Longibramides A-E, Peptaibols Isolated from a Mushroom Derived Fungus Trichoderma longibrachiatum Rifai DMG-3-1-1.

Five new peptaibols, longibramides A-E (1-5) with 11 amino acid residues, were isolated from a fungus Trichoderma longibrachiatum Rifai DMG-3-1-1, which was isolated from a mushroom Clitocybe nebularis (Batsch) P. Kumm collected from coniferous forest in the subboreal area of northeast China. The structures of longibramides A-E were determined by their spectroscopic data (NMR and MS-MS spectra), their absolute configurations were determined by X-ray diffractions and Marfey's analyses. The X-ray diffractions of longibramides A, B, and the similar CD spectra of A-E showed that they all had α-helix conformations. Longibramides B and E showed moderate cytotoxicities against BV2 and MCF-7 cells and also showed some inhibitory effects against methicillin-resistant Staphylococcus aureus MRSA T144. L-trans-Hyp was not commonly found in natural peptaibols, which was the 6th or 10th amino acid residue in longibramides C-E. The X-ray diffractions of longibramides A and B afforded the accuracy conformations of their secondary structures, which maybe help to interpret the structure-activity relationships of the family of peptaibols in the future.

Penicimutanin C, a New Alkaloidal Compound, Isolated from a Neomycin-Resistant Mutant 3-f-31of Penicillium purpurogenum G59.

Penicimutanin C, a new alkaloidal compound, was isolated from the neomycin-resistant mutant strain 3-f-31 of the marine-derived fungus, Penicillium purpurogenum G59, together with four known compounds. The structure of penicimutanin C, including the absolute configuration, was determined by spectroscopic and chemical methods. The absolute configuration of penicimutanin A was also re-confirmed by Marfey's and chiral HPLC analyses of the hydrolyzed products. Penicimutanins C and A inhibited the proliferation of five human cancer cell lines to some extent. Penicimutanin C is the third dimer of diketopiperazine and penicimutanolone, which are only produced by mutants of P. purpurogenum G59 isolated to date, and it showed cytotoxic activity against human cancer cell lines. The neomycin-resistant screening strategy has been previously successfully used to discover new compounds by activating silent metabolites in fungi, and the present results provide an additional example of the effectiveness of this method.

Choerosponins A and B, Two New Cytotoxic Bridged-Ring Ketones and the Determination of Their Absolute Configurations.

Bioactivity-directed fractionation of antitumor compounds from the stem barks of Choerospondias axillaries (Roxb.) Burtt et Hill (Anacardiaceae) afforded two new cytotoxic bridged-ring ketones, choerosponins A (1) and B (2), and their structures were elucidated by spectroscopic methods; their stereochemistry was determined by NOE difference experiments, CD spectra and the modified Mosher's method. Compound 1 has a rare dioxatricyclo skeleton. Flow cytometry and SRB methods were employed to evaluate the antitumor activity of the two compounds against tsFT210, HCT-15, HeLa, A2780 and MCF-7 cell lines, and both of them showed strong cytotoxicity. MTT and paper disc methods were also used to evaluate their anti-hypoxia and antibacterial activities, and both of them showed no apparent activities.

Pseudoverticin B, a novel geldanamycin analog obtained as new cell cycle inhibitor from Streptomyces pseudoverticillus YN17707.

Pseudoverticin B (1), a novel naturally occurring geldanamycin analog with cell cycle inhibitory activity, was isolated from the fermentation broth of Streptomyces pseudoverticillus YN17707, together with the known ansamycin antibiotic, hydroquinone geldanamycin (2), through bioassay-guided fractionation procedures. The structure of compound 1 was elucidated by spectroscopic methods, being characterized by an ansa bridge, same as that in geldanamycin and a novel hydroquinone-derived moiety. Compounds 1 and 2 arrested the cell cycle of tsFT210 cells at the G0/G1 phase with the minimum inhibitory concentration values of 10.1 and 20.2 µmolL(-1), respectively.

Activation of the silent secondary metabolite production by introducing neomycin-resistance in a marine-derived Penicillium purpurogenum G59.

