Advances in Pharmacological Studies of Calycosin-7-O-β-Dglucoside

Liang-Liang Li, Jin-Zhi Huang

Guangdong Medical University, Zhanjiang 524023

Keywords:

ABSTRACT

1. Introduction

As a natural drug storage, traditional Chinese herb is an indispensable part of the development of China's medical undertakings. It is also the wealth that our ancestors have accumulated and enriched in long-term life and clinical practice for thousands of years, which has made outstanding contributions to the great achievements in medicine field of China. With the increasing status of Chinese economic globalization, Chinese herb research has become a major subject in the world of medicine. The use of modern technology to extract one or several effective monomeric components with medicinal activity for clinical disease treatment has become a new field and direction in modern pharmaceutical industry. At the same time, people's demand and requirements for Chinese herb are also getting stronger and stronger. In order to meet requirements of three effects (high efficiency, quick-acting, longacting) and three “slight” (slight dosage, slight toxicity, and slight side effects), three “easy” (easy to store, easy to carry, and easy to take)[1], relying on modern technology to improve the mechanism of action of natural drugs, as well as extracting and purifying monomeric components with pharmacological activity are inevitable requirements for the modernization and internationalization of traditional Chinese herb.

Astragalus is a leguminous herb produced in Inner Mongolia and Gansu. Its medicinal effect is the dry root of Astragalus mongolicus or Astragalus membranaceus, sweet taste, and warm nature, with effects on lung, liver and kidney meridians. It has a wide range of pharmacological effects such as enhancing immune function, liver protection, diuresis, anti-aging and anti-tumor.[2,3]Astragalus is used in the treatment of hypertension, ischemic heart disease, acute glomerulonephritis, gastric ulcer, diabetes, psoriasis,chronic rhinitis, osteoporosis and so on.[4]. Saponins, flavonoids,polysaccharides and other compounds in Astragalus have a wide range of medicinal properties, the flavonoids include flavones,flavanols, flavanones and isoflavones, have significant action to anti-oxidation and promoting cell proliferation. Isoflavones have estrogen-like effects and antiviral, antibacterial, hypolipidemic and anti-oxygen free radicals.

Calycosin-7-O-β-D-glucoside (CG) is an isoflavone compound,which is an active monomer component with wide pharmacological activity and extracts from the dry roots of the leguminous plant Astragalus. The molecular formula is C22H22O10, and the molecular weight is 446.4, one of the most active main ingredients of the traditional Chinese herb Astragalus. CG content is the main indicator for the evaluation and control of Astragalus quality in the pharmacopoeia. CG has obvious effects of anti-oxidation, anti-virus,inhibition of melanin formation and immunomodulatory activity. In recent years, research has been conducted on the anti-tumor effect of CG and the treatment of cerebrovascular diseases, but more functions and mechanisms have yet to be developed. This article gives a brief review of the pharmacological effects and the latest research progress of CG.

2. Extraction, separation and purification of CG

Kou Zhao[5]compared the four methods of CG extraction, namely hot reflux extraction method, Soxhlet reflux extraction method, cold immersion method and ultrasonic extraction method, and at the same time, the extraction process with high yield and convenient operation was selected which laid the foundation on the efficient extraction of CG. Among the four extraction methods, the cold soaking method has the lowest yield but the Soxhlet extraction method is the highest,however, the Soxhlet extraction method is complicated and the cycle is long. Although the ultrasonic method is slightly lower than the Soxhlet extraction method, it is easy to operate and is the preferred method. . The orthogonal test[6]proved that the best process for extracting CG was to extract twice with refluxed 80% ethanol in a solvent amount of 10 times, for one hour each time. Astragalus has traditionally been heated and refluxed to remove impurity with petroleum ether, which affects the CG content due to the long heating time. The static adsorption performance of macroporous resin,especially AB-8 resin, makes the enrichment purity and biological activity of isoflavones including CG significantly improved, which lays a good foundation for its large-scale preparation[7], and Liu Lifeng[8]uses the near-infrared spectroscopy (NIR) technology to quickly predict the content of CG in the extraction process of Astragalus, which is the cornerstone of its application in the field of medicine.

3. Pharmacokinetics of CG

After oral administration, the drug is absorbed into the blood circulation through the absorption and metabolism of the gastrointestinal tract, and reaches various target organs and then exerts pharmacological effects. Previous studies have shown that although most of the CG given to rats by oral administration, tail vein injection, and intraperitoneal injection etc are metabolized with low bioavailability, but the absorption rate is very fast, reaching a peak in ten minutes, making it high pharmacological effect. Ruan et al found that CG[9,10]interacts with intestinal flora after oral administration, not only as an angiogenesis prodrug, but also as a regulator of intestinal flora. With the deglycosylation in the body,veratium isoflavones and verruflone-3 '-O-glucoside are the main metabolites. The CG metabolic pathway is divided into phase I metabolism and phase II metabolism[11]. The former includes deglycosylation, dehydroxylation and demethylation reactions;the latter includes sulfation, methylation, glucuronidation and glycosylation as well as acylation, hydroxylation and hydrogenation reactions. Chen Shuoji[12]found that CG was metabolized to the hairy isoflavones under the action of intestinal bacteria, and widely identified in target organs such as liver, kidney and heart, and then exerts its pharmacological effects. Therefore, CG can be used as a prodrug of hairy isoflavones. The intestine may be the main absorption and metabolism site after oral administration of CG in rats. With the rat intestinal perfusion model, Shi et al[13]performed pharmacokinetic studies and vitro experiments on CG and found that through sodium-dependent glucose transporter 1 (SGLT) CG can be intactly transported to small intestinal cells, which is an important part of CG absorption, de-sugar and metabolism in rats, different absorption rates in different intestinal segments, and the duodenum is the fastest[14].

