Protective effect of Trigonella foenum-graecum Linn. on monosodium glutamate-induced dyslipidemia and oxidative stress in rats.

OBJECTIVES: The present study was designed to evaluate the effect of aqueous extract of Trigonella foenum-graecum(AqE-TFG) seeds on monosodium glutamate (MSG)-induced dyslipidemia and oxidative stress in Wistar rats.
MATERIALS AND METHODS: Neonatal Wistar rats were treated subcutaneously with MSG (4 g/kg b.w.) from day 2 to 14 after birth, on alternate days. After attaining six-weeks of age, MSG-treated rats were administered with AqE-TFG (0.5 and 1 g/kg b.w., orally) or orlistat (10 mg/kg b.w., orally) for 28 days, respectively. Serum chemistry and relevant enzymes in hepato-cardiac tissues were assessed on day 29.
RESULTS: AqE-TFG produced significant reduction in serum total cholesterol (TC), triglycerides (TGs), lactate dehydrogenase (LDH), aspartate amino transferase (AST), alanine amino transferase (ALT), hepatic and cardiac lipid peroxides (MDA) levels and elevation in serum high density lipoprotein cholesterol (HDL-C), hepatic and cardiac antioxidant enzymes [glutathione (GSH), and superoxide dismutase (SOD) and catalase (CAT)] levels.
CONCLUSION: Results were comparable with orlistat, a standard anti-obesity drug, and provide clear evidence that the AqE-TFG treatment offered significant protection against MSG-induced dyslipidemia and oxidative stress, and may play an important role in amelioration of the free radical generated consequences like dyslipidemia and atherosclerosis.

Introduction

Monosodium glutamate, one of the most abundant naturally occurring amino acids, is frequently added as a flavor enhancer. MSG is known to have some adverse effects in humans and experimental animals. These include the Chinese restaurant syndrome,[1] neuroexcitotoxicity,[23] and obesity.[4] Chronic administration of MSG (4 g/kg b.w.) induces oxidative stress in hepatic and cardiac tissues in experimental animals due to metabolic shifting.[5-7] Increased oxidative stress brings change in the membrane lipids and proteins, which could be responsible for the initiation of metabolic disorders. Oxidative stress is abiochemical disequilibrium occurring due to excessive production of free radicals and reactive oxygen species, which aggravates oxidative damage to biomolecules that cannot becounteracted by antioxidative defense systems.[89]

Although some information is available on MSG-induced dyslipidemia and oxidative stress, the studies on the effect of antioxidants, especially those consumed in food, in MSG-induced dyslipidemia and oxidative stress are lacking. Trigonella foenum-graecum Linn. (Fenugreek, family - Fabaceae) has been shown to possess antioxidant activity in different experimental animal models.[10] The purpose of the present study was to investigate the protective effect of aqueous extract of Trigonella foenum-graecum on MSG-induced dyslipidemia and oxidative stress in rats. The dose of aqueous extract of Trigonella foenum-graecum was based on previous inquiries that were held to establish and assess the effect of aqueous extract of Trigonella foenum-graecum(AqE-TFG) and soluble dietary fibre of TFG (Tf-sdf) in different metabolic disorders (i.e. diabetes and dyslipidemia), respectively.[1112]

Materials and Methods

Trigonella foenum-graecum seeds were purchased from Indian Council for Agricultural Research, New Delhi. The seeds were botanically identified and authenticated by Head, Raw Materials Herbarium and Museum, NISCAIR, New Delhi and a voucher specimen (ref. NISCAIR/RHMD/Consult/-2011-12/1743/43) has been deposited in the herbarium of the Institute.

