lactate dehydrogenase
Creatine Kinase
Superoxide Dismutase
Maleic dialdehyde
Centrifugation at 1200g for
10 minute to get the serum
EKSPERIMEN ANTI ISCHEMIA
CONCLUSION
The 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).
MDA
0
0.5
1
1.5
2
2.5
3
3.5
4
Control Model MTF+H LTF + M DA
M
D
A
(
n
M
/
m
l
)
#
*
#p<0.05 vs.control;*p<0.05 vs. model
Experiment on lipid accumulation at
aorta vessel
Aim:
To study the effect of flavonoids
on lipid accumulation at aorta
vessel
Sample:
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
diet
Group II: high cholesterol diet
+ sukun fraction 150 mg/kg BW
Group III: high cholesterol diet
+ sukun fraction 300 mg/kg BW
Group IV (Negative control): standard diet
On Day 31:
Blood sample taken from the tail
Laparatomi an histology of aorta blood
vessel to observe the lipid accumulation
on every group
Aorta Histology
Positive Control Negative control
Flavonoids Total 150
mg/kg
Flavonoids Total 300
mg/kg
Cholesterol content assay
p<0.05 vs positvie control
Pengaruh ekstrak etil asetat daun sukun
terhadap kadar kolesterol pada tikus
0
20
40
60
80
100
120
140
160
180
200
Kontrol positif Sukun 150mg/kg Sukun 300mg/kg Kontrol negatif
K
a
d
a
r

k
o
l
e
s
t
e
r
o
l

(
m
g
/
d
l
)
Effect of Flavonoids on cholesterol
content in rat
Positive control
Negative control
C
h
o
l
e
s
t
e
r
o
l

c
o
n
t
e
n
t

(
m
g
/
d
l
)


Toxicity studies
Acute toxicity: using ICR mice
Subcronic toxicity: using Wistar rat
ACUTE TOXICITY EXPERIMENT
FEEDING
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 weigh
Death rate
Behaviour: walking, drinking and eating
(total water and food taken was recorded),
brightness of eyes and fur/hairs
CONCLUSION:
HighTF and BS is not toxic to animal
model (no weight reduction and
show normal behaviour )
ICR mice:
10 males
10 females
SUBCRONIC TOXICITY STUDY
Dosage:
flavonoid= 50 mg/kg
bb/hari.
Total flavonoid
content in A. Altilis
fraction=30%.
Route: oral, 1xdayly
for 90 days
Group Treatment No. male
rats (n)
Numbe of
female
rats(n)
1 Given Flavonoids
Total 83,33 mg/kg bw/
day
10 10
2 Given sukun fraction
166,67 mg/kg bw/ day
10 10
3 Given Flavonoids
Total 333,33 mg/kg
bw/ day

10 10
4 Normal control, given
1% CMC solution
10 10
Analysis on
Day 91
Heart Kidney Liver HAEMATOLOGY
PLASM:
Aspartate amino
transferase
Creatine kinase

Urea
Creatinin
Content
Alanin amino
transferase
Alkali
phosphatase


Number of
erythrocyte
leucocyte
thrombocyte
haemoglobin

HEART
6
0
.
9
1
6
7
.
7
7
7
7
.
1
9
7
7
.
8
5
7
7
.
1
8
7
1
.
9
2
6
7
.
4
8
7
7
.
6
8
0
10
20
30
40
50
60
70
80
A
k
t
i
v
i
t
a
s

(
U
I
/
I
)
1 2 3 4
Kelompok Percobaan
Jantan
Betina
4
5
.
9
4
6
.
6
5
4
3
.
8
1
3
8
.
7
3
9
.
1
9
4
7
.
4
3
4
5
.
0
3
4
3
.
0
1
0
5
10
15
20
25
30
35
40
45
50
A
k
t
i
v
i
t
a
s

(
U
/
I
)
1 2 3 4
Kelompok Percobaan
Jantan
Betina
ACTIVITY OF ASPARTATE AMINO
TRANSFERASE
ACTIVITY OF CREATINE KINASE
Group Repetiti
on
polymorpho
nuclear
infiltration
Lipid formation
on chamber
between
Fibrosis
I 6 - - -
II 6 - - -
III 6 - - -
IV 6 - - -
HEART
HISTOLOGY
ON MALE
AND
FEMALE
RAT
One 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)
JANTUNG (HISTOLOGI)
(A)nucleus, (B) fibroblast
GROUP
1
GROUP 2
GROUP 4 GROUP 3
Comparison between control and treated groups showed that flavonoids treatment
did not show any effect on heart of male and female rats.
KIDNEY
3
7
.
8
4
3
4
.
4
2
3
7
.
3
1
3
5
.
6
0
0
8
16
24
32
40
K
a
d
a
r

