Antihyperlipidemic activity of Clitoria ternatea and Vigna mungo in rats

Abstract

Context: Hyperlipidemia is one of the major risk factors for atherosclerosis and cardiovascular diseases. Some plants are effective in controlling hyperlipidemia.

Objective: To investigate the anti-hyperlipidemic effect of Clitoria ternatea L. and Vigna mungo L. (Fabaceae) on experimentally induced hyperlipidemia in rats.

Materials and methods: The poloxamer 407-induced acute hyperlipidemia and diet-induced hyperlipidemia models were used for this investigation.

Results: Oral administration of the hydroalcoholic extract of the roots and seeds of C. ternatea and the hydroalcoholic extract of the seeds of V. mungo resulted in a significant (p < 0.05) reduction of serum total cholesterol, triglycerides, very low-density lipoprotein cholesterol, and low-density lipoprotein cholesterol levels. The atherogenic index and the HDL/LDL ratio were also normalized after treatment in diet-induced hyperlipidemic rats. The effects were compared with atorvastatin (50 mg/kg, p.o.) and gemfibrozil (50 mg/kg, p.o.), reference standards.

Discussion: The cholesterol-lowering effect of C. ternatea might be attributed to increased biliary excretion and decreased absorption of dietary cholesterol. The cholesterol-lowering effects of V. mungo seeds might be because of decreased HMG-CoA reductase activity, increased biliary excretion, and decreased absorption of dietary cholesterol. Additionally, they improved natural antioxidant defense mechanisms.

Conclusion: The findings of the investigation suggest that C. ternatea and V. mungo have significant antihyperlipidemic action against experimentally-induced hyperlipidemia.

Keywords::

• Antihyperlipidemic activity
• Clitoria ternatea
• Vigna mungo
• poloxamer 407

Introduction

Hyperlipidemia plays a significant role in the manifestation and development of atherosclerosis, leading to cardiovascular diseases (Ross, 1990). Atherosclerosis involves the interplay of several factors (Singh et al., 2002). There are three major factors, viz., hyperlipidemia, clotting factors, and oxidation of lipoproteins, which play a crucial role in atherosclerosis and collectively contribute to the development and rupture of atherosclerotic plaques. It is the oxidation of low-density lipoprotein (LDL) that plays a major role in atherosclerotic plaque development (Steinberg et al., 1989).

Various indigenous plants are used for antihyperlipidemic effects in the Ayurvedic system of medicine in India. Two such plants, Clitoria ternatea L. and Vigna mungo L., both belonging to the family Fabaceae, were reported to be used in a variety of disease conditions of the liver (Chopra et al., 1956; Kirtikar & Basu, 1976). Various parts of C. ternatea have been reported to have nootropic activity, anxiolytic activity, tranquilizing property, anti-inflammatory and analgesic activity, antipyretic activity, and antimicrobial activity (Mukherjee et al., 2008). C. ternatea has been reported to contain kaempferol and related glycosides, aparajitin, anthocyanins (Shrivastava & Pande, 1977), γ-sitosterol and related sterols (Sinha, 1960), hexacosanol, β-sitosterol, and anthoxanthins (Gupta & Lal, 1968). V. mungo is a rich source of protein, carbohydrates, oil, iron, potassium, and vitamin B (Swaminathan & Jain, 1975). It also contains phenolics (vitexin and isovitexin) (Peng et al., 2008), polyphenolics (phytic acid and tannic acid), trypsin inhibitors, and aromatic constituents such as hexanol, benzyl alcohol, γ-butyrolacetone, methyl-2-propenal, and pentanol (Lee & Shibamoto, 2000). High-protein diets have been reported to reduce the risk of atherosclerosis (Meeker & Kesten, 1941; Wolfe & Giovannetti, 1992; Wolfe, 1995). Legumes are a rich source of dietary proteins. Hence, we investigated two leguminous plants, C. ternatea and V. mungo, for their possible antihyperlipidemic activity using poloxamer 407-induced acute hyperlipidemia and diet-induced hyperlipidemia models.

Materials and methods

Plant collection and identification

The plant C. ternatea is available in two varieties – blue and white. Since the blue variety is medicinally important, it was used for the present investigation. The plant was collected during April–May, 2007, from the fields and roadside of the Charotar region of Gujarat state, India. The seeds of V. mungo were purchased from the local market of the same region. Both plants were botanically identified by Dr. G. C. Jadeja, Professor and Head of Agricultural Botany Department, B. A. College of Agriculture, Anand, India. Specimens of each were stored in the museum of the department (specimen nos. 0701 and 0702). The quality of the plants was ascertained as per the Ayurvedic formulary of India by determining foreign matter, total ash, acid-insoluble ash, alcohol-soluble extractive, and water-soluble extractive values (Anonymous, 2003).

