Study on Pharmacological Effects of Calycosin in Astragali Radix

Yu ZHANG, Tong ZHANG, Shinong WANG, Yannan LI, Hui XUE, Wenbo ZUO, Chenghao JIN,2,3*

1. College of Life Science & Technology, Heilongjiang Bayi Agricultural University, Daqing 163319, China; 2. College of Food Science & Technology, Heilongjiang Bayi Agricultural University, Daqing 163319, China; 3. National Coarse Cereals Engineering Research Center, Daqing 163319, China

Abstract Calycosin is an important isoflavone compound in traditional Chinese medicine Astragali Radix. It not only has remarkable anti-oxidation, anti-inflammatory and anti-tumor functions, but also has the characteristics of small adverse reactions and low toxicity, so it has attracted wide attention of scholars and researchers. This paper reviewed the pharmacological effects and mechanisms of calycosin in recent years, in the hope of providing a theoretical basis for its clinical application.

Key words Calycosin, Pharmacological effect, Anti-oxidation, Anti-inflammatory, Anti-tumor

1 Introduction

Calycosin is a fat-soluble component in the dried root ofAstragalusmembranaceus. Its molecular formula is C16H12O5. Its appearance is a light yellow powder. It is easily soluble in organic solvents such as ethanol and methanol, and is not easily soluble in water. Calycosin has functions of reinforcing middle-warmer and replenishing qi, strengthening spleen and tonifying lung, detoxication and tissue generation, and has been widely applied in poor appetite and sloppy diarrhea, shortness of breath and panting, long term ulcerating of sore and ulcer, and prolapse of the rectum due to frequent diarrhea[1]. In bothinvitroandinvivoexperiments, calycosin has shown various biological activities such as anti-oxidation, anti-inflammatory and anti-tumor[2]. In this study, we reviewed the pharmacological effects and mechanisms of calycosin in recent years, in the hope of providing a theoretical basis for the development of anti-tumor clinical application.

2 Pharmacological effects

2.1 Anti-oxidant effectAt present,invivoanti-oxidant research is mainly carried out from two aspects: oxidative stress and free radical metabolism. Some studies have shown that excessive production of reactive oxygen free radicals or decreased biological clearance can lead to peroxidative damage to biofilms[3]. Through data statistics, Zhang Xinetal.[4]evaluated the anti-oxidant capacity of four isoflavone compounds fromA.membranaceus. The results show that the phenolic hydroxyl groups at the C3′ and C7′ positions of the calycosin molecules may be active sites for eliminating oxidative free radicals, which can improve the ability of the calycosin molecules to scavenge active oxygen radicals. According to the study of Wen Xiaodongetal.[5], the polarity of calycosin molecules is small and can not only remove free O2-C and ·OH free radicals, but also penetrate biofilm lipid bilayers to remove bound active oxygen and delay lipid peroxidation of biofilm. Besides, the addition of ferulic acid can increase the free concentration of calycosin, and accelerate the removal of free reactive oxygen free radicals, and the two substances can produce a synergistic antioxidant effect, thereby improving the body’s anti-oxidative stress ability. Gao Jianetal.[6]found that calycosin can activate Nrf2 through the protein kinase C (PKC) pathway, thereby reducing oxidative stress and inflammatory reactions, and improving cognitive function of amyloid precursor protein/presenilin-1 (APP/PS1) transgenic mice and relieving symptoms of Alzheimer’s disease.

2.2 Anti-inflammatory effectsCalycosin can exert anti-inflammatory effects by inhibiting the activation of inflammatory signaling pathways and infiltration of inflammatory cells. According to the study of Zhao Haipengetal.[7], calycosin is able to activate the protein kinase (AMPK) pathway and inhibit the transcription factor protein (NF-κB) pathway, thereby reducing PM 2.5-induced cell damage and inflammatory reaction. Chen Hanqingetal.[8]found that there is structural similarity between calycosin and estrogen: through interacting with estrogen receptors α and β on microglia, they inhibit LPS-induced microglial activation and inflammatory cytokine formation, and have a protective effect on microglial-induced dopaminergic neuron damage. Recent years of studies have shown that hypoxia-inducible factors are closely related to inflammatory reactions. Jiaetal.[9]found that calycosin reversed degradation of tight junction protein (TJ) caused by IL-1β through regulating hypoxia-inducible factor (HIF-1α) in AD in Alzheimer’s disease (cell) model to reduce the allergic inflammatory reactions in mice.

