New paraconic acids from the endophytic fungus Xylariaceae sp. J4.

Three new paraconic acids, xylariacinics A-C (1-3), were isolated from the endophyte Xylariaceae sp. J4 harbored in the medicinal plant Blumea balsamifera. Their structures were elucidated on the basis of extensive spectroscopic data including HRMS, and NMR. The antibacterial efficacies of compounds 1-3 were evaluated against a panel of bacteria such as Staphylococcus aureus, Bacillus subtilis, Escherichia coli and Pseudomonas aeruginosa. Their antifungal activities were also tested against Colletotrichum gloeosporioides. Unfortunately, all of them were inactive.

[Antibacterial secondary metabolites of Clonostachys rosea, an endophytic fungus from Blumea balsamifera (L.) DC.]

Endophytic fungus is an important treasure trove for discovery of structurally unusual and biologically diverse compounds. A phytochemical investigation on a fungus Clonostachys rosea inhabits inner tissue of Blumea balsamifera (L.) DC. was initiatedrecently in our lab. Six pure compounds were isolated through silica gel column chromatography, sephadex LH-20, and semi-preparative HPLC techniques, with bio-guided strategy. Their structures were characterized as verticillin A (1), (S)-(+)-fusarinolic acid (2), 8-hydroxyfusaric acid (3), cerebroside C (4), 3-Maleimide-5-oxime (5), and bionectriol A (6) by analyses of NMR and MS data. All compounds were tested in vitro antibacterial activities against four strains of bacteria, Escherichia coli, Staphylococcus aureus, Bacillus subtilis and Pseudomonas aeruginosa, and results revealed that 1, 4 and 6 display notableinhibition againstthree bacteria, with MIC values ranging from 2 to 16 µg/mL. Our findings provide references for mining novel antibiotics from endophytes originated from Li Minority medicinal plant B. balsamifera (L.) DC.

[Genome survey and characteristic analysis of SSR in Callicarpa nudiflora].

Callicarpa nudiflora,which is a big brand of Li nationality medicine with Hainan characteristics,has the effects of dissolving stasis,hemostasis,anti-inflammatory and antibacterial. At present,there is a lack of information about the reference genome of C. nudiflora. The study of the genome size,heterozygosity rate and characteristics of SSR of C. nudiflora,can provide an effective basis for the formulation of the whole genome de novo sequencing strategy and development of SSR molecular markers of C. nudiflora. To realize this purpose,high throughput sequencing platform Illumina Hiseq was used to sequence the genome structure of C. nudiflora and K-mer analysis was applied to estimate genome size,repeat sequences and heterozygosity rate. Simple-sequence repeat( SSR) loci that are suitable as markers were identified by MISA software. The results showed the estimated genome size of C. nudiflora was 822. 43 Mb,with a 0. 85% heterozygosity rate and 71. 67% repeats,and the GC content of genome was about 49. 20%. Therefore,C. nudiflora belongs to a complex genome with high heterozygosity and repetition. SSR molecular genetic markers were analyzed in the genome sequence,and a total of 206 049 SSRs were identified,among which mono-nucleotide,di-nucleotide and tri-nucleotide repetitive motifs summed up to 198 993,accounting for 96. 57% of the total SSRs. Among the 2-6 nucleotide repeats,AT/AT,AAT/ATT,AGCC/CTGG,AAAAT/ATTTT and AGATAT/ATATCT have the largest number,respectively. This report represents the first genome-wide characterization of C. nudiflora,and provides a reference for the construction of the library for the fine sequencing of the genome,and a molecular basis for the development of SSR molecular markers as well as for the protection and utilization of gene resources.

[Secondary metabolites from Epicoccum nigrum 14one,an endophytic fungus isolated from plant Leptogium masiaticum].

Phytochemical investigation of the culture of Epicoccum nigrum,an endolichenic fungus inhabiting Leptogium masiaticum,led to the isolation of 11 compounds. Based on NMR spectroscopy and HRESIMS data,their structures were determined as one alkaloid fusaricide( 1),and seven benzofuran derivatives including epicoccone( 2),4,6-dihydroxy-5-methoxy-7-methyl-1,3-dihydro isobenzofuran( 3),5-methyl-epicoccone B( 4),3,6,7-trihydroxy-5-methoxy-4-methylisobenzo furan-1( 3 H)-one( 5),3-methoxyepicoccone B( 6),2,3,4-trihydroxy-6-( hydroxymethyl)-5-methylbenzyl-alcohol( 7),and isoochracinic acid( 8),together with three epicoccolide analogs epicocconigrones A( 9),epicoccolide B( 10),and epicocconigrones B( 11). Compounds 1,9 and 10 showed potent microorganism inhibitory effects. These results indicated the potential perspective of this endophytic fungus as an eco-friendly biocide.

New α-pyrones from an endophytic fungus, Hypoxylon investiens J2.

