Comparative evaluation of flavone from Mucuna pruriens and coumarin from Ionidium suffruticosum for hypolipidemic activity in rats fed with high Fat diet
© Kumar and Muthu; licensee BioMed Central Ltd. 2012
Received: 6 July 2012
Accepted: 29 September 2012
Published: 2 October 2012
The objective of the study is a comparative evaluation of flavone isolated from Mucuna pruriens and coumarin isolated from Ionidium suffruticosum was assessed for the hypolipidemic activity in rats fed with high fat diet. The acute toxicity study was found that flavone (M.pruriens) and coumarin (I.suffruticosum) are safe up to 100mg/kg, so one tenth of this dose (10mg/kg) was consider as a evaluation dose. High fat diet group of rats showed significant (p<0.001) elevation in plasma total and LDL-cholesterol, triglycerides and phospholipids. Administration of flavone (M. pruriens) and coumarin isolated from (I.suffruticosum) at the dose of 10mg/kg b.wt/day along with high fat diet significantly (p<0.001) prevented the rise in the plasma total and LDL-cholesterol, triglycerides and phospholipids than that of other extracts. However, treatment of coumarin isolated from (I.suffruticosum) had showed more cardio protective effect against hyperlipidemia than that of flavone (M.pruriens).
Cardiovascular disease is a major problem worldwide. The World Health Organization estimates that this disease is responsible for the deaths of approximately 30,000 people each day. Hyperlipidaemia is characterized by elevated serum total cholesterol, low-density lipoprotein cholesterol, very low density lipoprotein cholesterol and decreased high-density lipoprotein cholesterol levels. As reported in literature synthetic drugs may be having serious side effects. Statins may also be associated with some other rarely occurring side effects like nausea, abdominal pain, dyspepsia, diarrhoea or constipation and flatulence. Medicinal plants, on the other hand, have been reported safer as compared topharmaceutically derived remedies.
Mucuna pruriens Linn belongs to the family fabaceae, traditionally in India the seeds of Mucuna pruriens are used as a tonic and aphrodisiac for male virility. It has been reported to be antidiabetic, analgesic and anti-inflammatory. Its different preparations (from seeds) are used for the management of several free radical-mediated diseases such as ageing, rheumatoid arthritis, diabetes, atherosclerosis, male infertility and nervous disorders. Ionidium suffruticosum (Ging.) it belongs to the family Violaceae, it is widely used as traditional healers for the treatment of diseases like diabetes, male sterility, urinary tract infections and water retention. Hence, the attempt is made for the comparative evaluation of flavone from Mucuna pruriens and Coumarin isolated from Ionidium suffruticosum for hypolipidemic activity in rats fed with high fat diet.
Materials and methods
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.
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 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.
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%. The diet was placed in the cage carefully and was administered for 14 days.
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 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.
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.
Results and discussion
Balance between synthesis and degradation of biological tissues is maintained by Lipid metabolism. Development of hyperlipidemia disease is a complicated process involving accumulation of lipid containing particles in the walls of coronary arteries & other major arteries within the body. A high-fat diet causes cholesterol levels to increase in susceptible people, which leads to obesity.
Average Body weight changes in control and experimental rats
Initial Weight (g)
Final Weight (g)
Average Body weight gain (g)
Effect of flavone from Mucuna pruriens and coumarin from Ionidium suffruticosum on plasma lipid profile in control and experimental rats
Total cholesterol (mg/dl)
Free cholesterol (mg/dl)
Ester cholesterol (mg/dl)
Effect of flavone from Mucuna pruriens and coumarin from Ionidium suffruticosum on tissues ester cholesterol profile in control and experimental rats
Ester cholesterol (mg/g tissue)
0.67 ± 0.02b*
Effect of flavone from Mucuna pruriens and coumarin from Ionidium suffruticosum on tissues free cholesterol profile in control and experimental rats
Free cholesterol (mg/g tissue)
Effect of flavone from Mucuna pruriens and coumarin from Ionidium suffruticosum on tissues Triglyceride level in control and experimental rats
Triglyceride (mg/g tissue)
15.47 ± 0.12a*,b*
12.08 ± 0.12a*,b*
12.15 ± 0.01a*,b*
Effect of flavone from Mucuna pruriens and coumarin from Ionidium suffruticosum on tissues Phospholipids level in control and experimental rats
Phospholipids (mg/g tissue)
17.81 ± 0.01a*,b*
Atherogenic Index (AI) indicates the deposition of foam cells or plaque or fatty infiltration or lipids in heart, coronaries, aorta, liver and kidney. The higher the AI, higher is the risk of above organs for oxidative damage. As shown in Table2, the atherogenic index (TG/HDL-C ratio) used to predict risk of CHD and marker of small, dense LDL-C (an atherogenic lipoprotein)[28, 29] were significantly reduced by the flavone from M.pruriens and coumarin from I.suffruticosum, indicating the beneficial effect of flavone and coumarin in cardiovascular diseases.
Effect of flavone from Mucuna pruriens and coumarin from Ionidium suffruticosum on plasma lipoprotein in control and experimental rats
HDL cholesterol (mg/dl)
LDL cholesterol (mg/dl)
VLDL cholesterol (mg/dl)
As shown in Table7, the elevated levels of LDL and VLDL-cholesterol in rats fed with HFD (group II) was significant (P<0.001) in comparison with control rats (group I). High cholesterol diet increased serum cholesterol and LDL-C level significantly. Clinical and epidemiological studies have proved that individuals with elevated LDL show an increased risk for cardiovascular diseases. Treatment of flavone from M.pruriens (Group III) and coumarin from I.suffruticosum (Group IV) markedly reduced the level of plasma LDL-cholesterol and VLDL-cholesterol when compared to HFD rats (group II). In comparison of flavone from Mucuna pruriens (Group III) and coumarin from Ionidium suffruticosum (Group IV) with HFD (Group II) rats, the coumarin from Ionidium suffruticosum (Group IV) was showed significant reduction on both LDL-cholesterol and VLDL-cholesterol than that of group III rats. Recent research has revealed that a 4-5% decrease in LDL-cholesterol results in a 5-10% decrease in the occurrence of coronary heart disease (CHD).
On the basis of the present investigation was observed as the Coumarin derivatives isolated from Ionidium suffruticosum had showed a better antihyperlipidemic activity in comparison with flavone isolated from Mucuna pruriens. The Coumarin derivatives isolated from Ionidium suffruticosum is beneficial in preventing hypercholesterolemic atherosclerosis and reducing risk factors for coronary artery disease.
We are thanking to the Department of Pharmacy, Annamalai University for providing the necessary facilities for carrying out the research work.
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