Pharmaceutical properties of calycosin, the major bioactive isoflavonoid in the dry root extract of Radix astragali

Abstract

Context: Radix astragali (Fabaceae astragalus propinquus Schischkin) is a Chinese medicinal herb traditionally used for the treatment of several diseases. Calycosin is the major bioactive chemical in the dry root extract of this medical plant.

Objective: This work presents a brief overview of recent reports on the potential effects of calycosin on several diseases and the possible mechanisms of action of this chemical.

Materials and methods: This review gathers information from the scientific literature (before 1 June 2013) that was compiled from various databases, such as Science Direct, PubMed, Google Scholar, and Scopus.

Results: The potential pharmaceutical properties of calycosin in the treatment of tumors, inflammation, stroke, and cardiovascular diseases have gained increasing attention in the recent years. The literature survey showed that calycosin exhibits promising effects for the treatment of several diseases and that these effects may be due to its isoflavonoid and phytoestrogenic properties. The effects of calycosin most likely result from its interaction with the ER receptors on the cell membrane and the modulation of the MAPK signaling pathway.

Conclusion: Calycosin exhibits great potential as a therapeutic drug and may be a successful example of the standardization and modernization of traditional Chinese herbal medicine.

• Anti-inflammatory
• antioxidative
• antitumor
• cardiovascular
• herbal medicine
• neuro-protective

Introduction

Herbal medicines are valuable sources of medicinally important substances. Plant-derived compounds play increasingly important roles in drug development, as exemplified by the successful commercialization of taxol and camptothecin (anticancer agents), artemisinin (Chinese antimalarial drug), and forskolin (East Indian ayurvedic drug). Globally accepted herbal drugs are thought to be safe and effective; however, herbal medicines are mixtures of different chemicals with different or even opposite functions. Thus, the purified key medical compounds in herbal medicines are more suitable as therapeutic drugs and should be given priority in research. In addition, mechanistic studies of drug efficacy and more evidence-based confirmation in controlled and validated trials are needed for the standardization and commercialization of traditional Chinese herbal medicines.

Radix astragali (Fabaceae astragalus propinquus Schischkin; syn: Astragalus membranaceus (Fisch.) Bunge and Astragalus membranaceus (Fisch.) Bunge var. mongholicus) is one of the most famous traditional Chinese herbal medicines. This herb has been used as a medicine for more than 2000 years. Radix astragali is widely used for the treatment of hypertension (Zhang et al., 2009), diabetes, cirrhosis (Gui et al., 2006), nephritis, cancer (Hong-Fen et al., 2001), and many other disorders (Guo et al., 2012; Tang et al., 2009). Its dry root extracts have shown anti-inflammatory, antidiabetic, hepatoprotective, neuroprotective, and anticarcinogenic properties (Chan et al., 2009; Chen et al., 2008; Romagnolo & Selmin, 2012). The composition of the dry root extract of Radix astragali was recently successfully analyzed. It contains multiple components, such as calycosin, saponins, polysaccharides, and some other isoflavonoids and astragalosides (Qi et al., 2008; Xiao et al., 2009; Zeng et al., 2012; Zhang et al., 2013). Among these, calycosin exerts the major function of the dry root extract and has been used as the representative bioactive chemical of Radix astragali. Technologies for the industrial-scale preparation of calycosin were recently rudimentarily established using macroporous resins or negative-pressure cavitation extraction (NPCE) (Chen et al., 2011a; Zhao et al., 2011). To promote the development of this potential drug, detailed studies on calycosin, such as its functional mechanism and its performance in preclinical and clinical trials, are very important.

However, the effects of pure calycosin were only studied in recent years, and an overview of its efficacy and pharmacology is lacking. This review emphasizes the effects of calycosin in several diseases. The mechanisms of its effects are also discussed and are summarized in Figure 1.

Figure 1. The interaction of calycosin (indicated by yellow hexagon) with cell. (A) The mechanism of the antitumor effects (upper-left area, indicated by blue lines). *The calycosin induced apoptosis of tumor cell at dosage higher than 7 mg L⁻¹, but induced the cell growth at dosages lower than that. Calycosin induced the expression of RASD1, which mediated the cell apoptosis signaling pathways. (B) The mechanism of neuroprotective effects (anti-oxidative effects, down-right area, indicated by yellow lines). The binding of calycosin on cell membrane prevented the diffusions of free radical and resulted in lower oxidative stress inside the cell. (C) The mechanism of the anti-inflammatory effects (down-left area, indicated by orange lines). Calycosin inhibited the phosphorylation of ERK1/2 and NF-κB, which blocked the pro-inflammatory cytokine expression and prevented the inflammation. (D) The mechanism of the cardiovascular effects (upper-right area, indicated by red lines). Calycosin induced the expression of VEGF, which further induced the angiogenesis process.

