Studies On The Chemical Constituents Of Hedyotis Diffusa Willd

Baihuasheshecao, the whole plant of Hedyotis Diffusa Willd(HD) is used as a traditional Chinese medicine, distributed in Jiangsu, Anhui, Fujian, Zhejiang, Guang xi, etc in China, picked in summer and autumn. It is cold, slightly bitter and sweet in nature. It has been used for the clinical treatment of carbuncle swollen, snake bite, sore throat, etc. The main chemical constituents in it include flavonoids, anthraquinones, iridoids, etc. Its pharmacological activities such as anti-tumor, immune regulation have been confirmed by modern research. In order to develop and utilize the medicinal resource rationally, isolation and structure identification of the chemical compounds from HD were carried out in this paper. Extraction process of total iridoid constituents of HD was optimized by orthogonal design, and the content of 6 constituents in HD was determined by HPLC-DAD. The results can provide a theory reference for the research of HD on definiting chemical constituents and quality control.The research is described as follows:1. Literature reviewAdvances in the studies on chemical constituents, pharmacological activities, chemical analysis, extraction technology of HD were reviewed.2. The isolation and structure identification of the chemical compounds of HD.20 compounds were isolated from HD by silica gel, Sephadex LH-20、C18、MCI column chromatography and HPLC. Based on physical and spectroscopic techniques, 17 compounds were identified:(E)-6-O-β-coumaroyl scandoside methyl ester (compound 1), (Z)-6-O-β-methoxy cinnamoyl scandoside methyl ester (compound 2), (E)-6-O-p-methoxy cinnamoyl scandoside methyl ester (compound 3), scandoside methyl ester (compound 4), deacetyl asperulosidic acid methyl ester (compound 5), 2-methyl-3-hydroxy-4-methoxy-anthraquinone (compound 6), quercetin-3-O-[2-O-(6-O-E-sinapoyl)-β-D-glucopyranoside]-β-D-glucopyranoside (compound 7), kaenpferot3-O-(2-O-β-D-glucopyranoside)-β-D-galactopyranoside (compound 8), quercetin-3-O-[2-O-(6-O-E-feruloyl)-β-D-glucopyranoside]-β-D-glucopyranoside (compound 9), oleanic acid (compound 10), kaempferol(compound 11), quercetin (compound 12),p-coumarie acid (compound 13),β-daucosterol (compound 14), tetradecanoic acid (compound 15), tetracosanol acetate (compound 16), 1-hentetracontanol (compound 17)3. Extraction technology of total iridoids from HDOrthogonal design was utilized to optimize the extraction conditions of iridoid glycoside in HD. The composite score of the content of total iridoids and extract yield was taken as investigation index, and the content of total iridoids was determined by spectrophotometry. L9 (34) orthogonal design was applied to study the effects of ethanol concentration, extraction time, extraction times and liquid-to-solid ratio.The optimal parameters of the extraction technology were that the concentration of ethanol was 90%, the ratio of extraction solution to sample was 8:1, the extraction times was 2 and the extraction time was 2 hours. Three batches of the extracts obtained under the optimal parameters of the extraction technology gave the result that the average extracting rate was 10.43%(RSD 1.64%), average total iridoid glycoside content was 22.85%(RSD 1.80%)3. The determination of six chemical compositions in HD. by HPLC-DAD.A method for simultaneous quantitative determination of six chemical compositions in HD, including deacetyl asperulosidic acid, asperuloside, quercetin, kaempferol,2-hydroxy-3-methyl-1-methoxy anthraquinone,2-hydroxy-3-methyl anthraquinone, was developed by HPLC-DAD. The procedure of HPLC-DAD was performed on the chromatographic column of Kromasil C18 (250 mm×4.6 mm,5μm), and the mobile phase was methanol (A)-acetonitrile (B)-0.5‰ phosphoric acid solution (D) with gradient elution at a flow rate of 0.8 ml/min. The detection wavelengths were 236 nm,367 nm and 274nm.Four factors, the extraction mode, extraction solvent, liquid-to-solid ratio, extraction time, were investigated. The best extraction process was that the extraction mode was ultrasonic extraction, extraction time was 45 min, solvent volumn was 50 times the amount of sample. The results showed that the contents of 6 compositions were differences between 10 batches of HD from different areas. The content of iridoids is higher than other compounds, and the content of iridoids in HD from Jiangsu and Henan is higher than other areas, the content of flavonoids in HD from Shandong and Neimeng is higher than other areas, the content of anthraquinone in HD from Henan is higher than other areas.