Potential Protective Effects of Equol (Soy Isoflavone Metabolite) on Coronary Heart Diseases—From Molecular Mechanisms to Studies in Humans

Equol, a soy isoflavone-derived metabolite of the gut microbiome, may be the key cardioprotective component of soy isoflavones. Systematic reviews have reported that soy isoflavones have no to very small effects on traditional cardiovascular disease risk factors. However, the potential mechanistic mode of action of equol on non-traditional cardiovascular risk factors has not been systematically reviewed. We searched the PubMed through to July 2021 by using terms for equol and each of the following markers: inflammation, oxidation, endothelial function, vasodilation, atherosclerosis, arterial stiffness, and coronary heart disease. Of the 231 records identified, 69 articles met the inclusion criteria and were summarized. Our review suggests that equol is more lipophilic, bioavailable, and generally more potent compared to soy isoflavones. Cell culture, animal, and human studies show that equol possesses antioxidative, anti-inflammatory, and vasodilatory properties and improves arterial stiffness and atherosclerosis. Many of these actions are mediated through the estrogen receptor β. Overall, equol may have a greater cardioprotective benefit than soy isoflavones. Clinical studies of equol are warranted because equol is available as a dietary supplement.


Introduction
Coronary heart disease (CHD) is the leading cause of morbidity and mortality worldwide [1]. Soy is a potential nutritional source for preventing CHD [2] and is a standard part of a traditional diet in East Asia [3]. The main components of soy that may exert cardioprotective effects are soy isoflavones (ISFs), mainly daidzein and genistein [4]. ISFs are phytoestrogens and are structurally similar to estradiol [5]. Estradiol exerts its biological action by binding both the estrogen receptor α (ERα), expressed in the reproductive, central nervous, and cardiovascular systems, as well as other systems [6], and the estrogen receptor β (ERβ), expressed in the cardiovascular and central nervous systems, as well as other systems. ISFs, however, preferentially bind to ERβ [7]. Among the flavonoid family of flavones, flavonols, flavanones, and ISFs, ISFs have the highest lipophilicity, which could contribute to their greater absorption from the gut [8]. ISFs may lower the risk of CHD by reducing inflammation [9][10][11] and oxidative stress [12][13][14]; the latter may prevent the oxidative damage to low-density lipoprotein (LDL) that contributes to atherogenesis [15].
Equol has the following characteristics that may grant it with possibly greater cardioprotective properties than ISFs: (1) Equol has higher antioxidant activity than ISFs [33,39,51], and its antioxidant properties are even greater than vitamins C and E in in vitro studies [52]; (2) the affinity of equol to ERβ is much higher than daidzein, though it is similar to genistein [53]; (3) equol is more lipophilic than ISFs; therefore, it has a higher absorption rate from the gut than ISFs, resulting in greater bioavailability [8]; (4) equol has a longer plasma half-life than ISFs, being retained in the body for a greater period following soy consumption [39]; (5) equol has a lower ability to bind to serum proteins (i.e., albumin, sex hormone-binding globulin, and alpha-fetoprotein) than ISFs and thus has a greater availability for receptor occupancy [27].

ERβ
Estrogen receptors (ERs) are intracellular transcription factors whose activity is modulated by estrogens, non-steroidal estrogen antagonists, or agonists such as ISFs. ERs belong to the nuclear hormone receptor family with two subtypes, ERα and ERβ [6]. Accumulating evidence has suggested that ERβ may play a more dominant role in mediating the cardioprotective effects of estradiol [6]. ERβ knock-out mice have an increased mortality and exacerbated heart failure after MI [54]. Studies of ERβ knock-out mice exhibited poor functional recovery compared to the wild-type or ERα knock-out mice in an ex vivo model of global ischemia-reperfusion [55]. Consistently, cardiomyocyte-specific ERβ overexpression improved cardiac function and survival after MI induced by left anterior descending coronary artery ligation [6].
Equol is a selective ERβ agonist [7]. Studies demonstrated that ERβ agonists are cardioprotective. For example, one study demonstrated that a selective ERβ agonist led to a significant recovery of cardiac functionality in ovariectomized mice due to the ERβdependent upregulation of cardioprotective genes [56]. Moreover, the exposure of human macrophages to an ERβ-selective agonist led to a robust decrease in the level of extracellular heat shock protein 27, a biomarker of atherosclerosis [57], while the ERα-selective agonists did not show such an effect [58]. Subjects receiving ERβ-selective agonists may retain the benefits of hormone therapy without the severe adverse effects.

