Oxidative stress is an unbalanced state of ROS production and abnormal regulation of the endogenous antioxidant mechanism . ROS production is affected by multiple factors, including dysfunction of oxidases, such as xanthinoxidase and nicotinamide adenine dinucleotide phosphate (NADPH), transport dysregulation in mitochondria, microsomes and/or the nucleus, neutrophil activation, arachidonic acid metabolism, and auto-oxidation of catecholamines, flavonoids, quinones, and proteins, etc. . Mitochondria are considered the main source of ROS production in high-metabolic organs such as the heart. Mitochondrial calcium activates the dehydrogenases in the Krebs cycle involved in oxidative phosphorylation and maintains a low level of nicotinamide adenine dinucleotide (NADH) accumulation, thus contributing to cellular energy balance and maintaining adequate cardiac function . In cardiac diseases, especially in heart failure, this process is disrupted, and mitochondrial Ca2+ uptake disorders eventually lead to NADPH oxidation and ROS production and accumulation [26, 27]. In addition, ROS can also be produced through the metabolism of drugs and exogenous substances, such as anticancer drugs, among which doxorubicin, which is related to the development of cardiomyopathy, is the most representative . Oxidative stress is an important common mechanism of myocardial injury caused by various aetiologies, such as hypertension, hyperglycaemia, ischaemia-reperfusion injury, and chemotherapy drugs, and is closely related to the subsequent occurrence and development of cardiac failure. Abnormally elevated ROS can cause many negative effects on cardiac function . In myocardial tissues, ROS directly damages the electrophysiology and contractile structure of myocardial cells by modifying the core proteins of the excitation-contraction coupling mechanism, including L-type calcium channels, sodium channels, potassium channels and sodium/calcium exchangers, and causes an energy deficiency in cardiac myocytes by affecting the function of proteins involved in energy metabolism . ROS leads to the activation of multiple signalling pathways related to cell death, cardiac fibroblast proliferation, matrix metalloproteinase activation, mitochondrial deoxyribonucleic acid (DNA) damage, mitochondrial dysfunction, impaired calcium regulation and cardiac hypertrophy [30-33]. These effects eventually lead to abnormal adaptive myocardial remodelling and cardiac dysfunction . In addition, recent studies have proposed that ROS can directly modify micro ribonucleic acid (microRNA), leading to changes in protein expression levels and the disruption of downstream gene regulation in corresponding tissues, thus leading to proteome remodelling and metabolic changes in cardiac tissues [35-37]. In addition to abnormally regulating related biomolecules and signaling pathways, ROS can also trigger cascade reactions of oxidative stress and expand the oxidative stress injury mentioned above, through producing other types of bioactive molecules (aldehydes), ROS induced ROS release and ROS mediated paracrine signals [36, 38].
The abnormal increase in ROS and clearance barriers are the main factors of oxidative stress injury, while flavonoids have been shown to be the main effective components in Astragalus membranaceus responsible for antioxidation and free radical elimination . Recently, studies have adopted electron spin resonance, chemiluminescence and other methods to test the bioactivity of anti-superoxide anion radicals in a few major types of flavonoids. Studies have shown that the antioxidant activity of different flavonoids by the order of strength is quercetin > kaempferol > isorhamnetin > isoquercitrin > rutin > calycosin > vitamin C > (3R)-8, 2′-dihydroxy 7, 4′-dimethoxyisoflavanone > formononetin > ononin > 2′, 4′-dimethoxy-3′-hydroxyl-isoflavanone-6-O-β-glucoside > (6aR, 11aR)-10-hydroxyl-3, 9-dimethylnissolin > (6aR, 11aR) 9, 10-dimethylnissolin-3-O-β-D-glycoside . These results suggest that calycosin has relatively strong antioxidant activity and is also superior to standard antioxidant vitamin C. Recent studies have more accurately discriminated, from the perspective of quantum chemistry, the antioxidant activity of four major flavonoids, from which it was confirmed that the antioxidant activity of calycosin is stronger than that of three other antioxidants (calycosin > calycosin-7-glucoside > formononetin > ononin) . The antioxidant bioactivity of calycosin is related to its chemical structure. The C-ring Δ2, 3-double bond and 4-carbonyl group in calycosin are all bioactive antioxidant groups. They can remove oxidative free radicals, reduce malondialdehyde (MDA) (lipid peroxidation product), protein carbonyl (amino acid oxidation product) and ROS levels, and increase superoxide dismutase (SOD), catalase and glutathione peroxidase (GSH-Px) levels. It can also enter the lipid bilayer of biological