INTRODUCTION:

Increased plasma lipid levels, mainly total cholesterol (TC), triglycerides (TG) and low density lipoproteins (LDL) along with decrease in high density lipoproteins (HDL) are known to cause hyperlipidemia which is core in initiation and progression of arteriosclerosis impasse. Therefore, prime consideration in therapy for hyperlipidemia and arteriosclerosis is to enervate the elevated plasma levels of TC, TG and LDL along with increase in HDL lipids levels¹.

However, despite such interesting health virtue of this plant a perusal of literature reveals that no scientific study has been carried out to screen the pharmacological activity of the extracts from the same in terms of their antihyperlipidemic or hypolipidemic effects. This investigation is an attempt to find out the possible effects of four different extracts from Rhynchosia beddomei whole plant in progesterone and MSG induced hyperlipidemic rats as well as in normocholesteremic rats, by measuring the changes in different parameters of blood-lipid profile, viz.TC, TG, LDL and HDL Rats administered progestin formulation (0.667 mg of synthetic progestin/kg body wt) showed increased hepatic cholesterogenesis, as evidenced by an increased activity of HMG-CoA reductase and increased incorporation of labeled acetate into liver cholesterol. Hepatic degradation of cholesterol to bile acids, however, was decreased. There was increased release of lipoproteins into the circulation but their clearance from the circulation was lower as revealed by a decreased activity of lipoprotein lipase of the extra-hepatic tissues. Activity of plasma LCAT, which is involved in the transport of cholesterol from the tissues, was also decreased. The increase in serum and aortic cholesterol levels, increase in LDL cholesterol and decrease in HDL cholesterol in rats administered estrogen-progestin formulation suggest that prolonged administration of this formulation may predispose towards atherosclerosis.² Oral ingestion of monosodium glutamate (MSG) to chronic alcoholic adult male mice at dose levels of 4 and 8 mg/g body weight for seven consecutive days caused a significant increase in lipid fractions, lipid peroxidation, xanthine oxidase, whereas the levels of superoxide dismutase, catalase, glutathione, and its metabolizing enzymes like glutathione peroxidase and glutathione reductase were significantly decreased in the arterial tissue. These observations suggested that ingestion of MSG to chronic alcoholic animals had no beneficial effect and thereby, could act as an additional factor for the initiation of atherosclerosis³.

The plant was collected during the march 2014 from Tirumalla forest area of Chitoor district of Andhra Pradesh India. The plant was authenticated by Dr. Madhava Chetty, Department of Botany, Sri Venkateshwara University, Tirupati and voucher specimen of the plant was preserved at institute herbarium library. The coarsely powdered plant material was then successively extracted in a Soxhlet with Methanol, Chloroform, and 90% (v/v) alcohol and then cold macerated with water to obtain the respective extracts viz; MET, CHE, ALE and AQE. All the extracts were filtered, distilled and the last traces of solvents were removed in vacuum The yields obtained were 1.3, 2.6, 10.1, and 34.5% (w/w) of dried, coarsely powdered plant material for methanol (MET), chloroform (CHE), alcohol (ALE) and water (AQE), extracts respectively⁴. The extracts were administered in the form of suspension p.o. at three dose levels of 200,300and 400 mg/kg body weight to rats as 1 % (w/v) sodium carboxy methyl cellulose (SCMC) for evaluating their hypolipidemic and antihyperlipidemic effects⁵.All the extracts were subjected to preliminary phytochemical screening for the detection of various phyto constituents. All chemicals used were of AR grade. Kits for lipid profile analyses were purchased from a local supplier, viz; Sibutramine was gifted by the Ranbaxy Laboratory Ltd, Progesterone Sun Pharmaceutical Ltd., Mumbai, Monosodium glutamate was purchased from SRL Pvt. LTD., Mumbai. TC and HDL (Span Diagnostics), TG (Diassays, Germany). 36 (06 x 6) Wistar rats (180-250 g) were used in this second model. They were housed six per cage under standard laboratory conditions at a room temperature at 22 ± 2^OC with 12h light/dark cycle. The animals were maintained under standard nutritional and environmental conditions throughout the experiment. The experiment ware carried out between 9:00–16:00 hours at ambient temperature⁶. All the pharmacological experimental protocols were approved by the Institutional Animals Ethics Committee (Ref: CPCSEA/769/2010) of Sigma Institute of Clinical Research and Administration Pvt. Ltd, Hyderabad, India.

Effects in Progesterone induced Obesity in Mice:

Animal Model no.1

Animal Grouping:

The animals were randomly divided into following 6 groups; each group consisting of six animals. Animal grouping and their treatment was as follows:

Group- I: Negative control (Effect of arachis oil on mice)

Group- II: Positive control (Effect of Progesterone on mice) Remaining groups have received different types of extracts at various doses as follows.