Anti-Inflammatory Effects
Inflammation contributes to atherosclerosis from its inception to its development into thrombotic complications [59,60]. The accumulation of oxidized lipids triggers atherosclerosis mediated by proinflammatory cytokines, such as interleukin-1β, tumor necrosis factor-α (TNF-α), and nuclear factor-κB (NF-κB). Proinflammatory cytokines initiate the expression of vascular cell adhesion molecule-1 (VCAM-1) by endothelial cells (ECs) and allow the attachment of leukocytes, monocytes, and T lymphocytes to the arterial wall. Once adhered to the arterial endothelium, the monocytes penetrate the endothelial lining and enter the intima of the vessel wall by diapedesis between the ECs, a process triggered by chemokine monocyte chemoattractant protein-1 (MCP-1) [60]. Within the intima, monocytes mature into macrophages, exhibit increased expression of scavenger receptors, and engulf modified lipoproteins. Cholesterol esters accumulate in the cytoplasm, and the macrophages become foam cells [59,60].
Equol inhibits the overproduction of inflammatory biomarkers expressed by macrophages, microglia cells, and adipose tissues. In addition, equol ameliorates the inflammatory processes important to the pathogenesis of rheumatoid arthritis, metabolic syndrome, and intracranial aneurysm (Table 1, Figure 3). Equol exhibited protective effects against pro-inflammatory cytokines (IL-6 and TNF-α) and NO production in murine microglia cells. Equol also showed greater permeability through artificial gut and blood-brain barriers compared to daidzein. In microglial cells, equol inhibited TLR4 activation, MAPK activation, NF-kB-mediated transcription of inflammatory mediators, production of NO, release of PGE-2, and secretion of TNF-α and IL-6 in LPS-activated murine microglia cells.

Yes 6
Moriyama [66] In vitro Equol attenuated LPS-induced NO production with a concomitant decrease in the expression of iNOS. Equol did not affect the LPS-induced increase in intracellular ROS production. Increased NO production is a well-known inflammatory change in astrocytes stimulated by LPS. Attenuation of NO production by equol may mitigate LPS-induced neuroinflammation in astrocytes.

Yes 7
Lin [67] In vivo Equol-administered collagen-induced arthritis mice had a lower severity of arthritis symptoms. Equol administration suppressed the expression of IL-6 and its receptor in the inflamed area of collagen-induced arthritis mice.

Yes 8
Yokosuka [34] In vivo In ovariectomized mice induced to have intracranial aneurysms, equol protected against aneurysm formation; the disruption of the intestinal microbial conversion of daidzein to equol abolished daidzein's protective effect against aneurysm formation. Moreover, mice treated with equol had lower inflammatory cytokines in their cerebral arteries.
Yes 9 van der Velpen [35] Human In the adipose tissue of postmenopausal women, the expression of inflammation-related genes was upregulated in equol producers but downregulated in non-producers.

Yes 10 Törmälä [68] Human
ISFs caused a decrease in the VCAM-1 and platelet-selectin. The fall in platelet-selectin was more marked in equol producers. No changes appeared in SHBG, CRP, or ICAM-1.

