Indian Journal of Pharmacology 1997; 29: 105-109 RESEARCH PAPER

CELL CULTURE DERIVED HEMIDESMUS lNDICUS IN THE PREVENTION OF

HYPERCHOLESTEROLEMIA IN NORMAL AND HYPERLIPIDEMIC RATS

K.N. BOPANNA, N. BHAGYALAKSHMI*, S.P. RATHOD, R. BALARAMAN, J. KANNAN

Pharmacy Department, Faculty of Technology and Engineering, M.S. University of Baroda, Baroda - 390 001
and *Plant Cell Biotechnology Department, Central Food Technological Research Institute, Mysore - 570 013.

Manuscript Received: 8-4-1996 Revised: 5-7-l 996 Accepted: 5-8-l 996

SUMMARY Objectives: To study the effect of cell culture extract of Hemidesmus indicus (CCH) on normal and
hypercholesterolemic rats for various lipid profile in serum, tissues and fecal matter.
Methods: Twelve groups of eight animals were subgrouped into three set of experiments. First group
received normal and CCH (2, 4 and 16 mg/kg, p.o.) for 30 days, second group received atherogenic diet
for 30 (l-30) days and CCH (2, 4 and 16 mg/kg, p.o.) for next 30 (31-60) days, third group received
atherogenic diet and CCH (2,4 and 16 mg/kg, p.o.) for 60 days concurrently. Serum tissue and faecal lipid
profile were analysed.
Results: It was observed that the CCH (2, 4, and 16 mg/kg, p.o.) had prevented hypercholesterolemia in
rats. Rats which received atherogenic diet for first 30 (l-30) days when subjected to CCH (2, 4 and 16
mg/kg, p.o.) for next 30 (31-60) days showed significant treatment reduction (p<0.01) of lipid levels in
serum. When atherogenic diet and CCH were given together for 60 (l-60) days there was significant
(p<0.01) reduction of lipid in liver, heart and serum fecal excretion of cholesterol and phospholipids were
significantly increased (p<0.01 in rats fed with CCH.
Conclusion: Atherogenic diet along with CCH concurrently lowered the levels of serum, tissue and fecal
lipid levels.

KEY WORDS Hemidesmus indicus atherosclerosis cholesterol triglycerides phospholipids HDL ratio
faecal cholesterol

INTRODUCTION MATERIALS AND METHODS

Hyperlipidemia is the major riskfactor in the initiation
and progression of the atherosclerotic lesions. Evi-
dence from studies both in animals and humans
indicates that progression can be slowed if elevated
serum concentration of the atherogenic lipoprotein
and triglycerides are reduced, which in turn prevents
coronary heart diseasel.
It is known that Indian Sarasaparilla (Hemidesmus
indicus R. Br.,) is a blood purifier, demulcent, dia-
phoretic and tonic2. It is also employed in nutritional
disorders, syphilis, chronic rheumatism, and other
infections3,4.
It mainly consists of essential oils and phytosterols
like hemidesmol, hemidesterol and saponins. Due
to its wide pharmacological actions, and recent in-
teres@, the present study was undertaken to es-
tablish the hypolipidemic effect of cell culture extract
of H.indicus (CCH) on normal and hyperlipidemic
rats.
Male Wistar rats weighing 175-200g were used for
all the experiments. They were divided into 12 groups
of eight animals each. In the first set of experiment,
a control group with normal diet received arachis
oil emulsion 2 hrs after diet administration for 30
days. The other three groups (group 2, 3 and 4)
received CCH dispersed in arachis oil emulsion (2,
4 and 16mg/kg, p.o) for 30 days.
In the second set of experiment, group 5 received
atherogenic diet (Table 1) for 30 (l-30) days and
normal diet for the next 30 (31-60) days. Groups
6, 7 and 8 received atherogenic diet for the first
30 (l-30) days and CCH (2, 4 and 16mg/kg, p.o)
respectively for the next 30 (31-60) days.
In the third set of experiment, Group 9 received
atherogenic diet for 60 days. The other groups 10,
11 and 12 received CCH (2, 4 and 16mg/kg p.o)
as well as atherogenic diet for 60 days concurrently.