Introduction of neomycin-resistance into a marine-derived, wild-type Penicillium purpurogenum G59 resulted in activation of silent biosynthetic pathways for the secondary metabolite production. Upon treatment of G59 spores with neomycin and dimethyl sulfoxide (DMSO), a total of 56 mutants were obtained by single colony isolation. The acquired resistance of mutants to neomycin was testified by the resistance test. In contrast to the G59 strain, the EtOAc extracts of 28 mutants inhibited the human cancer K562 cells, indicating that the 28 mutants have acquired the capability to produce bioactive metabolites. HPLC-photodiode array detector (PDAD)-UV and HPLC-electron spray ionization (ESI)-MS analyses further indicated that diverse secondary metabolites have been newly produced in the bioactive mutant extracts. Followed isolation and characterization demonstrated that five bioactive secondary metabolites, curvularin (1), citrinin (2), penicitrinone A (3), erythro-23-O-methylneocyclocitrinol (4) and 22E-7α-methoxy-5α, 6α-epoxyergosta-8(14),22-dien-3ß-ol (5), were newly produced by a mutant, 4-30, compared to the G59 strain. All 1-5 were also not yet found in the secondary metabolites of other wild type P. purpurogenum strains. Compounds 1-5 inhibited human cancer K562, HL-60, HeLa and BGC-823 cells to varying extents. Both present bioassays and chemical investigations demonstrated that the introduction of neomycin-resistance into the marine-derived fungal G59 strain could activate silent secondary metabolite production. The present work not only extended the previous DMSO-mediated method for introducing drug-resistance in fungi both in DMSO concentrations and antibiotics, but also additionally exemplified effectiveness of this method for activating silent fungal secondary metabolites. This method could be applied to other fungal isolates to elicit their metabolic potentials to investigate secondary metabolites from silent biosynthetic pathways.

Rare Chromones from a Fungal Mutant of the Marine-Derived Penicillium purpurogenum G59.

Three new and rare chromones, named epiremisporine B (2), epiremisporine B1 (3) and isoconiochaetone C (4), along with three known remisporine B (1), coniochaetone A (5) and methyl 8-hydroxy-6-methyl-9-oxo-9H-xanthene-1-carboxylate (6) were isolated from a mutant from the diethyl sulfate (DES) mutagenesis of a marine-derived Penicillium purpurogenum G59. The structures of 2-4 including the absolute configurations were determined by spectroscopic methods, especially by NMR analysis and electronic circular dichroism (ECD) experiments in conjunction with calculations. The absolute configuration of the known remisporine B (1) was determined for the first time. Compounds 2 and 3 have a rare feature that has only been reported in one example so far. The compounds 1-6 were evaluated for their cytotoxicity against several human cancer cell lines. The present work explored the great potential of our previous DES mutagenesis strategy for activating silent fungal pathways, which has accelerated the discovery of new bioactive compounds.

Polyanthumin, a novel cyclobutane chalcone trimmer from Memecylon polyanthum.

A novel unusual trimmer chalcone, polyanthumin (1), together with five known compounds myricetin 3-O-(3″-O-galloyl)-α-l-rhamnopyranoside (2), sulfuretin (3), fustin (4), gallic acid (5), and ethyl gallate (6), was isolated from the dry stems of Memecylon polyanthum H.L. Li. Among them, compound 1 is a new chalcone trimmer with a novel cyclobutane skeleton in nature. Compounds 3 and 4 are flavonoids carrying a single 7-OH in A ring, which provided the first example of these class flavonoids from the family Melastomataceae. In addition, the antitumor activities for 2-4 were reported for the first time in this study. The antitumor effects of the isolated compounds 1-6 in vitro were assayed by the SRB method using human cancer K562 cells, with the inhibition rates ranging from 39.4% to 54.5% at 100 µg/ml. The IC50 values of compounds 1 and 3 for the inhibition of K562 cell proliferation were determined to be 45.4 and 30.5 µg/ml, respectively. To the best of our knowledge, compound 1 was the second sample as chalcone trimer. In addition, the antitumor activities for 2-4 were reported for the first time in this study.

The synthesis and antitumor activity of twelve galloyl glucosides.