Antitumor effects

The therapeutic effects of Radix astragali root extracts in cancer were observed years ago (Hong-Fen et al., 2001; Wang et al., 2005). Calycosin, a phytoestrogenic compound in the dry root extract of Radix astragali, shows promising antitumor properties. It may modulate the ER-associated signaling pathway and transcriptional factors that control cell cycle and apoptosis (Jin et al., 2010). Zhang et al. (2012) found the potential effects of calycosin in leukemia, a malignant tumor in the hematopoietic system. In their in vitro experiments, calycosin inhibited the proliferation and induced the apoptosis of the K562 red leukemia cell line in a dose-dependent manner. The IC₅₀ of calycosin toward K562 cells was approximately 130 mg L⁻¹. Treatment with calycosin increased the percentage of cells in the G0/G1 stage and decreased the percentage of cells in the S phase. Furthermore, the expression of cyclin D1, which is a sensor for extracellular growth signals, was reduced by 39% after treatment with 100 mg L⁻¹ calycosin. More detailed studies were conducted by other researchers (Chen et al., 2011b,c; Tian et al., 2013), who found that calycosin induces the apoptosis of human breast cancer cells MCF-7 at a dosage higher than 7 mg L⁻¹.

The antitumor function of calycosin may be related to its induction of the RSD1 protein, which belongs to the Ras superfamily and regulates the mitogen-activated protein kinase (MAPK) signaling pathway (Tian et al., 2013). The MAPK signaling pathway regulates both cell proliferation and apoptosis. This superfamily is composed of ERK, JNK, and p38 kinase, which regulate the downstream genes, such as those belonging to the B-cell CLL/lymphoma 2 (Bcl-2) family. The proteins in the Bcl-2 family of proteins regulate the mitochondrial-mediated apoptotic pathway, which is one of the major apoptotic pathways in mammalian cells (Burz et al., 2009; Davids & Letai, 2012). This Bcl-2 family is further divided into two superfamilies: Bcl-2, which is a repressor of apoptosis, and BCL2-associated X protein (Bax), which is a promoter of apoptosis (Kang & Reynolds, 2009). In MCF-7 cells, calycosin induced the expression of RASD1 by binding the ER receptor and consequentially reduced the expression of Bcl-2 and increased the expression of Bax. By shifting the Bcl-2/Bax balance toward apoptosis, calycosin promotes apoptosis and exerts its antitumor effects (Tian et al., 2013).

The antitumor effect of calycosin is dose dependent and, at lower dosages, may promote the proliferation of tumor cells (Chen et al., 2011b,c). Thus, to further verify its function as a pharmaceutical agent, more in vivo studies are required to further clarify its regulatory mechanisms.

Neuroprotective effects

Ischemic stroke is caused by a transient or permanent reduction in the cerebral blood flow in a major brain artery. Approximately 60–70% of strokes are due to ischemic stroke that causes death and leads to disability in adults (Mahajan et al., 2004; Silvestrelli et al., 2002; Zhou et al., 2008). The re-canalization of occluded cerebral blood vessels was proven to efficiently treat ischemic stroke; however, reperfusion causes cerebral ischemia/reperfusion (I/R) injury. Recent findings in an animal model suggest that isoflavonoids can attenuate I/R injury (Sato et al., 2011; Watanabe et al., 2007). The effects of calycosin on cerebral I/R injury were studied by Guo et al. (2012). In a transient rat middle cerebral artery occlusion (MCAO) model, 30 mg/kg calycosin can induce neuro-protection by attenuating the infarct volume and improving neurological function. Similar protective effects of calycosin were observed in the PC12 neuronal cell line after damage was induced by l-glutamate or xanthine (XA)/xanthine oxidase (XO) (Yu et al., 2005, 2009). Calycosin effectively protected the neuronal cells and inhibited the injury induced by glutamate or XA/XO with a 50% effective concentration (EC₅₀) of 0.05 mg L⁻¹ and an IC₅₀ of approximately 50 mg L⁻¹.