( Correspondence: R. Balaraman )
 HYPOCHOLESTEROLEMIC EFFECT OF CELL CULTURE DERIVED HEMIDESMUS INDICUS 106

Table 1. Composition of control and atherogenic diet. The biomass was soxhleted with chloroform for 14
hours at 7O°C. Dried in vacuo until no traces of
Ingredients Control Atherogenic chloroform was present. The dried sample was kept
in an oven for 1 hr at 1 O O ° C and the extract obtained
Protein
(Milk powder) was dispersed in 5% w/v arachis oil emulsion. It
was prepared freshly before administration. CCH
Sucrose 3g - was administered orally by gastric intubation.
Carbohydrate Statistical evaluation of the analytical data was done
65 g 57.6 g
(Wheat flour) using Student’s t-test and a “P” value of < 0.05
was considered to be significant14115.
Fat
(Butter) 5g 15g

Salts 4g 4g
Vitamin mix 1g 1g
Fibre 2g 2g
Coconut oil - 5 ml
Cholesterol - 0.4 g
Total weight 1OOg 100g
At the end of the experiment the rats were an-
aesthetised by ether, blood was directly collected
from the heart without any anticoagulant and allowed
to clot at room temperature for 10 min. Centrifuged
at 1000 x g for 10 min the serum was kept at
4 ° C until used. VLDLg, LDLg, total cholestero18,
triglyceridelo, phospholipidsll a n d H D L choles-
terolJ2 were analysed from the serum. HDL ratio
was derived from the formula HDLchol/(total chol-
HDL chol). Lipid extraction13 of liver and heart mus-
cle were performed. Faecal samples were collected
from individual rats for the last 15 days of 60 days
treatment period. Feces were extracted with chlo-
roform, methanol mixture, homogenised and
freeze dried and stored at -2O°C. Faecal cholesterol8
and phospholipidll were estimated.
Preparation of cell culture extract of CCH
Initiation and maintenance of CCH was carried out
on surface sterilised leaf explants of 2-3 mm. This
was inoculated on to Murashige and Skoog’s me-
dium6 supplemented w i t h 2,4-dichlorophenoxy-
acetic acid (2,4 D, 1mg/l) and benzylaminopurine
(BAP, 2mg/l) aseptically. Callus produced after three
weeks were cut and transferred to liquid medium
(MS, 2,4-D 1mg/l + BAP, 2mg/l). Biomass was ob-
tained after the fourth week (incubating conditions;
90 rpm gyratory shaker, 25 ± 2°C, 2000 lux pho-
toperiod) and was used for further experiments.
RESULTS
CCH (16 mg/kg) administered to normal animals
(without atherogenic diet) showed significant
(P<0.01) decrease in LDL and VLDL cholesterol
and a significant (P<0.01) increase in HDL cholest-
erol ratio. There was no significant change in lipid
profile in rats treated with lower dose (2 and 4 mg/kg)
of CCH (Table 2). When the second set of rats
which receiving atherogenic diet for the first 30
(l-30) days were subjected to treatment with CCH
(2, 4 and 16 mg/kg, p.o) for the next 30 (31-60)
days, there was a significant (P<0.01) decrease
in the total cholesterol and LDL (2, 4 and 16 mg/kg
groups), triglycerides (4 and 16 mg/kg groups) and
phospholipids (16 mg/kg group). Cholesterol to
phospholipid (C/P) ratio decreased significantly
(P<0.01) with the dose of 16mg/kg of CCH. There
was a significant (P<0.01) increase in HDL ratio
in the rats treated with 4 and 16mg/kg of CCH
(Table 2).
The third set of rats which received CCH (2, 4
and 16 mg/kg, p.o) and atherogenic diet together
for 60 (l-60) days, showed significant (P<0.01) re-
duction in total cholesterol, triglycerides and LDL
cholesterol (2, 4 and 16 mg/kg groups). There was
a significant decrease in phospholipids in rats treated
with 4 and 16 mg/kg of CCH while HDL ratio in-
creased significantly (P<0.01) in all the three doses
of CCH. In all the three doses of CCH, C/P ratio
decreased significantly (P<0.01) (Table 2).
When CCH (4 and 16 mg/kg) and atherogenic diet
were given togetherfor days, there was significant
(P<0.01) reduction of cholesterol, triglycerides and
phospholipids (Table 3) in heart and liver. Faecal
excretion of cholesterol and phospholipids were sig-
nificantly (P<0.01) increased in rats treated with
CCH (4 and 16 mg/kg) and atherogenic diet for
60 days (Table 3).
107 K.N. BOPANNA et al.