Preparation of Aqueous Extract of Trigonella foenum-graecum (AqE-TFG) Seeds

Trigonella foenum-graecum seeds were dried at 40°C and finely powdered. Twenty five grams of powdered seeds were extracted with 500 ml of boiling distilled water for 45 min. The heated decoction was left overnight for complete socking, at room temperature. The decoction was filtered; the filtrate was lyophilized and stored in the refrigerator. On lyophilization, the resulting material weighed 1.133 g (4.53% yield). The extract was subjected to the preliminary phytochemical screening as per standard procedure.[13]

Drugs and Chemicals

Monosodium glutamate (MSG) (Sigma, St. Louis, USA), orlistat (Biocon Lab., Bangalore, India), LDH kit (Reckon diagnostics Pvt. Ltd., Vadodara, Gujarat, India), total cholesterol (TC), triglycerides (TGs), high-density lipoprotein cholesterol (HDL-C), aspartate amino transferase (AST), and alanine amino transferase (ALT) kits (Span Diagnostics Ltd., Vadodara, Gujarat, India) were used in the study. All chemicals were of analytical grade and those required for sensitive tissue assays were purchased from Sigma Chemical Co., St. Louis, USA, HiMedia, and SD Fine Chemicals.

Experimental Design

The experimental protocol was approved by the Institutional Animal Ethics Committee of Hamdard University, New Delhi, which is registered with Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), Government of India (registration no. 173/CPCSEA, dated January 28, 2000).

Newborn Wistar rat pups were injected subcutaneously with MSG at a dose of 4 g/kg body weight dissolved in normal saline on alternate days seven times, i.e. on postnatal day 2, 4, 6, 8, 10, 12, and 14, respectively.[14] Normal control animals received only normal saline on these days. After weaning on postnatal day 21, the female rat pups were excluded from the study and the male rat pups were housed in polypropylene cages under controlled conditions (room temperature 25 ± 2°C, air humidity 50 ± 15%, and photoperiod of 12 h Light:Dark cycle)[15] and had free access to commercial pellet diet (Amrut Rat Feed, Nav Maharashtra Chakan Oil Mills Ltd., Delhi, India) and water ad libitum. After attaining six-weeks of age, normal control rats (n=10) were treated with normal saline (2 ml/kg b.w., orally), while MSG-treated rats (n=10 pups in each group) were administered with AqE-TFG (0.5 and 1 g/kg b.w., orally) or orlistat (10 mg/kg b.w., orally) dissolved in normal saline for 28 days, respectively.

Determination of Biochemical Parameters

On the 29th day, blood was collected from the retro-orbital plexus of overnight fasted rats under light ether anesthesia and serum was separated by centrifugation at 3000 rpm for 15 min and frozen at –20°C for estimation of TC and HDL-C,[16] TGs,[17] LDH,[18] AST, and ALT[19] with commercially available kits using ultra violet (UV)-visible spectrophotometer (Shimadzu, UV-1601, Japan). The liver and heart were isolated and washed in ice-cold physiological saline and stored at –20°C for biochemical estimations of hepatic and cardiac lipid peroxides (MDA), glutathione (GSH), superoxide dismutase (SOD), and catalase (CAT).

MDA, a product of membrane lipid peroxidation, was estimated by the method of Okhawa et al.,[20] and expressed as nmoles/mg protein. Protein was estimated by the method of Lowry et al.[21] GSH content was estimated by the method of Sedlack and Lindsay.[22] The absorbance of reaction mixture was read within five min of addition of DTNB at 412 nm using UV-spectrophotometer, against a reagent blank. GSH activity was expressed as μmol of P liberated/min/mg protein. SOD content was estimated by the method of Marklund and Marklund.[23] The absorbance of reaction mixture was read at 420 nm at one-minute intervals for three minutes after addition of pyrogallol, which was inhibited by the presence of SOD. The results were expressed as U/mg protein. CAT content was estimated by the method of Clairborne.[24] The disappearance of H2O2was monitored at 240-nm wavelength at one-minute interval for three minutes. Catalase activity was expressed as nmol H2O2/min/mg protein.

All statistical analyses were performed using GraphPad Prism 3.0 (GraphPad, San Diego, CA). All results were expressed as mean ± S.E.M. The data were analyzed with one-way ANOVA followed by Dunnett's test. A statistical difference of P< 0.05 was considered significant in all cases.

