ABSTRACT
In the research and development of new drugs, it is very important to investigate the in vitro metabolism of candidate drugs. Traditional models such as liver microsomes have many limitations, while the in vitro model of recombinant human drug metabolizing enzymes is considered as an important and useful approach because of its convenient access, stable activity and low cost. In this study, six major human UDP-glucuronosyltransferases (UGTs) genes (UGT1A1, 1A3, 1A4, 1A6, 1A9 and 2B7) were cloned from human liver cDNA and heterologously expressed in Saccharomyces cerevisiae and baculovirus-infected insect cell. UGT1A1, 1A3, 1A6 and 1A9 were successfully expressed in yeast and showed glucuronidation activity against a variety of different structural types of substrates, but their activities were low. All six UGTs were successfully expressed and exhibited significantly improved glucuronidation activity when Trichopolusia ni cells BTI-TN5B1-4 (High Five) were used as the host. The recombinant human UGTs expressed in insect cells can catalyze the glucuronidation of their specific substrates, and the glucuronidation products were synthesized at milligram-scale with yields of 13%-66% for the first time, of which the structures were identified via MS, 1H NMR, and 13C NMR spectroscopic analysis. Above all, the recombinant human UGTs yeast and insect cell expression systems constructed in this study can be used for in vitro metabolism evaluation in the early stage of new drugs research and development, and also provide a new tool for the synthesis of glucuronide metabolites.
ABSTRACT
The glycosides of 4'-demethylepipodophyllotoxin (DMEP) possess various pharmacological activities; however, the chemical synthesis of these glycosides faces challenges in regioselectivity, stereoselectivity, and the protection and de-protection of functional groups. In this work, a novel glycosyltransferase (GT) gene AbGT5 from Aloe barbadensis was successfully cloned, heterogeneously expressed and purified. Recombinant AbGT5 was able to catalyze the glycosylation of DMEP and the glycosylated product, which was separated from the preparative scale reaction, was characterized as DMEP 4'-O-β-D-glucoside via MS, 1H-NMR, 13C-NMR, HSQC and HMBC. According to the investigations of enzyme properties, AbGT5 show the highest activity around 20 ℃ in the buffer of pH 9.0, and it was independent of divalent metal ions. Under the optimum conditions, the conversion rate of DMEP can reach 80%. Above all, in this work the enzymatic glycosylation of DMEP was achieved with high efficiency by the novel GT AbGT5.
ABSTRACT
Secoisolariciresinol dehydrogenase (SDH) is a key enzyme involved in the biosynthetic pathway of podophyllotoxin.In this study, two SDH candidate genes,SO282 and SO1223, were cloned from callus of Dysosma versipellis by homology-based PCR and rapid amplification of cDNA end (RACE).The SDH candidate genes were expressed in Escherichia coli and the subsequent enzyme assay in vitro showed that recombinant SO282 had the SDH activity. These results pave the way to the follow-up investigation of the biosynthetic of podophyllotoxin.
ABSTRACT
Various chromatographic techniques, including silica gel column chromatography, Sephadex LH-20, preparative thin-layer chromatography, and preparative HPLC, were employed to isolate the chemical constituents from callus cultures of Dysosma versipellis. Structures of the compounds were elucidated based on UV, IR, MS and NMR spectroscopic data analysis. Totally, seven flavonoid glycosides were isolated from the 95% ethanol extract of the callus cultures and identified as kaempferol-3-O-[6″-(3″'-methoxy)-malonyl]-β-D-glucopyranoside(1), kaempferol-3-O-(6″-O-acetyl)-β-D-glucopyranoside(2), kaempferide-3-O-β-D-glucopyranoside(3), kaempferol-3-O-β-D-glucopyranoside(4), isoquercitrin(5), quercetin-4'-O-β-D-glucopyranoside(6) and kaempferol-3-(6″-malonyl)-β-D-glucopyranoside(7), respectively.All these compounds were isolated from callus cultures of D. versipellis for the first time.Compounds 1, 2, 3, 6 and 7 were firstly obtained from plant materials of D. versipellis, and compound 1 was a new compound.
