Extraction & Isolation, Synthesis And Antitumor Activities Study Of Alkaloids In Tylophora Ovata And Analogues

Tylophora ovata (Lindl.) Hook.ex Steud., a herb of perennial creeper vine of Ascle-piadaceae family, Tylophora genus, has been a Chinese traditional toxic herbal medicine. It was frequently used as folk medicine in the south and south west of China. The tylophorine that widely existed in the plant like Tylophora ovata (Lindl.) Hook.ex Steud. and Cynanch-um komarrovii A1. lljinski, etc., belonged to phenanthro-indolizidine type alkloids, which were a series of compounds with remarkable antitumor activity by strongly inhibiting the growth of tumour cells. In this paper, the technology of extracting total alkaloid from Tylo-phora ovata (Lindl.) Hook.ex Steud., and the synthesis and structural modification of the antitumor active tylophorine and its analogs were investigated. The preliminary research of antitumor activity of these compounds by mean of cytology were also carried out.1. The solvent extraction technology, CO₂ supercritical fluid extraction technology and microwave-assist extraction technology of total alkaloids in Tylophora ovata (Lindl.) Hook.ex Steud. were optimized by orthogonal experiments respectively. The optimal expe-rimental parameters in solvent extraction technology were obtained as 90% ethanol, extrac-tion temperature 80℃, solid/liquid ratio 1:25, extraction time 2h (twice). The extraction yi-eld of total alkaloids from Tylophora ovata (Lindl.) Hook.ex Steud. reached 0.388%. The optimal experimental parameters in CO₂ supercritical fluid extraction technology were obtained as extraction temperature 50℃, extraction pressure 25 MPa, extraction time 90 min, entrainer (95% ethanol) dosage 2.5mL/g. The extraction yield of total alkaloids from Tylophora ovata (Lindl.) Hook.ex Steud. was 0.25%. The optimal experimental paramet-ers in microwave-assist extraction technology were obtained as 90% ethanol, extraction temperature 70℃, solid/liquid ratio 1:20, extraction time 15min. The extraction yield of to-tal alkaloids from Tylophora ovata (Lindl.) Hook.ex Steud. reached 0.438%. In comparison, the relatively high extraction rate was found in microwave-assist extraction technology, sol-vent extraction technology the second, and CO₂ supercritical fluid extraction technology the last.2. By using available raw materials as veratraldehyde, piperonal aldehyde,3,4-dime-thoxyphenylacetic acid and (S)-L-proline, and making the classical Friedel-Crafts reaction as the key step, the S-(+)-tylophorine and its analogs which containing methylenedioxy, symmetric and asymmetric phenanthrene were designed and synthesized as follows: α,β-diarylacrylic acid (acrylonitrile) was first synthesized and was ring-closed with PIDA to form multi-substituent 9-carboxyl phenanthrene. After decarboxylation and Friedel-Crafts reaction with (S)-N-(Trifluoroacetyl)-L-prolyl Chloride, the multi-substituent 9-L-prolyl phenanthrene was obtained. The intermediate derived was reacted with triethylsi-lane in the presence of boron trifluoride etherate to reduce the carbonyl group, and follow-ed by the detrifluoroacetylation. Finally, the target compounds were obtained by ring-closing reaction with formaldehyde in the presence of hydrochloric acid. By selecting the factors such as reaction temperature, reaction time, ratio of reactants, catalyst and dosage etc., the experiment conditions of synthetic process in each of key steps were optimi-zed. The influences on the reaction yield by the oxidative coupling reaction conditions and decarboxylation were also studied. By using PIDA to replace VOF₃,VOC1₃ and ultraviolet ray in the coupled ring-closing step, there were many advantages such as easy preparation of reagents, mild synthetic conditions, good operability, and improved yield. The target compounds and some intermediates were characterized by IR,¹H NMR,MS and X-ray single crystal diffraction.3. The preliminary study of the in vitro antitumour effect of S-(+)-tylophorine, the analogs and the total alkaloids (TOHa) were carried out and the in vitro antitumour mecha-nism in these compounds were investigated from multiple point-of-view. The inhibiting effects of TOHa, S-(+)-tylophorine and the analogs against five tumor cell lines, A549, HeLa, MGC-803, SGC-7091 and SMMC-7721, were determined by MTT assay.The influences of compound 2,3,6,7-Bis(methylenedioxy)-phenanthrene-9-[9,10-b]-indolizidine (BMPI) on the growth of normal cells was observeded with SPL. The inhibiting effect of compound BMPI on the proliferation of A549 cell line was determined by colony formation assay. The influences of compound BMPI on the morphology and ultrastructure of A549 cell line were observeded by inverted microscope, electron microscope and Gimesa staining method. The effects of compound BMPI on the apoptosis of A549 cell line was determined by AnnexinV FITC/PI assay. The influences of compound BMPI on the cycle distribution of A549 cell line were determined through flow cytometry. The results showed that S-(+)-tylophorine and its analogs had remarkable in vitro inhibiting effects on the proliferation of human tumour cell lines, and there were dose-effect relationships. While all the S-(+)-tylophorine and its analogs showed strong antitumor activities, BMPI was even better, which could be used as precursor of further research. By probing into the mechanism of induced apoptosis on tumour cell lines with compound BMPI and TOHa, the rationale of developing S-(+)-tylophorine analogues antitumour drugs may be provided.