Atherosclerosis is one of the most important causative factors of certain CVDs such as myocardial infarction (MI), heart failure, stroke and claudication (Frostegård, 2013). Atherosclerosis refers to a specific form of arteriosclerosis in which an artery-wall thickens due to invasion and accumulation of lipid laden white blood cells (foam cells) and proliferation of intimal-smooth-muscle cell forming a fibrofatty plaque (Lucas and Greaves, 2001). There have also been studies supporting the anti-atherosclerotic effects of RC. For example, a study on the anti-atherosclerotic potential of Ger-Gen-Chyn-Lian-Tang (a traditional mixture which contains berberine as one of its major active components) has demonstrated reduced atherosclerotic lesions and collagen expression within the atheroma plaques in mice. In addition, in vitro work has also shown a reduction in migration of vascular smooth muscle cells, which is significant as pathological migration of these cells represents a key step in the pathogenesis of atherosclerosis (Ho et al., 2012).

Reactive oxygen species (ROS) play a crucial role in the development and progression of atherosclerotic lesions because the formations of lesions are related to several ROS-regulated events. It was found that uncoupling protein 2 (UCP-2), the mitochondrial inner membrane protein can provide antioxidant mediated defense by negatively regulating ROS production (Moukdar et al., 2009). Utilizing a mouse model, Wang et al. (2011) reported that chronic administration of berberine resulted in significant reduction of aortic lesions, diminished oxidative stress and lowered expression of adhesion molecules in the aorta; this anti-atherogenic activity was due to AMPK-dependent ROS suppression through enhanced expression of UCP-2. Oxidative modification of LDL is also believed to be one of the main risk factors leading to atherosclerosis (Itabe, 2009); berberine—one of the major bioactive components of RC—has been reported to inhibit oxidation of LDL by virtue of its significant antioxidant activity (Hsieh et al., 2007). Berberine also appears to protect against oxidized-LDL induced apoptosis as demonstrated by reduced levels of mitochondrial cytochrome C, cleaved apoptotic effectors, caspase 3 and poly(ADP-ribose) polymerase in human umbilical vein endothelial cells treated with this compound (Hsieh et al., 2007). Magnoflorine, another alkaloid isolated from RC extract also possesses similarly beneficial effects; an effective antioxidant, it prevents the oxidation of various forms of LDL, including native, glycated and glycoxidated LDL (Hung et al., 2007a). Aside from this, magnoflorine also significantly inhibits Cu²⁺ and thermo-labile radical initiator (AAPH)-induced lipid peroxidation of high density lipoprotein (HDL); thus resulting in HDL retaining its ability to protect LDL from oxidation (Hung et al., 2007b).

Chronic inflammatory processes are a key additional factor leading to the development of atherosclerotic lesions in the vessel wall (Remppis et al., 2010). Hence, substances with anti-inflammatory property may have the potential to prevent atherosclerosis. Remppis et al. (2010) studied the effect of RC extract and berberine on lipopolysaccharide (LPS)-induced inflammatory activity in a murine macrophage cell line. The results of this study demonstrated that RC extract and berberine inhibit the production of monocyte chemoattractant protein 1 (MCP-1/CCL2) in macrophages, likely via inhibition of the transcription factors activator protein 1 (AP-1) and nuclear factor-kappaB (NFκB). Coptisine, another of the bioactive compounds in RC, also possesses demonstrable anti-inflammatory property. It resulted in decreased nitric oxide (NO) production in LPS-stimulated macrophages via inhibited protein and mRNA expression of inducible nitro oxide synthase (iNOS); additionally, the expression of pro-inflammatory cytokines interleukin (IL)-1β and IL-6 was inhibited at transcriptional level. The proposed underlying mechanism for these observations was blockade of NFκB, mitogen-activated protein kinases (MAPK), and phosphoinositide-3-kinase (PI3K)/Akt activation, which are the intracellular inflammation signaling pathways in macrophages (Supriady et al., 2015; Wu et al., 2016).

