Pharmacological Effects of Genistein on Cardiovascular Diseases

Cardiovascular diseases (CVDs) are a group of disorders that involve the heart or blood vessels and are the leading cause of mortality worldwide. Natural products have several pharmacological activities, such as anti-inflammatory, antioxidant, and immunoregulatory properties. This review summarizes the possible therapeutic effects of Genistein on CVD. The information from the current review study was obtained by searching for the keywords such as “Genistein”, “Cardiac dysfunction”, “hypertrophy”, and “Ischemia” “lipid profile” in different online database such as PubMed, Scopus, and Google Scholar, until February 2022. The results of the studies showed that genistein intake has a promising effect on improving cardiac dysfunction, ischemia, and reperfusion of the heart, decreasing cardiac toxicity, modulating lipid profile, and lowering blood pressure. The preventive effects of genistein on experimental models of studies were shown through mechanisms such as anti-inflammatory, antioxidant, and immunomodulatory effects. Pharmacological effects of genistein on cardiac dysfunction, cardiac toxicity, lipid profile, and hypertension indicate the possible remedy effect of this agent in the treatment of CVD.


Introduction
Cardiovascular diseases (CVDs) are a group of disorders of the heart and blood vessels that lead to the cause of mortality worldwide, approximately 31% of all global deaths [1]. CVDs consist of many types of diseases such as coronary heart disease (CHD), acute coronary syndrome (ACS), heart failure, stroke, cardiomyopathies, peripheral vascular diseases, hypertension, and dyslipidemias [2].
Diabetes, dyslipidemias, hypertension, smoking, lack of physical activity, and obesity are modifable cardiovascularrelated risk factors [3]. Prevalence of CVDs can be decreased by recognition and prevention of these risk factors. Besides lifestyle modifcations, antihypertensive drugs, lipid lowering, and antithrombotic therapies are the main regimes for the prevention and treatment of CVDs [4].
For thousands of years, plants have been used for medicinal purposes and as an origin of the new drug discoveries which help the management of disorders. For example, aspirin, digoxin, and lovastatin are extracted from salix alba L. tree, digitalis purpurea, and Monascus purpureus, respectively [5,6].
Phytoestrogens are a class of plant which has similar structures to estrogen, which allows them binding to estrogen receptor. Coumestans, isofavones, and lignans are the main classes of phytoestrogens [7,8].
Genistein is a phytoestrogen that belongs to the isofavones family of Leguminosae plants. Genistein is a good choice for the management of some diseases because of its pharmacological and physiological properties [9]. Also, due to the pharmacological and biological properties such as antioxidant, anti-infammatory, and anti-infective efects, isofavones are considered for therapy/prevention purposes in medicine [10][11][12].
Genistein selectivity to estrogen receptor β is 20 times more than α, which is an acceptable substitute for estrogen replacement in postmenopausal women [13]. Genistein has both estrogenic and antiestrogenic activities by binding to estrogen receptor and sex hormone-binding proteins [14]. Genistein consumption reduces the incidence of prostate and breast malignancy by decreasing the testosterone level and competition with natural estrogen and, fnally, inhibition of cancer cell growth and tumor development [15,16].
Te notable efect of genistein on bone density and bone turnover makes it as a principal option for the prevention of osteoporosis [17]. Genistein has safety and tolerability and has no signifcant change on liver function and hematology test result, and it could be a good reason to use genistein as medicine [8].
Previous studies revealed that dietary intake of genistein was related to lesser risk of cardiovascular diseases; therefore, it is time to assess the evidence of various efects of genistein on cardiovascular diseases. So, evaluating the effects of genistein on cardiovascular disease is the purpose of this systematic review.

Method
Te information of this study was obtained by searching the keywords such as "Genistein", "Cardiac dysfunction", "hypertrophy", and "Ischemia" "lipid profle" in diferent online database such as PubMed, Scopus, and Google Scholar until February 2022. Articles published in languages other than English were excluded. A total of 140 articles were selected from the above databases. 22 articles were duplicates, 65 including; reviews, letter to editor, and some articles with poorly and unsuitable information were removed, and at least 53 original articles related to the topic of this review were included in the current study as shown in Figure 1.