4. Study on pharmacological effects of CG

4.1 CG has strong antioxidant activity

Flavonoids are the main effective ingredients of Astragalus to exert anti-oxidation and eliminate free radicals and anti-aging effects[15]. Many studies have shown that active components of hairy isoflavones, hairy isoflavone glucosides, formononetin and ononin can be isolated from Astragalus. Qi Yunyun and Liu Jingli et al[16,17]studied their antioxidant activities, and concluded that:hairy isoflavones＞hairy isoflavone＞formononetin＞ononin. CG has antioxidant activity, inhibits oxidative modification of low-density lipoprotein and cyclooxygenase-2 (COX-2) activity, and clears 1,1-diphenyl-2-trinitrophenylhydrazine (DPPH free) Base)[18]. Plants with high CG content have higher activity of superoxide dismutase(SOD) and catalase (CAT) and have strong antioxidant capacity[19].Li Jian and Lin Xin[20,21]found that CG has protective effects on H2O2-induced oxidative stress in BRL-3A cells induced by Chang Liver cells and thioacetamide (TAA) in human normal hepatocytes and can obviously alleviate oxidative damage.

4.2 The role of CG in the prevention and treatment of bone and joint related diseases

CG has an anti-inflammatory effect and is a potential natural anti-osteoarthritis drug. CG was injected to the knee joint in the anterior cruciate ligament and meniscus amputation (ACLMT)-induced rabbit osteoarthritis(OA) model. After 4 weeks, the total protein, glycosaminoglycan(GAG) and prostaglandin (PGE2)levels in synovial fluid were detected. It was found that the total amount of synovial membrane and the content of PGE2 as well as total protein in synovial fluid was obviously reduced. The results demonstrated that CG could directly inhibit the degradation of cartilage and release degraded molecules like GAG from the damaged tissue, which coincided with the decrease of synovial volume, and then played the role of bone and joint protection.[22]Experiment[23]found that it had protective effects on the degradation of articular cartilage and chondrocyte matrix induced by hyaluronidase(HAase) and recombinant human interleukin-1β(IL-1β), which could effectively prevent protein degradation and promote cell differentiation into osteoblasts. Jing Jian et al[24]found in human studies that in osteogenic differentiation medium, after CG treatment of mouse bone marrow stromal ST2 cells, alkaline phosphatase (ALP) activity increased, Ocal mRNA expression increased, and osteoblast mineralization increased. CG may be a useful lead compound that stimulates osteoblast differentiation of ST2 cells by increasing expression of the regulatory factors BMP-2,o-Smad 1/5/8, β-catenin, and Runx 2 in the BMP/WNT signaling pathway. It ultimately improves the treatment of bone degeneration and promotes bone regeneration.

4.3 The antiviral and improving myocardial function of CG

Viral infection is likely to cause serious consequences such as myocardial cell damage, necrosis, cardiac dysfunction and even sudden death. The influencing factors are related to viral invasion,immune damage and nitric oxide (NO). In recent years, studies have shown that Astragalus has the functions of enhancing immunity, antimyocardial ischemia and anti-virus. CG has an anti-viral effect as an effective monomer component of Astragalus. CG[11,25]could not only treate myocarditis caused by Coxsackievirus B3(CVB3), but also evidently improve the survival rate and left ventricular function of animals infected with myocarditis, inhibit viral replication, improve cardiac function, and regulate cellular immune function. Zhang Yuanyuan's group[26,27]established cell models and animal models by CVB3 infecting SD rat primary cardiac cells and Ba1b/c mice, using serum pharmacology, pharmacokinetics and molecular biology, it is for the first time demonstrated that CG had significant anti-CVB3 and therapeutic effects on viral myocarditis (VMC) and applied for a patent (CN101940584A). Ren et al[28] found that CG could attenuate ischemia-reperfusion (I/R) injury and improve cardiac function by establishing a PI3K/Akt signaling pathway, inhibiting oxidative stress and pro-apoptotic factors.

