Antituberculosis compounds from a deep-sea-derived fungus Aspergillus sp. SCSIO Ind09F01.

Eleven diketopiperazine and fumiquinazoline alkaloids (1-11) together with a tetracyclic triterpenoid helvolic acid (12) were obtained from the cultures of a deep-sea derived fungus Aspergillus sp. SCSIO Ind09F01. The structures of these compounds (1-12) were determined mainly by the extensive NMR, ESIMS spectra data and by comparison with previously described compounds. Besides, anti-tuberculosis, cytotoxic, antibacterial, COX-2 inhibitory and antiviral activities of these compounds were evaluated. Gliotoxin (3), 12,13-dihydroxy-fumitremorgin C (11) and helvolic acid (12) exhibited very strong anti-tuberculosis activity towards Mycobacterium tuberculosis with the prominent MIC50 values of <0.03, 2.41 and 0.894 µM, respectively, which was here reported for the first time. Meanwhile gliotoxin also displayed significant selective cytotoxicities against K562, A549 and Huh-7 cell lines with the IC50 values of 0.191, 0.015 and 95.4 µM, respectively.

Cryptic antifungal compounds active by synergism with polyene antibiotics.

The majority of antifungal compounds reported so far target the cell wall or cell membrane of fungi, suggesting that other types of antibiotics cannot exert their activity because they cannot penetrate into the cells. Therefore, if the permeability of the cell membrane could be enhanced, many antibiotics might be found to have antifungal activity. We here used the polyene antibiotic nystatin, which binds to ergosterol and forms pores at the cell membrane, to enhance the cellular permeability. In the presence of nystatin, many culture extracts from entomopathogenic fungi displayed antifungal activity. Among all the active extracts, two active components were purified and identified as helvolic acid and terramide A. Because the minimum inhibitory concentration of either compound was reduced four-fold in the presence of nystatin, it can be concluded that this screening method is useful for detecting novel antifungal activity.

Preparative separation of helvolic acid from the endophytic fungus Pichia guilliermondii Ppf9 by high-speed counter-current chromatography.

High-speed counter-current chromatography (HSCCC) was applied for preparative separation of helvolic acid from the crude extract of the endophytic fungus Pichia guilliermondii Ppf9, associated with the medicinal plant Paris polyphylla var. yunnanensis for the first time. The two-phase solvent system consisted of n-hexane-ethyl acetate-methanol-water (4.5:4.5:5.0:5.0, v/v) appending with phosphoric acid (0.2%, v/v) was employed. The revolution speed of the separation column, flow rate of the mobile phase and separation temperature of the apparatus were 800 rpm, 3 ml min(-1) and 25°C, respectively. About 6.8 mg of helvolic acid was successfully obtained from 450 mg of the crude extract by HSCCC within 4 h separation procedure, and its purity reached to 93.2% according to the HPLC analysis. The product was further characterized by MS, (1)H-NMR and (13)C-NMR spectra.

Antimicrobial metabolites from the endophytic fungus Pichia guilliermondii isolated from Paris polyphylla var. yunnanensis.

Three steroids and one nordammarane triterpenoid were isolated for the first time from the endophytic fungus Pichia guilliermondii Ppf9 derived from the medicinal plant Paris polyphylla var. yunnanensis. By means of physicochemical and spectrometric analysis, they were identified as ergosta-5,7,22-trienol (1), 5α,8α-epidioxyergosta-6,22-dien-3ß-ol (2), ergosta-7,22-dien-3ß,5α,6ß-triol (3), and helvolic acid (4). Both micro-dilution-colorimetric and spore germination assays were employed to evaluate their antimicrobial activity. Among them, helvolic acid (4) exhibited the strongest antibacterial activity against all test bacteria, with MIC values ranging from 1.56 µg/mL to 50 µg/mL, and IC(50) values from 0.98 µg/mL to 33.19 µg/mL. It also showed strong inhibitory activity on the spore germination of Magnaporthe oryzae with an IC(50) value of 7.20 µg/mL. Among the three steroids, 5α,8α-epidioxyergosta-6,22-dien-3ß-ol (2) exhibited relatively strong antimicrobial activity. The results suggest that the endophytic fungus Pichia guillermondii Ppf9 could be a candidate for producing helvolic acid, and the metabolites from this fungus could be potentially developed as antimicrobial agents in the future.

In search of antioxidants and anti-atherosclerotic agents from herbal medicines.

