Antiproliferative ent-abietane diterpenoids from Euphorbia fischeriana.

Euphorfinoids M and N (1 and 2), two previously undescribed ent-abietane diterpenoids, together with seven known analogues (3-9), were isolated from the roots of wild Euphorbia fischeriana. Their structures were elucidated by spectroscopic analysis, including extensive NMR, HR-ESIMS, ECD, and comparison with structurally related known analogues. Bioassays against proliferative effects of HeLa cell line showed that compound 1 was the most active with IC50 3.62 ± 0.31 µM.

Euphorfinoids A and B, a pair of ent-atisane diterpenoid epimers from the roots of Euphorbia fischeriana, and their bioactivities.

Euphorfinoids A and B (1 and 2), a pair of ent-atisane diterpenoid epimers with a vicinal 2,3-diol moiety, together with four known analogues (3-6), were isolated from the roots of wild Euphorbia fischeriana. Their structures were elucidated by spectroscopic analysis, including extensive NMR, HR-ESIMS, NMR calculations, X-ray diffraction, and comparison with structurally related known analogues. Our bioassays have established that compound 1 displayed moderate anti-proliferative effects on Hcc1806 cell line with IC50 15.53 ± 0.21 µM, and compound 5 showed remarkable inhibitory effects against AChE with IC50 32.56 ± 2.74 µM by an in vitro screened experiment.

Morphological and Phylogenetic Characterizations Reveal Five New Species of Astrothelium (Trypetheliales, Ascomycota) from China.

The lichenized fungal genus Astrothelium is an important element of crustose lichen communities in tropical to subtropical forests. Morphological and molecular phylogenetic approaches to investigate species diversity of Astrothelium (Trypetheliaceae) from Southern China were carried out in this study. Bayesian and maximum-likelihood (ML) analyses were generated based on the combined data set of internal transcribed spacer (ITS), partial regions of the nuclear ribosomal large subunit (LSU), and the largest subunit of RNA polymerase II gene sequences (RPB1). The morphological comparison with the known Astrothelium taxa and molecular phylogeny support five new species: Astrothelium jiangxiense sp. nov., A. luminothallinum sp. nov., A. pseudocrassum sp. nov., A. subeustominspersum sp. nov., and A. subrufescens sp. nov. All these species are described and illustrated in detail.

Euphorfiatnoids A-I: Diterpenoids from the roots of Euphorbia fischeriana with cytotoxic effects.

Nine previously undescribed diterpenoids, euphorfiatnoids A-I, together with seven known diterpenoids, were isolated from the roots of wild Euphorbia fischeriana. Their structures were elucidated by the interpretation of HRESIMS, UV, and NMR data. Their configurations were determined by electronic circular dichroism (ECD) spectroscopy analysis and the structure of euphorfiatnoid A was confirmed by X-ray crystallography. To further understand the antitumor effects of E. fischeriana, we tested the cytotoxicity of these compounds against H460, HepG2, and MCF-7 cell lines in vitro using MTT assays. Euphorfiatnoid B exhibited the most promising inhibitory effect against H460 cells with an IC50 value of 9.97 µM. Euphorfiatnoid A and C also exhibited moderate cytotoxicity against HepG2 cells with IC50 values of 11.64 and 13.10 µM, respectively.

Molecular cloning and characterization of three phenylalanine ammonia-lyase genes from Schisandra chinensis.

Phenylalanine ammonia-lyase (PAL), which catalyzes the conversion from L-phenylalanine to trans-cinnamic acid, is a well-known key enzyme and a connecting step between primary and secondary metabolisms in the phenylpropanoid biosynthetic pathway of plants and microbes. Schisandra chinensis, a woody vine plant belonging to the family of Magnoliaceae, is a rich source of dibenzocyclooctadiene lignans exhibiting potent activity. However, the functional role of PAL in the biosynthesis of lignan is relatively limited, compared with those in lignin and flavonoids biosynthesis. Therefore, it is essential to clone and characterize the PAL genes from this valuable medicinal plant. In this study, molecular cloning and characterization of three PAL genes (ScPAL1-3) from S. chinensis was carried out. ScPALs were cloned using RACE PCR. The sequence analysis of the three ScPALs was carried out to give basic characteristics followed by docking analysis. In order to determine their catalytic activity, recombinant protein was obtained by heterologous expression in pCold-TF vector in Escherichia coli (BL21-DE3), followed by Ni-affinity purification. The catalytic product of the purified recombinant proteins was verified using RP-HPLC through comparing with standard compounds. The optimal temperature, pH value and effects of different metal ions were determined. Vmax, Kcat and Km values were determined under the optimal conditions. The expression of three ScPALs in different tissues was also determined. Our work provided essential information for the function of ScPALs.

