| Cancer continues to represent the largest cause of mortality in the world and claims over 6 million lives each year. Chemotherapeutics is still playing an important role in the treatment for malignant tumor. Although, there were majorities of anti-tumor drugs in clinical, the toxicity of this drugs can not be ignored. Thus search of new compound with high therapeutic effect and low side effect is concerned by most researchers. The screenig for new anti-tumor drugs from bioactve component of folklore medicine is a hot pot in this area.In this study, a single polysaccharide were isolated from corm of saffron (Crocus sativus L.) by homogenized in 50 mM Tris-HCl buffer, pH 8.0, containing 1 mM phenylmethyl sulfonylfluoride (PhMeSO2F) and 5 mM 1,4-dithio-DL-threitol (DTT) and purified by ion exchange chromatography with Macro-Prep DEAE column (16mmx300 mm) and reverse-phase high performance liquid chromatography with Supelcosil LC-304 C-4 column (4.6x250 mm). The polysaccharide was consisted of 94.88 % polysaccharide and 5.12 % protein, analyzed with Lowry method and phenol-sulfuric acid method. This result showed PG was belong to proteoglycan. The result of PMP labeled monosaccharide analysis and m-hydroxydiphenyl method analysis of uronic proteoglycan (PG) showed the polysaccharide part of PG was composed of rhamnose (28.03 %), galactose (13.14 %), fucose (8.20 %), xylose (19.31 %), mannose (23.40 %) and uronic acid (7.92 %). while the protein of PG is mainly consist of Asp, Glu, Gly, Ala and Leu (account to more than 50 % ) analyzed with auto amino acid analyzer. FTIR and 13C NMR spectra showed main functional group of polysacharide and protein in PG, and the main skeletons was acertained to beβ-(1,4)-D linkaged mannose accompanied with branched chain ofβ-(1,6)-D mannose and other monosaccharide, such as rhamnose.The antitumor effect of PG were studied both in vitro and in vivo. PG has servere cytotoxic effect on 5 human tumor cell lines (Hela, A549, MCF7, SGC-7901 and SMMC-7721 cell) and one mouse cell line (S180 cell) in vitro, with IC50 ranged form 10 to 40μg/ml. Further study showed the mechanism of PG induced cytotoxicity was: PG inhibit prduction of intracellular reactive oxygen species (ROS) S180 cell, which resulted in cell cycle arrest in G0/G1 phase, and followed by destory of cell membran intergrity, and cell apoptosis. The transplanted S180 tumor mice model was established, The transplanted S180 tumor mice model was established, and the tumor growth inhibition effect in vivo was detected through 3 different PG administration way (intraperitoneally, intravenously and intratumorally injection). All the results showed the tumor growth inhibition in vivo. According to the inhibition effect on mouse growth, the intravenously administration of PG at dose of 0.4 mg kg-1 day-1 were recommanded for further study.Callus Induction System in corm of saffron was Established. And the study showed the most effective culture condition was: use epidermis from corm of saffron around buds as exoplant and cultured in MS meida contain with 5.0 mg/L 6-BA, 8.0 mg/L NAA and 30 g/L surcrose at 22℃, in dark. Analyzed subculture conditon use callus derived from the media mentioned as above, and find calluse were accumulate PG most in the following conditon: MS+2.5 mg/L 2,4-D+0.5 mg/L 6-BA+30 g/L surcose, cultured in a sterile room with controlled ligeht illumination of 3000 lux, 12h/12h, 22±2℃. In vitro cytotoxicity test domenstrated the cytotoxicity of of PG isolated from callus and its similarity with corm of saffron. |