Twelve galloyl glucosides 1-12, showing diverse substitution patterns with two or three galloyl groups, were synthesized using commercially available, low-cost D-glucose and gallic acid as starting materials. Among them, three compounds, methyl 3,6-di-O-galloyl-α-D-glucopyranoside (9), ethyl 2,3-di-O-galloyl-α-D-glucopyranoside (11) and ethyl 2,3-di-O-galloyl-ß-D-glucopyranoside (12), are new compounds and other six, 1,6-di-O-galloyl-ß-D-glucopyranose (1), 1,4,6-tri-O-galloyl-ß-D-glucopyranose (2), 1,2-di-O-galloyl-ß-D-glucopyranose (3), 1,3-di-O-galloyl-ß-D-glucopyranose (4), 1,2,3-tri-O-galloyl-α-D-glucopyranose (6) and methyl 3,4,6-tri-O-galloyl-α-D-glucopyranoside (10), were synthesized for the first time in the present study. In in vitro MTT assay, 1-12 inhibited human cancer K562, HL-60 and HeLa cells with inhibition rates ranging from 64.2% to 92.9% at 100 µg/mL, and their IC50 values were determined to be varied in 17.2-124.7 µM on the tested three human cancer cell lines. In addition, compounds 1-12 inhibited murine sarcoma S180 cells with inhibition rates ranging from 38.7% to 52.8% at 100 µg/mL in the in vitro MTT assay, and in vivo antitumor activity of 1 and 2 was also detected in murine sarcoma S180 tumor-bearing Kunming mice using taxol as positive control.

Seven new and two known lipopeptides as well as five known polyketides: the activated production of silent metabolites in a marine-derived fungus by chemical mutagenesis strategy using diethyl sulphate.

AD-2-1 is an antitumor fungal mutant obtained by diethyl sulfate mutagenesis of a marine-derived Penicillium purpurogenum G59. The G59 strain originally did not produce any metabolites with antitumor activities in MTT assays using K562 cells. Tracing newly produced metabolites under guidance of MTT assay and TLC analysis by direct comparison with control G59 extract, seven new (1-7) and two known (8-9) lipopeptides were isolated together with five known polyketides 10-14 from the extract of mutant AD-2-1. Structures of the seven new compounds including their absolute configurations were determined by spectroscopic and chemical evidences and named as penicimutalides A-G (1-7). Seven known compounds were identified as fellutamide B (8), fellutamide C (9), 1'-O-methylaverantin (10), averantin (11), averufin (12), nidurufin (13), and sterigmatocystin (14). In the MTT assay, 1-14 inhibited several human cancer cell lines to varying extents. All the bioassays and HPLC-photodiode array detector (PDAD)-UV and HPLC-electron spray ionization (ESI)-MS analyses demonstrated that the production of 1-14 in the mutant AD-2-1 was caused by the activated production of silent metabolites in the original G59 fungal strain. Present results provided additional examples for effectiveness of the chemical mutagenesis strategy using diethyl sulphate mutagenesis to discover new compounds by activating silent metabolites in fungal isolates.

A practical strategy to discover new antitumor compounds by activating silent metabolite production in fungi by diethyl sulphate mutagenesis.

Many fungal biosynthetic pathways are silent in standard culture conditions, and activation of the silent pathways may enable access to new metabolites with antitumor activities. The aim of the present study was to develop a practical strategy for microbial chemists to access silent metabolites in fungi. We demonstrated this strategy using a marine-derived fungus Penicillium purpurogenum G59 and a modified diethyl sulphate mutagenesis procedure. Using this strategy, we discovered four new antitumor compounds named penicimutanolone (1), penicimutanin A (2), penicimutanin B (3), and penicimutatin (4). Structures of the new compounds were elucidated by spectroscopic methods, especially extensive 2D NMR analysis. Antitumor activities were assayed by the MTT method using human cancer cell lines. Bioassays and HPLC-photodiode array detector (PDAD)-UV and HPLC-electron spray ionization (ESI)-MS analyses were used to estimate the activated secondary metabolite production. Compounds 2 and 3 had novel structures, and 1 was a new compound belonging to a class of very rare natural products from which only four members are so far known. Compounds 1-3 inhibited several human cancer cell lines with IC50 values lower than 20 µM, and 4 inhibited the cell lines to some extent. These results demonstrated the effectiveness of this strategy to discover new compounds by activating silent fungal metabolic pathways. These discoveries provide rationale for the increased use of chemical mutagenesis strategies in silent fungal metabolite studies.