U
r
e
a

P
l
a
s
m
a

(
m
g
/
d
l
)
I II III IV
Kelompok Perlakuan
3
7
.
4
0
3
6
.
3
5
3
7
.
4
7
3
7
.
2
7
0
8
16
24
32
40
K
a
d
a
r

U
r
e
a

P
l
a
s
m
a

(
m
g
/
d
l
)
I II III IV
Kelompok Perlakuan
UREA CONTENT
0
.
7
9
0
.
7
9
0
.
8
0
0
.
8
0
0.00
0.20
0.40
0.60
0.80
1.00
K
a
d
a
r

K
r
e
a
t
i
n
i
n

P
l
a
s
m
a

(
m
g
/
d
l
)
I II III IV
Kelompok Perlakuan
CREATINE PLASMA CONTENT
0
.
8
3
0
.
8
1
0
.
8
3
0
.
8
2
0.00
0.20
0.40
0.60
0.80
1.00
K
a
d
a
r

K
r
e
a
t
i
n
i
n

P
l
a
s
m
a

(
m
g
/
d
l
)
I II III IV
Kelompok Perlakuan
One 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)
DIAMETER OF KAPSULA BOWMAN
114.47 114.43
113.28 114.51
0
30
60
90
120
D
i
a
m
e
t
e
r

k
a
p
s
u
l
a

b
o
w
m
a
n

(

m
)
I II III IV
KEL OMP OK
116.78 114.32 114.25 117.06
0
30
60
90
120
D
i
a
m
e
t
e
r

k
a
p
s
u
l
a

b
o
w
m
a
n

(

m
)
I II III IV
KEL OMP OK
DISTANCE BETWEEN GLOMERULUS CHAMBER AND KAPSULA BOWMAN
19.74
18.45
16.26
18.86
0
5
10
15
20
J
a
r
a
k

r
u
a
n
g

a
n
t
a
r
a

g
l
o
m
e
r
u
l
u
s

d
e
n
g
a
n

k
a
p
s
u
l
a

b
o
w
m
a
n

(

m
)
I II III IV
KEL OMP OK
19.90
16.48 17.08
20.13
0
5
10
15
20
J
a
r
a
k

r
u
a
n
g

a
n
t
a
r
a

g
l
o
m
e
r
u
l
u
s

d
e
n
g
a
n

k
a
p
s
u
l
a

b
o
w
m
a
n

(

m
)
I II III IV
KEL OMP OK
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)

LIVER
ACTIVITY OF ALANIN AMINO TRANSFERASE
ACTIVITY OF ALKALI PHOSPHATASE
One 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 HISTOLOGY
Group Degree of Damage Cell
0%
(without
damage)
20% - 40%
(medium
necrosis)
> 40%
(heavy
necrosis)
I
II
III
IV
100%
100%
100%
100%
0%
0%
0%
0%
0%
0%
0%
0%
Average number of damage cells on
male and female rats after 90 days of
treament
Diameter of Vena Centralis
Group Male Rat Female Rat
I
II
III
IV
62,86 1,29
63,39 1,05
64,32 1,25
63,21 1,56
63,50 2,64
63,73 2,51
64,32 1,93
63,05 2,29
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)
Haematology
Jumlah Eritrosit Darah
0
1
2
3
4
5
6
7
I II III IV
Kelompok
J
u
m
l
a
h

E
r
i
t
r
o
s
i
t

(
1
0
6
/
m
m
3

D
a
r
a
h
)
Jantan
Betina
Jumlah Leukosit Darah
7500
8000
8500
9000
9500
10000
I II III IV
Kelompok
J
u
m
l
a
h

L
e
u
k
o
s
i
t
/
m
m
3

D
a
r
a
h
Jantan
Betina
Jumlah Trombosit Darah
4.4
4.6
4.8
5
5.2
5.4
5.6
I II III IV
Kelompok
J
u
m
l
a
h

T
r
o
m
b
o
s
i
t

(
1
0
5
/
m
m
3

D
a
r
a
h
)
Jantan
Betina
Kadar Hemoglobin Darah
11.45
11.5
11.55
11.6
11.65
11.7
11.75
11.8
11.85
11.9
I II III IV
Kelompok
K
a
d
a
r

H
e
m
o
g
l
o
b
i
n

(
g
/
1
0
0

m
l

D
a
r
a
h
)
Jantan
Betina
ANAVA 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)
CONCLUSION
Total 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 blood
Hyperthrophy (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