Promoting cardioprotection with fenugreek: Insights from CoCl2-induced hypoxia in neonatal rat cardiomyocytes

Document Type : Original Article


1 Faculty of Agro-Based Industry, Universiti Malaysia Kelantan, Jeli, Kelantan, Malaysia

2 Faculty of Veterinary Medicine, Universiti Malaysia Kelantan, Pengkalan Chepa, Kelantan, Malaysia

3 Department of Basic Medical Sciences, Kulliyyah of Medicine, International Islamic University Malaysia (IIUM), Bandar Indera Mahkota, Kuantan, Pahang, Malaysia

4 School of Pharmacy, Monash University Malaysia, Jalan Lagoon Selatan, 47500 Bandar Sunway, Selangor, Malaysia

5 Department of Biomedical Sciences and Therapeutics, Faculty of Medicine and Health Sciences, Universiti Malaysia Sabah, Kota Kinabalu, 88400, Sabah, Malaysia

6 Department of Biochemistry, Faculty of Medicine and Health Sciences, Abdurrab University, Pekanbaru, Riau, Indonesia

7 Centre for International Relations and Research Collaborations, Reva University, Rukmini Knowledge Park, Kattigenahalli, Yelahanka, Bangalore, 560064, Karnataka, India


Objective(s): This study aimed to investigate the protective effects of fenugreek on CoCl2-induced hypoxia in neonatal rat cardiomyocytes.
Materials and Methods: Primary cardiomyocytes were isolated from Sprague Dawley rats aged 0–2 days and incubated with various concentrations of fenugreek (10-320 µg/ml) and CoCl2-induced hypoxia for different durations (24, 48, and 72 hr). Cell viability, calcium signaling, beating rate, and gene expression were evaluated. 
Results: Fenugreek treatments did not cause any toxicity in cardiomyocytes. At a concentration of 160 µg/ml for 24 hr, fenugreek protected the heart against CoCl2-induced hypoxia, as evidenced by reduced expression of caspases (-3, -6, -8, and -9) and other functional genes markers, such as HIF-1α, Bcl-2, IP3R, ERK5, and GLP-1r. Calcium signaling and beating rate were also improved in fenugreek-treated cardiomyocytes. In contrast, CoCl2 treatment resulted in up-regulation of the hypoxia gene HIF-1α and apoptotic caspases gene (-3, -9, -8, -12), and down-regulation of Bcl-2 activity.
Conclusion: Fenugreek treatment at a concentration of 160 µg/ml was not toxic to neonatal rat cardiomyocytes and protected against CoCl2-induced hypoxia. Furthermore, fenugreek improved calcium signaling and beating rate and altered gene expression. Fenugreek may be a potential therapeutic agent for promoting cardioprotection against hypoxia-induced injuries.


