The endophytic fungi of Salvia miltiorrhiza Bge.f. alba are a potential source of natural antioxidants.

BACKGROUND: Salvia miltiorrhiza Bge. f. alba is a traditional Chinese herbal drug with special pharmacological effect on thromboangiitis obliterans. However, the nature source of S.miltiorrhiza Bge.f.alba is now in short supply because of the over-collection of the wild plant. To better utilize this resource, the diversity and antioxidant activity of endophytic fungi isolated from S. miltiorrhiza Bge. f. alba were investigated. RESULTS: A total of 14 endophytic fungi were isolated from different parts of S. miltiorrhiza Bge.f.alba. Based on morphological and molecular identification, the endophytic fungi isolated were classified into four genera (Alternaria sp., Fusarium sp., Schizophyllum sp. and Trametes sp.). These fungal extracts were prepared using ethanol and evaluated for their phytochemical compounds and antioxidant activity. Alternaria alternata SaF-2 and Fusarium proliferatum SaR-2 are of particular interest because they yielded all of nine phytochemicals including saponins, phenol, flavonoids, cardiac glycosides, steroids, tannins, alkaloids, anthroquinone and terpenoids. F. proliferatum SaR-2 and A. alternata SaF-2 also exhibited stronger antioxidant activities by FRAP and DPPH method, having the higher levels of phenol and flavonoid than those of plant root. The total amount of phenol and flavonoid quantified were of 21.75, 20.53 gallic acid equivalent per gram and 8.27 and 7.36 µg/mg of quercetin equivalent respectively. These two endophytic fungi (SaR-2 and SaF-2) were found to have comparable scavenging abilities on both FRAP (1682.21 and 1659.05 µmol/mg, respectively) and DPPH-free radicals (90.14% and 83.25%, respectively, at 0.1 mg/mL). This is the first report about isolation of endophytic fungi from S. miltiorrhiza Bge.f.alba and their antioxidant activities. CONCLUSIONS: These results indicate that the endophytic fungi associated with S. miltiorrhiza Bge.f. alba can be a potential source of novel natural antioxidants.

[Construction of plant expression vectors with PMI gene as selection marker and their utilization in transformation of Salvia miltiorrhiza f. alba].

OBJECTIVE: To construct plant expression pCAMBIA1301-PMI by substituting PMI for hygromycin resistance gene in pCAMBIA1301 and obtain transgenic Salvia miltiorrhiza f. alba using PMI-mannose selection system. METHOD: The 6-phosphomannose isomerase gene (PMI) of Escherichia coli was amplified by PCR. Sequence analysis showed that it shared 100% amino acids identities with the sequences of PMI genes isolates reported in the NCBI. Based on pCAMBIA1305, the plant expression pCAMBIA1305-PMI was constructed successfully by substituting PMI for hygromycin resistance gene in pCAMBIA1305. pCAMBIA1305-PMI was transformed into Agrobacterium tumefaciens LBA4404, and then the leaves of S. miltiorrhiza f. alba were inoculated in LBA4404 with pCAMBIA1305-PMI. RESULT: Plant expression pCAMBIA1301-PMI was successfully constructed and the leaves of S. miltiorrhiza f. alba inoculated in LBA4404 with pCAMBIA1305-PMI were selected on medium supplemented with a combination of 20 g x L(-1) mannose and 10 g x L(-1) sucrose as a carbon source. The transformation efficiency rate was 23.7%. CONCLUSION: Genetic transformation was confirmed by PCR, indicating that a new method for obtaining transgenic S. miltiorrhiza f. alba plants was developed using PMI-mannose selection system.

Cloning, molecular characterization and functional analysis of 1-hydroxy-2-methyl-2-(E)-butenyl-4-diphosphate reductase (HDR) gene for diterpenoid tanshinone biosynthesis in Salvia miltiorrhiza Bge. f. alba.

The enzyme 1-hydroxy-2-methyl-2-(E)-butenyl-4-diphosphate reductase (HDR) is a terminal-acting enzyme in the plastid MEP pathway, which produce isoprenoid precursors. The full-length cDNA of HDR, designated SmHDR1 (Genbank Accession No. JX516088), was isolated for the first time from Salvia miltiorrhiza Bge. f. alba. SmHDR1 contains a 1389-bp open reading frame encoding 463 amino acids. The deduced SmHDR1 protein, which shows high identity to HDRs of other plant species, is predicted to possess a chloroplast transit peptide at the N-terminus and four conserved cysteine residues. Transcription pattern analysis revealed that SmHDR1 has high levels of transcription in leaves and low levels of transcription in roots and stems. The expression of SmHDR1 was induced by 0.1 mM methyl-jasmonate (MeJA) and salicylic acid (SA), but not by 0.1 mM abscisic acid (ABA), in the hairy roots of S. miltiorrhiza Bge. f. alba. Complementation of SmHDR1 in the Escherichia coli HDR mutant MG1655 ara < > ispH demonstrated the function of this enzyme. A functional color assay in E. coli showed that SmHDR1 accelerates the biosynthesis of ß-carotene, indicating that SmHDR1 encodes a functional protein. Overexpression of SmHDR1 enhanced the production of tanshinones in cultured hairy roots of S. miltiorrhiza Bge. f. alba. These results indicate that SmHDR1 is a novel and important enzyme involved in the biosynthesis of diterpenoid tanshinones in S. miltiorrhiza Bge. f. alba.