Four new α-pyrones, hypotiens A-D (1-4), were isolated from a fungal endophyte, Hypoxylon investiens J2, harbored in the medicinal plant Blumea balsamifera. Their structures were determined through detailed HRMS and NMR spectroscopic data. Compounds 1-4 are new α-pyrone derivatives containing an unusual dimethyl substitution in the highly unsaturated side chain. Their plausible biosynthetic pathway was discussed. Biological assay indicated that compounds 1-4 showed no antimicrobial, quorum sensing inhibitory, and cytotoxic activities. The specific side chain in α-pyrone derivatives 1-4 might be responsible for the weak pharmacological activities.

Effects and Mechanisms of Total Flavonoids from Blumea balsamifera (L.) DC. on Skin Wound in Rats.

Chinese herbal medicine (CHM) evolved through thousands of years of practice and was popular not only among the Chinese population, but also most countries in the world. Blumea balsamifera (L.) DC. as a traditional treatment for wound healing in Li Nationality Medicine has a long history of nearly 2000 years. This study was to evaluate the effects of total flavonoids from Blumea balsamifera (L.) DC. on skin excisional wound on the back of Sprague-Dawley rats, reveal its chemical constitution, and postulate its action mechanism. The rats were divided into five groups and the model groups were treated with 30% glycerol, the positive control groups with Jing Wan Hong (JWH) ointment, and three treatment groups with high dose (2.52 g·kg-1), medium dose (1.26 g·kg-1), and low dose (0.63 g·kg-1) of total flavonoids from B. balsamifera. During 10 consecutive days of treatment, the therapeutic effects of rates were evaluated. On day 1, day 3, day 5, day 7, and day 10 after treatment, skin samples were taken from all the rats for further study. Significant increases of granulation tissue, fibroblast, and capillary vessel proliferation were observed at day 7 in the high dose and positive control groups, compared with the model group, with the method of 4% paraformaldehyde for histopathological examination and immunofluorescence staining. To reveal the action mechanisms of total flavonoids on wound healing, the levels of CD68, vascular endothelial growth factor (VEGF), transforming growth factor-ß1 (TGF-ß1), and hydroxyproline were measured at different days. Results showed that total flavonoids had significant effects on rat skin excisional wound healing compared with controls, especially high dose ones (p < 0.05). Furthermore, the total flavonoid extract was investigated phytochemically, and twenty-seven compounds were identified from the total flavonoid sample by ultra-high-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry/diode array detector (UPLC-Q-TOF-MS/DAD), including 16 flavonoid aglucons, five flavonoid glycosides (main peaks in chromatogram), five chlorogenic acid analogs, and 1 coumarin. Reports show that flavonoid glycoside possesses therapeutic effects of curing wounds by inducing neovascularization, and chlorogenic acid also has anti-inflammatory and wound healing activities; we postulated that all the ingredients in total flavonoids sample maybe exert a synergetic effect on wound curing. Accompanied with detection of four growth factors, the upregulation of these key growth factors may be the mechanism of therapeutic activities of total flavonoids. The present study confirmed undoubtedly that flavonoids were the main active constituents that contribute to excisional wound healing, and suggested its action mechanism of improving expression levels of growth factors at different healing phases.

Variations in Essential Oil Yield, Composition, and Antioxidant Activity of Different Plant Organs from Blumea balsamifera (L.) DC. at Different Growth Times.

Blumea balsamifera, also named Ainaxiang, is widely used as an ancient medicinal herb in tropical and subtropical Asia. It is rich in essential oils. In this work the essential oils of B. balsamifera from different plant organs and in different months were extracted, and then analyzed by gas chromatography-mass spectrometry. The results showed that essential oil yield of young leaves was the highest (0.65 mL/100 g), followed by mature leaves (0.57 mL/100 g), and the oil yield was higher in October (0.47 mL/100 g) than other months. A total of 44 compounds were identified, representing 92.64%-96.71% of the oil. Eighteen common chemical components were found among the six plant organs, representing >80% of the oil constituents. l-borneol was the main ingredient in leaves, and its content was the highest in senescent leaves and in December. In the essential oils of young shoots and young stems, the main component was dimethoxydurene. Antioxidant activity was also determined using the 2,2-diphenyl-1-picrylhydrazyl (DPPH) and ß-carotene bleaching (BCB) assays. The results indicated that the ß-carotene bleaching activity was far stronger than the DPPH radical-scavenging capacity, and the young leaves and young shoots showed stronger antioxidant activity. Dimethoxydurene, ß-caryophyllene, and α-caryophyllene play a positive role in good antioxidant activity, while ß-eudesmol, phytol, and tetradecanal play a negative role. The antioxidant activity revealed in this study might help in developing this promising bioresource for use in the medicinal and cosmetic industries.

Hydroxysafflor yellow A attenuates carbon tetrachloride-induced hepatic fibrosis in rats by inhibiting ERK5 signaling.