membranes to ameliorate lipid peroxidation induced by free radicals . Further studies suggest that calycosin may stabilize the membrane structure and enhance membrane integrity, which can reduce the lipid fluidity of the cell membrane and hinder the diffusion of free radicals, thereby reducing oxidative damage in cells [19, 41]. Using biological experiments, it was also shown that calycosin has a good antioxidant effect and that its cellular protective effect in multiple pathological states is mainly antioxidative. Li et al. showed that calycosin has a significant inhibitory effect on H2O2-induced oxidative stress in human liver parenchymal cells and can significantly alleviate oxidative damage in hepatic cells . Recent studies on the neuronal protective effects of calycosin indicate that calycosin can alleviate oxidative stress and the inflammatory response of neurons in the hippocampus of a mouse model of Alzheimer′s disease by activating the protein kinase C pathway, thereby improving cognitive function . In addition, in myocardial injury caused by ischaemia and hypoxia, calycosin can activate the Akt/PI3K and Nrf2/HO-1 signalling pathways, with myocardial protective effects; these pathways are important in organism oxidation and antioxidative regulation . Therefore, the antioxidative activity of calycosin may play an important role in its cardioprotective potential.
Cardiovascular diseases are a large group of pathophysiological states with multiple risk factors and aetiologies, but at the cellular level, various cardiovascular diseases often have a common basis of cell damage, such as oxidative stress in cardiomyocytes or endothelial cells, substrate metabolism and energy consumption, cell survival and apoptosis, and autophagy . Therefore, the treatment of cardiovascular diseases mainly includes two directions: intervention for the aetiologies of cardiovascular diseases and protective measures for the injury effects (organ level, cellular level or molecular level). Based on the multiple biological effects of calycosin and its derivatives, the protective effect of calycosin on cardiovascular cells has been studied.
For in vitro cardiomyocytes affected by different injury factors (H2O2, hypoglycaemia, hypoxia, and viruses, etc.), calycosin and its derivatives exhibit a common effect, that is, to reduce cell damage, predominantly indicated by the release of myocardial enzymes, and to reduce apoptosis-dominated cardiomyocyte death levels; furthermore, calycosin may play an anti-injury role through multiple mechanisms. For example, in H9C2 cells under hypoxic and hypoglycaemic conditions, pretreatment with calycosin can reduce the released level of the cell damage indicator lactate dehydrogenase (LDH) (Table 1), improve the cell survival rate, and increase the protein expression levels of phosphorylated Akt/PI3K, Nrf-l, Nrf-2, and HO-1, which suggests that the myocardial protective effect of calycosin in this process is related to the direct activation of the PI3K/Akt and Nrf-2/HO-1 pathways . In addition, these pathways are important in cell proliferation and in the balance of oxidation and antioxidation. In another study, the cellular protective effect of calycosin is considered to be related to its phytoestrogen-like effects and the study first demonstrates that calycosin can inhibit the oxidative stress-induced apoptosis of cardiac muscle cells (H9C2 cells) in vitro and the protective effect on the heart occur through oestrogen receptor α (ERα
) binding to ERβ and upregulating its expression, thereby activating the Akt signalling pathway . For infective myocardial injury, calycosin can inhibit coxsackie virus replication in African green monkey kidney heteroploid cell (Vero cells), and its antiviral activity and specificity are all superior to the traditional antiviral drug ribavirin (antiviral activity analysis showed that its half-maximal inhibition (IC50) was 25 μg·mL−1 and that the ratio of cytotoxicity to antiviral activity was 5.7), suggesting that it plays a role in cell protection through direct antiviral effects . Chemical
Cell type In vitro effect Signalling
References Calycosin H2O2 5, 10, 20 μmol·L−1 pretreatment for 24 hours H9C2 cells Cardiomyocyte apoptosis ↓
/β expression levels ↑
 Calycosin -7-O-β-D-glucopyranoside Coxsackie virus B3 25 μg·mL−1 African green monkey kidney heteroploid
cells (Vero cells)
Virus replication ↓  Calycosin Hypoglycaemia and hypoxia 0.01,0.1,1,10 μmol·L−1 pretreatment
for 2 hours
H9C2 cells LDH↓
Cell survival rate↑
Phosphorylated Akt, PI3K proteins↑
Nrf2, HO-1 proteins↑
PI3K/Akt signalling pathway
Nrf2/HO-1 signalling pathway
Table 1. In vitro myocardial protective effects of calycosin and its derivatives
In recent years, to further explore the myocardial protective effect and mechanism of calycosin, increasingly more in vivo experiments have provided more accurate and reliable evidence from different aspects.