Table 2. Effect of cell culture extract of H.indicus (CCH).

Treatment
Total HDL LDL VLDL HDL
Dose Trigly- Phos- Chol /
Groups choles- choles- Choles- choles- ratio
of CCH cerides pholipids Phosp
Diet terol terol terol terol (%)
(mg/kg,
P.O.)

Study I

Group 1 88.9 54.4 110.2 32.8 42.6 13.5

0 0.81 58.46
(control) (l%d) f 1.4 *l.O i2.1 * 1.6 f 1.2 f 0.7

88.2 54.1 109.8 32.9 42.4 12.9

Group 2 ND 2a 0.80 59.49
f 1.5 f 1.9 zt 1.8 f 1.7 f 2.4 f 0.3

87.8 53.8 109.6 33.2 42.0 11.6

Group 3 ND 4a 0.80 60.80
f 1.2 f 1.3 f 1.2 f 1.9 i 1.8 f 0.4

81.4 49.2 108.1 37.0 36.0* 8.4*

Group 4 ND 16a 0.77 83.33’
f 1.7 f 1.2 f 1.4 f 2.2 i 1.6 f 0.3

Study II

(l%d)
Group 5 495.2 169.3 268.8 98.2 354.1 42.9
+ND 0 1.86 24.71
(Control) f 11.3 f 8.6 f 12.1 f 8.3 i 18.1 f 4.3
(31-60d)

392.5’ 129.2 234.95 97.1 262.9’ 32.4

Group 6 ND 2b 1.74 32.87
*11.5 ill.2 *11.2 f 7.4 zt 14.2 f 4.2

364.2” 112.1’ 212.2 104.1 234.12’ 26.1”

Group 7 ND 4b 1.71 40.06’
f 9.2 i 2.8 zt 12.8 k6.2 f 12.1 i3.1

164.2’ 82.4* 138.1’ 54.4 90.6’ 21.2’

Group 8 ND 16b 1.18’ 46.86
* 8.4 f 2.4 * 4.2 k4.2 zt 2.3 * 1.8

Study Ill

Group 9 544.9 278.2 275.4 179.3 412.2 53.4

0 1.97 27.20
(control) (1 fsDod, i 14.8 f 10.7 f 8.6 i 4.2 .+z 6.2 f 2.2

381.4’ 162.5* 213.2 103.4 242.6’ 35.4

Group 10 AD 2c 1.39 37.19’
f 6.8 i 5.2 i 2.4 f 1.4 f 4.2 f 3.2

304.2’ 112.5* 176.8” 94.2 180.8” 29.2’

Group 11 AD 4c 1.18 44.87’
i 8.2 i 2.6 f 6.4 i2.1 f 3.6 i 4.2

102.4” 60.2’ 114.6’ 38.4 52.6’ 11.4*

Group 12 AD 16c 0.87 60.0’
i 4.2 f 3.9 i 1.8 f 1.9 f 3.7 f 1.9

*P < 0.01 as compared to corresponding control group, n = 8, mean (± SEM).

