l-Phenylalanine Alters the Privileged Secondary Metabolite Production in the Marine-Derived Fungus Trichoderma erinaceum F1-1.

The effects of a single-amino-acid culture strategy on secondary metabolite production in the marine-derived fungus Trichoderma erinaceum F1-1 were investigated by culturing the fungus in GPY medium supplemented or not supplemented with l-phenylalanine. A suite of secondary metabolites, including seven terpenoids (1-7) and one polyketide (8), among which are four new compounds, harziandione A (1), cyclonerodiols A and B (3, 4), and trichodermaerin A (6), were isolated from the GPY medium without l-phenylanine, whereas 18 aromatic compounds (9-26), including six new compounds, trichoderolides A-F (9, 10, and 14-17), were isolated from the culture grown in the GPY medium with l-phenylalanine. The structures of the new compounds were determined by high-resolution mass spectrometry, NMR spectroscopic analysis, optical rotation calculations, chemical methods, and X-ray crystallography. Compounds 10, 12, 13, and 26 exhibited cytotoxic activities against MDA-MB-435 human melanocyte cancer cells. Compound 26 was cytotoxic to A549 adenocarcinomic human alveolar basal epithelial cells.

Chloraserrtone A, a Sesquiterpenoid Dimer from Chloranthus serratus.

Chloraserrtone A (1), a new sesquiterpenoid dimer with two lindenane-type sesquiterpenoid monomers bridged by two six-membered rings, was obtained from Chloranthus serratus. A combination of UV, IR, NMR, HRESIMS, and X-ray diffraction data were used to elucidate the structure of 1. Compound 1 represents the first lindenane-type sesquiterpenoid dimer with extremely unique C-15-C-15', C-4-C-6', and C-6-C-11' linkages to form two six-membered rings between the monomeric units. A plausible biosynthesis toward chloraserrtone A is proposed. This new compound (1), together with the known lindenane dimers (2-11), were assessed for their inhibitory effects on lipopolysaccharide-induced NO production in RAW264.7 cells. Compound 6 showed activity with an IC50 value of 3.7 µM.

A new labdane-type diterpenoid from Leonurus japonicus.

Five fractions prepared from the crude extract of Leonurus japonicus were examined in order to determine their cytotoxic potential. Under the bioassay guidance, a new labdane-type diterpenoid (1), and nine known ones (2-10) along with a seco-labdane (11) were isolated from the aerial parts of Leonurus japonicus. The structure elucidation was primarily based on comprehensive spectroscopic analyses, including HRESIMS, IR, and 1D and 2D NMR methods. Compound 4 (6ß-hydroxy-15,16-epoxylabda-8,13(16),14-trien-7-one) exhibited potential cytotoxicity against HeLa cell line (IC50 = 23.75 µM).

Polyketides and Alkaloids from the Marine-Derived Fungus Dichotomomyces cejpii F31-1 and the Antiviral Activity of Scequinadoline A against Dengue Virus.

In our continuous chemical investigation on the marine-derived fungus Dichotomomyces cejpii F31-1, two new polyketides dichocetides B-C (1, 2), two new alkaloids dichotomocejs E-F (3, 4), and three known fumiquinozalines: scequinadoline A (5), quinadoline A (6), and scequinadoline E (7) were discovered from the culture broth and the mycelium in the culture medium, by the addition of l-tryptophan and l-phenylalanine. Their chemical structures were established by one dimensional (1D), two dimensional (2D) nuclear magnetic resonance (NMR) and high resolution mass spectrometry (HR-MS) data. Among them, scequinadoline A (5) exhibited significant inhibitory activity against dengue virus serotype 2 production by standard plaque assay, equivalent to the positive control andrographlide. Scequinadoline A (5) possesses the potential for further development as a dengue virus inhibitor.

Diverse Secondary Metabolites from the Marine-Derived Fungus Dichotomomyces cejpii F31-1.