Oxidative stress is a major pathogenic trigger in neurodegeneration and leads to oxidative molecular damage of the tissue (Emerit et al., 2004; Sohal et al., 2002; Zhu et al., 2004). Many flavonoids exhibit greater antioxidant activity than antioxidant vitamins, vitamin C, vitamin E, and β-carotene (Chopra & Thurnham, 1999; Rice-Evans et al., 1995). Calycosin exhibited very effective antioxidative activity, likely due to the presence of a 7-hydroxyl group in its A ring and a 3′-hydroxyl group in its B ring (Shirataki et al., 1997; Yu et al., 2005). Treatment with calycosin reduced the malondialdehyde (MDA), protein carbonyl, and reactive oxygen species (ROS) levels and upregulated the activities of radical scavenging, as represented by the increase in the levels of superoxide dismutase (SOD), catalase, and glutathione peroxidase (GSH-Px). It is possible that calycosin stabilizes membrane structures and improves membrane integrity. This activity decreases the lipid fluidity of membrane and hinders the diffusions of free radicals, thereby lessening the cellular oxidative damage (Guo et al., 2002). In an in vivo study, in addition to a similar induction of SOD and GSH-Px, calycosin was also found to inhibit the expression of 4-hydroxy-2-nonenal (4-HNE), which indicates the breakdown of lipid hydroperoxide in response to oxidative stress and may be produced as a result of I/R injury in the brain (Eaton et al., 1999; Cindric et al., 2012). Thus, its reduction further proved the antioxidative effects and therapeutic potential of calycosin.

The above-mentioned studies suggest that the neuroprotective effects of calycosin effects are largely due to its antioxidant effects and that calycosin may be a possible therapeutic drug for the treatment of ischemic cerebrovascular diseases.

Anti-inflammatory effects

The use of Radix astragali for the treatment of immune-related diseases has been documented in the literature (Hoo et al., 2010; Ryu et al., 2008). Two Radix astragali-containing herbal formulae have been found to rescue 85% of legs that have been condemned to amputation due to non-healing chronic diabetic ulcers (Leung et al., 2008). Additionally, the active fraction of Radix astragali may alleviate obesity-induced metabolic damage through the inhibition of inflammation (Hoo et al., 2010). The levels of both fed and fasting glucose in db/db obese mice were markedly decreased after treatment with the active fraction of Radix astragali. Reduced serum triglyceride levels, alleviated insulin resistance, and glucose intolerance were observed in these mice compared with the vehicle-treated controls.

Macrophage infiltration and the aberrant production of pro-inflammatory cytokines, which is also recognized as chronic inflammation in white adipose tissue, play essential roles in the linking of obesity with diabetes and diabetic complications (Weisberg et al., 2003; Xu et al., 2003). In obese subjects, the infiltrated macrophages secrete pro-inflammatory cytokines, such as TNF-α and IL-6 (Hotamisligil et al., 1993; Uysal et al., 1997). These cytokines then stimulate the secretion of chemokines, such as MCP-1, which further enhance the recruitment of macrophages and amplify the inflammatory response (Christiansen et al., 2005; Kanda et al., 2006; Weisberg et al., 2006). Calycosin significantly reduced the secretion of pro-inflammatory cytokines and chemokines (TNF-α, IL-6, and MCP-1) in human THP-1 macrophages and the lipopolysaccharide (LPS)-induced activation of NF-κB in mouse RAW-Blue macrophages in a dose-dependent manner (Hoo et al., 2010). In a study, treatment with calycosin was shown to significantly reduce the mRNA expression levels of the inflammatory cell markers CD68 and F4/80 and the cytokines MCP-1, TNF-α, and IL-6 in epididymal adipose tissue and to significantly increase the levels of the activated macrophage marker arginase I.

Some findings suggest that calycosin can relieve the local inflammation induced by advanced glycation end products (AGEs) (Xu et al., 2011). AGEs play a pivotal role in vasculitis, such as atherosclerosis and diabetic retinopathy (Orasanu & Plutzky, 2009), by significantly elevating both the mRNA and the protein expression of receptor for AGE (RAGE). RAGE is a member of the immunoglobulin (Ig) superfamily of proteins (Neeper et al., 1992) and induces the ERK1/2 and NF-κB pathways, which mediate AGEs-induced cell damage, such as inflammation and vascular injury (Goldin et al., 2006; Lin et al., 2009; Sakaguchi et al., 2003; Schmidt et al., 2001; Zhou et al., 2003). Calycosin significantly attenuates vasculitis development by downregulating the AGEs-induced overexpression of RAGE and pro-inflammatory cytokines (Figarola et al., 2007). This reduction may be related to the ERK1/2 and NF-κB pathway because calycosin decreases AGEs-induced ERK1/2 and NF-κB phosphorylation (Cheng et al., 2010; Kim et al., 2009).

The anti-inflammatory effect of calycosin is aided by its inhibition of pro-inflammatory cytokine formation. Although the current literature suggests that calycosin inhibits the phosphorylation of ERK 1/2 and NF-κB, more convincing evidence is required. Moreover, because it can interact with ER receptors, the direct involvement of calycosin in the NF-κB signaling pathway may be responsible for the inhibition of cytokine production.