HDL ratio = HDL chol / (Total chol-HDL chol); ND = normal diet: AD = atherogenic diet
afrom l-30 days; bfrom 31-60 days; Cfrom l-60 days.
 HYPOCHOLESTEROLEMIC EFFECT OF CELL CULTURE DERIVED HEMIDESMUS INDICUS 108

Table 3. Effect of cell culture extract of H.indicus (CCH) treatment for 60 days (l-80d) on tissue and faecal lipid profile (mg/g) of rats
which received atherogenic diet for 60 (l-60 days).

Liver Heart Faeces

Group=
C T P CIP C T P C/P C P

3.9 6.4 2.1 3.4 5.2 45.2 26.9

1 5.5
* 0.36 0.85 0.40
f 0.24 50.18 f 0.1 f 0.2 f 0.32 zt 3.2 i 1.6

11.8 7.2 8.4 72.8 24.6

9 f16.2
0.29 9.8 10.2 1.50 1.40
f 0.02 *O.ll f 0.43 f 0.48 kO.16 f 5.6 i 1.8

7.2 8.4 8.2 5.1 6.5 79.2 31.4

10 f12.2
0.71 1.45 1.26
f 0.03 f 0.20 f 0.09 f 0.04 f 0.70 f 6.9 f 1.3

10.9’ 6.1’ 7.7’ 7.2’ 4.9’ 5.9* 86.4’ 38.6”

11 f 0.54 f 0.04 f 0.20 1.41 1.22
f 0.21 zt 0.23 f 0.38 zt 3.8 f 1.7

6.6’ 4.6’ 6.2’ 1.06 3.4” 3.8 5.4’ 0.89 142.1” 42.1’
12 zt 0.24 f 0.14 kO.16 f 0.31 i 0.28 it.16 i 2.8 f 3.1

aDetails of diet and CCH treatment are given in Table 2. l P < 0.01 as compared to group 9.
C = Cholesterol; T = Triglycerides; P = Phospholipids.

DISCUSSION useful in the treatment of type II hyperlipoprote-

The present study shows that CCH prevents hy-
percholesterolemia. High fat diet in combination with
cholesterol feeding raised the serum cholesterol,
LDL and VLDL cholesterol. After treatment with CCH
all the above parameters were lowered. This could
be possibly due to an increase in the liver-LDL re-
ceptor activity16p17 a n d d e c r e a s e d h e p a t i c
triglycerides synthesis18. It was further shown that
high C/P ratio decreased significantly in contrast
to increase in HDL ratio after treatment with CCH
in rats fed with atherogenic diet for 60 days.
High C/P ratios are usually associated with
atherosclerosis1g120.
CCH significantly increased HDL cholesterol con-
centration and HDL ratio; thus it would be useful
in diseases like diabetes mellitus and coronary heart
diseases because of their inverse relationship. Hy-
per-triglyceridemia is also associated in metabolic
consequences of hypercoagulability, hyperin-
sulinemia, insulin resistance and glucose toler-
ance21. CCH reduces triglycerides in rats fed with
atherogenic diet and may prevent the progression
of atherosclerosis21l22 and complications due to hy-
pertriglycerdemia.
inemia23. It is also reported that beta sitosterol in-
hibits cholesterol absorption23. Phytosterols present
in CCH may be beta sitosterol type or similar type
which may be responsible for the hypolipidemic
effect.
It can be conclude from the data that the levels
of total serum cholesterol, triglycerides, VLDL and
LDL cholesterol which are actually raised in athero-
genic diet, can be lowered significantly with con-
current feeding of CCH. Moreover its hypolipidemic
effect may have a protective mechanism against
the development of atherosclerosis24. CCH can be
utilised for providing dietary management in the pre-
vention of atherosclerosis in hyperlipidemic patients.
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