[Study on effective constituents extracted from fibrous roots of Salvia miltiorrhiza with degrading multi-enzymes from taishan Ganoderma lucidum].

OBJECTIVE: To study the application of degrading multi-enzymes from Ganoderma lucidum in extracting effective constituents from fibrous roots of Salvia miltiorrhiza. METHOD: Effective constituents were extracted from fibrous roots by degrading multi-enzymes of wood fiber. The enzymatic parameters were optimized by the orthogonal design. RESULT: The extraction efficiencies of total tanshinones and total salvianolic acids in the extracts of fibrous roots of S. miltiorrhiza was obtained using optimum enzymolysis process reached 11.923%, 12.465%, respectively, which were 62.794%, 56.086% more than that by conventional non-enzymatic hydrolysis. CONCLUSION: Degrading multi-enzymes of wood fiber can be used to fully extract effective constituents from fibrous roots of S. miltiorrhiza, which provides a new approach for recycling wastes of traditional Chinese medicines.

Perifosine as potential anti-cancer agent inhibits proliferation, migration, and tube formation of human umbilical vein endothelial cells.

Targeting angiogenesis is considered an effective strategy for treating the expansion and metastasis of tumors. The aim of this study is to assess the effects of perifosine, an inhibitor of Akt, on cell proliferation, apoptosis, angiogenesis, and VEGF-induced cell migration in cultured human umbilical vein endothelial cells (HUVECs) in vitro. MTT and cell cycle analysis results indicated that perifosine inhibited the growth of HUVECs in a dose-dependent manner, arrested cell cycle progression at the G(2) phase with regulation the expression of p21 and cyclinB1. Apoptosis induced by the higher concentrations of perifosine in HUVECs was also observed. In addition, tube formation of HUVECs and VEGF-induced cell migration were markedly inhibited by perifosine. Western blotting analysis of cell signaling molecules indicated that perifosine inhibited ERK and p38 phosphorylation in HUVECs. These results suggest that perifosine exerts anti-angiogenic activity in HUVECs and is a promising agent for treatment of angiogenesis related-diseases.

[Cloning, molecular characterization and expression of acireductone dioxygenase (ARD) gene from Salvia miltiorrhiza].

OBJECTIVE: To study the acireductone dioxygenase (designated as SmARD) gene of Salvia miltiorrhiza through bioinformatics and characterization of its tissue expression and response expression on stress in shoot. METHOD: SmARD gene was obtained by sequencing cDNA library constructed by us. BLAST was used for alignment, ORF finder software was applied to find open reading frame, prosite was used to analyze the protein characterization. Semi-quantitative RT-PCR was used to detect the gene expression level. RESULT: The full -length cDNA of SmRAD was 688 bp long with a 591 bp ORF (open reading frame) that putatively encoded a polypeptide of 196 amino acids; with a predicted molecular mass of 23.27 kDa. The deduced amino acid sequence of SmRAD of gene shared high homology with other known RADs. Semi-quantitative RT-PCR analysis indicated that SmRAD was constitutively expressed in roots, stems, flower and leaves of S. miltiorrhiza, with the high expression in roots. In addition, SmRAD expression level under different stress condition was also analyzed in root, including signaling components for plant defence responses, such as methyl jasmonate, salicylic acid and ABA, as well as drought, cold and salt abiotic stress. The expression of SmRAD was suppressed by water deficit treatment for 3 d, 150 mmol x L(-1) NaCl, 4 degrees C cold and 100 mmol x L(-1) ABA treatment for 1 d, but induced by 100 mmol x L(-1) MJ and 10 mmol x L(-1) ETH. CONCLUSION: A novel SmARD gene was cloned from S. miltiorrhiza. This study will enable us to further understand the role of SmARD in the defense response under different abiotic stress and in synthesis of active cmpounds in S. miltiorrhiza at molecular level.

[Exogenous nitric oxide accelerates soluble sugar, proline and secondary metabolite synthesis in Ginkgo biloba under drought stress].

The effects of exogenous nitric oxide donor sodium nitroprusside (SNP) on substance metabolism of Ginkgo biloba leaves under drought stress were studied. The results showed that 250 micromol/L SNP (Fig.2) treatment under 35% relative soil water content (RSWC) stress (Fig.1) raised remarkably soluble sugar content (Fig.3), proline content (Fig.4), phenylalanine ammonia lyase (PAL) activity (Fig.5), flavonoids (Fig.6) and ginkgolides content (Fig.7) of G. biloba leaves. Hemoglobin, used as NO scavenger, counteracted the effects of SNP in raising the soluble sugar (Fig.3), proline (Fig.4), flavonoid (Fig.6), ginkgolide content (Fig.7) and PAL activities (Fig.5), which indicates that the effects of sodium nitroprusside were through the nitric oxide released from sodium nitroprusside. We propose from these results that the roles of flavonoids and ginkgolides are the same as those of soluble sugars and proline under drought stress. NO may alleviate the damage caused by drought stress through raising soluble sugar, proline, flavonoid and ginkgolide content.