By adding l-tryptophan and l-phenylalanine to GPY medium, twenty-eight compounds, including amides, polyketides, a sesquiterpenoid, a diterpenoid, a meroterpenoid, diketopiperazines, ß-carbolines, fumiquinazolines, and indole alkaloids, were discovered from the marine-derived fungus Dichotomomyces cejpii F31-1, demonstrating the tremendous biosynthetic potential of this fungal strain. Among these compounds, four amides dichotomocejs A-D (1-4), one polyketide dichocetide A (5), and two diketopiperazines dichocerazines A-B (15 and 16) are new. The structures of these new compounds were determined by interpreting detailed spectroscopic data as well as calculating optical rotation values and ECD spectra. Obviously, Dichotomomyces cejpii can effectively use an amino acid-directed strategy to enhance the production of nitrogen-containing compounds. Dichotomocej A (1) displayed moderate cytotoxicity against the human rhabdomyosarcoma cell line RD with an IC50 value of 39.1 µM, and pityriacitrin (22) showed moderate cytotoxicity against the human colon carcinoma cell line HCT116 with an IC50 value of 35.1 µM.

Secondary Metabolites from the Marine-Derived Fungus Dichotomomyces sp. L-8 and Their Cytotoxic Activity.

Bioassay-guided isolation of the secondary metabolites from the fungus Dichotomomyces sp. L-8 associated with the soft coral Lobophytum crassum led to the discovery of two new compounds, dichotones A and B (1 and 2), together with four known compounds including dichotocejpin C (3), bis-N-norgliovictin (4), bassiatin (5) and (3R,6R)-bassiatin (6). The structures of these compounds were determined by 1D, 2D NMR and mass spectrometry. (3R,6R)-bassiatin (6) displayed significant cytotoxic activities against the human breast cancer cell line MDA-MB-435 and the human lung cancer cell line Calu3 with IC50 values of 7.34 ± 0.20 and 14.54 ± 0.01 µM, respectively, while bassiatin (5), the diastereomer of compound 6, was not cytotoxic.

Chlojaponilactone B from Chloranthus japonicus: Suppression of Inflammatory Responses via Inhibition of the NF-κB Signaling Pathway.

Bioassay-guided fractionation of an ethanolic extract of Chloranthus japonicus led to the isolation of the known lindenane-type sesquiterpenoid chlojaponilactone B (1). This compound exhibited pronounced inhibition of nitric oxide (NO) production in lipopolysaccharide (LPS)-induced RAW 264.7 macrophages. Further anti-inflammatory assays showed that 1 suppressed the levels of some key inflammation mediators, such as iNOS, TNF-α, and IL-6, in a dose-dependent manner, and reduced the ear thickness and neutrophil infiltration in 12-O-tetradecanoylphorbol-13-acetate (TPA)-stimulated mice. A mechanistic study revealed that compound 1 exerted its anti-inflammatory effects via the suppression of the NF-κB signaling pathway, which inhibited NF-κB-dependent transcriptional activity, IκBα phosphorylation, and p65 nuclear translocation. In contrast, chlojaponilactone B (1) was found to exert little influence on the MAPK signaling pathway.

Enantiomeric Neolignans and a Sesquiterpene from Solanum erianthum and Their Absolute Configuration Assignment.

One pair of new C-8-C-3'/C-7-O-C-4' linked neolignan enantiomers (1a/1b) and one new guaiane sesquiterpene (2) first featuring the 1(2),9(10)-conjugated double bond were isolated from the stems of Solanum erianthum (Solanceae). Their structures were characterized on the basis of extensive spectroscopic analyses, especially from their 2D nuclear magnetic resonance (NMR) spectra. The absolute configurations of 1a/2b were rigorously elucidated by electronic circular dichroism (ECD) experiments combined with the reversed helicity rule for the 2,3-dihydrobenzo[b]furan chromophore, and compound 2 is the first report on the sterochemical assignment of a guaiane sesquiterpene by using the allylic axial chirality rule for the conjugated diene chromophore in combination with the calculated ECD spectrum.

