Isolation and identification of flavonoid-producing endophytic fungi from Loranthus tanakae Franch. & Sav that exhibit antioxidant and antibacterial activities.

AIMS: Loranthus tanakae Franch. & Sav is a medicinal plant that has a variety of pharmacological properties. However, its study is currently limited because of its relative shortage of natural abundance. The objective of this work was to find an alternative resource from this plant that could produce its bioactive ingredients. METHODS AND RESULTS: We isolated endophytic fungi from the twigs of Loranthus tanakae Franch. & Sav and eight flavonoid-producing endophytic fungi were selected. The eight endophytic fungi meeting the criteria were identified as Alternaria tenuissima, Dothiorella gregaria, Penicillium aethiopicum, Nothophoma quercina and Hypoxylon perforatum by morphological and molecular methods. The antioxidant and antibacterial activities of the flavonoid-producing endophytic fungi were investigated in vitro, where Alternaria tenuissima ZP28 and ZM148 demonstrated greater activities than the other six strains. Flavonoids of ZP28 and ZM148 were preliminarily identified by liquid chromatography-mass spectrometry (LC-MS). CONCLUSION: After screening the flavonoid-producing endophytic fungi, Alternaria tenuissima ZP28 and ZM148 were found to have good antioxidant and antibacterial activities. Overall, this study provided new direction and resources for the acquisition of flavonoids. SIGNIFICANCE AND IMPACT OF THE STUDY: Endophytic fungi are a promising alternative approach for the large-scale production of flavonoids from Loranthus tanakae Franch. & Sav.

Taxonomic Structure of Rhizosphere Bacterial Communities and Its Association With the Accumulation of Alkaloidal Metabolites in Sophora flavescens.

Plant secondary metabolites (SMs) play a crucial role in plant defense against pathogens and adaptation to environmental stresses, some of which are produced from medicinal plants and are the material basis of clinical efficacy and vital indicators for quality evaluation of corresponding medicinal materials. The influence of plant microbiota on plant nutrient uptake, production, and stress tolerance has been revealed, but the associations between plant microbiota and the accumulation of SMs in medicinal plants remain largely unknown. Plant SMs can vary among individuals, which could be partly ascribed to the shift in microbial community associated with the plant host. In the present study, we sampled fine roots and rhizosphere soils of Sophora flavescens grown in four well-separated cities/counties in China and determined the taxonomic composition of rhizosphere bacterial communities using Illumina 16S amplicon sequencing. In addition, the association of the rhizosphere bacterial microbiota with the accumulation of alkaloids in the roots of S. flavescens was analyzed. The results showed that S. flavescens hosted distinct bacterial communities in the rhizosphere across geographic locations and plant ages, also indicating that geographic location was a larger source of variation than plant age. Moreover, redundancy analysis revealed that spatial, climatic (mean annual temperature and precipitation), and edaphic factors (pH and available N and P) were the key drivers that shape the rhizosphere bacterial communities. Furthermore, the results of the Mantel test demonstrated that the rhizosphere bacterial microbiota was remarkably correlated with the contents of oxymatrine, sophoridine, and matrine + oxymatrine in roots. Specific taxa belonging to Actinobacteria and Chloroflexi were identified as potential beneficial bacteria associated with the total accumulation of matrine and oxymatrine by a random forest machine learning algorithm. Finally, the structural equation modeling indicated that the Actinobacteria phylum had a direct effect on the total accumulation of matrine and oxymatrine. The present study addresses the association between the rhizosphere bacterial communities and the accumulation of alkaloids in the medicinal plant S. flavescens. Our findings may provide a basis for the quality improvement and sustainable utilization of this medicinal plant thorough rhizosphere microbiota manipulation.

α­rhamnrtin­3­α­rhamnoside exerts anti­inflammatory effects on lipopolysaccharide­stimulated RAW264.7 cells by abrogating NF­κB and activating the Nrf2 signaling pathway.