Three new and eleven known unusual C25 steroids: activated production of silent metabolites in a marine-derived fungus by chemical mutagenesis strategy using diethyl sulphate.

Three new (1-3) and 11 known (4-14) C25 steroids with an unusual bicyclo[4.4.1]A/B ring system were isolated by tracing newly produced metabolites in the EtOAc extract of an antitumor mutant AD-1-2 obtained by the diethyl sulphate (DES) mutagenesis of a marine-derived Penicillium purpurogenum G59. HPLC-PDAD-UV and HPLC-ESI-MS analyses indicated that the G59 strain did not produce these metabolites and the production of 1-14 in the mutant AD-1-2 extract was caused by the activation of silent metabolites in the original G59 strain by DES mutagenesis. The structures of the new compounds, named antineocyclocitrinols A (1) and B (2) and 23-O-methylantineocyclocitrinol (3), including their absolute configurations were determined by various spectroscopic methods, especially the NMR and Mo2-induced CD analyses. Compounds 1-3 provide the first examples of the C25 bicyclo[4.4.1]A/B ring steroids with the Z-configuration of 20,22-double bond. All of 1-14 weakly inhibited several human cancer cell lines to varying extents. These results provided additional examples for the successful application of the chemical mutagenesis strategy using DES to discover new compounds by activating silent metabolites in fungal isolates and supported also the effectiveness and usefulness of this new strategy.

Nine new and five known polyketides derived from a deep sea-sourced Aspergillus sp. 16-02-1.

Nine new C9 polyketides, named aspiketolactonol (1), aspilactonols A-F (2-7), aspyronol (9) and epiaspinonediol (11), were isolated together with five known polyketides, (S)-2-(2'-hydroxyethyl)-4-methyl-γ-butyrolactone (8), dihydroaspyrone (10), aspinotriol A (12), aspinotriol B (13) and chaetoquadrin F (14), from the secondary metabolites of an Aspergillus sp. 16-02-1 that was isolated from a deep-sea sediment sample. Structures of the new compounds, including their absolute configurations, were determined by spectroscopic methods, especially the 2D NMR, circular dichroism (CD), Mo2-induced CD and Mosher's 1H NMR analyses. Compound 8 was isolated from natural sources for the first time, and the possible biosynthetic pathways for 1-14 were also proposed and discussed. Compounds 1-14 inhibited human cancer cell lines, K562, HL-60, HeLa and BGC-823, to varying extents.

Activation of dormant secondary metabolite production by introducing neomycin resistance into the deep-sea fungus, Aspergillus versicolor ZBY-3.

A new ultrasound-mediated approach has been developed to introduce neomycin-resistance to activate silent pathways for secondary metabolite production in a bio-inactive, deep-sea fungus, Aspergillus versicolor ZBY-3. Upon treatment of the ZBY-3 spores with a high concentration of neomycin by proper ultrasound irradiation, a total of 30 mutants were obtained by single colony isolation. The acquired resistance of the mutants to neomycin was confirmed by a resistance test. In contrast to the ZBY-3 strain, the EtOAc extracts of 22 of the 30 mutants inhibited the human cancer K562 cells, indicating that these mutants acquired a capability to produce antitumor metabolites. HPLC-photodiode array detector (PDAD)-UV and HPLC-electron spray ionization (ESI)-MS analyses of the EtOAc extracts of seven bioactive mutants and the ZBY-3 strain indicated that diverse secondary metabolites have been newly produced in the mutant extracts in contrast to the ZBY-3 extract. The followed isolation and characterization demonstrated that six metabolites, cyclo(D-Pro-D-Phe) (1), cyclo(D-Tyr-D-Pro) (2), phenethyl 5-oxo-L-prolinate (3), cyclo(L-Ile-L-Pro) (4), cyclo(L-Leu-L-Pro) (5) and 3ß,5α,9α-trihydroxy-(22E,24R)-ergosta-7,22-dien-6-one (6), were newly produced by the mutant u2n2h3-3 compared to the parent ZBY-3 strain. Compound 3 was a new compound; 2 was isolated from a natural source for the first time, and all of these compounds were also not yet found in the metabolites of other A. versicolor strains. Compounds 1-6 inhibited the K562 cells, with inhibition rates of 54.6% (1), 72.9% (2), 23.5% (3), 29.6% (4), 30.9% (5) and 51.1% (6) at 100 µg/mL, and inhibited also other human cancer HL-60, BGC-823 and HeLa cells, to some extent. The present study demonstrated the effectiveness of the ultrasound-mediated approach to activate silent metabolite production in fungi by introducing acquired resistance to aminoglycosides and its potential for discovering new compounds from silent fungal metabolic pathways. This approach could be applied to elicit the metabolic potentials of other fungal isolates to discover new compounds from cryptic secondary metabolites.