2.3 Protective effects on endothelial cellsStudies have shown that endothelial cell damage is the root cause of many cardiovascular diseases. Song Ruixiaetal.[10]found that calycosin can enhance the expression of angiotensin converting enzyme II (ACE II), reduce the expression of angiotensin converting enzyme (ACE), and then reduce the damage of human umbilical vein endothelial cells (HUVECs). According to study of Guo Cetal.[11], calycosin can activate the TRPC6-CREB signaling pathway, enhance the expression of transient receptor potential cation channel protein 6 (TRPC6) and cyclophosphine adenosine response element binding protein (P-CREB), and inhibit calpain expression, thereby reducing the damage to endothelial cells. In addition, according to findings of Jiangetal.[12], calycosin can promote the production of NO, reduce the expression of vascular endothelial growth factor (VEGF), vascular endothelial growth factor receptor-2 (VEGFR-2) and phosphatidylinositol 3-kinase (PI3K), and activate myosin light chain phosphatase (MLCP), inhibit the AKT pathway, reduce the protein expression of phosphorylated myosin light chain phosphatase (PMLC), and thus protect HUVEC cells from LPS-induced endothelial damage. Calycosin can promote the release of NO from HUVECs cells, significantly inhibit the apoptosis of endothelial cells induced by angiotensin II (Ang II) and the secretion of endothelin-1 (ET-1), and reduce the injury of human umbilical vein endothelial HUVECs cells[13]. Shu Hongetal.[14]found that calycosin can significantly reduce the expression of α-smooth muscle actin (α-SMA) and inhibit the transformation of endothelial cells (ECV-304) induced by transforming growth factor-β1 (TGF-β1), thereby preventing kidney fibrosis. Renetal.[15]found that calycosin can inhibit the activity of caspase-3 and caspase-9, enhance the expression of PI3K p85 and AKT, and reduce myocardial ischemia-reperfusion injury to endothelial cells.

2.4 Anti-tumor effectsAccording to theGlobalCancerStatistics2018, malignant tumors are a serious threat to human health, so the research and clinical application of anti-tumor drugs has become a hot spot in international research[16]. Calycosin can block the growth cycle of tumor cells, regulate reactive oxygen levels and related signaling pathways, inhibit tumor cell proliferation and induce apoptosis.

2.4.1Inhibiting the proliferation of cancer cells. Calycosin can inhibit the proliferation of a variety of cancer cells, including lung cancer, liver cancer, breast cancer, and osteosarcoma. In mouse experiment, Quanetal.[17]found that calycosin can reduce the formation of bone marrow derived macrophages (BMMs) and osteoclasts in a dose-dependent manner. Besides, studies on osteoclast differentiation factor (RANKL) induced bone model systemsinvitrohave shown that calycosin significantly increases the osteoclasts gene expression levels of tartrate resistnt acid phosphatase (TRAP), matrix metalloproteinase (MMP-9), cathepsin K (CTSK), down-regulates proto-oncogene (C-fos), up-regulates protein expression of nuclear factor of activated T-cells cytoplasmic 1 (NFATC1), and inhibits osteoclastogenesis. Zhangetal.[18]found that calycosin can significantly up-regulate protein kinase (CDC2), cyclin-dependent protein kinase 7 (CDK7) and recombinant human cyclin B1 (CCNB1), and down-regulate the expression levels of TFDP1, SKP2 and CDKN2D proteins. According to findings of Zhang Dongqingetal.[1], calycosin can reduce the mRNA expression level of Cyclin D1, and arrest cycle of red leukemia cells (K562) in the G0/G1 phase, thereby inhibiting the number of cells. These indicate that calycosin can induce cell cycle arrest in different cell lines G0/G1, thereby inhibiting the proliferation of cancer cells.

2.4.2Inducing apoptosis of cancer cells. Inducing tumor cell apoptosis is a hot spot of current research. Wang Deqingetal.[19]found that the growth curve and mitotic index of hepatoma cells (BEL-7402) were significantly inhibited and decreased in a dose-dependent manner. Electron microscopy showed that cytoplasmic vacuoles and nuclear shrinkage appeared in BEL-7402 cells, indicating that the total flavonoids of Astragalus (TFA) had substances that promote apoptosis of BEL-7402 cells. According to findings of Sunetal.[20], calycosin can significantly down-regulate the anti-apoptotic protein Bcl-2, increase the expression of the pro-apoptotic proteins caspase-3 and PARP, and induce the estrogen receptor-positive (ER) human osteosarcoma MG-63 apoptosis by regulating the PI3K/AKT/mTOR signaling pathway, and the apoptosis of cells showed time-concentration-dependent change. Chenetal.[21]found that calycosin only promotes the apoptosis of estrogen receptor (ER) positive cells (especially MCF-7 cells) in a dose-dependent manner, but this phenomenon was not observed in ER negative cells. Studies have shown that calycosin conducted selective regulation through ERβ-mediated IGF-1R/MAPK and IGF-1R/PI3K/AKT pathways. According to findings of Zhouetal.[22], calycosin can significantly inhibit the expression levels of p-PI3K, glycogen synthase kinase 3β (p-GSK3β), p-AKT and rapamycin target protein (p-mTOR), indicating that calycosin can induce the apoptosis of breast cancer MCF-7, SK-BR-3 and MDA-MB-231 cells through regulating the PI3K/AKT/mTOR signaling pathway.