Five New Cytotoxic Metabolites from the Marine Fungus Neosartorya pseudofischeri.

The marine fungus Neosartorya pseudofischeri was isolated from Acanthaster planci from the South China Sea. In a preliminary bioactivity screening, the crude methanol extract of the fungal mycelia showed significant inhibitory activity against the Sf9 cell line from the fall armyworm Spodoptera frugiperda. Five novel compounds, including 5-olefin phenylpyropene A (1), 13-dehydroxylpyripyropene A (4), deacetylsesquiterpene (7), 5-formyl-6-hydroxy-8-isopropyl-2- naphthoic acid (9) and 6,8-dihydroxy-3-((1E,3E)-penta-1,3-dien-1-yl)isochroman-1-one (10), together with eleven known compounds, phenylpyropene A (2) and C (3), pyripyropene A (5), 7-deacetylpyripyropene A (6), (1S,2R,4aR,5R,8R,8aR)-1,8a-dihydroxy-2-acetoxy-3,8-dimethyl-5- (prop-1-en-2-yl)-1,2,4a, 5,6,7,8,8a-octahydronaphthalene (8), isochaetominine C (11), trichodermamide A (12), indolyl-3-acetic acid methyl ester (13), 1-acetyl-ß-carboline (14), 1,2,3,4-tetrahydro-6-hydroxyl-2-methyl-l,3,4-trioxopyrazino[l,2-a]-indole (15) and fumiquinazoline F (16), were obtained. The structures of these compounds were determined mainly by MS and NMR data. The absolute configuration of 9 was assigned by the single-crystal X-ray diffraction studies. Compounds 1-11 and 15 showed significant cytotoxicity against the Sf9 cells from S. frugiperda.

Two Additional New Compounds from the Marine-Derived Fungus Pseudallescheria ellipsoidea F42-3.

Two additional new compounds, pseudellone D (1) and (5S,6S)-dihydroxylasiodiplodin (3), along with the two known compounds lasiodipline F (2), (5S)-hydroxylasiodiplodin (4) were isolated from the marine-derived fungus Pseudallescheria ellipsoidea F42-3 associated with the soft coral Lobophytum crassum. Their structures, including absolute configurations, were elucidated on the basis of the corresponding spectroscopic data and electronic circular dichroism (ECD) spectra.

Pseudaboydins A and B: novel isobenzofuranone derivatives from marine fungus Pseudallescheria boydii associated with starfish Acanthaster planci.

Two novel isobenzofuranone derivatives, pseudaboydins A (1) and B (2), along with five known compounds, including (R)-2-(2-hydroxypropan-2-yl)-2,3-dihydro-5-hydroxybenzofuran (3), (R)-2-(2-hydroxypropan-2-yl)-2,3-dihydro-5-methoxybenzofuran (4), 3,3'-dihydroxy-5,5'-dimethyldiphenyl ether (5), 3-(3-methoxy-5-methylphenoxy)-5-methylphenol (6) and (-)-regiolone (7), were isolated from the culture broth of the marine fungus, Pseudallescheria boydii, associated with the starfish, Acanthaster planci. Their structures were elucidated primarily based on NMR and MS data. The absolute configurations of 1-4 were determined by CD spectroscopy and single-crystal X-ray diffraction studies. The cytotoxic and antibacterial activities of 1-4 were evaluated. Pseudaboydin A (1) showed moderate cytotoxic activity against human nasopharyngeal carcinoma cell line HONE1, human nasopharyngeal carcinoma cell line SUNE1 and human glandular lung cancer cell line GLC82 with IC50 values of 37.1, 46.5 and 87.2 µM, respectively.

Exploring the chemodiversity and biological activities of the secondary metabolites from the marine fungus Neosartorya pseudofischeri.