Pro-angiogenesis effects

The vasorelaxant effect of calycosin was observed in pre-contracted rat thoracic aortic rings (Wu et al., 2006). A calycosin concentration of 80 mg L⁻¹ produced a maximum relaxation. Calycosin decreased the extracellular Ca²⁺ influx through voltage-operated Ca²⁺ channels (VOC) and receptor-operated Ca²⁺ channels (ROC), which resulted in the inhibition of KCl- or PHE-induced contraction.

Additionally, as a phytoestrogen, calycosin may play beneficial roles in the prevention of osteoporosis and exert protective effects against cardiovascular diseases (Bitto et al., 2010; Fan et al., 2003; Pilšáková et al., 2010). The study conducted by Tang et al. (2010) showed that calycosin induces angiogenesis in human umbilical vein endothelial cells (HUVEC) in vitro and in vivo in zebrafish embryos. The potential involvement of the VEGF(R) and FGF(R) signaling pathways was proposed to be important for the induction of angiogenic activities by calycosin treatment.

It was demonstrated that calycosin acts similarly to other selective estrogen receptor modulators (SERMs), such as raloxifene and tamoxifen, by displaying selective potency and affinity to the estrogen receptors ERα and ERβ. Transcriptome information further indicated that calycosin promotes angiogenesis via the activation of the MAPK signaling pathway (Li et al., 2011).

In a recent study conducted by our group, the effect of calycosin on the myocardial ischemia (MI) rat model was observed (paper submitted). Calycosin treatment effectively prevented infarction in the rat MI model and promoted cardiovascular angiogenesis at a dosage of 4 mg/kg. The acceleration of angiogenesis was found to be crucial for the prevention of cardiology function in the MI model. Similar to the findings in the zebrafish model, the induction of vascular endothelial growth factor (VEGF) by calycosin was found to be dose-dependent.

The rescue of an impaired myocardium after surgery is important in MI treatments, such as percutaneous coronary intervention (PCI), because the promotion of angiogenesis activity in MI patients remains an essential task for the achievement of a successful treatment. Both in vivo and in vitro studies have shown that calycosin exerts a promising effect in the promotion of angiogenesis. Thus, calycosin has great potential for MI treatment.

Conclusions

As summarized in Figure 1, the pharmaceutical properties of calycosin are largely due to its role as an isoflavonoid and a phytoestrogen. This chemical interacts with the ER receptors on the cellular membrane and modulates the MAPK signaling pathway. Through its effect on this pathway, calycosin can regulate several cell activities, such as apoptosis and angiogenesis, which enables its therapeutic functions. The dosage is crucial for the function of calycosin. As an antitumor drug, the dosage of calycosin should be higher than 7 mg L⁻¹. Dosages lower than this value may inversely induce the proliferation of cancer cells. In contrast, a concentration as high as 28 mg L⁻¹ improved angiogenesis in zebrafish and HUVECs. This phenomenon indicates that the same dosage may exert opposite effects under different conditions. Thus, more detailed information on how calycosin interacts with the ER is required, and the use of calycosin as a therapeutically drug in pre-clinical and clinical studies should be carefully studied.

In addition to the medical effects mentioned above, many other effects of Radix astragali have not yet been studied using calycosin. The flavonoids from Astragalus have been shown to exhibit anti-lipid peroxidation, blood pressure lowering, and liver protective effects (Liu et al., 2005, 2007). The work conducted by Qi et al. (2011) showed that the total flavonoids protected the mice against radiation damage induced by ⁶⁰Co γ-irradiation (Qi et al., 2011). Wang et al. (2012) demonstrated the anti-atherosclerosis effects of the total flavonoids of Astragalus. Because it is the major isoflavonoid of Astragalus, calycosin is most likely responsible for these important pharmaceutical effects.

Based on the results of this review, the efficacy and pharmacology of calycosin have been recently receiving increasing attention. However, additional studies are still needed to make calycosin an applicable drug. The first problem is that the effects have only been studied in animal models to date. Reliable and feasible human trials should thus be conducted. Because calycosin is separated from the medical plant Radix astragali, it is expected to be safe and inexpensive. Second, the pharmacokinetics of calycosin should be clarified. Currently, the conjugation and oxidation of calycosin have been observed in zebrafish larvae (Hu et al., 2012). More sophisticated and thorough studies on the in vivo metabolism of calycosin are important and required for the wider and improved application of this traditional drug. Finally, although the effectiveness of this drug was verified, its mechanism has only been preliminarily established. More detailed information and molecular-level evidence, particularly on the interaction of calycosin with signaling cascades related to cell apoptosis and angiogenesis, are required.

Declaration of interest

The authors report no declarations of interest. This work was financially supported by the Shanghai key medical specialties construction project, the project of National Natural Science Foundation [81303145], the project of the Science and Technology Commission of the Shanghai Municipality [124119b1600] and the special project of the State Administration of traditional Chinese Medicine.