Study On The Therapeutic Basis Of Traditional Chinese Medicine Luying

Sambucus chinensis L. is a native perennial herb distributed throughout China. All parts of the plant can be used in traditional Chinese medicine (TCM) as Luying. Luying is one of the important folk medicines in TCM. It has been used to treat hepatitis over a very long period of time and has produced quite a good effect.The therapeutic basis of Luying was studied in this paper. Luying extract by different extracting methods were prepared. Effects of Luying extracts of different processes on alanine transaminase (ALT) and aspartate transaminase (AST) in hepatic injury mice were studied. As a result, 75% alcohol was selected as the optimal extraction reagent. The fractions of various polarities fractionated and purified with macroporous resin from 75% alcohol extract were further investigated. The ethyl acetate fraction and the fraction eluted from the macroporous resin with 30% alcohol were found to be primarily active.In order to isolate the active constituents in these two main effect parts of Luying, various Chromatographic techniques were performed. Six compounds were isolated and identified with IR, NMR and MS data and physical-chemical properties. They wereβ-sitosterol, stigmasterol, oleanolic acid, ursolic acid, kaempferol-3-O-β-D-(6-O-acetylglucopyranosid)-7-O-β-D-glucopyranoside, and kaempferol-3-O-β-D-glucopyranosid-7-O-β-D-glucopyranoside. Two of the compounds, which were named as kaempferol-3-O-β-D-(6-O-acetylglucopyran-osid)-7-O-β-D-glucopyranoside and kaempferol-3-O-β-D-glucopyranosid-7-O-β-D-glucopyranoside, were isolated from these plants for the first time. The in vivo antitumour effects of these compounds were investigated. It was suggested that ursolic acid and oleanolic acid possessed inhibition activity to Bel-7402 human liver cancer cell line. RP-HPLC method was developed for the determination of ursolic acid and oleanolic acid in Luying. The linear ranges for ursolic acid and oleanolic acid were 2.3～92.0μg·mL^-1(r=0.9999)and 2.6～104.0μg.mL^-1(r=0.9999), respectively. The average recoveries were 97.6％(RSD=2.1％) and 102.1％(RSD=1.7％), respectively. RP-HPLC method for the simultaneous determination of chlorogenic acid (CGA), eaffeic acid (CFA), kaempferol-3-O-β-D-glucopyranosid-7-O-β-D- glucopyranoside (KG), and (kaempferol-3-O-β-D-(6-O-acetylglucopyranosid)-7- O-β-D-glucopyranoside) (KAG)in Luying was developed and validated. The linear ranges for CGA, CFA, KG and KAG were 12.00～240.00 (r=0.9992), 0.40～8.00 (r=0.9992), 1.18～23.60 (r=0.9982), and 1.19～23.80μg·mL^-1 (r=0.9993). The average recoveries were 100.9％(RSD=3.8％), 98.3％(RSD=2.8％), 99.2％(RSD=3.3％), and 96.3％(RSD=3.2％), respectively. The essential oil in Luying was analyzed by GC-MS. 25 compounds were got and identifed in the oil. There total content of the identified compounds was 92.8％. The content of methyl salicylate was the highest (19.48％).The optimal alcohol extraction process of Luying extract was studied by orthogonal design with extraction yield and the contents of ursolic acid, oleanolic acid, and chlorogenic acid as quality assessment indices. Three factors were studied in this experiment, including the solvent consumption, duration of extraction and times of extraction. The result showed that the optimal extracting condition was 75％alcohol consumed ten times the amount of material, and three times for 1 hour each time.A rapid, sensitive, and accurate liquid chromatography-mass spectrometry (LC-MS) method for the determination of ursolic acid in rat plasma was developed and validated. Plasma samples taken from rats that had received Luying extract orally were acidified with acetic acid and then extracted with a mixture of hexane-dichloromethane-2-propanol (20:10:1, v/v/v). Atmospheric pressure chemical ionization was operated in negative-ion mode. Using selected ion-monitoring mode, the deprotonated molecules [M-H]^- at m/z 455 and 469 were used to quantify ursolic acid and glycyrrhetic acid (internal standard), respectively. The assay was shown to be linear over the range of 10～1000 ng·mL^-1 (r≥0.9960) with a lower limit of quantification of 10 ng·mL^-1. The method was shown to be reproducible and reliable with intraday precision below 7.8％, interday precision below 8.1％, accuracy within±4.3％, and mean extraction recovery excess of 83.6％, which were all calculated from the blank plasma sample spiked with ursolic acid at three concentrations of 20, 200, and 800 ng.mL^-1. The LC-MS method has been successfully applied to pharmacokinetic studies of ursolic acid after oral administration of Luying ethanolic extract to rats. The main pharmacokinetic parameters were: t_1/2, 4.3 h; C_max, 294.8 ng·mL^-1; and t_max, 1.0 h, respectively.A simple and sensitive high-performance liquid chromatographic method has been developed for the determination of chlorogenic acid in plasma and applied to its pharmacokinetic study in rats after administration of Luying decoction. After deproteinized by methanol, the plasma samples were analyzed with puerarin as internal standards. The assay was shown to be linear over the range of 42～2100 ng·mL^-1 (r≥0.9957) with a lower limit of quantification 42 ng·mL^-1. The method has shown to be reproducible and reliable with intra-day precision blow 6.7％, inter-day precision blow 7.2％, accuracy within±2.6％, and the mean extraction recovery excess 84.4％. The validated method was used to take a limited view of the pharmacokinetic profile of chlorogenic acid in rat plasma after taken Luying extract. The main pharmacokinetic parameters were: t_max, 0.36 h; C_max, 670.9 ng·mL^-1; and t_1/2, 4.85 h, respectively.Under the direction of theory and clinical practice of TCM, the therapeutic material basis was elucidated, from which quality control indices were selected. The extraction process was optimized and quality assessment methods for Luying were developed. Pharmacokinetic study of ursolic acid and chlorogenic acid was carried out in rats to take a limited view of pharmacokinetic profiles of Luying extract. This research provided an exploration for the modernization of TCM.