One new and nine known flavonoids from Choerospondias axillaries and their in vitro antitumor, anti-hypoxia and antibacterial activities.

In the present study, a new flavanoid 1, together with nine known ones 2-10 were isolated from the stem bark of Choerospondias axillaries, the fruit of which was used mainly for treatment of cardiovascular diseases in China. The structure of 1 was established on the basis of its extensive spectral data, and the absolute structures of 1 and 10 were determined by their CD data. The absolute structure of 10 was established for the first time. Among the obtained compounds, 5-8 inhibited the proliferation of K562 cells with inhibition rates of 26.6%, 65.7%, 40.4% and 45.6% at 100 µg/mL; 1 and 4-10 showed significant protective effects on anoxia-induced injury in cultured ECV304 or PC12 cells at 50 µg/mL; 8 and 9 showed antibacterial effects on Staphylococcus aureus ATCC6538 at the tested concentration of 150 µg/8 mm paper disc. Compounds 2 and 4-10 were isolated for the first time from this genus. The proliferation inhibiting activities of 7 and 8, the anti-hypoxia activities of 1 and 4-10, and the antibacterial effect of 8 and 9 on Staphylococcus aureus ATCC6538 are reported here for the first time.

A sterol and spiroditerpenoids from a Penicillium sp. isolated from a deep sea sediment sample.

A new polyoxygenated sterol, sterolic acid (1), three new breviane spiroditerpenoids, breviones I-K (2-4), and the known breviones (5-8), were isolated from the crude extract of a Penicillium sp. obtained from a deep sea sediment sample that was collected at a depth of 5115 m. The structures of 1-4 were elucidated primarily by NMR experiments, and 1 was further confirmed by X-ray crystallography. The absolute configurations of 2 and 3 were deduced by comparison of their CD spectra with those of the model compounds. Compounds 2 and 5 showed significant cytotoxicity against MCF-7 cells, which is comparable to the positive control cisplatin.

Activation of the dormant secondary metabolite production by introducing gentamicin-resistance in a marine-derived Penicillium purpurogenum G59.

A new approach to activate silent gene clusters for dormant secondary metabolite production has been developed by introducing gentamicin-resistance to an originally inactive, marine-derived fungal strain Penicillium purpurogenum G59. Upon treatment of the G59 spores with a high concentration of gentamicin in aqueous DMSO, a total of 181 mutants were obtained by single colony isolation. In contrast to the strain G59, the EtOAc extracts of nine mutant cultures showed inhibitory effects on K562 cells, indicating that the nine mutants had acquired capability to produce antitumor metabolites. This was evidenced by TLC and HPLC analysis of EtOAc extracts of G59 and the nine mutants. Further isolation and characterization demonstrated that four antitumor secondary metabolites, janthinone (1), fructigenine A (2), aspterric acid methyl ester (3) and citrinin (4), were newly produced by mutant 5-1-4 compared to the parent strain G59, and which were also not found in the secondary metabolites of other Penicillium purpurogenum strains. However, Compounds 1-4 inhibited the proliferation of K562 cells with inhibition rates of 34.6% (1), 60.8% (2), 31.7% (3) and 67.1% (4) at 100 µg/mL, respectively. The present study demonstrated the effectiveness of a simple, yet practical approach to activate the production of dormant fungal secondary metabolites by introducing acquired resistance to aminoglycoside antibiotics, which could be applied to the studies for eliciting dormant metabolic potential of fungi to obtain cryptic secondary metabolites.

Purpurogemutantin and purpurogemutantidin, new drimenyl cyclohexenone derivatives produced by a mutant obtained by diethyl sulfate mutagenesis of a marine-derived Penicillium purpurogenum G59.

