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fenugreek are in Brăila and Constanţa counties. The flowers develop in elongated pods enclosing 10-20 yellow-to-brown prismatic seeds, with a strong smell of.

Annals of RSCB

Vol. XV, Issue 2

TRIGONELLA FOENUM-GRAECUM (SICKLEFRUIT FENUGREEK) SEEDS – A NATURAL HEPATOPROTECTOR THAT PREVENTS ETHANOL-INDUCED TOXICITY Corina Rosioru1, G. Pribac2, Iuliana Simeoni1, C. Craciun1, A. Ardelean2 1

SCHOOL OF BIOLOGY AND GEOLOGY, BABEŞ-BOLYAI UNIVERSITY, CLUJNAPOCA, ROMANIA; 2SCHOOL OF MEDICINE, PHARMACY AND DENTAL MEDICINE, „VASILE GOLDIŞ” WESTERN UNIVERSITY, ARAD, ROMANIA

Summary Fenugreek has been used in empirical medicine and as a food additive since ancient times. Lately, its antioxidant and hypoglycemiant properties are intensely studied. This paper reports an investigation of hepatoprotective action of fenugreek seed flour in ethanolintoxicated rats. Animals received ethanol (EtOH) 10% v/v in drinking water, or EtOH and 5% (EtOH+T5%) and 10% (EtOH+T10%) respectively, fenugreek flour (w/w) in a standard diet. After 30 days of treatment, rats were sacrificed, and blood and liver tissue samples were collected for morphological and biochemical assays. The results were compared to values obtained from control animals, and from animals which received EtOH only. Most of the investigated parameters altered by EtOH intoxication were modified by fenugreek seeds in a beneficicial manner. Good effects were obtained especially for WBC count and enzymatic activities (ASAT, ALAT, LDH), and were more concludent with the higher (10%) fenugreek dose. The obtained results are discussed in comparison with data in the litterature. Key words: ethanol intoxication, fenugreek seeds, immunity, glycemia, transaminases.

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They are also a source of saponins such as diosgenin, yamogenin, gitogenin, and tigogenin. Seeds are rich in proteins and free amino acids (25%), alkaloids such as trigonelline and gentianine, and vitamins: B1, niacin (PP provitamin), and caroten (provitamin A). Triglycerides and phytosterols (9%) consist of lecithin, monoand polyunsaturated fatty acids. Fenugreek volatile oils have a high content of coumarin (the core skeleton of flavonoid compounds). Seeds also contain organic substances with sulfur, and mineral salts with iron, phosphorus and magnesium. The curative properties of fenugreek seeds are due in part to their polyphenolic flavonoid content: vitexin, tricin, naringenin, quercetin and tricin-7-O-β-Dglucopyranoside (Shang et al., 1998), with powerful antioxidant properties. 4Hydroxy-isoleucine, an amino acid extracted from fenugreek seeds, exhibits

Introduction Trigonella foenum-graecum (sicklefruit fenugreek) belongs to the family Leguminosae. It is one of the oldest medicinal plants, dating back to the ancient Egyptians, Greeks and Romans, who used it as a culinary and medicinal herb. Fenugreek is used both as a herb (the leaves) and as a spice (the seeds). In the Indian subcontinent, it is a component of curry. It is cultivated worldwide in semi-arid regions, especially in Europe and Asia. In our country, optimal areas to grow fenugreek are in Brăila and Constanţa counties. The flowers develop in elongated pods enclosing 10-20 yellow-to-brown prismatic seeds, with a strong smell of coumarin. The seeds contain water (12%), carbohydrates and mucosugars (30%): stachyose, starch, galactomannan, cellulose, with energetic and structural functions. 390

