Therapeutic potential of ethanolic extract of Solanum nigrum for lipofundin-induced hyperlipidemia in Rabbits

Attenuation of hyperlipidemia with extract of medicinally important plant and reversal of lipid disorders is clinically very important. The aim of present study was to identify the therapeutic potential of Solanum nigrum ethanolic extract in lowering the cholesterol level in lipofundin treated hyperlipidimic rabbits in vivo. 20% lipofundin was used to induce hyperlipidmia in rabbits @ 2 ml/kg body weight through slow intravenous administration in the marginal ear vein for 7 days. For next 2 weeks the control group was placed at standard diet, whereas the test group was given the ethanolic crude extract of Solanum nigrum at the dose of 300 mg/kg body weight. On the completion of treatment blood samples were collected from both control and test groups and were analyzed for the lipid profile values. It was observed in the test group after treatment with ethanolic extract of S. nigrum, the raised level of serum total cholesterol, triglyceride, highdensity lipoprotein, low-density lipoprotein were decreased towards normal values. Thus the present study demonstrated that S. nigrum possessed significant anti-hyper lipidimic activity in lipofundin induced hyperlipidimic rabbits.


Introduction
Hyperlipidemia, including high serum total cholesterol (TC) and low-density lipoprotein cholesterol (LDL-C) is an independent menace to atherosclerosis which can lead to coronary heart disease and ischemic stroke [1, 2].Therefore, prevention and treatment of hyperlipidemia is very important.Commonly hyperlipidemia is treated with lipid-lowering drugs such as Hydroxy methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors (statins), phenoxy aromatic acids (fibrates), nicotinic acid and its derivatives.All of the main lipidlowering drugs including statins and fibrates have certain side effects.Moreover, the combination of statins and fibrates has a high risk of side effects such as muscle damage and liver damage.The use of plant based medicines has been increasing gradually world-wide in recent years, as well as the search for new phytochemicals that could be potentially developed as useful drugs for the treatment of various diseases.More than 2000 plants are used in traditional herbal or alternative medical systems and some may provide relief to patients with cardiovascular diseases, especially those with hyperlipidemia and ischemic heart disease.Solanum nigrum, a weed, also known as black-nightshade is a member of Solanaceae family found in the dry parts of Pakistan, India and other parts of the world.

Plants materials
Solanum nigrum L. plant was collected from rural area from the vicinity of Jhelum River, District Jhelum, Pakistan.The herbs were shade dried at room temperature and at normal humidity level.After complete drying, the aerial parts of the herb were spliced carefully and then pulverized into powder.The powder was stored in air tight amber glass bottle.

Extract preparation
About 200gm of dried powder of aerial parts of Solanum nigrum was extracted by maceration with 1000ml of absolute ethyl alcohol and kept at room temperature for 10 days.The macerate was shaken by manual stirring 3 to 4 times daily.In order to remove the vegetative plant material, the soaked solution was passed through dried muslin cloth and the filtrate was obtained.The pooled extract of plant was concentrated in vacuum by using a rotary evaporator.A semi-solid material was obtained which was air dried and weighed.The extract thus obtained was stored in an air tight closed container.

Animal used
A local breed of rabbits weighing between 1 to 2 kg was used in the present study.The animals were housed in specially designed well ventilated animal cage and the animals had free access to water and food.A 12 Hr light and dark cycle was maintained.All the experiments on animals were performed in compliance with the guidelines of NIH.

Induction of hyperlipidemia
Hyperlipidemia was induced in all the rabbits with intravenous administration of lipofundin 20% as previously reported [13], Briefly, lipofundin was slowly administered by intravenous injection over approximately 10 minutes @ 2 ml/kg body weight of the animal.This procedure is repeated for seven consecutive days.

Experimental design
Animals were divided in following three groups (n=5): 1.Control group: Lipofundin induced + No treatment 2. SN Treatment group: Lipofundin induced + Treated with S. nigrum L. extract All the rabbits were weighed and tagged separately.To establish baseline values of different markers of lipid profile, about 3ml blood samples from marginal ear vein of overnight fasted rabbits of both groups were collected and immediately placed in EDTA tubes.After centrifugation, the serum was separated and stored at -20 °C for further analysis.Hyperlipidemia was induced in all the rabbits..For next two weeks the control group was placed at standard diet whereas the treatment groups were given the semisolid plant extract at the dose of 300mg/kg body weight.At the end of treatment that is at 23 rd day, the blood samples of each rabbit were taken for lipid profile.Computerized Metro Lab 1600 DR Semi Automated Chemistry Analyzer along with the commercial kits available for determining serum total cholesterol, serum triglycerides, high density lipoprotein cholesterol and low density lipoprotein cholesterol was used to determine lipid profiles.

Statistical analysis
All the data collected was transferred to Microsoft Excel 2010 sheet and was tabulated.All the graphs were plotted in Microsoft Excel 2010.The values were calculated for their Mean, Standard Error of Mean and Standard Deviation.P-value was calculated for each group to measure the degree of change occurring in parameters observed, for each animal of control group and test group as well.

