Food and Chemical Toxicology 37 (1999) 831838

Research Section

www.elsevier.com/locate/foodchemtox

Acute and Subchronic Toxicity Assessment of Debitterized Fenugreek Powder in the Mouse and Rat
MURALIDHARA1*, K. NARASIMHAMURTHY1, S. VISWANATHA1 and B. S. RAMESH2
1

Department of Biochemistry & Nutrition and 2Technology Transfer & Business Development, Central Food Technological Research Institute, Mysore-570 013, India (Accepted 8 March 1999)

AbstractIncreased human use of fenugreek seeds (Trigonella foenum graecum) entails the generation of toxicity data in experimental animals. In this investigation, toxic eects of debitterized fenugreek (DFG) powder have been assessed following acute and subchronic regimens in mice and rats. In the acute study, DFG powder intragastrically administered to albino mice (CFT-Swiss, Mus musculus) and albino rats (CFT-Wistar, Rattus norvegicus) of both sexes failed to induce any signs of toxicity or mortality up to a maximum practical dosage of 2 and 5 g/kg body weight, respectively. Further, no signicant alterations either in relative organ weights or their histology were discernible at terminal autopsy. In the 90-day subchronic study, DFG fed to weanling rats of both sexes at dietary doses of 0, 1, 5 and 10% in a pure diet had no eect either on the daily food intake or growth. Terminal autopsy revealed no alterations in relative organ weights of various vital organs, or their histoarchitecture. Haematological constants in DFG-fed rats were on par with those of controls. Further, biochemical measurements in serum and liver of DFG-fed rats revealed no appreciable changes in various parameters such as enzyme levels of GPT , GOT and ALP, as well as many serum constituents such as proteins, cholesterol, urea and creatinine at any of the dietary levels. From these results, it may be concluded that DFG does not produce any signicant acute and cumulative toxicity at the doses administered, as reected by the various parameters investigated. # 1999 Published by Elsevier Science Ltd. All rights reserved Keywords: debitterized fenugreek; acute/subchronic toxicity; rats and mice.

INTRODUCTION

Fenugreek (Trigonella foenum graceum) is a leguminous herb grown widely in India, Egypt and Middle Eastern countries, the seeds of which are commonly used as a condiment. Fenugreek seeds, like other seeds, are not cotyledenous but endospermic in nature, and are eshy, rich in bre and gum (Wealth Of India, 1976). In general, the nutrient composition of fenugreek seeds is (in g%): moisture 2.4; protein 25.4; fat 7.9; saponins 4.8; total dietary bre 48.0; insoluble bre 28; soluble bre 20; and ash 3.9. Fenugreek leaves are also consumed widely in India as a green leafy vegetable and are a rich source of calcium, b-carotene and many other vitamins.

*Corresponding author.

Primarily two pharmacological properties of fenugreek seeds, namely antidiabetic and hypocholesterolaemic activities in both experimental animals (Ajabnoor and Tilmisany, 1988; Madar, 1984; Ribes et al., 1984; Shani et al., 1974; Sharma, 1986a; Valette et al., 1984) and human subjects (Madar et al., 1988; Sharma, 1986b; Sharma and Raghuram, 1990) have been adequately demonstrated. These benecial properties of fenugreek seeds have attracted wide attention in the recent past (Sharma, 1991) due to their therapeutic potential. In their attempts to identify the component(s) responsible for the observed hypocholesterolaemic and antidiabetic activities, researchers have employed defatted fenugreek our (Ribes, 1986; Sharma, 1986a), fractions rich in testa and endosperm (Ribes et al., 1984), gum fractions (Madar et al., 1988) and isolated galactomannan (Madar and Shomer, 1990). The reported chemical analysis