Yes 11 Reverri [69] Human
Consuming soy improved arterial stiffness as was assessed by the augmentation index, but did not improve the inflammatory biomarkers (CRP, TNF-α, IL-6, IL-18, and IL-10). The addition of equol-producing status as a covariate did not significantly change these results. In women who received the ISFs intervention, there was no significant differences in percent change in the serum inflammatory markers between equol producers and non-producers. tween equol producers and non-producers.
Several in vivo studies in mice reported the effect of equol on inflammatory diseases and inflammation. Equol administration in a rheumatoid arthritis mouse model improved arthritis-induced bone mineral density and suppressed the expression of IL-6 and its receptor in inflamed areas [67]. In ovariectomized mice induced to have intracranial aneurysms, daidzein with equol supplementations protected against aneurysm formation, whereas the disruption of the intestinal microbial conversion of daidzein to equol abolished daidzein's protective effect against aneurysm formation, indicating that equol alone had a protective effect [34]. The same study also suggested that mice treated with equol had lower inflammatory cytokines in their cerebral arteries [34]. All of the above effects were exerted through the activation of ERβ.
In some of the human RCTs of ISFs, the anti-inflammatory effects of the ISFs were more pronounced in the equol producers than in the non-producers, while in other RCTs, the anti-inflammatory effects of the ISFs were only observed in equol producers. One RCT of ISFs among 110 postmenopausal women on tibolone showed that the equol producers had a larger reduction in platelet-selectin levels than the non-producers after being treated with ISFs [73]. Platelet-selectin is a cell adhesion molecule on the surface of activated ECs [74]. In the peripheral blood mononuclear cells in 30 equol-producing postmenopausal women, ISF intervention down-regulated clusters of genes that were involved in inflammation, oxidative phosphorylation, and cell-cycle regulation, as was suggested by the analysis in the whole-genome gene expression profiles [75]. Further analysis in the same group of participants later reported that ISF supplements upregulated the expression of the anti-inflammatory genes in the adipose tissue of equol producers but down-regulated the expression in non-producers [35].
An RCT of ISFs with 34 women found that neither the ISFs nor the equol-producing status was associated with CRP, VCAM-1, and ICAM-1 [71]. This null finding may be due to the small number of participants.
Cell culture studies show that equol is a potent antioxidant in numerous models (Table 2, Figure 4). The equol protected macrophages from oxidative stress induced by L-lactate dehydrogenase or oxLDL by reducing the lipid peroxidation product malondialdehyde (MDA), enhancing the antioxidant glutathione, or increasing the activities of the antioxidant enzyme superoxide dismutase (SOD) [89,90]. Equol treatment on neutrophils that were activated with various stimulants to produce ROS caused reduced toxic action of the ROS; for example, equol decreased the phosphorylation of the proteins regulating NADPH oxidase [91]. Phagocytic cells, after being incubated with equol for 1 h, showed a significant reduction in the intracellular production of superoxide anion and hydrogen peroxide [92]. In the intestinal epithelial cells with oxidative stress induced by hydrogen peroxide, equol promoted the expression of antioxidant genes, increased the activities of SOD, and increased the abundance of nuclear factor erythroid 2 (Nrf2) transcripts [93]. Finally, equol decreased the ratio of reduced/oxidized glutathione in primary cortical neuron cells [94]. Although some antioxidant effects are independent of ERβ [95], these effects are generally considered to be mediated through ERβ.  Animal studies also demonstrated the antioxidant properties of equol. Two mice studies demonstrated that equol improved vascular health in the brain [33,105]. It was suggested that equol elevated the activities of SOD, catalase, acetylcholinesterase, and glutathione peroxidase, and decreased the MDA levels in mice that had deoxycorticosterone acetate salt-induced hypertension and associated vascular dementia [105]. Equol also reduced NADPH-induced superoxide production in the cerebral arteries of normotensive rats and hypertensive rats that were induced by angiotensin II [33]. However, the antioxidant effect was not observed in daidzein [33]. One study showed that equol suppressed tumor formation in rats, presumably through decreasing the concentrations of thiobarbituric acid-reactive substances and 8-hydroxy-2-deoxyguanosine, and increasing the activity of catalase, SOD, and glutathione peroxidase [97].
One RCT in humans demonstrated that equol-producing individuals could receive the antioxidant benefits from ISFs, but non-producers could not. An RCT of 190 postmenopausal women found that after 6 months of supplementation with ISFs, blood MDA concentrations were significantly lower in the equol producers compared with the non-producers in the ISFs group [36].
At the normal physiological concentrations, equol is a more potent antioxidant compared to ISFs, probably due to the absence of 2,3-double bond, the 4-oxo group, and a 5,7dihydroxyl [96,[101][102][103]106]. The antioxidant property of equol is even more potent than ascorbic acid, alpha-tocopherol, and 17β-estradiol. Comparative studies found that the potency of inhibiting LDL lipid peroxidation was in this order: equol > genistein > daidzein [96,106].

