| Purpose: To establish the method of simultaneous determination of 3 kinds of flavonoid aglycones: apigenin, genkwanin and 3`-hydroxyl genkwanin in genkwa. To explore the preparation proess for the monomer components and optimize part of procee . To study the parmacological action of preventing cough and eliminating phlegm of monomer components and genkwa before and after processing, and the antioxidant activity of extracts of different polarity and monomer components, to provide basics for effectually using of genkwa .To provide evidence for structural transformation of flavonoids and selection of lead compounds by exploring the structure-activity relationships of monomer.Materials and Methods:Materials:Genkwa medicines purchasing from anguo, apigenin reference substance from Kunming Ke Xiang Biotechnology Co., Ltd., genkwanin reference substance provided by Mao Shujie (the research fellow at China Academy of Traditional Chinese Medicine Research Institute); HP-20 resin from Mitsubishi Chemical Corporation, D-312 resin from Shandong Lukang Record Pharmaceuticals Co., Ltd., DM-301,HPD-600 resin from Changzhou Bo Yan adsorption material Technology Co., Ltd., AB-8 resin from Nankai University Chemical Plant; polyamide (100-200 mesh , Cangzhou Technology Co., Ltd. Bo Yan adsorbent material), silica gel (200-300 mesh, Qingdao Ocean Chemical Industry); Kunming mice from the Experimental Animal Center of Dalian Medical University.Methods:1,The content of aigenin, genkwanin and 3`-hydroxyl genkwanin in genkwa were determined by HPLC.2,3 kind of flavonoids in genkwa were extracted by ultrasound, solvent-heat and cold soak. The extraction process was optimized throngh the single factor and orthogonal experiment. 3,Resin was used to purify flavonoid aglycones, and the purify process was optimized by the single factor experiment.4,flavonoid aglycones were preparated by column chromatography and silica gel column chromatography.5,The antioxidant role and structure-activity relationship of genkwa and monomer components was studied by hydroxyl radicals and superoxide radical scavenging experiment in vitro.6,Parmacological action of preventing cough and eliminating phlegm was research by ammonia stimulation and tracheal section of phenol red on whole animal experiment.Results:1,The method was established for determination of apigenin, genkwanin, and 3`-hydroxyl genkwanin in genkwa by HPLC, the results meet the requirements. The content of aigenin, 3`-hydroxyl genkwanin and genkwanin in two batches of genkwa were 4.7974 mg·g-1, 2.8910 mg·g-1, 3.4094 mg·g-1 and 3.7922 mg·g-1, 2.3771 mg·g-1, 2.6956 mg·g-1.2,The optimal extraction process by solvent heated was that: crushed herbs was extracted at 60℃, with material ratio of 1:10 of 70% ethanol, 1h one time, extracted 3 times; The optimal extraction process by ultrasound was that : crushed herbs was extracted by 70% ethanol, ratio of 1:15, 30min one time, extracted 3 times. By comparison, effects of different extraction methods followed by solvent heat extraction> ultrasonic extraction> cold soak extraction.3,The optimal purity condition by resin was that : AB-8 resin was choosed, medicinal extract was dissolved in 35% ethanol, with the concentration of 0.1g crude drug ? mL-1 and pH at 4～5, volume of 16BV. 20BV 20% ethanol and 9BV 95% ethanol elution pH at 5～6 was usd to remove impurities and get products. The purity of 3 flavonoid aglycones was raised from 4.3% to 8.9%, with the purification rate of 206.4%.4,The purity of aigenin, genkwanin and 3`-hydroxyl genkwanin obtained by polyamide column chromatography was 97.0%, 82.2% and 86.3%, with the yield of 49.5%, 45.4% and 36.9%. The purity of them obtained by silica gel column chromatography was 93.7%,78.0% and 74.9% , with the yield of 35.3%,49.6% and 30.1%. 5It was proved that different polar extracts of genkwa showed antioxidant activity, in which ethyl acetate and methanol extracts be with the strongest antioxidant activity. The IC50 of scavenging activity on hydroxyl radicals of aigenin, genkwanin and 3`-hydroxyl genkwanin were 0.096 mg·mL-1, 0.072 mg·mL-1 and 0.063 mg·mL-1. The IC50 of scavenging activity on superoxide anion radicals of them were 0.375 mg·mL-1, 0.362 mg·mL-1 , 0.147 mg·mL-1 ,which showd obvious concentration-response relations. Compared the structure and activity, there was a certain relationship between flavonoid antioxidant molecules and the number of phenolic hydroxy on B ring, 7- phenolic hydroxy on A ring has the contributions to the effect of antioxidant, the effect of phenolic hydroxy on B ring may better than on A ring . The affect above was more remarkable for superoxide anion scavenging than hydroxyl radical scavenging. 6,It was proved by pharmacological experiments through oral administration that there was no difference about preventing cough and eliminating phlegm activity between genkwa before and after processing. All of 3 kind of flavonoid aglycones had preventing cough and eliminating phlegm activity with a certain structure-activity relationship in the middle dose (300mg·kg-1) and low dose (150mg·kg-1 ): the methoxylation on the 7- phenolic hydroxyl on A ring could help to improve their preventing cough effect; the increase in the number of phenolic hydroxy on B ring and methoxylation on the 7- phenolic hydroxyl on A ring could help to improve their eliminating phlegm activity.Conclusions:1,The method for determination by HPLC was established of 3 main flavonoid aglycones in genkwa, which was simple, fast and provide basis for quality control of herbs and preparation process research foundation.2,The best preparation method and craft of 3 flavonoid aglycones were determined by comparision and optimization. 3,It was found that genkwa has good antioxidant activity, which provided experimental basis for development and utilization of herbs.4,It provided the basis by the pharmacological experiments for the research on processing mechanism about genkwa. It was clear that part the structure-activity relationship between 3 kind of flavonoid aglycones, which further added the research on structure-activity relationship of the antioxidant activity and provided experimental basis about that of preventing cough and eliminating phlegm activity of flavonoid. |
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