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of the fenugreek seeds has shown the presence of antinutritional factors such as saponins (Rao and Sharma, 1987) and alkaloids (Shani et al., 1974). Human use of fenugreek is expected to increase in view of its benecial properties. Earlier researchers (Sharma et al., 1996) fed patients fenugreek in the range of 25 to 100 g/subject for 24 wk during their clinical studies. As defatted seeds are known to be devoid of bitter taste, recently debitterized fenugreek (DFG) powder is being incorporated as a bre source in certain dietary formulations targeted for the general population. One such formulated 1 product, (Fibernat ) has 49% DFG, and the recommended dose for general human consumption is one to three measures (one measure =10 g) per day. Thus, because of the widespread use of fenugreek seeds by the general population, and the anticipated increase in therapeutic use for both diabetic and hyperlipidaemic patients at higher doses, the presence of antinutritional/bitter factors and their incorporation into new dietary formulations necessitates the generation of comprehensive toxicological data to ascertain its safety. Hence, a programme was initiated to comprehensively assess the toxicological potency of DFG in experimental animals. In this paper, we report the studies conducted to evaluate the acute and subchronic oral toxicity in mice and rats.

groundnut oil, 9.0%; mineral mixture, 2.0%; vitaminized premix, 1.0% and vitaminized oil, 1.0%) was used (Muralidhara and Krishnakumari, 1983; Rajini and Krishnakumari, 1989). DFG powder was incorporated into the diet at three levels, 1, 5 and 10 %. Fresh 6-kg batches of each diet were prepared twice a week and the control diets were prepared weekly. Acute toxicity in mice and rats In general, graded doses of DFG powder were administered intragastrically as a single dose to mice (0.252.0 g/kg body weight) or rats (1, 2, 4 and 5 g/kg body weight). The exact amount of DFG (in mg/kg) was calculated for each animal and administered by oral gavage. With rats, since the higher two doses were too large, the individual doses were divided into two equal doses and dosed at 2-hourly intervals. Prior to dosing, both mice and rats were fasted overnight. Mice and rats were observed thoroughly for the onset of any immediate toxic signs and also during the observation period to record any delayed acute eects. All animals were killed humanely after 14 days and selected vital organs were excised, blotted, weighed and processed for routine microscopic examination. Subchronic toxicity in rats Weanling rats of both sexes were randomly assigned to control and treatment groups. The rats were fed diets containing 0, 1, 5 and 10% DFG. Diets and water were given ad lib. for a continuous period of 90 days (9095 because of phased terminal autopsy). Monitoring daily food intake and weekly body weights, all the animals were observed thoroughly for the onset of any signs of toxicity. Terminally, all the rats were killed humanely (over a 5-day period) under light ether anaesthesia. Organ weights and histopathological studies. The following vital organs of each rat were excised, blotted and weighed and the organ/body weight ratios calculated: adrenals, brain, heart, kidneys, liver, lungs, ovaries, spleen and testes. Samples of these organs were xed in Bouins' xative, embedded in paran wax, sectioned at 5 mm and stained with haematoxylin and eosin. Detailed microscopic examination was carried out on all organs of both control and treatment groups of both sexes. Haematological constants. Blood samples were collected from a minimum of six rats from each group, and were examined for haemoglobin concentration, packed cell volume, erythrocyte, leucocyte and dierential counts. Biochemical analysis of serum and liver. During the second phase of the autopsy, whole blood taken from a minimum of six rats from each group was allowed to clot and the serum separated and analysed for alkaline phosphatase (ALP), aspartate aminotransferase (GPT), alanine aminotransferase

MATERIALS AND METHODS

Debitterized fenugreek powder Debitterized fenugreek powder (DFG) was supplied (single batch) by M/s Sterling Home Products (Chennai, India). The compound was in the form of a ne powder. Animals and maintenance Mice. Adult mice (CFT-Swiss strain, 8 wk old) of both sexes drawn from the stock colony were housed in groups of six in rectangular polypropylene cages with dust-free paddy husk as bedding material. Prior to the study, they were acclimatized for 1 wk by feeding on commercial pellet diet ad lib. Rats. Adult rats (CFT-Wistar strain, 810 wk old) of both sexes were used for the acute study, whereas weanling rats (28 days old) of both sexes were employed for the subchronic toxicity. Randomly drawn from the stock colony, they were held in separate individual cages, acclimatized for 1 wk on control diet prior to the start of the study. Diets and their preparation Mice and rats used for acute studies were fed the commercial pellet diet (M/s Gold Mohur, Lipton India Ltd). For the subchronic study in rats, a puried diet (corn starch, 69.4%; casein, 17.6%;

