INTRODUCTION:

Hyperlipidemia is considered as a risk factor involved in the development of various disorders worldwide ¹. Over-weight and obesity are defined as abnormal or excessive accumulation of fat occurs due to disproportion of energy intake and expenditure ^2-4. Obesity has been found to be associated with various disorders like atherosclerosis, osteoarthritis, diabetes and hypertension ^5-8.Curently, no pharmacological treatment is available to reduce the weight with lesser side effects ⁹. In recent years, interest is going on the ingredients of natural plants, vegetables and cereals, not only for their free radical-scavenging activities, but also dueto their negligible side effects. There is availability of numerous medicinal plants exerting good hypolipidemic actions with few side effects in Asian countries. In literature review vast number of plants have been shown to lower plasma lipid levels such as Moringa oleifera¹⁰, Nigella sativa ¹¹, Cinnamomum tamala¹², Bauhinia variegate¹³and so on as antihyperlipidemics. In current researches, it is proved that hyperlipidemia is associated with oxidative stress. Hence the plants with good antioxidant activity acts as antihyperlipidemics. Hence the study was aimed to evaluate antihyperlipidemic and antioxidant activity methanolic leaf extract of Ochna obtusata (MEOOL).

Ochna obtusata is a small tree up to 8m tall belongs to the family Ochnaceae. The family is characterised by the presence of secondary metabolites like flavonoids and terpenoids and it is extensively used in the treatment of epilepsy, menstrual complaints, lumbago, asthma, ulcers and as an antidote to snake bite¹⁴. The leaves and roots of Ochnaobtusata was proved of having diuretic activity¹⁵, anti convulsant¹⁶, antibacterial¹⁷, antidiabetic¹⁸ and antiulcerogenic activity¹⁹.

MATERIALS AND METHODS:

Collection and Preparation of Plant extract:

The leaves of Ochna obtusta was collected from Tirumala Hills, Tirupati, India. The plant was authenticated by Dr. Madhava Chetty, Professor of Botany, Sri Venkateshwara University, Tirupati and voucher specimen of the plant were preserved at institute herbarium library. The fresh leaves are washed, shade dried and powdered. The powder was subjected to soxhlet extractor using methanol. The extract was filtered and then solvent was evaporated under reduced pressure to a solvent free concentrated mass, which was then stored in air-tight container in a cool and dry condition.

Preliminary Phytochemical Screening:

The preliminary phytochemical of Ochna obtusata was carried out according to the methods described by Kokate and Khandelwal²⁰. Phytochemical analysis of the extract was performed for the identification of phytochemicals such as alkaloids, carbohydrates, proteins and amino acids, tannins, flavanoids, steroids, resins.

Experimental Animals:

Wistar albino adult male rats weighing 200-250g were obtained from the animal house. The animal were grouped and housed in polyacrylic cages (38x 23x 10 cm). Each group consisting of six animals and maintained under standard laboratory (temperature 25±2^oC) with dark and light cycle (14/10 hour). They were allowed free access to standard dry pellet diet and water ad libitum. The rats were acclimatized to laboratory condition for 10 days before commencement of experiment. The experimental protocol was approved by Institutional Animal Ethical Committee (IAEC) constituted under CPCSEA. (Reg no 769/ 2011/ CPCSEA).

Dose Selection:

In the present study, two doses of Ochna obtusata leaf extract were selected as 200 mg and 400mg, p.o. The doses was selected on basis of previous reports of the acute toxicity study performed using the single dose of orally administered 2g/kg of methalonic extracts of O. obtusata (leaf) which shows no signs of toxicity in rats¹⁹

Composition of High Fat Diet (HFD):

HFD consist of Protein Milk powder (10%) Carbohydrates Wheat flour (61%) Sugar (05%), Fat Butter (16%), salts (04% ), vitamins (02%) , fibers ( 01% ), cholesterol (01%) as percentage of total kcal ad libitum, respectively was administered every day. Food intake was calculated every day and body weights were measured once in every two days .The composition of normal pellet diet and (NFD) and HFD diet was shown in table 1.

Methodology:

High fat diet (HFD) induced obesity in rats is considered to be a reliable tool for the evaluation of antiobesity activity. The animals were divided into five groups. Each group contains six animals. The study was carried out for 28 days. Group 1 represented normal control in which the animals were feed on normal diet and free access of watered libitum. Group 2 represented negative control in which the rats feed on high fat diet. Group 3 represented standard control in which the rats were treated with Atorvastatin (10 mg/kg, p.o). Group 4 represented test treatment in which rats were treated with the dose of methonalic exrtract of Ochna obtusata (200 mg/kg, p.o.) along with high fat diet. Group 5 represented test treatment in which rats were treated with dose of methanolic extract of Ochna obtusata (400 mg/kg, p.o.) along with high fat diet²¹.