4.4 CG protects vascular endothelium

Complex and diverse pathological factors lead to dysfunction of the vascular endothelium. The cytoskeleton plays a role in the vascular endothelial permeability barrier and inflammatory response.There were experimental studies to investigate the effects of CG for lipopolysaccharide (LPS)-induced cell viability, apoptotic rate,cell migration, nitric oxide synthase (NOS), intracellular reactive oxygen species (ROS) production, and cytoskeletal organization[28,29]. The result demonstrated that CG significantly inhibited the damage by induced LPS in human umbilical vein endothelial cells (HUVECs). Liang Liansheng pointed out in the study[30]that CG can inhibit angiotensin II (AngII)-induced endothelial cell apoptosis, reduce the expression of apoptosis gene Fas, and reduce the leakage of endothelial cell lactate dehydrogenase (LDH) and the secretion of endothelin 1 (ET-1), increase NO level, thereby counteract endothelial cell damage. CG can protect vascular endothelial cells by inhibiting the Rho/ROCK pathway to upregulate AKT pathway, improve apoptosis and inhibit inflammation,thus protecting endothelial cell damage caused by LPS Ang and AGEs[23,25-27,31]. Vascular endothelial cell injury is a pivotal process in the development of vascular lesions in patients with diabetic nephropathy. CG had a protective effect on vascular endothelial cells, suggesting that it might be a potential drug for the treatment of diabetic-nephropathy and diabetes-cardiovascular disease[30,31].CG[32]promoted the formation of ROS, caused oxidative stress, and increased the mRNA expression of sod-1, sod-2, sod-3, sod-5, ctl-1,ctl-2, ctl-3, induced the antioxidant enzyme activities of superoxide dismutase and catalase, and scavenges free radicals to inhibit the growth of Caenorhabditis elegans. TANG et al[33]found that flavonoids and CG can inhibit the early proliferation of glomerular mesangial cells induced by high glucose, and also attenuate the apoptosis of glomerular endothelial cells mediated by AGES. The result indicated that hairy isoflavones and CG have significant regulation in the development of diabetic nephropathy.

4.5 The anti-tumor effect of CG

Because of its slight side effects, Chinese herb has a large development space and has become the focus of research by oncologists. Astragalus extract and its monomer components can not only directly inhibit the proliferation of tumor cells, induce apoptosis of tumor cells, but also enhance the body's immunity to resist the side effects of chemotherapy and improve survival rate. Zhang Dongqing[34]suggested that CG has anti-hepatocarcinoma effect, and can down-regulate CCNA2, CDK2, CCNB1, and block the cell cycle of liver cancer cells (BEL-7402) in G0/G1 phase, and then induce apoptosis. Zhang Dongmei[35]examined the effects of CG on its proliferation, cell cycle, and expression of apoptosis-related proteins by in vitro culture of human cervical cancer Hela cells using MTT,flow cytometry, and Western Blot techniques, and discussed the molecular mechanisms it may involve. Experiments have shown that CG can inhibit the proliferation of cervical cancer cells and induce its apoptosis to achieve therapeutic goals by increasing the activity of caspase-3, down-regulating the expression of B lymphocyte-2 gene(Bcl-2) protein and up-regulating Bax protein.

4.6 Other effects

CG protects the integrity of the blood-brain barrier in experimental reperfusion cerebral ischemia by regulating the NO/cav-1/MMPs pathway[36]. It also has a good therapeutic effect on cardiovascular diseases. The survival rate of heatstroke rats treated with CG is significantly improved, the body temperature is lowered, myocardial ischemia, inflammation and oxidative damage are significantly reduced, and it can be concluded that heatstroke can be prevented by reducing myocardial damage[37]. CG and flavonoids also enhance the activity of neuronal SOD and glutathione peroxidase (GSHPx), inhibit the release of LDH, and have neuroprotective effects.CG[38]can significantly reduce the melanin formation of Melana cells without cytotoxic, suitable for skin whitening agents. Xu Feng et al[39]found that CG can also significantly inhibit the proliferation of T lymphocytes induced by concanavalin A (ConA) and the macrophage toxicity induced by LPS. It has anti-inflammatory and immunosuppressive effects and can be used to prepare antiinflammatory drugs and immunosuppressive agents. CG[40]also has an anti-AIDS effect. After establishing the 3T3-L1 fatty insulin resistance (IR) model, Liu Li's study group[41]can significantly increase the glucose consumption of IR fat cells, reduce the TG level, and improve the IR by affecting glycolipid metabolism after 48 hours of CG intervention, however, its mechanism of action needs further exploration.

5. Summary and outlook

In summary, the research progress of CG extraction technology and CG in pharmacokinetics has laid a solid foundation for the research and development of a wide range of pharmacological effects of CG.Studies have confirmed that CG has multiple medicinal properties such as antibacterial, antiviral, and anti-tumor, especially in antitumor, and related researches have been increasing and some progress has been made, which is of great significance for guiding clinical drug use. Through the above review, we can find that CG involves multiple signaling pathways in the mechanism of treating diseases, but the specific mechanisms are different due to different diseases, which also confirms the multi-targeting of traditional Chinese herb in treatment. It is certain that CG can inhibit the proliferation of tumor tissues or tumor cells, promote tumor cell apoptosis, and have a synergistic effect on existing commonly used anti-tumor drugs, enhance the sensitivity of tumor cells to chemotherapy drugs. Therefore, the role of CG in tumors deserves further study, and it is believed that its prospects are very broad.