Toxicity assessment of debitterized fenugreek

Table 1. Acute dosing of debitterized fenugreek (DFG) to albino mice and its eect on body weight and organ weights Body weight (g) Dose (g/kg body weight) Male Control 0.25 0.50 1.00 2.00 Female Control 0.25 0.50 1.00 2.00 Initial 34.5 34.0 33.5 34.0 33.8 33.5 33.6 33.5 33.6 33.6 Final 40.5 39.5 40.0 38.5 37.6 21.07 32.4 32.5 31.5 32.6 32.5 20.83 Liver 4.96 5.20 5.25 5.07 4.95 20.17 4.75 4.70 4.57 4.98 4.52 20.12 Relative organ weight (g/100 g body weight) Lungs 0.53 0.55 0.54 0.51 0.55 20.04 0.70 0.64 0.62 0.61 0.61 20.05 Kidney 1.35 1.36 1.28 1.43 1.38 20.06 1.06 1.10 1.18 1.13 1.03 20.05

833

Spleen 0.26 0.30 0.33 0.34 0.30 20.03 0.39 0.40 0.38 0.39 0.40 20.03

No signicant dierences between control and DFG-fed groups.

(GOT), and constituentscholesterol, creatinine and urea employing standard kits (Span Diagnostics, Gujarat, India). Serum samples were also analysed for total proteins (Gornall et al., 1949). Enzyme activities, ALP, GOT, GPT and constituents such as proteins and cholesterol were also determined in frozen liver samples of all groups. Statistical analysis Data on weekly body weights, food intake, relative organ weights and biochemical determinations were analysed by one-way analysis of variance (Snedecor and Cochran, 1980). All comparison tests were two-tailed, and a probability level of P > 0.05 was considered signicant.
RESULTS

weight. Dosing of DFG to rats also failed to induce any signs of toxicity even at the highest dose of 5 g/ kg body weight. No mortality occurred in both species either immediately or during the 14-day observation period. Food intake, body weight gain, organ weights and pathology. Oral administration of DFG did not cause any appreciable alterations in the feed intake (data not shown) during 2 wk in both the species. Further, body weight gain during the observation period and the relative organ weights among the treated animals were comparable to their respective controls (Tables 1 and 2). No sex-related dierences were evident in either species. Various tissues such as liver, lungs, kidney and spleen of DFG-dosed animals showed no pathological alterations under light microscopy. Subchronic toxicity in rats Clinical signs and mortality. Dietary feeding of DFG for 3 months produced no obvious signs of toxicity or mortality during the experimental period at any of the concentrations in both sexes.

Acute toxicity in mice and rats Clinical signs and mortality. Mice administered DFG did not develop any clinical signs of toxicity either immediately or during the post-treatment period even at the highest dose of 2 g/kg body

Table 2. Acute dosing of DFG to albino rats and its eect on body weight and organ weights Body weight Dose (g/kg body weight) Male Control 1.0 2.0 4.0 5.0 Female Control 1.0 2.0 4.0 5.0 Initial 183.8 183.6 182.8 183.4 184.5 142.5 139.4 140.0 139.5 137.0 Final 240.0 231.0 232.0 226.0 228.6 25.46 167.6 166.0 169.4 165.6 159.0 26.93 Liver 5.07 4.76 4.62 4.39 5.04 20.14 4.40 4.23 4.68 4.14 4.77 20.10 Relative organ weight (g/100 g body weight) Lungs 0.47 0.56 0.46 0.50 0.49 20.03 0.59 0.56 0.56 0.56 0.56 20.03 Kidney 0.78 0.86 0.82 0.77 0.77 20.46 0.81 0.77 0.78 0.84 0.84 20.04 Spleen 0.33 0.22 0.28 0.28 0.27 20.03 0.29 0.31 0.30 0.31 0.32 20.04

No signicant dierences between control and DFG-fed groups.

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Fig. 1. Mean absolute body weights of male rats fed dietary debitterized fenugreek (DFG) for 90 days.

Body weights and growth. Data on absolute body weights of weanling rats of both sexes fed DFG at various concentrations (1, 5 and 10%) are presented in Figs 1 and 2. As such, DFG did not cause any signicant change in body weight gain in either sex. While the pattern of weight gain was similar in both sexes, the total average weight gain in females was lower compared with males.