α­rhamnrtin­3­α­rhamnoside (ARR) is the principal compound extracted from Loranthus tanakae Franch. & Sav. However, its underlying pharmacological properties remain undetermined. Inflammation is a defense mechanism of the body; however, the excessive activation of the inflammatory response can result in physical injury. The present study aimed to investigate the effects of ARR on lipopolysaccharide (LPS)­induced RAW264.7 macrophages and to determine the underlying molecular mechanism. A Cell Counting Kit­8 assay was performed to assess cytotoxicity. Nitric oxide (NO) production was measured via a NO colorimetric kit. Levels of prostaglandin E2 (PGE2) and proinflammatory cytokines, IL­1ß and IL­6, were detected using ELISAs. Reverse transcription­quantitative (RT­q)PCR analysis was performed to detect the mRNA expression levels of inducible nitric oxide synthase (iNOS), cyclooxygenase­2 (COX­2), IL­6 and IL­1ß in LPS­induced RAW246.7 cells. Western blotting, immunofluorescence and immunohistochemistry analyses were performed to measure the expression levels of NF­κB and nuclear factor­erythroid 2­related factor 2 (Nrf2) signaling pathway­related proteins to elucidate the molecular mechanisms of the inflammatory response. The results of the cytotoxicity assay revealed that doses of ARR ≤200 µg/ml exhibited no significant effect on the viability of RAW264.7 cells. The results of the Griess assay demonstrated that ARR inhibited the production of NO. In addition, the results of the ELISAs and RT­qPCR analysis discovered that ARR reduced the production of the proinflammatory cytokines, IL­1ß and IL­6, as well as the proinflammatory mediators, PGE2, iNOS and COX­2, in LPS­induced RAW264.7 cells. Immunohistochemical analysis demonstrated that ARR inhibited LPS­induced activation of TNF­associated factor 6 (TRAF6) and NF­κB p65 signaling molecules, while reversing the downregulation of the NOD­like receptor family CARD domain containing 3 (NLRC3) signaling molecule, which was consistent with the results of the western blotting analysis. Immunofluorescence results indicated that ARR reduced the increase of NF­κB p65 nuclear expression induced by LPS. Furthermore, the results of the western blotting experiments also revealed that ARR upregulated heme oxygenase­1, NAD(P)H quinone dehydrogenase 1 and Nrf2 pathway molecules. In conclusion, the results of the present study suggested that ARR may exert anti­inflammatory effects by downregulating NF­κB and activating Nrf2­mediated inflammatory responses, suggesting that ARR may be an attractive anti­inflammatory candidate drug.

Biotransformation of Rutin Using Crude Enzyme from Rhodopseudomonas palustris.

Rhodopseudomonas palustris was selected for the ability to grow in diglycosylated flavonoids-based media, exhibited deglycosylation activity. The present study aimed to investigate the ability of a crude enzyme from Rhodopseudomonas palustris to transform rutin. Our results showed the crude enzyme was found to transform rutin to quercetin via isoquercitrin. The maximum enzyme activities were observed at pH 7.0, 25 °C, and rutin concentration of 1.0 mg mL- 1. Under optimal conditions, 13.11 µM rutin was biotransformed into 6.86 µM isoquercitrin and 11.64 µM quercetin after 11 and 21 h, respectively. The study demonstrates an eco-friendly and potential economically viable 'green' conversion route to convert rutin to isoquercitrin and quercetin, which is of great interest, considering the therapeutic applications of isoquercitrin and quercetin. The specific biotransformation of rutin to isoquercitrin and quercetin, using the crude enzyme from Rhodopseudomonas palustris, may potentially serve as a new method for industrial production of isoquercitrin and quercetin.

High-throughput transcriptome sequencing analysis provides preliminary insights into the biotransformation mechanism of Rhodopseudomonas palustris treated with alpha-rhamnetin-3-rhamnoside.