Endothelial Function and Vasodilation
The endothelium regulates vascular tone, carefully balancing vasoconstriction and vasodilation to provide adequate perfusion to target organs [111]. All of the above inflammatory and oxidative processes cause damage to the vascular endothelium, leading to the formation of arteriosclerotic plaques [112]. Flow-mediated dilatation (FMD) of the brachial arteries provides a non-invasive measurement of endothelial function [113].
It has been observed in several categories of vessels (umbilical vein, aorta, pulmonary artery, and cerebral basilar artery) that equol stimulated endothelial redox signaling and increased NO production in the ECs [114][115][116] (Table 3). Studies indicated that equol, by binding to ERβ, could rapidly stimulate the phosphorylation of the extracellular signal-  Equol acted as an antioxidant in the brains of rats. The ratio of GSH/GSSG in primary cortical neuron cells exposed to equol for 24 and 72 h significantly decreased in a time-and dose-dependent manner. Moreover, equol treatment significantly increased the LDH release in a time-and dose-dependent manner.

Yes 15
Gou [90] In vitro Equol protected chicken macrophages from oxidative stress induced by lipopolysaccharide through reducing lipid peroxidation products such as MDA and enhancing the contents of antioxidants such as glutathione and the activities of relevant antioxidase enzymes such as total SOD; effects were also seen in gene expression related to the immune response and increased contents of cytokines.

Yes 16
Liu [105] In vitro Equol elevated brain antioxidant activity by increasing SOD, CAT, and GPx levels. MDA levels and AChE activity were decreased in hypertensive and vascular dementia rats. Equol further improved the long-and short-term memory of the rats.

Yes 17
Vedavanam [106] In vivo The order of the half-maximal inhibitory concentration values, the indication of the potency of inhibiting glucose-induced LDL lipid peroxidation observed for the compounds, was equol > genistein > daidzein.

Yes 18
Choi [89] In vivo Equol might act as an antioxidant through an inhibition of oxidative stress and the stimulation of CAT and SOD, but could also cause pro-oxidant effects, such as the reduction of the GSH/GSSG ratio, depending on the treatment period. A study in mice showed that equol administration significantly inhibited biomarkers of oxidative stress (thiobarbituric acid-reactive substances value, carbonyl content, and serum 8-hydroxydeoxyguanosine). Moreover, the CAT and total SOD activities and their transcripts were significantly increased by equol. Although equol increased the glutathione peroxidase activity in mice treated with equol for 1-week, long-term administration of equol (7 weeks) caused a decrease in the ratio of GSH/GSSG and the activities of GPx and glutathione reductase. Ma [107] In vivo A study in male and ovariectomized female rats with transient middle cerebral artery occlusion revealed that the pretreatment of equol significantly reduced infarct size in both sexes. This neuroprotection was accompanied by a decrease in the NADPH oxidase activity and superoxide levels in the brain. In addition, equol reduced plasma thiobarbituric acid reactive substances and neurological deficits up to 7 days after injury.

Yes 20
Horiuchi [108] In vitro The study demonstrated that equol had suppressive effects against oxidative stress in pancreatic β-cells in a dose-dependent manner and presumably through activating PKA signaling.

Yes 21
Jackman [33] In vivo Equol exerted weak antioxidant effects in cerebral arteries, whereas the effects of daidzein were insignificant. Antioxidant activity was assessed as the reduction in NADPH-induced superoxide levels.

Yes 22
Widyarini [109] In vivo In addition to the activation of estrogenic signaling pathways for photoprotection, equol also provided UV-protective antioxidant effects that depend partially on HO-1 induction. Equol dose-dependently inhibited the oxidative stress measured as UVA-induced lipid peroxidation on mouse skin. A component of the equol lipid protection capacity is attributed to endogenous cutaneous antioxidant enzymes, including the inducible stress protein HO-1.

Yes 23 Nhan [110] Human
Urinary equol was not associated with the secretion of urinary F2 isoprostane, a measure of cellular lipid peroxidation, after ISF treatment in postmenopausal women. However, the observations on the effect of equol were limited because only two of the eight subjects were equol producers, one of whom experienced a large increase in the biomarker excretion, whereas the other experienced small decreases.

