Biomedical Research and Therapy Biomedical Research and Therapy Hepatoprotective and nephroprotective effects of Trigonella foenum-graecum L. (Fenugreek) seed extract against sodium nitrite toxicity in rats ATİLA USLU Gözde gzd.gozde@hotmail.com 0000-0002-2328-9164 1 USLU Hamit hamit_uslu@hotmail.com 0000-0002-3974-5814 2 ADALI Yasemen yasemenadali@hotmail.com 0000-0002-8004-7364 3 Department of Physiology, Faculty of Veterinary Medicine, University of Kafkas, Kars – Turkey Department of Health Care Services, Atatürk Vocational School of Health Services, University of Kafkas, Kars – Turkey Department of Pathology, Faculty of Medicine, University of Çanakkale Onsekiz Mart, Çanakkale – Turkey Abstract

Introduction: Feeding habits and environmental factors may rival genetic susceptibility as etiological factors related to various cancers. Humans are continuously exposed to many synthetic food additives, one of which is sodium nitrite (NaNO2). There is a direct correlation between increases in consumption of nitrite-treated products and incidence of tissue damage, hepatotoxicity, nephrotoxicity and some types of cancer. The objective of this study was to investigate the protective effects of Trigonella foenum-graecum (TFG) on NaNO2-induced hepatotoxicity and nephrotoxicity. Methods: Forty rats were randomly assigned (10 per group) to control (physiological saline solution), TFG (150 mg/kg/day), NaNO2 (80 mg/kg/day), and NaNO2+TFG (80 mg/kg/day + 150 mg/kg/day) groups. This group was offered TFG seed extract two hours before NaNO2. At the end of three months, the rats were decapitated, and blood, kidney and liver tissues were removed. Results: Three months of oral administration of NaNO2 increased serum aspartate aminotransferase (AST), alanine aminotransferase (ALT), urea, creatinine, and pro-inflammatory cytokine levels in the liver and kidney tissues [except for liver Interleukin 1 alpha (IL-1α)] of rats. Serum AST, ALT, urea, creatinine, liver IL-6, and kidney tumor necrosis factor-α (TNF-α), IL-6, IL-1α levels significantly decreased in the NaNO2+TFG group compared to the NaNO2 group. Pathological examinations, it was determined show that exogenously administered TFG could alleviate the effects of NaNO2 hepatotoxicity and nephrotoxicity. Conclusions: Our results suggest that exogenous TFG mitigates NaNO2-administration induced hepatotoxicity and nephrotoxicity. TFG extract exerted antioxidative and anti-inflammatory effects, and played a significant role in preventing hepatic and renal damage induced by chronic NaNO2 administration.

Keywords Hepatotoxicity Nephrotoxicity Pro-inflammatory cytokine Sodium nitrite Trigonella foenum-graecum Introduction

Much research has been devoted to determining the relationship between diet and chronic diseases like diabetes, cancer, coronary heart disease, metabolic syndrome and osteoporosis. Humans are continuously exposed to many synthetic food additives, one of which is sodium nitrite (NaNO2). NaNO2 inhibits bacterial growth secondary to colour preservatives, improves flavour, and extends the shelf-life of meat and fish products1,2. Unfortunately, high levels of NaNO2 intake may increase the risks of cancer, hepatotoxicity, nephrotoxicity, inflammation, oxidative stress, and tissue injury3,4. The toxic effects of NaNO2 are due to the production of nitrosamines and nitroamides when nitrites react with amines and amides in food5,6,7. The resulting nitrosamines and nitroamides also increase the production of free radicals8. Intestinal bacteria with nitrite reductase activity may increase the production of nitric oxide (NO) from nitrites9,10. Akhzari et al. (2019) reported that alanine aminotransferase (ALT), alkaline phosphatase (ALP), caspase-3 activities, malondialdehyde (MDA), TNF-α and transforming growth factor-β1 levels increased and reduced glutathione (GSH), glutathione reductase (GR) and glutathione peroxidase (GPx) levels decreased in rats treated with sodium nitrite11. Another study determined that NaNO2 increased lipid peroxidation, MDA, NO levels, arginase activity but decreased activity of GSH and catalase (CAT) toxicity induced by sodium nitrite in rats6. Also, Ansari et al. (2019) indicated that NaNO2-induced oxidative stress causes a decrease in the activity of antioxidant defence system, brush border membrane and metabolic enzymes in kidneys12.