The production of fungal metabolites can be remarkably influenced by various cultivation parameters. To explore the biosynthetic potentials of the marine fungus, Neosartorya pseudofischeri, which was isolated from the inner tissue of starfish Acanthaster planci, glycerol-peptone-yeast extract (GlyPY) and glucose-peptone-yeast extract (GluPY) media were used to culture this fungus. When cultured in GlyPY medium, this fungus produced two novel diketopiperazines, neosartins A and B (1 and 2), together with six biogenetically-related known diketopiperazines,1,2,3,4-tetrahydro-2, 3-dimethyl-1,4-dioxopyrazino[1,2-a]indole (3), 1,2,3,4-tetrahydro-2-methyl-3-methylen e-1,4-dioxopyrazino[1,2-a]indole (4), 1,2,3,4-tetrahydro-2-methyl-1,3,4-trioxopyrazino[1,2-a] indole (5), 6-acetylbis(methylthio)gliotoxin (10), bisdethiobis(methylthio)gliotoxin (11), didehydrobisdethiobis(methylthio)gliotoxin (12) and N-methyl-1H-indole-2-carboxamide (6). However, a novel tetracyclic-fused alkaloid, neosartin C (14), a meroterpenoid, pyripyropene A (15), gliotoxin (7) and five known gliotoxin analogues, acetylgliotoxin (8), reduced gliotoxin (9), 6-acetylbis(methylthio)gliotoxin (10), bisdethiobis(methylthio) gliotoxin (11) and bis-N-norgliovictin (13), were obtained when grown in glucose-containing medium (GluPY medium). This is the first report of compounds 3, 4, 6, 9, 10 and 12 as naturally occurring. Their structures were determined mainly by MS, 1D and 2D NMR data. The possible biosynthetic pathways of gliotoxin-related analogues and neosartin C were proposed. The antibacterial activity of compounds 2-14 and the cytotoxic activity of compounds 4, 5 and 7-13 were evaluated. Their structure-activity relationships are also preliminarily discussed.

Thin-layer chromatographic identification of Chinese propolis using chemometric fingerprinting.

INTRODUCTION: Poplar tree gum has a similar chemical composition and appearance to Chinese propolis (bee glue) and has been widely used as a counterfeit propolis because Chinese propolis is typically the poplar-type propolis, the chemical composition of which is determined mainly by the resin of poplar trees. The discrimination of Chinese propolis from poplar tree gum is a challenging task. OBJECTIVE: To develop a rapid thin-layer chromatographic (TLC) identification method using chemometric fingerprinting to discriminate Chinese propolis from poplar tree gum. METHODS: A new TLC method using a combination of ammonia and hydrogen peroxide vapours as the visualisation reagent was developed to characterise the chemical profile of Chinese propolis. Three separate people performed TLC on eight Chinese propolis samples and three poplar tree gum samples of varying origins. Five chemometric methods, including similarity analysis, hierarchical clustering, k-means clustering, neural network and support vector machine, were compared for use in classifying the samples based on their densitograms obtained from the TLC chromatograms via image analysis. RESULTS: Hierarchical clustering, neural network and support vector machine analyses achieved a correct classification rate of 100% in classifying the samples. A strategy for TLC identification of Chinese propolis using chemometric fingerprinting was proposed and it provided accurate sample classification. CONCLUSION: The study has shown that the TLC identification method using chemometric fingerprinting is a rapid, low-cost method for the discrimination of Chinese propolis from poplar tree gum and may be used for the quality control of Chinese propolis.

Norditerpenoids from Flickingeria fimbriata and their inhibitory activities on nitric oxide and tumor necrosis factor-α production in mouse macrophages.