Two new drimenyl cyclohexenone derivatives, named purpurogemutantin (1) and purpurogemutantidin (2), and the known macrophorin A (3) were isolated from a bioactive mutant BD-1-6 obtained by random diethyl sulfate (DES) mutagenesis of a marine-derived Penicillium purpurogenum G59. Structures and absolute configurations of 1 and 2 were determined by extensive spectroscopic methods, especially 2D NMR and electronic circular dichroism (ECD) analysis. Possible biosynthetic pathways for 1-3 were also proposed and discussed. Compounds 1 and 2 significantly inhibited human cancer K562, HL-60, HeLa, BGC-823 and MCF-7 cells, and compound 3 also inhibited the K562 and HL-60 cells. Both bioassay and chemical analysis (HPLC, LC-ESIMS) demonstrated that the parent strain G59 did not produce 1-3, and that DES-induced mutation(s) in the mutant BD-1-6 activated some silent biosynthetic pathways in the parent strain G59, including one set for 1-3 production.

Chrysomutanin and related meroterpenoids from a DES mutant of the marine-derived fungus Penicillium chrysogenum S-3-25.

A new meroterpene, chrysomutanin (1), two new meroterpenoids (4 and 5) together with nine known ones were isolated from the diethyl sulphate (DES) mutant 3d10-01 of the marine-derived fungus Penicillium chrysogenum S-3-25. The structures of the isolated compounds were determined by their spectroscopic data, and the absolute configuration of 1 was determined by Rh2-induced electrical circular dichroism (ECD) analysis or by comparison of the measured ECD with that of the known compounds. The cytotoxic activity was preliminarily evaluated against five human cancer cell lines. HPLC-UV analysis showed that compounds 1-12 were all newly produced by the mutant, and were not detected from the initial strain S-3-25. Chrysomutanin (1) is a new member with a chain sesquiterpene unit to the family of meroterpenes. Present results confirm that DES mutagenesis strategy is an effective method to exploit the dormant metabolites of fungi.

Surfactin isoforms isolated from a mushroom derived Bacillus halotolerans DMG-7-2.

A new iso-C14 [Val2, Val7] surfactin isoform (1) together with eight known ones (2-9), was isolated from the culture of a mushroom derived bacterium, Bacillus halotolerans DMG-7-2. The structures of them were mainly elucidated by NMR and MS data, and the NMR data of 5 also was reported for the first time. The absolute configuration of 1 was determined by Marfey's analysis (for amino acid residues) and the 13C NMR calculation of the two plausible epimers of 1 (for fatty acid). Compounds 1-9 showed moderate cytotoxicity against two human cancer cell lines (A549, MCF-7) and mice microglial BV2 cells, the IC50 values ranged from 8.91 to 33.00 µM, and the IC50 values of the positive control 5-FU were 99.94, 71.49 and 0.12 µM, respectively.[Formula: see text].

Insoluble dietary fiber derived from brown seaweed Laminaria japonica ameliorate obesity-related features via modulating gut microbiota dysbiosis in high-fat diet-fed mice.

Gut microbiota (GM) is considered to play an important role in obesity. Additionally, the impact of dietary fiber (DF) consumption on GM has been well established. Brown seaweeds are known to be a rich source of DF. However, the effect of insoluble DFs (IDFs) alone from brown seaweed on obesity and GM remains to be determined. This study investigated the effect of IDFs prepared from Laminaria japonica Aresch on high-fat diet (HFD)-induced obesity and GM dysbiosis in mice. Although HFD-induced body weight gain was not significantly attenuated by the IDF treatment, HFD-induced liver injury was ameliorated, and the HFD-elevated serum cholesterol concentration and glucose level of obese mice were significantly lowered. IDF treatment significantly modulated the GM composition disturbed by the HFD. It was found that 5% IDFs restored the GM to a very similar composition to that in the normal mice. The relative abundance of Akkermansia genus was decreased by >300-fold in HFD-fed mice, and it was fully restored by 5% IDF administration. Akkermansia muciniphila, a short-chain fatty acid producer, was identified as a marker species in both control and high-dose IDF groups. Furthermore, IDFs significantly restored the HFD-reduced acetate and propionate levels in the cecal content. In conclusion, the beneficial effect of IDFs derived from L. japonica on obesity was confirmed in mice, and the underlying mechanism may be associated with the modulation of GM composition, possibly through the enrichment of Akkermansia.