There is a growing interest in the use of plant-derived agents for treating diseases and/or preventing chronic health conditions. Trigonella foenum-graecum L. (fenugreek or TFG) is an annual plant that belongs to the Leguminosae family13. Its seeds and leaves are commonly used as a condiment and seasoning, and a wheat and maize flour supplement for bread-making. Additionally, TFG is a staple food in Asian and North African regions14 and is also used in traditional medicine owing to its antidiabetic, hypoglycaemic, antioxidant, hypolipidemic and immunomodulatory effects15,16. The most important phytochemicals isolated from TFG are saponins, trigonelline alkaloids, trigocoumarin, phosphates, potassium, proteins (4-hydroxyisoleucine), choline, vitamin C, beta-carotene, nicotinic acid, and folic acid17,15. TFG may mitigate the adverse effects of NaNO2 administration. This study investigate the protective effects of TFG on NaNO2-induced hepatotoxicity and nephrotoxicity were investigated.

Methods <bold id="s-06c284ba5a90">Extraction of plant material</bold>

TFG seeds were purchased at a local herbal market. Mature fruits were collected between August and September and then sundried. After this process the seeds become yellow and light brown. Each seed is4-6 mm long and 2-3 mm width. They are slightly rough, squared or rectangular and unevenly shaped. We pulverated 1000 g of the seeds into a fine powder and dissolved this powder into a mixture of ethyl alcohol and water (2:8) at a ratio of 1:5 (seed weight: solvent volume)18. The mixture was kept at room temperature in a shaking water bath for 2 days. It was filtered through filter paper, and the excess solvents were evaporated under pressure at 50°C. We obtained 9 g of the extract from 250 ml of macerated seeds.

<bold id="s-c7b8e5fac2da">Experimental design</bold>

We used 40 female Wistar Albino rats, aged around 2 months. The rats were divided equally into four groups (10 rats per group) for the experiments. The animals were maintained under standard conditions [temperatures (23±2ºC), humidity (55±5%) and 12 hrs light: 12 hrs dark cycle] and were fed standard laboratory chow (Moisture: 12.8%, crude protein: 23.1%, crude cellulose: 5%, crude oil: 2.8%, crude ash: 7.1%, and sodium 0.60%). The rats were allowed tap water ad libitum throughout the experiment. The animal protocol was accepted by the ethical committee for Animal Experiments at Kafkas University.

Control group (C): This group received oral physiological saline solution (300 µL/day).

Trigonella foenum-graecum group (TFG): This group received 150 mg/kg/day TFG seed extract dissolved in water.

Sodium nitrite group (NaNO2): This group received 80 mg/kg/day NaNO2 dissolved in water.

Sodium nitrite + Trigonella foenum-graecum group (NaNO2+TFG): This group received 150 mg/kg/day TFG seed extract 60 minutes prior to 80 mg/kg/day NaNO2.

Rats were administered with NaNO2, TFG extract and physiological saline solution by oral gavage over three months. The dose and duration of NaNO2 and TFG extract used in this study were in the range of those used in other studies, and of the same animal species19,20,21.

 <bold id="strong-8">Biochemical analysis and Histopathological evaluations</bold>

At the end of the experiment, blood samples were collected under 0.4 ml/kg pentobarbital sodium anaesthesia via the intracardiac route. Blood samples were taken with a non-anticoagulant tube and then centrifuged at 1000xg for 15 min at 4°C to produce serum. The serum aspartate aminotransferase (AST), alanine aminotransferase (ALT) (Mindray BS 120), urea and creatinine levels (Architect c16000 Abbott Diagnostic–USA model) were measured using an autoanalyser. The rat livers and kidneys were removed quickly and cleaned with cold physiological saline solution. Pieces of the livers and kidneys were homogenised in a 10-fold volume of phosphate buffered saline. We used ELISA kits to determine pro-inflammatory cytokine (IL-6, IL-1α, TNF-α) levels in tissue homogenates (Elabscience WuHan-P.R.C). Moreover, some tissues were fixed in 10% formalin and processed according to standard procedures. We cut 4-μm-thick paraffin sections, stained them with haematoxylin-eosin and evaluated the results using a light microscope.

