Studies On The Chemical Constituents And Bioactivity Of Chirita Eburnea Hance Of Gesneriaceae

Niu′erduo, Latin name: Chirita eburnea Hance is belong to Gesneriaceae family. As a folk herb, Niu′erduo is also called shisanqi and shihu′er etc. It distributes mainly in Guangxi, Guangdong, Hunan, Hubei, Sichuan and Guizhou Province. The rhizome or whole plant of C. eburnea was used as folk medicine for clearing lung; hemostasis; relieving cough; e1iminating dampness; detoxifying; treating pulmonary tuberculosis and so on. In the folk of Guangxi province, people often used the C. eburnea to treat diseases such as pulmonary tuberculosis; hypertension, etc. Due to lack of chemical studying of C. eburnea, till now, it only limited to use as a folk medicine, the medical value of C. eburnea is not be developed and utilized enough. Here, we study on the detail chemical constituents and its cytotoxic activity of this plant. It provides some chemical theoretic evidences for its further utilization, it also provides a quality control method for raw material and extract of C. eburnea. The main research and achivement are as follow:1. Non-polar liposoluble components of C. eburneaTo analyze liposoluble components of C. eburnea by GC-MS for the first time. The components were identified by analysis of MS spectra and indexing of NIST05a.L spectral database. Their relative contents were quantified by peak area normalization, 30 components were separated and 24 components were identified, which comprising 87.38% of the total liposoluble constituents. The major liposoluble constituents of C. eburnea are Linoleic acid ethyl ester(10.86%), Ethyl Oleate(9.46%), Tetradecanoic acid, hydroxy ethyl(9.15%), beta-Sitosterol (6.99%) and n-Hexadecanoic acid(4.89%).2.Polar chemical constituents of C. eburneaIn our detail phytochemical research of C. eburnea, solvent partition, column chromatography and HPLC preparation were used to isolated method, eleven compounds have been isolated and nine of them have been identified by spectrum and chemical method, such as MS, 1H-NMR, 13C-NMR and so on. They are:β-stiosterol(CE-1); 1-Hydroxy-2-methoxy -7-methylanthraquinone(Peganone 2)(CE-2); 1, 7-Dihydroxy-6-methoxy-2-methylanthraquinone (Robustaquinone D)(CE-5); 5, 7, 4′-trihydroxyflavone (Apigenin)(CE-6); 3, 4-dihydroxyphenyl alcohol-3-O-cafeoyl-β-D-glucopyranoside(PlantainosideΑ)(CE-7); 3, 4-dihydroxyphenyl alcohol-6-O-glucopyranosylcafeoyl-β-D-glucopyranoside(Chiritoside C)(CE-8); 3, 4-dihydro- xyphenyl alcohol-2-O-cafeoyl-β-D-glucopyranoside(Plantaninoside B)(CE-9); 3, 4-dihydroxy- phenyl alcohol-β-D-glucopyranosyl-(1→3)-4-O-cafeoyl-β-D-glucopyranoside(Plantamajoside) (CE-10) and 3, 4-dihydroxyphenyl alcohol-4-O-cafeoyl-β-D-glucopyranoside(Desrhamnosylver- bascoside)(CE-11). They including five phenylethanoids: CE-7-11; two anthraquinones: CE-2; CE-5; one sterol: CE-1. Compound CE-5 and CE-10 were isolated for the first time from the family Gesneriaceae, and compounds CE-1, CE-2, CE-6, CE-7, CE-8, CE-9 and CE-11 were isolated for the first time from C. eburnea.3.To establish a method of preparation five phenylethanoid glycoside components by Semi-Preparative HPLCA method was established for the isolation and preparation of five phenylethanoid glycoside components from C. eburnea using MCI column chromatography and semi- preparative HPLC. The HPLC preparation condition: A semi-preparative Eclipse XDB-C18 (9.4×250 mm, 5μm) was used as a column, by gradient elution with methanol-water as the mobile phase at a flow rate of 4 ml/min, five phenylethanoid glycoside components were obtained from C. eburnean , and on the basis of spectral data(EI-MS, 1H-NMR, 13C- NMR), these compounds were identified as Plantainoside A(CE-7), Chiritoside C(CE-8), Plantaninoside B(CE-9), Plantamajoside(CE-10) and Desrhamnosylverbascoside (CE-11), the purities of these five were all beyond of 98% which analyzed by HPLC. The method is efficient, simple and high resolution for five phenylethanoid glycosides at one time.4.To establish a quantitative method of simultaneous determination of four phenylethanoid glycoside components in C. eburnea by HPLC.Samples were ultrasonic extracted with 50% methanol. The chromatographic analysis was carried out on a ZORBAX SB-C18 column (250 mm×4.6 mm,5μm), The mobile phase was acetonitrile and water (0.05% acetic acid) with gradient elution at the flow rate of 1 ml/min, the detection wavelength was set at 330 nm and the column temperature was controlled at 30℃. The sample injection volume was 10μl. The result indicated that Plantainoside A, Chiritoside C, Plantaninoside B and Desrhamnosylverbascoside had linear relationship at the range of 1.0~8μg; 0.4~3.2μg; 0.4~3.2μg and 0.4~3.2μg respectively. All compounds were stable within 72 h. This simple, accurate, stable and reproducible method is suitable for quality control and determination of raw materials and extract of C. eburnea.5.Cytotoxic activity of eight pure compounds from C. eburneaEight compounds were tested for cytotoxic activity in vitro by used a MTT method.The result indicated that compound CE-4 showed cytotoxic activity against human lung cancer cell lines NCI -H460 with IC50 of 34.2μg/ml.Compound CE-5 showed moderate cytotoxic activity against human lung carcinoma cell lines SPC-A1 with IC50 of 50.1μg/ml. Compounds CE-9 and CE-10 showed weak cytotoxic activities against human lung carcinoma cell lines SPC-A1 with IC50 of 88.2μg/ml and 97.3μg/ml respectively. Compounds CE-2, CE-7, CE-8 and CE-11 hadn′t any growth inhibition to four tumor cell lines. It indicates that compounds CE-4, CE-5, CE-9 and CE-10 have anti-tumor activities in vitro.