Myocardial ischaemia-reperfusion injury is the main pathological of ischaemic heart disease develops and is associated with the development of myocyte necrosis, arrhythmia, myocardial stunning, endothelial dysfunction, and microvascular complications, etc [48-50]. Calycosin has been shown to have a good protective effect on ischaemic and reperfused myocardium: On the one hand, calycosin can improve cardiac function after ischaemia. For example, it increases the ejection fraction (EF), fractional shortening (FS), and left ventricular end systolic pressure (LVESP) and decreases left ventricular end diastolic pressure (LVEDP) (Table 2). Furthermore, it can induce the expression of vascular endothelial growth factor (VEGF) and CD31 and promote the angiogenesis of ischaemic myocardium . On the other hand, oxidative stress is an important mechanism for myocardial ischaemia-reperfusion injury. Calycosin can significantly reduce the level of MDA, the lipid oxidation product of the cell membrane. It also increases the level of the protective antioxidant product SOD, thus inhibiting the release of myocardial enzymes including creatine kinase (CK) and LDH, etc [52-54]. The P13K/Akt signalling pathway is considered an important pathway involved in the regulation of cardiomyocyte apoptosis and myocardium tissue protection. In addition, calycosin can increase the protein phosphorylation level of P13K and Akt during ischaemia-reperfusion injury, suggesting that calycosin may exert a myocardial protective effect through activating the P13K/Akt signalling pathway in ischaemia-reperfusion injury .
Drug administration methods Dose Animal Model Protective effects Signalling
References Calycosin Intraperitoneal injection 40 mg·kg−1·d−1
Isoproterenol-induced myocardial infarction mouse model MPO↓ (Neutrophils seepage reduction)
MDA↓ (Lipid peroxidation attenuation)
 Calycosin Intraperitoneal injection 0.5 mg·kg−1
myocardial ischaemia rat model Cardiac function↑
 Calycosin-7-O-β-d glucoside Intravenous injection 30 mg·kg−1
(low dose) pretreatment once
Myocardial ischaemia-reperfusion rat model Cardiac function↑
Area of myocardial infarction ↓
CK, LDH, MDA↓
phosphorylated PI3K p85/Akt↑
PI3K/Akt signalling pathway  Calycosin-7-O-β-D-glucopyranoside Intragastric gavage 24 mg·kg−1
Coxsackie virus B3 viral myocarditis mouse model Survival rate, cardiac function↑
Mononuclear cell infiltration, Virus replication↓
 Calycosin Ex vivo perfusion 0.1 μmol·L−1 pretreatment continuous perfusion
for 10 min
Ex vivo cardiac ischaemia-reperfusion rat model CK, LDH↓
 Calycosin Intragastric gavage 50 mg·kg−1
Isoproterenol intraperitoneal injection and aorta constriction myocardial hypertrophy mouse model Cardiac function↑
Marker of myocardial hypertrophy↓
Akt/GSK3β signalling pathway
ERK signalling pathway
 Calycosin Intraperitoneal injection 1 mg·kg−1·d−1
Abdominal aorta constrictive pressure overload rat model of myocardial hypertrophy Cardiac function↑
JAK1, STAT3 protein expression↓
JAK/STAT signalling pathway 
Table 2. In vivo myocardial protective effects of calycosin and its derivatives
In addition to its protective effect on acute ischaemic injury, calycosin also has a protective effect on some maladaptive changes in myocardial tissue. In a mouse model of myocardial hypertrophy constructed by isoproterenol induction and aortic constriction, calycosin significantly reduced markers of cardiac hypertrophy (atrial natriuretic peptide and myosin heavy chain-β) and inhibited myocardial remodelling. Additionally, the activation of the mitogen-activated protein kinase (MAPK) and AKT signalling pathways in mouse myocardium tissue was significantly attenuated, suggesting that calycosin may reduce cardiac hypertrophy induced by isoproterenol or aortic coarctation by inhibiting AKT/GSK3β and extracellular signal-regulated kinase (ERK) signalling .
