Antihyperlipidemic activity of Achyranthes aspera Linn leaves on cholesterol induced hyperlipidemia in rats

Sarvesh. C.N¹*, Jennifer Fernandes², Suresh Janadri³, Yogesh H.S¹, Shivakumar Swamy¹

¹Department of Pharmacology, Mallige College of Pharmacy, Bengaluru, Karnataka, India.

²Department of Pharmacology, Nitte Gulabi Shetty Memorial Institute of Pharmaceutical Sciences, Deralakatte, Mangalore.

³Department of Pharmacology, Acharya and B M Reddy College of Pharmacy, Bengaluru, Karnataka, India

*Corresponding Author E-mail: cnsarvesh@yahoo.co.in

ABSTRACT:

Plants have been one of the important sources of medicines since the beginning of human civilization. Achyranthes aspera Linn. (A.aspera) belonging to family Amaranthaceae, commonly found in India. The plant is used as antiarthritic, purgative, diuretic and antimalarial etc. The antihyperlipidemic activity of alcoholic and aqueous extracts of leaves of A.aspera was evaluated using cholesterol induced hyperlipidemia in albino wistar rats. In this model, 8 groups consist of 6 animals per group. Group I and II were considered as saline and hyperlipidemic controls respectively, and groups III-VIII received respective treatment (ethanolic extract 250 mg, 500 mg, aqueous extract 250mg, 500 mg, Ayurvedic preparation and atorvastatin). All the groups received cholesterol (400 mg/kg b.w. p.o) except saline control for 4 weeks. The serum HDL, LDL, TG, and TC levels were analyzed in cholesterol induced hyperlipidemia where both the extracts of ethanolic and aqueous extract were shown antihyperlipidemic activity compared to hyperlipidemic control.

KEYWORDS: Achyranthes aspera, cholesterol, HDL, LDL, TG, TC, Antihyperlipidemic.

INTRODUCTION:

Hyperlipidemia is abnormal high levels of lipids, elevated serum levels of one or more of total cholesterol, low density lipoprotein cholesterol, triglycerides, or both total cholesterol and total triglycerides, very low density lipoprotein and chylomicron.¹ Hyperlipidemia is a lifestyle lipid disorder which fatally affects the human health.² It leads to various cardiovascular disorders like arteriosclerosis, angina pectoris, myocardial infarction, hypertension, atherosclerosis, congestive heart failure, cerebrovascular diseases like ischemic stroke and hemorrhagic stroke.³

Although the incidence of the atherosclerosis related events has declined in the united states, these condition still accounts for the majority of morbidity and mortality among middle aged and older adults, the incidence and absolute number of annual events will increase over the next decade because of epidemic of obesity and ageing of the U.S population.^4,5

Reduction in serum cholesterol levels reduces the risk for cardiac diseases. The main aim of treatment in patients with hyperlipidemia is to reduce the risk of developing ischemic heart disease or the occurrence of further cardiovascular or cerebrovascular disease.⁶ Currently available hypolipidemic drugs have been associated with a number of side effects.⁷ The consumption of synthetic drugs leads to hyperuricemia, diarrhoea, nausea, myositis, gastric irritation, flushing, dry skin and abnormal liver function.⁸ To avoid the side effects produced by synthetic drugs usage of plant based drugs better choice for the treatment of lipid disorders. In this present study we selected the Achyranthes aspera Linn leaves on cholesterol induced hyperlipidemia in rats.

MATERIAL AND METHODS:

Collection and authentication of plant material

The leaves of “Achyranthes aspera Linn.” (A.aspera) were collected from local area in and around Mangalore in the month of June- July. It was authenticated by Dr. Noeline J.Pinto, HOD, Dept of Botany, St Agnes College, Mangalore-2. Thus obtained leaves were cleaned from dust and other materials, and it was dried under the shade for 15 days. And the dried leaves were pulverized in an electric grinder. And passed through sieve No- 40. The powdered materials were subjected to maceration.

