The Separation And Purification Of Effective Constituent In Fruit Of Ehretia Macrophylla Wall, And The Bioactivity Study

Fruit of Ehretia macrophylla Wall,commonly known as yepipa in Hunan and so on,Eating and drinking extractions of this fruit shows high effectiveness.Fresh fruit is commonly used for the treatment of coughing and sphagitis.The effective components were extracted and purified from the fruit.Antioxidative,anti proliferative and anti-inflammatory activities were studied.The research contents and results of the paper are as following:Separation and purification of active ingredients.The effective constituents were extracted from the dry fruit of Ehretia macrophylla Wall by 70% ethyl alcohol,and fractional leaching with four different polar organic solvents(ethyl acetate,n-butyl alcohol,trichloromethane and petroleum ether)were carried out,and EA,n-Bu,TCM and PE fractions were obtained.Tested by Folin-reagent method,the total polyphenol of EA was 938.72±9.41 mg GAE/100 g DW,which was higher than others significantly,and followed by n-Bu fraction(204.3±3.33 mg GAE/100 g DW).By HPLC and comparing with 20 various standards,vanillic acid,catechin hydrate,quercetin,o-Anisic acid and naringin were found in EA.While n-Bu shows o-Anisic acid and naringin.These compounds were separated by polyamide column chromatography and sephadex LH-20,following two compounds(rosmarinic acid and saikolignanoside A)were separated from n-Bu which was identified by NMR.Saikolignanoside A in Boraginaceae plant was detected for the first time in this research.Antioxidative and anti-proliferative activity of effective components.The antioxidation of six samples,EA,n-Bu,TCM,PE fractions,methyl caffeate and methyl rosmarinate,were estimated by methods of ORAC,PSC and CAA.It was found that the antioxidation of two compounds were stronger than the four fractions,methyl rosmarinate was found best,it's ORAC values,PSC values and CAA values(wash)were 28976.58±3589.36 ?mol TE/g DW),32139.75±3979.76 ?mol EVc/g DW and 317.77±36.69 ?mol QE/100?mol,respectively.EA had an excellent antioxdant activity among four fractions,and it's ORAC and PSC was higher than methyl caffeate,but it's CAA was similar.HepG2 and Caco-2 cellular model was adopted to estimate the anti-proliferation of six samples.It was noted that the anti-proliferation of EA and TCM was better in tested dosage,while the activity of n-Bu and PE was poor.Two compounds showed great anti-proliferation than other extractives,EC50 value of methyl rosmarinate to Caco-2 was 132.73±18.19?M,while EC50 value of Methyl caffeate to HepG2 was 48.35±0.61?M.Anti-inflammation and its mechanism.After LPS stimulated cell RAW264.7 generated inflammation,NO,an inflammation index,can be detected the secretion using Griess reagent to judge the anti-inflammtion result initially.The anti-inflammtion of EA was the best in the four extractions,which could be attributed to IC50 value of NO release suppression,19.10±0.31 ?g/mL,and following by TCM(19.48±0.25 ?g/mL).The anti-inflammative activity of two compounds was stronger than all other extractives.To study how to regulate genes expression,RT-QPCR was adopted to appraise three inflammation factors(TNF-?,IL-1? and iNOS)in EA,n-Bu and two compounds.It was found that EA,n-Bu only decrease the expression of iNOS but have no influence on TNF-? and IL-1?,however the low concentrations of Methyl caffeate and methyl rosmarinate showed remarkable effect on three inflammation factors.,described these three genes were associated with its signal regulation pathway to achieve antiinflammatory purposes.