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 ± 2C 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.

 

6. REFERENCES:

1.       Ghatak A, Asthana OP. Recent Trends in hyperlipoproteinemias and its pharmacotherapy. Indian Journal of Pharmacology. 27(1); 1995: 14-29.

2.       Ford ES, Giles WH, Dietz WH. Prevalence of metabolic syndrome among US adults: Findings from third National Health and Nutrition Examination Survey. Journal of the American Medical Association. 287(3); 2002: 356-9.

3.       Avery Graeme S, Speight Trevor M. Avery’s Drug treatment: Principles and practices of clinical pharmacology and therapeutics. Williams and Wilkins, Baltimore.1987. 

4.       WHO Guidelines for the assessment of herbal medicines. Programme on traditional medicines, Geneva: World health Organization, 1991. Available from: http://whqlibdoc.who.int/hq/1991/WHO_TRM_91.4.pdf.

5.       Robert WM, Thomas PB, Laurence LB, John SL, Keith LP. Anti hyperlipidemic agent. In: Goodman and Gillman’s The Pharmacological Basis of Therapeutics, Edited by Laurence L. Brunton. Mc Graw-Hill, New York. 2006; 11th ed: pp. 933-81.

6.       Kirtikar KR and Basu BD. Indian Medicinal Plants. Vol III. Edited by Basu LM, Allahabad, India. 1933; pp. 1647.

7.       Kirtikar KR and Basu BD. Illustrated Indian Medicinal Plants. Edited by Mhaskar KS, Blatter E and Cains JF. Sir Satguru's Publications, Delhi, 2000.

8.       Anonymous, Wealth of India- Raw Materials, Sp-W. Vol. 10, Publications and Information Directorate, CSIR, New Delhi, 1976; pp: 66-67.

9.       Singh H, Kapoor VK, Phillipson JD and Bisset N.G. Diaboline from Strychnos potatorum. Phytochemistry. 14 (2); 1975: 587-588.

10.     Singh A and Bajpai RK. Chemical examination of fixed oil from the seeds of Strychnos potatorum "Linn. f." I mixed fatty acid. Journal of Indian Chemical Society. 52(8); 1975: 768-769.

11.     Singh H, Kapoor VK and Manhas MS. Investigation of Strychnos Spp. III. Study of triterpenes and sterol of Strychnos potatorum seeds. Planta Medica. 28 (4); 1975: 392-396.

12.     Singh H, Kapoor VK. Investigation of Strychnos spp. VI - Pharmacological studies of alkaloids of Strychnos potatorum seeds. Planta Medica. 38(2); 1980: 133-137.

13.     Singh H, Kapoor VK. Investigation of Strychnos spp. IV. Pharmacological studies of alkaloids of Strychnos potatorum. Planta Medica. 29(3); 1976: 226-33.

14.     Biswas  S, Murugesan T, Maiti K, Ghosh L, Pal M, Saha BP. Study on the diuretic activity of Strychnos potatorum Linn. seed extract in albino rats. Phytomedicine. 8(6); 2001: 469-471.

15.     Biswas S, Murugesan T, Sinha S, Maiti K, Gayen JR, Pal M, Saha BP. Antidiarrhoeal activity of Strychnos potatorum seed extract in rats. Fitoterapia. 73 (1); 2002: 43–47.

16.     Sanmugapriya E, Senthamil SP and Venkataraman S. Evaluation of antiarthritic activity of Strychnos potatorum Linn. seeds in Freund's adjuvant induced arthritic rat model. BMC Complementary and Alternative Medicine. 10(56); 2010: 1-9. 

17.     Sanmugapriya E, Venkataraman S. Studies on hepatoprotective and antioxidant actions of Strychnos potatorum Linn. seeds on CCl4-induced acute hepatic injury in experimental rats. Journal of Ethnopharmacology. 105 (1–2); 2006: 154–160.

18.     The Ayurvedic Pharmacopoeia of India, Part I, Vol-IV. Government of India, Ministry of Health and Family Welfare,  Department of Ayurveda, Yoga-Naturopathy, Unani, Siddha & Homeopathy (AYUSH), New Delhi. pp. 158-165.

19.     Sanmugapriya E and Venkataraman S. Toxicological investigations on Strychnos potatorum Linn. Seeds in experimental animal models. Journal of Health Science. 52(4); 2006: 339-343.

20.     Kellner A, Correll, JW, Ladd AT. 1951. Sustained hyperlipidaemia induced in rabbits by means of intravenously injected surface-active agents. Journal of Experimental Medicine. 93(4); 1951: 373-383.

21.     Asima C, Satyesh CP. The Treatise on Indian Medicinal Plants, Vol 4, New Delhi, Publications and Information Directorate, CSIR. 2001: pp. 85–87.

22.     Steinbreg D, Parthasarathy S, Carew TE, Khoo JC, Wititum JL. Beyond cholesterol: modification of LDL that increase it’s atherogenicity. New England Journal of Medicine. 1989; 320: 915-924.

23.     Kaamanen M, Adlecreutz H, Jauhiainen M, Tikkanen MJ. Accumulation of genistein and lipophilic genistein derivatives in lipoproteins during incubation with human plasma in vitro. Biochemica et Biophysica Acta. 2003; 1631: 147-152.

 

Received on 28.06.2012

Modified on 04.07.2012

Accepted on 07.07.2012

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Research J. Pharmacology and Pharmacodynamics. 4(4): July –August, 2012, 213-217