The accumulation of lipid-laden foam cells is also an important step in the progression of atherosclerosis (Chi et al., 2014). The authors demonstrated that berberine effectively inhibited oxidized-LDL-induced foam cell formation through the activation of adenosine 5′-monophosphate-activated protein kinase (AMPK)-sirtuin 1-peroxisome proliferator-activated receptor gamma (PPAR-γ) pathway as well as through reduced uptake of oxidized LDL by foam cells. Furthermore, the same study showed that combination therapy of berberine and atorvastatin (a well-established anti-atherosclerotic drug) was more effective in preventing atherosclerotic processes compared to administration of atorvastatin alone.

Clinically, the major concern when artherosclerosis is present is the occurrence of plaque disruption which may then lead to unstable angina, or an outright MI. Plaque progression and destabilization is due to the over-expression of matrix metalloproteinases-9 (MMP-9) and extracellular matrix metalloproteinase inducer (EMMPRIN) in monocytes/macrophages (Cao et al., 2014). Berberine inhibits the up-regulation of MMP-9 and EMMPRIN in phorbol 12-myristate 13-acetate-induced macrophages by suppressing the activation of p38 pathway, showing its ability to stabilize atherosclerotic plaque (Huang et al., 2011).

RC seems to have multiple active components that target varying stages of the development of atherosclerosis through a variety of pathways, thus making it an effective anti-atherosclerotic agent.

Hyperlipidemia or dyslipidemia, which is characterized by increased plasma triglyceride concentration and lowered HDL cholesterol, is one of the major risk factor contributing to the progression of CVDs such as CHD and peripheral artery disease. Hence, maintenance of total cholesterol (TC) level within the normal range can be an effective strategy to lower the risk of cardiovascular events (Zou et al., 2016). The liver is the main site of cholesterol homeostasis maintenance, which involves several distinct processes including biosynthesis via the activity of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGCR), uptake by low density lipoprotein receptors (LDLR), release of lipoprotein in the blood, storage, degradation and conversion into bile acids (Trapani et al., 2012). RC possesses lipid-lowering effects as shown by the lowered cholesterol levels in different animal models treated with RC extract (Yokozawa et al., 2003; Cao et al., 2012; Kou et al., 2016). Studies have indicated that the five main bioactive compounds in RC (berberine, coptisine, palmatine, epiberberine, and jatrorrhizine) exert their lipid-lowering effects through distinct mechanisms. Berberine's hypolipidemic effect was the result of up-regulation of LDLR mRNA and protein expression (Zhou et al., 2014). Coptisine treatment also resulted in increased expression of LDLR, and additionally was associated with up-regulated expression of cholesterol 7-alpha-hydroxylase (CYP7A1) and down-regulated expression of HMGCR (Chen et al., 2015; He et al., 2015b). In addition to up-regulated expression of LDLR and CYP7A1, treatment with palmatine and epiberberine also caused down-regulation of apical sodium-dependent bile salt transporter (ASBT) mRNA and protein expression, as well as enhanced fecal excretion of TC and total bile acids (Ning et al., 2015; Zou et al., 2016). Jatrorrhizine has been linked to up-regulated mRNA and protein expression of LDLR and CYP7A1 but it has no effect on the expression of ASBT and HMGCR (Wu et al., 2014a). Comparative studies have been done on the lipid-lowering effect of individual alkaloids and among those tested, coptisine was reported to have the highest lipid-lowering effect (Wu et al., 2014c). More recently, research involving combined treatment with the alkaloids in RC extract has also been carried out. While each of the compounds administered in isolation did demonstrate lipid-lowering effect, the administration of a combination of these alkaloids resulted in synergistic effects, with combination therapy resulting in a significantly greater lipid-lowering effect compared to treatment with a single alkaloid. The main mechanisms associated with this effect are thought to be the retardation of cholesterol synthesis and accelerated clearance of cholesterol (He et al., 2015a; Kou et al., 2016).