Many recent studies have suggested that low-density lipoprotein (LDL) oxidation, endothelial dysfunction, and inflammation are involved in the pathogenesis of atherosclerosis. Herbal regimens in the treatment of blood stasis, a counterpart of atherosclerosis, commonly use medicinal plants of leguminosae and labiatae. We have developed disease-oriented screening methods to search for bioactive components, particularly isoflavones in leguminosae and polyphenols in labiatae from Chinese herbal medicines. Many bioactive components and active fractions capable of inhibiting a. Cu(II)-induced LDL oxidation, b. oxidized LDL-induced endothelial damage, c. uptake of oxidized LDL by macrophages (J774A.1), and d. expression of cell adhesion molecules (CAMs) have been identified. A polyphenol, namely salvianolic acid B from Salvia miltiorrhiza was identified to be a potent antioxidant, endothelial-protecting agent, and an inhibitor to suppress the expression of ICAM and VCAM. This review also briefly describes the strategy for developing herbal medicines as anti-atherosclerotic agents.

Aspergillus fumigatus CY018, an endophytic fungus in Cynodon dactylon as a versatile producer of new and bioactive metabolites.

Aspergillus fumigatus CY018 was recognized as an endophytic fungus for the first time in the leaf of Cynodon dactylon. By bioassay-guided fractionation, the EtOAc extract of a solid-matrix steady culture of this fungus afforded two new metabolites, named asperfumoid (1) and asperfumin (2), together with six known bioactive compounds including monomethylsulochrin, fumigaclavine C, fumitremorgin C, physcion, helvolic acid and 5alpha,8alpha-epidioxy-ergosta-6,22-diene-3beta-ol as well as other four known compounds ergosta-4,22-diene-3beta-ol, ergosterol, cyclo(Ala-Leu) and cyclo(Ala-Ile). Through detailed spectroscopic analyses including HRESI-MS, homo- and hetero-nuclear correlation NMR experiments (HMQC, COSY, NOESY and HMBC), the structures of asperfumoid and asperfumin were established to be spiro-(3-hydroxyl-2,6-dimethoxyl-2,5-diene-4-cyclohexone-(1,3')-5'-methoxyl-7'-methyl-(1'H, 2'H, 4'H)-quinoline-2',4'-dione) and 5-hydroxyl-2-(6-hydroxyl-2-methoxyl-4-methylbenzoyl)-3,6-dimethoxyl-benzoic methyl ester, respectively. All of the 12 isolates were subjected to in vitro bioactive assays against three human pathogenic fungi Candida albicans, Tricophyton rubrum and Aspergillus niger. As a result, asperfumoid, fumigaclavine C, fumitremorgin C, physcion and helvolic acid were shown to inhibit C. albicans with MICs of 75.0, 31.5, 62.5, 125.0 and 31.5 microg/mL, respectively.

Detection and quantification of phytotoxic metabolites of Sarocladium oryzae in sheath rot-infected grains of rice.

AIMS: The present study describes the detection and quantification of the Sarocladium oryzae metabolites, helvolic acid and cerulenin in extracts of rice grains collected from plants infected with sheath rot. It also describes the phytotoxicity of these metabolites on rice seedlings. METHODS AND RESULTS: Helvolic acid and cerulenin in sheath rot-infected rice grains were detected using thin layer chromatography (TLC) and nuclear magnetic resonance (NMR) analyses. On the TLC plates helvolic acid and cerulenin moved as brownish yellow spots and showed R(F) values of 0.61 and 0.49, respectively. A standard assay curve was developed on the basis of selective toxicity of helvolic acid towards Calvibacter michiganensis ATCC 2140 and cerulenin towards Candida albicans 1150. The amounts of helvolic acid and cerulenin on the basis of standard assay curve were 2.2 and 1.75 microg g(-1) of infected seeds. Treatment of IR 36 rice seedlings with metabolites induced chlorosis and reduced shoot length by 20%, root length by 30% and root number by 7% relative to control. CONCLUSIONS: Helvolic acid and cerulenin were detected in infected rice grains and these metabolites induced chlorosis and reduced the seed viability and seedling health of rice. SIGNIFICANCE AND IMPACT OF THE STUDY: Antimicrobial and phytotoxic metabolites, helvolic acid and cerulenin are present in infected grains and reduce the seed viability and seedling health. These metabolites may increase the pathogenic potential and survival of S. oryzae in rice seed by competing with other seed-borne fungi.