Group- III: MERB (100mg/kg) + Progesterone

Group- IV: MERB (200mg/kg) + Progesterone

Group- V: MERB (400mg/kg) + Progesterone

Group- VI: Sibutramine 10 mg/kg + Progesterone

Test Drug Preparation:

All plant extracts was soluble in water so distilled water was used as media to dissolve the extracts and preparation of test drugs. Standard Sibutramine is also soluble in water. While preparation of test drug concentration precaution has been taken that all animal groups should receive approximate same volume of dose around 400μl. All the test drugs and standard were given by oral gavaging.

Induction of Progesterone Induced Obesity:

rogesterone vial contents were dissolved in arachis oil and dose of 10 mg/ kg was administered subcutaneously in the dorsal neck region to mice for 28 days, control group received the vehicle. All drugs were given at a dose of 0.4 ml/100 gm body weight. The test drugs were injected 30 minutes before to progesterone administration.

Evaluation and Biochemical Parameters:

Change in Body weight: The body weight of mice (g) was recorded every week for 28 days in each group just before dosing on 29th day of study after the last test drug administration animals were anaesthetized under light ether anaesthesia and blood for plasma preparation was collected by retro orbital puncture, using 10μl x 20mm (L) x 0.8 mm (2R) glass capillary into sterile EDTA-coated tube (3 mg/ml) for the estimation. Blood was kept in wet ice for 30 min, centrifuged for 5 min at 4000g at 4 o C and plasma was aspirated out for the analysis of lipid profile and glucose. The plasma was stored in the refrigerator for the analysis of biochemical parameters⁷. All analyses on plasma were completed within 24 h of sample collection. Plasma samples were analyzed for glucose, SGOT, SGPT, Triglyceride and total cholesterol, using biochemical kits.

Effects in Monosodium Glutamate induced Obesity in Rats:

Animal Model no.2

Animal Grouping:

The animals were randomly divided into following 6 groups; each group consists of six animals. Animal grouping and their treatment is as follows⁸:

Group- I: Sham operated Normal (Normal Saline 0.9%)

Group- II: Control (Monosodium glutamate)

Remaining groups have received different types of extracts at various doses as follows.

Group- III: RB (100mg/kg) + MSG

Group- IV: RB (200mg/kg) +MSG

Group- V: RB (400mg/kg) + MSG

Group- VI : Finofibrate 25 mg/kg + MSG

Induction of Monosodium Glutamate induced obesity:

Group I animals were administered with distilled water once daily by oral gavaging technique and served as negative control. For the Group II, III, IV, V and VI, in addition to normal diet and water, prepared monosodium glutamate solution was administered by gavaging to induce obesity. Monosodium glutamate was gavaged at the dose rate of 8mg/kg body weight to each animal orally daily up to 7 days. Once the obesity was induced between 1st to 7th days of the experiment, from 8th to 28th day various extract treatment was carried out¹⁰.

Evaluation and Biochemical Parameters:

Change in Body weight: The body weight of rat (g) was recorded every week for 28 days in each group just before dosing. On 29th day of study after the last test drug administration animals were anaesthetized under light ether anaesthesia and blood for plasma preparation was collected by retro orbital puncture, using 10μl x 20mm (L) x 0.8 mm (2R) glass capillary into sterile EDTA-coated tube (3 mg/ml) for the estimation. Blood was kept in wet ice for 30 min, centrifuged for 5 min at 4000g at 4⁰C and plasma was aspirated out for the analysis of lipid profile and glucose. The plasma was stored in the refrigerator for the analysis of biochemical parameters. All analyses on plasma were completed within 24 h of sample collection. Plasma samples were analyzed for glucose, SGOT, SGPT, Triglyceride and total cholesterol, using biochemical kits⁹.

RESULTS AND DISCUSSION:

Table-1: Results of preliminary phytochemical screening of the MERB-methanolic extract of Rhynchosia beddomei

PRIVATE Extractive	Glycosides	Flavonoid	Alkaloid	Sterols	Tannins	Saponins	Tri-terpeniods
Methanol	-ve	+ve	-ve	+ve	+ve	+ve	+ve
Ethanol	-ve	-ve	+ve	+ve	+ve	+ve	+ve
Chloroform	+ve	-ve	-ve	-ve	-ve	+ve	+ve
Water	+ve	-ve	-ve	-ve	+ve	+ve	+ve

Table-2: Effect of Methanoilc extract of Rhynchosia beddomei on body weight in Progesterone induced obesity in albino mice. MERB- Methanoilc Extract of Rhynchosia beddomei

