Protective
effects of Strychnos potatorum Linn.
seeds extract in hyperlipidemic rat model
Urvi S. Shah1*,
K.N.Patel2
1Singhania
University, Jhunjhunu, Rajasthan, India.
2SAL
Institute of Pharmacy, Ahmadabad, Gujarat, India.
ABSTRACT:
Objective: To investigate the antihyperlipidemic activity of aqueous extract of Strychnos potatorum Linn. seeds in experimentally
induced hyperlipidemia in rats.
Materials and
Methods: Hyperlipidemia was induced by single intraperitoneal
injection of triton WR 1339 (Tyloxapol) at a dose of
400mg/kg in rats. The doses of 100mg/kg and 200mg/kg of aqueous extract of Strychnos potatorum
Linn. seeds were evaluated for their actions on serum lipid profile. Atorvastatin at a dose of 10mg/kg was used as standard
drug. Aqueous extract doses and Atorvastatin were
given orally, once immediately and after 24 hours of triton WR 1339
administration. Serum total cholesterol, triglycerides, low density lipoprotein
cholesterol, very low density lipoprotein cholesterol and high density
lipoprotein cholesterol levels were estimated after 6 hour, 24 hour and 48 hour
of triton WR 1339 administration.
Results: Aqueous extracts of Strychnos potatorum
significantly (p<0.05) reduced
serum total cholesterol, triglycerides, low density lipoprotein cholesterol and
very low density lipoprotein cholesterol levels in dose dependent and time
dependent manner when compared to hyperlipidemic
group.
Conclusion: The findings of the
investigation suggest that aqueous extract of Strychnos potatorum seeds exhibited quite
competitive potential when compared with reference drug Atorvastatin
against experimentally induced hyperlipidemia.
KEYWORDS: Antihyperlipidemic activity, Triton WR 1339, Strychnos potatorum
seed extract, Atorvastatin
1. INTRODUCTION:
Experimental and epidemiological studies
have suggested that hyperlipidemia is a highly
predictive risk factor for atherosclerosis, coronary artery diseases (CAD) and
cerebral vascular diseases; the primary causes of mortality in the developing
countries like India.1 Secondly, prevalence of metabolic syndrome
has been increasing rapidly in the past decade in most western countries also.
Complications of metabolic syndrome are defined as a cluster of three of five
criteria: insulin resistance and glucose tolerance, abdominal obesity,
hypertension, low high-density lipoprotein cholesterol (HDL-C) and
hypertriglyceridemia.2 So it is very important to pay attention to
early stage prevention and control of hyperlipidemia
in a comprehensive way. The allopathic hypolipidemic
drugs, although available at large in the market, their popularity has been
marred by numerous side effects, severe contraindications and exuberant cost.3
This has further necessitated the search for alternatives. Plants are important
sources of medicinal compounds and major portion of population in developing
countries is dependent on traditional folk medicinal therapies for treating
their ailments. This fact has been recognized by WHO and its recommendations
include evaluation of traditional medicines in primary health care of these
countries.4
The investigation on plant drugs will be a
useful strategy in the discovery of new lead molecules eliciting improved
activity by regulating different mechanisms that maintains the lipid metabolism
and thus can be used in treating hyperlipidemia of
varied etiology.5 Traditional system of medicine like Ayurveda, Unani and Chinese
prescribe numerous herbal drugs for cardiovascular disorders including
atherosclerosis. Recently herbal hypolipidemics have
gained importance to fill the lacunae created by the allopathic drugs.
Strychnos potatorum Linn. (Family: Loganiaceae) is a moderate sized tree found in southern and
central parts of India, Srilanka and Burma.6
Selected parts of the tree likes seeds, ripe fruits and roots are used in
traditional system of medicine for the treatment of various ailments like
tumors, pain, inflammation, anaemia and jaundice.7
In English, it is commonly known as clearing nut tree. The ripe seeds are
used for clearing muddy water. They are reported to be very effective as
coagulant aids. They are effective in removing the suspended impurities.8
The clarification is due to the combined action of colloids and alkaloids in
the seeds. The albumin and other colloids sensitize the suspension and the
coagulation is then caused by the alkaloidal ions.