Extraction

The dried, powdered (mesh no. 40) seeds of both plants were defatted with petroleum ether and then extracted with 50% v/v alcohol by maceration. The solvents were evaporated at 60°C to a pasty mass referred to as C. ternatea and V. mungo seed extracts, respectively. The roots of C. ternatea were directly extracted with 50% v/v alcohol by maceration and the solvents were evaporated at 60°C to yield the hydroalcoholic extract of the root, referred to as C. ternatea root extract.

Chemicals and reagents

All the chemicals used were of analytical grade. Poloxamer 407 (P-407) was obtained from Sun Pharmaceutical Ltd., Vadodara, Gujarat. Cholesterol and cholic acid were obtained from S. D. Fine Chemicals Ltd., Mumbai. Atorvastatin and gemfibrozil were received as gift samples from Zydus Research Center, Ahmedabad, Gujarat. The solvents and reagents were also from S. D. Fine Chemicals Ltd.

Pharmacological evaluation

Animals

Albino rats (SD strain) weighing 150–200 g of either sex were divided into different groups, each consisting of six animals. Animals were maintained on a commercial pellet diet (Pranav Agro Industries Ltd., Sangli, India) and water ad libitum throughout the study period. This study was approved by the institutional animal ethics committee in accordance with the guidelines of the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA, 2003).

Drugs and extracts

All the extracts were suspended in distilled water using 1% w/v gum acacia. The reference drugs atorvastatin and gemfibrozil were suspended in distilled water using 1% carboxymethylcellulose (CMC). The control group received 1% w/v gum acacia in distilled water and 1% CMC solution as vehicles.

Acute toxicity studies

Animals were treated with different doses up to 1500 mg/kg, p.o. of each extract. After single-dose administration, animals were observed for mortality or any other deformities up to 72 h.

Poloxamer 407-induced hyperlipidemia in rats

Acute hyperlipidemia was induced in rats using poloxamer 407 (Johnston & Palmer, 1993). Animals were divided into different groups (Table 1). All the extracts were administered orally about 1 h before the i.p. injection of 1 ml of 30% w/v solution of P-407. Animals were fed a normal chow diet throughout the study. Blood samples were collected at 15 and 24 h after P-407 injection and investigated for lipid profiles.

Table 1.  Poloxamer 407-induced acute hyperlipidemia model: summary of animal groups and treatments.

Group	Treatments
Normal	Vehicles (1 ml of 1% gum acacia and 1% CMC)
Hyperlipidemic control	Poloxamer 407 (1 ml of 30% w/v solution, i.p.)
Atorvastatin	Poloxamer 407 (1 ml of 30% w/v solution, i.p.) + atorvastatin (50 mg/kg, p.o.)
Gemfibrozil	Poloxamer 407 (1 ml of 30% w/v solution, i.p.) + gemfibrozil (50 mg/kg, p.o.)
CT-seed extract	Poloxamer 407 (1 mL of 30% w/v solution, i.p.) + C. ternatea seed extract (500 mg/kg, p.o.)
CT-root extract	Poloxamer 407 (1 ml of 30% w/v solution, i.p.) + C. ternatea root extract (500 mg/kg, p.o.)
VM-seed extract	Poloxamer 407 (1 ml of 30% w/v solution, i.p.) + V. mungo seed extract (500 mg/kg, p.o.)

Note. CT, C. ternatea; VM, V. mungo; CMC, carboxymethylcellulose.

Diet-induced hyperlipidemia in rats

The method of Blank et al. (1963), with modification, was used to produce diet-induced hyperlipidemia. Animals were divided into different groups (Table 2). Briefly, the normal group received a standard chow diet and all other groups received a high-cholesterol diet consisting of normal chow diet 92%, cholesterol 2%, cholic acid 1%, and coconut oil 5% for 7 days. The reference drugs and extracts were administered once daily between 8:00 and 9:00 a.m. for 7 days. The daily food intakes were determined before treatments.

Table 2.  Diet-induced hyperlipidemia model: summary of animal groups and treatments.

Group	Treatments
Normal	Vehicles (1 ml of 1% gum acacia and 1% CMC)
Hyperlipidemic control	High cholesterol diet
Atorvastatin	High cholesterol diet + atorvastatin (50 mg/kg, p.o.)
Gemfibrozil	High cholesterol diet + gemfibrozil (50 mg/kg, p.o.)
CT-seed extract	High cholesterol diet + C. ternatea seed extract (500 mg/kg, p.o.)
CT-root extract	High cholesterol diet + C. ternatea root extract (500 mg/kg, p.o.)
VM-seed extract	High cholesterol diet + V. mungo seed extract (500 mg/kg, p.o.)
VM-seed powder	High cholesterol diet + V. mungo seed powder (10% of diet)

Note. CT, C. ternatea; VM, V. mungo; CMC, carboxymethylcellulose.