[A new norsesquiterpenoid from Schisandra chinensis].

This study investigated the chemical components from the leaves and stems of Schisandra chinensis. Three norsesquiterpenoids were isolated from S. chinensis by various column chromatographies(silica gel, Sephadex LH-20, and MCI), reversed-phase medium-pressure preparative, and semi-preparative high-performance liquid chromatography(HPLC). Their structures were identified based on physicochemical properties, mass spectrometry(MS), nuclear magnetic resonance(NMR), ultraviolet(UV), and electro-nic circular dichroism(ECD) as(3R,4R,5R,6S,7E)-3,4,5,6-tetrahydroxy-7-megastigmen-9-one(1),(3S,5R,6R,7E)-3,5,6-trihydroxy-7-megastigmen-9-one(2), and(3S,4R,9R)-3,4,9-trihydroxymegastigman-5-ene(3). Compound 1 was a new compound, and its absolute configuration was determined by ECD. Compounds 2 and 3 were isolated from the Schisandra plant for the first time.

Two novel polyketones from the leaves and twigs of Clerodendrum trichotomum.

Two novel polyketones, along with four known compounds, were isolated from the leaves and twigs of Clerodendrum trichotomum. Their structures were determined to be clerodendruketone A (1), clerodendruketone B (2), ecdysanols E (3), ecdysanols D (4), 5,5'-dimethoxy-7-oxolariciresinol (5), and (-)-(7'S,8S,8'R)-4,4'-dihydroxy-3,3',5,5'-tetramethoxy-7',9-epoxy-lignan-9'-ol-7-one (6) through the methods of NMR, HRESIMS and ECD data analyses. The antioxidant effects against free radical were tested by DPPH assay. The antibacterial activity against Escherichia coli and Staphylococcus aureus were tested by turbidimetry assay. The results demonstrated that compounds 1 and 2 had significative antibacterial activity, and compound 3 had moderate antioxidant activity.

Euphorfistrines A-G, cytotoxic and AChE inhibiting triterpenoids from the roots of Euphorbia fischeriana.

Seven new triterpenoids including two cycloartanes (1-2), a lanostane (3), a tirucallane (4), a dammarane (5), an ursane (6), and an oleanane (7), along with nineteen known triterpenoids (8-26), have been obtained from the roots of Euphorbia fischeriana. Their structures were established by NMR, HRESIMS, single-crystal X-ray diffraction analysis, Mosher's method, NMR calculations, ECD analysis, and comparison with structurally related known analogues. Among them, compounds 1 and 8 were a pair of cycloartane-type triterpenoids epimers. Our bioassays have established that compounds 1-5 and 10 displayed moderate cytotoxic effects, and the structure-activity relationships of cycloartane-type triterpenoids (CTTs) were further examined. Notably, some triterpenoids displayed moderate inhibitory effects against AChE by an in vitro screened experiment. Triterpenoid 7 (Euphorfistrine G, ETG) displayed the potent inhibitory effect with IC50 = 2.45 and Ki = 2.30 µM (inhibition kinetic). And, in silico docking analyses have been performed to investigate the inhibitory mechanism of compound 7.

Euphorfinoids E-L: Diterpenoids from the roots of Euphorbia fischeriana with acetylcholinesterase inhibitory activity.

Eight undescribed diterpenoids, euphorfinoids E-L, together with twelve known analogues, were isolated from the roots of wild Euphorbia fischeriana. Their structures and absolute configurations were elucidated by a combination of NMR, MS, ECD, and X-ray diffraction analyses. The plausible biosynthetic pathway of 1 was also proposed. The isolated compounds displayed moderate inhibitory activity against acetylcholinesterase (AChE) with 50% inhibiting concentration (IC50) values of 6.23-192.38 µM.

