Dose-dependent effects, safety and tolerability of fenugreek in diet-induced metabolic disorders in rats
© Muraki et al.; licensee BioMed Central Ltd. 2011
Received: 24 November 2011
Accepted: 21 December 2011
Published: 21 December 2011
We previously reported that fenugreek (Trigonella foenum-graecum L.) improved diet-induced metabolic disorders in rats. The purpose of the present study was to examine the dose-dependent effects, safety and tolerability of fenugreek.
The diets used in this study were the high-fat high-sucrose diet (HFS; lard 50%kcal, sucrose 25%kcal) as a control (Ctrl group) or the HFS containing 0.25% (VL group), 1.25% (L group), 2.50% (M group), 5.00% (H group) or 12.30% (VH group) fenugreek based on the modified version of the AIN-93G purified diet.
Fenugreek dose-dependently reduced the hepatic triglyceride and total cholesterol levels. Fenugreek also dose-dependently increased the excretion of cholesterol and total bile acids into the feces. However, the glucose tolerance showed no significant change by fenugreek administration. The VL and L groups did not significantly change triglyceride or total cholesterol levels in the liver. The VL group showed no increase in excretion of triglyceride, total cholesterol or bile acids in the feces. The VH group showed appetite reduction and diarrhea, while no adverse effect or symptoms were observed in the M group.
These results suggest that fenugreek inhibited lipid accumulation in the liver by increasing the lipid excretion in the feces. The effective, safe and tolerable dose of fenugreek was found to be around 2.50% (w/w).
Recently, the incidence of lifestyle-related diseases including metabolic disorders has been expanding in parallel with rapid economic development in advanced countries. The principle therapeutic policy for these diseases lies in the improvement of dietary habits and the increase of physical activity. However, it is difficult to control and modify one's usual life style. In that case, functional foods with preventive and therapeutic effects on metabolic disorders are very helpful for the improvement of lifestyle-related diseases. On the other hand, the excessive intake of some functional foods may result in adverse effects. However, a lot of such functional foods are actually available at the market, though their safety has not been confirmed .
It has been reported that the active components of fenugreek, 4-hydroxyisoleucine and galactomannan, lowered blood glucose levels and improved lipid metabolism in vivo[2–5]. Fenugreek also contains dioscin, a steroidal saponin, and trigonelline, an alkaloid. These phytoestrogens have estrogen-like activities in vitro[6, 7]. In addition, fenugreek contains a large quantity of insoluble dietary fibers. These active components might cause several adverse reactions including hypoglycemia, diarrhea and galactorrhea, when an excessive amount of fenugreek was ingested [2, 8, 9].
The purpose of this study was to examine the dose-dependent effects, safety and tolerability of fenugreek.
Animals and housing
Three-week-old male, Sprague-Dawley rats were obtained from CLEA (Tokyo, Japan). Rats were housed individually in stainless steel wire-bottom cages in a room maintained at 22 ± 2 °C and 55 ± 5% relative humidity with a 12 h cycle of light and dark. Rats were given free access to tap water throughout the experiment. They were fed a commercial diet (CE-2; CLEA) for 1 wk prior to the experiment. The experimental protocol was approved by the Institutional Animal Care and Use Committee of Josai University.
Oral glucose tolerance tests and intraperitoneal insulin tolerance tests
At the 10th wk, the rats were deprived of food for 12 h. Then they were given oral glucose (2 g/kg body weight) for the oral glucose tolerance test (OGTT) and an intraperitoneal injection of human regular insulin (0.75 U/kg body weight) for the intraperitoneal insulin tolerance test (IPITT). Blood glucose concentrations were measured by Ascensia™ DexterZII (Bayer Medical, Tokyo, Japan) using tail blood samples at 0, 30, 60 and 120 min after glucose administration and at 0, 15, 30, 45, 60 and 120 min after insulin administration. Plasma insulin concentrations were measured using a commercial kit (Rat Insulin ELISA kit, Shibayagi Co., Gunma, Japan) using separated tail plasma samples at 0, 30, 60 and 120 min after glucose administration.
Plasma triglyceride, total cholesterol, AST and ALT were measured by colorimetric slides using the Fuji Dri-Chem 3500 (Fujifilm Corp., Tokyo, Japan).
