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1.
Acta Pharmaceutica Sinica ; (12): 1727-1738, 2021.
Article in Chinese | WPRIM | ID: wpr-881568

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.

2.
Acta Pharmaceutica Sinica ; (12): 598-602, 2017.
Article in Chinese | WPRIM | ID: wpr-779634

ABSTRACT

A new phomapyrone derivative (1), and 9 known compounds were isolated from the ethyl acetate fraction of solid fermentation of Fusarium redolens, the endophytic fungus from Edgeworthia chrysantha, by using various isolation techniques such as Sephadex LH-20, MCI GEL and Semi pre-HPLC, etc. Their structures were identified by spectroscopic analysis, including MS, UV, CD, specific rotation, IR, 1D and 2D NMR, as (+)-7S-4-deoxy-9-hydroxyphomapyrone C (1), uracil (2), uridine (3), 2'-deoxyuridine (4), adenosine (5), 2'-deoxyadenosine (6), cordysinin B (7), ergosterol (8), ergosta-5α, 8β-epidioxy-6, 22-dien-3β-ol (9), and (22E, 24S)-3α-hydroxy-24-methylcholesta-5, 8, 22-trien-7-one (10).

3.
Article in Chinese | WPRIM | ID: wpr-275130

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.

4.
Article in Chinese | WPRIM | ID: wpr-304890

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.

5.
Article in Chinese | WPRIM | ID: wpr-231019

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.

6.
Article in Chinese | WPRIM | ID: wpr-337931

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 Structure
7.
Article in Chinese | WPRIM | ID: wpr-279259

ABSTRACT

A study on the microbial transformation of glycyrrhetinic acid (GA) was conducted by a fungus, Cunninghamella blakesleeana CGMCC 3.970 systematically. After incubation with the cell cultures of C. blakesleeana CGMCC 3.970 at 25 degrees C for 7 days on a rotary shaker operating at 135 r x min(-1), GA was converted into one major product and five minor products. The products were extracted and purified by solvent extraction, macroporous adsorbent resin, silica gel column chromatography, and semi-preparative RP-HPLC chromatography. Their structures were identified as 3-oxo-15α-hydroxy-18β-glycyrrhetinic acid(1), 3-oxo-15β-hydroxy-18β-glycyrrhetinic acid (2), 7β,15α-dihydroxy-18β-glycyrrhetinic acid (3), 3-oxo-7β, 15α-dihydroxy-18β-glycyrrhetinic acid (4), 7β-hydroxy-18β-glycyrrhetinic acid(5) and 15α-hydroxy-18β-glycyrrhetinic acid(6) by the analyses of MS, 1H-NMR and 13C-NMR spectroscopic data respectively. Among them, 2 was a new compound. These results suggest that C. blakesleeana CGMCC 3.970 has the capability of selective ketonization and hydroxylation for GA. [Key words] glycyrrhetinic acid; Cunninghamella blakesleeana CGMCC 3. 970; microbial transformation


Subject(s)
Biotransformation , Cunninghamella , Metabolism , Glycyrrhetinic Acid , Chemistry , Metabolism , Molecular Structure , Spectrometry, Mass, Electrospray Ionization
8.
Acta Pharmaceutica Sinica ; (12): 913-920, 2014.
Article in English | WPRIM | ID: wpr-244995

ABSTRACT

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)
Berberidaceae , Microbiology , Cell Line , Cell Line, Tumor , Humans , Lactones , Pharmacology , Monoterpenes , Pharmacology , Penicillium , Chemistry
9.
Article in Chinese | WPRIM | ID: wpr-330307

ABSTRACT

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 , Metabolism
10.
Acta Pharmaceutica Sinica ; (12): 1279-1288, 2014.
Article in Chinese | WPRIM | ID: wpr-299138

ABSTRACT

The tumor multidrug resistance reversal effect of NPB304, a novel taxane, was studied. MTT assay was used to determine the IC50 of chemotherapy drugs. Western blotting assay was applied to analyze the expression of P-glycoprotein (P-gp). The effect of compounds on the P-gp function and P-gp ATPase activity was determined by rhodamine 123 (Rh123) accumulation assay and analysis kit, respectively. Molecular docking was employed to predict the binding force between compounds and P-gp. Transmembrane transport of NPB304 was analyzed using MDCK II and MDR1-MDCK II cell model. NPB304 displayed multidrug resistance reversal effect on KBV cells and MCF-7/paclitaxel cells, NPB304 collaborative with P-glycoprotein (P-gp) inhibitors verapamil enhanced the reversal activity, specifically, 10 μmol x L(-1) verapamil in combination with paclitaxel reversed resistance by 56.5-fold, while combined with NPB304 increased the reversal fold; NPB304 synergistically increased Rh123 accumulation in the resistant cells when combined with verapamil, and NPB304 at 0-1 μmol x L(-1) enhanced the ATPase activity activated by verapamil was observed. NPB304 existed the hydrophobic interactions with the TM regions of P-gp, and the binding force between NPB304 and the A chain of the TM region was stronger. P-gp ATPase activity assay demonstrated NPB304 at lower concentrations (0-1.5 μmol x L(-1)) could activate the P-gp ATPase, playing a role on inhibition of P-gp function. However, NPB304 did not have an obvious feature of P-gp substrate. NPB304 exerted itself and synergy with verapamil activity on reversing tumor resistance via inhibiting the P-gp function.


