Studies On The Phytochemical Constituents And Pharmacological Activities Of Trollius Ledebouri Reichb And Croton Tiglium L.

Traditional Chinese herbal medicine has a long history of application in our country. It is the product of the wisdom of Chinese people's, gathered in the past thousands of years. Based on this fact, to discover novel lead compounds from Chinese herbs seems to be not only a quite feasible, but also a timesaving way for developing new drugs.In search of phytochemicals with anti-inflammatory, analgesic or antibacterial effects, the extracts of hundreds of medicinal plants had been evaluated on several pharmacological models in our laboratory. Some of the medicinal plants were found biologically active. In my study, Trollius ledebouri Reichb and Croton tiglium L. were chosen for further phytochemical and pharmacological studies. The bioactive constituents of these two plants have been isolated by modern phytochemical methods under the guidance of pharmacological evaluation. HPLC methods for analyzing the bioactive constituents have also been established.1. The chemical constituents and bioactivities of T. ledebouri ReichbT. ledebouri Reichb, belonging to the Trollius genus, was widely used for treating upper respiratory infection, pharyngitis, tonsillitis and bronchitis in Chinese folk medicine. Previous studies exhibited thai this plant is abundant in flavone C-glycosides, which were probably responsible for the pharmacological activities of this plant. Theaim of my study is to isolate the chemical constituents from T. ledebouri, especially the flavone C-glycosides, and evaluate their anti-inflammatory and antibacterial activities using the TPA-induced mice ear edema, C0X2 binding and bacteria inhibiting tests.The flowers of T. ledebouri Reichb were extracted with 60% EtOH. The 60% EtOH extract was distributed with solvents of different polarity into four fractions, namely the petroleum ether fraction (PE), EtOAc fraction, BuOH fraction and H2O fraction. Each of the fractions was further separated by repeated column chromatography, selectively using silica gel, polymide, HP20 resin, Sephadex LH-20, C-18 ODS as absorbent. About forty compounds were obtained, including mostly flavonoids and phenyiethanoid, as well as small amounts of steroids, coumarin glycosides, terpenoids, organic acids and amino acids. The structures of thirty compounds were identified by chemical and spectroscopic methods (IR^ UV, ORD, 1H-NMR, 13C-NMR, DEPT, 1H-1H COSY, HMQC, HMBC, NOESY, EI-MS, ESI-MS, HRMS). Six compounds were found to be new compounds; one is a new natural product; fifteen were isolated from the trollius genus for the first time. The essential oil of the flowers of T. ledebouri Reichb was analyzed by GC-MS. The majority of the volatile constituents were found to be fatty acid derivatives.The new compounds include two acylated flavone C-glycosides: 7-methoxyl- 2"-0-(2"'-methylbutyryl)-orientin and 7-methoxyl-2"-O-(2"'-methylbutyryl-6m-met-hryi)-vitexin; three phenylethanol glycosides: 2-(3-O-P- Z)-glucosyl-4-methoxyl phenyl)ethanol, 2-(3-hydroxy-4-methoxylphenyl)ethanol-/?-.D-glucoside, 2-(3,4-dimethoxylphenyl)ethanoll-/?-￡>-glucoside; one coumarin: 6-O-rha-8-O-glucosyl-coumarin. The compound 2-(4-hydroxyphenyl)ethanoll-/?-Z)-glucoside is a new natural product. Several compounds were isolated from the trollius genus for the first time, including luteolin, quercetin, 6"'-glc-vitexin, 6"'-glc-orientin, 4'-methoxy-7-0-glc(l-6)rha-apigenin, 3',4'-O-dimethylene-orientin, apigenin, 2-(3,4-dihydroxyphenyl)ethanol-/?-D-glucoside, 2- (4-hydroxy-3-O-glucosyl-phenyl) ethanol, phenylethanol-/?-Z)-glucoside, tryprophan,phenylalanine, veratric acid, and 5-O-rha-coumarin, respectively. Some known compounds have also been isolated and identified as orientin, vitexin, 7-methoxyl-2"-O-(2I"-methylbutyryl)-vitexin, 2"-O-(3"\ 4"'-dimethoxyl-benzoyl)-vitexin, 2"-O-(3"', 4"'-dimethoxyl,benzoyl)-orientin, 2"-0-(2'"-methylbutyryl)-vite-xin, 2"-O-(2"'-methy 1 buty r y 1 )o r ientin, 0- sitosterol and sti gmastero 1.In our investigation, T. ledebouri Reichb possessed good anti-inflammatory and moderate anti-bacterial effects. Pharmacological effects of individual fractions have been evaluated to tracing the bioactive principles. The anti-inflammatory activities of the total extract and the fractions have been evaluated on TPA-induced mice ear oedema. The results showed that the 60% EtOH extract and all the fractions could inhibit the TPA-induced ear edema, especially the EtOAc and BuOH fractions, which showed about 60% inhibitory rates. These two fractions were rich in flavonoids and several flavone C-glycosides have been isolated from this tow fractions. Tile anti-inflammatory