 <bold id="strong-9">Statistical analysis</bold>

We used a one-way ANOVA to determine whether there were significant differences among the groups. The Tukey multiple range test was used to detect significant pairwise differences between the groups. A value of p<0.05 was considered significant. All statistical tests were performed using SPSS 18.

Results

We evaluated the hepatoprotective and nephroprotective effects of TFG seed extract by measuring the serum activities of ALT, AST, urea and creatinine. Rats administered NaNO2 showed significant increases in serum ALT compared to the C and TFG groups (p<0.05). We found a significant increase in the AST levels in the NaNO2 group relatively to the C and TFG groups (p<0.001). Moreover, there was a significant decrease in AST levels in the TFG group when compared to the C group (P<0.01). Administration of NaNO2 with TFG seed extract (NaNO2+TFG) significantly decreased serum AST and ALT levels compared to the NaNO2 group (p<0.001, p<0.05 respectively). A significant increase in urea levels were observed in the NaNO2 and NaNO2+TFG groups compared to the C and TFG groups (p<0.001, p<0.01 respectively). Serum urea levels were also significantly lower in the NaNO2+TFG group, compared to the NaNO2 group (p<0.05). Both the NaNO2 and NaNO2+TFG groups exhibited significantly higher serum creatinine levels compared to the C and TFG groups (p<0.001, p<0.05 respectively). Moreover, the serum creatinine levels were significantly decreased in the NaNO2+TFG group, as compared to the NaNO2 group (p<0.05) (Figure 1).

<bold id="s-a02981a734df">Effect of TFG extract on serum Urea (mg/dL) and Creatinine (mg/dL) levels, AST (U/L), ALT (U/L) mean±SD of animals in each group (n:10).</bold> a-b, a-e: p<0.01, a-c, c-e, c-ae: p<0.001, a-d, b-c, c-d: p<0.05.

NaNO2 significantly increased liver TNF-α and IL-6 levels compared to the C and TFG groups (p<0.05, p<0.001 respectively), yet, did not affect the IL-1α. Chronic treatment with TFG+NaNO2 significantly decreased IL-6 in liver tissues of rats, compared with the NaNO2 group (p<0.01). However, IL-6 increased in TFG+NaNO2 group compared to C group (p<0.05) (Figure 2). NaNO2 administration increased kidney TNF-α, IL-6 and IL-1α levels when compared to the control group (p<0.05, p<0.001 and p<0.01 respectively). Our results showed that the TFG+NaNO2 group exhibited significantly decreased kidney TNF-α and IL-6 levels comparing to with the NaNO2 group (p<0.05) (Figure 3).

<bold id="s-4455a5476666">Effect of TFG extract on liver proinflammatory cytokines (IL-6, IL-1α, TNF-α) levels (pg/mL), mean±SD of animals in each group. </bold>a-b:p<0.05, b-c: p<0.01, a-c, ab-c: p<0.001

<bold id="s-ba90360ea2c3">Effect of TFG extract on kidney proinflammatory cytokines (IL-6, IL-1α, TNF-α) levels (pg/mL), mean±SD of animals in each group. </bold>a-b,b-e: p<0.05, a-d, ab-e: p<0.01, a-e: p<0.001.