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hypoglycemiant, hypocholesterolemiant and antidiabetic effects (Vijayakumar et al., 2005; Handa et al., 2005). Numerous papers demonstrated the utility of fenugreek, as a dietary supplement, in the treatment of type 2 diabetes (Broca et al., 1999; Broca et al., 2004; Srinivasan, 2006; Salimuddin et al., 2003); only two studies, one on human Chang liver cells (Kaviarasan et al., 2006) and one in rats (Thirunavukkarasu et al., 2003) investigated the implications fenugreek might have in preventing and treating alcoholic liver disease. Vanadates represent a largely investigated auxiliary therapy in type 2 diabetes; however, vanadium compounds treatment has important side effects, induced especially by free radical generation. Because of its antioxidant properties, fenugreek is a good candidate to be used, together with vanadates: it can not only reduce the oxidative damages, but also may act as a hypoglycemiant agent (Yadav et al., 2004; Siddiqui et al., 2005; Preet et al., 2005). It is wellknown that an exagerated alcohol consumption induces a massive proliferation of free radicals in the hepatic tissue, resulting in alterations of membrane structures (Lieber, 2000), depletion of the glutathione-dependent antioxidant system (Tsukamoto and Lu, 2001; Lieber, 2002), together with impaired synthesis and export of liver proteins (Willis et al., 2002). Hyperglycemia and hypercholesterolemia (Carrasco et al., 2001) also accompany ethylic intoxication. In this context, we aimed to investigate the hepatoprotective potential of fenugreek seeds flour, in mid-term alcohol intoxication.

Plant Material Dried fenugreek seeds were purchased from S.C. ADSERV S.R.L., Braşov, Romania and ground into a fine powder. The powder was added to animals fodder as 5% (w/w), and 10% respectively. Animals Animals were white male Wistar rats, weighing 180±20 g, housed in the zoobase of Experimental Biology Chair, School of Biology and Geology. They were kept in hygienic conditions, under 12/12 h light/dark cycle, received a standard diet (S.C. Siamond Prod. S.R.L., Cluj Napoca, Romania), with or without added fenugreek seeds flower, and had ad libitum access to tap water. Rats were divided in four experimental groups. The control group (C) received only standard diet and tap water. The ethanol-treated group (EtOH) received the standard diet and 10% ethanol in the water. The next two groups (EtOH+T5% and EtOH+T10%) received the same volume of ethanol in the water, and the standard diet supplemented with fenugreek seed flour 5% (w/w), and 10% respectively. The experiment lasted 30 days. Animals were gently handled, without causing them stress or pain, and at the end of experiment, they were sacrificed under anesthesia. Assays Blood was collected from jugular vein and processed for morphological and biochemical examinations. Whole blood was used for red and white blood cell count, hematocrit (packed red cell volume) determination by Wintrobe micromethod, and blood smears to assess white blood cells differential. Hemoglobin concentration was determined with Drabkin reagent. Glycemia was also measured from whole blood. Serum was used for the determination of protein (Gornall et al., 1949) and cholesterol (Oser, 1965) concentrations, and for assessing the activities of lactate dehydrogenase (LDH) (Bergmeyer and Bernt, 1974) aspartate aminotransferase (ASAT) and alanine

Materials and Methods Chemicals All chemicals used in this study were of analytical grade and were purchased from Sigma-Aldrich Chemie GmBH, and Nordic Invest S.R.L., Cluj Napoca, Romania. 391

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aminotransferase (ALAT) (Reitman and Frankel, 1957). Liver samples were collected for measurement of glycogen content (Lo et al., 1970) and for preparation of homogenate in PBS. Homogenate was used for estimation of glucose (Nelson, 1944), total proteins, total cholesterol and activities of LDH, ALAT and ASAT. Data analysis The results are presented as means±SE. The data were analysed for statistical significance using Student’s t test. A value of P < 0.05 was considered significant: *. Significant at P < 0.01: ** ; significant at P < 0.001: ***.

previously documented the fluidizing effect of alcohol on RBC membranes (Hill et al., 1991; Verine et al., 1991), and the impaired deformability of RBC, necessary to maintain microcirculation systems (Oonishi and Sakashita, 2000). Alteration of RBC plasma membrane composition and architecture also impairs the capacity of these cells to bind and release respiratory gases. Therefore, a rise in RBC count may appear as an adaptative reaction of the organism, which needs more cells to vehiculate the same amounts of gases. On the other hand, the protective effect of fenugreek flavonoids on plasma membranes accounts for the lowering tendency of RBC count in EtOH+T groups. However, in animals which received fenugreek RBC count remained elevated, due to the antioxidant activity of flavonoids (Bravo, 1998) present in fenugreek seeds, thereby elevating the antioxidant capacity of the blood. Our results are consistent with the findings of other researchers (Esomonu et al., 2005), showing the influence of flavonoid compounds on RBC count and related parameters, in Wistar rats.

