| Lu xian cao（Herba Pyrolae） is commonly used herbal drug as tonics, sedatives, hemostatics, anti-inflammation, and analgesics against rheumatoid arthritis in China since the antiquity. According to the Pharmacopoeia of the PRC（edition 2005）, lu xian cao is the dried whole plant of Pyrola decorata H. Andres. In this dissertation, the chemical components, therapeutic basis, quality control method, as well as pharmacokinetics of lu xian cao were studied systematically.From the whole plant of Pyrola decorata H. Andres, 22 compounds were isolated by various chromatographic techniques and structurally elucidated by chemical and spectroscopic methods. One of them was a new compound, 6,6’-dihydroxy-4,5’-dimethyl-[1,1’-biphenyl]-3,3’-diyl bis-β-D-glucopyranoside, and sixteen of them were isolated from this plant for the first time: chimaphilin, oleanolic acid（OA）, ursolic acid（UA）, toluhydroquinone, vanillic acid, pomolic acid（PA）, maslinic acid（MA）, colosic acid（CA）, quercetin（QG1）, isoquercitrin（QRh）, quercitrin, pyrolaside A, isohomoarbutin, gallic acid, 3-β-O-α-L-arabinopyranosylsiaresinolic acid-28-O-β-D-glucopyranosyl ester（ASGE） and ziyuglycoside I（APGE）. Five known ones, hyperin（QGa）, quercetin-3-O-α-L-arabinofuranoside（QAr）, homoarbutin, 2"-O-galloylhyperin（GQGa） and pirolatin were also isolated.The therapeutic bases of lu xian cao were explored by antitumor and antibacterial activity evaluation.①The antitumor activity of lu xian cao was firstly studied on screening of active ingredients with their cytotoxicity against Bel-7402 hepatoma cell line by using MTT method. As a result, the ethyl acetate soluble extract of lu xian cao showed intense activity. Further fractionation of the ethyl acetate soluble extract on silica gel column lead to two main parts, namely fraction eluted with chloroform and fraction eluted with menthol-chloroform（1:9）. These two fractions acted only when they were combined and the activity of the combination was not significantly different from the acetate soluble extract by ANOVA test. The cytotoxicitiy of compounds isolated from the two main effect parts was further investigated using five human tumor cell lines, namely, Bel-7402 hepatoma ceil line, HepG-2 hepatoma cell line, Hela epithelial carcinoma cell line, SGC-7901 gastric carcinoma cell line, and HT-1080 fibrosarcoma cell line. Chimaphilin, toluhydroquinone, ursolic acid, pomolic acid, and colosic acid showed significant cytotoxicities against the aforementioned cell lines and gave IC50 values in the range of 7.64～19.14μg·mL-1, 14.87～28.52μg·mL-1, 1.36～5.84μg·mL-1, 17.34～23.99μg·mL-1 and 12.69～28.75μg·mL-1, respectively. Maslinic acid showed slelective activity on three tumor cell lines with IC50 values in the range of 24.24～47.29μg·mL-1.②The in vitro antimicrobial activity of the isolated compounds from lu xian cao was tested against gram-positive（Staphylococcus aureus and Streptococcus pneumoniae）, gram-negative（Escherichia coli and Pseudomonas aeruginosa） bacteria and the yeast（Candida albicans）. The MIC was estimated by filter paper mensuration and the broth micro-dilution method in 96-well microtiter plates. As a result, chimaphiiin and toluhydroquinone exhibited significant inhibitory effect against S. aureus, S. pneumoniae and E. coli with MIC values in the range of 4～64μg·mL-1 and 8～32μg·mL-1, respectively. 2"-O-galloylhyperin exhibited significant inhibitory effect against C. albicans and the MIC was 32μg·mL-1Simultaneous quantification methods of multi-constituents in lu xian cao were developed and validated in this paper. The selected indices involved three main kinds of constituents in lu xian cao. A LC-MS method was established for the determination of seven major triterpenoids ASGE, APGE, PA, MA, CA, OA and UA. LC separation was performed on Hypersil C18 column using gradient elution of methanol-water as mobile phase. The analytes were ionized by APCI source and determined on SIM mode. All analytes showed good linearity within