[Screening of drugs inhibiting cholesterol synthesis]. / Poisk preparatov, ingibiruiushchix sintez xolesterina

The biosynthesis of fusidin, an antibiotic of steroid structure, was used as a model of steroid synthesis. Screening of compounds modifying the fusidin synthesis included 80 strains of mycelial fungi. A high frequency of such fungal cultures was observed. 9 cultures forming compounds which inhibited the synthesis of cholesterol in the cell culture of human hepatocytes were screened. A method for biological estimation of the activity of cholesterol synthesis inhibitors was developed on rabbits with high blood levels of cholesterol. It was shown that the cholesterol synthesis inhibitors, isolated as a result of the specific screening were not toxic and markedly lowered the cholesterol blood levels.

Evaluation of enhancers to increase nasal absorption using Ussing chamber technique.

The effects of eight prospective absorption enhancers on the nasal mucosa in rabbit have been assessed using an in vitro Ussing chamber technique. Sodium taurodihydrofusidate (STDHF), sodium deoxycholate (DC), polyoxyethylene-9-lauryl ether (BL-9), lysophosphatidylcholine (LPC) and sodium dodecyl sulfate (SDS) were found to possess relatively high protein leaching activity, while sodium glycocholate (GC), sodium taurocholate (TC) and EDTA had relatively low activity. The permeation of fluorescein isothiocyanate-labeled dextran (FD, M.W. 9400) as a model drug across the nasal mucosa was found to be greater in the presence of these enhancers. Their enhancement ratio was found to be in the order of BL-9 > STDHF > SDS > LPC > DC > EDTA > GC > TC, which correlated with the protein leaching activity. The differences in protein leaching and enhancement ratio dependent on the magnitude of change of membrane resistance (delta Rm), indicating that these enhancers damaged the membrane and increased FD permeation. delta Rm thus appears to be a useful indicator by which one can estimate nasal mucosa damage by the enhancers.

The adjuvant effect of bacitracin on nasal absorption of gonadorelin and buserelin in rats.

Nasal absorption of gonadorelin (luteinizing hormone-releasing hormone; LH-RH) and buserelin, an LH-RH agonist, was studied in anesthetized rats. Administration of peptides was by nasal instillation of aqueous peptide/buffer solutions. Peptide absorption was monitored using different techniques: (a) by specific radioimmunoassays for serum levels of lutropin (LH), (b) by the cumulative urinary excretion of buserelin, and (c) by the ovulatory activity after nasal LH-RH and buserelin, respectively. Without adjuvant the nasal absorption of LH-RH and buserelin was relatively poor compared to subcutaneous or intravenous injection. Using absorption adjuvants of different types, e.g., sodium taurodihydrofusidate (STDHF) and bacitracin, marked increases in nasal absorption and, therefore, significant nasal adjuvant activity were found, as demonstrated by an increase in the biological response after nasal administration of the peptides. The mucosal compatibility of bacitracin at the concentrations used for enhancement of absorption was confirmed by an in vitro investigation using isolated gastric mucosa of guinea pigs as a test model.

Effects of sodium taurodihydrofusidate on nasal absorption of insulin in sheep.

To investigate the utility of a novel adjuvant, sodium taurodihydrofusidate (STDHF), as an enhancer of mucosal permeation of drugs, experiments involving intranasal insulin:STDHF administration in sheep were performed. Rabbit erythrocyte lysis assays were employed to assess the relative membrane lytic activity of STDHF, as well as that of its glycine-conjugated analogue, compared with a nonionic detergent and a common bile salt. Equivalent weight concentrations of the fusidates were found to be 5- to 10-fold less lytic than the bile salt and at least 100-fold less lytic than the nonionic detergent laureth-9. Provided the concentration of STDHF was greater than its critical micellar concentration, formulations of insulin with STDHF greatly enhanced intranasal insulin absorption. Optimal nasal insulin absorption was attained at a molar ratio of STDHF to insulin of 5:1. In addition, intranasal absorption was linearly related to insulin dose. Compared with intravenous administration, the mean bioavailability of intranasal insulin was 16.4%. Interovine variability was low, with a coefficient of variation of 14% for 12 animals. It was found that intranasal absorption of sodium insulin was not significantly different from that of zinc insulin. However, formulations of both crystalline insulin preparations were absorbed more efficiently than a formulation prepared using commercially available solutions of U-500 insulin. The results taken together indicate that STDHF is an excellent enhancer of insulin absorption from the nasal mucosa.