Results and discussion Red blood cell count (Table 1) was not modified significantly in any experimental group, as compared to the control. However, a 22% rise in erythrocyte number was noticed in the EtOH group, and a tendency to restore the control value (8.2% and -7.4%, respectively, as compared to the EtOH group), in the EtOH+T5% and EtOH+T10% groups. It have been

Table 1. RBC count, hematocrit, and hemoglobin concentration in alcohol and fenugreek treated rats

Parameters RBC (no. x 106/mL) Hematocrit (%) Hemoglobin (g/dL)

m±SE Sa Sb m±SE Sa Sb m±SE Sa Sb

Control 8.74±0.68

EtOH 10.66±1.16 NS

43.26±1.23

47.26±0.98 *

15.77±0.65

17.63±0.06 NS

EtOH+T5% 9.78±0.29 NS NS 46.19±0.97 * NS 16.88±0.19 NS NS

EtOH+T10% 9.87±0.61 NS NS 47.23±0.72 * NS 19.48±0.14 * ***

m±SE: mean value±standard error; Sa: statistical significance against the control group; Sb: statistical significance of fenugreek-treated groups against the EtOH group.

The hematocrit significantly increased in all experimental groups, as compared to the control (Table 1), paralleling the high RBC count. As the hematocrit increase in ethanol-treated animals (9.25%) only accounts for half of RBC count modification, a shrinkage of cells should be suspected, due to their

contact with the alcohol. Our data show that fenugreek did not induce any significant modification on the background of ethanol consumption. Hemoglobin concentration varied almost indentically to RBC count in EtOH and EtOH+T5% groups; in animals that received 10% fenugreek, values were 392

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significantly higer than in both control and EtOH groups (Table 1). The high iron content of fenugreek seed flour (Ibrahium and Hegazy, 2009) stimulated hemoglobin synthesis, even if rats were intoxicated with ethanol. WBC count was significantly elevated in animals that received ethanol only (Table 2), as it is wellknown that alcohol intoxication is associated with inflammatory processes in the hepatic tissue, triggered by activation of Kupffer cells and continued with neutrophils and lymphocytes invasion. There was a slow decrease of WBC count in the group receiving T5%, but only T10% restored this parameter to control value. Numerous papers reported an immunodeficiency produced by alcohol abuse, in both humans and animals (Thiele et al., 2005; Szabo, 1997; 1998). On the other hand, it is equaly known that organ damage by alcohol is exacerbated by autoimmune response to proteins biotransformed by alcohol

metabolites acetaldehyde and malondialdehyde (Klassen and Thiele, 1998). The two reactions seem to by contradictory, however each of these immune responses depend on many interacting factors (various cytokine production and their interaction, membrane receptors and their functionality, the ability of naive lymphocytes to differentiate into mature cells) which make it possible to have two separate manifestations. Differential WBC count, in blood smears stained with May Grünwald-Giemsa coloration and expressed as percentage of total WBC, showed different ratios in the experimental groups (Table 2). Ethanol treatment alone induced a considerable decrease of circulant neutrophils, and a spectacular rise of lymphocytes, while addition of fenugreek flour in the diet of alcoholic animals had an opposite effect: it strongly increased the cells of unspecific immunity and restored to the control value the cells of specific immunity.

Table 2. Total and differential WBC count in control, ethanol and fenugreek treated rats

Parameters WBC (no. x 103/mL)

Neutrophils (%)

Lymphocytes (%)

Monocytes (%)

Eosinophils (%)

Basophils (%)

m±SE Sa Sb m±SE Sa Sb m±SE Sa Sb m±SE Sa Sb m±SE Sa Sb m±SE Sa Sb

Control 13.53±0.75

EtOH 18.56±1.88 *

37.6±2.78

29.4±1.75 *

35.8±2.03

50.6±2.31 ***

15.4±0.51

7.6±0.81 ***

8.0±1.14

8.4±0.51 NS

2.8±0.73

4.0±0.70 NS

EtOH+T5% EtOH+T10% 17.67±1.99 14.04±1.17 * NS NS * 49.4±1.50 44.8±1.80 ** * *** *** 35.0±0.95 32.6±3.07 NS NS *** *** 5.2±0.58 9.0±1.22 *** *** * NS 7.6±1.21 11.2±0.58 NS * NS ** 2.8±0.20 3.0±0.32 NS NS NS NS

m±SE: mean value±standard error; Sa: statistical significance against the control group; Sb: statistical significance of fenugreek-treated groups against the EtOH group.

