Study On Preparation And Preliminary Quality Standard Of Total Flavonoids From Trollius Altaicus

Objective:To study chemical components,optimize the extraction and purification process of total flavonoids from Trolius altaicus,set up the preliminary quality standard and evaluate the activity of anti-inflammatory,analgesic of total flavonoids.Methods:1)The 70%ethanol extracts from T.altaicus were prepared by refluxing method.Active constituents were separated by HSCCC.Solvent system of ethyl acetate:n-butanol:water(2:1:3)was employed,and the chemical composition were determined by MS.2)The contents of orientin-2"-O-?-L-galactopyranosylide(OGA)were selected as index,the extraction process of OGA from T.altaicus was optimized by L9(34)repeated orthogonal design.The macroporous adsorption resin was selected by inspecting the adsorption and desorption ability of six candidate macroporous resins.The static adsorption kinetics and dynamic gradient elution of the selected macroporous resin was investigated.To optimization of purification process of OGA from T.altaicus,single factor test was used to optimize the sample concentration,the sample rate,the volume of elution solvent.Finally,the OGA monomers were prepared by MCI column.3)The content of total flavonoids and the scavenging rate of DPPH free radical were used as the evaluation index,the extraction of total flavones from T.altaicus(AJTF)was optimized by L9(34)repeated orthogonal design.The macroporous adsorption resin for AJTF was selected by inspecting the adsorption and desorption ability of six candidate macroporous resins.To optimization of purification process of AJTF,single factor test was used to optimize the sample concentration,the sample rate,the volume of elution solvent.4)The quality control systems for AJTF were established.A preliminary study of its stability was carried out.5)The anti-inflammatory and analgesic effects of AJTF and OGA were preliminarily evaluated by ear swelling model induced by xylene and acetic acid-induced writhing model.Results:1)Three compounds were separated from T.altaicus which include orientin-2"-O-?-L-galactopyranosylide(?),quercetin-3-O-?-D-xylopyranosyl(1?2)-?-D-glucopyranoside(?)and oientin(?),compound(?)and(?)were separated from this plant for the first time.2)The optimum extraction process for OGA was as following:70%ethanol was selected as solvent,solid-liquid ratio was 1:30(g·mL-1)and the extract 2 times and every time last 1.5 h.The content of OGA from T.altaicus were 29.02 ± 0.46 mg·g-1.D101 macroporous resin was selected to purify the OGA.Equilibrium adsorption date were fitted Langmuir isotherm equation which was belonging to multi-molecular layer adsorption,low temperature was advantageous to adsorption.Adsorption dynamic behavior was well described by quasi-second-order kinetics equation,adsorption rate was mainly controlled by liquid film diffusion and intraparticle diffusion.Optimum purification process was as following:the concentration of sample solution of 0.2779 mg·mL-1,adsorbed by D101 macroporous resin column with a flow rate of 2mL/min,washed by 5BV water,and eluted by 7BV 30%ethanol with a flow rate of 1mL·min-1.The purity of OGA in dry extract was 33.20%,transferring rate of OGA was 90.23%.After MCI column,the content of OGA was 968.9437 mg·g-1.3)The optimum extraction process of AJTF was as following:70%ethanol was used as solvent,solid-liquid ratio was 1:30(g·mL-1),extract 2 times and every time last 1.5 h.The contents of AJTF were 170.531±3.30mg·g-1.D101 macroporous resin was selected to purify the AJTF.Optimum purification process was as following:the concentration of sample solution of 1.47 mg/mL,adsorbed by D101 macroporous resin column with a flow rate of 2mL·min-1,washed by 4BV water,and eluted by 7BV 30%ethanol with a flow rate of 2 mL·min-1.The purity of AJTF in dry extracts was 76.52%.transferring rate of AJTF was 66 49%.4)The physicochemical identification and TLC,HPLC method for AJTF were established.According to the results,moisture content in AJTF should be less than 5%,burn residue should be less than 1%,heavy metal should be less than 20ppm,arsenic salt content should be under 0.5ppm and dry weight loss should be less than 1%,total flavonoid content in AJTF should be above 624mg·g-1,the content of OGA in AJTF should be above 236mg·g-1.According to the results of stability test,the appearance and color of AJTF were not obviously affected by high temperature and strong light,and the contents of AJTF and identify component is stable.But high humidity could affect the stability of AJTF.It should be kept in dry place and sealed.5)Compared with the model group,AJTF(125,250,500mg·kg-1)and OGA(25,50,100 mg·kg-1)had significantly suppressed xylene induced ear swelling of mice(p<0.01),the inhibition rates were AJTF(25.52%,40.61%,48.34%)and OGA(33.77%,43.11%,50.16%).AJTF and OGA in each dose group can significantly reduce writhing reaction caused by acetic acid(p<0.01).Conclusion:Chemical composition of T.altaicus was studied,AJTF extraction process was optimized,the quality control systems for AJTF were established preliminarily,AJTF and OGA from T.altaicus had certain anti-inflammatory and analgesic effects.The results provides the research foundation for the development and utilization of T.altaicus.