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The fruits extract of Prosopis cineria, herb has been used traditionally in India for medicinal purposes. The plant has been reported to treat hyperglycemia and associated hyperlipidemia. Hyperlipidemia and oxidative stress hastens coronary arterial diseases and develops atherosclerotic lesions. This study was to evaluate the hypolipidemic and antioxidant potential of Prosopis cineria, in hyperlipidaemic rats. In single iv (200 mg/kg b.w.) Triton WR 1339 induced hyperlipidaemic rats, it showed persistent elevated levels of serum total cholesterol (TC), low-density lipoprotein cholesterol (LDL-c), tri-glyceride (TG), and decreased levels of high-density lipoprotein cholesterol (HDL-c). The ethanolic extract of Prosopis cineria fruits (Et-PCF) (200, 400, 600 mg/kg b.w./day) was administered to normal and hyperlipidemic rats. Serum and liver tissue were analysed for lipid proï¬le, lipid peroxidation products, antioxidants enzymes and the activity was compared to the standard, Simvastatin (4 mg/kg/b.w.). Parameters during hyperlipidemia reverted back to near normal values after Et-PCF treatment or standard drug Simvastatin. The current study provides a strong indication that Et-PCF has a beneï¬cial effect in treating hyperlipidemia and might provide protection against cardiovascular diseases.
Keywords: hypolipidemic activity, hyperlipidemia, Prosopis cineria, Triton WR 1339, Antioxidant activity.
Hypercholesterolemia is often associated with obesity, diabetes mellitus and hypertension, each and all contribute to elevated cardiovascular mortality (Who, 1994). There is a general consent that these metabolic disorders share hyperinsulinemia and insulin resistance as a common link (Modan et al., 1985; Paolissa et al., 1992) leading to both micro- and macro-angiopathies (Bennani-Kabchi, 1999). During the last two decades, statins have been in clinical use as selective inhibitors of the key enzyme, Hydroxy methyl glutaryl Coenzyme-A (HMG-Co A)-reductase that determines the rate of cholesterol synthesis in hepatocytes. In population studies, statins have been evidenced to reduce cholesterol levels by about 25% and mortality due to myocardial infarction by about 42 percent. Atherogenesis is a multifactor process that includes oxidative modification of LDL which triggers pathological events through multiple pathways leading to atherosclerosis (Berliner and Heinecke, 1996). Research in recent years has been directed towards dietary antioxidants from plant-derived sources to normalize the elevated levels of cholesterol atherogenous fractions, mainly LDL, and of glucose in an attempt to reduce the cardiovascular risk (Gingliano, 2000; Anderson et al., 2001).
Nowadays most widely used drugs for the management of hyperlipidemia are "Statins" which suffer from limitations like, intolerance and adverse effects, Statins are only partially effective in lowering of cholesterol levels & only up to maximum 40% risk reduction & finally "Cost". Therefore, there is an urgent need to have drugs with lipid lowering and antioxidant activities with no side effects and natural products are the best claimed option. Several medicinal plant have been found to possess antioxidant properties, lipid lowering properties had beneficial effects can be used to control hyperlipidemia and related complications in patients.
Prosopis cineraria (Fabaceae), locally known as 'Khejri', has a long history of use in herbal medicine in greater parts of India, Burma and Sri Lanka. Since all parts of the tree are useful, it is called 'Kalptaru' (Burkart, 1976). Bark of the tree is used in the treatment of asthma, bronchitis, dysentery, leucoderma, leprosy, muscle tremors and piles (Shalini, 1997; Toky, 1999). Several bioactive compounds such as flavonoids, alkaloids, phenolic contents, free amino acids, spicigerin, prosogerin A,B,C,D, b-sitosterol and vitamins have been isolated from different parts of the plant (Purohit et al., 1979; Rhoades, 1979). But till date, the hypolipidemic and antioxidant potential of P. cineraria has not been scientifically evaluated. Hence, in the present study, the effect of oral treatment with P. cineraria leaves extract with a dose dependent manner on hyperlipidemia and resultant oxidative stress was investigated by evaluating its hypolipidemic activity in triton induced hyperlipidaemic rats along with the antioxidative properties.
Material and Methods
The study was conducted on Sprague Dawley rats weighing 180-200 g and were housed in polypropylene cages and maintained under standard conditions (12 hrs light and dark cycles, at 25 Â± 30C and 35-60 % humidity). Standard palletized feed and tap water were provided ad libitum. The study was approved by Institutional Animal Ethical Committee of R. C. Patel Institute of pharmaceutical Education and Research, Shirpur, India, registered under CPCSEA, India (Registration No.651/02/C/CPCSEA).