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Liver glycogen and glucose concentrations (Table 3) had a parallel evolution. Depletion of glycogen stores in alcohol intoxicated rats, also revealed in ultrastructure images, maintained in the group which received EtOH+T5%, with a slight tendency of restore only in the EtOH+T10% group. In the parenchymal cells of this group, electron microscopy images also showed the recurrence of glycogen granules (Pribac et al., in press). Liver glucose was significantly decreased (with 34%) in EtOH group, the tendency of return towards normal values being evident in the EtOH+T10% group (a decrease of only 17% as compared to the control). Our data are consistent with those reported by others (Fisher et al., 2002). Van Horn et al. ( 2001) showed that a low liver glycogen content in chronically ethanol intoxicated rats is the result of an impaired glycogen synthesis and loss of glycogen synthase. These authors, and Nanji et al. (1995) also reported alterations in glucose transporter proteins (especially GLUT-1) in the perivenous region of the liver lobule,

which may account for a low liver glucose concentration. Glycemia did not show significant changes in any experimental group, except a slight decrease in ethanol treated animals, due to impaired intestinal absorption of glucose and a decreased gluconeogenesis (Lieber, 2000; 2001). Numerous papers (Yadav et al., 2004; Siddiqui et al., 2005; Preet et al., 2005) highlighted the hypoglycemiant action of fenugreek seeds, suggesting their use as a nutraceutic in the treatment of diabetes; however, our experiment did not demonstrate such an effect. This might have two different reasons: on one hand, it may be possible that hypoglycemia is induced only in response to a high blood glucose level, as the one encountered in diabetes, and not on the background of a normal glucose concentration; on the other hand, the fenugreek hypoglycemiant effect might have been minimized by the short duration of the experiment (30 days). Both hypotheses are going to be checked out in further experiments.

Table 3. Glycemia, liver glycogen stores and glucose concentration in the liver of control, ethanol and fenugreek treated rats

Parameters Glycemia (mg/dL)

Liver glucose (mg/g) Liver glycogen (mg/100 mg)

Control m±SE 177.31±7.93 Sa Sb m±SE 31.52±0.75 Sa Sb m±SE 5.12±0.53 Sa Sb

EtOH 149.12±4.32 NS 20.73±1.32 * 3.34±1.06 NS

EtOH+T5% EtOH+T10% 177.27±4.15 165.45±4.00 NS NS NS NS 21.39±2.22 26.02±1.52 NS NS NS NS 3.28±0.81 3.79±0.75 * NS NS NS

m±SE: mean value±standard error; Sa: statistical significance against the control group; Sb: statistical significance of fenugreek-treated groups against the EtOH group.

Cholesterolemia and liver cholesterol concentration (Table 4) did not confirm, in our experimental model, the hypocholesterolemiant effect of fenugreek reported by other authors (Srinivasan, 2006; Reddy and Srinivasan, 2009; Belguith-Hadriche et al., 2010). On the contrary, the elevation of plasma cholesterol noticed in the EtOH group was amplified by the presence in the diet of

fenugreek flour (statistically significant higher values in the EtOH+T5% and EtOH+T10% groups). In animals which received 10% fenugreek in their diet, cholesterol concentration in the liver was also increased. It is worth to be mentioned that the above cited authors worked on animals in which a high level of cholesterol was experimentally induced, previously to fenugreek treatment. 394

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Table 4. Cholesterolemia, proteinemia and liver total cholesterol concentration in control, ethanol and fenugreek treated rats

Parameters

Control 76.05±1.97

m±SE Sa Sb m±SE Sa Sb m±SE Sa Sb

Cholesterolemia (mg/dL) Liver cholesterol (mg/g) Proteinemia (mg/dL)

8.33±0.65

7.03±0.02

EtOH EtOH+T5% EtOH+T10% 87.17±2.35 92.28±0.51 90.14±1.56 NS ** * NS NS 8.98±0.40 8.55±0.53 9.25±0.31 NS NS * NS NS 7.13±0.03 6.91±0.05 7.15±0.04 NS NS NS NS NS

m±SE: mean value±standard error; Sa: statistical significance against the control group; Sb: statistical significance of fenugreek-treated groups against the EtOH group.