Biochemical Estimations:

On the 28^th day of experiment blood was withdrawn from the retro-orbital plexus and the serum was separated and used for biochemical estimations of TG, TC, HDL, VLDL, LDL, blood glucose and atherogenic index. Then the animals were sacrificed by cervical dislocation and liver were removed to measure the In vivo antioxidant parameters like SOD, catalase, LPO and reduced GSH.

The friedewal formula was used to calculate serum low density lipoprotein (LDL) values and atherogenic index follows

LD L=TC –HDL –TG/5 and

Atherogenic index=TC – HDL/HDL.

Table-1: Composition of Normal and High fat diet.

Composition	Normal diet (%)	High Fat diet (%)
Protein(Milk powder)	12	10
Carbohydrates(Wheat flour)	71	61
Sugar	05	05
Fat(Butter)	05	16
Salts	04	04
Vitamins	01	02
Fibers	02	01
Cholesterol	--	01
Total Weight	100g	100g

Statistical Analysis:

The results were expressed as Mean±SEM comparison between treatment group and control group were performed by one way analysis of variance (ANOVA) followed dunnetts multiple comparison test.

RESULTS:

Preliminary Phytochemical Screening:

The result of the preliminary phytochemical screening of methanolic extract of Ochna obtusata leaves (MEOOL) shows the presence of alkaloids, carbohydrates, steroids, glycosides, saponins, resins, fixed oils, proteins and amino acids, flavonoids.

Effect of High Fat Diet and Meool on Body Weight:

At the end of the study the animals fed with high fat diet for 28 days produced a significant increase (p<0.001) in the body weight compared to the animals fed with normal diet. The group of animals treated with MEOOL (200 mg/kg and 400 mg/kg) showed a significant at decrease in the bodyweight at the end of the study as represented in table-2.

Effect of High Fat Diet and MEOOL on Biochemical Parameters:

As shown in table-3, the animals fed with high fat diet showed a significant increase (p< 0.05) in the serum TC, LDL-C, VLDL-C, triglycerides and blood glucose levels as compared to normal group. However the animals treated with atorvastatin and MEOOL showed a significant decrease in the biochemical parameters compared to hyperlipidemic control group.

Effect of High Fat Diet and MEOOL on Antioxidant Parameters:

From the results presented in table-4, HFD increased the MDA level in plasma significantly (p< 0.05) when compared with that of the normal group. HFD group treatment with AL extracts showed a significant reduction (p< 0.05) in MDA levels. The levels of reduced GSH and catalase were raised significantly (p< 0.05) after treatment with MEOOL when compared to HFD treated rats.

Table-2 Change in body weights before treatment and after treatment with MEOOL.

HFD diet	NORMAL	CONTROL	STANDARD	T1	T2
B. w B.T	228.83±0.8	228±1.07^a	227.83±1^b	227.5±0.71^b	226.16±0.91^b
B. w A.T	262.5±0.77	311.83±1.6^a	281.33±0.9^b	293.83±1.27^b	290±1.19^b

Values are expressed as mean ± SEM for six animals a=P<0.05ascomparedtoGroup-I b=P<0.05as compared to Group-I

Table-3. Effect of MEOOL on biochemical parameters

TEST	NORMAL	CONTROL	STANDARD	T1	T2
HDL	27.00±0.81	22.7±0.71^a	31.55±0.54^b	31.04±0.85^b	37.98±0.76^b
LDL	25.20±0.82	61.88±0.95^a	35.60±0.78^b	41.31±1.11^b	24.81±1.09^b
VLDL	11.32±0.68	16.11±0.85^a	12.31±0.58^b	14.32±0.39^b	12.37±0.58^b
GLUCOSE	73.51±0.90	146.4±0.85^a	104.2±0.91^b	121.2±0.7^b	106.2±0.72^b
TC	65.03±0.97	106.0±0.63^a	84.7±0.80^b	83.49±1.17^b	67.36±0.57^b
TG	54.08±1.35	94.04±0.68^a	63.94±1.16^b	73.41±1.31^b	54.91±0.93^b
AI	1.41±0.574	3.67±0.529^a	1.66±0.619^b	1.69±0.46^b	0.77±0.98^b
CRR	1.42±0.574	3.69±0.529^a	1.65±0.528^b	1.70±0.46^b	0.75±0.98^b