Results

The preliminary phytochemical screening of AqE-TFG, revealed the presence of saponin glycoside, carbohydrates, and proteins.

Effect of AqE-TFG on Serum Biochemical Parameters

The serum TC, TGs, and HDL-C levelsare depicted in Table 1. MSG control group showed a significant (P< 0.01) increase in serum TC and TGs levels and a significant (P< 0.01) decrease in HDL-C level when compared with the normal control group. Administration of AqE-TFG (0.5 and 1 g/kg b.w., orally) or orlistat (10 mg/kg b.w., orally) for a period of 28 days caused a significant (P< 0.01) decrease in serum TC and TGs and a significant (P< 0.01) increase in serum HDL-C as compared to the MSG control group. Also, a significant (P < 0.01) decrease in serum TC and TGs levels was observed in the orlistat (10 mg/kg b.w., orally) treated groupas compared to the AqE-TFG (0.5 g/kg b.w., orally) treated group, while there was no significant (P > 0.05) difference between orlistat (10 mg/kg b.w., orally)and the AqE-TFG (1 g/kg b.w., orally) treated groups for serum TC and TGs levels in MSG-treated rats. Furthermore, there was no significant (P > 0.05) difference between serum HDL levels in the orlistat (10 mg/kg b.w., orally) and the AqE-TFG (0.5 and 1 g/kg b.w., orally) treated groups.

Table 1

Effect of aqueous extract of Trigonella foenum-graecum (AqE-TFG) on serum lipid levels in monosodium glutamate (MSG) treated rats

The serum LDH, AST, and ALT levels are depicted in Table 2. MSG control group showed a significant (P < 0.01) increase in serum LDH, AST, and ALT levels as compared to the normal control group. AqE-TFG (0.5 and 1 g/kg b.w., orally) or orlistat (10 mg/kg b.w., orally) administration caused a significant (P < 0.05 and P< 0.01) decrease in these levels as compared to the MSG control group. Also, there was no significant (P > 0.05) difference between orlistat(10 mg/kg b.w., orally)and AqE-TFG (0.5 and 1 g/kg b.w., orally) treated groups for serum LDH, AST, and ALT levels in MSG-treated rats.

Table 2

Effect of aqueous extract of Trigonella foenum-graecum(AqE-TFG) on serum lactate dehydrogenase (LDH), aspartate transaminase (AST), and alanine transaminase (ALT) levels in monosodium glutamate (MSG) treated rats

Effect of AqE-TFG on lipid peroxides (MDA), GSH, SOD and CAT levels

MSG control rats showed a significant (P< 0.01) increase in hepatic and cardiac MDA levels as compared to the normal control group. Administration of AqE-TFG (0.5 and 1 g/kg b.w., orally) or orlistat (10 mg/kg b.w., orally) caused a significant (P < 0.05 and P< 0.01) decrease in both hepatic and cardiac MDA levels as compared to the MSG control group [Figure 1]. Also, there was no significant (P > 0.05) difference between the orlistat (10 mg/kg b.w., orally) and AqE-TFG (0.5 and 1 g/kg b.w., orally) treated groups for hepatic and cardiac MDA levels in MSG-treated rats.

Figure 1

Effect of aqueous extract of Trigonella foenum-graecum (AqE-TFG) on hepatic and cardiac lipid peroxides (MDA) levels in monosodium glutamate (MSG) treated rats

The MSG control group showed a significant (P < 0.01) depletion in hepatic and cardiac GSH, SOD, and CAT levels as compared to normal control group. Administration of AqE-TFG (0.5 and 1 g/kg b.w., orally) or orlistat (10 mg/kg b.w., orally) caused a significant (P < 0.05 and P < 0.01) increase in these levels as compared to MSG control group [Table 3]. Also, there was no significant (P > 0.05) difference between orlistat (10 mg/kg b.w., orally) and AqE-TFG (0.5 and 1 g/kg b.w., orally) treated groups for hepatic and cardiac GSH, SOD and CAT levels in MSG-treated rats.

