INTRODUCTION:

Hyperlipidemia is one of the major causes for various cardiovascular and central nervous system disorders. It is a silent killer. Both genetic disorders and diet enriched in saturated fat and cholesterol, contribute to the elevated lipid levels in our population and in many other developed countries around the world. It is a major cause of atherosclerosis and is associated with conditions such as coronary heart disease, cerebrovascular diseases and peripheral vascular diseases¹.

A recent survey carried out by WHO indicates that coronary heart disease (CHD) alone accounts for more than half of the total mortalities associated with cardiovascular diseases. Atherosclerosis is the focal point of pathogenesis of these diseases. The American Heart Association identified the primary risk factors associated with atherosclerosis as elevated levels of cholesterol and triglycerides in the blood².

Atherosclerosis remains the major cause of death and premature disability in developed societies. Current predictions estimate that by the year 2020 cardiovascular diseases notably atherosclerosis will become the leading global cause of total disease burden. Coronary atherosclerosis causes myocardial infarction and angina pectoris; strokes and transient cerebral ischemia are due to cerebral atherosclerosis. Intermittent Claudication and gangrene are dreaded squeal of atherosclerosis occurring peripherally. Abnormalities in plasma lipoproteins and derangements in lipid metabolism rank among the most firmly established and the best understood risk factors for atherosclerosis³.

Lipid lowering drugs, mostly statins and fibric acid derivatives have been widely used to manage the elevated levels of various forms of lipids in hyperlipidemic patients. Due to its serious complications, these drugs have to be used safely or avoided when possible⁴. In recent years, the development of lipid lowering drug or formulations from natural source has gained importance. Hence an attempt was made to develop a polyherbal formulation (PHF) containing the extracts of Embilica officinalis, Zingiber officinalis, Luffa aegyptiaca, Moringa oleiferia, Hibiscus canabinus, Rhinacanthus nasutus (Linn) which are known to possess anti-hyperlipidemic effect and the formulation was used, to evaluate its efficacy on lipid levels using high fat diet induced hyperlipidemia in rats.

MATERIALS AND METHOD:

Drugs and chemicals:

Atorvastatin was received as a gift sample from Biocon pharmaceuticals, Bangalore (India). The coconut oil (Marico Industries Ltd., Mumbai) and vanaspati ghee (Ruchi industries, Mumbai) were procured from the local market. The raw plants material was obtained from a local herbal drug dealer stores in Hyderabad, Andhra Pradesh, India.

Preparation of Polyherbal Formulation⁵:

All the plants material used in the formulation were collected from an authorized raw drug dealer, Hyderabad, India. The plants material was properly authenticated and shade dried, coarsely powdered using mechanical grinder. The Zingiber officinale rhizomes, fruits of Embilica officinalis and the whole plant of Rhinacanthus nasutus (Linn) were extracted with distilled water individually by cold maceration process for 05 days. The fruits and leaves of Luffa aegyptiaca, Moringa oleiferia and leaves and flowers of Hibiscus canabinus were soaked individually with ethyl alcohol for 05 days and extracts were obtained by cold maceration method. The extracts were filtered and concentrated under vacuum. The crude extracts obtained were stored in refrigerator for further use. The formulation was prepared by mixing 16.6% of each dried extracts of 06 plants in equal proportions.

Preliminary phytochemical analysis^{6, 7, 8, 9}:

The literature survey for all the 06 herbs used in the preparation of polyherbal formulation revealed the presence of saponins, tannins, glycosides, alkaloids, volatile oils, flavonoids, steroids, terpenoids, vitamin C, amino acids and carbohydrates imparting them with the needed lipid lowering property.

Experimental animals:

Wistar albino rats of either sex weighing between 200 - 250g were procured from the licensed animal dealer, Hyderabad (India). The animals were acclimatized for 07 days and housed under standard conditions of temperature (25±20ºC) and relative humidity (30-70%) with a 12:12 Light-Dark cycle. The animals were fed with standard pellet diet (Hindustan Lever Pvt. Ltd., Mumbai) and water ad libitum. Approval from the institutional animal ethics committee (IAEC) was obtained for conducting animal experiments and the animal studies were performed in accordance to guidelines of CPCSEA.

Method of Preparation and administration of High Fat Diet¹⁰:

Edible coconut oil and vanaspati ghee were mixed together in the ratio of 2:3 v/v respectively. High fat diet in a dose of 10ml/kg was administered to the animals orally for 08 consecutive weeks in addition to normal diet.