Genistein Efects on Cardiac Dysfunction and Ischemia/ Reperfusion
3.1.1. In Vitro Studies. Te cardioprotective efect of genistein against cobalt chloride (CoCl2) induced hypoxia in H9c2, up-regulated the expression of Notch-1, reduced cell death, and suppressed HIF-1α expression. Furthermore, genistein could antagonize CoCl2-induced apoptosis through inhibiting caspase-3 expression and up-regulating Bcl-2/Bax ratio [18]. Genistein treatment (10 −5 to 10 −10 μmol) in an acute myocardial ischemia model, increased the endothelial colony-forming cell (ECFC) migration and proliferation, and parallel increased the expression of ILK, α-parvin, Factin, and phosphorylation of ERK 1/2 signaling. ECFCs induction into myocardial ischemic sites, cellular expansion stimulation, and angiogenic modulators secretion at the damaged tissues are the result of genistein treatment. Genistein also improved cardiac function, neovascularization, and decreased myocardial fbrosis. Te genistein administration to ECFCs before transplantation improved the regenerative potential in ischemic tissues; therefore, this is a new approach for stem cell therapy in ischemic diseases [19].
Genistein outcome on lipopolysaccharide (LPS)-induced myocarditis in H9c2 cells enhanced the potential of cells growth and prevented cells apoptosis and infammatory reaction. Te results also showed that MAPK as the signaling pathway and Myc (a well-target gene of genistein) as the potential target of genistein in myocarditis [21].
Genistein protective efects against oxidative stress on cardiomyoblasts (H9C2) were studied. Treatment of cells with genistein (10 pM and 100 nM; for 24 h) modulated NO release, induced endothelial NOS-dependent NO production through modulation of intracellular signaling related to Akt, p38MAPK, and ERK1/2, increased the content of glutathione, and decreased reactive oxygen species production [22].
Genistein administration (250 mg/kg) in ovariectomized (OVX) rats increased contractility (259 mm Hg/sec) and cardiac output (7 mL/min) above baseline. Genistein-treated hearts (n � 5) demonstrated ischemic tolerance, greater cardiac output, and markedly improvements of contractility after reperfusion. Also, genistein decreased mean glucose transporter protein 4 content [24].  Once daily injection of genistein (0.1 and 0.2 mg·kg) in male, Wistar rats showed that genistein prevents increase in heart weight to body weight ratio induced by isoproterenol, fbrosis, left ventricular mass, myocardial oxidative stress, myocyte size, and myocardial 1-OH proline. Inducible nitric oxide synthase (iNOS) inhibition and improvement of endothelial nitric oxide synthase (Enos) activities are the important efects of genistein on isoproterenol-induced cardiac hypertrophy [25].
Intraperitoneal administration of genistein improved pressure and left ventricular pressure/total heat rate in aged female and young male rats but not in young female rats in stunning induced by ischemia/reperfusion (I/R) in isolated hearts. In young male rats, genistein and estradiol have synergistic cardioprotective efect. After ischemia, SR Ca leak causing diastolic contracture was increased due to genistein. Results suggested that genistein was more cardioprotective and synergistic with estradiol, which depends on the mKATP and protein-kinase C channel pathway activation [26].
Te efects of genistein (as tyrosine kinase inhibitor) on hypertrophy induced by phenylephrine in ventricular myocytes cultured from neonatal rats prevented activation of three promoters, such as atrial natriuretic factor (ANF), Fos, and the myosin light chain 2 (MLC-2) in induced cells. Inhibition of the GTP loading of the Ras protein and activation of the mitogen-activated protein (MAP) kinases Erk1 and Erk2 in phenylephrine-induced cells were another efect of genistein [28].
Te results demonstrated that for the activation of the Ras-MAP kinase pathway, which is a critical pathway in hypertrophic response regulation, genistein is important.
In pulmonary hypertension (PH) rats, genistein (1 mg/ kg/day) administration showed decreased right ventricular ejection fraction to 41.99 ± 1.27% and increased peak systolic right ventricular pressure to 66.35 ± 1.03 mm Hg. Similar to the control group, right ventricular pressure was reduced, and right ventricular ejection fraction was completely restored to 65.67 ± 1.08%. Proliferation of the smooth muscle cell of the human pulmonary artery through estrogen receptor-β was inhibited by genistein [30].
In hypersensitive necrotic area, genistein IV administration attenuated the necrosis of myocardia and myocardial myeloperoxidase activity (MPO). It also decreased serum levels of TNF-α, creatinine phosphokinase activity (CPK), and ventricular arrhythmias and slowed intercellular adhesion molecule-1 (ICAM-1) expression in the injured myocardium [32]. Tese data suggested that myocardial ischemia-reperfusion injury protection and infammatory response inhibition are some efects of genistein.
Genistein administration as dietary supplement (54 mg/ day) in postmenopausal women for one year showed a signifcant development of left ventricular ejection fraction (LVEF) and left atrial (LA) area fractional change that followed 1 year of treatment. LA longitudinal strain peak and body surface area indexed LA systolic volume were changed by genistein. In women with metabolic syndrome (MetS), genistein intake (54 mg/day/year) improved both LA remodeling and function and LV ejection fraction [33]. Administration of dietary intake of the genistein in humans showed that genistein mitigates ischemic stroke-induced damage by alterations in molecular pathways. Genistein treatment blocked targeting estrogen, nuclear factor (NF)kappa B, direct antioxidant action, androgen-mediated molecular, and Akt signaling pathways which are important for stroke damages decreasing and cell survival increasing [34]. Subcutaneous genistein administration of 0.2 mg/kg/day in ovariectomized New Zealand white female rabbits did not have a protection efect on ischemic myocardium in both nonovariectomized or ovariectomized animals for 4 weeks [35]. Compared to control groups, genistein treatment (600 mg/kg, diet) for 4 weeks in both sexes of C57BL/6J mice made echocardiographic changes in function, increased left ventricle internal dimension (LVIDs), cardiac GLUT4 protein expression, and signifcantly decreased fractional shortening and whole heart surface area [36].
Genistein efects on cardiac dysfunction and ischemia/ reperfusion are shown in Table 1.