Main Subjects

1. Hasler CM, Kundrat S, Wool D. Functional foods and cardiovascular disease. Curr Atheroscler Rep 2000; 2: 467-475.
2.    World Health Organization (WHO). Obesity and overweight 9 June 2021. Available from
3.    Porez G, Prawitt J, Gross B, Staels B. Bile acid receptors as targets for the treatment of dyslipidemia and cardiovascular disease. J Lipid Res 2012; 53: 1723-1737. 
4.    Hadi A, Arab A, Hajianfar H, Talaei B, Miraghajani M, Babajafari S, et al. The effect of fenugreek seed supplementation on serum irisin levels, blood pressure, and liver and kidney function in patients with type 2 diabetes mellitus: A parallel randomized clinical trial. Complement Ther Med 2020; 49: 102315. 
5.    Snehlata HS, Payal DR. Fenugreek (Trigonella foenum-graecum L.): An overview. Int J Curr Pharm Rev Res 2012; 2: 169-187.
6.    Marine EB, Tasneem IA, Hanan E, Ahmed MES, Diana GM, Marwa TB, et al. Anti-diabetic effects of fenugreek (Trigonella foenum-graecum): A comparison between oral and intraperitoneal administration - an animal study. Int J Funct Nutr 2020: 1-9. 
7.    Bafadam S, Mahmoudabady M, Niazmand S, Rezaee SA, Soukhtanloo M. Cardioprotective effects of Fenugreek (Trigonella foenum-graceum) seed extract in streptozotocin induced diabetic rats. J Cardiovasc Thorac Res 2021; 13: 28-36. 
8.    Prema A, Justin Thenmozhi A, Manivasagam T, Mohamed Essa M, Guillemin GJ. Fenugreek seed powder attenuated aluminum chloride-induced tau pathology, oxidative stress, and inflammation in a rat model of Alzheimer’s disease. J Alzheimer’s Dis 2017; 60: S209-S220. 
9.    Kassaian N, Azadbakht L, Forghani B, Amini M. Effect of fenugreek seeds on blood glucose and lipid profiles in type 2 diabetic  patients. Int J Vitam Nutr Res Int Zeitschrift  fur Vitamin- und Ernahrungsforschung J Int Vitaminol Nutr 2009; 79: 34-39. 
10.    Mukthamba P, Srinivasan K. Dietary fenugreek (Trigonella foenum-graecum) seeds and garlic (Allium sativum) alleviates oxidative stress in experimental myocardial infarction. Food Sci Hum Wellness. 2017; 6: 77-87. 
11.    Zemzmi J, Ródenas L, Blas E, Najar T, Pascual JJ. Characterisation and in vitro evaluation of fenugreek (Trigonella foenum-graecum) seed gum as a potential prebiotic in growing rabbit nutrition. Animals 2020; 10: 1-15. 
12.    Kandhare AD, Bodhankar SL, Mohan V, Thakurdesai PA. Effect of glycosides based standardized fenugreek seed extract in bleomycin-induced pulmonary fibrosis in rats: Decisive role of Bax, Nrf2, NF-κB, Muc5ac, TNF-α and IL-1β. Chem Biol Interact 2015; 237: 151-165. 
13.    Hassani SS, Fallahi Arezodar F, Esmaeli SS, Gholami-Fesharaki M. Effect of fenugreek use on fasting blood quality of life in patients with type 2 diabetes. Galen Med J 2019;  8: 1-8.
14.    Knott EJ, Richard AJ, Mynatt RL, Ribnicky D, Stephens JM, Bruce-Keller A. Fenugreek supplementation during high-fat feeding improves specific markers of metabolic health. Sci Rep 2017; 7: 1-15.
15.    Najdi RA, Hagras MM, Kamel FO, Magadmi RM. A randomized controlled clinical trial evaluating the effect of Trigonella foenum-graecum (fenugreek) versus glibenclamide in patients with diabetes. Afr Health Sci 2019; 19: 1594-1601.
16.    Sheikh Abdul Kadir SH, Miragoli M, Abu-Hayyeh S, Moshkov AV, Xie Q, Keitel V, et al. Bile acid-induced arrhythmia is mediated by muscarinic M2 receptors in neonatal rat cardiomyocytes. PLoS One 2010; 5: e9689.
17.    Kumaravel S, Muthukumaran P, Shanmugapriya K. Chemical composition of Trigonella foenum- graecum through gas chromatography mass spectrometry analysis. J Med Plants Stud Compd 2017; 5: 1-3.
18.    Clark WA, Rudnick SJ, LaPres  JJ, Lesch M, Decker RS. Hypertrophy of isolated adult feline heart cells following beta-adrenergic-induced beating. Am J Physiol 1991; 261: C530-C542.
19.    Noorul Izzati H, Noor Hafizoh S, Maizan M, KNS Sirajudeen, Siew Hua G, Khomaizon AKPZ, Rao PV. Review: Ischemic heart disease and the potential role of fenugreek (Trigonella foenum graecum Linn.) in cardioprotection. J Teknol 2020; 84: 183-197.
