| The chinese crude drug 'Tong-guang-teng' is the stems of Marsdenia tenacissima(Roxb.) Wight et Arn.(Asclepiadaceae). Xiaoaiping preparation of Marsdenia tenacissima has been used for the treatment of gastric cancer, hepatoma,lung cancer and others late malignant tumor. In this research we determined that C21 steroidal fraction of Marsdenia tenacissima had evident effect on proliferation of SGC-7901 human gastric cancinoma cell line by targeting separation method of activity indicatrixt. On the basic of the effect, we adopted modern separating and characterization to study and research chemical constituents of C21 steroidal fraction Marsdenia tenacissima; reference substance of C21 steroidal fraction; we optimized preparation method of C21 steroidal fraction by horizontal experiment and orthogonal experiment and established determination method of C21 steroidal fraction by modern chromatographic technique.1. Effects of extract and C21 steroidal fraction of Marsdenia tenacissima on proliferation of human tumor cell: we adopted MTT method to detect the effects of extract and C21 steroidal fraction of Marsdenia tenacissima on proliferation of SGC-7901 human gastric cancinoma cell line and A375 human melanocytoma, the results indicated: C21 steroidal fraction of Marsdenia tenacissima had evident effect on proliferation of SGC-7901 human gastric cancinoma cell line, better to the effect of extract of Marsdenia tenacissima.2. The research of chemical constituent of C21 steroidal fraction of Marsdenia tenacissima: we separated compounds from C21 steroidal fraction of Marsdenia tenacissima by ODS column chromatography and HPLC separation technique and elucidated their structures by the data of UV,ESI-MS,1H-NMR,13C-NMR,DEPT,HMBC, six compounds were isolated and accredited from C21 steroidal fraction of Marsdenia tenacissima, 12β-O-2-methylbutyryl-tenacigenin A was a natural compound that isolated from Marsdenia tenacissima for the first time. We took tenacissoside H and 11α-O-tigloyl-12β-O-acetyl-tenacigenin B as reference substance in order to establish material foundation of preparation method and quality specification of C21 steroidal fraction from Marsdenia tenacissima.3. The research of determination method of C21 steroidal fraction from Marsdenia tenacissima: we determined content of C21 steroid taking high-content tenacissosides H as reference substance with colorimetric method. We considerated temperature of coloration,time of coloration,proportion of developer and decided the optimal conditions as followed: temperature of coloration is 60℃,time of coloration is 60 minutes',developer is H2SO4- methanol(6: 1). The concentration of tenacissoside H in the range of 9.59-47.95μg/mL submitted good linear correlation with absorbability(r=0.9999). The average recovery was 100.01％(RSD=2.32％, n=6). Determination results of C21 steroids in Marsdenia tenacissima from different regions indicated, the discrepancy of C21 steroidal content in Marsdenia tenacissima was obviously, C21 steroidal content was highest in Linchang of Yunnan province, and C21 steroidal content was lowest in Zhaotong of Yunnan province.4. The research of preparation method of C21 steroidal fraction from Marsdenia tenacissima: we optimized preparation method of C21 steroidal fraction by horizontal experiment and orthogonal experiment; the results of the optimal conditions were as followed: 90％ethanol, extracting for 3 times, ratio of solvent volumn and the stems weight was 6: 1, and the extracting time was 90 minutes. We enriched C21 steroid from Marsdenia tenacissima with macroporous adsorptive resins method, the results of the optimal conditions were as followed: the type of was D101, the concentration of sample was 1g/mL, the flow velocity of sample was 1BV/h, the eluents were 10％ethanol,30％ethanol and ethanol, the quantity of ethanolis 6BV, collecting ethanol elutriant.5. The research of determination method of C21 steroidal fraction from Marsdenia tenacissima: we determined content of C21 steroids in C21 steroidal fraction from Marsdenia tenacissima by colorimetric method. The average content of six different samples was 0.8568-0.9371g/g, and determined content of 11α-O-tigloyl-12β-O-acetyl-tenacigenin B in C21 steroidal fraction from Marsdenia tenacissima by HPLC, the sample size of 11α-O-tigloyl-12β-O-acetyl-tenacigenin B in the range of 1.016-5.080μg submitted good linear correlation with score of peak area(r=0.9999). The average recovery was 100.16％(RSD=1.97％, n=6), the average content of six different samples was 45.09-53.62mg/g.This paper studied and researched systematically effect of C21 steroidal fraction of Marsdenia tenacissima on proliferation of SGC-7901 human gastric cancinoma cell line,chemical constituents,preparation method and quality control specification, in order to establish foundation of exploitation of innovative drugs of chinese medicine of the five category registration with independence intellectual property rights for treatment gastric cancer, the results has important academic value and application prospect. |
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