| Lu Ying, the dried whole herb of the plant Sambucus Chinensis Lind1., belonging to the plant family Caprifoliaceae Sambucus, is a kind of commonly used medicinal herb in folk medicine. In this study, Lu Ying was investigated systematically with regard to chemical constituents, quality control, optimization of extraction process, and the pharmacokinetic studies of the main components.The chemical constituents of Lu Ying were thoroughly studied with liquid-liquid extra-ction, adsorption with macroporous resin, and various chromatographic separation techniques. Totally,23 compounds were isolated. Among them, the structures of 17 compounds listed here were identified and proved with chromatographic data, physicochemical properties, and spectral data, such as Kaempferol(1), Kaempferol-3-O-β-D-galactopyranoside(2), Kaempferol-3-O-β-D-gl ucopyranoside(3), Kaempferol-3-O-(6"-acetyl)-β-D-galactopyranoside(4), Kaempferol-7-O-β-D-glucopyranosid e-3-O-(6"-acetyl)-β-D-glucopyranoside(5),2-hydroxy-3-methyl anthraxquinone(6), Ferulic acid(7), Ethylf erulate(8), Caffeic acid(9),Ethyl caffeate(10), P-coumaric acid(11), Chlorogenic acid(12), Ursolic Acid(1 3), Oleanolic acid(14), Syringaresinol(15),β-sitosterol(16) and Daucosterol(17). Notably, it is the first time for compounds with numbers as 1,2,3,4,6,7,8,9,11,15 and 17 in parentheses s hown above to be isolated from this plant, which provides the material basis for quality co ntrol of Lu Ying and the further pharmacokinetic studies.Main chemical constituents of Lu Ying were quantitatively analyzed using HPLC method and the evaluation of Lu Ying with fingerprint analysis method was established. Using gradient elution, HPLC methods were established for simultaneously determination of seven active ingredients and for simultaneously determination of four kinds of organic acids in Lu Ying, repectively. After optimization of separation conditions and extraction process, the analysis methods were validated to meet the requirements of drug content determination. HPLC method for kaempferol content determination in Lu Ying was also established with isocratic elution method. All the analysis methods established in this study are simple, rapid, accurate, and were successfully used for quantitative analysis of the main chemical constituents of Lu Ying from different sources.Using HPLC method, fingerprint for six batches of Lu Ying herbs from different sources were studied. With chlorogenic acid as reference compound, totally 23 common peaks for Lu Ying were verified; by comparison with the reference substances, among which 8 peaks were identified. Mutual mode of chromatographic fingerprint for Lu Ying was established to evaluate the quality of the herbs according to similarity between the fingerprint of sample and the common pattern of fingerprint.With the established HPLC method for chromatographic fingerprint of Lu Ying, different chromatographic fingerprints for extracts with different extraction solvents and methods, which showed anti-hepatic injury activity (Lu Ying water extract,75% ethanol extract, alcohol solution of ethyl acetate extract and 30% ethanol eluate of macroporous resin column) were analyzed. Through comparison of the fingerprints, the main active ingredients of anti-hepatitis were initially confirmed as organic acids and flavonoids. Different types of macroporous resins were screened applying chlorogenic acid and flavonoids index. The eluting solvents, sample volume, pH of the sample, pH of eluate, and influence of salt addition on the content of active ingredient in extracts were systematically investigated applying total flavonoids as index. The optimized process was primarily validated. This optimized process provides an experimental basis for extraction of the effective parts of Lu Ying, and presents the technical parameters for application of macroporous resin in the large-scale production.UPLC-MS/MS method for the determination of ferulic acid in rat plasma was established using docetaxel as the internal standard. Plasma was acidized by formic acid, and extracted with liquid-liquid extraction method with ether as the extraction solvent. Analysis was performed using the multiple reaction monitoring (MRM) mode with an electrospray ionization (ESI) interface. The results show that:endogenous substances in blank plasma do not interfere with the determination of internal standard (docetaxel) and ferulic acid; a good linear relationship was found for ferulic acid in the range of 0.50-800.00 ng·mL-1. Precision, accuracy and extraction recovery of the method were satisfactory. With this established method, concentration of ferulic acid in rat plasma was determined after intragastral and intravenous administration of ferulic acid, as well as after intragastral administration of Lu Ying extracts. Pharmacokinetic behaviors of ferulic acid were examined. The results show that:for intragastral and intravenous administration of ferulic acid, and intragastral administration of Lu Ying extracts, Cmax is 948±73 ng·mL-1,602.4±111.8 ng·mL-1 and 245.2±123.2 ng·mL-1;t1/2 is 1.65±0.53 h,2.59±0.44 h and 2.81±0.88 h; AUC0-t is 123.1±12.2 ng·h·mL-1, 129.8±28.4 ng·h·mL-1 and 295.4±50.8 ng·h·mL-1; AUC0-∞is 128.4±8.9 ng·h·mL-1, 144.5±27.4 ng·h·mL-1 and 513.0±124.5 ng·h·mL-1, respectively.In this study, guided by the theory and practice of Traditional Chinese Medicine, with the combination of traditional Chinese pharmacology, analytical chemistry, biopharmaceutics, statistics of chemistry and computer technology, the chemical constituents of Lu Ying were throughly separated; quality control method was established; the extraction process of the effective parts was optimized; with the established analytical methods of its related components in biological samples, the pharmacokinetics were studied. This work supplies a material foundation for quality control of the traditional Chinese medicine, and is a meaningful exploration for the modernization of Traditional Chinese Medicine. |
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