Hepatic and fecal lipids concentrations
The hepatic and fecal lipids were extracted in accordance with the method of Bligh and Dyer  and the method of Folch , respectively. Each extract was solubilized by Triton-X100 (Wako Pure Chemical Industries, Osaka, Japan), and the lipids concentrations were determined enzymatically by means of commercial kits (Triglycerides E-Test Wako, Cholesterol E-Test Wako, Total Bile Acid Test Wako; Wako Pure Chemical Industries).
All data are expressed as the mean ± SEM. Statistical analyses were carried out using Statistical Package for Social Sciences (SPSS12.0J for Windows; SPSS Japan, Tokyo, Japan). In the three-group comparison, the effects of treatment were analyzed using one-way analysis of variance (ANOVA), and the differences among means were tested by means of the Tukey's honestly significant difference (HSD) test. In the two-group comparison, the effects of treatment were analyzed through a T-test. In OGTT and IPITT, the effects of treatment were analyzed by means of repeated ANOVA. Differences were considered significant at P < 0.05.
Effect of fenugreek on the body weight, energy intake, tissue weights and plasma parameters
Effect of fenugreek on the body weight, energy intake, tissue weights and plasma
Body weight gain (g)
500.6 ± 18.5a
491.2 ± 10.2a
486.7 ± 18.5a
463.9 ± 10.2a
480.6 ± 18.5a
408.4 ± 10.2b
15.6 ± 0.3a
15.3 ± 0.1a
15.6 ± 0.3a
14.6 ± 0.2ab
15.4 ± 0.3a
13.4 ± 0.5b
Liver weight (g)
19.3 ± 1.1
20.0 ± 0.6
18.3 ± 0.7
17.2 ± 0.6
18.0 ± 0.8
17.3 ± 1.0
EWAT2 weight (g)
15.3 ± 0.5a
15.9 ± 0.9a
13.6 ± 0.7ab
14.5 ± 0.7a
16.3 ± 1.4a
10.1 ± 1.0b
IBAT3 weight (g)
0.77 ± 0.04
0.76 ± 0.04
0.74 ± 0.05
0.83 ± 0.05
0.78 ± 0.08
0.55 ± 0.03*
Plasma TG4 (mmol/L)
1.64 ± 0.51
1.98 ± 0.61
2.06 ± 0.29
1.79 ± 0.29
3.63 ± 0.73
2.47 ± 0.46*
Plasma TC5 (mmol/L)
1.50 ± 0.14
1.94 ± 0.14
1.73 ± 0.17
1.83 ± 0.17*
1.72 ± 0.13
1.70 ± 0.12
66.2 ± 8.0
76.2 ± 10.6
94.3 ± 21.7
98.5 ± 25.7
67.7 ± 9.1
74.8 ± 8.2
17.3 ± 0.7
21.2 ± 3.2
22.5 ± 3.9
25.0 ± 4.6
19.2 ± 2.4
22.2 ± 1.4
1.08 ± 0.11
1.03 ± 0.04
1.14 ± 0.06
1.05 ± 0.08
1.02 ± 0.09
0.95 ± 0.07
No significant difference was found in AST, ALT levels or AST/ALT in the liver among the groups (Table 2). Thus fenugreek intake did not show any liver injury.
Effects of fenugreek on glucose tolerance
Insulin resistance index
Fasting blood glucose (mmol/L)
6.27 ± 0.24a
6.19 ± 0.22ac
5.50 ± 0.28ab
5.20 ± 0.15bc
5.60 ± 0.34ab
5.03 ± 0.11b
Fasting blood insulin (pmol/L)
129 ± 37
167 ± 35
56 ± 27
117 ± 29
94 ± 33
54 ± 10
1.00 ± 0.28
1.69 ± 0.49
0.85 ± 0.52
0.78 ± 0.20
0.98 ± 0.36
0.35 ± 0.06
Lipids and bile acid levels in plasma, liver and feces
Hepatic steatosis is a risk factor for liver cirrhosis and atherosclerosis. Both the liver and adipocytes play a major role in the regulation of cellular and circulating serum lipids, predominantly triglyceride and cholesterol. In our previous studies, fenugreek decreased the hepatic triglyceride and total cholesterol levels in normal rats fed with a high-fat high-sucrose diet [13, 14]. The present study investigated dose-dependent effects of fenugreek and its safety and tolerability.
