Methods of Determination
Determination of Astragaloside IV by HPLC
Chromatographic conditions: Column: C18 5u 4.6mm × 250mm; mobile phase: acetonitrile-water (1: 2); flow rate: 0.8ml/min; column temperature: 25℃; detection wavelength: 200nm.
Preparation of the test solution: Accurately weigh 0.1g of the extract powder, and add appropriate amount of water to do ultrasound dissolution, and then shake and extract it in 15ml × 4 n-butanol (water-saturated) to get a butanol layer until it is evaporated to dryness, and melt the residue with methanol and set the volume to 10ml, after mixing evenly, flit it through 0.45μm micro porous membrane.
Preparation of the standard solution: Accurately weigh an appropriate amount of standard astragaloside IV and dissolve it in methanol, and prepare the solution with the concentration of 0.1mg/ml.
Determination: draw the test solution and standard solution 20μl respectively by using the micro-injectors, and inject it to the liquid chromatograph, and record the peak area and calculate the content by external standard method.
1. Immunomodulatory effect: Astragaloside IV, mouse peritoneal macrophages and Mycobacterium tuberculosis were co-cultured to test the phagocytic ability to Mycobacterium tuberculosis of mouse peritoneal macrophages, and the content of γ-interferon (IFN-γ) and interleukin-1β (IL-1β) in the culture medium was detected as well. The results showed when the dose of astragaloside IV were 0.2,0.6,1.5,4.0 g L-1, the number of DNA copies (TB-DNA) of Mycobacterium tuberculosis that phagocyted by macrophages, and the IFN-γ and IL-1β levels in the supernatant were significantly higher than the control. Astragaloside IV can enhance macrophage’s phagocytic ability to Mycobacterium tuberculosis. Astragaloside IV could promote proliferation of T, B lymphocyte in vitro and in vivo and production of antibody in mice, and promote B cells proliferation in the thymus-independent area and promote the formation of a large number of plasma cells and synthesis of antibodies, but it had no significant effect on T cells in the thymus-dependent area.
2.Organ protection effect (1) Brain protection: astragaloside IV has a protective effect on brain injury in mice that induced by instant focal ischemia, which is related to its antioxidant effects, and astragaloside IV is expected to become the clinical drug in treatment of stroke. Astragaloside IV has a protective effect on blood-brain barrier after cerebral ischemia and reperfusion in rats.(2) Kidney protection: astragaloside IV has a protective effect on renal injury that induced by ischemia and reperfusion, and it can protect the kidney effectively when doing a kidney transplant, thus improve the success rate of transplant. Besides, astragaloside IV can down-regulate the content of monocyte chemoattractant protein-1 (MCP-1) in kidney tissues and the overexpression of mRNA.(3) Lung protection: astragaloside IV has a protective effect on lung injury that induced by ischemia and reperfusion, and it can reduce pulmonary bleeding and focal pulmonary hemorrhage in rats. Astragaloside IV can effectively inhibit ovalbumin-induced chronic asthma.(4) Myocardial protection: astragaloside IV can significantly improve myocardial ischemia and cardiac function in rats with myocardial infarction, and this effect is positively correlated with the dose and time. After the intraperitoneal injection of sterile Coxsackie virus (CVB3) to Balb/C mice, causing the myocarditis mice model, the mice were gavaged with 9% astragaloside IV for 7 days, the survival rate of mice with myocarditis increased, and the collagen synthesis and myocardium cell apoptosis reduced.(5) Liver protection: Astragaloside IV inhibits the collagen synthesis and proliferation of hepatic stellate cells, and has significant inhibition in fibrosis of liver cells.
3. Hypoglycemic effect: Taking rats with type-2 diabetes as research objects to study the regulation of astragaloside IV to hepatic glucose enzymes in rats with that streptomycin and high-fat diet induced diabetes, the results showed when the dose of astragaloside IV was 25, 50 mg/kg, the levels of blood glucose, and triglyceride (TG) and insulin in rats were significantly reduced, and related mRNA and protein expression were inhibited as well. In addition, astragaloside IV can inhibit TNF-α-induced 3T3-L1 adipocytes lipolysis, thereby reducing the level of free fatty acid (FFA), increasing insulin sensitivity, and having the hypoglycemic and hypolipidemic effect.
4. Anti-apoptotic effect: astragaloside IV can significantly reduce the apoptosis index of myocardial cells with CVB3 viral myocarditis. Astragaloside IV had a certain inhibition on Adriamycin-induced apoptosis of bone mesenchymal stem cells (BMSCs) in mice, but there was no significant dose-dependent manner.
5. Anti-inflammatory and antiviral effect: astragaloside IV significantly inhibited the xylene-induced ear edema in mice, with a strong anti-inflammatory effect. Astragaloside IV has the effect of anti-hepatitis B virus. After 10 days’ medical treatment, with the dose of 120,40 and 10 mg/kg astragaloside, its inhibition rate on HBV was 64.0%, 49.6% and 41.7%, respectively. It was also observed that the level of Hepatitis B Virus (DHBV) DNA decreased.
6. Anti-aging effect: the anti-aging effect of astragaloside IV is related to its functions in scavenging free radical and anti-lipid peroxidation, promoting protein turnover, eliminating nucleic acid metabolic disorders and promoting the proliferation and apoptosis of human skin fibroblasts. At lower concentrations (5~20 mg/L), astragaloside IV can promote proliferation of wrinkle and wrinkle-free skin fibroblasts and synthesis of type Ⅰ collagen in wrinkles, wrinkle-free and aged-skin fibroblasts, reducing the apoptosis rate of wrinkle and wrinkle-free skin fibroblasts.