In Vitro Studies.
Dietary intake of genistein efect (20 microM) on ventricular myocytes in high glucose medium induced diabetic cardiomyopathy in adult rats reduced cardiac mechanical dysfunction, so it concluded that genistein had a signifcant efect against cardiac dysfunction induced by diabetes [38].   Evidence-Based Complementary and Alternative Medicine treatment part (s.i And i.p, respectively) showed that genistein elevated the myocardial guanosine 3′,5′-cyclic monophosphate (cGMP) and concentration of plasma nitrate after 6-hour endotoxic shock. Genistein signifcantly attenuated the cardiac action potential duration (APD) shortening in endotoxic shock by lowering the plasma nitrate and the cardiac cGMP production in both kinds of administration [39]. Te protective efects of genistein (5 mg/kg) against cardio toxicity induced by doxorubicin (DOX) in the mice model signifcantly reduced cardiac troponin and redox markers such as lipid peroxidation (LPO) and reactive oxygen species (ROS) in serum that increased by DOX. In addition, genistein reduced the expressions of interleukin (IL)-6, IL-8, and tumor necrosis factor (TNF)-α which increased during DOX-induced infammatory and regulated antioxidant response by increased protein expressions of Nrf-2, HO-1, nitroquinoline oxide (NQO1) [40].