Experimental Design:

The experiment was carried out for a period of 08 consecutive weeks. 30 healthy wistar albino rats of either sex, weighing 200 - 250g were used in the study. The animals were weighed, numbered and randomly divided into 05 groups of 06 animals in each. The treatment pattern followed was –

Group I	-	Normal diet
Group II	-	High fat diet (Control)
Group III	-	Standard group - High fat diet (10ml/kg) + Atorvastatin (7.2 mg/kg)
Group IV	-	Test group I - High fat diet (10ml/kg) + PHF (100mg/Kg)
Group V	-	Test group II - High Fat diet (10ml/kg) + PHF (200mg/Kg)

All the animals used for the experiment were kept under observation for daily food intake. The drugs were administered to the animals for a period of 08 weeks, by means of an intragastric feeding tube. At the end of the 08^th week, 24h after the last dose, blood samples were collected from all the animals of each group for assessment of lipid profile. Blood samples were collected from the retro-orbital plexus and allowed to coagulate at room temperature then centrifuged at 3000rpm for 10 mins. The serum was separated and used for the biochemical estimations. The collected serum was analyzed for Serum Total Cholesterol, Triglycerides, High Density Lipoprotein Cholesterol (HDL), Low Density Lipoprotein Cholesterol (LDL) and Very Low Density Lipoprotein Cholesterol (VLDL). Serum lipid profile was carried out using standard protocols^{11, 12.}

Statistical Analysis:

Results were analyzed and interpreted by one way ANOVA, followed by Dunnett’s test. P values ≤ 0.05 were considered as significant. Results are expressed as Mean±SEM of 06 animals from each group.

RESULTS:

The data obtained from the evaluation of serum lipid profile of each treatment group is given in the Table.1. The PHF in doses of 100 and 200mg/kg significantly reduced the serum cholesterol, triglycerides, VLDL, LDL and increased the levels of HDL in a dose dependent manner in comparison with control and standard groups.

Table.1. Effect of PHF on serum lipid profile in high fat diet induced hyperlipidemia.

Treatment groups	Biochemical Parameters
Treatment groups	Total Cholesterol (mg/dl)	Triglycerides (mg/dl)	VLDL (mg/dl)	LDL (mg/dl)	HDL (mg/dl)
Normal Diet	177.5±0.42	108.17±1.66	102.08±0.30	53.00±0.36	11.33±0.35
Control Group	422.5±1.11	34.33±0.33	1082.5±0.0808	608.50±0.42	5.66±0.23
Standard Group	202.3±0.66	22.15±0.51	110.2±0.30	55.33±0.21	12.33±0.33
Test group 1	20808.08±1.75 ***	33.17±0.40 ***	150.2±0.47 ***	64.17±0.30 ***	8.33±0.21 ***
Test group 2	217.5±1.25 ***	25.33±0.21 *	119.0±0.25 ***	59.67±0.33 ***	9.608±0.33 ***

Values are mean ± SEM; n=6; *P < 0.05 compared with Standard

DISCUSSION:

The purpose of the present study was to evaluate the effect of polyherbal formulation containing herbs with known hypolipidemic effect on serum lipid profile in adult albino rats fed with high fat diet in comparison to a standard hypolipidemic agent Atorvastatin.

Hyperlipidemia is a major contributor for health problems worldwide and leads especially to atherosclerosis, resulting in coronary heart diseases (CHD). According to WHO, by 2020, 60% of the cardiovascular cases will be of Indian origin. Hyperlipidemia induces the damages in various tissues, which in turn, alters the cellular functions leading to cell damage and many pathological conditions. A high fat diet may cause elevated levels of cholesterol, which ultimately leads to obesity. Elevated cholesterol levels particularly LDL, VLDL increases the risk of cardiovascular diseases particularly coronary heart disease (CHD). Increase in HDL cholesterol reduces the risk of CHD. Reduction of 1% cholesterol can lead to 2-3% reduction of CHD risk. The importance of medicinal plants in the treatment of hyperlipidemia was experimentally studied in recent years, where oxidative stress induced apoptosis in adipose tissue was noticed⁵.

In-vitro and in-vivo studies on flavonoids from Emblica officinalis revealed reduction in serum and tissue lipid levels of hyperlipidemic rats. Besides they also possess good antioxidant and cardio protective properties. Preclinical evaluation with ginger has revealed antioxidant and hypolipidemic effects. Previous studies have also confirmed that the presence of phytoconstituents like flavonoids, alkaloids, saponins and tannins in extract or as isolated compound might contribute towards hypolipidemic activity⁵.