2.4.3Inhibiting metastasis and invasion of cancer cells. The metastasis and invasion of tumor cells are closely related to high clinical mortality. Therefore, inhibiting the metastasis and invasion of cancer cells is an important method to prolong the life of patients. Through scratch test and Transwell test, Liuetal.[23]found that calycosin can significantly down-regulate the protein expression of MMP-2 and MMP-9, inhibit the degradation of human melanoma A375 extracellular matrix, and significantly inhibit the proliferation, metastasis and invasion of human melanoma A375 cells. Ouyang Jiaetal.[24]found that by regulating TGF-β1 protein, calycosin inhibits expression of vimentin, N-cadherin, Snai 1, MMP-2 and MMP -9, thereby inhibiting the metastasis and invasion of glioma cells. Yangetal.[25]found that estrogen receptor ERβ inhibits the reproduction, metastasis and invasion of osteosarcoma U2-OS cells through regulating the apoptosis inhibitory protein (IAP), NF-κB/Bcl-2 and PI3K/AKT signaling pathways, showing good anti-tumor effect. Chengetal.[26]found that calycosin can regulate the expression of E-cadherin (E-Cad), integrin β1 and MMPs through the PKC-α/ERK1/2 signaling pathway, thereby inhibiting the metastasis and invasion of A549 cells. Nieetal.[27]found that calycosin can significantly down-regulate the expression of N-cadherin, Snai 1, Vimentin, MMP-2 and MMP-9. In addition, U87 cells are more sensitive than U251 cells in terms of proliferation, metastasis, and invasion, and calycosin has no significant toxic effect on normal tissues.

2.4.4Eliminating immunosuppression. Immunosuppression can weaken the response of various agents. Therefore, reducing immunosuppression is an important means in tumor treatment. In the study of immunosuppressive treatment, it was found that the plasma afterA.membranaceustreatment mainly contains two components, flavonoids and saponins. These two types of compounds have higher response values, faster absorption and longer retention time, indicating that flavonoids and saponins play an important role in immunosuppressive treatment[28]. The tumor tissue that is wrapped inside is severely hypoxic, while promoting microangiogenesis and alleviating the hypoxic environment, it can also promote the infiltration of immune cells, improve the tumor microenvironment, and achieve anti-cancer purposes, but its mechanism needs further research. According to findings of Yu Hetal.[29], flavonoids can promote the transcriptional activity of hypoxia response element (HRE), significantly increase the expression of hypoxia inducible factor-1α (HIF-1α), and then activate erythropoietin (EPO). These indicate that flavonoids can improve hematopoietic function through regulating the expression of EPO, and then promote the infiltration of immune cells. In studying the functions of Danggui Buxue Decoction, Dengetal.[30]found that calycosin can regulate Treg cell-mediated immunosuppressive functions through regulating the proliferation and differentiation of effector T cells; it can also inhibit the activity of the JAK/STAT signaling pathway, repair the abnormal immune response and defects of hematopoietic cells in AA mice. Besides,A.membranaceuscan reduce the levels of two miRNAs (miR-146b and miR-155) with immunomodulatory effects, and can improve the immune imbalance of regulatory T cells (Treg) around viral myocarditis.

3 Conclusions and prospects

As a flavonoid compound extracted fromA.membranaceus, calycosin has the characteristics of wide source, low toxicity and small molecular weight. Due to its multiple biological activities, it has become a hot medicine in both scientific research and clinical research. A number of experimental studies have found that calycosin has good anti-oxidant, anti-inflammatory, anti-tumor and immune activity regulation effects, but as a traditional Chinese medicine compound, the research and clinical application of its drug functional mechanism is still in its infancy. For example, through binding membrane surface receptors calycosin enters cells to exert anti-oxidant and anti-inflammatory effects and what are the ways to inhibit tumor cell development and regulate immune activity? Based on this, it is necessary to carry out deeper research on calycosin at the animal level (such as constructing nude mouse tumor models and zebrafish models), cells (using various cell experiments), and molecular levels in combination with relevant Chinese medicine theories, in order to lay a new foundation for promoting the development of calycosin TCM preparations.