In Vivo
Oral administration of genistein (300 mg/kg/day) in streptozotocin (STZ)-induced diabetic rats showed decreasing in C-reactive protein, HbA1c, blood glucose, and expression of TGF-β1 and NF-α proteins that were increased in STZ-induced diabetes. In addition, compared to control groups, genistein treatment increased total antioxidant reserves of the hearts and reduced the ultrastructural degenerative changes in the cardiac tissues [41]. Tese results indicated that genistein had an anti-infammatory and antioxidant efect.
Intraperitoneal injection of genistein (0.25, 0.5, or 1 mg/ kg) in mice revealed cardioprotective and antinitrative/oxidative efects after burning injury in mice. Genistein reduced myocardial injury such as improved left ventricle ejection fraction, reduced creatine kinase levels, and lactate dehydrogenase in the serum. It also reduced expressions of inducible nitric oxide (NO) synthase, NO and superoxide anions production, and reduced peroxynitrite. Treatment with genistein signifcantly up-regulated the expression of the enhancer of split (Hes-1) and Notch-1 intracellular domain (NICD1) after burning injury [42]. Genistein efects on cardio toxicity are summarized in Table 2.
Genistein (1000 nM) administration in human umbilical vein endothelial cells (HUVECs) for 30 min showed inhibition of ox-LDL-induced lowering of the activity of SAβ-gal levels and P16 and P21 proteins. Te genistein efect was bound up with elevating autophagic fux [44].

In Vivo
Studies. L-carnitine and genistein (50 mg/kg) administration on serum lipid and cytokine profles in induced nephrotic syndrome demonstrated genistein efect on cardiovascular diseases by inducing alterations in metabolism of lipid and production of cytokine. In addition, after treatment with genistein, low-density lipoproteins (LDL) and interleukin-6 markedly decreased. Te efect of genistein on triglyceride and high-density lipoproteins (HDL) levels was less than L-carnitine [45].
Genistein (20 mg/kg) administration for 42 days on polycystic ovary syndrome (PCOS) rats signifcantly increased insulin level. Genistein also signifcantly lower the levels of MDA TNF-α and higher SOD activity in the serum. Furthermore, in the histopathological analysis, genistein led to fewer cysts development and increase in luteinization [46]. Compare to control groups, estradiol (E2) and genistein (Gen) administration to ovariectomized (OVX) rats showed that treatment with E2 had signifcantly increased LDL chol, total cholesterol (Total-C), total oxidant status, and oxidative stress index. Also, in the genistein group, LDL chol and total-C decreased more than in the E2 treatment group. So we could conclude that Gen treatment might be preferred to E2 treatment for alleviating the menopausal symptoms and those who are at risk for cardiovascular diseases [47].
Genistein (600 mg/kg diet) supplement and soy protein (200 g/kg diet) in STZ-induced diabetic rat increased the glucokinase level and plasma insulin level but decreased glucose-6-phosphatase, thiobarbituric acid reactive substances, and HbA(IC) level of the STZ-induced diabetic rats. Tis combination administration signifcantly increased hepatic SOD, glutathione peroxidase, and catalase activities compared to the control groups [48].
Administration of 200 ppm genistein in rats by a 4-week feeding decreased cholesterol levels and improved calcium absorption efciency. In addition, in OVX rats receiving an AIN-93M diet supplement with 200 ppm genistein, restored total and trabecular bone mineral density at the distal femur similar to the levels of sham rats [49].
Subcutaneous administration of Gen (10 mg/kg) in orchidectomized (Orx) and intact (IA) middle-aged male rats signifcantly reduced lipoprotein cholesterol and TC levels. Gen showed signifcant cholesterol-lowering efect but increased total triglycerides only in the Orx group [50]. Dietary of genistein on induced hyperlipidemia in male hamsters showed genistein (2 g/kg) signifcantly lowered plasma cholesterol, triglyceride, LDL cholesterol, MDA, and liver cholesterol than those in the high-fat diet (HFD) group. In addition, genistein signifcantly up-regulated the expression of α and β mRNA estrogen and hepatic LDL receptors compared to the HFD group [51].
Administration of 250 μmol/L genistein that dissolved in dimethyl sulfoxide in humans showed that by decreasing lipid elements, genistein has benefcial efects on Evidence-Based Complementary and Alternative Medicine  Evidence-Based Complementary and Alternative Medicine atherosclerotic risk and lowering platelet aggregation. Genistein decreased collagen-and ADP-dependent platelet activation and inhibited agonist-induced platelet aggregation by dose-dependent manner [52]. In a human study, administration of Gen (0.5-2.5 microM) inhibited Cu ( ++ )-induced lipid peroxidation (LP) of HDL and increased the levels of conjugated dienes in lipoproteins oxidized. In addition, genistein blocked the alterations of the structure and physico-chemical properties of apoprotein, related to Cu ( ++ ) triggered LP of lipoproteins [53]. Supplementation with genistein (90 mg/day), vitamin D, and calcium in healthy postmenopausal women in clinical double blind study indicated variant allele rs9340799, rs928554, and rs4986938, and estrogen receptors (ERs) polymorphisms were associated with a greater reduction in LDL cholesterol, total cholesterol, and triglyceride levels. Tese results indicated the relationship between ER genes polymorphisms and improvement in the serum lipid profle after treatment with genistein in postmenopausal women [54].
Te results of a placebo-controlled trial on postmenopausal women (n � 389) with low bone mass showed treatment with of genistein aglycone (54 mg) for 24 months signifcantly declined fasting glucose and insulin, homocysteine, and fbrinogen. Genistein did not afect HDL cholesterol and triglycerides through serum osteoprotegerin. Tese results showed that a healthy diet and treatment with genistein aglycone plus calcium showed benefcial efects on some CVD risk factors and homocysteine levels in low bone mass postmenopausal women [55].
Treatment of hyperlipidemic postmenopausal women in China with genistein (60 mg/day) for six months signifcantly lowered TC, TG, and LDL cholesterol levels, while HDL cholesterol levels as well as mRNA expression and protein levels of LDLR, and liver X receptor α (LXRα) were markedly increased [56]. Te efects of genistein on lipids profle are shown in Table 3.