Twelve New Species Reveal Cryptic Diversification in Foliicolous Lichens of Strigula s.lat. (Strigulales, Ascomycota).

We employed a molecular phylogenetic approach using five markers (ITS, nuSSU, nuLSU, TEF1-α, and RPB2) to assess potential cryptic speciation in foliicolous members of Strigula s.lat. (Strigulaceae), including the recently segregated genera Phylloporis, Puiggariella, Raciborskiella, Racoplaca, and Serusiauxiella, from tropical areas in Asia, with selected materials from the Neotropics as reference. On the basis of combined molecular and phenotypic datasets, two new species of Racoplaca and 10 new species of Strigula s.str. are described: Racoplaca macrospora sp. nov., R. maculatoides sp. nov., Strigula guangdongensis sp. nov., S. intermedia sp. nov., S. laevis sp. nov., S. microcarpa sp. nov., S. pseudoantillarum sp. nov., S. pseudosubtilissima sp. nov., S. pycnoradians sp. nov., S. sinoconcreta sp. nov., S. stenoloba sp. nov., and S. subtilissimoides sp. nov. In addition, we propose the new combination Phylloporis palmae comb. nov. (≡ =Manaustrum palmae) and we validate the earlier combination Racoplaca melanobapha comb. nov. (≡ Verrucaria melanobapha; Strigula melanobapha). Our data clearly indicate a considerable degree of cryptic diversification in foliicolous representatives of Strigula s.lat., particularly in the presumably widespread taxa Strigula antillarum, S. concreta, S. nitidula, and S. smaragdula. Given that these phylogenetic revisions are thus far limited to few regions, we predict that our findings only represent the proverbial tip of the iceberg in this group of lichenized fungi.

A new genus and species of foliicolous lichen in a new family of Strigulales (Ascomycota: Dothideomycetes) reveals remarkable class-level homoplasy.

Phylogenetic analysis of some foliicolous lichens collected in Hainan Province, China, revealed a new lineage morphologically similar to Porina but phylogenetically related to Strigulaceae (Dothideomycetes), differing from the latter in ascus type. The monospecific genus Tenuitholiascus gen. nov. is introduced for the single species, T. porinoides sp. nov., which is placed in the new, monogeneric family Tenuitholiascaceae, sister to Strigulaceae in Strigulales. The new taxon closely resembles the genus Porina in external morphology and ascospore type, as well as the thin-walled asci and unbranched paraphyses. Yet, it is entirely unrelated to the latter, which belongs in class Lecanoromycetes in the order Gyalectales.

Chemical characteristics of the fungus Ganoderma lucidum and their inhibitory effects on acetylcholinesterase.

The chemical characteristic of a well-known folk medicine Ganoderma lucidum has been investigated by a series of chromatographic technologies, which displayed the presences of 45 lanostane type triterpenoids, including two new nor-lanostane triterpenoids (40, 41). Their structures were identified on the basis of spectroscopic data analysis (UV, IR, HRESIMS, 1D, and 2D NMR). Notably, some triterpenoids displayed moderate inhibitory effects against AChE (acetylcholinesterase) by an in vitro screened experiment. Triterpenoid 2 displayed the potent inhibitory effect with IC50 10.8 and Ki 14.95 µM (inhibition kinetic). The preliminary SAR has been discussed by the docking analyses between ganoderic acids (1, 2) and AChE.

Two new species of Endocarpon (Verrucariaceae, Ascomycota) from China.

Endocarpon species are key components of biological soil crusts. Phenotypic and systematic molecular analyses were carried out to identify samples of Endocarpon collected from the southeast edge of the Tengger Desert in China. These morphological and molecular analyses revealed two previously undescribed species that form highly supported independent monophyletic clades within Endocarpon. The new taxa were named Endocarpon deserticola sp. nov. and E. unifoliatum sp. nov. Furthermore, our results indicated that the newly developed protein coding markers adenylate kinase (ADK) and ubiquitin-conjugating enzyme h (UCEH) are useful for assessing species boundaries in phylogenic analyses.