Liver protein content (% control)

Serum protein concentration (Table 4) did not show notable modifications, values being similar in all experimental groups. Ethanol administered to rats for one month significantly increased protein concentration in the liver (Fig. 1). It has been demonstrated that acetaldehyde produced in the alcohol dehydrogenase reaction, and malondialdehyde that results from lipid peroxidation, have a pronounced tendency to form stable adducts with intracellular proteins (Niemelä et al., 1998;

Freeman et al., 2005). Immobilization of proteins in these adducts, together with altered plasma membrane permeability, impairs protein export from parenchymal cells and contributes to elevated liver protein content, in early stages of ethanol intoxication. In a more advanced stage of intoxication the entire energy metabolism is depressed, a shortage of available ATP occurs and both synthesis and export of liver proteins are altered. Our subchronic experiment revealed only the first phase of protein metabolism alteration.

70 60

**

Fig. 1. Total protein concentrations in the livers of control, ethanol intoxicated and fenugreek treated rats. **Significantly different from control (p < 0.01); xSignificantly different from EtOH (p < 0.05).

50 **

40 30 x

20 10 0 EtOH

EtOH+T5%

EtOH+T10%

The fenugreek seed flour added to the diet of ethanol intoxicated rats as 5%, significantly lowered hepatic protein concentration. The higher fenugreek dose had the same lowering effect, but less pronounced, so that in the EtOH+T10% protein concentration was still elevated, as compared to the control value. Serum and liver enzymes. Aspartate aminotransferase (ASAT) and alanine

aminotransferase (ALAT), together with lactate dehydrogenase (LDH) are common markers of plasma membrane integrity. Their concentrations in the serum are normally low, as they are considered „liver enzymes”, although they can be also found in lower concentrations in other tissues. A rise of the seric transaminases is usually considered a sign of altered plasma membrane structure, causing the „leaking” of enzymes outside the cells. In our experiment, 395

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seric ALAT was not modified (Fig. 2), in all groups its activity being in the normal range. ASAT activity in the serum (Fig. 3) was significantly increased in the EtOH group, as compared to the control. In animals which received a diet supplemented with seed flour ASAT level in the serum was lowered, as compared to both EtOH and control group, demonstrating a protective action of fenugreek on cellular membrane system. The main function of liver ALAT is alanine conversion to glucose: alanine serves as a carrier of ammonia and pyruvate carbon chain from muscle to liver, where ammonia enters the urea cycle and pyruvate is used as a gluconeogenic substrate. A low level of the enzyme was noticed only in EtOH+T5% group (Fig. 5).

Liver ASAT (Fig. 6) had considerably lower activities, as compared to both control and EtOH animals, in the fenugreek treated groups. This enzyme participates in malate-aspartate shuttle, the principal mechanism for the transport of reducing equivalents (in the form of NADH) from cytoplasm to mitochondria, thus maintaining a physiological NAD+/NADH ratio. The shuttle is usually very active in the alcoholic liver, where cytosolic oxidation of ethanol in the ADH reaction lowers this ratio, by raising NADH concentration. Our data did not emphasize any modification of ASAT activity in the EtOH group as compared to the control, which suggest that the short duration of intoxication only produced mild alterations of liver cells functions. However, the lowered ASAT activity in the groups receiving fenugreek (Fig. 7) may be considered benefical, as an indirect proof of unaltered redox status.

ALAT serum

ASAT serum 800

µg pyruvate/ml serum/h

µg pyruvate/ml serum/h

1200 1000 800 600 400 200

***

700

xxx

*

EtOH+T5%

EtOH+T10%

500 400 300 200 100 0

0 Control

EtOH

EtOH+T5%

Control

EtOH+T10%

ALAT liver

ASAT liver 9 8 7 6 5 4 3 2 1 0

30

*

15 10 5 0 Control

EtOH

EtOH+T5%

x

µg pyruvate/g/h

25 20

EtOH

Fig. 3. ASAT activity in the serum of ethanol and fenugreek treated rats. ***Significantly different from control (p < 0.001); *Significantly different from control (p < 0.05); XXXSignificantly different from EtOH (p < 0.001).

Fig. 2. ALAT activity in the serum of ethanol and fenugreek treated rats.