On the last day, animals were deprived of food but not water. Blood samples were collected by retroorbital puncture technique under light anesthesia. The animals were sacrificed and liver tissues were collected and preserved at −40°C for further analysis. The fecal matters of the last 24 h before fasting were collected, immediately dried in an oven at 80°C for 1 h, and stored at −40°C for further analysis.

Estimation of biochemical parameters

Lipid profile

The serum lipid profile was determined at 15 and 24 h after P-407 injection and on day 8 in the case of diet-induced hyperlipidemia. The total cholesterol (TC), triglycerides (TG), and high-density lipoprotein cholesterol (HDL-C) levels were estimated using commercially available kits (Erba; Transasia Bio-Medicals Ltd., Daman, India). Very low-density lipoprotein cholesterol (VLDL-C) was calculated as TG/5. LDL-cholesterol (LDL-C) levels were calculated using Friedewald’s formula (Friedewald et al., 1972). The atherogenic index was calculated using the formula: atherogenic index (AI) = (VLDL-C + LDL-C)/HDL-C.

HMG-CoA reductase activity

The activity of the enzyme HMG-CoA (3-hydroxy-3-methyl-glutaryl-coenzyme-A) reductase was determined by an indirect method (Rao & Ramakrishnan, 1975). The method estimates the HMG-CoA/mevalonate ratio as an index of the activity of HMG-CoA reductase. The liver tissue was removed as quickly as possible and a 10% homogenate was prepared in saline arsenate solution. The homogenate was deproteinized using an equal volume of dilute perchloric acid and allowed to stand for 5 min, before centrifugation. To 1 ml of the filtrate, 0.5 ml of freshly prepared hydroxylamine reagent (alkaline hydroxylamine reagent in the case of HMG-CoA) was added. This was mixed, and 1.5 ml of ferric chloride reagent was added after 5 min. The absorbance was read after 10 min at 540 nm against a similarly treated saline arsenate blank. The ratio of HMG-CoA/mevalonate was calculated.

Fecal cholesterol and bile acid excretion

Fecal matter was collected during the last 24 h before fasting in the diet-induced hyperlipidemia model. The dried and powdered fecal matter was extracted with alkaline methanol. The resultant extract was then analyzed for cholesterol content in a manner similar to that of the serum. The cholesterol excreted in the fecal matter was calculated and expressed as mg/g of fecal matter. The method of Evrard and Janssen (1968) modified by Manes and Schneider (1971) was used for fecal bile acid extraction, and bile acid levels were estimated by the colorimetric method (Snell & Snell, 1954) and expressed as cholic acid equivalent per g of fecal matter.

Phenobarbitone-induced sleeping time

To investigate the hepatic HMG-CoA reductase enzyme inhibition, potentiation of the phenobarbitone-induced sleeping time was measured in rats (Walker & Parry, 1949). Animals were divided into different groups, each consisting of six. The control group received a single dose of phenobarbitone (80 mg/kg, i.p.). The treatment groups received 500 mg/kg, p.o. of each test extract 1 h before the phenobarbitone injection. The animals were observed for righting reflex. If the animals failed to maintain normal posture when placed on one side within 30 s, it was considered as a loss of righting reflex.

Lipid peroxidation and antioxidant parameters in the liver

In the diet-induced hyperlipidemia model, animals were dissected at the end of the study and the liver was collected, washed thoroughly in normal saline, bloated, and preserved at −40°C for further analysis. The liver homogenates were prepared in a Tris-hydrochloride buffer (0.1 M, pH 7). They were subjected to protein (Lowry et al., 1951), malondialdehyde (Okhawa et al., 1979), superoxide dismutase (SOD) (Misra & Fridovich, 1973), catalase (Aebi, 1974), reduced glutathione (GSH) (Beutler et al., 1963), NO scavenging (Sreejayan & Rao, 1997; Nakagawa & Yokozawa, 2002), and myeloperoxidase activity (Zhang et al., 2001) estimation.

Serum ascorbic acid levels

Serum total ascorbic acid (TAA), l-ascorbic acid (LAA), and dehydroascorbic acid (DAA) levels were estimated according to the method of Schaffert and Kingsley (1955).

Statistical analysis

Statistical analysis was carried out using one-way analysis of variance (ANOVA) followed by Tukey’s test. The level p < 0.05 was considered statistically significant.

Results

Acute toxicity study

No death or deformities were observed in any of the treatment groups up to 72 h. All of the extracts were found to be safe up to the dose level of 1 g/kg body weight.