Triton WR 1339 (Loba- chemie Ltd), cholesterol, triglyceride, HDL kits (BEACON Diagnostic Pvt. Ltd), ethanol were used in this study.
Preparation of Prosopis cineria extract
The plant material was collected from Satpuda region, Maharastra, India. At the time of collection whole plant including fruits, leaves, stem and flower were collected and authentication was made from Department of Botany S.S.V.P.S's College of science; Dhule (Maharashtra). A voucher specimen has been kept in department for further reference.
Fruits of the plant were removed and dried under shade in a room. After 10 days of drying fruits were pulverized and sieved with a 40 # sieve. Prosopis cineria powder was extracted in ethanol by using Soxhlet extractor. The extract was concentrated in vacuum evaporator below 40Â°C.
Phytochemical Investigation of Extract:
Different chemical constituents present in ethanolic extract were subjected to the tests by Kokate (1994).
Acute oral toxicity study:
Albino female mice weighing 25-30 gm were used for acute oral toxicity study. Acute toxicity study was carried out as per "Up & Down" method. The test drug was found to be safe up to the dose 5000 mg/kg body weight hence 1/10th of dose was taken as an effective dose (500 mg/kg). The doses were selected 200 mg/kg, 400 mg/kg and 600 mg/kg.
Triton WR 1339 induced hyperlipidemia:
Sprague Dawley rats were selected for the experimental model. The rats were fed with the standard diet and water ad libitum before the experiment. Room temperature was maintained between 20-30oC along with the humidity 40-60%. The weight of selected rats were the 180-200gm. 36 rats were divided into 6 groups each group contain 6 rats. According to the body weight of individual rat 200 mg/kg Triton was injected intraperitoneally (Kourounakis et al, 2002)
Group 1 - Normal saline 1ml/animal.
Group 2 - Triton WR 1339 (200 mg/kg) B.W
Group 3 - Triton WR 1339 (200 mg/kg) B.W. + Simvastatin 4 mg/kg orally B.W.
Group 4 - Triton WR 1339 (200 mg/kg) B.W. + Et-PCF 200 mg/kg B.W.
Group 5 - Triton WR 1339 (200 mg/kg) B.W. + Et-PCF 400 mg/kg B.W.
Group 6 - Triton WR 1339 (200 mg/kg) B.W. + Et-PCF 600 mg/kg B.W.
Group 2nd received Triton solution 200 mg/kg intraperitoneally and vehicle while Group 4th, 5th and 6th received Triton oil solution 200 mg/kg along with extract 200 mg/kg, 400 mg/kg and 600 mg/kg respectively. Group III received Simvastatin the standard drug in the dose of 4 mg/kg. Group 1st normal rats were taken for the analysis. In treatment groups the extract was given orally 30 minutes before the Triton injection. Blood was collected from all the rats before Triton injection at 0 hours and 24 hours after Triton injection (Hemlata et al., 2006).
The mixture containing 0.5ml of homogenate, 1ml of 0.15 ml KCl and 0.5ml of different concentrations of drug extract were prepared. Lipid peroxidation was initiated by adding 100Âµlof 1mM ferric chloride. The reaction mixture was incubated for 30min.at 37Â°C. After incubation 2ml of ice-cold 0.25N HCl containing 15% TCA & 0.38% TBA & 0.2ml of 0.05% BHT was added in the reaction mixture. This reaction mixture was heated for 60min at 80Â°C, cooled and centrifuged at 5000-x g for 15min. The absorbance of the supernatant was measured at 532nm against a blank, which contained all reagents except liver homogenate and drug. Identical experiments were performed to determine the normal (without drug and ferric chloride) and induced (without drug) lipid peroxidation leveling the tissue. The % of anti-lipid peroxidation effect (%ALP) was calculated by following formula (Umamaheswari et al., 2007).
Ferric chloride O.D. - Sample O.D
Ferric chloride O.D. - Normal O.D.
Reducing power assay:
Reducing power of ethanolic extract of Prosopis cineria was determined on the ability of antioxidants to form colored complex with potassium ferricyanide. Different concentration of the extract (10, 20, 40, 60, 80Âµg/ml) were mixed with 2.5ml phosphate buffer (pH 6.6) and 2.5ml potassium ferricyanide (1%). The mixture was incubated at 50Â°C for 20 min. 2.5ml TCA (10%) was added to it and centrifuged at 3000rpm for 10min. 2.5ml of supernatant was mixed with 2.5ml of water and 0.5ml of FeCl3 (0.1%) were added to it and absorbance was measured at 700nm. Increased absorbance of the reaction mixture indicated increased reducing power (Oyaizu, 1986).