20.    World Health Organisation (WHO). WHO Traditional Medicine Strategy 2014-2023. World Heal Organ 2013: 1-76.
21.    Dumolt JH, Rideout TC. The lipid-lowering effects and associated mechanisms of dietary phytosterol supplementation. Curr Pharm Des 2017; 23: 5077-5085. 
22.    Jessup W, Herman A, Chapman J, Jessup W, Herman A, Chapman MJ. Phytosterols in cardiovascular disease: Innocuous dietary components, or accelerators of atherosclerosis? Futur Lipidol 2017; 3: 301-310.
23.    Reaver A, Hewlings S, Westerman K, Blander G, Schmeller T, Heer M, et al. A randomized, placebo-controlled, double-blind crossover study to assess a unique phytosterol ester formulation in lowering LDL cholesterol utilizing a novel virtual tracking tool. Nutrients. 2019; 11: 1-13.
24.    Kandhare AD, Thakurdesai PA, Wangikar P, Bodhankar SL. A systematic literature review of fenugreek seed toxicity by using ToxRTool : evidence from preclinical and clinical studies. Heliyon 2019; e01536.
25.    Szabó K, Gesztelyi R, Lampé N, Kiss R, Remenyik J, Pesti-Asboth G, et al. Fenugreek (Trigonella Foenum-Graecum) seed flour and diosgenin preserve endothelium-dependent arterial relaxation in a rat model of early-stage metabolic syndrome. Int J Mol Sci 2018; 19: 1-21.
26.    Oliveira ALMB, Rohan P de A, Gonçalves TR, Soares PP da S. Effects of hypoxia on heart rate variability in healthy individuals: A systematic review. Int J Cardiovasc Sci 2017; 30: 251-261.
27.    Zhang D, She J, Zhang Z, Yu M. Effects of acute hypoxia on heart rate variability, sample entropy and cardiorespiratory phase synchronization. Biomed Eng Online 2014; 13: 1-12.
28.    Oyagbemi AA, Omobowale TO, Awoyomi O V. Ajibade TO, Falayi OO, Ogunpolu BS, et al. Cobalt chloride toxicity elicited hypertension and cardiac complication via induction of oxidative stress and up-regulation of COX-2/Bax signaling pathway. Hum Exp Toxicol 2019; 38: 519-532.
29.    Nguyen PD, Hsiao ST, Sivakumaran P, Lim SY, Dilley RJ. Enrichment of neonatal rat cardiomyocytes in primary culture facilitates long-term maintenance of contractility in vitro. Am J Physiol - Cell Physiol 2012; 303: 1220-1228.
30.    Li Y, Zhang Z, Zhou X, Li R, Cheng Y, Shang B, et al. Histone deacetylase 1 inhibition protects against hypoxia-induced swelling in H9c2 cardiomyocytes through regulating cell stiffness. Circ J 2018; 82: 192-202.
31.    Niu N, Li Z, Zhu M, Sun H, Yang J, Xu S, et al. Effects of nuclear respiratory factor-1 on apoptosis and mitochondrial dysfunction induced by cobalt chloride in H9C2 cells. Mol Med Rep 2019; 19: 2153-2163.
32.    Chang JC, Hu WF, Lee WS, Lin JH, Ting PC, Chang HR, et al. Intermittent hypoxia induces autophagy to protect cardiomyocytes from endoplasmic reticulum stress and apoptosis. Front Physiol 2019; 10: 995.
33.    Cheng BC, Chen JT, Yang ST, Chio CC, Liu SH, Chen RM. Cobalt chloride treatment induces autophagic apoptosis in human glioma cells via a p53-dependent pathway. Int J Oncol 2017; 50: 964-974.
34.    Wani SA, Khan LA, Basir SF. Cobalt-induced hypercontraction is mediated by generation of reactive oxygen species and influx of calcium in isolated rat aorta. Biol Trace Element Res 2020; 196: 110-118.
35.    Akbari S, Abdurahman NH, Yunus RM. Optimization of saponins, phenolics, and antioxidants extracted from fenugreek seeds using microwave-assisted extraction and response surface methodology as an optimizing tool. Comptes Rendus Chim 2019; 22: 714-727.
36.    Jayachandran KS, Rachel Vasanthi AH, Gurusamy N. Steroidal saponin diosgenin from Dioscorea bulbifera protects cardiac cells from hypoxia-reoxygenation injury through modulation of pro-survival and pro-death molecules. Pharmacogn Mag. 2016; 12: S14-S20.
37.    Wu FC, Jiang JG. Effects of diosgenin and its derivatives on atherosclerosis. Food Funct 2019; 10: 7022-7036.
38.    Fuller S, Stephens JM. Diosgenin, 4-Hydroxyisoleucine, and fiber from Fenugreek: Mechanisms of actions and potential effects on metabolic syndrome. Adv Nutr An Int Rev J 2015; 6: 189-197.
39.    Kassouf T, Sumara G. Impact of conventional and atypical mapks on the development of metabolic diseases. Biomolecules 2020; 10: 1-34.