<bold id="s-83d7e4c2f9da">Focal moderate hepatocyte damage (white arrow), interhepatocytic hemorrhage (blue arrow) and vascular congestion (green arrow), H & E-200x. </bold>a-b:p<0.001, a-c, b-c: p<0.05

<bold id="s-a197febf4330">Tubular epithelial cell damage (white arrow) andinter tubular bleeding areas (blue arrow), H & E 200x.</bold> a-b: p<0.01, b-c: p<0.05, a-c: p>0.05

Histopathological examination revealed hepatocyte damage of variable prevalence and intensities in all NaNO2 group rats. Also, in the TFG+NaNO2 group, half of the rats had hepatocyte damage (Figure 4). In the C group, we observed mild congestion in only one kidney tissue sample. In the NaNO2 group, most of the cases had focal tubular epithelial damage, mild vascular congestion in one of these damaged cases and the least severe inter and intratubular haemorrhage in the damaged cases. There was one case of mild violent intratubular haemorrhage in the TFG group. In the TFG+NaNO2 group, mild severe vascular congestion and mild severe intermittent haemorrhage was detected in one of the cases with mild severe focal tubular epithelial damage in most of the cases (Figure 5). We therefore conclude that exogenously-administered TFG may alleviate the effects of NaNO2 hepatotoxicity and nephrotoxicity.

Discussion

NaNO2 is an important food additive with antibacterial properties for pathogens such as Cl. botulinum, Listeria monocytogenes, Escherichia coli O157:H7 and Salmonella22,23,24. However, because high doses and chronic NaNO2 consumption increase the levels of carcinogenic N-nitrosamines, restricted dietary intake should be considered25,26. Prolonged oral administration of NaNO2 has been shown to result in widespread and moderate inflammation and damage in rats. Previous studies support our findings2,4,27. However, oral administration of TFG+NaNO2 significantly reduced hepatic and renal damage and inflammation compared to the NaNO2 group. TFG mitigates various toxicities, but there is no published literature on the effects of TFG extract on NaNO2 toxicity. This study presented an important step in this regard. TFG extract exerted positive effects on alcohol28, acrylamide29, and cyclophosphamide + buthionine-SR-sulfoximine30 toxicity in previous studies. The seeds also have hypoglycaemic31, anti-inflammatory32, antiulcerogenic33, and antinociceptive34 effects. In another study, both hepatic and testicular dysfunction were found in male rats exposed to chronic cadmium, but the application of fenugreek seed powder was beneficial for these disorders35. Thirunavukkarasu et al. (2003) found that aqueous extract of TFG may protect against damage to lipid peroxidation and liver and brain tissue damage36. Belaïd-Nouira et al. (2013) found that treatment with fenugreek seed powder normalises plasma markers and alleviates histopathological changes in cases with aluminium-induced kidney damage37.

Serum enzymes including AST, ALT, urea and creatinine are used to evaluate patients with hepatic and renal disorders. This enzymes activity increases in cases of liver and kidney damage38. NaNO2 reacts with amines in the stomach, producing nitrosamines that cause serious damage to the liver and kidneys39. NaNO2 administration can alter liver and kidney function, as evidenced by significant increases in AST, ALT, urea and creatinine, compared to group C. TFG seed extract lowers levels of key enzymes that characterise liver and kidney damage.

NaNO2 initiates peroxidative damage in cells and increases the activation of pro-inflammatory cytokines40. Therefore, pro-inflammatory cytokines may play an important role in the formation of liver and kidney damage. Al-Gayyar et al. (2014) found that NaNO2 increased levels of cardiac pro-inflammatory cytokines and decreased levels of anti-inflammatory cytokines1. Another study determined that NaNO2 caused renal toxicity and significantly increased oxidative stress, inflammation, and apoptosis markers41. In the present study, we determined that NaNO2 caused serious damage to liver and kidney tissues and led to significant increases in pro-inflammatory cytokine levels in these tissues. TFG seed extract reduced liver and kidney damage induced by NaNO2, and reduced pro-inflammatory cytokine levels, especially in the kidneys. Previous studies showed that TFG seeds are a rich source of polyphenols. Polyphenols such as apigenin, kaempferol, quercetin, vitexin and tricine, were identified by HPLC42,43. Also, sapogenins, diosgenin, 4-hydroxy-isoleucine, trigonelline, alkaloids, phosphate, potassium, copper, protein, choline, vitamin C, beta carotene, nicotinic acid and folic acid are active chemical components of TFG44,45,46. Bin-Hafeez et al. (2013) reported that the immunostimulatory activity of TFG is mediated by the saponins, fibres and flavonoids it contains15. The toxic effects of NaNO2 are caused by the production of nitrosamines and nitrosamides, which are known to cause tissue damage due to inflammation and oxidative stress. In this study, TFG extract was found to cause significant decreases in liver and kidney damage markers and pro-inflammatory cytokine levels in NaNO2-induced hepatotoxicity and nephrotoxicity. It is proposed that TFG performs hepato and nephroprotective effects with immunostimulatory factors found in previous studies.