Hibiscus cannabis has been used as an antidote for chemicals (acid, alkali, pesticides) poisoning and venomous mushrooms, to treat bruises, bilious conditions, fever, etc. The stem peelings are being used in treating dysentery and blood and throat disorders. It is also having aphrodisiac, as well as fattening, purgative and stomachic. The effect of oral administration of 50% hydroalcoholic extract of Hibiscus canabinus L. leaves (HC) on experimentally induced hyperlipidemia in rats was investigated. The extract exhibited a strong dose dependent (100mg/kg, 200mg/kg, 400mg/kg) antihyperlipidemic activity and at dose level 400mg/kg showed a significant decrease in the levels of serum TC, TG, LDL-C, VLDL-C and TBARS. In addition, the extract markedly prevented the liver micro vesicular steatosis in hyperlipidemic rats. The presence of lignans, alkaloids and flavonoids as pharmacologically active compounds in HC may likely to have contributed for the observed hypolipidemic activity⁶.

Moringa oleifera Lam had been studied for several pharmacological actions like wound healing, anti-urolithiasis activity, antibacterial activity, antifungal, anti-tubercular activity and hypotensive actions. Pankaj G. Jain et al. studied the hypolipidemic activity of Moringa oleifera Lam., on high fat diet induced hyperlipidemia in albino rats with simvastatin as standard drug. Methanolic extract of this plant in the dose of 150, 300 and 600 mg/kg, was given p.o., for thirty days. The hypolipidemic activity was assessed by faecal cholesterol excretion, HMGCoA reductase activity and lipid profile in serum. Treatment with methanolic extract, at three different doses significantly decreased the levels of total cholesterol and LDL as compare to the controls. The cholesterol lowering effect may be due to inhibition in reabsorption of cholesterol from endogenous sources in association with a simultaneous increase in its excretion into faeces in the form of neutral steroids^{13, 14}.

The medicinal uses of Luffa are as Dyslipidemic, Anti-Diabetic, Hepatoprotective, Anti-Hypertensive and Diuretic. British people are extensively using Luffa aegyptiaca Mill fruits along with egg preparations as a food habit, which is possibly reduces the cholesterol from the egg. Luffa aegyptiaca tender fruit is taken as vegetable, the course sponge of mature fruit is used as a bath scrub and juice of leaves cures conjunctivitis. Abdul hameed thayyil et al evaluated hypolipidemic activity of Luffa aegyptiaca fruits in cholesterol fed hypercholesterolemic rabbits. Methanolic extract of Luffa aegyptiaca fruits at 300mg/kg significantly reduced serum lipid profile. This hypolipidemic activity is attributed to the presence of chemical ingredients in Luffa aegyptiaca fruit such as Ascorbic acid, Niacin, Fiber, MUFAs (Linoleic acid, Oleanolic acid, Oleic acid) and stearic acid¹⁵.

Phytochemical analysis of the Rhinacanthus nasutus (Linn) extract showed different phytoconstituents viz. glycosides, phytosterols, triterpinoids, alkaloids and flavonoids. Several phytoconstituents like glycosides, triterpinoids, Saponins, alkaloids and flavonoids are known to have anti-hyperlipidemic properties⁷.

Based on the above information, the polyherbal formulation was prepared using extracts of above mentioned 06 medicinal plants which are known to possess hypolipidemic potentials and evaluated scientifically for its potentials using Wistar rats as experimental animal models in high fat diet induced hyperlipidemia.

The results obtained from the study revealed that upon analysis of lipid profile of animals from various PHF treatment groups showed significant results. The animals treated with PHF in a dose of 100mg/kg showed significant increase in the serum HDL values upon comparison with control, it also exhibited a significant decrease in the serum total cholesterol, VLDL, LDL, triglycerides considerably. On the other hand, treatment with PHF in a dose of 200mg/kg, showed much impressively significant results when compared to group treated with 100mg/kg and control group. Thus from the results obtained, it can be concluded that the PHF in a dose of 100mg/kg and200mg/kg reduces the elevated lipid levels in a dose dependent manner upon comparison with control and standard groups (Table.1).

CONCLUSION:

From the results obtained (Table.1), it can be concluded that PHF in dose of 100 and 200mg/kg have significant hypolipidemic activity, comparable to that of Atorvastatin. The levels of lipid parameters i.e. total serum cholesterol, serum triglycerides, LDL and VLDL which were raised with high fat diet, were significantly reduced and serum HDL was increased with administration of PHF (100 and 200mg/kg). Thus, PHF is a potent hypolipidemic formulation.

