Chemical synthesis and mechanism of a natural product from endolichenic fungus with a broad-spectrum anti microorganism activity.

Background: The antibiotic resistance in various bacteria is consistently increasing and is posing a serious threat to human health, prompting the need for the discovery of novel structurally featured natural products with promising biological activities in drug research and development. Endolichenic microbes have been proven to be a fertile source to produce various chemical components, and therefore these microbes have been on a prime focus for exploring natural products. In this study, to explore potential biological resources and antibacterial natural products, the secondary metabolites of an endolichenic fungus have been investigated. Methods: The antimicrobial products were isolated from the endolichenic fungus using various chromatographic methods, and the antibacterial and antifungal activities of the compounds were evaluated by the broth microdilution method under in vitro conditions. The antimicrobial mechanism has been discussed with measuring the dissolution of nucleic acid and protein, as well as the activity of alkaline phosphatase (AKP) in preliminary manner. Chemical synthesis of the active product compound 5 was also performed, starting from commercially available 2,6-dihydroxybenzaldehyde through a sequence of transformations that included methylation, the addition of propylmagnesium bromide on formyl group, the oxidation of secondary alcohol, and the deprotection of methyl ether motif. Results: Among the 19 secondary metabolites of the endolichenic fungus, Daldinia childiae (compound 5) showed attractive antimicrobial activities on 10 of the 15 tested pathogenic strains, including Gram-positive bacteria, Gram-negative bacteria, and fungus. The Minimum Inhibitory Concentration (MIC) of compound 5 for Candida albicans 10213, Micrococcus luteus 261, Proteus vulgaris Z12, Shigella sonnet, and Staphylococcus aureus 6538 was identified as 16 µg/ml, whereas the Minimum Bactericidal Concentration (MBC) of other strains was identified as 64 µg/ml. Compound 5 could dramatically inhibit the growth of S. aureus 6538, P. vulgaris Z12, and C. albicans 10213 at the MBC, likely affecting the permeability of the cell wall and cell membrane. These results enriched the library of active strains and metabolites resources of endolichenic microorganisms. The chemical synthesis of the active compound was also performed in four steps, providing an alternative pathway to explore antimicrobial agents.

Dihydrophenanthrofurans and bisbibenzyl derivatives from the stems of Dendrobium nobile.

Two new dihydrophenanthrofurans (1 and 2) and two new bisbibenzyl derivatives (3 and 4) were isolated from the traditional Chinese medicinal plant Dendrobium nobile, along with four known compounds (5-8). The absolute configurations of compounds 1 and 4 were elucidated through extensive NMR and ECD spectroscopic analyses. New compounds showed no antimicrobial activity against four gram-positive bacterial strains and four gram-negative bacteria at the concentration of 1 mg/mL, but displayed significant cytotoxic activity against HepG2 human hepatic cell line with the IC50 values ranging from 1.25 µM to 19.47 µM.

Two new echinocystic acid derivatives catalyzed by filamentous fungus Gliocladium roseum CGMCC 3.3657.

Biotransformation of Echinocystic acid (EA,1) using G. roseum CGMCC 3.3657 has been investigated, which leads to the isolation and identification of two novel Echinocystic acid derivatives, 4, 16α-dihydroxy-3,4-seco-olean-12-en-3,28-dioic acid (2) and 16α-hydroxy, A-homo-3α-oxa-olean-12-en-3-one-28-oic acid (3). Their structures have been elucidated by analysis of spectroscopic data. This biocatalysis could serve as an efficient tool complementary to classical chemical methods for the transformation of EA.

Mahimbrine A, a Novel Isoquinoline Alkaloid Bearing a Benzotropolone Moiety from Mahonia imbricata.

A novel isoquinoline alkaloid, mahimbrine A, possessing a rare benzotropolone framing scaffold, was isolated from the endemic plant of Mahonia imbricata. Its structure was established on the basis of extensive spectroscopic analysis. A plausible biosynthetic route of mahimbrine A was proposed. Mahimbrine A showed no antimicrobial activity at the concentration of 1 mg/mL.