No 24 Hidayat [36] Human
The level of MDA, an oxidative stress marker, was lower in equol producers than non-producers. This RCT was conducted with 190 postmenopausal women aged 47-60 who received 100 mg ISFs for 6 months. The random allocation of ISFs intervention was carried out separately by equol-producing status.

Yes 25
Richardson [95] In vitro Equol might have a beneficial effect in delaying the onset and decreasing the severity of symptoms in Friedreich's ataxia patients by an antioxidant mechanism, such as reducing the ROS-induced modification of proteins and lipids and impaired mitochondrial function. These effects were independent of the ERβ.
Animal studies also demonstrated the antioxidant properties of equol. Two mice studies demonstrated that equol improved vascular health in the brain [33,105]. It was suggested that equol elevated the activities of SOD, catalase, acetylcholinesterase, and glutathione peroxidase, and decreased the MDA levels in mice that had deoxycorticosterone acetate salt-induced hypertension and associated vascular dementia [105]. Equol also reduced NADPH-induced superoxide production in the cerebral arteries of normotensive rats and hypertensive rats that were induced by angiotensin II [33]. However, the antioxidant effect was not observed in daidzein [33]. One study showed that equol suppressed tumor formation in rats, presumably through decreasing the concentrations of thiobarbituric acid-reactive substances and 8-hydroxy-2-deoxyguanosine, and increasing the activity of catalase, SOD, and glutathione peroxidase [97].
One RCT in humans demonstrated that equol-producing individuals could receive the antioxidant benefits from ISFs, but non-producers could not. An RCT of 190 postmenopausal women found that after 6 months of supplementation with ISFs, blood MDA concentrations were significantly lower in the equol producers compared with the nonproducers in the ISFs group [36].

Endothelial Function and Vasodilation
The endothelium regulates vascular tone, carefully balancing vasoconstriction and vasodilation to provide adequate perfusion to target organs [111]. All of the above inflammatory and oxidative processes cause damage to the vascular endothelium, leading to the formation of arteriosclerotic plaques [112]. Flow-mediated dilatation (FMD) of the brachial arteries provides a non-invasive measurement of endothelial function [113].
It has been observed in several categories of vessels (umbilical vein, aorta, pulmonary artery, and cerebral basilar artery) that equol stimulated endothelial redox signaling and increased NO production in the ECs [114][115][116] (Table 3). Studies indicated that equol, by binding to ERβ, could rapidly stimulate the phosphorylation of the extracellular signal-regulated protein kinase 1/2 and phosphatidylinositol 3-kinase/protein kinase B (PI3K/Akt), leading to the activation of endothelial NO synthase (eNOS) [114,117]. NO can react with superoxide anions to form peroxynitrite. NO and peroxynitrite then, in turn, enhance the nuclear accumulation of Nrf2, which binds to an antioxidant response element in target genes to enhance the transcription of the phase II antioxidant defense enzymes, such as superoxide dismutase, catalase, glutathione-S-transferase, glutathione peroxidase, and heme oxygenase-1 [118]. A second pathway by which equol improved endothelial function was through the transactivation of the epidermal growth factor receptor kinase, resulting in a reorganized F-actin cytoskeleton [119]. A third pathway in which equol was involved was through the direct upregulation of eNOS, resulting in reduced oxidative stress in the ECs [115,120]. The target gene eNOS contains an estrogen-response element. Thus, it can be reasoned that the binding of equol to ERβ may be responsible for enhanced eNOS expression. In a fourth pathway, equol could increase the expression of phospho-p38 MAPK and B-cell lymphoma-2 to reduce intracellular ROS production in the ECs [121]. Furthermore, equol directly inhibited the apoptosis of the ECs [122], presumably through stimulating the thymidine incorporation, which is important for the deoxyribonucleic acid (DNA) synthesis of the ECs [122]. This stimulatory effect on cell growth was not observed for daidzein or genistein [122]. Equol had a significant antioxidant effect on the bAECs that were exposed to hydrogen peroxide. Equol pretreatment effectively inhibited the hydrogen peroxide-induced cell death by the reduction in intracellular ROS production, probably through increasing phospho-p38 MAPK. Zhang [123] In vitro

Yes
The improvement of atherosclerosis by equol through attenuation of endoplasmic reticulum stress is mediated by activating the Nrf2 signaling pathway. Equol treatment inhibited cell apoptosis and attenuated upregulation of endoplasmic reticulum stress markers in HUVECs. In an oxidative stress environment, equol treatment dose-dependently activated the Nrf2 signaling pathway.