Conclusions

In conclusion, chronic NaNO2 consumption causes severe physiological and histopathological changes in liver and kidney tissues in rats. The TFG extract showed antioxidant and anti-inflammatory effects, and prevented hepatic and renal damage induced by chronic NaNO2 administration.

<bold id="s-2602f78940fe">Abbreviations</bold>

ALP: Alkaline phosphatase

ALT : Alanine aminotransferase

AST : Aspartate aminotransferase

CAT: Catalase

GPx: Glutathione peroxidase

GR: Glutathione reductase

GSH: Reduced glutathione

IL-1α : Interleukin 1 alpha

IL-6 : Interleukin-6

MDA: Malondialdehyde

NO: Nitric oxide

TFG : Trigonella foenum-graecum

TNF-α : Tumor necrosis factor alpha

Competing Interests

The authors declare no conflict of interest.

 Authors' Contributions

The design, experimental part and biochemical analysis of the study were carried out by Dr. Gözde ATİLA USLU and Dr. Hamit USLU. The histopathological evaluations were performed by Dr. Yasemen ADALI. All the authors took equal roles in writing the research.

Acknowledgments

This study was awarded the best oral presentation award in the field of veterinary medicine in the second international science symposium (science festival) held in Tbilisi / Georgia on 05-08 September 2017.