The seeds are reported to have various phytochemical constituents like alkaloids - diaboline and its acetate, brucine,
loganine, mannose, sucrose, arachidonic
acid, lignoceric acid, linoleic
acid, oleic acid, palmitic acid, stearic
acid, triterpenes and polysaccharides (mannogalactans).9,10,11
Various pharmacological activities of the
seeds like antidiarrhoeal, CNS effects, antidiabetic, diuretic, anti-inflammatory, antiulcerogenic and hepatoprotective have also been reported.12-17
Taking into consideration the folkloric use of seeds reported, the present
study was designed to evaluate the antihyperlipidemic
properties of Strychnos potatorum
Linn. seeds in triton-1339 induced hyperlipidemia in
rats.
2. MATERIALS AND METHODS:
2.1 Plant
collection and identification:
Strychnos potatorum seeds were purchased from M/S Lalubhai Vrajlal Gandhi, Gandhi
Road, Ahmadabad-Local market and were botanically identified and authenticated
by Dr. H. B. Singh, Head, Raw Materials Herbarium & Museum, NISCAIR, New
Delhi. A voucher specimen has been kept in our laboratory for further reference
(Specimen no. 0101). The quality of seeds was ascertained as per the Ayurvedic Pharmacopoeia of India by determining foreign
matter, total ash, acid-insoluble ash, alcohol-soluble extractive and
water-soluble extractive values.18
2.2 Chemicals:
All the chemicals used were of analytical
grade. Triton WR-1339 (a non- ionic detergent, isooctyl
polyoxy ethylene phenol, formaldehyde polymer/ Tyloxapol) was obtained from Sigma Laboratories Ltd. Atorvastatin (Atocor) was of Dr.
Reddy’s Laboratories Ltd.
2.3 Animals used:
Adult Wistar
albino rats weighing 150-200 gm of either sex were procured from Cadila Pharmaceuticals Ltd, Dholka.
The animals were housed at animal house in standard polypropylene cages with 12
h: 12 h light dark cycle at 22 ± 2⁰C
temperature and 50-60% relative humidity. The animals were fed with commercial
standard pellet diet (Pranav Agro Industries Ltd., Sangli, India) and water ad libitum. This study was approved by
the Institutional Animal Ethics Committee (Protocol No: SALIP/ Pharmacology/
IAEC/ CPCSEA/ 2011/02) in accordance
with the guidance of Committee for the Purpose of Control and
Supervision of Experiments on Animals (CPCSEA, 2003).
2.4 Drugs and herb
The
air-dried seeds were powdered and passed through 40- mesh sieve to get uniform
particle size. The seeds were subjected to hot water decoction for 3 hrs, it
was then filtered and the filtrate was evaporated to dryness. A dark brownish
colored semisolid mass was obtained which was dried under vacuum. The
percentage yield of the aqueous extract (SPSE) was 18.2% w/w from the starting
crude seed powder. SPSE was triturated with 1% gum acacia solution for the
experimental treatments. The reference drug Atorvastatin
was suspended in distilled water using 1% carboxy
methyl cellulose (CMC). The suspensions were prepared fresh each time before
dosing.
2.5 Acute toxicity
studies
The
acute toxicity studies of aqueous Strychnos
potatorum seeds extract (SPSE) were carried out
in mice on a graded dose of 500, 1000, 1500 and 2000 mg/kg b.w.
(p.o.) as per OECD-423 guidelines for acute oral
toxicity. The treated mice of all groups were normal and healthy throughout the
study period. Animals did not show any toxic effects or mortality upto the dose of 2000mg/kg orally even seven days after the
treatment. Thus, the oral aqueous extract of S. potatorum
is safer upto the dose of 2000 mg/kg b.w.19
2.6 Experimental
protocol:
After 10 days of acclimation, overnight
fasted rats were randomly divided into five groups containing six rats in each
(Table 1). Acute hyperlipidemia was induced in rats
using triton WR 1339.20 Atorvastatin and
SPSE were administered once immediately and again after 24 h of i.p. injection of 10% solution of triton WR 1339 in normal
saline. During the period of study, animals had access to water only.
Table 1. Triton WR 1339 induced acute hyperlipidemia
model: summary of animal groups and treatments.