Preparation of extracts

The powder was soaked in 90 % ethanol and distilled water separately for six days, stirring of the mixture was done thrice daily. The syrupy consistency materials obtained, were concentrated by evaporating using the water bath until semisolid consistancy was obtained.These ethanolic extract (reddish brown colur) and aqueous extracts (dark greenish colour) were having the weight about 35.5 g and 101 g respectively and stored in a dessicator. The ethanolic extract was dissolved in water using tween 80 and the aqueous extract was freely soluble in the water.

Preliminary qualitative phytochemical analysis

The ethanolic and aqueous extract of leaves of A.aspera were subjected to qualitative examination for different phytoconstituents like alkaloids, carbohydrates, flavonoids, glycosides, saponins, terpenoids and steroids by using standard methods.

Selection of animals

Wistar albino rats of either sex weighing between 150-200 g were obtained from NGSMIPS, Paneer, Deralakatte, Mangalore. These animals were used for the evaluation of antihyperlipidemic activity. The animals were stabilized for 1 week; they were maintained in standard condition at room temp; 60 ± 5 % relative humidity and 12 h light dark cycle. They had been given standard pellet diet supplied by Hindustan Lever Co. Mumbai. And water ad libitum throughout the course of the study. The cleaning and sanitation work done alternative days and paddy husk was provided as bedding material, which was changed everyday. The cages and water bottles were regularly cleaned. The animals were handled gently to avoid giving them too much stress, which could result in an increased adrenal out put. Institutional Animal Ethics Committee for the project has approved (KSHEMA/AEC/072/2008).

Acute toxicity test

The limit test is a sequential test that uses a maximum of 5 animals. A test dose of 2,000 mg/kg, or exceptionally 5,000 mg/kg, may be used. One animal dosed at the test dose. If the animal survived, four additional animals dosed sequentially so that a total of five animals were tested. In case three animals die, the limit test is terminated and main test is performed, if LD50 is greater than the test dose three or more animals will survive. However, all the dosed five animals were survived at 2,000 mg/kg. The alcoholic extract was dissolved in water using tween 80 and prepared a concentration of 200mg/ml and aqueous extract was dissolved in water and prepared a concentration of 500mg/ml The test dose is given to over night fasted animals and continuously observed for 2-3 hours for general behavioral, neurological, autonomic profiles then occasionally checked for the next 24 hours and after 48 hours (initial observation period). Further, animals were observed for 8 days (to check any late deaths). The limit test procedure of 5,000 mg/kg was carried out according to OECD guidelines and found all the animals were well tolerated and survived. From this one can conclude that the LD50 is more than 5,000 mg/kg body weight.^9,10

Induction of hyperlipidemia using cholesterol

The animals were dividing into different groups, were kept in separate labelled cages. Group I Served as control (saline only) Group II served as hyperlipidemic control, Group III received ethanolic extract 250 mg/kg b.w./day for 4 weeks. Group IV received ethanolic extract 500 mg/kg b.w./day for 4 weeks. Group V received aqueous extract 250 mg/kg b.w./day for 4 weeks. Group VI received aqueous extract 500 mg/kg b.w./day for 4 weeks. Group VII received Ayurvedic preparation (LEAN HEAL) 468 mg/kg b.w./day for 4 weeks. Group VIII received atorvastatin 2.70 mg/kg b.w./day for 4 weeks. Oral cholesterol feeding (400 mg/kg b.w./day) dissolved in 5ml conut oil for 4 weeks for all groups except saline control.

