Collection and identification of plant materials
The whole plant of Mucuna pruriens (Linn), were collected from Neiyur dam, Kanyakumari District of Tamil Nadu, India and the whole plant of Ionidium suffruticosum (Ging) were collected from Kilikulam, Tirunelveli District of Tamil Nadu, India. The taxonomic identification was made from Botanical Survey of Medicinal Plants Unit Siddha, Government of India, Palayamkottai. The whole plant of Mucuna pruriens (Linn) and Ionidium suffruticosum (Ging) were dried under shade, segregated, pulverized by a mechanical grinder and passed through a 40 mesh sieve. The powdered plant materials were stored in an airtight container.
Chemicals
All the chemicals used in the study were of analytical grade, procured from the credible concerns e.g.: Sigma, Merck and Qualigens. Atorvastatin was provided as a generous gift sample by Ranbaxy Pharmaceuticals, India.
Preparation of various extracts from Mucuna pruriens and ionidium suffruticosum
The whole plant of Mucuna pruriens (Linn) and Ionidium suffruticosum (Ging) were dried in shade and powdered. The powdered plant materials were successively extracted with petroleum ether (40-60°C) by hot continuous percolation method in Soxhlet apparatus[10] for 24 hrs. Then the marc was dried and then subjected to ethyl acetate (76-78°C) for 24 hrs, then marc was dried and then it was subjected to methanol (80°C) for 24 hrs. The solvent from the extracts was recovered under reduced pressure using rotary evaporator and subjected to freeze drying in a lyophilizer till dry powder was obtained.
Isolation of flavone from methanol extract of Mucuna pruriens
The methanol extract of Mucuna pruriens was subjected to column chromatographic separation using normal phase silica gel column. The dark brown solid (20 g methanol extract of Mucuna pruriens) was adsorbed on silica gel (20 g) and transferred to a column of silica gel (200g equilibrated with benzene). Flavone (265mg) was eluted with ethyl acetate: methanol, 80:20 v/v from methanol extract of Mucuna pruriens.
Isolation of coumarin from methanolic extract of ionidium suffruticosum
The methanolic extract of Ionidium suffruticosum was subjected to column chromatographic separation using normal phase silica gel column. The dark brown solid (20 g methanolic extract of Ionidium suffruticosum) was adsorbed on silica gel (20 g) and transferred to a column of silica gel (200g equilibrated with benzene). The Coumarin derivative (245 mg) was eluted with benzene: Chloroform 70:30, v/v from methanolic extract of Ionidium suffruticosum.
Experimental animals
Healthy male Wistar rats (weighing 120-150g) were procured from Central Animal House, Rajah Muthiah Medical College, Annamalai University. The animals were kept in cages, 2 per cage, with relative humidity (55%) in a 12 hour light/dark cycle at 25±2°C. They were given access to water and a commercial diet ad libitum. The experiment were carried out as per the guidelines of Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), New Delhi, India, and approved by the Institutional Animal Ethics Committee (IAEC), Annamalai University (Approved number: 160/1999/CPCSEA/745).
Acute toxicity studies
Acute oral toxicity study was performed as per Organization and Economic Cooperation and Development (OECD) guidelines 423 adopted on 17th December 2001 received from Committee for the purpose of Control and Supervision of Experimental Animals (CPCSEA). The rats were fed with flavone from Mucuna pruriens and Coumarin derivatives from Ionidium suffruticosum suspended in 1% gum acacia at the dose of 100mg/kg body weight. The animals were observed individually every 30 minutes after dosing the first 24hrs and thereafter daily for a total of 14 days. The time at which signs of toxicity appear and disappear was observed systematically and recorded for each animal.
Experimental induction of hyperlipidemia
High fat diet was prepared by mixing Wheat flour 20.5%, roasted bengal gram 52.6%, skimmed milk powder 5%, casein 4%, refined oil 4%, coconut oil 9%, salt mixture with starch 4% and vitamin & choline mixture 0.5%, cholesterol 0.4%[11]. The diet was placed in the cage carefully and was administered for 14 days[12].
Protocol for antihyperlipidemic activity
In the experiment a total number of 36 rats were used. The rats were divided into five groups of six each. Group I: control, Group II: High Fat Diet (HFD), Group III: HFD plus Flavone (M. pruriens) at the dose of 10mg/kg b.wt, Group IV: HFD plus Coumarin (I. suffruticosum) at the dose of 10mg/kg b.wt, Group V: HFD + standard drug atorvastatin (1.2 mg/kg b.wt). The drugs were suspended in 2% tween 80[13] separately and fed to the respective rats by oral intubation. At the end of the study all the rats were sacrificed by cervical dislocation after overnight fasting. Just before sacrifice, blood was collected from the retro-orbital sinus plexus under mild ether anaesthesia and blood sample collected in heparinised tubes and plasma was separated. Liver, heart and aorta were cleared of adhering fat, weighed accurately and used for the preparation of homogenate. Animals were given enough care as per the Animal Ethical Committee’s recommendations.
Biochemical analysis
Plasma samples were analyzed for total cholesterol, HDL-cholesterol and triglycerides using Boehringer Mannheim kits by Erba Smart Lab analyzer USA. LDL-cholesterol and VLDL-cholesterol were calculated by using Friedwald method[14]. Ester cholesterol[15] and free cholesterol[15] were analyzed by using digitonin. Portions of liver, heart and aorta tissues were blotted, weighed and homogenized with methanol (3 volumes) and the lipid extracts were obtained by the method of Folch et al. (1957)[16]. Extracts were used for the estimation of ester cholesterol and free cholesterol, triglycerides[17], and phospholipids[18]. The atherogenic Index was calculated by using the following formula.
(1)
Statistical analysis
Results were expressed as mean ± SE of 6 rats in each group. The data were also analysed by one way analysis of variance (ANOVA) followed by Dunnet’s t-test. ‘P, value less than 0.05 is considered significant.