Group |
Treatments |
Control
|
Vehicles
(1% w/v gum acacia, 1% w/v CMC p.o. + normal saline
i.p.) |
Hyperlipidaemic |
Triton
WR 1339 (400mg/kg i.p.) |
Atorvastatin |
Triton
WR 1339 (400mg/kg i.p.) + Atorvastatin
(10mg/kg. p.o.) |
SPSE100 |
Triton
WR 1339 (400mg/kg i.p.) + Aqueous Strychnos potatorum
seed extract (100mg/kg p.o.) |
SPSE200 |
Triton
WR 1339 (400mg/kg i.p.) + Aqueous Strychnos potatorum seed extract (200mg/kg p.o.) |
2.7 Collection of
Serum and Estimation of Lipid Profile
After 6, 24 and 48 h from the treatments,
animals were anaesthetized with diethyl ether and blood was withdrawn by retro-orbital
puncture. The blood samples were kept aside for 30 mins
and were centrifuged at 2000rpm for 15 min and serum was used for lipid profile
analysis.
The serum total cholesterol (TC),
triglycerides (TG), high density lipoproteins cholesterol (HDL-C) levels were
quantified using standard kits. (Span Diagnostics Ltd, Surat,
Gujarat, India). TC levels were measured by CHOD-PAP colorimetric assay. HDL-C
levels were measured by PEG precipitation and CHOD-PAP colorimetric assay. TG
levels were measured by GPO-PAP end point colorimetric assay. The very low
density lipoproteins cholesterol (VLDL-C) levels were calculated by formula:
VLDL-C = TG/5. The low density lipoproteins cholesterol (LDL-C) levels were
calculated using Friedewald’s formula: LDL-C = TC –
HDL-C – Triglycerides /5.
2.8 Statistical
analysis
Data obtained were analyzed using one-way
analysis of variance (ANOVA) followed by Dunnet’s
test and p values less than 0.05 was
considered significant. Results are expressed as mean±S.E.M.
3. RESULTS:
3.1 Induction of hyperlipidemia with triton WR-1339 in rats
After 6 h of treatment: The serum TC was
increased by 96%, TG by 277%, LDL by 141%, VLDL by 283% and HDL-C was decreased
by 31% (Table 2). After 24 h of treatment: The serum TC was increased by 275%,
TG by 383%, LDL-C by 526%, VLDL-C by 383% and HDL-C was decreased by 45% (Table
3). After 48 h of treatment: The serum TC was increased by 160%, TG by 235%,
LDL-C by 322%, VLDL-C by 254% and HDL-C was decreased by 15% (Table 4).The
maximum hyperlipidemic state was achieved at 24h
after triton administration compared to 6h and 48 h.
3.2 Effects of
aqueous extract of Strychnos potatorum
seeds and Atorvastatin on serum lipid profile in rats
After 6 h of treatment: Atorvastatin
decreased serum TC levels by 32%, TG by 30%, LDL-C by 45% and VLDL-C by 30%. It
increased HDL-C levels by 20%. SPSE100 decreased serum TC by 27%, TG by 14%,
LDL-C by 40% and VLDL-C by 15% when compared to hyperlipidemic
group. It didn’t produce any significant effect in raising the HDL-C. SPSE200
decreased serum TC levels by 28%, TG by 17%, LDL-C by 42% and VLDL-C by 17%. It
increased HDL-C levels by 5%.
After 24 h treatment, Atorvastatin
decreased serum TC levels by 43%, TG by 62%, LDL-C by 49% and VLDL-C by 51%. It
increased HDL-C levels by 52%. SPSE100 decreased serum TC levels by 38%, TG by
36%, LDL-C by 43% and VLDL-C by 36% when compared to hyperlipidemic
group. It increased HDL-C by 23%. SPSE200 decreased serum TC and TG levels by
39%, LDL-C by 45% and VLDL-C by 40%. It increased HDL-C levels by 22%.