Food intake

No significant difference in food intake among the different groups was observed (Table 3).

Table 3.  Effects of different treatments on food intake of diet-induced hyperlipidemic rats.

Group (n = 6)	Daily food intake (g)
	Day 1	Day 2	Day 3	Day 4	Day 5	Day 6	Day 7
Normal	19.58 ± 0.44	20.58 ± 0.23	19.75 ± 0.21	20.92 ± 0.44	20.15 ± 0.40	19.78 ± 0.45	19.91 ± 0.55
Hyperlipidemiccontrol	19.79 ± 0.21	19.47 ± 0.41	19.52 ± 0.39	19.68 ± 0.78	20.75 ± 0.31	20.13 ± 0.50	19.48 ± 0.54
Atorvastatin	18.95 ± 0.52	19.48 ± 0.42	20.12 ± 0.34	20.03 ± 0.32	21.77 ± 0.75	19.41 ± 0.41	21.58 ± 0.48
Gemfibrozil	20.83 ± 0.32	19.91 ± 0.45	18.69 ± 0.50	20.12 ± 0.65	21.83 ± 0.52	22.18 ± 0.37	19.58 ± 0.70
CT-seed extract	19.22 ± 0.31	20.03 ± 0.52	20.66 ± 0.32	19.17 ± 0.42	21.65 ± 0.58	21.15 ± 0.29	21.21 ± 0.54
CT-root extract	20.08 ± 0.35	21.08 ± 0.35	19.43 ± 0.42	21.52 ± 0.47	19.20 ± 0.45	19.19 ± 0.62	19.30 ± 0.52
VM-seed extract	19.72 ± 0.46	20.12 ± 0.33	20.65 ± 0.48	19.17 ± 0.55	22.08 ± 0.35	19.42 ± 0.59	21.34 ± 0.28
VM-seed powder	19.65 ± 0.38	21.08 ± 0.45	20.44 ± 0.37	19.33 ± 0.53	22.10 ± 0.47	20.20 ± 0.45	20.75 ± 0.31

Note. All values represent mean ± SEM from six animals. Statistical analysis was carried out using one-way ANOVA followed by Tukey’s test. p < 0.05 was considered statistically significant. CT, Clitoria ternatea; VM, Vigna mungo.

Poloxamer 407-induced acute hyperlipidemia

The serum TC, TG, LDL-C, and VLDL-C levels were significantly increased in the hyperlipidemic control group at 15 h (Table 4) and 24 h (Table 5), when compared with the normal group. Both C. ternatea root extract and V. mungo seed extract significantly reduced serum TC and TG levels at 15 and 24 h. These effects were comparable to those of the reference standard, atorvastatin. The V. mungo seed powder significantly increased serum HDL-C levels, and serum VLDL-C levels were significantly decreased in all the treatment groups at 24 h. The serum LDL-C levels were significantly decreased by gemfibrozil only at 15 h. However, the HDL-C/LDL-C ratio was not altered by any of the treatment groups (Table 6).

Table 4.  Effect of C. ternatea and V. mungo on serum lipid profile 15 h after poloxamer 407-induced acute hyperlipidemia in rats.

Group	TC (mg/dL)	TG (mg/dL)	HDL (mg/dL)	LDL (mg/dL)	VLDL (mg/dL)
Normal	66.15 ± 2.35	54.56 ± 1.72	23.72 ± 0.92	31.52 ± 2.82	10.91 ± 0.34
Control	244.30 ± 5.89*	398.01 ± 4.82*	82.60 ± 4.14*	82.09 ± 8.02*	79.60 ± 0.96*
Atorvastatin	198.14 ± 7.47**	315.99 ± 6.02**	70.14 ± 2.40	64.80 ± 9.42	63.20 ± 1.20
Gemfibrozil	183.53 ± 4.67**	352.83 ± 2.53**	77.61 ± 1.79	35.35 ± 2.37**	70.57 ± 0.51
CT-seed extract	230.36 ± 11.44	327.12 ± 7.53**	80.84 ± 2.52	84.10 ± 10.43	65.42 ± 0.51
CT-root extract	203.29 ± 4.91**	332.95 ± 9.03**	61.19 ± 3.10**	75.52 ± 5.41	66.59 ± 1.81
VM-seed extract	194.80 ± 5.14**	300.30 ± 14.74**	85.31 ± 5.99	49.43 ± 11.91	60.06 ± 2.95

Note. All values represent mean ± SEM from six animals.*Compared with normal group (p < 0.05), **compared with hyperlipidemic control group (p < 0.05). TC, total cholesterol; TG, triglycerides; HDL-C, high-density lipoprotein cholesterol; LDL-C, low-density lipoprotein cholesterol; VLDL-C, very low-density lipoprotein cholesterol; CT, Clitoria ternatea; VM, Vigna mungo.