Type 2 diabetes is strongly associated with significant morbidity and mortality due to cardiovascular complications. This is in part due to the impaired utilization of carbohydrate that is part of the metabolic profile of diabetes, which results in accelerated lipolysis and thus hyperlipidemia or dyslipidemia (Vijayaraghavan, 2010; Ma et al., 2016). RC has been shown to have the ability to attenuate the impaired lipid metabolism associated with diabetes. JiuHuangLian (RC steamed with rice wine), a traditional Chinese medicine preparation has been reported to restore the disordered lipid metabolism of type 2 diabetic rats, by decreasing the TC, total glyceride and free fatty acid levels (Li et al., 2013). The anti-hyperlipidemic effect of the alkaloids isolated from RC have been further tested on diabetic mice. Palmatine and jatrorrhizine were shown to decrease the concentrations of serum TC and triglyceride; while elevated HDL cholesterol levels were found in diabetic mice administered with palmatine, jatrorrhizine and berberine (Ma et al., 2016). In another study, there were decreased serum TC, triglyceride, LDL cholesterol and increased HDL cholesterol in diabetic rats treated with berberine (Tang et al., 2006), showing the ability of RC to attenuate the impaired lipid metabolism in diabetic conditions.

In short, RC has remarkable lipid-lowering effects, mainly through inhibition of cholesterol synthesis, in combination with increased usage and excretion of cholesterol.

Obesity has become a global epidemic and is strongly associated with occurrence of CVDs including heart failure, CHD, sudden cardiac death and atrial fibrillation (Lavie et al., 2009). As the condition is characterized by accumulation of extra body fat into adipocytes, the typical feature of obesity is an increase in the number and size of adipocytes. The etiology of obesity is rooted in the process of differentiation of preadipocytes into adipocytes, known as adipogenesis (Kwak et al., 2013; Choi et al., 2014). The key transcription regulator genes involved in adipogenesis are PPAR-γ and CCAAT/enhancer binding protein alpha (C/EBP-α). The inhibition of these two transcription factors could represent an effective method of inhibiting adipogenesis (Choi et al., 2014). RC has potential as an anti-obesity agent as the extract of RC as well as its isolated alkaloids have demonstrated the ability to inhibit cellular triglyceride accumulation in 3T3-L1 adipocytes. Significant down-regulation of expression of PPAR-γ and C/EBP-α by the alkaloids was also demonstrated, which is consistent with the reported mechanism for the anti-adipogenic activity of berberine (Huang et al., 2006).

The anti-obesity potential of RC has also been demonstrated in in vivo studies. The administration of RC and berberine to mice fed a high fat diet has resulted in significantly lower body and visceral adipose weight. Interestingly, this effect has been related to the antimicrobial activities of both RC and berberine; with reduced fecal microbes resulting in decreased degradation of dietary polysaccharides, lower calorie intake and de novo lipogenesis. This was followed by activation of fasting-induced adipose factor (Fiaf) protein and related gene expressions of mitochondrial energy metabolism such as AMPK, PGC1α, UCP2, CPT1α, and Hadhb in visceral adipose tissue (Xie et al., 2011). Recently, the gut microbiota of obese mice treated with RC alkaloids has been further studied, demonstrating an increased abundance of microbial species negatively associated with obesity namely Sporobacter termitidis, Alcaligenes faecalis, and Akkermansia muciniphila, and a decreased abundance of the microbial species positively associated with obesity namely Escherichia coli, Desulfovibrio C21 _c20 and Parabacteroides distasonis. This indicates the ability of RC to prevent the development of obesity by restoring the balance of disrupted gut microflora associated with the obese condition (He et al., 2016). RC appears to be effective in preventing obesity as it is able to inhibit adipogenesis and modulate the gut microflora that plays important role for the development of obesity.