After 48 h of treatment: Atorvastatin decreased serum TC levels by 53%, TG by 49%,
LDL-C by 67% and VLDL-C by 48%. It increased HDL-C levels by 7%. SPSE100
decreased serum TC by 44%, TG by 23%, LDL-C by 58% and VLDL-C by 23% when
compared to hyperlipidemic group. It didn’t produce
any significant effect in raising the HDL-C. SPSE200 decreased serum TC levels
by 46%, TG by 31%, LDL-C by 60% and VLDL-C by 30%. It increased HDL-C levels by
2%.
4. DISCUSSION:
In
Indian system of medicine, Strychnos potatorum seeds have been used as an astringent,
demulcent, emetic, diuretic, stomachic and to purify water. They are used in
vitiated conditions of kapha and vata, hepatopathy,
nephropathy, gonorrhea, leucorrhoea, gastropathy,
bronchitis, chronic diarrhoea, dysentery, strangury, renal and vesicle calculi, for burning
sensation, conjunctivitis, scleritis and eye
diseases.21 In prior studies, seeds have been reported to have
antioxidant, hepatoprotective and antidiabetic
activities. To verify the feature of seeds as antihyperlipidemic,
triton WR 1339, a non ionic surfactant was used in this study to generate acute
hyperlipidemic model. It is well known that high
serum TC, TG and LDL-C levels are primary risk factor for vascular diseases and
high serum level of HDL-C confers a protective effect against its development.
The experimental data in the current study shows that all the changes in serum
lipid levels induced by triton WR 1339 can be resisted by SPSE. The amount of
TC, TG, LDL-C, VLDL-C were reduced by aqueous extract of seeds in a dose
dependent and time dependent manner. Meanwhile, the proportion of cholesterol
component was also changed under the action of SPSE. These results prove antihyperlipidemic activity of SPSE. SPSE may become a kind
of herbal drug remedy in future.
Table 2. Effect of aqueous extract of Strychnos Potatorum
seeds on serum lipid profile 6 h after triton WR 1339 induced acute hyperlipidemia in
rats.
Group |
TC (mg/dL) |
TG (mg/dL) |
LDL (mg/dL) |
VLDL (mg/dL) |
HDL (mg/dL) |
Control |
78.66 ± 1.8 |
61.39 ± 2.02 |
36.95 ± 2.5 |
12.28 ± 0.40 |
29.42 ± 0.7 |
Hyperlipidaemic |
153.77 ± 3.31* |
230.64 ± 2.99* |
87.37 ± 3.09* |
46.13 ± 0.59* |
20.28 ± 0.56* |
Atorvastatin |
104.48 ± 1.55** |
160.60 ± 2.85** |
47.60 ± 1.81** |
32.12 ± 0.57** |
24.76 ± 0.57** |
SPSE100 |
112.41 ± 0.97 ** |
198.36 ± 1.10** |
52.06 ± 1.86** |
39.67 ± 0.22** |
20.67 ± 1.09 |
SPSE200 |
110.15 ± 3.94** |
191.44 ± 2.05** |
50.58 ± 3.33** |
38.29 ± 0.41** |
21.28± 0.69 |
Note: All values represent mean ± S.E.M.
from six animals. *compared with normal group (p<0.05), **compared with hyperlipidemic group (p<0.05). TC: total cholesterol,
TG: triglycerides, HDL-C: high density lipoproteins cholesterol, VLDL-C: very
low density lipoproteins cholesterol, LDL-C: low density lipoproteins
cholesterol, SPSE100: aqueous extract of Strychnos potatorum
seeds 100mg/kg, SPSP200: aqueous extract of Strychnos potatorum seeds 200mg/kg.
Table 3. Effect of aqueous extract of Strychnos Potatorum
seeds on serum lipid profile 24 h after triton WR 1339 induced acute hyperlipidemia in rats.