Biotransformation of ursolic acid by Syncephalastrum racemosum CGMCC 3.2500 and anti-HCV activity.

Microbial transformation of ursolic acid (UA, 3ß-hydroxy-urs-12-en-28-oic acid, 1) by filamentous fungus Syncephalastrum racemosum CGMCC 3.2500 was conducted. Five metabolites 3ß, 7ß, 21ß-trihydroxy-urs-12-en-28-oic acid (2); 3ß, 21ß-dihydroxy-urs-11-en-28-oic acid-13-lactone (3); 1ß, 3ß, 21ß-trihydroxy-urs-12-en-28-oic acid (4); 3ß, 7ß, 21ß-trihydroxy-urs-1-en-28-oic acid-13-lactone (5); and 21-oxo-1ß, 3ß-dihydroxy-urs-12-en-28-oic acid (6) were afforded. Elucidation of the structures of these metabolites was primarily based on 1D and 2D NMR and HR-MS data. Metabolite 2 was a new compound. In addition, the anti-HCV activity of compounds 1-6 was evaluated.

Biotransformation of ursolic acid by an endophytic fungus from medicinal plant Huperzia serrata.

Endophytic fungi were used not only for their producing bioactive products but also for their ability to transform natural compounds. An endophytic fungus, isolated from medicinal plant Huperzia serrata, was identified as Umbelopsis isabellina based on the internal transcribed spacer of ribosomal DNA (rDNA-ITS) region. It was used to transform ursolic acid (1), a pentacyclic triterpene. Incubation of ursolic acid with U. isabellina afforded three products, 3ß-hydroxy-urs-11-en-28,13-lactone (2), 3ß,7ß-dihydroxy-urs-11-en-28,13-lactone (3), 1ß,3ß-dihydroxy-urs-11-en-28,13-lactone (4). Although product 2 was a known compound, it was first obtained by microbial transformation. Products 3 and 4 were new compounds. The structural elucidation of the three compounds was achieved mainly by the 1D- and 2D-NMR, MS, IR data. The endophytic fungus U. isabellina can hydroxyate the C12-C13 double bond at position 13 of ursolic acid 1 and form a five-member lactone effectively. In the meantime, this fungus can also introduce the hydroxyl group at C-1 or C-7 of ursolic acid 1.

Multihydroxylation of ursolic acid by Pestalotiopsis microspora isolated from the medicinal plant Huperzia serrata.

The structural modification of ursolic acid by an endophytic fungus Pestalotiopsis microspora, isolated from medicinal plant Huperzia serrata was reported for the first time. The structure diversity was very important for the SAR study of ursolic acid and its derivatives. Incubation of ursolic acid 1 with P. microspora afforded four metabolites: 3-oxo-15α, 30-dihydroxy-urs-12-en-28-oic acid (2), 3ß, 15α-dihydroxy-urs-12-en-28-oic acid (3), 3ß, 15α, 30- trihydroxy-urs-12-en-28-oic acid (4) and 3,4-seco-ursan-4,30-dihydroxy-12-en-3,28-dioic acid (5). All products were new compounds and their structures elucidation was mainly based on the spectroscopic data.

Microbial transformation of diosgenin by filamentous fungus Cunninghamella echinulata.

Microbial transformation of diosgenin (1) by suspended-cell cultures of the filamentous fungus Cunninghamella echinulata CGMCC 3.2000 was investigated. Incubation of the substrate diosgenin (1) with this fungus led to the isolation of three products: two known compounds, (25R)-spirost-5-en-3ß,7ß,12ß-triol and (25R)-spirost-5-en-3ß,7ß,11α-triol, and a new compound (25R)-spirost-5-en-3ß,7α,11α-triol. The structural elucidations of the three compounds were achieved mainly by the MS, 1D and 2D NMR spectroscopic methods and comparison with known compounds. C. echinulata CGMCC 3.2000 has not been used before in the biotransformation of diosgenin.