Food intake, total DFG intake and organ weights. In general, incorporation of DFG even at the 10% level did not signicantly aect the food intake in treatment groups (Tables 3 and 4). No meaningful dierences in the actual food consumption was observed in the treatment groups in both sexes throughout the 13-wk experimental period. As a result, growth of DFG-fed rats was consistently

Fig. 2. Mean absolute body weights of female rats fed dietary debitterized fenugreek (DFG) for 90 days.

Toxicity assessment of debitterized fenugreek

Table 3. Weekly food intake pattern among growing rats fed dietary DFG for 90 days Food intake (g/rat/wk) Group/wk Male C 1% DFG 5% DFG 10 % DFG Female C 1% DFG 5% DFG 10% DFG 1 64.8 66.1 65.5 63.4 59.6 62.3 61.8 64.0 2 69.9 69.9 69.8 69.5 67.0 69.5 68.2 69.6 3 91.6 96.9 96.1 95.7 83.6 80.1 76.5 84.1 4 100 103 101 100 78.4 75.9 73.7 78.2 5 100 96.0 94.6 102 79.8 74.0 73.9 76.3 6 75.5 68.5 68.0 76.3 55.5 55.1 51.0 49.5 7 96.6 90.3 91.0 91.5 81.5 75.8 75.0 76.3 8 89.9 84.5 85.6 94.5 69.0 67.3 78.0 73.5 9 91.8 89.6 88.9 91.2 65.5 73.7 67.5 68.8 10 98.6 102 98.2 100 76.7 75.6 74.6 78.5 11 89.9 95.0 91.4 94.9 71.2 70.8 69.1 67.7 12 93.1 93.8 89.6 89.2 65.6 68.7 66.6 65.5

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13 93.2 92.7 92.8 93.6 82.4 78.3 76.5 82.3

C = control; DFG = debitterized fenugreek.Each value is mean of 12 rats. Data analysed by analysis of variance. No signicant dierences between control and DFG-fed groups.

similar to those of respective controls. The total DFG consumed calculated based on the total average food intake/rat/90 days is as follows. Males: 1% level, 11.48 g/rat; 5% level, 56.60 g/rat; 10% level, 116.2 g/rat: females: 1% level, 9.28 g/rat; 5 % level, 45.6 g/rat and 10% level, 93.5 g/rat. There were no marked dierences in the group mean absolute or relative weights of various vital organs in the DFG-treated rats compared with the controls (Table 4). Haematological prole. Data on the various haematological parameters measured in rats fed DFG at all levels are presented in Table 5. Feeding DFG even at 10% in the diet caused no signicant alterations in Hb, RBC, WBC and dierential counts. Histopathology. Gross examination did not reveal any abnormalities that could be ascribed to DFG feeding in rats of both sexes. Further, on microscopic examination, no treatment-related pathological lesions were evident in any of the vital organs irrespective of the DFG level. Serum biochemical parameters. Data on various biochemical parameters measured in the serum of control and DFG-fed rats of both sexes is presented in Table 6. There were no marked alterations in any of the specic activities of enzymes, GPT, GOT

and ALP in DFG-fed rats. A similar trend of results was observed with regard to the levels of serum constituents, proteins, cholesterol, creatinine and urea. Hepatic enzymes and constituents. The specic activities of hepatic enzymes, GPT, GOT and ALP in the DFG-fed rats were highly comparable to those of controls among both sexes. Hepatic concentrations of protein and cholesterol were in the normal range in rats fed DFG at all levels (Table 7).
DISCUSSION

Although commonly used as a condiment in various part of the world, there are no available data or estimates on the actual consumption of fenugreek seeds by humans. However, with human subjects, fenugreek seed powder at doses ranging from 25 to100 g/subject have been employed while testing its ecacy in the management of non-insulin-dependent diabetes (NIDD) (Sharma and Raghuram, 1990; Sharma et al., 1996). The basis for these doses stems possibly from the earlier data in experimental animals. The antidiabetic as well as the hypolipidaemic properties of fenugreek seeds have been adequately