Table 3

Effect of aqueous extract of Trigonella foenum-graecum (AqE-TFG) on hepatic and cardiac reduced glutathione (GSH), superoxide dismutase (SOD), and catalase (CAT) levels in monosodium glutamate (MSG) treated rats

Discussion

In the present study, the MSG control rats showed dyslipidemia and oxidative stress perhaps due to the destruction of hypothalamic arcuate nucleus,which is in agreement with previous reports.[25-28] The increased total cholesterol levels and hypertriglyceridemia in MSG-treated animals is a high risk dysmetabolic situation. Metabolic disturbance is the main cause of dyslipidemia, which is a major risk factor for cardiovascular diseases.[29] Administration of AqE-TFG or orlistat resulted in significant reduction in levels of TC, TGs and elevation in HDL-C which is similar to other findings.[30] It is well known that hyperlipidemia decreases the strength of the antioxidative defense system.[31] Thus, the present study hypothesizes that the possible explanation for improvement in dyslipidemia following administration of AqE-TFG or orlistat may be due to reduction in oxidative stress in MSG-treated rats.

As per earlier reports,[32] the ALT enzyme is a sensitive marker of liver damage and AST levels are predictive of damage to the liver and other organs with high metabolic activity (brain, heart, and lungs). Moreover, LDH is a sensitive biomarker for cardiac diseases. Hence any necrosis or membrane damage to the liver and heart leads to leakage of these enzymes into the blood circulation.[2833-35] The results of our study showed that MSG-treated rats were more prone to hepatotoxicity and cardiotoxicity as evidenced by increased levels of serum LDH, AST, and ALT. Administration of AqE-TFG significantly reduced the elevated LDH, AST, and ALT levels, which could be attributed to the protective effect on hepatic and cardiac tissues.

The serum malondialdehyde (MDA) concentration, a marker of lipid peroxidation, increased in MSG-treated rats. GSH play an important role in catalysis, metabolism, and transport. It protects the cells against free radical peroxides and other toxic compounds.[3637] The reduction in GSH levels in our study are in agreement with Onyema et al.,[28] who suggested that MSG-induced lipid peroxidation contributed to the depletion of tissue levels of GSH. SOD catalyzes the breakdown of superoxide anion into oxygen and hydrogen peroxide,[38] and CAT is a key component of the antioxidant defense system, which catalyzes the conversion of hydrogen peroxide to water and oxygen, using an iron or manganese cofactor.[39] In our study, MSG control rats showed decreased activities of SOD and CAT enzymes, which is again in agreement with earlier reports.[56] Therefore, it appears that MSG causes the induction of oxidative stress in the liver and heart, which as a consequence, may lead to liver disorders (like dyslipidemia) and cardiac complications (like atherosclerosis).

Treatment with AqE-TFG caused significant reduction in lipid peroxides levels and elevation in antioxidant enzymes GSH, SOD, and CAT indicating the protection of the hepatic and cardiac tissues from the damaging effect of MSG. The results of our study show that the indices of hepatic and cardiac risk predictor (TC, TGs, HDL-C), hepatic and cardiac risk biomarkers (LDH, AST, and ALT), indices of lipid peroxidation (MDA) and the hepatic and cardiac tissue antioxidant enzymes (GSH, SOD and CAT) were significantly restored to normal levels with AqE-TFG treatment. Results were also comparable with orlistat, a standard antiobesity drug.

The major constituents of AqE-TFG are saponin glycoside, carbohydrates, and proteins. It is well-established that saponins are known to be hypocholesterolemic and antioxidant in animal species.[4041] Therefore, it is possible that the presence of saponins in AqE-TFG is responsible for the observed lipid lowering and antioxidant activity. In conclusion, Trigonella foenum-graecum seems to have an important role in preventing the development of MSG-induced dyslipidemia and oxidative stress. The protective effect of Trigonella foenum-graecum may be related to its free radical scavenging and membrane stabilizing property, and may be helpful in protection from the metabolic disorders like dyslipidemia and atherosclerosis.