The body weight gain and EWAT and IBAT weights in the VH group decreased significantly as compared with all of the other groups. In a pair feeding procedure, the VH group showed slightly less food intake than the other groups. It has been reported that the reduced food intake in the presence of soluble fibers such as galactomannan contained in fenugreek was caused by delaying gastric emptying and promoting satiety. In our study, the ratio of body weight gain to food intake was similar among the groups . However, the VH group showed significant lower levels of the energy efficiency ratio (EER) compared to the Ctrl group. Therefore, we suspect that the decreases in body and tissue weight were caused by the decrease of EER rather than the decrease in energy intake.
Fenugreek dose-dependently reduced the hepatic triglyceride and total cholesterol levels. Whereas the fecal triglyceride excretion levels rose significantly only in the VH group, fenugreek dose-dependently augmented the fecal total cholesterol and the bile acid excretion levels. The plasma triglyceride and total cholesterol levels were not significantly different among the groups. These results suggest that the mechanism underlying the inhibition of lipid accumulation in the liver and the adipose tissue would have enhanced the total cholesterol and the bile acid excretion in feces. Likewise, the increase of triglyceride excretion led to these results in the VH group.
Saponins, such as diosgenin contained in fenugreek, form large micells from bile acid and saponin molecules in the small intestine, and these micelles inhibit the cholesterol absorption by directly excreting cholesterol in feces [15, 16]. Diosgenin also reduces the triglyceride content and mRNA expression levels of lipogenic genes (FAS, SCD-1 and ACC) and suppresses LXRα transactivation. This leads to down-regulation of both the mRNA and protein expression levels of SREBP-1c in HepG2 cells . These results obtained from the previous reports supported that fenugreek attenuated lipid accumulation in the liver by down-regulating lipid synthesis as well as increasing lipid excretion in the feces.
The fasting blood glucose levels decreased significantly in the M and VH groups as compared with the Ctrl group, but the fasting plasma insulin levels did not differ significantly among the fenugreek-administered groups as compared with the Ctrl group. HOMA-IR was not significantly different among the groups. Therefore, in these experiments, fenugreek did not affect the glucose tolerance. However, some improving effects on the glucose metabolism and glucose tolerance of fenugreek have been reported. For example, 4-hydroxyisoleucin contained in fenugreek stimulates insulin secretion [2, 18]. In OGTT in our experiment, the fenugreek administration did not significantly change the plasma insulin levels or blood glucose levels except at 120 min in the VH group. Further, fenugreek inhibited insulin secretion only in the VH group. It has been reported that fenugreek also enhances insulin sensitivity through the activation of insulin signaling at an early stage in peripheral tissues and liver . This effect is brought by the activation of glucose and lipid metabolism with up-regulation of several enzymes [19–23] and the increase of glycogen synthesis in the muscle and liver [22–24]. In addition, diosgenin enhances the peroxisome proliferator-activated receptor-γ (PPARγ) level in EWAT and promotes both the adipocyte differentiation and the size reduction. As a result, the secretion of monocyte chemoattractant protein-1 (MCP-1) in adipocytes is suppressed, while the secretion of adiponectin is promoted, and the inflammation in adipose tissue is inhibited [18, 25]. Therefore, we assume that fenugreek activated firstly insulin sensitivity rather than insulin secretion in the relatively mild metabolic disorders we generated in rats through high-fat high-sucrose diets. The VH group showed appetite reduction and diarrhea, while no rats in the M group showed any adverse effects or symptoms.
These results suggest that fenugreek dose-dependently inhibited lipid accumulation in the liver by increasing the lipid and bile acids excretion in the feces, and that an effective, safe and tolerable dose of fenugreek was around 2.50% (w/w).
Department of Clinical Dietetics & Human Nutrition, Faculty of Pharmaceutical Sciences, Josai University, Saitama, Japan.
This study was supported by Urakami Foundation (Tokyo, Japan) and House Foods Corporation (Tokyo, Japan).
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