µg pyruvate/g/h

xxx

***

600

Control

EtOH+T10%

Fig. 4. ALAT activity in the liver of ethanol and fenugreek treated rats. *Significantly different from control (p < 0.05).

EtOH

xx

*

**

EtOH+T5%

EtOH+T10%

Fig. 5. ASAT activity in the liver of ethanol and fenugreek treated rats. *Significantly different from control (p < 0.05); **Significantly different from control (p < 0.01); X Significantly different from EtOH (p < 0.05); XXSignificantly different from EtOH (p < 0.01).

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Serum and liver LDH. Similar to serum transaminases, LDH may be considered a marker of plasma membrane integrity. The activity of the enzyme was markedly elevated by ethanol treatment (Fig. 6), while 10% fenugreek in the diet produced a consistent lowering of the enzyme in the serum. Hepatic LDH catalyse the reversible reduction of pyruvate to lactate. Its activity

usually increases in alcohol intoxication, when oxidative catabolism of glucose is impaired. This also represents a temporary and less effective modality to regenerate NAD+ which was reduced in ethanol oxidation. As shown in Fig. 7, there was a slight, statistically unsignificant increase in enzyme’s activity in the EtOH group. Fenugreek added to the diet in 10% massively decreased liver LDH activity. LDH liver

46 14

µmoles pyruvate/g/min

µmoles pyruvate/dL serum/min

LDH serum

*

44

12

42 x

40

10

38 36 34 32 30

8

xx

***

6 4 2 0

Control

EtOH

EtOH+T5%

EtOH+T10%

Fig. 6. LDH activity in the serum of ethanol and fenugreek treated rats. *Significantly different from control (p < 0.05). XSignificantly different from EtOH (p < 0.05).

Control

EtOH

EtOH+T5%

EtOH+T10%

Fig. 7. LDH activity in the liver of ethanol and fenugreek treated rats. ***Significantly different from control (p < 0.001). XXSignificantly different from EtOH (p < 0.01).

We may conclude that the majority of the investigated parameters were modified by fenugreek flour, administered on the background of ethilic intoxication, in a beneficial manner. Edifying effects were recorded especially in the case of differential WBC count and enzymatic activities, and were more concludent with the higher (10%) fenugreek dose. Our in vivo results are consistent with data obtained in vitro by others (Kaviarasan, 2006), demonstrating the hepatoprotective properties of fenugreek seed flour in preventing/improving the condition of ethanol intoxicated liver.

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Acknowledgements This research was supported by the Cross-Border Cooperation Program HungaryRomania, no. HURO/0801/110. We thank Mrs. Ana-Maria Răbulea for her excellent technical support.

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Annals of RSCB

Vol. XV, Issue 2

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seeds in experimental ethanol toxicity, Phitoter. Res. 17, 737-743, 2003. Tsukamoto, H. and Lu, S. C.: Current Concepts in the Pathogenesis of Alcoholic Liver Injury, FASEB J., 15, 1335-1349, 2001. Van Horn, C.G., Ivester, P., Cunningham, C.C.: Chronic ethanol consumption and liver glycogen synthesis, Arch. Biochem. Biophys., 392, 1, 145-152, 2001. Vérine, A., Valette, A., Richard, D., Boyer, J.: Acute ethanol treatment induces a bimodal response of phospholipid acylation rates in rat red blood cells, Life Sci., 49, 17, PL125PL128, 1991. Vijayakumar, M.V., Singh, S., Chippa, R.R., Bhat, M.K.: The hypoglycaemic activity of fenugreek seed extract is mediated through the stimulation of an insulin signalling pathway, Brit. J. Pharmacol., 146, 41-48, 2005. Willis, M.S., Klassen, L.W., Tuma, D.J., Sorrell, M.F., Thiele, G.M.: In Vitro Exposure to Malondialdehyde-Acetaldehyde Adducted Protein Inhibits Cell Proliferation and Viability, Alcoholism: Clin. Exp. Res., 26 (2), 158-164, 2002. Yadav, U.C.S., Moorthy, K., Baquer, N.Z.: Effects of sodium-ortovanadate and Trigonella foenum-graecum seeds on hepatic and renal lipogenic enzymes and lipid profile during alloxan diabetes, J. Biosci., 29 (1), 8191, 2004.

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