Nonalcoholic fatty liver disease (NAFLD)—a common liver disease with a disease spectrum ranging from simple steatosis to steatohepatitis—is regarded as the hepatic manifestation of metabolic syndrome (Liu and Lu, 2014). Recently, hepatic steatosis has been identified as one of the associated risk factors closely related to the morbidity and mortality of CVDs. It has been reported that patients with NAFLD have higher risk of getting CHD compared to the general population (Pisto et al., 2014). There are several possible mechanisms by which hepatic steatosis may contribute to the risk of CVDs including oxidative stress, inflammation, dyslipidemia, ischemia reperfusion injury, visceral fat, low adiponectin, ectopic adipose tissue distribution, endothelial dysfunction and postprandial dyslipidemia (Liu and Lu, 2014). In spite of the many advances in modern medicine, there is as yet no effective treatment of NAFLD (Huang et al., 2013). Herbal medicine has been an area of interest for scientists seeking new modalities of treatment of hepatic steatosis due to the low incidence of side effects and promising therapeutic benefits (Xiao et al., 2013; Tan et al., 2016). For example, Jiao Tai Wan, a classic traditional Chinese prescription consisting of RC and Cortex Cinamomi was able to inhibit hepatic lipid accumulation in diabetic rats and humans, due to down-regulation of lipogenic gene expression and inhibition of lipogenesis in liver tissue (Huang et al., 2013). Sam-Hwang-Sa-Sim-Tang, a Korean traditional preparation which contains RC, Scutellaria radix and Rhei rhizoma also exhibited protective effect against hepatic steatosis by inhibiting the expression of hepatic molecules that modulate cholesterol metabolism (Ahn et al., 2013). While RC extract clearly does have significant potential as an anti-hepatic steatosis agent, there has been interest in identifying which of the extract's active components exert the greatest anti-hepatic steatosis effect. Using a free fatty-acid induced hepatic steatosis HepG2 cell assay, Fan et al. (2013) demonstrated that berberine and coptisine are the alkaloids with strongest inhibitory effect on triglyceride accumulation. This is consistent with the findings of other studies that proposed berberine as the effective compound against hepatic steatosis (Yuan et al., 2015; Guo et al., 2016; Zhang et al., 2016). In short, RC possesses anti-hepatic steatosis effect which is related to its ability to inhibit hepatic lipogenesis.

Coronary artery disease resulting in compromised oxygen supply to the myocardium is a common cause of morbidity and mortality. Medical intervention now makes it possible to achieve rapid restoration of blood flow to the ischemic myocardium following an acute event, which should produce better clinical outcomes as prompt restoration of blood circulation helps to prevent further tissue injury. However, the restoration of blood flow or reperfusion, will also cause injury, which is known as myocardial ischemia reperfusion injury (I/R) (Yu et al., 2015). Apoptosis is an important cellular mechanism resulting in I/R injury, with oxidative stress representing one of the major contributing factors promoting apoptosis. The excessive production of ROS which are mainly produced by mitochondria will initiate apoptotic signaling (Chan et al., 2012, 2015). Hence, reducing oxidative stress may help inhibit apoptosis thus potentially providing an effective strategy to attenuate I/R injury (Fan et al., 2012). Traditional Chinese herbal treatment have been commonly used to treat MI for hundreds of years and recently, it has also been reported to exert protective effect against myocardial I/R injury (Liu et al., 2013; Li et al., 2014; Zhao et al., 2016a).

One example of the traditional Chinese medications is San-Huang-Xie-Xin-Tang (SHXT), which consists of three herbs: RC, Scutellariae radix and Rhei rhizome. Liou et al. (2011) showed that pre-treatment with SHXT was associated with significantly lower arrhythmia scores, reduced mortality rates and reduced levels of cardiac enzymes such as creatine kinase (CK), lactate dehydrogenase (LDH) and troponin I in rats with myocardial I/R injury. Additionally, there was reduced infarct size and decreased apoptosis in myocytes, partly due to modulation of endothelial nitric oxide synthase (eNOS) and MAPK pathways. While it is commonly used as a component of herbal mixtures, the extract of RC alone has been shown to possess cardioprotective effect against I/R damage. Administration of crude extract of RC for 7 days preceding acute myocardial injury in rats was associated with reduced myocardial injury as evidenced by decreased ST-T elevation on electrocardiogram (ECG) and biochemically by decreased levels of CK and LDH. Other beneficial effects were reduction in oxidative stress reflected by reduced levels of malondiealdehyde (MDA) and increased activity of superoxide dismutase (SOD) (Liu et al., 2010).