In Vitro Studies.
Te efects of genistein on the vascular calcifcation in rat monocytes, osteoblasts cultures, and aortic vascular cell (in vitro) were evaluated. Te mRNA expression of intercellular adhesion molecule-1 was reduced in the cells exposed to genistein (10 nM or 1 μM). Also, the cell proliferation and migration on vascular muscle cells were also markedly reduced.
Both osteoblastic markers such as deposition of calcium nodules and alkaline phosphatase activity were markedly reduced after genistein treatment [57].
Te acute treatment of thoracic aortae was isolated from adult male SHR rats with genistein (10 μM) restores the impaired endothelial functions in the aorta.
Genistein transactivates epidermal growth factor receptor (EGFR) through membrane ERα via G proteincoupled pathways [58]. Tis is enhanced eNOS phosphorylation and hence endothelial function in the aorta. Genistein treatment (50 micromol/l) inhibited the expressions of p22phox NADPH oxidase subunit and angiotensin II type 1 receptor (AT1) in aortic endothelial cells from stroke-prone SHR. Genistein also signifcantly reduced the angiotensin II induced superoxide by reduction of nitroblue tetrazolium, endothelin-1 production, and inhibition of nitrotyrosine formation [59].

In Vivo Studies.
Te efects of genistein (0.1-30 micromol/L) on aortic smooth muscle cells (SMC) from hypertensive rats showed inhibitory efects on proliferation and DNA synthesis of SMC in a dose-dependent manner. It also showed inhibitory efects on platelet-derived growth factor (PDGF) induced aortic SMC proliferation [60]. Tese results indicated that genistein treatment may be useful for reducing cell proliferation that involved in atherosclerotic vascular changes. Administration of genistein for 10 days (1 mg/kg) signifcantly improved lung and heart function by reduction in right ventricular pressure and restored right ventricular ejection fraction similar to the control group. Genistein also reversed pulmonary hypertension (PH)-induced pulmonary vascular remodeling (in vivo) and inhibited human pulmonary artery SMC proliferation (in vitro) likely through treatment with estrogen receptor-β. It also reversed right ventricular (RV) hypertrophy, inhibited neonatal rat ventricular myocyte hypertrophy, and restored PH-induced loss of capillaries in the RV [30]. Te results indicated that genistein prevents the progression of severe PH to right heart failure.
Efects of genistein on pulmonary arterial hypertension (PAH) in monocrotaline-induced rat model were also studied. Genistein (20,80, and 200 μg/kg) signifcantly improved diameter of pulmonary artery, speed of tricuspid regurgitation, mean pulmonary artery pressure, and RV hypertrophy index. Treatment with genistein also improved proliferation of smooth muscle, stenosis of pulmonary artery, RV hypertrophy, and myocardial hypertrophy. In rat lung tissue, phosphorylated protein kinase B (P-Akt), phosphorylated eNOS, and expressions of nitric oxide (NO) were remarkably increased in the genistein treatment group compared to the PAH group [62].
Te genistein fed diet (600 mg/kg food) remarkably less weight than male's mice fed the genistein-free diet (0G). Te Genistein fed diet also exhibited remarkably elevated serum insulin and decreased serum glucose levels. Basal vascular reactivity of isolated aortic rings (IAR) was increased remarkably by the genistein diet in both males and females mice [63].