The phytochemical screening of ethanolic extract showed positive test for Flavonoids, Tannins, Saponins, Phenolic compounds and Protein etc
In vivo hypolipidemic effect of Et-PCF on lipid proï¬le
The effect of Et-PCF on serum lipid proï¬le in control and experimental rats at different doses were shown in Table 1. The acute injection of TWR caused a signiï¬cant increase in lipid levels when compared with Group I rats. Group VI showed a signiï¬cant (P<0.01) reduction in cholesterol (26.67%), triglyceride (26.52%), LDL (54.93%) and VLDL (26.60%) levels, where as signiï¬cant increase (P<0.05) was observed in HDL (11.53%) after treatment of Et-PCF when compared to Group II. The AI was also signiï¬cantly (P < 0.01) decreased (53.34%) in Group VI after treatment. We observed dose dependent reduction of lipid proï¬le and maximum antihyperlipidemic effect (P<0.01) was in Group VI (600 mg/kg/b.w. of Et-PCF administered) when compared to Group II. Simvastatin signiï¬cantly reduced the lipid parameters; Et-PCF however, was more effective. There was no signiï¬cant difference in serum lipid proï¬les, in Group IV rats.
Table No: 1
Effect of Ethanolic Extract of Prosopis cineria on lipid profile in triton induced Hyperlipidemia:
Normal saline 1ml/animal.
Triton only 200 mg/kg
Values are expressed as mean Â±SEM n=6
Data was analyzed by one way ANOVA followed by Dunnet's test
P<0.01=very significant ** P<0.05=significant* compared with Triton treated rats (Group II).
In the present study ethanol extract showed potent inhibition of lipid peroxidation induced by Iron/ADP/Ascorbate complex in rat liver homogenate, IC50 value was 58.33 Î¼g/ml. It showed dose dependent inhibition of lipid peroxidation. Standard ascorbic acid showed IC50 value 50.92 Î¼g/ml (Fig. 1).
The measurement of reductive ability was done by Fe3+-Fe2+ transformation in the presence of ethanol extract and standard antioxidant, ascorbic acid (Shimazaki et al., 1994). The reducing power is associated with antioxidant activity. As shown in Fig. 2, ethanolic extract showed reducing power comparable with standard at higher concentration i.e. at 80 Î¼g/ml.
Fig. 1 Effect of Prosopis cineria on lipid peroxidation activity by using rat liver homogenate:
Fig. 2 Effect of Prosopis cineria on in-vitro reducing power assay:
The Triton WR-1339, a non-ionic detergent has been widely used to block the clearance of triglyceride-rich lipoproteins by inhibiting the lipoprotein lipase and to induce an acute hyperlipidemia in experimental animals. This model was used for a number of different objectives including studies of lipid metabolism and screening for natural and chemical hypolipidemic drugs. Hence, many plants for example Prosopis cineria have been investigated for their acute hypolipidemic activity in Triton WR-1339-induced hyperlipidaemic animals. The highest levels of plasma triglycerides and total cholesterol were reached 20 h after Triton administration.
In the present study we have to estimated the effect of ethanolic extract of P. cineria on experimentally induced hyperlipidemia in rats. There was significant increase in the levels of serum cholesterol, triglyceride and LDL-c with significant decrease in level of HDL-c in experimental animals. Elevated level of blood cholesterol especially LDL-c is a known major risk factor for CHD whereas HDL-c is cardio protective. Treatment with ethanolic extract, at three different doses (200, 400 and 600mg/kg) significantly decreased the levels of total cholesterol and LDL-c as compare to the Triton treated (control) group and compared to standard drug Simvastatin. Atherogenic index indicates the deposition of foam cells or plaque or fatty infiltration or lipids in heart, coronaries, aorta, liver and kidneys. The higher the atherogenic index, the higher is the risk of oxidative damage to above organs. (Jain et al., 2010). Atherogenic index was significantly reduced in the P. cineria treated groups. Since the results of the study indicated that extract has beneficial effect on lipid profile.
In present antioxidant study showed that the Prosopis cineria fruits extract possessed strong free radical scavenging activity and antioxidant activity. Polyphenols other than vitamin E have been known to exert powerful antioxidant effect in vitro. They inhibit lipid peroxidation by acting as chain-breaking peroxyl-radical scavengers, and can protect LDL from oxidation (Pilaipark et al., 2008). Phytochemical investigation showed presence of polyphenols, in Prosopis cineria fruits extract, therefore, the antioxidant effects could be due to presence of Phenolic components in the extract (Pilaipark et al., 2008).