Shanxi The Geoherbs Astragalus Flavonoid Fingerprint Of The Active Ingredient And In Vitro Metabolism Studies

Astrgalus membranaceus(Fisch.)Bge. var. mongholicus(Bge.)Hsiao, one species of Astragalus which belong to Family Leguminosae, the roots of A. mongholicus, known as Huangqi in China, is the most important tonic in the Traditional Chinese Medicine to reinforce "qi" (vital egergy), to strengthen the superficial resistance, and to promote the discharge of pus and the growth of new tissue. A. mongholicus contains many active constituents, in which flavonoid constituents, astragaloside and polysaccharids were three main active constituents. Recently, the chemical constituents, finger prints spectra and metabolism of chemical constituents were deeply studied. In this paper, two methods were developed for quatification of two main flavonoid constituents, the high performance liquid chromatography (HPLC) finger prints of the flavonoid constituents and the metabolism of flavonoid constituents were studied.According to characteristic ultraviolet absorption of flavonoid constituents, UV detection wave length was set at 254nm, reverse-phase high performance liquid chromatography (RP-HPLC) methods were developed to determine formononetin and calycosin. Linear range of formononetin concentration was 1⁴0μg/mL, the RSD of precision was less than 2%, the mean recovery was between 98.0¹02.0%. The repeatability and specificity met with the requirements of Chinese Pharmacopoeia (edition 2005). Linear range of calycosin concentration was 0.65～65μg/mL, the RSD of precision was less than 2%, the mean recovery was between 98.0¹02.0%. The repeatability and specificity met with the requirements of Chinese Pharmacopoeia (edition 2005). The HPLC method established in this paper could be used to determine the content of flavonoid constituents in A. mongholicus.On the base of one-effect test, the effects on ultrasonic extraction of flavonoid constituents such as assumption of ethanol, times of extraction, ultrasonic time and concentration of ethanol were investigated by using orthogonal design table and formononetin was used as a target index, the optimized extraction method was determined according to the experiment results. The optimized extraction conditions were: adding 15 times the volume of 75% ethanol, three successive extractions, 20 minutes every time.The finger prints chromatogram of flavonoid constituents from five samples were established by HPLC-UV detection and in a gradient elution mode. The profile of calycosin, calycosin-7-O-β- D-glucoside and formononetin were qualitationed. The results suggested that there were 8 common peaks in chromatography graph of each sample. The profile of HPLC finger prints and the relative ratios of the common peak retention time of the 5 samples were approximately the same. The sum of the common peak area was more than 90% of total peaks area of each sample, but there were some differences among the relative ratios of the common peak areas of the 5 samples.The metabolism mechanism and the degradation rate of flavonoid constituents in rat liver microsomes and in gastrointestinal contents were studied. The metabolic rates of calycosin-7-O-β-D-glucoside, calycosin and formononetin in rat liver microsomes were 22.64%, 31.18% and 55.42%, the perseudo first-order degradation rate constants were 0.267 h^-1(r = 0.992), 0.386 h^-1(r = 0.991) and 0.826 h^-1(r = 0.996); Respectively, The metabolic rates of calycosin-7-O-β-D-glucoside and formononetin in gastro contents were 33.45% and 38.06%, the perseudo first-order degradation rate constants of were 0.198 h^-1(r = 0.962) and 0.232 h^-1(r = 0.938); The metabolic rates of calycosin-7-O-β-D-glucoside and formononetin in duodenum contents were 100% and 30.47%, The perseudo first-order degradation rate constants were 0.944 h^-1(r = 1) and 0.188 h^-1(r = 0.963). The results suggested that calycosin-7-O-β-D-glucoside was hydrolyzed to calycosin in rat liver microsomes and in gastrointestinal contents.