Metabolic engineering of the phenylpropanoid pathway enhances the antioxidant capacity of Saussurea involucrata.

The rare wild species of snow lotus Saussurea involucrata is a commonly used medicinal herb with great pharmacological value for human health, resulting from its uniquely high level of phenylpropanoid compound production. To gain information on the phenylpropanid biosynthetic pathway genes in this critically important medicinal plant, global transcriptome sequencing was performed. It revealed that the phenylpropanoid pathway genes were well represented in S. involucrata. In addition, we introduced two key phenylpropanoid pathway inducing transcription factors (PAP1 and Lc) into this medicinal plant. Transgenic S. involucrata co-expressing PAP1 and Lc exhibited purple pigments due to a massive accumulation of anthocyanins. The over-expression of PAP1 and Lc largely activated most of the phenylpropanoid pathway genes, and increased accumulation of several phenylpropanoid compounds significantly, including chlorogenic acid, syringin, cyanrine and rutin. Both ABTS (2,2'-azinobis-3-ethylbenzotiazo-line-6-sulfonic acid) and FRAP (ferric reducing anti-oxidant power) assays revealed that the antioxidant capacity of transgenic S. involucrata lines was greatly enhanced over controls. In addition to providing a deeper understanding of the molecular basis of phenylpropanoid metabolism, our results potentially enable an alternation of bioactive compound production in S. involucrata through metabolic engineering.

Molecular characterization and expression analysis of dihydroflavonol 4-reductase (DFR) gene in Saussurea medusa.

Dihydroflavonol 4-reductase (DFR), which catalyzes the reduction of dihydroflavonols to leucoanthocyanins, is a key enzyme in the biosynthesis of anthocyanidins, proanthocyanidins, and other flavonoids of importance in plant development and human nutrition. This study isolated a full length cDNA encoding DFR, designated as SmDFR (GenBank Accession No. EF600682), by screening a cDNA library from a red callus line of Saussurea medusa, which is an endangered, traditional Chinese medicinal plant with high pharmacological value. SmDFR was functionally expressed in yeast (Saccharomyces cerevisiae) to confirm that SmDFR can readily reduce dihydroquercetin (DHQ) and dihydrokampferol (DHK), but it could not reduce dihydromyricetin (DHM). The deduced SmDFR structure shared extensive sequence similarity with previously characterized plant DFRs and phylogenetic analysis showed that it belonged to the plant DFR super-family. SmDFR also possessed flavanone 4-reductase (FNR) activity and can catalyze the conversion of eridictyol to luteoforol. Real-time PCR analysis showed that the expression level of SmDFR was higher in flowers compared with both leaves and roots. This work greatly enhances our knowledge of flavonoid biosynthesis in S. medusa and marks a major advance that could facilitate future genetic modification of S. medusa.

[Phenolic acid derivatives from Bauhinia glauca subsp. pernervosa].

To study the chemical constituents of Bauhinia glauca subsp. pernervosa, eleven phenolic acids were isolated from a 95% ethanol extract by using a combination of various chromatographic techniques including column chromatography over silica gel, ODS, MCI, Sephadex LH-20, and semi-preparative HPLC. By spectroscopic techniques including 1H NMR, 13C NMR, 2D NMR, and HR-ESI-MS, these compounds were identified as isopropyl O-beta-(6'-O-galloyl)-glucopyranoside (1), ethyl O-beta-(6'-O-galloyl)-glucopyranoside (2), 3, 4, 5-trimethoxyphenyl-(6'-O-galloyl)-O-beta-D-glucopyranoside (3), 3, 4, 5-trimethoxyphenyl-beta-D-glucopyranoside (4), gallic acid (5), methyl gallate (6), ethyl gallate (7), protocatechuic acid (8), 3, 5-dimethoxy-4-hydroxybenzoic acid (9), erigeside C (10) and glucosyringic acid (11). Among them, compound 1 is a new polyhydroxyl compound; compounds 2, 10, and 11 were isolated from the genus Bauhinia for the first time, and the other compounds were isolated from the plant for the first time. Compounds 6 and 8 showed significant protein tyrosine phosphatase1B (PTP1B) inhibitory activity in vitro with the IC50 values of 72.3 and 54.1 micromol x L(-1), respectively.

Quality evaluation of snow lotus (Saussurea): quantitative chemical analysis and antioxidant activity assessment.

Snow lotus is commonly used as a medicinal plant and has great pharmacological value. To protect these endangered plants, in vitro propagation and cell cultures have been established in order to meet the growing market demand. The phenolic composition, antioxidant activities, total phenolic content (TPC) and total flavonoid content (TFC) from three most commonly used species, in vitro propagated lines and the cell cultures were investigated to qualify their pharmacological value. Quantitative analysis showed that the phenolics varied greatly among different species and the same species at different habitats. From this it can be inferred that the phenolics were influenced by genetic background and the environmental conditions. Significant correlations were observed between the antioxidant activity and several phenolics/TPC/TFC, suggesting that the phenolics are a major contributor of the antioxidant activity and are important for quality evaluation of snow lotus. Based on the abundance of phenolics, TPC, TFC and antioxidant activity, the order of the quality for wild species would be Saussurea involucrata > Saussurea medusa > Saussurea gossypiphora. For S. medusa, its quality judged by origin would be Shigatse > Lhasa > Nagqu. For in vitro propagated plants, the matured plants could be a reliable substitute for wild plants, and the dynamics of phenolics is critical for quality control of this monocarpic species. We provide the first report of quality comparison between the wild plants and the cell cultures. The advantages of developing cell cultures as alternatives for plants collected from the wild are discussed.