Study On The Extraction, Purification And Biological Activity Of Flavonoids From Hypericum Ascyron L.

Hypericum ascyron L was mainly distributed in Northeast China and the Yellow River, Yangtze River, Pearl River Basin, it has been used to stop bleeding, cure jaundice, hepatitis, and sore boil, etc., with a long history. According to the precious research, the main active ingredient of Hypericum ascyron was flavonoids and xanthene, e.g, ketonesquercetin, kaempferol, Hyperoside, extract of which exert a role as anti-inflammatory, analgesic, anti-oxidation, histamine release inhibiting.So far, no detailed research has been reported about the extraction, purification and the biological activity of the flavonoids from Hypericum ascyron, which hampered the widely application of the active ingredient of Hypericum ascyron. The present research was designed to study the methods of extraction and purification and estimate the relative biological activity of the flavonoids from Hypericum ascyron L, which may help us to fully understand the basic knowledge of this medicinal resources, and provide the evidence for the further application of the Hypericum ascyron L The main results are as follows:1. Three extraction methods (refluxing, ultrasonic assisted extraction, and microwave assisted extraction method) had been compared preliminarily. The result showed that the method of microwave assisted extraction was better than the other two. Combined with response surface, we optimized the extraction conditions of the total flavonoids of Hypericum ascyron L, the conditions were as follows:microwave power was700W, microwave time was4min, ethanol concentration was56%, liquid-solid ratio was42:1, under these conditions, extraction yield of the total flavonoids could reach to5.91%.2. The separating and purifying purpose of the flavonoids from Hypericum ascyron L was studied and compared with six kinds of macroporous resins including ncluding X-5, S-8, D101, AB-8, NKA-9and NKA-Ⅱ. The adsorption rate of NKA-9resin was79.41%, and the resolutional ability of NKA-9resin was98.35%. Because of good adsorption and analytical ability, NKA-9resin could be considered as the best adsorbent of the total flavonoids. Next, it had been studied that the optimum conditions of the separation and purification of the total flavonoids of Hypericum ascyron with NKA-9resin, the result as follows:best adsorption time was6h, the concentration of the total flavonoids of Hypericum ascyron was3.5mg/mL, pH was6.0, adsorption temperature was35℃; the desorption conditions were as follows: eluent was60%ethanol, desorption of liquid to solid ratio of10:1(60%ethanol solution: macroporous resin, mL/g). Under these conditions, the purity of the total flavonoids increased from13.78%to67.41%。3. HPLC analysis showed that the major components of total flavonoids of Hypericum ascyron were quercetin, kaempferol, rutin, hyperin and isoquercetin and total flavonoids content was47.7%.4. In order to evaluate the antioxidant capacity of the total flavonoids of Hypericum ascyron, experiments were performed to determine the ability to scavenge DPPH and hydroxyl radical, the total reducing capacity, the total antioxidant capacity, the bleaching trials of beta-carotene, and the protection of DNA oxidative damage. The results showed that the total flavonoids of Hypericum ascyron exerted good antioxidant capacity and could be applied as a new type of natural antioxidants.5. The inhibition determination, reversible inhibition kinetics studies of the tyrosinase, xanthine oxidase and a-glucosidase suggested that the total flavonoids of Hypericum ascyron was a moderate reversible competitive tyrosinase inhibitor, as the half inhibition rate IC50was46.99μg/mL, the inhibition constant Ki was34.07μg/mL. The total flavonoids of Hypericum ascyron could also be excellent reversible competitive inhibitor of xanthine oxidase, as the half inhibitory constant IC50was11.73μg/mL, the inhibition constant Ki was0.61μg/mL. Additionally, the total flavonoids of Hypericum ascyron may also be preferably reversible mixed a-glucosidase inhibitors, and its half inhibition constant IC50was0.237mg/mL, the inhibition constant Ki was0.149mg/mL, and inhibition constant to the enzyme-substrate complex Kis was0.123mg/mL.