Anti-tumor target prediction and activity verification of Ganoderma lucidum triterpenoids / 中国中药杂志

It has reported that Ganoderma lucidum triterpenoids had anti-tumor activity. However, the anti-tumor target is still unclear. The present study was designed to investigate the anti-tumor activity of G. lucidum triterpenoids on different tumor cells, and predict their potential targets by virtual screening. In this experiment, molecular docking was used to simulate the interactions of 26 triterpenoids isolated from G. lucidum and 11 target proteins by LibDock module of Discovery Studio2016 software, then the anti-tumor targets of triterpenoids were predicted. In addition, the in vitro anti-tumor effects of triterpenoids were evaluated by MTT assay by determining the inhibition of proliferation in 5 tumor cell lines. The docking results showed that the poses were greater than five, and Libdock Scores higher than 100, which can be used to determine whether compounds were activity. Eight triterpenoids might have anti-tumor activity as a result of good docking, five of which had multiple targets. MTT experiments demonstrated that the ganoderic acid Y had a certain inhibitory activity on lung cancer cell H460, with IC₅₀ of 22.4 μmol•L ⁻¹, followed by 7-oxo-ganoderic acid Z2, with IC₅₀ of 43.1 μmol•L ⁻¹. However, the other triterpenoids had no anti-tumor activity in the detected tumor cell lines. Taking together, molecular docking approach established here can be used for preliminary screening of anti-tumor activity of G.lucidum ingredients. Through this screening method, combined with the MTT assay, we can conclude that ganoderic acid Y had antitumor activity, especially anti-lung cancer, and 7-oxo-ganoderic acid Z2 as well as ganoderon B, to a certain extent, had anti-tumor activity. These findings can provide basis for the development of anti-tumor drugs. However, the anti-tumor mechanisms need to be further studied.

Downregulation of lanosterol synthase gene expression by antisense RNA technology in Saccharomyces cerevisiae / 药学学报

The cyclization of 2,3-oxidosqualene is the key branch point of ergosterol and triterpenoid biosynthesis. Downregulation of 2,3-oxidosqualene metabolic flux to ergosterol in Saccharomyces cerevisiae may redirect the metabolic flux toward the triterpenoid synthetic pathway. In our study, primers were designed according to erg7 gene sequence of S. cerevisiae. Three fragments including 5' long fragment, 5' short fragment and erg7 coding region fragment were amplified by PCR. 5' long fragment consists of the promoter and a part of erg7 coding region sequence. 5' short fragment consists of a part of promoter and a part of erg7 coding region sequence. These fragments were inserted reversely into pESC-URA to construct antisense expression plasmids. The recombinant plasmids were transformed into S. cerevisiae INVSc1 and recombinant strains were screened on the nutritional deficient medium SD-URA. The erg7 expression level of recombinant strains, which harbored antisense expression plasmid of erg7 coding region, was similar to that of INVScl by semi-quantitative PCR detection. But erg7 expression level of recombinant strains, which harbored 5' long antisense fragment and 5' short antisense fragment, was significantly lower than that of the control. The results of TLC and HPLC showed that the ergosterol content of recombinant strains, which harbored 5' long antisense fragment, decreased obviously. The ergosterol contents of the others were almost equal to that of INVSc1. Lanosterol synthase gene expression was downregulated by antisense RNA technology in S. cerevisiae, which lays a foundation for reconstructing triterpenoid metabolic pathway in S. cerevisiae by synthetic biology technology.

The basic strategies and research advances in the studies on glycosyltransferases involved in ginsenoside biosynthesis / 药学学报

Traditional herbal medicines, Panax ginseng, Panax quinquefolium and Panax notoginseng, attract our attention for their extensive and powerful pharmacological activities. Ginsenosides are the main active constituents of these medicinal herbs. The related glycosyltransferases involved in ginsenoside biosynthesis are the key enzymes which catalyze the last important step. Modification of ginsenoside aglycones by glycosyltransferases produces the complexity and diversity of ginsenosides, which have more extensive pharmacological activity. At present, ginsenoside aglycones and compound K have been obtained by synthetic biology. As the last step of ginsenoside biosynthesis, glycosylation of ginsenoside aglycones has been studied intensively in recent years. This review summarizes the basic strategies and research advances in studies on glycosyltransferases involved in ginsenoside biosynthesis, which is expected to lay the theoretical foundation for the in-depth research of biosynthetic pathway of ginsenosides and their production by synthetic biology.

Elucidating the structure of two cyclotides of Viola tianshanica maxim by MALDI TOF/TOF MS analysis / 药学学报

The cyclotides are a family of cyclic "mini" proteins that occur in Violaceae, Rubiaceae and Cucurbitaceae plant families and contain a head-to-tail cyclic backbone and a cystine knot arranged by three disulfide bonds. To study the natural cyclotides of V tianshanica, dried herb was extracted with 50% ethanol, and the concentrated aqueous extract was subjected to a solvent-solvent partitioning between water and hexane, ethyl acetate and n-butanol, separately. The n-butanol extract containing cyclotides was subjected to column chromatography over Sephadex LH-20, eluted with 30% methanol. The subfractions were directly reduced by DTT and analyzed by reverse-phase HPLC. The peaks with different retention times were shown on the profile of RP-HPLC and collected. The cyclotides were speculated based on masses range from 3 000 to 3 500 Da. The purified cyclotides were reduced with DTT, alkylated with iodoacetamide, and then were cleaved with endoproteinase Glu-C, endoproteinase Lys-C and Trypsin, separately. The digested peptides were purified on RP-HPLC and analyzed on MALDI TOF/TOF analyzer. A new cyclotide, cycloviolacin T1 and a reported cyclotide varv E were systemically determined using MALDI TOF/TOF system. So the method for the isolation and characterization of cyclotides was quickly built up in succession.