activities of the flavone C-glycoside were estimated on TPA-induced mice era edema and using a COX2 binding assay based on ultrafiltration and HPLC-DAD-ESIMS analysis. The flavone C-glycosides were proved to be the main anti-inflammatory constituents of T. ledebouri Reichb. Interestingly, two flavone C-glycosides, each with a disaccharide moiety, showed not only remarkable inhibiting effect on TPA-induce mice ear edema, but also strong COX2-binding activity as well. The inhibitory rates on TPA-induce mice ear edema was about 70%, comparatively, the positive control, Leigongtengduogan, showed only 47% inhibition under the same experiment condition. In the COX2-binding assay, the 60% EtOH extract was incubated together with COX2 enzyme, the constituents without binding activity were washed out by, and then the compounds bound to the enzyme were released by changing the pH value of the solution and analysed by HPLC-DAD-ESIMS. The compounds with COX2-binding activities were identified to be flavone C-glycosides, and this result was further proved using purified standard compounds. This is the first report about the anti-inflammatory constituents of T. ledebouri Reichb, and the anti-inflammatory effect of flavoneC-glycoside A HPLC method had been set up for qualitative and quantitative analysis of the main flavone C-glycosides of T. ledebouri Reichb, using a MeOH: H2O gradient (30:70 to 70:30, in 40 minutes) and UV absorbance at 335 nm. The two flavone C-glycosides with disaccharide substituent, compound 11 and compound 12, were set as the aims of quantitative determination. The regress equations for both of the compounds were y = 109.46x + 19.634(r = 0.9958) and y = 217.96x + 19.098(r = 0.9949), respectively, and their detective limits were 4.5ng and 9.Ing. This method was good repeatable, easy to carry out and sensitive. Above results provided good bases for further R & D of T. ledebouri Reichb, which exhibited promising pharmacological characteristics as a anti-inflammatory agent. The anti-bacterial effect of T. ledebouri Reichb has also been evaluated. The previously reported antibacterial effect of the flavon C-glyccsides was not detected in our experiments. But the phenylethanoid glycosides isolated in our study showed somewhat antibacterial effect, especially compound 14, which showed moderate antibacterial activity against Staphylococcus aureus with a MIC of 64 ug/ml, but no effect on other microorganisms tested simultaneously. Synergetic effect has not been found when the extract of T. ledebouri Reichb was used together with classic antibiotics.In the course of elucidating the structures of the flavone C-glycosides, some rules of ionization of this kind of compound under ESI-MS-MS-condition were revealed. The ESI-MS spectrum often give [M— 1] and [M+Cl] signals in negative model and [M+ H], [M + Na] and [M + K] in positive model. The response of negative model is normally better than that of positive model. Further ionization of negative ions by MS-MS showed regularly the ion signals of [M—H—60], [M—H -90] and [M-H-120]. Appling these rules to study the LC-ESI-MS-MS spectrum of the total extract of T. ledebouri Reichb, a possibly new flavone C-glycoside was detected, whose structure seems to be 7-methoxyl- 2"-O-(2"'-methryl )-vitexin.2. The chemical constituents and analgesic effect of C. tiglium L.C. tiglium L. is a plant of the croton genus. According to Flora Reipublicae Popularis Sinicae, C. tiglium L. was used as cathartic, insecticide, or wound healing and antimicrobial agent in Chinese folk medicine. Some diterpenoids have been isolated from the croton genus previously. In the course of screening analgesic Chinese herb, it was found that the extracts of the leaves of C. tiglium L. exhibited remarkable analgesic effect in acetic acid induced writhing and hot plate experiments on mice.In order to isolate and identify the bioactive constituents, dry fresh leaves of C. tiglium L were extracted with 95% EtOH. By solvent extraction, the extract was divided into three fractions: the PE, EtOAc and H2O fractions. Under the guidance of activity evaluation, fifteen compounds, including steroids, terpenoids and alkaloids, were isolated from the EtOAc nd the H2O fractions by column chromatography, using silica gel, HP 20 resin, Sephadex LH-20, and preparative thin layer chromatography (TLC). Their structures were determined by spectral analysis and physico-chemical properties. Three of the fifteen compounds were new compounds, named 2-(furan-2-yl)-5-(2,3,4-trihydroxy-butyl)-1,4-diazine, 2-[(2-methoxypyridin-5-yl)-methyl]-hex-4-3n-3-one and Tiglium C, respectively. The other compounds, p-sitosterol, stigmasterol, octacosanol, 24-ethyl-5, 22-cholest-dien-3