References Al-Gayyar M.M. Al Youssef A. Sherif I.O. Shams M.E. Abbas A. Protective effects of arjunolic acid against cardiac toxicity induced by oral sodium nitrite: effects on cytokine balance and apoptosis Life Sci 2014 111 1-2 18 26 0024-3205 10.1016/j.lfs.2014.07.002 25064822 Salama M.F. Abbas A. Darweish M.M. El-Hawwary A.A. Al-Gayyar M.M. Hepatoprotective effects of cod liver oil against sodium nitrite toxicity in rats Pharm Biol 2013 51 11 1435 43 1388-0209 10.3109/13880209.2013.796564 23862714 Alyoussef A. Al-Gayyar M.M. Thymoquinone ameliorated elevated inflammatory cytokines in testicular tissue and sex hormones imbalance induced by oral chronic toxicity with sodium nitrite Cytokine 2016 83 64 74 1043-4666 10.1016/j.cyto.2016.03.018 27038016 Al-Gayyar M.M. Hassan H.M. Alyoussef A. Abbas A. Darweish M.M. El-Hawwary A.A. Nigella sativa oil attenuates chronic nephrotoxicity induced by oral sodium nitrite: effects on tissue fibrosis and apoptosis Redox Rep 2016 21 2 50 60 1743-2928 26221999 Abdeen A.M. El-Shayeb A.F. Hassan H.A. El-Agamy S.A. The Protective Role of Garlic Oil Against the Histopathological and Histochemical Changes Induced by Sodium Nitrite Toxicity in Kidneys of Albino Rats J Jazan Univ Appl Sci Branch. 2015 4 1 57 67 El-Sheikh N.M. Khalil F.A. L-arginine and L-glutamine as immunonutrients and modulating agents for oxidative stress and toxicity induced by sodium nitrite in rats Food Chem Toxicol 2011 49 4 758 62 0278-6915 10.1016/j.fct.2010.11.039 21130833 Tong M. Longato L. de la Monte S.M. Early limited nitrosamine exposures exacerbate high fat diet-mediated type 2 diabetes and neurodegeneration BMC Endocr Disord 2010 10 1 4 1472-6823 10.1186/1472-6823-10-4 20302640 Hassan H.A. Yousef M.I. Ameliorating effect of chicory (Cichorium intybus L.)-supplemented diet against nitrosamine precursors-induced liver injury and oxidative stress in male rats Food Chem Toxicol 2010 48 8-9 2163 9 0278-6915 10.1016/j.fct.2010.05.023 20478349 Duncan C. Dougall H. Johnston P. Green S. Brogan R. Leifert C. Chemical generation of nitric oxide in the mouth from the enterosalivary circulation of dietary nitrate Nat Med 1995 1 6 546 51 1078-8956 10.1038/nm0695-546 7585121 El-Wakf A.M. Hassan H.A. Mahmoud A.Z. Habza M.N. Fenugreek potent activity against nitrate-induced diabetes in young and adult male rats Cytotechnology 2015 67 3 437 47 0920-9069 10.1007/s10616-014-9702-7 24615531 Akhzari M. Shafiee S.M. Rashno S. Akmali M. Berberine Attenuated Oxidative Stress Induced by Sodium Nitrite in Rat Liver Jundishapur J Nat Pharm Prod 2019 14 1 e68532 1735-7780 10.5812/jjnpp.68532 Ansari F.A. Khan A.A. Mahmood R. Ameliorative effect of carnosine and N-acetylcysteine against sodium nitrite induced nephrotoxicity in rats J Cell Biochem 2018 120 5 7032 44 0730-2312 10.1002/jcb.27971 30368897 Eidi A. Eidi M. Sokhteh M. Effect of fenugreek (Trigonella foenum graecum L.) seeds on serum parameters in normal and streptozotocin-induced diabetic rats Nutr Res 2007 27 11 728 33 0271-5317 10.1016/j.nutres.2007.09.006 Xue W.L. Li X.S. Zhang J. Liu Y.H. Wang Z.L. Zhang R.J. Effect of Trigonella foenum-graecum (fenugreek) extract on blood glucose, blood lipid and hemorheological properties in streptozotocin-induced diabetic rats Asia Pac J Clin Nutr 2007 16 1 422 6 0964-7058 17392143 Bin-Hafeez B. Haque R. Parvez S. Pandey S. Sayeed I. Raisuddin S. Immunomodulatory effects of fenugreek (Trigonella foenum graecum L.) extract in mice Int Immunopharmacol 2003 3 2 257 65 1567-5769 10.1016/S1567-5769(02)00292-8 12586606 Renuka C. Ramesh N. Saravanan K. Evaluation of the antidiabetic effect