the test ranges and the recovery rates were 94.5～103.3%（RSD<4.6%）. A HPLC-UV method was developed for the determination of five major flavonoids QGa, QGl, GQGa, QAr and QRh, the separation was carried on Zobax Extend C18 column using acetonitrile-water（14:86, v/v） as mobile phase. The absorbance was monitored at 350 nm. All analytes show good linearity within the test ranges and the recovery rates were 96.3～104.2%（RSD<4.2%）. A HPLC-UV method was developed for the determination of major phonolic compounds toluhydroquinone, isohomoarbutin and homoarbutin. Separation was achieved on Zobax Extend C18 column using gradient elution of methanol-water as mobile phase. The absorbance was monitored at 280 nm. All analytes show good linearity within the test ranges and the recovery rates were 97.2～101.3%（RSD<2.7%）. The established assay methods were simple, accurate and performed well in application to the determination of twenty commercial samples of lu xian cao collected from different regions of China. It can be further used for the quality control of both plant materials and preparations of lu xian cao.Pharmacokinetic study of lu xian cao was accomplished on the constituent toluhydroquinone, isohomoarbutin and homoarbutin, oleanolic acid and ursolic acid. Chimaphilin: The LC separation was carried on Cromasil C18 column using methanol-water（75:25, v/v） as mobile phase after the plasma sample was extracted with diethyl ether. APCI-MS in SIM mode was used to determine[M]-at 186 and 210 for chimaphilin and benzyl（internal standard）. Toluhydroquinone: The GC separation was achieved on HP-5 capillary column. After extracted with diethyl ether-isopropanol（9:1, v/v）, the plasma sample was analyzed by FID with p-nitroacetophenone as internal standard. Isohomoarbutin and homoarbutin: The HPLC separation was achieved on Zobax Extend C18 column using methanol-water（6:94, v/v） as mobile phase after the plasma sample was deproteinized by methanol, acyclovir was used as internal standard and the UV detector was set at 280 nm. Oleanolic acid and ursolic acid: The LC separation was carried on Hypersil C18 column using methanol-water（82:12, v/v） as mobile phase after the plasma sample was extracted with ethyl acetate. APCI-MS in SIM mode was used to determine[M-H]-at 455 for oleanolic acid, ursolic acid and betulinic acid（internal standard）. Validation assays indicated that the established methods were simple and performs well in terms of selectivity, linearity, precision, and accuracy.After oral administration of Lu Xian Cao Decoction, the main pharmacokinetic parameters for aforementioned constituents were as follows: chimaphilin(30 mg·kg-1): Tmax, 1.2 h; MRT, 5.0 h; V, 44.5 L·kg（-1）, AUC0-∞, 3467 ng·h·mL（-1）; isohomoarbutin（20.2 mg·kg（-1））: Tmax, 0.6 h; Cmax, 18.5μg·mL-1; AUC0-∞, 37.7μg·h·mL-1; homoarbutin（20.0 mg·kg（-1））: Tmax, 0.5 h; Cmax, 35.1μg·mL-1; AUC0-∞, 52.7μg·h·mL-1; oleanolic acid（88.4 mg·Kg（-1））: Cmax, 93.5 ng·mL（-1）;AUC0-∞, 215.2 ng·h·mL（-1）; t（1/2）, 1.7 h; ursolic acid（20.8 mg·Kg（-1））: Cmax, 178.9 ng·mL（-1）; AUC0-∞, 1685.9 ng·h·mL（-1）; t1/2,6.9 h; toluhydroquinone was not founded in plasma due to its minor content in lu xian cao. After intravenous administration of toluhydroquinone（20 mg·Kg（-1）） injection, the pharmacokinetic behavior of toluhydroquinone was found to be in line with the two-compartment intravenous model. The t1/2α, t1/2β,Cmax and AUC0-∞ were 1.6 min, 31.6 min, 23.9μg·mL-1,37.7μg·h·mL-1, respectively.Conclusively, under the theory and methodologies of traditional Chinese medicine, utilizing the knowledge of phytochemistry, analytical chemistry, pharmacology, microbiology, pharmacokinetics and chemometrics, the quality control methods for lu xian cao was developed, the pharmacokinetic study of this medicinal herb was also investigated to some extend. The present research provided valuable academic evidences for the future research and development of lu xian cao. |
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