Measurement of total cholesterol
Measurement of total cholesterol (TC) is an important biomarker for the evaluation of hyperlipidemia and it was measured in this study to evaluate anti-hyperlipidemic potential of S. nigrum ethanolic extract.The results showed TC values of normal (baseline), control group at day1, control group at day23 and treatment group as 52.4±3.2, 76±3.7,71.4±4.4 and 48.6±3.1 respectively as shown in figure 1.There was a decrease in the ethanolic extract of S. nigrum in serumTG levels of treated group as compared to the control group.

Measurement of high-density lipoproteincholesterol
The high-density lipoprotein-cholesterol is another important biomarker of hyperlipidemia.The results showed HDL-C values of normal (baseline), control group at day1, control group at day23 and treatment group 15.8±0.8,25±1.1, 22.8±0.9 and 14.4±1.3respectively as shown in figure 3. A significant decrease in high-density lipoprotein-cholesterol was observed in treated group as compared to the control group.

Measurement of low-density lipoproteincholesterol
The results showed LDL-C values of normal (baseline), control group at day1, control group at day23 and treatment group 25.2±2.1,34±2.6,30.8±2.3 and 22.2±1.2respectively as shown in figure 4. A significant decrease in low-density lipoprotein-cholesterol was observed in treated group as compared to the control group.

Discussion
The present study showed that lipofundin 20% administration produces hyperlipidemia in rabbits and S.nigrum ethanolic extract supplementation decreased hyperlipidemia to the normal levels suggesting the therapeutic potential of S.nigrum ethanolic extract for hyperlipidemia.S. nigrum aerial parts ethanolic extract resulted in significant decrease in levels of biomarkers such as total cholesterol, troglycerides, HDLcholesterol and LDL-cholesterol.Antioxidant activity of the extract probably has this protective effect by blocking the free radicals production and lipid peroxidation inhibition.The anti-oxidant activity of drugs or other products is due to reduction of oxidative damage by inhibiting the generation of free radicals, ultimately leading to inhibition of lipid peroxidation [3].Thus, it can be concluded from the present study that S. nigrum aerial parts ethanolic extract has anti-oxidant activity as it neutralized the oxidant biomolecules involved in oxidative damage to lipid membranes.S. nigrum extract has been reported as a potent antioxidant against various diseases and oxidative stress induced degenerative processes [14, 15].This antioxidant potential of the extract is might be due to the presence of excess phenolic compounds like flavonoids, Vitamin C, steroids and β-carotene.Strong scavenging activity of glycoprotein from S. nigrum against lipid peroxidation peroxyl radicals has been studied

Figure 1 .
Figure 1.Effect of treatment of Solanum nigrum extract on total Cholesterol levels.Concentrations of total Cholesterol of normal, control and treatment groups are shown.There is a marked decrease in total Cholesterol of treatment group as compared to control group.All values are expressed as Mean ± SEM Thus, the ethanolic extract of S. nigrum has decreased TC levels of treated group significantly as compared to the control group.Measurement of serum triglycerides To assess the antihyperlipidemic activity of ethanolic extract of S. nigrum, the serum TG values were measured as a part of lipid profile of different groups.The serum TG values of normal (baseline), control group at day1, control group at day23 and treatment group were 48±2.5, 65.8±3.1,61.4±2.8 and 49.8±3.0respectively as shown in figure 2.

Figure 2 .
Figure 2. Potential of Solanum nigrum extract treatment for triglycerides.The values of triglycerides of normal, control and treatment groups are shown.A relatively lower value of triglycerides can be seen in treatment group as compared to control group.All values are expressed as Mean ± SEM

Figure 3 .
Figure 3.Effect of Solanum nigrum extract treatment on HDL-C levels.Concentrations of HDL-C of normal, control and treatment groups are shown.There is a significant decrease in HDL-C in treatment group as compared to control group.All values are expressed as Mean ± SEM.

Figure 4 .
Figure 4. Potential of Solanum nigrum extract treatment for LDL-C.The values of LDL-C of normal, control and treatment groups are shown.There is no prominent difference between treatment group and control group.All values are expressed as Mean ± SEM.
medicine research one of the most promising fields in biomedical research.There is a growing concern to find alternative sources of drugs to cure different diseases.Plant extracts have emerged as a new therapeutic tool in alternative medicine.In vivo study demonstrated reversal of hyperglipidemia in Solanum nigrum extract treated rabbits.Hydroxy methylglutaryl coenzyme A. SEM: Standard error of the mean, NIH: National health institute, USA, FFA: Free fatty acid.Authors' contributions Conceived and designed the experiments: I Ali, Performed the experiments: W Ahmed, Analyzed the data: I Ali, Contributed reagents/ materials/ analysis tools: M Tariq, R Asghar & MA Hussain, Wrote the paper: I Ali.References 1. Malloy MJ & Kane JP (2012).Hyperlipidemia and cardiovascular disease.Curr Opin Lipidol 23(6): 591-92.2. Liu J, Zhao D & Qiu L (2000).Association of low density iipoprotein cholesterol with cardiovascular disease in a prospective