| Objective 7-Demethoxylophorine(DEM), a main effect-toxic component in dried herb Cynanchum komarovii Al. Iljinski, was purified from 70% alcohol extraction. Extraction technology for DEM was optimized. Variation of the pivotal toxic components including DEM and essential oil between crude and vinegar-processing Cynanchum komarovii was analyzed to lay the base for toxicity attenuation mechanism by vinegar-processing. Toxicity attenuation technology was optimized by using DEM content as the index to ensure the safety of the medicine.Methods Total 16.8 kg of Cynanchum komarovii was microwave-assisted extracted by 70% ethyl alcohol and the solvent was evaporated to obtain the extraction. The total alkaloids were enriched on 001 × 7 cation-exchange resin. The aimed compound was isolated from the total alkaloids and the stucutre was elucidated by 1H-NMR, 13C-NMR, and 2D-NMR. HPLC-UV method was established to determine DEM content and extraction technology was optimized by L9(34) orthogonal design using DEM content as index. Difference of DEM between crude and vinegar processing Cynanchum komarovii was studied. Chemical compounds in petroleum ether and dichlormethane extractions were analyzed by GC-MS. Vinegar processing technology was optimized by L16(45) orthogonal design using DEM content as index.Results About 236.5 g of total alkaloids were obtained with the yield 1.41% and 200 mg of the aimed alkaloid was purified with the yield 0.25%. HPLC-UV assay of DEM was estabolished with a good linearity(Y = 93649X-20052, r = 0.9999) in the range from 0.11 to 70.4 μg/m L. LLOQ was 110 nm/m L and LLOD was 33.3 ng/m L with satisfied intra- and inter- precision and repeatality.The extraction technology conditions were optimized as the ratio of material-solvent 1:10, 80% of methanol as extraction solvent, and 30 min to ultrasonic-assisted extract. The content of DEM was determined to be(348.27 ± 2.8) μg/g(RSD=0.8%,n=3) under the above optimized technology which is higher than the obtained original values.DEM content in vinegar-processing Cynanchum komarovii by yellow and white vinegar decreased 0.23 fold and 0.18 fold(P<0.01,n=6) compared with crude Cynanchum komarovii. The former decreased more than the latter(P<0.01,n=6).GC-MS analysis results showed that the essential oil components in crude and vinegar processing Cynanchum komarovii are basically the same, but the peak area decreased significantly after vinegar-processing. The Cynanchum komarovii processed by white vinegar yield a lower area.Vinegar processing technology results showed that the concentration of vinegar influenced the DEM content signiciantly(P<0.05). The technology was optimized as A1B4C1D1, namely 30% of vinegar solution, soaking time 24 h, the temperature 60℃, and fried time 40 min.Conclusion The total alkaloids content is very low in herb of Cynanchum komarovii. Content of DEM is only about 0.034% with yield lower than 0.3%. So it is very difficult to enrich it. DEM content in vinegar processed Cynanchum komarovii decreased deeply compared with crude materials. Meanwhile, essential oil scattered when heated. This is the pivotal mechanism for toxicity attenuation after vinegar processing. The technologies for extraction and processing are very simple and reliable, which can lay the foundation for its effect and clinical safty. |
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