Ethanol Extract of Rosa laevigata Michx. Fruit Inhibits Inflammatory Responses through NF-κB/MAPK Signaling Pathways via AMPK Activation in RAW 264.7 Macrophages.

The fruit of Rosa laevigata Michx. (FR), a traditional Chinese herb utilized for the treatment of a variety diseases, has notably diverse pharmacological activities including hepatoprotective, anti-oxidant, and anti-inflammatory effects. Despite ongoing research on illustrating the underlying anti-inflammatory mechanism of FR, the principal mechanism remained inadequately understood. In this study, we investigated in depth the molecular mechanism of the anti-inflammatory actions of the ethanol extract of FR (EFR) and its potential targets using lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages in vitro. We showed that EFR effectively ameliorated the overproduction of inflammatory mediators and cytokines, as well as the expression of related genes. It was further demonstrated that LPS-induced activation of nuclear factor kappa B (NF-κB) and mitogen-activated protein kinases (MAPKs) were significantly inhibited by pretreatment with EFR, accompanied by a concomitant decrease in the nuclear translocation of the p65 subunit of NF-κB and activator protein 1 (AP-1). In addition, EFR pretreatment potently prevented LPS-induced decreased phosphorylation of adenosine monophosphate-activated protein kinase (AMPK). Our data also revealed that the activation of AMPK and subsequent inhibition of the mammalian target of the rapamycin (mTOR) signaling pathway was probably responsible for the inhibitory effect of EFR on LPS-induced inflammatory responses, evidenced by reverse changes observed under the condition of AMPK inactivation following co-treatment with the AMPK-specific inhibitor Compound C. Finally, the main components with an anti-inflammatory effect in EFR were identified as madecassic acid, ellagic acid, quinic acid, and procyanidin C1 by LC-MS and testified based on the inhibition of NO production and inflammatory mediator expression. Taken together, our results indicated that EFR was able to ameliorate inflammatory responses via the suppression of MAPKs/NF-κB signaling pathways following AMPK activation, suggesting the therapeutic potential of EFR for inflammatory diseases.

[Antifungal activity and genomic analysis of Bacillus velezensis X49].

Proteolytic enzymes and lipopeptides contain broad-spectrum antimicrobial activities, which have great potential for research and development. A microbial strain X49 obtained from protease screening plate showed antifungal activities against six fungi. Biochemical analysis, 16S rRNA sequencing, API identification system, and electron microscope analysis were carried out to identify the bacterium. Azocasein method was used to analyze the protease activity. Lipopeptides were extracted for antifungal analysis. The result indicated that strain X49 grew in the range of 10-50 ℃ and pH 4.0-9.0. Moreover, it survived in 10% NaCl, showing good halotolerance. Strain X49 was identified as Bacillus velezensis. Genomic analysis of B. velezensis X49 revealed eleven genes encoding serine protease. The ID 1_894 belonging to S8 subtilisin family was 99% similar to the serine protease with known antifungal ability. On the other hand, thirty genes encoding non-ribosomal peptide synthetase involved in the lipopeptide biosynthesis, including surfactin, iturin, fengycin, bacitracin, and gramicidin, were identified. Part of the extracellular proteolytic activity remained under high temperature. After co-fermentation of B. velezensis X49 with Zingiber officinale Rosc., the antifungal activity of the lipopeptide extract from the co-fermentation was greatly improved. In conclusion, B. velezensis X49 showed clear inhibitory effect on both plant and human pathogens. The active substances co-fermented with Chinese herbs and microbes can be utilized for further drug development.

Marixanthomonas spongiae sp. nov., isolated from marine sponge.

A novel bacterial strain, designated as HN-E44T, was isolated from marine sponge collected from Yangpu Bay, Hainan, PR China. Strain HN-E44T was Gram-stain-negative, non-motile, catalase-positive, oxidase-negative, rod-shaped and yellow-pigmented. Growth occurred at 4-37 °C (optimum, 28 °C), at pH 6-8 (pH 7) and in 0.5-14 % (w/v) NaCl (3-5 %). Phylogenetic analysis based on 16S rRNA gene sequences showed that strain HN-E44T formed an independent cluster with Marixanthomonas ophiurae JCM 14121T within the family Flavobacteriaceae and had the highest sequence similarity of 93.6 % to the closest type strain M. ophiurae JCM 14121T. The major fatty acids were iso-C15 : 0, anteiso-C15 : 0, iso-C17 : 0 3-OH, summed feature 3 (C16 : 1 ω7c and/or C16 : 1 ω6c) and iso-C15 : 1 G. The polar lipids comprised phosphatidylethanolamine, sphingolipid, four unidentified phospholipids, an unidentified aminophospholipid and an unidentified lipid. The respiratory quinone was identified as MK-6. The genomic DNA G+C content was determined to be 40.6 mol%. The average nucleotide identity (ANI) and average amino acid identity (AAI) values between strain HN-E44T and closest type strain M. ophiurae JCM 14121T were, respectively, 79.6 and 85.2 %, both of which were below thresholds for species delineation (95-96 % ANI and 95-96 % AAI), but were over thresholds for genus delineation (73.98 % ANI and 70-76 % AAI). The combined genotypic and phenotypic distinctiveness demonstrated that strain HN-E44T could be differentiated from closely related genera. Therefore, it is proposed that strain HN-E44T represents a novel species of the genus Marixanthomonas, for which the name Marixanthomonas spongiae sp. nov. is proposed, with the type strain HN-E44T (=MCCC 1K03332T=LMG 30459T).