The biochemical parameters of lipid profile have been investigated in serum once a week for four weeks during the treatment with alcoholic and aqueous extracts of the plant, atorvastatin and ayurvedic preparation (Lean heal). The alcoholic and aqueous extracts were prepared as 100 mg/ml concentration for the oral dosing to the animals. The ayurvedic preparation (Lean heal) had a free flowing syrup like consistancy, hence was easily dosed orally using the oral gavage. Atorvastatin was dissolved in water and orally dosed using the oral gavage. Blood was withdrawn using the heparanised capillaries from the retro-orbital sinus in the overnight fasted animals. The serum was obtained after centrifuging the blood, which was used to estimate the concentration of biochemical parameters using the semi autoanalyser and relevant lipid profile kits.^11,12

Statistical analysis

The data were represented as Mean ± SD. The data of antihyperlipidemic activity of leaves of A.aspera were analyzed by one way analysis of variance (ANOVA) followed by Dunnet’s test for comparison of data in between the groups. ‘P’ value less than 0.05 was considered as statistically significant.

RESULTS:

The serum cholesterol levels of different groups of animal during the study were presented in the Fig 1. The normal saline control rats maintained the serum cholesterol levels uniformly throughout the experiment. The mean (± SD) serum cholesterol levels in the cholesterol induced hyperlipidemic control rats was initially at 49.07±12.79 mg/dL and later gradually increased to129.7±11.08mg/dL over a period of 30 days. There was a significant rise in the serum cholesterol levels in this group when compared with the normal saline control group and atorvastatin treated group. The atorvastatin treated cholesterol induced hyperlipidemic rats had shown mean (± SD) serum cholesterol level initially at 46.87±11.73 mg/dL, which was later gradually increased to 69.75±9.364 mg/dL over a period of 30 days. There was a significant inhibition on the rise of serum cholesterol levels in this group when compared with the cholesterol induced hyperlipidemic control group. The ethanolic extract (250 and 500 mg/kg) treated cholesterol induced hyperlipidemic rats had shown mean (± SD) serum cholesterol levels initially 51.03±11.79 and 45.13±7.548 mg/dL and which was gradually increased to 114.4±13.92and 88.78±11.09mg/dL over a period of 30 days, respectively. This was found to be lower in serum cholesterol levels in these groups in comparison with hyperlipidemic control group. The ethanolic extract (500 mg/kg) treated rats had the serum cholesterol levels significantly lower than that of the hyperlipidemic control group and a slight more than the atorvastatin treated group. This shows the dose dependent action of extract on the serum cholesterol levels of the cholesterol induced hyperlipidemic rats.

The aqueous extract (250 and 500 mg/kg) treated cholesterol induced hyperlipidemic rats had shown mean (± SD) serum cholesterol levels initially 46.42±10.28 and 43.55±8.846 mg/dL and which was gradually increased to 112.1±11.28and 89.68±15.03 mg/dL over a period of 30 days, respectively. This was found to be lower in serum cholesterol levels in these groups in comparison with hyperlipidemic control group. The aqueous extract (500 mg/kg) treated rats had the serum cholesterol levels significantly lower than that of the hyperlipidemic control group and had a slight more than the atorvastatin treated group. This shows the dose dependent action of extract on the serum cholesterol levels of the cholesterol induced hyperlipidemic rats. The Ayurvedic preparation (LEANHEAL) treated rats showed mean (± SD) serum cholesterol level initially 41.08±9.460 mg/dL which was later increased to 91.30±4.178 mg/dL over a period of 30 days, which was found to be lower than that of hyperlipidemic control group. This indicates that Ayurvedic preparation was able to reduce the serum cholesterol level in cholesterol induced hyperlipidemic model.

The serum triglyceride levels of different group of animals during the study were presented in the Fig. 2. The normal saline control rats maintained the serum triglyceride levels uniformly throughout the experiment.

The mean (± SD) serum triglyceride levels in the cholesterol induced hyperlipidemic control rats was initially at 54.55±11.30 mg/dL and later gradually increased to119.7±14.30 mg/dL over a period of 30 days. There was a significant rise in the serum triglyceride levels in this group when compared with the normal saline control group and atorvastatin treated group. The Atorvastatin treated cholesterol induced hyperlipidemic rats had shown mean (± SD) serum triglyceride levels initially at 53.07±10.77 mg/dL, which was later gradually increased to 69.95±14.93 mg/dL over a period of 30 days. There was a significant inhibition on the rise of the serum triglyceride levels in this group when compared with the cholesterol induced hyperlipidemic control group.

