| Objective:To separate, screen and identify antitumor active endophytic fungus from the tissue of Paris polyphylla Smith var.chinensis Hara, and to primariy reveal its antitumor components as well as their mechanism, so as to provid theoretical and experimental basis for the endophytic fungus resources exploitation of Paris polyphylla Smith.Methods:(1) Streak plate method was carried out to separate and purify the endophytic fungus of Paris polyphylla Smith. ITS DNA sequencing method was adopted for fungus identification, and the genetic relationship between the endophytic funguses was analyzed by MGEA4.0software.(2) MTT method was conducted to screen antitumor active endophytic fungus, and the strongest candidate was selected to further analysis.(3) Extraction, thin layer chromatography (TLC), silic gel chromatography, gel column chromatography and high performance chromotagraphy (HPLC) methods were carried out to extract, separate, and purify the antitumor active components. The UV-VIS, MS and NMR spectrum methods were carried out to identify the active compounds.(4) In vitro antitumor assay, endothelial cell growth inhibiton assay, cell scratch wound healing assay, Boyden chamber assay, flow cytometry assay and microscopic assay were applied to evaluate the antitumor activity and to recover its probable mechanism of the active compounds and active fractions.Results:(1) The endophytic biodiversity in the stem tuber of Paris polyphylla Smith was analyzed, a total24strains were isolated, and assigned to8taxa:Fusarium, Alternaria, Aspergillus, Schizophyllum commune, Polyporus, Mucor, Trichoderma and Colletotrichum. Of which, Fusarium (41.7%) was the most prevalent genus, followed by Alternaria (16.7%). (2) MTT assay indicated3endophytic strains with antitumor activity; the one with the strongest activity strain was selected and named as RPF8. MTT results showed that the inhibition ratio against HepG2cell reached82.45±0.46%treated with30μL fermentation of RPF8; comparing with other strains we primary presumed the strain RPF8with great antitumor activity. DNA sequencing revealed RPF8shared high homology (96%) with Aspergillus fumigatus; however, significant differences were observed on morphologically, accordingly we assigned RPF8into unknown strain temporary.(3) MTT guided fractionation indicated that the acetoacetate extract and chloroform extract with antitumor activity, and these two parts were combined and separated by different kinds of column. Two compouds and one strong active sub-fraction were abtained, and the pure compounds were identified as ergosterin (M1) and ergosterin peroxide (M2) by UV-VIS, MS and NMR spectrum.(4) In vitro antitumor and endothelial cell inhibition assay indicated that the active sub-fraction manifested strong antitumor activity against HepG2cell lines and great cell growth inhibition activity against HUVEC cell lines. However, two pure compound M1and M2showed no significant antitumor activity and HUVEC cell growth inhibiton activity; but remarkable endothelial cell migration inhibition activity was observed by cell scratch wound healing assay and Boyden chamber assay. Additionally, flow cytometry assay indicated the active fraction and sub-fraction with strong apoptosis inducing activity against HepG2cells, which was reconfirmed by fluorescence microscopic assay. And we presumed that the extraction of RPF8could educe antitumor activity by direct killing tumor cell and by inhibiting angiogenesis ways.Conclusion:There are multiple endophytic funguses in Paris polyphylla Smith which showed high microbial diversity. Several antitumor active funguses were screened, and one strain RPF8showed significant antitumor activity. The active fraction and sub-fraction were located, and two pure compounds were identified. The active fractions showed induce cell apoptosis activity, and both the active fractions and pure compounds showed potential angiogenesis inhibiton activity. This research paved the way for further research of the endophytic fungus of Paris polyphylla Smith. |
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