REFERENCES:

1. Thomas P Bersot. Drug therapy for hypercholesterolemia and dyslipidemia. Goodman and Gilman’s the Pharmacological basis of therapeutic. 12^th ed., McGraw-Hill, New York. 2011; 877-904.

2. Sanjy Batra, Seema Srivastava, Kavita Singh, Ramesh Chander, Ashok K. Khanna, Bhadruri P. Synthesis and biological evaluation of 3‐substitued amino‐1‐aryl‐6‐hydroxy‐hex‐2‐ene‐1‐one as antioxidant and hypolipidemic agents. Bioorganic and medicinal chemistry.2000; 8:2195‐209.

3. Peter Libby. Harrison’s Principles of Internal Medicine. 17^th Ed., McGraw-Hill, New York. 2010; 2:1501-509.

4. Trease GE, Evans WC. Textbook of Pharmacognosy. 12^th ed. London: Balliere Tindall; 1908-9.

5. C. David Raj, V. Jayanthi, V.S. Manaswini, R. Gayathri, C. Ranjani, P.Brindha. Effect of polyherbal formulation (OB-6) on high fat diet induced hyperlipidemia in rats. Int. J Pharm. Pharm. Sci. 2012; 4(2):31-5.

6. Venu Gopala Rao Konda, Madhavi. E, Ruckmani. A, Venkataramana. Y. A Review on medicinal plants with potential Hypolipidemic activity. Int. J Pharm Bio Sci. 2013; 4(4):729-40.

7. Brahma Srinivasa Rao Desu, Ch. Saileela. Anti-Hyperlipidemic activity of methanolic extract of Rhinacanthus nasutus. IJRPC 2013; 3(3):708-11.

8. AK Meena*, Arjun Singh, Mm Rao. Evaluation of Physicochemical and Preliminary Phytochemical Studies on the fruit of Emblica officinalis Gaertn. Asian Journal of Pharmaceutical and Clinical Research.2010; 3(3): 242-43.

9. Hiba Ali Hasan, Ayad Mohammed Rasheed Raauf, Basama Monjd Abd Razik, Bassam Abdul Rasool Hassan. Chemical Composition and Antimicrobial Activity of the Crude Extracts Isolated from Zingiber officinale by Different Solvents. Pharmaceutca Analytica Acta. 2012; 3(9):1-5.

10. Shyamala MP, Venukumar MR, Latha MS. Antioxidant potential of the Syzygium aromaticum (Gaertn) Linn (Cloves) in rats fed with high fat diet. Indian Journal of Pharmacology 2003; 35:99-103.

11. Friedewald WT, Levy RI, Fredrickson DS. Estimation of the concentration of low-density lipoprotein cholesterol in plasma, without use of the preparative ultracentrifuge. Clin Chem 1972; 108:499-502.

12. Dobiasova M, Frohlich J. The plasma parameter log (TG/HDL-C) as an atherogenic index: correlation with lipoprotein particle size and esterification rate in apo B-lipoprotein-depleted plasma (FER (HDL)). Clin Biochem 2001; 34:583-88.

13. Subrata Kumar Biswa., Anusua Chowdhury, Joysree Das, Ajoy Roy,S.M Zahid Hosen, Pharmacological Potentials of Morringa Oleifera Lam. A review. International Journal of Pharmaceutical Sciences and Research. 2012; 3(2):305-10.

14. Pankaj G. Jain, Savita D. Patil, Nitin G. Haswani, Manoj V. Girase, Sanjay J. Surana, Hypolipidemic activity of Moringa oleifera Lam., Moringaceae, on high fat diet induced hyperlipidemia in albino rats. Brazilian Journal of Pharmacognosy. 2010 20(6):969-73.

15. Abdul Hameed Thayyil, M.K.M. Surulivel, Mohammed. Fazil Ahmed, G. Shaik Shafee Ahmed et al., Hypolipidemic Activity of Luffa Aegiptiaca Fruits in Cholesterol Fed Hypercholesterolemic Rabbits. International Journal of Pharmaceutical Applications. 2011; 2(1):81-88.

Received on 05.11.2014 Modified on 18.11.2014

Res. J. Pharm. Dosage Form. & Tech. 7(1): Jan.-Mar. 2015; Page 11-14

DOI: 10.5958/0975-4377.2015.00003.8