40.    Hernandez ARV. ERK5 as a Metabolic Regulator in the Heart. The University of Manchester (United Kingdom); 2019. 
41.    Liu W, Ruiz-Velasco A, Wang S, Khan S, Zi M, Jungmann A, et al. Metabolic stress-induced cardiomyopathy is caused by mitochondrial dysfunction due to attenuated Erk5 signaling. Nat Commun 2017; 8: 1-15.
42.    Ussher JR, Baggio LL, Campbell JE, Mulvihill EE, Kim M, Kabir MG, et al. Inactivation of the cardiomyocyte glucagon-like peptide-1 receptor (GLP-1R) unmasks cardiomyocyte-independent GLP-1R-mediated cardioprotection. Mol Metab 2014; 3: 507-517.
43.    Khan MS, Fonarow GC, McGuire DK, Hernandez AF, Muthiah V, Rosenstock J, et al. Glucagon-like peptide 1 receptor agonists and heart failure: The need for further evidence generation and practice guidelines optimization. Circulation 2020; 142: 1205-1218.       
44.    Eisner DA, Caldwell JL, Kistamás K, Trafford AW. Calcium and Excitation-Contraction Coupling in the Heart. Circ Res 2017; 121: 181-195.
45.    Garcia MI, Karlstaedt A, Chen JJ, Amione-Guerra J, Youker KA, Taegtmeyer H et al. Functionally redundant control of cardiac hypertrophic signaling by inositol 1,4,5-trisphosphate receptors. J Mol Cell Cardiol 2017; 112: 95-103.
46.    Kido M, Du L, Sullivan CC, Li X, Deutch R, Jamieson SW, et al. Hypoxia-inducible factor 1-alpha reduces infarction and attenuates progression of cardiac dysfunction after myocardial infarction in the mouse. J Am Coll Cardiol 2005; 46: 2116-2124.
47.    Liu H, Li S, Jiang W, Li Y. MiR-484 protects rat myocardial cells from ischemia-reperfusion injury by inhibiting caspase-3 and caspase-9 during apoptosis. Korean Circ J 2020; 50: 250-263.
48.    Sabbah HN. Targeting the mitochondria in heart failure: A translational perspective. JACC Basic to Transl Sci 2020; 5: 88-106.
49.    Kreckel J, Anany MA, Siegmund D, Wajant H. TRAF2 controls death receptor-induced caspase-8 processing and facilitates proinflammatory signaling. Front Immunol 2019; 10: 2024.
50.    Wang Y, Li M, Xu L, Liu J, Wang D, Li Q, et al. Expression of Bcl-2 and microRNAs in cardiac tissues of patients with dilated cardiomyopathy. Mol Med Rep 2017; 15: 359-365.
51.    Kang PM, Haunstetter A, Aoki H, Usheva A, Izumo S. Morphological and molecular characterization of adult cardiomyocyte apoptosis during hypoxia and reoxygenation. Circ Res 2000; 87: 118-125.
52.    Van Opdenbosch N, Lamkanfi M. Caspases in cell death, inflammation, and disease. Immunity 2019; 50: 1352-1364.
53.    Muhammad IF, Borné Y, Melander O, Orho-Melander M, Nilsson J, Soderholm M, et al. FADD (Fas-associated protein with death domain), caspase-3, and caspase-8 and incidence of ischemic stroke. Stroke 2018; 49: 2224-2226.
54.    Pop C, Timmer J, Sperandio S, Salvesen GS. The apoptosome activates Caspase-9 by dimerization. Mol Cell 2006; 22: 269-275.
55.    Li Y, Liang P, Jiang B, Tang Y, Lv Q, Hao H, et al. CARD9 inhibits mitochondria-dependent apoptosis of cardiomyocytes under oxidative stress via interacting with Apaf-1. Free Radic Biol Med 2019; 141: 172-181.
56.    Dong Y, Chen H, Gao J, Liu Y, Li J, Wang J. Molecular machinery and interplay of apoptosis and autophagy in coronary heart disease. J Mol Cell Cardiol 2019; 136: 27-41.
57.    Szegezdi E, Duffy A, O’Mahoney ME, Logue SE, Mylotte LA, O’Brien T, et al. ER stress contributes to ischemia-induced cardiomyocyte apoptosis. Biochem Biophys Res Commun 2006; 349: 1406-1411.
58.    Lamkanfi M, Kalai M, Vandenabeele P. Caspase-12: An overview. Cell Death Differ 2004; 11: 365-368. 
59.    Todor A, Sharov VG, Tanhehco EJ, Silverman N, Bernabei A, Sabbah HN. Hypoxia-induced cleavage of caspase-3 and DFF45/ICAD in human failed cardiomyocytes. Am J Physiol-Hear Circ Physiol 2002; 283:  H990-H995.
60.    Huang CY, Chen SY, Fu RH, Huang YC, Chen SY, Shyu WC, et al. Differentiation of embryonic stem cells into cardiomyocytes used to investigate the cardioprotective effect of salvianolic acid B through BNIP3 involved pathway. Cell Transplant 2015; 24: 561-571.