Table 5.  Effect of C. ternatea and V. mungo on serum lipid profile 24 h after poloxamer 407-induced acute hyperlipidemia in rats.

Group	TC (mg/dL)	TG (mg/dL)	HDL (mg/dL)	LDL (mg/dL)	VLDL (mg/dL)
Normal	68.53 ± 1.82	56.96 ± 1.72	25.23 ± 0.94	31.91 ± 1.40	11.39 ± 0.35
Control	254.20 ± 3.36*	552.05 ± 47.12*	39.22 ± 2.56*	114.07 ± 7.31*	110.41 ± 9.42**
Atorvastatin	217.31 ± 9.82**	386.87 ± 11.72**	43.42 ± 1.35	96.51 ± 10.91	77.37 ± 2.35**
Gemfibrozil	249.18 ± 2.68	360.76 ± 2.49**	41.41 ± 0.97	130.61 ± 2.76	72.15 ± 0.49**
CT-seed extract	235.98 ± 6.20	381.79 ± 2.39**	35.07 ± 2.71	124.56 ± 5.96	76.36 ± 0.48**
CT-root extract	213.43 ± 9.34**	339.47 ± 6.70**	36.63 ± 1.00	108.90 ± 8.10	67.89 ± 1.34**
VM-seed extract	212.07 ± 5.67**	332.97 ± 10.15**	48.56 ± 1.88**	96.91 ± 6.06	66.59 ± 2.03**

Note. All values represent mean ± SEM from six animals. *Compared with normal group (p < 0.05), **compared with hyperlipidemic control group (p < 0.05). TC, total cholesterol; TG, triglycerides; HDL-C, high-density lipoprotein cholesterol; LDL-C, low-density lipoprotein cholesterol; VLDL-C, very low-density lipoprotein cholesterol; CT, Clitoria ternatea; VM, Vigna mungo.

Table 6.  Effect of C. ternatea and V. mungo on atherogenic index and HDL/LDL ratio.

Group	Poloxamer 407-induced acute hyperlipidemia				Diet-induced hyperlipidemia
	Atherogenic index		HDL-C/LDL-C ratio		Atherogenic index	HDL-C/LDL-C ratio
	15 h	24 h	15 h	24 h
Normal	1.88 ± 0.16	1.84 ± 0.07	0.79 ± 0.10	0.79 ± 0.04	1.19 ± 0.06	1.18 ± 0.06
Hyperlipidemic control	1.76 ± 0.17	5.97 ± 0.48*	1.06 ± 0.12	0.38 ± 0.04*	3.42 ± 0.10*	0.32 ± 0.01*
Atorvastatin	1.61 ± 0.19	4.57 ± 0.23	1.35 ± 0.39	0.48 ± 0.06	1.43 ± 0.11**	1.13 ± 0.12**
Gemfibrozil	1.37 ± 0.09	5.04 ± 0.26	2.59 ± 0.52	0.31 ± 0.01	1.76 ± 0.09**	0.73 ± 0.05**
CT-seed extract	2.02 ± 0.10	6.36 ± 0.59	1.05 ± 0.16	0.29 ± 0.03	2.66 ± 0.12**	0.43 ± 0.02
CT-root extract	2.17 ± 0.20	5.40 ± 0.28	0.84 ± 0.09	0.35 ± 0.03	2.20 ± 0.12**	0.57 ± 0.03**
VM-seed extract	0.97 ± 0.20	3.88 ± 0.21**	3.28 ± 1.59	0.51 ± 0.04	2.33 ± 0.09**	0.50 ± 0.02

Note. All values represent mean ± SEM from six animals. *Compared with normal group (p < 0.05), **compared with hyperlipidemic control group (p < 0.05). CT, Clitoria ternatea; VM, Vigna mungo.

Diet-induced hyperlipidemia

There was a significant increase in the serum levels of TC, TG, LDL-C, VLDL-C, and HDL-C in the hyperlipidemic control group as compared with the normal control group. All the treatment groups produced a significant decrease in serum TC, TG, HDL-C, and VLDL-C levels (Table 7) and AI (Table 6). In addition to the above, the serum LDL-C levels were significantly decreased by the C. ternatea root extract and the V. mungo seed extract.

Table 7.  Effect of C. ternatea and V. mungo on serum lipid profile of diet-induced hyperlipidemia in rats.