Values are expressed as mean± SEM for six animals a=P<0.05as compared to Group-I ,b=P<0.05as compared to Group-II

Table-4. Effect of MEOOL on In vivo Antioxidant parameters

ANTI OXIDANT	NORMAL	CONTROL	STANDARD	T1	T2
LPO	0.13±1.59	0.31±1.51^a	0.23±0.52^b	0.20±0.46^b	0.13±1.27^b
GSH	1.31±0.20	0.62±0.20^a	1.09±0.68^b	0.82±0.90^b	1.05±0.96^b
CAT	0.36±2.28	0.02±1.38^a	0.14±2.14^b	0.15±2.50^b	0.17±1.40 ^b

Values are expressed as mean±SEM for six animals a=P<0.05as compared to Group-I, b=P<0.05 as compared to Group-II

DISCUSSION:

Hyperlipidemia is the metabolic disorder of carbohydrate, fat and protein. Hence, it is necessary to search for new drugs to treat this metabolic disorder without any side effect. Oxidative stress is suggested as a mechanism underlying hyperlipidemia, which is one of the major risk factor for coronary artery disease²². In our study, phytochemical investigation revealed the presence of flavonoids, glycosides, saponins and steroids. As flavonoids proved of having good antioxidant activity the decrease in the lipid levels may be due to the flavonoids. Elevated blood triglyceride and cholesterol, especially low-density-lipoprotein cholesterol (LDL-C), is a major risk factor for the development of cardiovascular disease ²³. In the present study apart from body weight reduction, animals treated with MEOOL significantly reduced the levels of TC, LDL and elevated the levels of HDL and the findings are in consonance with the earlier reports of Ghasi et al²⁴ and Aattar²⁵. Thus it can be concluded that leaves of O.obtusata possess cardioprotective activity. Further atherogenic index is regarded as a marker for various cardiovascular disorders; higher the value higher is the risk of developing cardiovascular disease and vice versa ^26-27. Treatment with higher dose of MEOOL (400 mg/kg) significantly reduced the atherogenic index supporting the cardioprotectant nature of O. obtusata. The results are similar in findings with Souravhbais et al ²⁸. Intake of HFD contributes to number of syndromes such as hyperlipidemia, hypertension, glucose intolerance and atherosclerosis ²⁹. Number of studies have proved high fat diet resulted in disturbance of glucose metabolism and glucose tolerance³⁰ and the present study demonstrate the reduction in blood glucose level those treated with MEOOL (200 mg/kg and 400mg/kg).

In the present study, catalase, MDA and reduced GSH levels were measured in high fat diet rats. Since atherogenic diet induces oxidative stress leading to generation of free radicals. These free radicals cause the peroxidation of lipids especially LDL there by producing oxidized LDL which is taken up by the endothelial cells and macrophages and thus elevating atherosclerotic process. The antioxidant enzymes like catalase is the first line defensive antioxidants against free radicals³¹.In hyperlipidemic condition, the levels of lipids and phospholipids are very high leading to increased production of arachindonic acid and prostaglandins. Oxy radicals are produced during the production of prostaglandins. MDA is the end product of lipid peroxidation. Therefore measurement of MDA gives an indirect evidence of LDL oxidation. From the results it is investigated that MEOOL treated groups have higher levels of catalase, reduced GSH and decreased levels of MDA indicating its efficacy to reduce LDL-oxidation³². It is already reported that flavonoids are potent natural antioxidants and also having significant increased SOD and catalase activities³³. High fat diet brings remarkable changes in the antioxidant defense mechanism against the process of lipid peroxidaton. A number of studies have investigated the ability of flavonoid rich fraction to acts as antioxidants and antihyperlipidemics. The elevated levels of catalase, reduced GSH and the declined levels of MDA with treatment of O. obtusata treatment could be due to the influence of flavonoids. Thus it can be concluded that the antihyperlipidemic and antioxidant activity of methanolic leaf extract of Ochna obtusata may be due to the presence of these phytoconstitents. The several reported studies of Ochna obtusata also evidenced the similar findings.^15-19

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Received on 25.10.2014 Modified on 21.11.2014

Res. J. Pharmacology & P’dynamics. 7(1): Jan.-Mar. 2015; Page 01-04

DOI: 10.5958/2321-5836.2015.00001.4