Inspecting the genome sequence and agarases of Microbulbifer pacificus LD25 from a saltwater hot spring.

Neoagaro-oligosaccharides prepared by agar hydrolysis have various application fields, including the pharmaceutical, cosmetic, and food industries. In this study, an agarolytic strain was isolated from a saltwater hot spring and identified as Microbulbifer pacificus LD25 by 16S rRNA. The whole genome sequence of M. pacificus LD25 was obtained. It had a size of 4.27 Mb and comprised 3062 predicted genes in 37 contigs with a G+C content of 58.0%. Six agarases were annotated and classified into three families, namely, GH16 (AgaL1), GH86 (AgaL2, AgaL3), and GH50 (AgaL4, AgaL5, AgaL6), which shared 75-96% identities with unpublished hypothetical proteins and agarases. AgaL1, AgaL4, and AgaL6 can be successfully expressed and purified in Escherichia coli. AgaL1 and AgaL4 displayed a significantly agarolytic capability, whereas AgaL6 exhibited a rarely detectable enzymatic activity. The optimal temperature and pH required for the activity of AgaL1 and AgaL4 was 50°C and 60°C, respectively, at pH 7. The specific activities of AgaL1 and AgaL4 were achieved at 16.8 and 9.6 U per mg of protein. Both agarases were significantly inhibited in the presence of EDTA, MgO, ZnCl2, and H2O2. However, AgaL1 was resistant to 0.1% SDS and AgaL4 was slightly activated by CaCl2. Substrate hydrolysis detected by LC-MS/MS analysis indicated that neoagarobiose was the main product during AgaL1 and AgaL4 catalysis. Furthermore, AgaL4 was thermostable and retained over 93% of its relative activity after pre-incubation at 70°C for 180 min. Consequently, M. pacificus LD25 has a potential for agarase production in E. coli and industrial applications.

Anti-Inflammatory Effect of a Polyphenol-Enriched Fraction from Acalypha wilkesiana on Lipopolysaccharide-Stimulated RAW 264.7 Macrophages and Acetaminophen-Induced Liver Injury in Mice.

A polyphenol-enriched fraction (PEF) from Acalypha wilkesiana, whose leaves have been traditionally utilized for the treatment of diverse medical ailments, was investigated for the anti-inflammatory effect and molecular mechanisms by using lipopolysaccharide- (LPS-) stimulated RAW 264.7 macrophages and acetaminophen- (APAP-) induced liver injury mouse model. Results showed that PEF significantly attenuated LPS-induced nitric oxide (NO) and prostaglandin E2 (PGE2) production and suppressed the expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase (COX-2) in RAW 264.7 macrophages. PEF also reduced the secretion of proinflammatory cytokines including tumor necrosis factor-α (TNF-α), interleukin- (IL-) 1ß, and IL-6 in LPS-stimulated RAW 264.7 macrophages. Moreover, PEF potently inhibited LPS-induced phosphorylation of mitogen-activated protein kinases (MAPKs) as well as the activation of nuclear factor-κB (NF-κB) by preventing the degradation of inhibitor κB-α (IκB-α). In vivo, PEF pretreatment ameliorated APAP-induced liver injury and hepatic inflammation, as presented by decreased hepatic damage indicators and proinflammatory factors at both plasma and gene levels. Additionally, PEF pretreatment remarkably diminished Toll-like receptor 3 (TLR3) and TLR4 expression and the subsequent MAPKs and NF-κB activation. HPLC analysis revealed that two predominantly polyphenolic compounds present in PEF were geraniin and corilagin. These results indicated that PEF has an anti-inflammatory effect, and its molecular mechanisms may be involved in the inactivation of the TLR/MAPK/NF-κB signaling pathway, suggesting the therapeutic potential of PEF for inflammatory diseases.