Yes 7
Somjen [122] In vitro Equol, but not daidzein and genistein, had a monophasic stimulatory effect on thymidine incorporation, which boosts DNA synthesis. In human endothelial cells, equol, daidzein, and genistein stimulated DNA synthesis in a dose-dependent manner. The administration of equol, daidzein, and genistein to immature and ovariectomized female rats resulted in increased creatine phosphokinase in the aorta and in the left ventricle of the heart.

Yes 8
Kim [124] In vitro Equol had a vasodilatory effect on human uterine arteries vascular smooth muscle, which was mediated through antagonistic action for a receptor-dependent Ca 2+ channel. Yes 9 Johnson [62] In vitro Equol exhibited protective effects against NO production in murine microglial cells.
Equol also showed greater permeability through artificial gut and blood-brain barriers compared to daidzein.

Yes 10
Chin-Dusting [125] In vivo Equol had a dose-dependent inhibition of the contractile responses to noradrenaline in rat isolated aortic rings. Equol independently increased the release of a vasoconstrictor prostanoid, such as thromboxane.

Yes 11
Jackman [33] In vivo In normotensive rats, equol displayed vasorelaxant activity similar to daidzein. The relaxant effect of equol was independent of intact endothelium, NOS activity, K + channels, and gender. In the basilar artery, where superoxide levels are higher, equol exerted weak antioxidant effects, whereas the effects of daidzein were insignificant. During hypertension, equol-induced vasorelaxation was preserved, whereas relaxant responses to daidzein were impaired.

Yes 12
Matsumoto [126] In vivo Contractions induced by a selective 5-HT receptor agonist increased with insulin treatment, but less so with equol + insulin. In the endothelium-denuded preparations, 5-HT-induced contractions were augmented with insulin treatment but less so by equol + insulin treatment. These differences in 5-HT-induced contractions were eliminated by a large-conductance of Ca 2+ -activated K + channel inhibitor.

Yes 13
Yu [116] In vivo Equol significantly increased regional cerebral blood flow in rats and produced an endothelium-independent relaxation in rat cerebral basilar arteries. Selective Ca 2+ -activated K + channel blockers significantly inhibited equol-induced vasodilation in cerebral arteries.

Yes 14
Ohkura [120] In vivo Ovariectomized rats were assigned to (1) an ISF-deficient but equol-sufficient group, (2) an ISFs-deficient and equol-deficient group. In the thoracic artery, endothelium-dependent relaxation, cyclic guanosine monophosphate levels in the tissue, and eNOS synthase expression and phosphorylation were significantly higher in the first group compared to the second group.

Yes 15 Törmälä [73] Human
Before ISF intervention, women with a 4-fold elevation in equol levels had a lower endothelial function index compared to women without this capacity. Soy supplementation had no effect on arterial stiffness or endothelial function in either group.

Yes 16
Kreijkamp-Kaspers [37] Human This RCT did not support the hypothesis that ISFs have beneficial effects on endothelial function in older postmenopausal women. However, in the soy-only group, systolic and diastolic blood pressure decreased, and endothelial function improved in the equol producers, whereas blood pressure increased, and endothelial function deteriorated in the non-producers.
Independent of an effect on the intact endothelium and NOS activity, equol exhibited vasodilator activity in vascular smooth muscle cells of various of arteries, which may be induced by the antagonism of the Ca 2+ -activated K + channel [33,116,124,126]. This vasodilatory effect was not observed with daidzein [33].
Equol-producing status in humans may be critical in unlocking the endothelial benefits of ISFs. In a long-term RCT of ISFs among 202 older postmenopausal women, endothelial function was significantly improved in equol producers, whereas it deteriorated in non-producers when both groups were treated with ISFs [37]. Secondary analysis of the RCT of ISFs with 110 women [73] reported significantly improved endothelial function in equol producers as compared to the controls, whereas the ISFs themselves had no significant effects on endothelial function. Another RCT of ISFs in 62 adults with hypercholesterolemia observed the vasodilatory effect of ISFs on the brachial artery and determined that improvement was larger in equol producers [127].