Group |
TC (mg/dL) |
TG (mg/dL) |
LDL (mg/dL) |
VLDL (mg/dL) |
HDL (mg/dL) |
Control |
77.54 ± 1.84 |
60.03 ± 3.36 |
34.34 ± 3.32 |
12.01± 0.67 |
31.20 ± 1.11 |
Hyperlipidaemic |
289.51± 3.26* |
290.45 ± 2.95* |
213.95± 4.13* |
58.09 ± 0.59* |
17.47± 0.74* |
Atorvastatin |
164.50 ± 1.69** |
140.64 ± 0.96** |
109.87± 1.54** |
28.13 ± 0.19** |
26.50± 1.19** |
SPSE100 |
180.76 ± 1.78** |
185.31 ± 1.09** |
122.12± 1.63** |
37.06 ± 0.21** |
21.58 ± 0.28 |
SPSE200 |
175.52 ± 1.51** |
177.28 ± 1.98** |
118.67± 1.83** |
35.46 ± 0.39** |
21.39 ± 0.69 |
Note: All values represent mean ± S.E.M.
from six animals. *compared with normal group (p<0.05), **compared with hyperlipidemic group (p<0.05). TC: total cholesterol,
TG: triglycerides, HDL-C: high density lipoproteins cholesterol, VLDL-C: very
low density lipoproteins cholesterol, LDL-C: low density lipoproteins
cholesterol, SPSP100: aqueous extract of
Strychnos potatorum
seeds 100mg/kg, SPSP200: aqueous extract of Strychnos potatorum seeds 200mg/kg.
Table 4. Effect of aqueous extract of Strychnos Potatorum
seeds on serum lipid profile 48 h after triton WR 1339 induced acute hyperlipidemia in rats.
Group |
TC (mg/dL) |
TG (mg/dL) |
LDL (mg/dL) |
VLDL (mg/dL) |
HDL (mg/dL) |
Control |
76.27 ± 1.7 |
59.55 ± 1.86 |
31.86± 1.71 |
11.91 ± 0.37 |
32.50
± 0.61 |
Hyperlipidaemic |
198.66 ± 2.22* |
198.37 ± 2.37* |
131.48± 2.81* |
39.67 ± 0.47* |
27.51
± 1.20* |
Atorvastatin |
92.98 ± 1.87** |
100.98 ± 1.69** |
43.38 ± 1.52** |
20.20 ± 0.34** |
29.40
± 1.19** |
SPSE100 |
110.53 ± 1.73** |
152.46 ± 2.29** |
55.43± 1.63** |
30.49 ± 0.46** |
27.84
± 1.31 |
SPSE200 |
107.48 ± 1.39** |
137.65 ± 1.38** |
53.59± 1.86** |
27.53 ± 0.27** |
28.02
± 1.36 |
Note: All values represent mean ± S.E.M.
from six animals. *compared with normal group (p<0.05), **compared with hyperlipidemic group (p<0.05). TC: total cholesterol,
TG: triglycerides, HDL-C: high density lipoproteins cholesterol, VLDL-C: very
low density lipoproteins cholesterol, LDL-C: low density lipoproteins
cholesterol, SPSE100: aqueous extract of
Strychnos potatorum
seeds 100mg/kg, SPSE200: aqueous extract of
Strychnos potatorum
seeds 200mg/kg.
The
oxidative modification of LDL-C and its accumulation in serum is a primary
event in the proceeding of atherosclerosis.22 It is generally
believed that the drug which increases LDL oxidation resistance of the body
could inhibit atherosclerosis, though there is no direct evidence yet.23
This study also proved the matter by decreasing the LDL-C levels in serum.
The
content of HDL-C in serum implies the activity of lecithin cholesterol acyltransferase (LCAT), which plays a key role in
lipoprotein metabolism and may contribute to the regulation of blood lipids.
SPSE may decrease the risk of cardiovascular disease by increasing the HDL-C
levels. Increase in HDL-C and decrease in LDL-C levels indicates that SPSE may
hasten removal of cholesterol from peripheral tissues to liver for catabolism
and excretion.
5. CONCLUSION:
The
results of the experiment above provide useful information regarding the lipid
lowering activity of aqueous extract of Strychnos potatorum seeds. The beneficial effects of seeds on
serum lipid levels are dose dependent and time dependent. The current study
supports, atleast partly, the traditional use of this
seed as antihyperlipidemic agent. The mechanisms
responsible for this hypolipidemic effect of seeds
should be explored further in future studies.
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Received on 28.06.2012
Modified on 04.07.2012
Accepted on 07.07.2012
© A&V Publication all right
reserved
Research J. Pharmacology and
Pharmacodynamics. 4(4): July –August, 2012, 213-217