ABSTRACT
To investigate the secondary metabolites of endophytic fungi Pericinia sp. F-31. Column chromatography on silica gel, Sephadex LH-20 and semi-preparative HPLC were used to separate and purify the compounds. Two compounds were isolated from the fermentation broth of Periconia sp. Their structures were identified as 5-(1-hydroxyhexyl) -6-methyl-2H-pyran-2-one (1) and 2-(3-hydroxy-4-methylphenyl) -propanoic acid (2). Compound 1 was a new lactone compound, compound 2 was new natural product, and the NMR data of compound 2 was reported for the first time.
Subject(s)
Annona , Microbiology , Ascomycota , Chemistry , Genetics , Metabolism , Drugs, Chinese Herbal , Chemistry , Metabolism , Endophytes , Chemistry , Genetics , Metabolism , Lactones , Chemistry , Metabolism , Mass Spectrometry , Molecular StructureABSTRACT
Seven meroterpenoids and five small-molecular precursors were isolated from Penicillium sp., an endophytic fungus from Dysosma versipellis. The structures of new compounds, 11beta-acetoxyisoaustinone (1) and isoberkedienolactone (2) were elucidated based on analysis of the spectral data, and the absolute configuration of 2 was established by TDDFT ECD calculation with satisfactory match to its experimental ECD data. Meroterpenoids originated tetraketide and pentaketide precursors, resepectively, were found to be simultaneously produced in specific fungus of Penicillium species. These compounds showed weak cytotoxicity in vitro against HCT-116, HepG2, BGC-823, NCI-H1650, and A2780 cell lines with IC 50 > 10 micromol x L(-1).
Subject(s)
Humans , Berberidaceae , Microbiology , Cell Line , Cell Line, Tumor , Lactones , Pharmacology , Monoterpenes , Pharmacology , Penicillium , ChemistryABSTRACT
Naringenin (1) was transformed to three metabolites (2-4) by Mucor sp. Based on LCMS(n)-IT-TOF and NMR spectroscopic data, 2-4 were identified as naringenin-7-O-sulphate, naringenin-4'-O-sulphate, and naringenin-5-O-sulphate, respectively. These results might provide hints to the mammalian/human metabolism of naringenin.
Subject(s)
Biotransformation , Drugs, Chinese Herbal , Chemistry , Metabolism , Flavanones , Chemistry , Metabolism , Magnetic Resonance Spectroscopy , Molecular Structure , Mucor , Metabolism , Sulfates , MetabolismABSTRACT
Syringin, chlorogenic acid and 1,5-dicaffeoylquinic acid are three main bioactive ingredients in herbs of Saussurea involucrata with various pharmacological properties, while their contents are very low. In this study, the biosynthesis of syringin, chlorogenic acid and 1,5-dicaffeoylquinic acid in the cell suspension cultures of S. involucrata were regulated by feeding carbon sources and precursors, which resulted in a great increase of the contents and yields of the above three bioactive ingredients. After 16 days of fermentation, the yields of syringin, chlorogenic acid and 1,5-dicaffeoylquinic acid reached 339.0, 225.3, 512.7 mg x L(-1), respectively. Meanwhile, their contents increased up to 67.9, 1.9, 10.6 times of wild medicinal material, respectively. The results provided a solid basis for further studies on application of cell suspension cultures of S. involucrata for large-scale production of bioactive compounds syringin, chlorogenic acid and 1,5-dicaffeoylquinic acid.