Several active compounds identified in RC have been suggested as possible candidates accounting for the cardioprotective activities against I/R injury as demonstrated. One of the compounds that appears to have cardioprotective activity is berberine. Although the cardioprotective activity of berberine is well recognized, the exact mechanism by which it protects from I/R injury remains unknown. Chang et al. (2012) found that pretreatment with berberine decreased infarct size and reduced the incidence as well as duration of arrhythmias in rats with I/R injury with demonstrable difference in AMPK activity in both non-ischemic areas (NIA) and myocardial areas at risk (AAR). Up-regulation of AMPK in the NIA helped to enhance cardiac function through increased uptake of glucose, glycolysis and oxidation of fatty acid whereas down-regulation of AMPK in the areas at risk helped to protect the cardiac myocytes from apoptosis. This protective effect of berberine was later specifically demonstrated in the CVD prone diabetic heart, as demonstrated by the regulation of myocardial energy metabolism by berberine during I/R injury (Chang et al., 2016). In myocardial I/R, there is always excessive autophagy which will lead to expansive degradation of cytosolic proteins and organelles, resulting in collapse of cellular functions. Huang et al. (2015) have reported the beneficial effect of berberine in lowering the cellular autophagy level in berberine-treated myocytes exposed to I/R injury which were found to have reduced expression of autophagy-related proteins, which then led to suppression of autophagy activation. Berberine's cardioprotective action may also be associated with its anti-apoptotic action. Based on both in vitro and in vivo results, berberine decreases I/R-induced myocardial apoptosis, as a result of modulation of the Notch1/ Hairy and enhancer of split 1(Hes1)- Phosphatase and tensin homolog deleted on chromosome 10 (PTEN)/Akt signaling pathway (Yu et al., 2015). In a diabetic rat model, berberine exerted anti-apoptotic activity through activation of AMPK and PI3K-Akt-eNOS signaling (Chen et al., 2014).

Mitochondrial dysfunction is an additional factor believed to be responsible for myocardial I/R injury. Treatment with berberine has been shown to improve mitochondrial dysfunction, as demonstrated by improved mitochondrial membrane potential, increased mitochondrial complex I activity and reduced release of cytochrome C from the mitochondria (Wang et al., 2015). Endoplasmic reticulum (ER) stress is also known to play an important part during I/R injury. Unfolded protein response (UPR) is initiated in the myocardium after I/R and the continuous UPR causes upregulation of proapoptotic proteins. Consequently, cellular apoptosis occurs, resulting in myocardial I/R injury (Wu et al., 2014b). The role of berberine in modulation of ER stress level during myocardial I/R injury has also been described. Pretreatment with berberine has resulted in suppressed myocardial I/R induced ER stress due to the activation of Janus kinase 2/signal transducer and activator of transcription 3 (JAK2/STAT3) signaling (Zhao et al., 2016b) An additional mechanism by which berberine is able to ameliorate myocardial I/R injury is via repression of the inflammatory response. Lower levels of inflammatory markers, namely the cytokines tumor necrosis factor (TNF)-α, IL-6 and IL-β were found in rats pre-treated with berberine prior to induction of myocardial ischemia; this was believed to be mediated by downregulation of PI3K/AKT signaling thus preventing myocardial I/R injury (Zhu and Li, 2016).

Coptisine is one of the other compounds in RC that might have a role in prevention of I/R injury. In a rat model, administration of coptisine alleviated I/R-induced arrhythmias and attenuated the reduction in ejection fraction as well as fractional shortening on echocardiography. Treatment with coptisine was also associated with reduced infarct size, suppressed myocardial apoptosis and reduced proinflammatory cytokines. Based on the reduced expression of Rho, Rho-kinase 1 (ROCK1), ROCK2 and attenuation of myosin phosphatase targeting subunit-1 phosphorylation, it was speculated that inhibition of the Rho/ROCK pathway was likely to be coptisine's mechanism of cardioprotection (Guo et al., 2013).

Palmatine, another component contained in RC extract, is also recognized as a potential cardioprotective agent. The administration of palmatine to rats prior to myocardial I/R injury was associated with reduced I/R–induced myocardial dysfunction as evidenced by inhibition of the expected increase in LDH, CK, and MDA, as well as significant reduction in cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) expression. Interestingly, in vitro studies using human aortic endothelial cells resulted in increased heme-oxygenase-1 induction, indicating the strong antioxidant and anti-inflammatory action of palmatine (Kim et al., 2009). Based on the studies, RC is a potential protective agent against myocardial I/R injury through its active compounds which exert different protection actions including regulation of cellular energy metabolism, anti-apoptosis effect, protection against mitochondrial dysfunction and ER stress as well as antioxidant and anti-inflammatory action.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.