For infective myocardial injury, calycosin showed multiple positive effects, such as anti-viral, anti-apoptosis and anti-inflammatory properties. In a mouse model of coxsackie virus B3 infection, calycosin-7-O-β-D-glucopyranoside (CCGR) treatment increased myocardial contractile force and cardiac EF. In addition, the progression of heart failure in infected mice was inhibited by increasing the systolic ventricular septal thickness, left ventricular posterior wall thickness and left ventricular volume. Overall, the symptoms of viral myocarditis were reduced, and the survival rate was improved (77.7% in the treatment group and 44.4% in the non-treatment group). This study further suggested that CCGR could play a protective role in myocardial tissue in viral myocarditis by significantly reducing viral titres, reducing myocardial cell oedema and inhibiting myocardial cell necrosis and monocyte infiltration caused by viral infection .
The influence of calycosin on vascular endothelial cells has multiple effects and multiple mechanisms. First, calycosin, as a phytoestrogen, can bind to ER-positive cells in the vascular endothelium and exert its role as a selective oestrogen receptor modulator (SERM) by increasing ERK1/2 phosphorylation and activating the MAPK signalling pathway, thereby promoting vascular endothelial cell proliferation . Second, calycosin can interfere with ion channels. It has been shown that calycosin has a vasodilating effect, which is not endothelium-dependent and independent of intracellular calcium ion release. Calycosin is a non-competitive calcium channel blocker that primarily blocks voltage-gated calcium ion channels and receptor-gated calcium channels . Tseng  et al. demonstrated that calycosin could activate the large-conductance Ca2+-activated K+ (BKCa) channels of human umbilical vein endothelial cells, thereby increasing the hyperpolarization of endothelial cells and the production of nitric oxide and mediating endothelium-dependent vasodilation. In addition, calycosin also has protective effects on endothelial cell injury induced by various factors. For endothelial cell injury induced by bacterial endotoxin, calycosin can increase the production of nitric oxide, reduce phosphorylated myosin light chain, reduce Rho/Rock signalling pathway activation-induced cytoskeleton remodelling by inhibiting the AKT signalling pathway, and exert protective effects on endothelial cells . Calycosin can also reduce the cytotoxicity and apoptosis of human umbilical vein endothelial cells induced by vascular endothelial growth factor receptor (VEGFR) tyrosine kinase inhibitor II (VRI), which is related to the activation of the PI3K/Akt/Bad and BRAF/MEK1/2/ERK1/2 signalling pathways . However, for human umbilical vein endothelial cell apoptosis induced by glycation end products, calycosin can directly increase the expression of anti-apoptotic protein Bcl-2 and simultaneously reduce the expression levels of pro-apoptotic proteins Bax and Bad, thereby inhibiting endothelial cell apoptosis [62, 63].
In addition to these protective effects on endothelial cells, calycosin can also directly affect endothelial cells (Fig.1). Calycosin-7-glucoside has therapeutic effect on angiotensin II (Ang II)-induced renin-angiotensin-aldosterone system (RAAS) disorder in human umbilical vein endothelial cells by down-regulation of angiotensin-converting enzyme (ACE) expression and increased ACE2 expression . Calycosin can inhibit the synthesis of Ang II-induced thromboxane A2 (TXA2) and prostacyclin (PGI2) in endothelial cells and reduce Ang II-induced endothelial cell injury. In addition, calycosin can inhibit the secretion and expression levels of endothelial cell inflammatory factors induced by tumor necrosis factor-α (TNF-α), such as intercellular adhesion molecule-1 (ICAM-1) and its receptor lymphocyte function-associated antigen 1 (LFA-1), thus protecting endothelial cells [65, 66].