Hepatic stellate cells (HSCs) undergo activation during the development of liver fibrosis. Transcription factor myocyte enhancer factor (MEF2) 2C plays a key role in this process. In the present study, we investigated the effect of hydroxysafflor yellow A (HSYA) on hepatic fibrosis and further investigated potential mechanisms in vivo. Sprague-Dawley rats were administered with CCl(4) together with or without HYSA for 12 weeks. The effect of HYSA on hepatic fibrosis was evaluated using hematoxylin-eosin and Van Gieson staining. Messenger RNA expression was quantified by real-time polymerase chain reaction, and protein was quantified by Western blot or immunohistochemistry. Our results revealed that CCl(4) treatment induced micronodular hepatic fibrosis with a pronounced deposition of collagen fibers. Treatment with HYSA resulted in a significant decrease in fibrosis, protein expression of α-SMA, and MEF-2C gene expression. This was accompanied by a decreased expression of Tß-RI, Tß-RII, MEKK3, MEK5, and phosphorylation of ERk5. HYSA alone had no effect on the measured parameters. Our findings demonstrate that HSYA protected, at least in part, the rat liver from CCl(4)-caused fibrogenesis through inhibition of hepatic stellate cell (HSC) activation, attenuation of transforming growth factor beta (TGF-ß) signaling. HSYA may become a novel and promising agent for the inhibition of hepatic fibrosis.

Hydroxysafflor yellow A protects against chronic carbon tetrachloride-induced liver fibrosis.

Hydroxysafflor yellow A (HSYA) was isolated from the dried flower of Carthamus tinctorius L. which was extensively used in traditional Chinese medicine to treat cirrhosis. However, the potential protective effect of HSYA in liver fibrosis is still unknown. In the present study, we investigated the effects of HSYA in rats with carbon tetrachloride (CCl4)-induced liver fibrosis. Sprague-Dawley (SD) rats were subjected to biweekly CCl4 injections over 12 weeks, while controls were given isovolumetric injections of olive oil. HSYA was given in a daily dose of 5 mg/kg by means of intraperitoneal concurrent with CCl4. Hepatic fibrosis was quantified by digital analysis of Masson's trichrome stained slides and hydroxyproline content. mRNA expression was quantified by real-time polymerase chain reaction (PCR), and protein was quantified by western blot or enzyme-linked immunosorbent assay (ELISA). CCl4 treatment induced micronodular liver fibrosis with a pronounced deposition of collagen fibers. HSYA significantly reduced liver fibrosis. HSYA down regulates α-smooth muscle actin (SMA), collagen α type I, matrix metalloproteinases (MMP)-9, and tissue inhibitors of metalloproteinases (TIMP)-1 gene expression. This was accompanied by a decreased expression of transforming growth factor (TGF)-ß1 and phosphorylation of Smad4. These results indicate that HSYA might be a promising antifibrotic agent in chronic liver disease.

Emodin protects rat liver from CCl(4)-induced fibrogenesis via inhibition of hepatic stellate cells activation.

AIM: To investigate the role of emodin in protecting the liver against fibrogenesis caused by carbon tetrachloride (CCl(4)) in rats and to further explore the underlying mechanisms. METHODS: Rat models of experimental hepatic fibrosis were established by injection with CCl(4); the treated rats received emodin via oral administration at a dosage of 20 mg/kg twice a week at the same time. Rats injected with olive oil served as a normal group. Histopathological changes were observed by hematoxylin and eosin staining. The activities of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in serum and hepatic hydroxyproline content were assayed by biochemical analyses. The mRNA and protein relevant to hepatic stellate cell (HSC) activation in the liver were assessed using real-time reverse transcription-polymerase chain reaction (RT-PCR), immunohistochemistry, western blotting and enzyme-linked immunosorbent assay. RESULTS: The degree of hepatic fibrosis increased markedly in the CCl(4) group compared to the normal group (P < 0.01), and decreased markedly in the emodin group compared to the CCl(4) group according to METAVIR scale (P < 0.01) compared with those in the normal control group (51.02 +/- 10.64 IU/L and 132.28 +/- 18.14 IU/L). The activities of serum ALT and AST were significantly higher in rats injected with CCl(4) (289.25 +/- 68.84 IU/L and 423.89 +/- 35.67 IU/L, both P < 0.05). The activities of serum ALT and AST were significantly reduced by administration of emodin (176.34 +/- 47.29 IU/L and 226.1 +/- 44.52 IU/L, both P < 0.05). Compared with the normal controls (54.53 +/- 13.46 mg/g), hepatic hydroxyproline content was significantly higher in rats injected with CCl(4) (120.27 +/- 28.47 mg/g, P < 0.05). Hepatic hydroxyproline content was significantly reduced in the rats treated with emodin at 20 mg/kg (71.25 +/- 17.02 mg/g, P < 0.05). Emodin significantly protected the liver from injury by reducing serum AST and ALT activities and reducing hepatic hydroxyproline content. The mRNA levels of transforming growth factor-beta1 (TGF-beta1), Smad4 and alpha-SMA in liver tissues were significantly down-regulated in SD rats that received emodin treatment. Furthermore, significant down-regulation of serum TGF-beta1 protein levels and protein expression of Smad4 and alpha-SMA in liver tissues was also observed in the rats. Emodin inhibited HSC activation by reducing the abundance of TGF-beta1 and Smad4. CONCLUSION: Emodin protects the rat liver from CCl(4)-induced fibrogenesis by inhibiting HSC activation. Emodin might be a therapeutic antifibrotic agent for the treatment of hepatic fibrosis.