Subject(s)
Cell Culture Techniques , Cells, Cultured , Chlorogenic Acid , Metabolism , Cinnamates , Metabolism , Glucosides , Phenylpropionates , Saussurea , Chemistry , MetabolismABSTRACT
The prenylation of aromatic compounds plays an important role in the natural product research because it not only gives rise to an astounding diversity of primary and secondary metabolites in plants, fungi and bacteria but also enhances the bioactivities and bioavailabilities of these compounds. However, further investigation of prenylated aromatic compounds is frequently hindered due to their low content in nature and difficulties in chemical synthesis. Cloning aromatic prenyltransferase genes followed by heterologous expression would be attractive tools for the chemoenzymatic synthesis of bioactive molecules. This review summarizes the classifications, structural investigations, enzymatic catalysis and other progress in aromatic prenyltransferases originated from microorganisms.
Subject(s)
Bacteria , Dimethylallyltranstransferase , Chemistry , Classification , Fungi , Molecular Structure , Substrate Specificity , Synthetic BiologyABSTRACT
A total of 24 biologically pure entophytic fungal strains were isolated from stems, leaves, and seed coats of Xylocarpus plants by repeated purification, and identified with Internal Transcribed Spacer (ITS) rDNA molecular method, which belonging to 14 genera, 11 families, 9 orders and 3 classes. There were differences in genus and species levels among three plant materials from different habitats and species, and it was found that the strains of Phomopsis and Colletotrichum existed in all three plant materials. In vitro assay of antitumor activity by MTT method revealed that the EtOAc extracts of 15 strains exhibited potent antitumor activity. These results suggest that it is of value for further investigation on the above fungal strains.
Subject(s)
Humans , Antineoplastic Agents , Pharmacology , Biodiversity , Cell Line, Tumor , Endophytes , Chemistry , Classification , Genetics , Fungi , Chemistry , Classification , Genetics , HCT116 Cells , Meliaceae , Microbiology , PhylogenyABSTRACT
Ten compounds were isolated and purified from cell suspension cultures of Cudrania tricuspidata with silica-gel column chromatography, semi-preparative HPLC and Sephadex LH-20. On the base of their physicochemical properties and spectral data, their structures were identified as 1, 3, 5-trihydroxy4-(3, 3-dimethylallyl) xanthone (1), wighteone (2), 6-prenylapigenin (3), licoflavone C(4), cudraflavanone C(5), erythrivarone A (6), derrone (7), carthamidin (8), genistein (9) and aromadendrin (10). Among them, compounds 2-10 were flavonoids, and compound 1 was a xanthone which was isolated from the plant for the first time.
Subject(s)
Cell Culture Techniques , Methods , Chromatography, High Pressure Liquid , Flavanones , Flavones , Flavonoids , Genistein , Moraceae , Chemistry , Cell Biology , Plant Leaves , Chemistry , Cell Biology , Plants, Medicinal , Chemistry , XanthonesABSTRACT
The column chromatography on silica gel, Sephadex LH-20 and semi-preparative HPLC were used to separate and purify the compounds from the EtOAc extract of medium and MeOH extract of cell cultures of Morus alba. Eight compounds were isolated. Based on physico-chemical properties and spectroscopic data, their structures were identified as isobavachalcone (1), genistein (2), norartocarpetin (3), albanin A (4), guangsangon E (5), mulberrofuran F (6), chalcomoracin (7), kuwanon J (8). Compounds 3-6 were isolated from the cell cultures of M. alba for the first time.
Subject(s)
Benzofurans , Cell Culture Techniques , Methods , Chalcones , Chromatography, Gel , Methods , Chromatography, High Pressure Liquid , Dextrans , Genistein , Morus , Chemistry , Cell Biology , Plant Leaves , Chemistry , Cell Biology , Plants, Medicinal , Chemistry , Cell Biology , Silica Gel , TerpenesABSTRACT
Syringin is one of the main bioactive ingredients in Saussurea involucrata. In this study, various chromatographic techniques were employed to isolate and purify syringin in the polar extraction of cell suspension cultures of S. involucrata. The structure of syringin was characterized by the analysis of spectroscopic data. A quantitative analytical method for the content of syringin in cultures of S. involucrata was established with RP-HPLC. The method is convenient, accurate and reliable. All this results provided a basis for further studies on application of cell suspension cultures of S. involucrata for large-scale production of bioactive compound syringin.