Pharmacology Investigation Of Rhizoma Sparaganii (Sparganium Stoloniferum Buch.-Ham) And Analysis Of Its Related Active Components

Rhizoma Sparganii (R.Spa.), a traditional Chinese medicine used on treatment of blood stasis, amenorrhea, functional dyspepsia and early stage tumors especially for hysteromyoma, is dried rhizome of Sparganium stoloniferum (San Leng). Dozens of components had been isolated from R. Spa. but the total yield was in trace (dry weight<0.009‰). For all the reported components isolated from R. Spa. were insoluble in water and could not be obtained by conventional R. Spa. preparation method. Thus the reported components have little effect on R.Spa. bioactivity. In present, pharmacology studies on R.Spa. and analysis of its components are both insufficiency. Therefore, this investigation targeted on endocrine/ angiogenesis functions that are common pathological mechanism of all R.Spa. indications, and studied reproductive toxicity of intaking 100,200,400 mg/kg/d R.Spa. during a 8.5-18.5dpc sensitive period of ICR mice teratogenesis. As a result, groups intaking 400 mg/kg/d R.Spa. extract had severely shorten pregnant times (0.5～1 dpc), and reduced mean offspring number, mean offspring birth weight as well as mean offspring weaning weight (21dpp) of the treated mice. A sex rate abnormal inside offspring has also been observed in 400 mg/kg/d group, and 2.9% offspring grow tumor on umbilical cord place meanwhile 5.1% offspring showed phalanx dysplasia in contrast to control group. Besides,13.3% female parents of 400 mg/kg/d group died with uterine glands hyperplasia inside 45d after the offspring weaning time, which is significantly higher than the control groups ones (0%).13.5 dpc embryos from 400 mg/kg/d group showed significantly depressed average FGF-1 (P<0.01) and VEGF levels (P<0.05), but boosted average ER (estrogen receptor) level as compared to controls. Dysplasia in the heart (12.9%), craniofacial region (18.3%) and vertebrae (32.5%) presented in 400 mg/kg/d group embryos, also, the ER-a expression was inversely proportional to FGF-1 levels in the same embryo (P<0.01). These results implicate that R.Spa. extract do effect notable toxicity during a special physiological period in vivo. The R.Spa. active compounds presented as novel natural plant estrogen antagonist, and, the toxicity mechanism of R.Spa. is anti-estrogen induced FGF signing abnormal, depressing VEGF and FGF expression induced anti-angiogenesis, crosstalked with mediating ER induced anti-estrogen function.For angiogenesis and endocrine regulation are all necessary for tumor development. The nutrition providing banded by anti-angiogensis and cell cycle bogged down by anti-estrogen all can severely depress the cancer cell growth. So, in a further study, R.Spa.-W-hydro was investigated on four different estrogen receptor-positive (ER+) cell lines with various concentrations. The results confirmed that, over 188μg/mL R. Spa.-W-hydro treatments showed clear cytotoxicity on all the four HeLa, MCF-7, A549, HepG-2 cells. In contrast to the 1% methanol medium treated cell control, R. Spa.-W-hydro treatments could severely depress the four ER+ cell lines activity inside 48h (P<0.01), produce cell morphology modification, remove contact vegetative state and induce MCF-7, A549 microfilament microtubule abnormal, moreover, it could also induce distinct HeLa cell apoptosis (P<0.01). 750μg/mL group 36h treatment had severely induced A549, MCF-7 cell S/G2 cell cycle arrest, HepG-2 G1 cell cycle arrest as well as HeLa cell M stage depression. We further characterized the active compounds of different prepared R.Spa. which had exactly cytotoxicity on HeLa cells by HPLC-fingerprint. R.Spa. were extracted respectively by water (R. Spa.-W-hydro), 75% methanol (R. Spa.-M75),100%mathanol (R. Spa.-M100), ethyl acetate (R. Spa.-W-lipo) and cultured with HeLa cells under a same Sparganium stoloniferum herb concentration. MTT and cell cytoskeleton observation results showed that, except the R. Spa.-W-lipo extracts, another three R. Spa.-W-hydro, R. Spa.-Me75, R. Spa.-Me100 extracts all led to obviously cell active depression, cell contact failure, cell microfilament reduction as well as cell morphology abnormal cytotoxicity of HeLa cells. The cytotoxicity strength of different R.Spa. extract were R. Spa.-W-hydro> R. Spa.-M75>R. Spa.-M100 on HeLa cells in this study. HPLC-fingerprint of R. Spa.-W-hydro and R. Spa.-M100 presented 18 peaks in all, 10 in common and 7 of them response for the anti-cancer bioactivity.A new A1 complex alkaloid (compound I), one nitrogen-containing carbohydrates (VI) and seven degradation products of compoundⅠ(Ⅲ,Ⅳ,Ⅴ,Ⅶ,Ⅷ,Ⅸ,Ⅹ) have been isolated from R. Spa.-W-hydro by normal phase column chromatography method. The compoundⅠis a new alkaloid-glucosides, meanwhile, it is also the first alkaloid that has been isolated from R.Spa., yield of 5.99%(dry weight/dry rhizome weight), account for 61.4% dry weight of R. Spa.-W-hydro extract. Further HPLC results have proved that, the compoundⅠhad a same peak time with the number 8 main peak (area rate 29.32%) of R. Spa.-W-hydro HPLC-fingerprint results. These results provide a necessary data for further pharmacology research of R.Spa. and improvment of the herb quality control method. In addition, the compoundⅠstructure identification has also proved an abundant A1 complex organic compound existence in higher plants, which has important academic value.