Group (n = 6)	TC (mg/dL)	TG (mg/dL)	HDL-C (mg/dL)	LDL-C (mg/dL)	VLDL-C (mg/dL)
Normal	133.64 ± 2.97	100.46 ± 4.16	61.12 ± 1.32	52.43 ± 2.45	20.09 ± 0.83
Hyperlipidemic control	328.23 ± 6.95*	303.80 ± 12.59*	104.56 ± 3.54*	162.91 ± 9.58	60.76 ± 2.52*
Atorvastatin	162.00 ± 6.97**	162.99 ± 1.95**	66.87 ± 2.47^†	62.53 ± 6.48**	32.60 ± 0.39**
Gemfibrozil	196.41 ± 6.32**	128.46 ± 4.23**	71.07 ± 1.04^†	99.64 ± 7.09**	25.69 ± 0.85**
CT-seed extract	242.26 ± 3.45**	108.42 ± 2.12**	66.50 ± 2.41^†	154.08 ± 3.61	21.68 ± 0.42**
CT-root extract	207.56 ± 8.91**	130.51 ± 5.43**	64.99 ± 2.31^†	116.46 ± 7.73**	26.10 ± 1.09**
VM-seed extract	238.56 ± 7.98**	127.67 ± 7.10**	73.37 ± 2.50^†	139.65 ± 7.14**	25.53 ± 1.42**
VM-seed powder	238.72 ± 2.48**	115.42 ± 9.37**	71.88 ± 1.98^†	143.75 ± 4.06	23.08 ± 1.87**

Note. All values represent mean ± SEM from six animals. *Compared with normal group (p < 0.05), **compared with hyperlipidemic control group (p < 0.05), ^†significant reduction compared with hyperlipidemic control group (p < 0.05). TC, total cholesterol; TG, triglycerides; HDL-C, high-density lipoprotein cholesterol; LDL-C, low-density lipoprotein cholesterol; VLDL-C, very low-density lipoprotein cholesterol; CT, Clitoria ternatea; VM, Vigna mungo.

Fecal bile acid and cholesterol excretion

The C. ternatea seed and root extracts and V. mungo seed extract significantly increased fecal cholesterol excretion (Table 8). The fecal bile acid excretion was significantly increased by all the treatment groups except atorvastatin.

Table 8.  Effect of C. ternatea and V. mungo on fecal cholesterol and bile acid excretion in diet-induced hyperlipidemic rats.

Group	Fecal cholesterol (mg/g of fecal matter)	Fecal bile acid^‡ (mg/g of fecal matter)
Normal	2.05 ± 0.09	1.29 ± 0.08
Hyperlipidemic control	3.18 ± 0.07*	1.12 ± 0.05
Atorvastatin	2.27 ± 0.07	2.56 ± 0.06
Gemfibrozil	4.44 ± 0.11**	3.93 ± 0.11**
CT-seed extract	4.83 ± 0.13**	3.69 ± 0.04**
CT-root extract	8.50 ± 0.13**^a	3.97 ± 0.10**
VM-seed extract	8.62 ± 0.16**^a	3.57 ± 0.04**
VM-seed powder	2.44 ± 0.10	4.35 ± 0.10**

Note. All values represent mean ± SEM from six animals. *Compared with normal group (p < 0.05), **compared with hyperlipidemic control group (p < 0.05), ^acompared with atorvastatin and gemfibrosil groups, ^‡as cholic acid equivalent. CT, Clitoria ternatea; VM, Vigna mungo.

Liver HMG-CoA reductase activity

The HMG-CoA/mevalonate ratio was significantly increased by the V. mungo seed extract as compared to the hyperlipidemic control group (Table 9).

Table 9.  Effect of C. ternatea and V. mungo on HMG-CoA activity in liver of diet-induced hyperlipidemic rats.

Group	HMG-CoA reductase^ψ activity in liver
Normal	1.68 ± 0.24
Hyperlipidemic control	1.19 ± 0.19
Atorvastatin	4.49 ± 0.36*
Gemfibrozil	1.93 ± 0.07
CT-seed extract	1.86 ± 0.30
CT-root extract	1.79 ± 0.09
VM-seed extract	2.37 ± 0.27*
VM-seed powder	1.69 ± 0.11

Note. All values represent mean ± SEM from six animals. *Compared with control group (p < 0.05), ^Ψexpressed as ratio of HMG-CoA/mevalonate. CT, Clitoria ternatea; VM, Vigna mungo.

Lipid peroxidation

Lipid peroxidation and antioxidant status were studied in the liver tissues of diet-induced hyperlipidemic rats. The liver MDA level, SOD level, NO scavenging activity, and myeloperoxidase activity were significantly increased in the hyperlipidemic control group (Table 10). The GSH level and catalase activities were decreased in the hyperlipidemic control group, suggesting the presence of significant oxidative stress. The liver MDA, SOD, myeloperoxidase, and NO scavenging activities were significantly reduced by pretreatment with all the extracts. The liver GSH and catalase activities were increased in all the treatment groups when compared with the hyperlipidemic control group. The V. mungo seed extract showed a significant decrease in myeloperoxidase activity compared with all other treatments, but the V. mungo seed powder did not produce an increase in serum GSH levels.