The pathophysiological mechanism of many cardiovascular diseases is closely related to the inflammatory response. Previous studies have shown that the inflammatory response plays an important role in the occurrence and progression of cardiovascular diseases . In recent years, it has been found that calycosin and its derivatives have potential anti-inflammatory effects. Recent studies have revealed that the inhibition of the NF-κB signalling pathway may be the main mechanism of calycosin’s anti-inflammatory effects [68, 69]. By inhibiting the expression of NF-κB and MAPK signalling pathway-related proteins, calycosin-7-glucoside can reduce the production of pro-inflammatory factors including Prostaglandin E2 (PGE2), TNF-α, interleukin-1β (IL-1β) and interleukin-6 (IL-6) in endotoxin-induced macrophagocytes and inhibit the mRNA expression levels of inflammatory mediators inducible Nitric Oxide Synthase (iNOS) and cyclooxygenase-2 (COX-2) . However, studies on the anti-inflammatory effects of calycosin mainly focus on the diseases of the haematologic system, immune system and neurological system. There is no definitive research on the effects of calycosin on the inflammatory response in cardiovascular diseases.
Ischemic heart disease and thromboembolic disease are common types of cardiovascular diseases. Platelets play a crucial role in the development of these diseases by participating in thromboembolism development . Thus, anti-platelet is often a crucial part of cardiovascular disease therapy . Studies have shown that calycosin and its derivatives are the main active ingredients in many traditional Chinese medicine compounds widely used in cardiovascular diseases [73-76]. For example, calycosin-7-O-β-D-glucoside as one of main active ingredients of BuyangHuanwu decoction (BHD) can inhibit adenosine diphosphate (ADP)-induced platelet aggregation in vitro . In addition, the core bioactive components of compound xueshuantong capsule (CXC) act on different aspects of the vascular system to promote blood circulation. Among them, calycosin-7-O-β-D-glucoside has been proved to mainly affect extrinsic clotting activity and to have negative effects on red blood cell (RBC) aggregation, RBC deformability, intrinsic clotting activity and platelet aggregation . Although the above studies reveal the effect of calycosin on vascular system, the mechanism underlying it functional activity requires further systematic research.
The cardiovascular protective effect and mechanism of calycosin and its derivatives
- Received Date: 2020-05-20
- Available Online: 2020-12-20
Abstract: Cardiovascular disease is the main cause of mortality and morbidity in the world, especially in developing countries. Drug therapy is one of the main ways to treat cardiovascular diseases. Among them, great progress has been made in the treatment of cardiovascular diseases with traditional Chinese medicine. In terms of experimental research, the mechanism of traditional Chinese medicine in the treatment of cardiovascular diseases has been thoroughly discussed in vitro and in vivo. In terms of clinical treatment, traditional Chinese medicine with flavonoids, saponins and alkaloids as the main effective components has a definite effect on the treatment of cardiovascular diseases such as arrhythmia, myocardial ischemia, angina pectoris and myocardial infarction, with high safety and good application prospects. With the further research on the effective ingredients, mechanism and adverse reactions of traditional Chinese medicine, it will be beneficial to the effectiveness of traditional Chinese medicine, reduce side effects and promote the modernization of traditional Chinese medicine. Calycosin and its derivatives, the main bioactive flavonoids in Astragalus membranaceus have multiple biological effects, such as antioxidant, pro-angiogenesis, anti-tumour, and anti-inflammatory effects. Based on the above biological effects, calycosin has been shown to have good potential for cardiovascular protection. The potent antioxidant effect of calycosin may play an important role in the cardiovascular protective potential. For injured cardiac myocytes, calycosin and its derivatives can alleviate the cell damage mainly marked by the release of myocardial enzymes and reduce the death level of cardiac myocytes mainly characterized by apoptosis through various mechanisms. For vascular endothelial cells, calycosin also has multiple effects and multiple mechanisms, such as promoting vascular endothelial cell proliferation, exerting vasodilating effect and directly affecting the synthesis function of endothelial cells. The present review will address the bioactivity of calycosin in cardiovascular diseases such as protective effects on cardiac myocytes and vascular endothelial cells and elucidate main mechanism of calycosin and its derivatives to exert the above biological effects.