Functional analysis of thioredoxin from the desert lichen-forming fungus, Endocarpon pusillum Hedwig, reveals its role in stress tolerance.

Endocarpon pusillum is a lichen-forming fungus with an outstanding stress resistance property closely related to its antioxidant system. In this study, thioredoxin (Trx), one of the main components of antioxidant defense systems in E. pusillum (EpTrx), was characterized and analyzed both in transgenic yeasts and in vitro. Our analyses identified that the heterologous expression of EpTrx in the yeast Pichia pastoris significantly enhanced its resistance to osmotic and oxidative stresses. Assays in vitro showed EpTrx acted as a disulfide reductase as well as a molecular chaperone by assembling into various polymeric structures. Upon exposure to heat-shock stress, EpTrx exhibited weaker disulfide reductase activity but stronger chaperone activity, which coincided with the switching of the protein complexes from low molecular weight forms to high molecular weight complexes. Specifically, we found that Cys31 near but not at the active site was crucial in promoting the structural and functional transitions, most likely by accelerating the formation of intermolecular disulfide bond. Transgenic Saccharomyces cerevisiae harboring the native EpTrx exhibited stronger tolerance to oxidative, osmotic and high temperature stresses than the corresponding yeast strain containing the mutant EpTrx (C31S). Our results provide the first molecular evidence on how Trx influences stress response in lichen-forming fungi.

Genome characteristics reveal the impact of lichenization on lichen-forming fungus Endocarpon pusillum Hedwig (Verrucariales, Ascomycota).

BACKGROUND: Lichen is a classic mutualistic organism and the lichenization is one of the fungal symbioses. The lichen-forming fungus Endocarpon pusillum is living in symbiosis with the green alga Diplosphaera chodatii Bialsuknia as a lichen in the arid regions. RESULTS: 454 and Illumina technologies were used to sequence the genome of E. pusillum. A total of 9,285 genes were annotated in the 37.5 Mb genome of E. pusillum. Analyses of the genes provided direct molecular evidence for certain natural characteristics, such as homothallic reproduction and drought-tolerance. Comparative genomics analysis indicated that the expansion and contraction of some protein families in the E. pusillum genome reflect the specific relationship with its photosynthetic partner (D. chodatii). Co-culture experiments using the lichen-forming fungus E. pusillum and its algal partner allowed the functional identification of genes involved in the nitrogen and carbon transfer between both symbionts, and three lectins without signal peptide domains were found to be essential for the symbiotic recognition in the lichen; interestingly, the ratio of the biomass of both lichen-forming fungus and its photosynthetic partner and their contact time were found to be important for the interaction between these two symbionts. CONCLUSIONS: The present study lays a genomic analysis of the lichen-forming fungus E. pusillum for demonstrating its general biological features and the traits of the interaction between this fungus and its photosynthetic partner D. chodatii, and will provide research basis for investigating the nature of its drought resistance and symbiosis.

A study of the genetic variability of Rhizoplaca chrysoleuca using DNA sequences and secondary metabolic substances.

Specimens of Rhizoplaca chrysoleuca from Mount Wuling can be divided into two distinct groups based on obvious differences in morphological characters. Here we investigated 26 specimens of R. chrysoleuca from Mount Wuling, 10 specimens of this species from other areas and seven specimens of other Rhizoplaca species by analyzing morphology, chemistry and genetics. Nine chemotypes were detected among the specimens of R. chrysoleuca from Mount Wuling, and five of them were reported for the first time. Based on the ITS phylogenetic analysis, the chemotypes and the insertion distribution patterns in SSU rDNA, the samples of R. chrysoleuca from Mount Wuling were grouped in two distinct clades corresponding to two phenotypic groups and no gene flow was detected between these two groups. Our results establish all individuals of Rhizoplaca chrysoleuca are conspecific although some populations have been isolated on Mount Wuling, indicating that they are in the process of speciation. Our study also reveals that the relationships between genotypes and chemotypes are complicated and should be avoided, and we instead recommend using single individuals or few individuals from the same site to represent the population or whole species in systematics study. The results also indicate that Rhizoplaca chrysoleuca might provide a good model for studying the speciation of saxicolous lichenized fungi.