Table 4. Relative organ weights of rats fed dietary DFG for 90 days Body weight Group Male C 1% DFG 5% DFG 10% DFG Female C 1% DFG 5% DFG 10% DFG Final 318.1 318.0 306.9 306.2 SEm (44 df) 200.7 195.33 187.92 195.33 SEm (44 df) Liver 3.24 3.22 3.20 3.20 20.09 3.28 3.16 3.22 3.10 20.10 Lungs 0.43 0.41 0.43 0.42 20.02 0.52 0.49 0.56 0.53 20.02 Relative weight (g/100 g body weight) Kidney 0.56 0.59 0.57 0.57 20.01 0.72 0.61 0.65 0.64 20.04 Heart 0.28 0.28 0.28 0.28 20.01 0.32 0.326 0.318 0.319 20.011 Spleen 0.21 0.22 0.21 0.22 20.01 0.28 0.26 0.28 0.24 20.02 Adrenal 0.023 0.024 0.024 0.025 20.002 0.034 0.028 0.036 0.034 20.002 Brain 0.56 0.56 0.58 0.58 20.01 0.84 0.84 0.79 0.84 20.04 Testis 0.95 0.95 0.98 0.99 20.02 Ovary 0.053 0.048 0.056 0.055 20.004

C = control; DFG = debitterized fenugreek. Each value is mean of 12 rats. Data analysed by analysis of variance. No signicant dierences between control and DFG-fed groups.

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Table 5. Haematological prole of rats fed dietary DFG for 90 days DC (%) Hb (g/dl) M F 15.1 14.4 14.0 14.3 20.4 RBC (106 ml) M 9.8 11.4 12.2 11.2 20.6 F 11.1 11.5 11.7 9.9 20.6 WBC (ml) M 11375 14017 8833 8408 21472 F 10483 10867 10567 7992 21215 PCV (%) M 38.7 38.5 36.7 36.5 21.02 F 35.3 36.2 35.0 36.3 21.1 L 85 85 85 86 P 12 13 12 11 M M 2 2 3 2 E 1 1 B L 85 84 85 82 P 12 13 13 15 F M 3 2 2 2 E 1 1 B

C 1% DFG 5% DFG 10% DFG SE m (20df)

14.6 14.3 14.1 14.0 20.3

C = control; DFG = debitterized fenugreek; M = male; F = female; Hb = haemoglobin; DC = dierential counts; L = lymphocytes; P= polymorphs; M = monocytes; E = eosinophils; RBC = red blood cells; WBC = white blood cells; PCV = packed cell volume. No signicant dierences (P > 0.05) was found between the control and DFG-fed groups.

Table 6. Serum enzymes and biochemical constituents of rats fed dietary DFG for 90 days IU/ml Group Male C 1% DFG 5% DFG 10%DFG SEm (12 df) Female C 1% DFG 5% DFG 10% DFG SEm (12 df) GPT 41.50 39.50 38.50 39.00 23.33 31.50 29.75 30.25 34.25 22.63 GOT 197.5 195.0 204.75 201.25 24.05 225.25 220.50 233.25 236.50 25.02 ALP 54.85 54.12 53.22 49.00 22.62 44.15 54.75 47.60 41.98 24.61 Protein (mg/ml) 56.75 64.00 60.50 54.75 22.94 65.25 68.75 62.75 70.00 23.79 Cholesterol (mg/100 ml) 77.62 77.62 79.25 80.62 21.27 78.88 77.62 77.75 80.38 20.92 Creatinine (mg/ml) 0.72 0.75 0.74 0.74 20.028 0.86 0.82 0.75 0.83 20.05 Urea (mg/ml) 30.72 25.41 31.99 24.50 22.58 30.18 28.17 23.13 25.66 23.38

C = control; DFG = debitterized fenugreek. Each value is mean of four rats. Data analysed by analysis of variance. No signicant dierences (P > 0.05) was found between the control and DFG-fed groups.

demonstrated in mice (Ajabnoor and Tilminsay, 1988), rats (Madar, 1984; Ribes et al., 1984; Shani et al., 1974; Sharma, 1984, 1986a), dogs (Ribes et al., 1987; Valette et al., 1984) and NIDD human subjects (Sharma, 1986b; Sharma and Raghuram, 1990; Sharma et al., 1996). Despite its recommended use at high doses, studies to elucidate

the possible toxicological eects following short- and long-term consumption in experimental animals are scarce (Bhat et al., 1985; Rao et al., 1996). It is in this context that the present study assumes relevance. Further, this is the rst study conducted employing a commercially debitterized fenugreek (DFG) powder for toxicity assessment in rodent species.