of Trigonella foenum graecum seed powder on alloxan induced diabetic albino rats Int J Pharm Tech Res 2009 1 4 1580 4 0974-4304 Petit P.R. Sauvaire Y.D. Hillaire-Buys D.M. Leconte O.M. Baissac Y.G. Ponsin G.R. Steroid saponins from fenugreek seeds: extraction, purification, and pharmacological investigation on feeding behavior and plasma cholesterol Steroids 1995 60 10 674 80 0039-128X 10.1016/0039-128X(95)00090-D 8539775 Hamza N. Berke B. Cheze C. Le Garrec R. Umar A. Agli A.N. Preventive and curative effect of Trigonella foenum-graecum L. seeds in C57BL/6J models of type 2 diabetes induced by high-fat diet J Ethnopharmacol 2012 142 2 516 22 0378-8741 10.1016/j.jep.2012.05.028 22633967 Hassan H.A. Hafez H.S. Zeghebar F.E. Garlic oil as a modulating agent for oxidative stress and neurotoxicity induced by sodium nitrite in male albino rats Food Chem Toxicol 2010 48 7 1980 5 0278-6915 10.1016/j.fct.2010.05.001 20457208 Mokhtari M. Shariatie M. Gharamanie R. Effect of Trigonella foenum-graecum L. seed extract on concentration of testosterone and spermatogenesis in rats JMP 2008 1 12 20 Morani A.S. Bodhankar S.L. Mohan V. Thakurdesai P.A. Ameliorative effects of standardized extract from Trigonella foenum-graecum L. seeds on painful peripheral neuropathy in rats Asian Pac J Trop Med 2012 5 5 385 90 2352-4146 10.1016/S1995-7645(12)60064-9 22546656 Duffy L.L. Vanderlinde P.B. Grau F.H. Growth of Listeria monocytogenes on vacuum-packed cooked meats: effects of pH, aw, nitrite and ascorbate Int J Food Microbiol 1994 23 3-4 377 90 0168-1605 10.1016/0168-1605(94)90164-3 7873338 Dykhuizen R.S. Frazer R. Duncan C. Smith C.C. Golden M. Benjamin N. Antimicrobial effect of acidified nitrite on gut pathogens: importance of dietary nitrate in host defense Antimicrob Agents Chemother 1996 40 6 1422 5 0066-4804 10.1128/AAC.40.6.1422 8726013 Milkowski A. Garg H.K. Coughlin J.R. Bryan N.S. Nutritional epidemiology in the context of nitric oxide biology: a risk-benefit evaluation for dietary nitrite and nitrate Nitric Oxide 2010 22 2 110 9 1089-8603 10.1016/j.niox.2009.08.004 19748594 Kozisek F. Influence of nitrate levels in drinking water on urological malignancies: a community-based cohort study BJU Int 2007 99 6 1550 1 1464-4096 10.1111/j.1464-410X.2007.06970_4.x 17537226 Ward M.H. Pan W.H. Cheng Y.J. Li F.H. Brinton L.A. Chen C.J. Dietary exposure to nitrite and nitrosamines and risk of nasopharyngeal carcinoma in Taiwan Int J Cancer 2000 86 5 603 9 0020-7136 10.1002/(SICI)1097-0215(20000601)86:5<603::AID-IJC1>3.0.CO;2-H 10797279 Sherif I.O. Al-Gayyar M.M. Antioxidant, anti-inflammatory and hepatoprotective effects of silymarin on hepatic dysfunction induced by sodium nitrite Eur Cytokine Netw 2013 24 3 114 21 1952-4005 24225033 Kaviarasan S. Ramamurty N. Gunasekaran P. Varalakshmi E. Anuradha C.V. Fenugreek (Trigonella foenum graecum) seed extract prevents ethanol-induced toxicity and apoptosis in Chang liver cells Alcohol Alcohol 2006 41 3 267 73 0735-0414 10.1093/alcalc/agl020 16574673 Abdel-Daim M.M. Abd Eldaim M.A. Hassan A.G. Trigonella foenum-graecum ameliorates acrylamide-induced toxicity in rats: roles of oxidative stress, proinflammatory cytokines, and DNA damage Biochem Cell Biol 2015 93 3 192 8 0829-8211 10.1139/bcb-2014-0122 25607344 Bhatia K. Kaur M. Atif F. Ali M. Rehman H. Rahman S. Aqueous extract of Trigonella foenum-graecum L. ameliorates additive urotoxicity of buthionine sulfoximine and cyclophosphamide in mice Food Chem Toxicol 2006 44 10 1744 50 0278-6915 10.1016/j.fct.2006.05.013 16828947 Puri D. Prabhu K.M. Murthy P.S. Mechanism of action of a hypoglycemic