Bioassay-guided fractionation of the ethanolic extract of the leaves of Flickingeria flimbriata led to the isolation of two new degraded diterpenoids 1 and 2, a new ent-pimarane type diterpenoid 3, and four known steroids 4-7. The structures of 1-3 were elucidated by spectroscopic analysis, and their absolute configurations were determined by chemical methods, TDDFT quantum chemical calculations of ECD spectra, and CD exiton chirality method. Compounds 1 and 2, named flickinflimilins A and B, possess a rare 15,16-dinor-ent-pimarane skeleton. Compounds 1-7 were screened for the inhibitory activity against lipopolysaccharide (LPS)-induced NO and TNF-α production in RAW264.7 cells. Compounds 1-3 exhibited potent inhibitory activities, with IC50 values of less than 10 µM.

Sarcanoids A and B, two new lindenane-type sesquiterpenoid dimers from the aerial parts of Sarcandra glabra.

Sarcanoids A and B (1 and 2), two new lindenane-type sesquiterpenoid dimers with a γ-hydroxysenecioate moiety at C-15', were isolated from the ethyl acetate extract of Sarcandra glabra. The structures were elucidated by extensive analysis of spectroscopic data, and their absolute configurations were determined by single-crystal X-ray crystallography. Compounds 1 and 2 showed moderate inhibitory activities on the nitric oxide (NO) production induced by lipopolysaccharide (LPS) in RAW264.7 macrophages.

(±)-Sarcanan A, a pair of new enantiomeric dihydrobenzofuran neolignans from the aerial parts of Sarcandra glabra.

(+)-Sarcanan A (1a) and (-)-Sarcanan A (1b), a pair of new dihydrobenzofuran neolignan enantiomers, together with six known compounds (2-7), were isolated from the aerial parts of Sarcandra glabra. Their structures were elucidated by spectroscopic analysis, and the absolute configurations of 1a and 1b were determined by analyses of the electronic circular dichroism (ECD) data. All compounds were evaluated for their inhibitory effects on the nitric oxide (NO) production induced by lipopolysaccharide (LPS) in RAW264.7 cells, and compounds 2-4 exhibited moderate inhibition against NO production.

Ellagic acid, a phenolic compound, exerts anti-angiogenesis effects via VEGFR-2 signaling pathway in breast cancer.

Anti-angiogenesis targeting VEGFR-2 has been considered as an important strategy for cancer therapy. Ellagic acid is a naturally existing polyphenol widely found in fruits and vegetables. It was reported that ellagic acid interfered with some angiogenesis-dependent pathologies. Yet the mechanisms involved were not fully understood. Thus, we analyzed its anti-angiogenesis effects and mechanisms on human breast cancer utilizing in-vitro and in-vivo methodologies. The in-silico analysis was also carried out to further analyze the structure-based interaction between ellagic acid and VEGFR-2. We found that ellagic acid significantly inhibited a series of VEGF-induced angiogenesis processes including proliferation, migration, and tube formation of endothelial cells. Besides, it directly inhibited VEGFR-2 tyrosine kinase activity and its downstream signaling pathways including MAPK and PI3K/Akt in endothelial cells. Ellagic acid also obviously inhibited neo-vessel formation in chick chorioallantoic membrane and sprouts formation of chicken aorta. Breast cancer xenografts study also revealed that ellagic acid significantly inhibited MDA-MB-231 cancer growth and P-VEGFR2 expression. Molecular docking simulation indicated that ellagic acid could form hydrogen bonds and aromatic interactions within the ATP-binding region of the VEGFR-2 kinase unit. Taken together, ellagic acid could exert anti-angiogenesis effects via VEGFR-2 signaling pathway in breast cancer.

LDH-A silencing suppresses breast cancer tumorigenicity through induction of oxidative stress mediated mitochondrial pathway apoptosis.