Treatment Groups	1^st Day	7^th Day	14^th Day	21^st Day	28^th Day
Sham operated Normal	22.93±0.85	23.24±0.43	24.36±0.30	25.56±0.23	25.95±0.25
Control(Progesterone 10mg/kg)	22.15±0.72	26.11±0.50^b	26.77±0.27^a	27.67±0.32^c	28.04±0.36^b
Standard(Sibutramine10mg/kg)	23.92±0.60^ns	24.12±0.52*	24.87±0.43*	25.42±0.43**	25.80±0.16**
MERB (100mg/kg)	25.06±0.45*	24.78±0.73^ns	24.73±0.17**	25.17±0.36**	25.00±0.46***
MERB (200mg/kg)	22.18±0.58^ns	24.79±0.28^ns	24.23±0.37***	24.32±0.64***	24.67±0.51***
MERB (400mg/kg)	22.67±0.43^ns	24.12±0.62*	24.58±0.61**	25.67±0.66*	24.98±0.64***

All the values are mean ± SEM ,n=6, ns=Not significant, One way Analysis of Variance (ANOVA) followed by multiple comparison Dunnett’s test,*p<0.05, **p<0.01, *** p<0.001 vs. control group (Progesterone), and ^ap<0.001and ^bp<0.01 vs Sham operated normal

Table-3: Effect of Methanoilc extract of Rhynchosia beddomei on Glucose, SGOT and SGPT levels on Progesterone induced obesity in Albino mice SGOT: Serum Glutamamic oxaloacetic transaminase, SGPT: Serum glutamic pyruvic transaminase

Treatment Groups	Glucose mg/dl	SGOT mg/dl	SGPT mg/dl
Sham operated Normal	77.83±4.42	12.84±0.82	34.12±2.35
Control(Progesterone 10mg/kg)	87.5±2.81	25.50±2.14	44.31 ±2.36
Standard(Sibutramine10mg/kg)	64.00±1.39***	12.70±0.87***	28.97±1.45***
MERB(100mg/kg)	85.17±1.24***	23.44±1.12ns	59.53±3.50**
MERB (200mg/kg)	92.17±4.28***	21.02±1.29ns	45.35±4.37***
MERB (400mg/kg)	63.83±1.49***	13.59±1.35***	38.98±4.26***

All the values are mean ± SEM ,n=6, ns=Not significant, One way Analysis of Variance (ANOVA) followed by multiple comparison Dunnett’s test, **p<0.01, *** p<0.001 vs. control group ,and ^ap<0.001.

Table-4: Effect of Methanoilc extract of Rhynchosia beddomei on Lipid profile levels on Progesterone induced obesity in Albino mice

Treatment Groups	TC (mg/dl)	TG (mg/dl)	HDL-C (mg/dl)	LDL-C (mg/dl)	VLDL-C (mg/dl)
Sham operated Normal	125.7±1.45	66.84±0.48	40.05±0.66	72.33±1.53	13.37±0.09
Control(Progesterone10mg/kg)	184.6±2.55	76.84±0.48	68.15±0.66	92.33±1.53	18.37±0.09
Standard(Sibutramine10mg/kg)	142.2±2.10***	68.00±1.77***	47.89±1.20***	80.40±3.28***	13.89±0.22***
MERB(100mg/kg)	185.7±2.96***	72.17±1.88***	39.34±1.67***	131.9±3.35ns	14.43±0.37***
MERB (200mg/kg)	172.1±1.23***	70.24±1.58***	54.15±1.08***	118.4±0.80ns	14.04±0.31***
MERB (400mg/kg)	162.7±1.03***	63.70±1.08***	56.31±1.52***	103.9±2.11*	12.73±0.21***

All the values are mean ± SEM, n=6, ns=Not significant, One way Analysis of Variance (ANOVA) followed by multiple comparison Dunnett’s test, **p<0.01, *** p<0.001 vs. control group ,and ^ap<0.001

Table-5: Effect of Methanolic extract of Rhyncosia beddomei on Body Weight in Monosodium glutamate induced obesity in rats.

Treatment Groups	1^st Day	7^th Day	14^th Day	21^st day	48^th Day
Sham operated Normal	157.5±2.81	162.5±2.14	164.3±2.36	169.8±1.662	174.5±1.258
MSG (control)	157.5±2.81	172.5±2.14	184.3±2.36	189.8±1.662	194.5±1.258
Standard(Fenofibrate25mg/kg)	155.0±1.82	165.2±3.13	168.5±3.05*	169.5±1.83***	170.2±2.19***
MERB(100mg/kg)	153.3±3.07	168.2±0.79	168.8±2.77*	171.0±4.25***	192.0±4.50***
MERB (200mg/kg)	153.3±3.07	169.0±1.52	167.5±0.84**	175.0±0.81***	183.2±2.72***
MERB (400mg/kg)	155.2±2.89	161.3±1.56	166.8±1.77**	172.8±1.68***	185.3±5.10***