Administration of genistein (1 mg/kg) on lipopolysaccharide (LPS)-induced rat cardiovascular abnormalities causes to reduction in contractile response to noradrenaline (NA) in vascular tissue, and inhibition in hyporesponsiveness to NA, increasing in nitrite plasma levels, and iNOS expression production by endotoxin. In addition, Evidence-Based Complementary and Alternative Medicine 11  [69] genistein restored lipid peroxidation, impaired aortic relaxation to acetylcholine, and suppressed long-term hypotension [64]. Tis result indicated that genistein prevented vascular alterations and hypotension induced by LPS that was mediated by its anti-infammatory and antioxidant efects as well as inhibitory efect on NO overproduction. Te efects of 10 nM genistein in rat aortic strips signifcantly increased nitric oxide synthesis. It also exhibited an antiaggregatory action, dependent on the NO release from vascular tissue, and suppressed the inhibition of platelet aggregation. Moreover, genistein inhibited platelet aggregation in aortic strips from ovariectomized rats [65]. Te vascular efects of genistein in ovariectomized spontaneously hypertensive rats (SHR) were evaluated. Animals treated with genistein (2.5 and 25 mg/kg), for 2 days or 2 weeks, and contractility of the renal arterial rings were studied. Te tissue contractions and tyrosine phosphorylation were reduced by the genistein (2.5 mg/kg) for 2 days [66].
Genistein treatment (20 and 80 µg/kg) on heme oxygenase-1 (HO-1) expression in PAH induced by monocrotaline (MCT) in rats as dose dependently improved the RV hypertrophy index, increased the expression of HO-1, and decreased the elevated mean PA pressure [67].
Dietary genistein (0.06%, wt/wt) decreased NaClsensitive hypertension in young, male stroke-prone spontaneously hypertensive rats (SHRs). Genistein also declined plasma insulin and insulin resistance in SHR on a high NaCl diet and reduced plasma triglycerides and cholesterol in SHR. Te antihypertensive efect of genistein was infuenced by the autonomic nervous system [68].
Treatment of 23-week-old rats with genistein (10 mg kg) reduced NADPH-induced O 2 ( − ) production and systolic blood pressure (SBP) and enhanced endothelium-dependent aortic relaxation to acetylcholine and increased aortic calmodulin-1 protein abundance and eNOS activity [69]. Te benefcial efects of genistein on blood pressure and endothelial dysfunction may be due to increased eNOS activity associated with increased calmodulin-1 expression and declined O 2 ( − ) generation. Te efects of genistein on hypertension are shown in Table 4. Te possible therapeutic efects of genistein on CVDs are shown in Figure 2

Conclusion
In vitro and in vivo studies reported potential therapeutic efects of genistein on cardiovascular disorders including improving cardiac dysfunction, ischemia and reperfusion of heart, decreasing cardiac toxicity, modulating lipid profle, and lowering the blood pressure which are reviewed in the current article. Te preventive or prophylactic efects of genistein on cardiovascular disorders may be due to antiinfammatory, antioxidant, and immunomodulatory efects. Although the efect of genistein on CVD is well shown in the reviewed studies, while further standard clinical trials and meta-analyzing the previous results are needed to be performed.

Data Availability
Te data used to support the fndings of this study are included within the article.

Conflicts of Interest
Te authors declare that there are no conficts of interest. Evidence-Based Complementary and Alternative Medicine 13 revised the manuscript. All authors read and approved the fnal version of the manuscript.