Subject(s)
ATP Binding Cassette Transporter, Subfamily B, Member 1 , Metabolism , Antineoplastic Agents , Pharmacology , Biological Transport , Drug Resistance, Multiple , Drug Resistance, Neoplasm , Drug Synergism , Humans , MCF-7 Cells , Rhodamine 123 , Taxoids , Pharmacology , Verapamil , Pharmacology
11.
Article in Chinese | WPRIM | ID: wpr-299835

ABSTRACT

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 , Metabolism
12.
Article in Chinese | WPRIM | ID: wpr-315042

ABSTRACT

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)
Antineoplastic Agents , Pharmacology , Biodiversity , Cell Line, Tumor , Endophytes , Chemistry , Classification , Genetics , Fungi , Chemistry , Classification , Genetics , HCT116 Cells , Humans , Meliaceae , Microbiology , Phylogeny
13.
Acta Pharmaceutica Sinica ; (12): 161-169, 2013.
Article in Chinese | WPRIM | ID: wpr-235687

ABSTRACT

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 Biology
14.
Article in Chinese | WPRIM | ID: wpr-346848

ABSTRACT

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 , Xanthones
15.
Article in Chinese | WPRIM | ID: wpr-346847

ABSTRACT

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 , Terpenes
16.
Acta Pharmaceutica Sinica ; (12): 436-439, 2009.
Article in Chinese | WPRIM | ID: wpr-278243

ABSTRACT

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.


Subject(s)
Cell Culture Techniques , Chromatography, High Pressure Liquid , Methods , Glucosides , Phenylpropionates , Plants, Medicinal , Chemistry , Cell Biology , Reproducibility of Results , Saussurea , Chemistry , Cell Biology
17.
Acta Pharmaceutica Sinica ; (12): 1314-1319, 2007.
Article in Chinese | WPRIM | ID: wpr-268185

ABSTRACT

The gene encoding amorpha-4, 11-diene synthase was cloned from Artemisia annua L. Other two genes encoding the FPP synthase (FPPS) and HMG-CoA reductase (HMGR) were cloned from Saccharomyces cerevisiae. The cloned cDNAs were confirmed by DNA sequencing. Two expression vectors were constructed, one is named pGBT9/A/HMG/FPP harboring genes for HMG-CoA reductase and FPP synthase and the other is pYeDP60/G/AS, containing the gene encoding amorpha-4,11-diene synthase. Two kinds of engineered yeast were constructed: the first was named WHT [AS], which contained the plasmid pYeDP60/G/AS; the second was WHT [HMG + FPP + AS], in which the vectors pGBT9/A/ HMG/FPP and pYeDP60/G/AS were introduced by cotransformation mediated with LiOAc and PEG4000. The positive clones were identified for further fermentations. The samples from fermentations were analyzed by GC-MS for amorpha-4,11-diene. The results show that engineered yeasts could produce amorpha-4,11-diene. Moreover, the amorpha-4,11-diene production of WHT[ HMG + FPP + AS] and WHT[ AS] were 23.6 mg x L(-1) and 10 microg x L(-1), respectively. Its concentrations were reported as equivalents of valencene. The results showed the copy number increase of HMGR and FPPS genes can improve the production of amorpha-4, 11-diene in the fermentation of engineered yeasts.


Subject(s)
Alkyl and Aryl Transferases , Genetics , Artemisia annua , Genetics , Fermentation , Gene Dosage , Genes, Plant , Genetic Engineering , Methods , Genetic Vectors , Geranyltranstransferase , Genetics , Metabolism , Hydroxymethylglutaryl CoA Reductases , Genetics , Metabolism , Saccharomyces cerevisiae , Genetics , Metabolism , Sesquiterpenes , Metabolism
18.
Acta Pharmaceutica Sinica ; (12): 834-837, 2005.
Article in English | WPRIM | ID: wpr-253558

ABSTRACT

<p><b>AIM</b>To obtain more valuable derivatives for the further structural modification of 6beta-santonin (1) via biotransformation by using cell suspension cultures of Phytolacca acinosa.</p><p><b>METHODS</b>The substrate 1 was incubated with cell suspension cultures of P. acinosa, the products were obtained by chromatography, and identified on the basis of their physical and spectral data (HRMS, 1D NMR, 2D NMR, NOE and IR).</p><p><b>RESULTS</b>After incubation with cell suspension cultures of P. acinosa, 1 was converted into five products. Among them, 3 is a new compound.</p><p><b>CONCLUSION</b>6beta-santonin could be selectively reduced and hydroxylated by the cell suspension cultures of P. acinosa, which would provide valuable intermediates for its further structural modification.</p>


Subject(s)
Biotransformation , Cell Culture Techniques , Methods , Cells, Cultured , Molecular Structure , Phytolacca , Cell Biology , Metabolism , Plants, Medicinal , Cell Biology , Metabolism , Santonin , Chemistry , Metabolism
19.
Acta Pharmaceutica Sinica ; (12): 555-558, 2003.
Article in Chinese | WPRIM | ID: wpr-266638

ABSTRACT

<p><b>AIM</b>To study the microbial transformation of sinenxan A.</p><p><b>METHODS</b>Choose two strains of Fungi (Mucor spinosus AS 3.3450 and Cunninghamella echinulata AS 3.3400) and a strain of bacterium (Proteus vulgaris AS 1.1208) to transform the substrate.</p><p><b>RESULTS</b>Three products were obtained and identified as 10-deacetylsinenxan A1, 6 alpha-hydroxy-10-deacetylsinenxan A2 and 9 alpha-hydroxy-10-deacetylsinenxan A3 respectively.</p><p><b>CONCLUSION</b>Sinenxan A is facile to be transformed by microorganisms, the 10-acetyl group of which is an active group.</p>


Subject(s)
Acetates , Metabolism , Biotransformation , Culture Techniques , Cunninghamella , Metabolism , Diterpenes , Metabolism , Mucor , Metabolism , Plants, Medicinal , Chemistry , Proteus vulgaris , Metabolism , Taxus , Chemistry
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