7. Promoting cell proliferation: appropriate concentration of astragaloside IV can promote the rapid proliferation of chondrocytes and maintain the activity of chondrocytes, providing a new way for cartilage tissue engineering to get a lot of seed cells and maintain chondrocyte activity in a short term.
8. Other effects: astragaloside IV has a certain anti-cancer effect by inhibiting the gene expression of Vav3.1. By inhibiting the flow of calcium into the cells through NO-cGMP pathway and the release of intracellular calcium and the activity of phenylephrine and angiotensin Ⅱ, astragaloside IV has a relaxing effect on the aortic ring blood vessels and the isolated mesenteric artery of rats.
Physical and Chemical Properties
White to pale yellow powders, melting point 295.0~296.0 ℃; Soluble in methanol, ethanol, acetone; Insoluble in chloroform, ethyl acetate and other weak polar organic solvents.
Astragaloside IV is a cycloartane-type triterpene glycosides, it is one of the main active ingredients of traditional Chinese medicine Astragalus membranaceus, whose content is the main criteria for evaluation of the quality merits of Astragalus membranaceus. Astragaloside IV has effects in anti-tumor, anti-inflammatory, antioxidant, hypoglycemic, myocardial protection, anti-viral myocarditis, protecting brain tissue and anti-hepatitis B virus etc., and it has a wide range of pharmacological effects and very bright application.
vasodilator, antihypertensive, antiaging
Astragalus extracts are the extraction of raw material products of dry roots of Astralgus membranceus (Fisch) Bge Var. Mongholicus (Bye).
[Crude Medicine Resources] There are about 1600 species under genus Astragalus in the world, and more than 200 species in China, among which about seven species used as medicines. Astragalus membranacus mainly distributes in Heilongjiang, Liaoning, Inner Mongolia, Hebei, Shandong, Shanxi, Shaanxi, Ningxia, Gansu, Qinghai, Xinjiang, Sichuan and Yunnan in China, and it also has distribution in Mongolia, North Korea and Russia. Radix Astragali mainly distributes in the Great Khingan, Nenjiang, Aihun, Sunwu, Morin Dawa banner in Inner Mongolia. Ninggu Astragali mainly distributes in Ning'an of Heilongjiang Province, Dongning, Linkou, Muling, Hailin. Zhenggu Astragali mainly distributes in Hebei, Zhangjiakou areas. Astragalus mongolicus mainly distributes in Jilin, Hebei, Shanxi and Inner Mongolia of China, and it also has distribution in Mongolia and Russia. The wild resources of Astragalus have been rare in China, with Astragalus membranacus and Astragalus mongolicus falling into the third-ranking national protection category. At present most Astragalus for medicinal use are cultivars, which are mainly produced in Hunyuan, Fanshi, Ying county, Dai county, Guangling of Shanxi, Guyang, Wuchuan, Zhuozi of Inner Mongolia, Heilongjiang and Jilin provinces.
[Main Components]Astragaloside IV, flavonoids, polysaccharides, amino acids, organic acids, trace elements, riboflavin, folic acid, vitamin P, sitosterol, lupeol, n-hexadecyl alcohol etc.
Figure 1. Astragalus membranaceus
The above information is edited by the Chemicalbook of Cheng Jingmin.
Astragaloside IV has a very low content in Astragalus. Currently, the traditional boiling water extraction and reflux boiling ethanol extraction used in astragaloside IV extraction have the shortcomings like high extraction temperature, low extraction rate and time consuming. The latest extraction techniques include high-speed centrifugal extraction, microwave assisted extraction, ultrasonic extraction, ultrafiltration, supercritical fluid extraction, high pressure extraction, high-speed countercurrent extraction. New extraction technology plays an important role in improving astragaloside IV extraction rate, shortening the extraction time and preventing damage of the active ingredient.
The process of astragaloside IV extraction by Supercritical CO2 Extraction: Weigh 15g of Astragalus IV and crush it to 40 meshes, put it into the extraction kettle and pour into quantitative entrainment agent (volume fraction 75% ethanol). Put the extraction kettle into the supercritical device. Adjust the temperature and pressure of the supercritical carbon dioxide fluid, and pump the supercritical carbon dioxide fluid into the kettle with a constant flow rate. Separate and collect the extracts by cyclone separation, and centrifuge, concentrate and dry the extracts.
The optimal conditions of astragaloside IV extraction by Supercritical CO2 Extraction: extraction pressure 40Mpa, temperature 45 ℃, extraction time 2 hours, entrainer 95% ethanol, entrainer amount 4ml (95% ethanol)/g (dry Astragalus powder), CO2 flow rate10kg/kg.h.
The results of astragaloside IV ’s effects on embryonic and fetal development of SD rats and New Zealand rabbits showed: when the dose was higher than 1 mg/kg, astragaloside IV showed maternal toxicity; when the dose was higher than 0.5 mg/kg, it showed fetal toxicity. Therefore, it is recommended that pregnant women should use astragaloside IV with caution. Further research showed astragaloside IV can significantly delay the development of rat hair, eye opening and Parry reflection. But astragaloside IV had no significant effect on memory and learning ability.