Table 10.  Effects of C. ternatea and V. mungo on lipid peroxidation and antioxidant parameters in liver tissues of diet-induced hyperlipidemic rats.

Group	Normal	Hyperlipid. control	Atorvastatin	Gemfibrozil	CT-seed extract	CT-root extract	VM-seed extract	VM-seed powder
MDA (nmol/mg protein)	2.39 ± 0.22	8.82 ± 0.68*	4.17 ± 0.35**	4.37 ± 0.29**	4.19 ± 0.31**	3.10 ± 0.33**	2.77 ± 0.38**	2.33 ± 0.35**
SOD (U/min/mg protein)	2.00 ± 0.20	31.01 ± 2.96*	11.98 ± 2.19**	12.87 ± 1.67**	9.54 ± 1.06**	3.94 ± 0.45**^‡	3.39 ± 0.34**^‡	4.48 ± 0.77**^‡
Catalase (U/min/mg protein)	0.36 ± 0.03	0.26 ± 0.02	0.30 ± 0.002	0.31 ± 0.02	0.39 ± 0.02**	0.34 ± 0.01**	0.48 ± 0.02**	0.35 ± 0.02**
GSH (μg/mg protein) × 10^3	0.0419 ± 0.0046	0.0119 ± 0.0002*	0.0125 ± 0.0002	0.0151 ± 0.0008	0.0240 ± 0.0012**	0.0248 ± 0.0016**	0.0346 ± 0.0049**	0.0201 ± 0.0011**
NO scav. activity (nmol/mg protein)	134.45 ± 5.73	1545.35 ± 160.72*	546.02 ± 34.66**	372.93 ± 36.15**	351.19 ± 32.90**	380.33 ± 27.89**	433.33 ± 26.35**	415.63 ± 20.30**
Myeloperoxidase activity × 10^−8 (U/mg protein)	2.18 ± 0.22	5.67 ± 0.64*	3.38 ± 0.31**	4.41 ± 0.24**	1.423 ± 0.10**	3.07 ± 0.29**	0.82 ± 0.04**	3.79 ± 0.47**

Note. All values represent mean ± SEM from six animals. *Compared with normal group (p < 0.05), **compared with control group (p < 0.05), ^‡compared with atorvastatin (p < 0.05).

Serum TAA, LAA, and DAA levels

The serum total ascorbic acid levels were significantly increased by all the treatment groups except CT root extract. Considering l- and d-ascorbic acids, l-ascorbic acid is biologically more active. The serum l-ascorbic acid levels were significantly increased by all the extracts except C. ternatea root extract. The dehydroascorbic acid levels were significantly increased by V. mungo seed powder (Table 11).

Table 11.  Effect of C. ternatea and V. mungo on serum total ascorbic acid (TAA), l-ascorbic acid (LAA), and dehydroascorbic acid (DAA) levels in diet-induced hyperlipidemic rats.

Group	TAA^‡	LAA^‡	DAA^‡
Normal	3.69 ± 0.07	2.00 ± 0.05	1.69 ± 0.11
Hyperlipidemic control	3.26 ± 0.13	2.58 ± 0.14	0.68 ± 0.13*
Atorvastatin	3.44 ± 0.07	2.66 ± 0.14	0.78 ± 0.12
Gemfibrozil	3.55 ± 0.17	2.504 ± 0.08	1.05 ± 0.12
CT-seed extract	4.51 ± 0.26**	4.05 ± 0.19**	0.62 ± 0.08
CT-root extract	3.05 ± 0.10	2.82 ± 0.08	0.23 ± 0.03
VM-seed extract	5.15 ± 0.43**	4.33 ± 0.33**	0.81 ± 0.11
VM-seed powder	5.86 ± 0.27**	4.22 ± 0.21**	1.63 ± 0.16**

Note. All values represent mean ± SEM of six animals. *Compared with normal (p < 0.05), **compared with hyperlipidemic control (p < 0.05), ^‡values are in mg/g of liver tissue. CT, C. ternatea; VM, V. mungo.

Potentiation of phenobarbitone-induced sleeping time

To investigate hepatic enzyme inhibition as a possible mechanism, we studied the effects of all the extracts on the phenobarbitone-induced sleeping time. The V. mungo seed extract significantly prolonged the phenobarbitone-induced sleeping time (Table 12). The C. ternatea seed extract significantly decreased the phenobarbitone-induced sleeping time. The C. ternatea root extract did not alter the phenobarbitone-induced sleeping time.