Table 7. Biochemical analysis in liver of rats fed dietary DFG for 90 days Protein (IU/ml) Group Male C 1% DFG 5% DFG 10% DFG SEm (12df) Female C 1% DFG 5% DFG 10% DFG SE m (12 df) GPT 128.50 105.75 135.0 114.50 212.78 150.50 150.25 172.00 158.25 210.31 GOT 296.25 277.50 295.25 296.75 230.04 246.00 298.25 286.25 253.75 215.96 ALP 0.478 0.546 0.585 0.468 20.04 0.346 0.378 0.339 0.288 20.038 Protein (mg/g) 135.00 133.00 120.50 132.50 25.91 108.50 110.50 104.00 108.50 26.65 Cholesterol (mg/g) 2.75 3.06 2.98 2.49 20.12 3.18 2.95 3.15 2.85 20.14

C = control; DFG = debitterized fenugreek. Each value is mean of four rats. Data analysed by analysis of variance. No signicant dierences (P > 0.05) was found between the control and DFG-fed groups.

Toxicity assessment of debitterized fenugreek

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Our results of the acute study suggest that DFG powder is well tolerated up to maximum practical doses of 2 and 5 g/kg body weight in mice and rats, respectively. These doses were considered as the `test limit' as per the recommended acute toxicity testing procedures, and administration of further higher doses was considered physiologically unsound and is not generally recommended (Hayes, 1989). The highest dose administered for rats (5 g/ kg), if extrapolated on a body weight basis to humans, corresponds to 250 g/50-kg adult, which is nearly 2.5- to 10-fold higher than is recommended for human use in the case of diabetic and hyperlipidaemic patients. In the acute study, the absence of symptoms, no negative impact on growth, and the normal state of vital organs in terms of weight/histology, suggest that DFG is practically non-toxic to both rodent species. In the subchronic study, weanling rats accepted the DFG-incorporated diet even at 10% level from the rst week (Table 4). This is in contrast to reported results with unprocessed fenugreek powder ( Rao et al., 1996) suggesting the advantages of debitterization process. The food eciency ratio was not altered and the growth of rats in the DFG group was comparable to controls, clearly indicating that DFG does not interfere in the normal growth process. At necropsy, we observed no signicant alterations in absolute/relative weights of any vital organs among the DFG-fed rats of both sexes. However, Rao et al. (1996) recently found increased hepatic weights (unaccompanied by pathological lesions) in only female rats fed fenugreek seeds at 10% and 20% levels. As such, no specic reason(s) have been ascribed by the earlier authors. The normal range of haematological constants such as Hb, RBC, WBC and dierential counts observed in all the treatment groups clearly suggested the absence of any haematotoxic potency of DFG. Except for minor occasional alterations in the serum biochemical parameters, no appreciable alterations were evident in DFG-fed rats in the specic activities of serum enzymes generally used as diagnostic indicators of liver damage. Further, no signicant alterations in the hepatic levels of certain enzymes and constituents in the DFG-fed rats clearly indicates an absence of hepatotoxicity. In conclusion, the results of the present study clearly show that debitterized fenugreek powder lacks the propensity to induce acute toxic eects in both mice and rats. Further, the compound is well tolerated up to a 10% dietary level as evidenced by the absence of any eect on growth, organ weights/ histology and haematological parameters. The normal range of biochemical parameters in the serum and liver of DFG-fed rats clearly suggests that DFG causes no disruption of normal physiological and biochemical homeostasis.

AcknowledgementsThis work was supported by a Grant from M/S Sterling Home Products, Chennai, India. We wish to thank Dr S. G. Bhat, Head of the Dept and Dr V. Prakash, Director of the Institute for their keen interest in this investigation.

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