principle isolated from fenugreek seeds Indian J Physiol Pharmacol 2002 46 4 457 62 0019-5499 12683221 Mandegary A. Pournamdari M. Sharififar F. Pournourmohammadi S. Fardiar R. Shooli S. Alkaloid and flavonoid rich fractions of fenugreek seeds (Trigonella foenum-graecum L.) with antinociceptive and anti-inflammatory effects Food Chem Toxicol 2012 50 7 2503 7 0278-6915 10.1016/j.fct.2012.04.020 22542922 Pandian R.S. Anuradha C.V. Viswanathan P. Gastroprotective effect of fenugreek seeds (Trigonella foenum graecum) on experimental gastric ulcer in rats J Ethnopharmacol 2002 81 3 393 7 0378-8741 10.1016/S0378-8741(02)00117-4 12127242 Puri D. Therapeutic potentials of fenugreek Indian J Physiol Pharmacol 1998 42 3 423 4 0019-5499 9741661 Arafa M.H. Mohammad N.S. Atteia H.H. Fenugreek seed powder mitigates cadmium-induced testicular damage and hepatotoxicity in male rats Exp Toxicol Pathol 2014 66 7 293 300 0940-2993 10.1016/j.etp.2014.04.001 24813645 Thirunavukkarasu V. Anuradha C.V. Viswanathan P. Protective effect of fenugreek (Trigonella foenum graecum) seeds in experimental ethanol toxicity Phytother Res 2003 17 7 737 43 0951-418X 10.1002/ptr.1198 12916070 Bela\"\id-Nouira Y. Bakhta H. Haouas Z. Flehi-Slim I. Ben Cheikh H. Fenugreek seeds reduce aluminum toxicity associated with renal failure in rats Nutr Res Pract 2013 7 6 466 74 1976-1457 10.4162/nrp.2013.7.6.466 24353832 Hassan H.A. El-Agmy S.M. Gaur R.L. Fernando A. Raj M.H. Ouhtit A. In vivo evidence of hepato- and reno-protective effect of garlic oil against sodium nitrite-induced oxidative stress Int J Biol Sci 2009 5 3 249 55 1449-2288 10.7150/ijbs.5.249 19305642 Choi S.Y. Chung M.J. Sung N.J. Volatile N-nitrosamine inhibition after intake of Korean green tea and Maesil extracts with an amine-rich diet in subjects ingesting nitrate Food Chem Toxicol 2002 40 7 949 57 0278-6915 10.1016/S0278-6915(02)00025-X 12065217 Mittal G. Brar A.P. Soni G. Impact of hypercholesterolemia on toxicity of N-nitrosodiethylamine: biochemical and histopathological effects Pharmacol Rep 2006 58 3 413 9 1734-1140 16845216 Elsherbiny N.M. Maysarah N.M. El-Sherbiny M. Al-Gayyar M.M. Renal protective effects of thymoquinone against sodium nitrite-induced chronic toxicity in rats: impact on inflammation and apoptosis Life Sci 2017 180 1 8 0024-3205 10.1016/j.lfs.2017.05.005 28495515 Kenny O. Smyth T.J. Hewage C.M. Brunton N.P. Antioxidant properties and quantitative UPLC-MS analysis of phenolic compounds from extracts of fenugreek (Trigonella foenum-graecum) seeds and bitter melon (Momordica charantia) fruit Food Chem 2013 141 4 4295 302 0308-8146 10.1016/j.foodchem.2013.07.016 23993618 Benayad Z. Gómez-Cordovés C. Es-Safi N.E. Characterization of flavonoid glycosides from fenugreek (Trigonella foenum-graecum) crude seeds by HPLC-DAD-ESI/MS analysis Int J Mol Sci 2014 15 11 20668 85 1661-6596 10.3390/ijms151120668 25393509 Hamadi S.A. Effect of trigonelline and ethanol extract of Iraqi Fenugreek seeds on oxidative stress in alloxan diabetic rabbits JAUBAS 2012 12 1 23 6 10.1016/j.jaubas.2012.02.003 Sauvaire Y. Petit P. Broca C. Manteghetti M. Baissac Y. Fernandez-Alvarez J. 4-Hydroxyisoleucine: a novel amino acid potentiator of insulin secretion Diabetes 1998 47 2 206 10 0012-1797 10.2337/diab.47.2.206 9519714 Yoshikawa M. Murakami T. Komatsu H. Murakami N. Yamahara J. Matsuda H. Medicinal foodstuffs. IV. Fenugreek seed. (1): structures of trigoneosides Ia, Ib, IIa, IIb, IIIa, and IIIb, new furostanol saponins from the seeds of Indian Trigonella foenum-graecum L Chem Pharm Bull (Tokyo) 1997 45 1 81 7 0009-2363 10.1248/cpb.45.81 9023970