BACKGROUND: The purple photosynthetic bacterium Rhodopseudomonas palustris has been widely applied to enhance the therapeutic effects of traditional Chinese medicine using novel biotransformation technology. However, comprehensive studies of the R. palustris biotransformation mechanism are rare. Therefore, investigation of the expression patterns of genes involved in metabolic pathways that are active during the biotransformation process is essential to elucidate this complicated mechanism. RESULTS: To promote further study of the biotransformation of R. palustris, we assembled all R. palustris transcripts using Trinity software and performed differential expression analysis of the resulting unigenes. A total of 9725, 7341 and 10,963 unigenes were obtained by assembling the alpha-rhamnetin-3-rhamnoside-treated R. palustris (RPB) reads, control R. palustris (RPS) reads and combined RPB&RPS reads, respectively. A total of 9971 unigenes assembled from the RPB&RPS reads were mapped to the nr, nt, Swiss-Prot, Gene Ontology (GO), Clusters of Orthologous Groups (COGs) and Kyoto Encyclopedia of Genes and Genomes (KEGG) (E-value <0.00001) databases using BLAST software. A total of 3360 unique differentially expressed genes (DEGs) in RPB versus RPS were identified, among which 922 unigenes were up-regulated and 2438 were down-regulated. The unigenes were mapped to the KEGG database, resulting in the identification of 7676 pathways among all annotated unigenes and 2586 pathways among the DEGs. Some sets of functional unigenes annotated to important metabolic pathways and environmental information processing were differentially expressed between the RPS and RPB samples, including those involved in energy metabolism (18.4% of total DEGs), carbohydrate metabolism (36.0% of total DEGs), ABC transport (6.0% of total DEGs), the two-component system (8.6% of total DEGs), cell motility (4.3% of total DEGs) and the cell cycle (1.5% of total DEGs). We also identified 19 transcripts annotated as hydrolytic enzymes and other enzymes involved in ARR catabolism in R. palustris. CONCLUSION: We present the first comparative transcriptome profiles of RPB and RPS samples to facilitate elucidation of the molecular mechanism of biotransformation in R. palustris. Furthermore, we propose two putative ARR biotransformation mechanisms in R. palustris. These analytical results represent a useful genomic resource for in-depth research into the molecular basis of biotransformation and genetic modification in R. palustris.

Cytotoxic activities of extracts and compounds from Viscum coloratum and its transformation products by Rhodobacter sphaeroides.

The bioassay-oriented fractionation of mistletoe crude extracts (MCEE) using 75% ethanol and culture products of mistletoe transformed by Rhodobacter sphaeroides, a photosynthetic bacterium (PSBT), revealed that the high cytotoxic activities were due to the petroleum ether extracts (PEs) and the acid-precipitated proteins from the aqueous extracts (AQs) of MCEE and PSBT. The isolated triterpenes may account for the activities of the PEs of MCEE and PSBT, respectively. Extraction of MCEE using petroleum ether led to the isolation of 3-epi-betulinic acid (1), betulonic acid (2), oleanolic acid (3), and beta-amyrin acetate (4), while petroleum ether extraction of PSBT led to the isolation of 1,3,4,betulinic acid (5), erythrodiol (6), and (3beta)-olean-12-ene-3,23-diol (7). The PE of PSBT exerted higher cytotoxicity than the PE of MCEE, which was due to the different triterpene contents of these two extracts. The cytotoxic activities of all compounds were tested, and the results revealed that compounds 1, 2, 3, 5, 6, and 7 contributed significantly to the cytotoxicities of both PEs. The AQ of the PSBT exerted almost the same cytotoxic activity and lower toxicity compared to the AQ of the MCEE. These findings indicate that mistletoe products biotransformed by R. sphaeroides could be used to treat cancers, since they have lower toxicities and higher antitumor activities compared to standard treatments.

Biotransformation of beta-amyrin acetate by Rhodobacter sphaeroides.

The microbial transformation of beta-amyrin acetate (compound 1) occurred in the culture of Rhodobacter sphaeroides producing three metabolites. The metabolites were purified and identified as beta-amyrin (compound 2), (3beta)-olean-12-ene-3, 23-diol (compound 3), and erythrodiol (compound 4). Initially, it was found that beta-amyrin acetate could be deacetylated and hydroxylated by microbial methods.

Bioremediation of cadmium by growing Rhodobacter sphaeroides: kinetic characteristic and mechanism studies.

The removal kinetic characteristic and mechanism of cadmium by growing Rhodobacter sphaeroides were investigated. The removal data were fitted to the second-order equation, with a correlation coefficient, R2=0.9790-0.9916. Furthermore, it was found that the removal mechanism of cadmium was predominantly governed by bioprecipitation as cadmium sulfide with biosorption contributing to a minor extent. Also, the results revealed that the activities of cysteine desulfhydrase in strains grown in the presence of 10 and 20 mg/l of cadmium were higher than in the control, while the activities in the presence of 30 and 40 mg/l of cadmium were lower than in the control. Content analysis of subcellular fractionation showed that cadmium was mostly removed and transformed by precipitation on the cell wall.