Antioxidant and anti-inflammatory capacities of collagen peptides from milkfish (Chanos chanos) scales.

Milkfish (Chanos chanos), which is resistant to water quality changes is the fourth largest aquaculture commodity. Abandoned wastes of fish scale and bones aggravate environmental pollution. In this study, the effect of collagen peptides isolated from milkfish scales (MSCP) by pepsin-soluble collagen method on cell viability was investigated. The antioxidant, anti-inflammatory, and DNA-protective activities of MSCP were also evaluated. Results revealed that more than 95% of viable cells were retained in human keratinocytes after addition of 100 mg/mL MSCP. Measurement of DPPH· and ABTS· + radical scavenging activities and cellular reactive oxygen species revealed the high antioxidant activities of MSCP. MSCP demonstrated anti-inflammatory activities by reducing lipoxygenase activity and nitric oxide (NO·) radicals. Moreover, DNA electrophoresis assay indicated that MSCP treatment can directly protect against cyclobutane di-pyrimidine production and DNA single-strand breaks, which are harmful effects of UV radiation and H2O2. Given its antioxidant, anti-inflammatory, and DNA-protective activities, MSCP has potential applications in cosmeceuticals and supplementary health food.

Hepatoprotective Effect of Polyphenol-Enriched Fraction from Folium Microcos on Oxidative Stress and Apoptosis in Acetaminophen-Induced Liver Injury in Mice.

Folium Microcos (FM), the leaves of Microcos paniculata L., shows various biological functions including antioxidant activity and α-glucosidase inhibitory effect. However, its therapeutic potential in acute liver injury is still unknown. This study investigated the hepatoprotective effect and underlying mechanisms of the polyphenol-enriched fraction (FMF) from Folium Microcos. FMF exhibited strong free radical scavenging activities and prevented HepG2/Hepa1-6 cells from hydrogen peroxide- (H2O2-) induced ROS production and apoptosis in vitro. Antioxidant activity and cytoprotective effects were further verified by alleviating APAP-induced hepatotoxicity in mice. Western blot analysis revealed that FMF pretreatment significantly abrogated APAP-mediated phosphorylation of MAPKs, activation of proapoptotic protein caspase-3/9 and Bax, and restored expression of antiapoptotic protein Bcl2. APAP-intoxicated mice pretreated with FMF showed increased nuclear accumulation of nuclear factor erythroid 2-related factor (Nrf2) and elevated hepatic expression of its target genes, NAD(P)H:quinine oxidoreductase 1 (NQO1) and hemeoxygenase-1(HO-1). HPLC analysis revealed the four predominantly phenolic compounds present in FMF: narcissin, isorhamnetin-3-O-ß-D-glucoside, isovitexin, and vitexin. Consequently, these findings indicate that FMF possesses a hepatoprotective effect against APAP-induced hepatotoxicity mainly through dual modification of ROS/MAPKs/apoptosis axis and Nrf2-mediated antioxidant response, which may be attributed to the strong antioxidant activity of phenolic components.

New apoptosis-inducing sesquiterpenoids from the mycelial culture of Chinese edible fungus Pleurotus cystidiosus.

Two new bisabolane-type sesquiterpenoids, pleuroton A (1) and pleuroton B (2), and three clitocybulol derivatives, clitocybulol D (3), clitocybulol E (4), and clitocybulol F (5), were obtained from the mycelial culture of edible fungus Pleurotus cystidiosus O. K. Mill by repeated column chromatography over RP-18, Sephadex LH-20, and silica gel. Their structures were determined according to nuclear magnetic resonance data, high-resolution electron impact mass spectrometry, and circular dichroism spectra. These new sesquiterpenoids exhibited significant cytotoxicity against two human prostate cancer DU-145 and C42B cells in the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. The median inhibitory concentration (IC50) of compounds 1, 2, 3, 4, and 5 was 174, 28, 233, 162, and 179 nM, respectively, against the DU-145 cell and was 104, 52, 163, 120, and 119 nM, respectively, against the C42B cell. Especially, pleuroton B (2) exhibited the strongest cytotoxity among these sesquiterpenoids, which was confirmed by the colony formation assay. Furthermore, pleuroton B (2) could trigger the apoptosis of DU-145 cells through the detection of apoptosis cells using annexin V-FITC staining by flow cytometry, the observation of condensed nuclei in the apoptosis cells, and the western blot analysis for the expression of apoptosis-related proteins Bcl-2, Bak, and Bax. Analysis of structure-activity relationships of these sesquiterpenoids revealed that the unusual functional moiety of pleuroton B should contribute to its significant bioactivity. These results display the pharmacological potential of P. cystidiosus.