The ethanolic extract (250 and 500 mg/kg) treated cholesterol induced hyperlipidemic rats had shown mean (± SD) serum triglyceride levels initially 59.67±10.55 and 56.90±10.50 mg/dL and which was gradually increased to 110.9±15.88and 78.90±13.48 mg/dL over a period of 30 days, respectively. This was found to be lower in serum triglyceride levels in comparison with hyperlipidemic control group. The ethanolic extract (500 mg/kg) treated rats had the serum triglyceride levels significantly lower than that of the hyperlipidemic control group and a slight more than the atorvastatin treated group. This shows the dose dependent action of extract on the serum triglyceride levels of the cholesterol induced hyperlipidemic rats. The aqueous extract (250 and 500 mg/kg) treated cholesterol induced hyperlipidemic rats had shown mean (± SD) serum triglyceride levels initially 51.75±10.66 and 57.22±8.498 mg/dL and which was gradually increased to 98.33±13.47and 76.77±11.41 mg/dL over a period of 30 days, respectively. This was found to be lower in serum triglyceride levels in these groups in comparison with hyperlipidemic control group. The aqueous extract (500 mg/kg) treated rats had the serum triglyceride levels significantly lower than that of the hyperlipidemic control group and had a slight more than the atorvastatin treated group. This shows the dose dependent action of extract on the serum triglyceride levels of the cholesterol induced hyperlipidemic rats. The Ayurvedic preparation (LEANHEAL) treated rats showed mean (± SD) serum triglyceride level initially 52.52±10.51 mg/dL which was later increased to 80.78±15.04 mg/dL over a period of 30 days, which was found to be lower than that of hyperlipidemic control group This indicates that Ayurvedic preparation was also able to reduce the serum triglyceride level in Cholesterol induced hyperlipidemic model.

DISCUSSION:

The dried and powdered of A. aspera Linn leaves was subjected to soxhlet extraction with 90 % ethanol .The yield of the extract was 10 % w/w. Phytochemical analysis of the ethanolic extract showed different phytoconstituents viz. glycosides, phytosterols,

triterpinoids, alkaloids and flavonoids. Glycosides, triterpinoids, Saponins, alkaloids and flavonoids are known to have antihyperlipidemic property. It has been well established that nutrition plays an important role in the etiology of hyperlipidemia and atherosclerosis.¹³ Cholesterol feeding has been often used to elevate serum cholesterol levels to assess the hypercholesterolemia related metabolic disturbances in animals.^14,15Cholesterol feeding alone however does not affect the serum TG level. It is assumed that a high level of saturated fat in addition to cholesterol is required to significantly elevate serum TG level in rat model.¹⁶

A.aspera has been shown to decrease the serum as well as liver levels of lipids and enhanced the excretion of bile acids. The alcoholic extract of A.aspera contains glycosides of oleanolic acid (saponin A and B) with steroidal nucleus. It has been reported that cardiac stimulant and diuretic activity of the saponins from this plant. The lipid lowering activity of A.aspera may contribute to these pharmacological properties. Hypocholesterolemic effect of A.aspera may be in part due to its action on faecal excretion of bile acids which may regulate cholesterol homeostasis.¹⁷ The detailed study is further required in clinical aspects and molecular level to prove the aqueous and ethanolic extract of A.aspera to reduce the cholesterol and triglyceride levels. Also to prevent the cardiovascular and cerebrovascular diseases in clinical is needed.

ACKNOWLEDGEMENT:

The authors are grateful to the management of Nitte Gulabi Shetty Memorial Institute of Pharmaceutical Sciences for providing the facility to carryout research work.

CONFLICT OF INTEREST:

The authors declare no conflict of interest.

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Received on 01.12.2016 Modified on 14.12.2016

Research J. Pharm. and Tech. 2017; 10(1): 200-204.

DOI: 10.5958/0974-360X.2017.00043.9