Table 12.  Effects on phenobarbitone-induced sleeping time.

Group (n = 10)	Treatments	Induction time (min)	Sleeping time (min)
Control	Vehicle + phenobarbitone (80 mg/kg, i.p.)	50.6 ± 1.69	78.6 ± 2.71
CT-seed extract	C. ternatea seed extract (500 mg/kg, p.o.) + phenobarbitone (80 mg/kg, i.p.)	51.2 ± 2.21	45.3 ± 1.78^†
CT-root extract	C. ternatea root extract (500 mg/kg, p.o.) + phenobarbitone (80 mg/kg, i.p.)	54.5 ± 1.99	79.7 ± 4.08
VM-seed extract	V. mungo seed extract (500 mg/kg, p.o.) + phenobarbitone (80 mg/kg, i.p.)	25.3 ± 3.91**	307.0 ± 4.18**

Note. All values represent mean ± SEM of 10 rats. p < 0.05 was considered statistically significant (one-way ANOVA followed by Tukey’s test), **significant increase when compared with control group, ^†significant decrease when compared with control group. CT, C. ternatea; VM, V. mungo.

Discussion

In the present study, we have investigated the effect of C. ternatea (seed and root extracts) and V. mungo (seed extract and powdered seeds) against experimentally induced hyperlipidemia in rats. All the extracts at the dose of 500 mg/kg, p.o. significantly reduced serum TC and TG levels. The C. ternatea seed extract mainly affected serum TG levels and the root extract affected both TC and TG levels in the P-407-induced acute hyperlipidemia model. The serum TC and TG levels were reduced by both extracts of C. ternatea in the diet-induced hyperlipidemia model. V. mungo seed extract as well as seed powder reduced both serum TC and TG levels in both hyperlipidemia models. Additionally, this decrease in TC levels corresponded significantly to a reduction in LDL-C levels. These findings were supported by a decrease in atherogenic index and an increase in the HDL-C/LDL-C ratio. Since P-407-induced hyperlipidemia is mainly due to inhibition of the extractable (heparin releasable) pool of lipoprotein lipase (Johnston & Palmer, 1993), the serum TG-lowering effects can be attributed to the activation of lipoprotein lipase.

It is reported that cholesterol homeostasis is maintained by the control of two processes, viz., cholesterol biosynthesis, in which HMG-CoA reductase catalyzes the rate-limiting process, and cholesterol absorption of both dietary cholesterol and cholesterol cleared from the liver through biliary secretion. The HMG-CoA/mevalonate ratio has an inverse relationship to the activity of HMG-CoA reductase (Rao & Ramakrishnan, 1975). The results of the study indicated that the activity of the enzyme was significantly depressed by the V. mungo seed extract as was evident by the increase in the ratio. Furthermore, there was also an increase in the cholesterol content of the fecal matter, indicating that all the extract either promoted the excretion of cholesterol or prevented the absorption of cholesterol. Since the fecal bile acid levels were significantly increased, they might have promoted the cholesterol excretion.

Lipid peroxidation is the key factor leading to atherosclerotic plaque formation. It is the oxidized LDL that is responsible for chemotaxis of macrophages and the subsequent cascade of events to form plaques (Schaffert & Kingsley, 1955). The results of the present study indicated that lipid peroxidation was significantly reduced by all the extracts. Additionally, they improved the liver antioxidant status by improving the activities of the various enzymes.

l-Ascorbic acid is the only biologically active form, playing a vital role as a natural antioxidant against a variety of stress conditions including lipid peroxidation (Frei et al., 1990). It is easily converted into dehydroascorbic acid, thereby regenerating vitamin E. It also maintains high intracellular levels of glutathione (Meister, 1994). The findings of this study showed significantly higher levels of the serum total and l-ascorbic acid levels in all the extract-pretreated groups, suggesting a marked reduction in oxidative stress. This can partly be supported by increased glutathione levels in the liver tissues.

In conclusion, C. ternatea seed and root extracts and V. mungo seed extract possess significant lipid-lowering activities against experimentally induced hyperlipidemia. The TG-lowering effects might be attributed to an increase in lipoprotein lipase activities. The cholesterol-lowering effects of V. mungo seeds can be related partly to the decreased activity of hepatic HMG-CoA reductase enzyme and partly to increased fecal excretion by promoting biliary excretion and preventing absorption of dietary cholesterol. The cholesterol-lowering effect of C. ternatea root extract might be because of increased fecal excretion by promoting biliary excretion and preventing absorption of dietary cholesterol. Further, the extracts improved the natural antioxidant status in the liver tissues.

Declaration of interest

This work is financially supported by a grant from the Gujarat Council of Science and Technology (GUJCOST), Gandhinagar, Gujarat, India.