| Artemisia argyi Folium,the dried leaves of Artemisia argyi Levl.Et Vant.,is a commonly used Chinese herbal medicine.Studies have shown that the main active ingredients are volatile oil,flavonoids,tannic acid and polysaccharide,etc.To date,most of the previous studies focused on the volatile oil of A.argyi,and the flavonoids is lack of systematic study.However,flavonoids in A.argyi are found to possess a variety of pharmacological activities and have great development value in medicinal and healthy industry.The aim of this experiment is to study the flavonoids of A.argyi from four aspects: chemical composition,quality control,purification process and pharmacological activity,so as to provide reference for further development of flavonoids of A.argyi.The specific research is divided into four parts,which are as follows: the first,in order to clarify the substance basis of A.argyi leaves and provide basis for quality control,we studied the chemical constituents of A.argyi leaves.Silica gel column chromatography,ODS column chromatography,Sephadex LH-20 column chromatography,preparative liquid chromatography were used to isolate and purify the ethyl acetate fraction,and the structure of the compound was identified according to its physicochemical properties and spectral data.Fourteencompounds were isolated and identified,namely(1)artemisolide,(2)casticine,(3)centaureidin,(4)eupatilin,(5)jaceosidin,(6)hispidulin,(7)5,7,3’-trihydroxy-6,4’,5’-trimethoxyflavone,(8)6-methoxytricin,(9)capillarisin,(10)apigenin,(11)5,7,3’,4’.-tetrahydroxy-6,5’-dimethoxyflavone,(12)eriodictyol,(13)luteolin-7-O-rutin,(14)apigenin-7-O-rutin.Compounds 13 and 14 have been isolated from this plant for the first time.The second,in order to evaluate the quality of A.argyi Folium comprehensively and accurately.The chemical constituents of A.argyi Folium were first qualitatively analyzed by UPLC/LTQ-Orbitrap-MS.Six flavonoids and six phenolic acids were identified ambiguously,and the possible structures of the other three flavonoids were deduced.UHPLC-DAD was then used to establish a method for the determination of multiple components in the herb.Xtimate UHPLC C18 column was used to elute with gradient elution of acetonitrile-0.1% formic acid aqueous solution at a flow rate of 0.4 ml·min-1 and a detection wavelength of 330 nm.Twelve main components were selected for content determination.Each component showed a good linearity,precision,accuracy,repeatability and stability.The method is novel,rapid and accurate,and can be used as a reliable quality control method for A.argyi Folium from different areas.It is found that the content of 7 components in Qi’ai,the genuine regional drug,are higher than samples collected from other areas.In the third part,the technology of separating and purifying the total flavonoids from the ethanol extract of A.argyi Folium by macroporous resins was optimized.The adsorption and desorption properties of 21 different types of macroporous resins were compared by static and dynamic adsorption-elution tests,and the optimum resin type was selected.The purification conditions were investigated by single factor test with the concentration of total flavonoids.Theresults showed that AB-8 macroporous resin had good adsorption and desorption capacity for total flavonoids.optimum separation and purification conditions for total flavonoids were as follows: the concentration of sample solution 0.74mg/m L,adsorption rate 2 BV/h,sample volume 6 BV,eluted by 6 BV of 55%ethanol at a flow rate of 2 BV/h The optimum process is stable and feasible,and is suitable for the separation and purification of total flavonoids from Artemisia argyi Folium.In the fourth part,on the basis of purifying total flavonoids of A.argyi with macroporous resin,the anti-inflammatory and antioxidant activity of purified total flavonoids of A.argyi(AATF)was studied in order to provide experimental data and theoretical basis for the development and utilization of health products of flavonoids of A.argyi.RAW264.7 cells were stimulated by LPS to construct an inflammation cell model in vitro.The contents of pro-inflammtory mediates and cytokines were detected by Griess and ELISA respectively.The elevated levels of NO,ROS,PGE2,TNF-a,IL-6,IL-10,and MCP-1 in RAW264.7 cells induced by LPS were suppressed significantly by AATF treatment.To evaluate the antioxidant activity of AATF,DPPH,ABTS,FRAP,hydroxyl radical and superoxide anion scavenging activity tests were carried out.The results showed that the EC50 of AATF was 34.0、98.6、59.2 、246.2、15.5 μg/m L respectively,indicating that the flavonoids of A.argyi also had good antioxidant activity.The results provided experimental data and theoretical basis for the development and utilization of flavonoids of A.argyi. |