Arterial Stiffness
A healthy aorta normally exerts a powerful cushioning function, which delivers a nearly steady flow of blood to the end organs [128]. Arterial stiffness impairs this cushioning function and is now recognized as a significant predictor of future cardiovascular events, independent of traditional risk factors [129][130][131]. Arterial stiffness is closely associated with the endothelial function and vasodilation [132] described in the previous section. Arterial stiffness can be determined by measuring carotid-femoral pulse wave velocity (cfPWV), brachial-ankle PWV (baPWV), and the cardio-ankle vascular index (CAVI) [129].
Many nutrients have been hypothesized to improve arterial stiffness, potentially through the modulation of endothelial function, and reduce oxidative stress and inflammatory processes [133]. Pase et al. conducted a systematic review of 38 RCTs of nutrients in 2011 and concluded that supplementation of ISFs and marine-derived omega-3 fatty acids improved arterial stiffness [133]. We recently updated this systematic review of the RCTs of ISFs and conducted a meta-analysis. We showed that supplementation of ISFs significantly improved arterial stiffness [134]. The effect of ISFs on arterial stiffness is independent of blood pressure. However, the duration of intervention in these RCTs was relatively short (23 h to 12 weeks) and the effects of ISFs in these RCTs are likely to result in functional rather than structural changes.
Equol supplements relieve the severity of arterial stiffness in the human body, as is suggested by the RCTs (Table 4). Usui et al. conducted a crossover RCT of 10 mg/day of equol on arterial stiffness assessed by the CAVI in 54 overweight or obese middle-aged men and women in Japan and showed that the supplementation of equol significantly improved arterial stiffness [135]. However, the sample size was small, and the duration of intervention was short (12 weeks). Several sources of evidence support the hypothesis that equol may have a long-term effect on arterial stiffness. A 12-month RCT of ISFs plus epicatechin in 35 patients with diabetes (mean age of 62) showed ISFs plus epicatechin significantly improved arterial stiffness assessed by cfPWV. Although the effect was not solely attributed to the ISFs, the improvement was more pronounced in the equol producers, suggesting that long-term exposure to equol may improve arterial stiffness [136]. Furthermore, the administration of 10 mg/day of equol in 74 women in Japan (mean age of 55) for 12 months significantly reduced arterial stiffness assessed by baPWV [137]. This study was not placebo-controlled; thus, the interpretation of the result is limited. In men prospectively recruited according to equol-producing status, acute (24 h) ISF treatment improved cfPWV in equol producers but had no effect in non-producers [138]. Compared with the placebo group, intervention with natural equol led to a significant decrease in the HbA1c, serum LDL-C levels and CAVI score. Furthermore, the effect was more prominent in a subgroup of female equol non-producers.

Yes 2 Curtis [136] Human
Overall, the ISF intervention did not significantly change the common carotid artery or augmentation index, but the pulse pressure variability improved. Equol producers had larger reductions in mean arterial pressure and PWV compared with non-producers.

Yes 3 Hazim [138] Human
In an RCT, acute ISF treatment (24 h) improved cfPWV in equol producers but had no effect on endothelial function and NO in non-producers. Yes Soy supplementation had no effect on arterial stiffness in either equol producers or non-producers. At baseline (before ISF treatment), women with a 4-fold elevation in equol level had a lower augmentation index compared to women without this capacity.
Observational studies in humans also show that equol-producing status or serum equol levels were associated with a lower degree of arterial stiffness. In a cross-sectional study of Japanese women aged 21-89 years, 67 equol-producing women in their 50s showed a significantly lower PWV than 147 non-producers [139]. Although soy had no effect on arterial stiffness measured by augmentation index, women with a 4-fold elevation in equol level cross-sectionally had a lower augmentation index compared to women without the capacity of producing equol [73]. It is worth noting that because the Japanese RCTs comprise a population with the habitual intake of soy ISFs and a high prevalence of equol producers, studies that showed a beneficial effect of ISFs may be attributed to equol. A cross-sectional study of 652 men in Japan (mean age of 49) showed that dietary intake of ISFs was inversely associated with arterial stiffness assessed by baPWV even after adjusting for age and traditional cardiovascular risk factors [140]. Although this study was cross-sectional, the result implies that long-term exposure to equol may improve arterial stiffness. In contrast, in one RCT, equol-producing status did not modify the effect of ISFs on arterial stiffness, measured by peripheral arterial tonometry [69].