All the values are mean ± SEM, n=6, ns=Not significant, One way Analysis of Variance (ANOVA) followed by multiple comparison Dunnett’s test,*p<0.05, **p<0.01, *** p<0.001 vs. control group (Progesterone), and ^ap<0.001and ^bp<0.01 vs Sham operated normal

Table -6: Effect of Methanolic extract of Rhynchosia beddomei on Glucose, SGOT and SGPT levels on Monosodium glutamate induced hyperlipidemia in rats

Treatment Groups	Glucose mg/dl	SGOT mg/dl	SGPT mg/dl
Sham operated Normal	77.83±4.42	12.84±0.82	34.12±2.35
MSG( control)	88.5±2.81	25.50±2.14	44.31 ±2.36
Standard(Fenofibrate25mg/kg)	64.00±1.39***	12.70±0.87***	28.97±1.45***
MERB (100mg/kg)	75.17±1.24***	23.44±1.12ns	56.53±3.50**
MERB (200mg/kg)	72.17±4.28***	21.02±1.29ns	45.35±4.37***
MERB (400mg/kg)	63.83±1.49***	13.59±1.35***	38.98±4.26***

All the values are mean ± SEM, n=6, ns=Not significant, One way Analysis of Variance (ANOVA) followed by multiple comparison Dennett’s test, **p<0.01, *** p<0.001 vs. control group and ^ap <0.001

Table no-7: Effect of Methanoilc extract of Rhynchosia beddomei on Lipid profile levels on Monosodium glutamate induced obesity in rats.

Treatment Groups	TC (mg/dl)	TG (mg/dl)	HDL-C (mg/dl)	LDL-C (mg/dl)	VLDL-C (mg/dl)
Sham operated Normal	125.7±1.45	66.84±0.48	40.05±0.66	72.33±1.53	13.37±0.09
MSG( CONTROL)	199.6±2.55	86.84±0.48	78.15±0.66	82.33±1.53	20.37±0.09
Standard(Fenofibrate 25mg/kg)	142.2±2.10***	68.00±1.77***	47.89±1.20***	80.40±3.28***	13.89±0.22***
MERB(100mg/kg)	185.7±2.96***	72.17±1.88***	39.34±1.67***	131.9±3.35ns	14.43±0.37***
MERB (200mg/kg	172.1±1.23***	70.24±1.58***	54.15±1.08***	118.4±0.80ns	14.04±0.31***
MERB (400mg/kg)	162.7±1.03***	63.70±1.08***	56.31±1.52***	103.9±2.11*	12.73±0.21***

All the values are mean ± SEM , n=6, ns=Not significant, One way Analysis of Variance (ANOVA) followed by multiple comparison Dunnett’s test, **p<0.01, *** p<0.001 vs. control group ,and ^ap<0.001

CONCLUSION:

The Phytochemical tests with the ethanol extract of R. beddomei indicated the presence of carbohydrates, glycosides, terpenes, saponins, proteins and amino acids. Hyperlipidemia is associated with heart disease, which is the leading cause of death in the world. The lowering of the levels of harmful lipids to satisfactory values has been confirmed by several experimental animal and interventional studies indicating lowered morbidity and mortality in coronary heart diseases. The results are discussed under the lipid profile in serum. Lipid profile in serum with high triglyceride (TG) and cholesterol levels were significantly reduced by treatment of 400mg//day R. Beddomei. LDL and VLDL levels were significantly increased in progesterone-injected animals compare to control mice. The results are shown in Tables 1 and 3. The R. Beddomei markedly lowers the levels of serum cholesterol and VLDL. The decrease in cholesterol may indicate increased oxidation of mobilized fatty acids of inhibition or lipolysis. The present investigation shows that all triton induced rats displayed hyperlipidemia as shown by their elevated levels of serum and liver cholesterol, triglyceride, PL, VLDL, LDL and the reduction in the HDL level. It can be concluded that 400mg/day of R. Beddomei treatment was effective in reduction of cholesterol, PL, TG, VLDL, LDL and HDL in a dose dependant manner. Histopathological studies revealed that group treated with 200mg/kg extract and 400 mg/kg extract showed mild cytoplasmic fatty infiltration and mild granular degeneration as compared to normal and control groups. It can be concluded that 200mg/kg extract and 400 mg/kg extract of R. Beddomei treatment was effective in reduction of cholesterol, PL, TG, VLDL, LDL and HDL in a dose dependant manner. In this way increased doses can increase the percentage of weight reduction

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Received on 07.03.2017 Modified on 19.03.2017

Asian J. Research Chem. 2017; 10(3):275-279.

DOI: 10.5958/0974-4150.2017.00045.1