LDH-A, as the critical enzyme accounting for the transformation from pyruvate into lactate, has been demonstrated to be highly expressed in various cancer cells and its silencing has also been approved relating to increased apoptosis in lymphoma cells. In this study, we intend to investigate the correlation between LDH-A and other clinicopathological factors of breast cancer and whether LDH-A silencing could suppress breast cancer growth, and if so the potential mechanisms. 46 breast cancer specimens were collected to study the relation between LDH-A expression and clinicopathological characteristics including menopause, tumor size, node involvement, differentiation, and pathological subtypes classified by ER, PR, and Her-2. shRNAs were designed and applied to silence LDH-A expression in breast cancer cell lines MCF-7 and MDA-MB-231. The effects of LDH-A reduction on cancer cells were studied by a series of in vitro and in vivo experiments, including cell growth assay, apoptosis evaluation, oxidative stress detection, transmission electron microscopy observation, and tumor formation assay on nude mice. LDH-A expression was found to correlate significantly with tumor size and to be independent for other clinicopathological factors. LDH-A reduction resulted in an inhibited cancer cell proliferation, elevated intracellular oxidative stress, and induction of mitochondrial pathway apoptosis. Meanwhile, the tumorigenic ability of LDH-A deficient cancer cells was significantly limited in both breast cancer xenografts. The Ki67 positive cancer cells were significantly reduced in LDH-A deficiency tumor samples, while the apoptosis ratio was enhanced. Our results suggested that LDH-A inhibition might offer a promising therapeutic strategy for breast cancer.

In-vitro antitumor activity evaluation of hyperforin derivatives.

The derivatives of hyperforin, namely hyperforin acetate (2), 17,18,22,23,27,28,32,33-octahydrohyperforin acetate (3), and N,N-dicyclohexylamine salt of hyperforin (4), have been investigated for their antitumor properties. In-vitro studies demonstrated that 2 and 4 were active against HeLa (human cervical cancer), A375 (human malignant melanoma), HepG2 (human hepatocellular carcinoma), MCF-7 (human breast cancer), A549 (human nonsmall cell lung cancer), K562 (human chronic myeloid leukemia), and K562/ADR (human adriamycin-resistant K562) cell lines with IC(50) values in the range of 3.2-64.1 µM. The energy differences between highest occupied molecular orbital and lowest unoccupied molecular orbital of 2-4 were calculated to be 0.39778, 0.43106, and 0.30900 a.u., respectively, using the Gaussian 03 software package and ab initio method with the HF/6-311 G* basis set. The result indicated that the biological activity of 4 might be the strongest and that of 3 might be the weakest, which was in accordance with their corresponding antiproliferative effects against the tested tumor cell lines. Compound 4 caused cell cycle arrest at G2/M phase in flow cytometry experiment and induced apoptosis by 4',6-diamidino-2-phenylindole staining and Annexin V-FITC/PI (propidium iodide) double-labeled staining in HepG2 cells. The results indicated a potential for N,N-dicyclohexylamine salt of hyperforin as a new antitumor drug.

[Isolation of alkaloids from Corydalis saxicola by high-speed counter-current chromatography].

OBJECTIVE: To develop a method for the rapid isolation of alkaloids from the crude extract of Corydalis saxicola. METHODS: High-speed counter-current chromatography (HSCCC) was applied for the separation of alkaloids from Corydalis saxicola, with the biphase solvent systems comprised n-butanol-ethyl acetate-water-formic acid (5: 1: 5: 0.01) and n-butanol-ethyl acetate-methanol-water-formic acid (5: 5: 1:9:0.05). RESULTS: About 300 mg of crude extract was isolated by HSCCC, yielding 3.6 mg of scoulerine, 9.2 mg of isocorydine, 5.5 mg of dehydrocheilanthifoline, 7.5 mg of dehydrocavidine, 20.4 mg of palmatine and 20.9 mg of berberine, with purities of 71%, 92%, 85%, 76%, 90%, 97%, respectively. CONCLUSION: It is time-saving and simple to isolate alkaloids from Corydalis saxicola by HSCCC with high yields and purities.