Atherosclerosis and CHD
An RCT with mice suggested that equol had anti-atherogenic effects, where equol intervention reduced atherosclerotic lesions in the aorta in apolipoprotein E-deficient mice fed a high-fat diet [123] (Table 5). Observational studies in humans have shown that equol producers, as compared to non-producers, had significantly lower levels of atherosclerosis. Our cross-sectional study in 272 Japanese men aged 40-49 years found that equol producers had 9-fold lower odds of coronary artery calcification (CAC) presence compared to non-producers, but the serum ISFs were not significantly associated with CAC [142]. The Guangzhou Nutrition and Health Study of 2,572 middle-aged and older adults found that the serum level of equol was prospectively associated with reduced progression of carotid intima-media thickness over 8.8 years [143].
The first and the only observational study that ever explored the association between the equol level and CHD incidence was a nested case-control study within a cohort in China (377 cases and 753 controls). Total urinary ISFs or their metabolites other than equol were not associated with CHD in either women or men. However, urinary equol excretion showed an inverse association with CHD in women. The adjusted hazard ratio (95% confidence intervals) for CHD across increasing quartiles of equol levels were 1.00 (reference), 0.61 (0.32, 1.15), 0.51 (0.26, 0.98), and 0.46 (0.24, 0.89) (p = 0.02) [30].

Discussion
Evidence in this present systematic review suggests that equol has anti-atherogenic properties which are more potent than ISFs. Previous studies investigating the association of the dietary intake of ISFs with CHD have observed controversial results. Our review suggests that the potential protective effects of ISFs on CHD may require the intestinal conversion of daidzein to equol. This systematic review demonstrates that the potential cardioprotective effects of equol are potentially through its anti-inflammatory, vasodilatory, antioxidative properties, as well as its association with the improvement in endothelial functions and arterial stiffness.
In general, cell culture and preclinical studies show that equol is more efficacious than ISFs. Some effects, such as reducing the secretion of inflammatory biomarkers in macrophage [61], reducing NADPH-induced superoxide levels [33], stimulating endothelial cell growth [122], and improving vasodilation [33] were observed with equol treatment only and not with daidzein or genistein. Moreover, the disruption of the intestinal microbial conversion of daidzein to equol abolished daidzein's protective effect against aneurysm formation [34]. In addition, observational studies showed that equol rather than the ISFs was significantly and inversely associated with CHD incidence [143] as well as the progression of atherosclerosis [142]. In the RCTs of ISFs in humans, the cardioprotective effects of the ISFs were either more pronounced in equol producers than non-producers [73] or were only present in equol producers [35,36]. These observations strongly support the equol hypothesis that equol rather than ISFs are cardioprotective.
Observational studies showed that dietary intake of antioxidants is significantly and inversely associated with the risk of CVD, whereas the RCTs of vitamin E and other antioxidants reported little or no observed risk reduction among participants of a variety of characteristics [144][145][146][147][148][149][150]. One explanation for this discrepancy is that synthetic versions of the antioxidants administered in these trials may not completely mimic the natural forms of the antioxidants [151]. Equol is available as a dietary supplement and can be administered as a natural form because it is developed by the fermentation of soy germ with Lactococcus [135].
A large number of studies in this systematic review focused on an estrogen-deprivation situation, e.g., cell models simulating a postmenopausal cellular environment and human studies among postmenopausal women. The effects of estrogen therapy on CVD are controversial. Generally, when blood vessels are healthy, estrogen appears to protect them from the development of atherosclerotic plaques [152]. However, if atherosclerosis is wellestablished, estrogen does not appear to be beneficial but rather increases the risk of clinical events [153,154]. Because equol has a greater affinity for ERβ than for ERα, equol may not have adverse effects associated with estrogen supplementation, which is considered to target ERα [155].
Overall, we thoroughly reviewed the potential protective effects of equol on CHD from a comprehensive collection of studies, including transcriptomics, whole-genome gene expression, in vitro, animal, and observational and clinical studies. Based on this systematic review, the clinical studies of equol on atherosclerosis and CHD are warranted.