Establishment of a genetic transformation system for Codonopsis pilosula callus.

Codonopsis pilosula, a traditional Chinese medicinal and edible plant, contains several bioactive components. However, the biosynthetic mechanism is unclear because of the difficulties associated with functional gene analysis. Therefore, it is important to establish an efficient genetic transformation system for gene function analysis. In this study, we established a highly efficient Agrobacterium-mediated callus genetic transformation system for C. pilosula using stems as explants. After being pre-cultured for 3 days, the explants were infected with Agrobacterium tumefaciens strain GV3101 harboring pCAMBIA1381-35S::GUS at an OD600 value of 0.3 for 15 min, followed by co-cultivation on MS induction medium for 1 day and delayed cultivation on medium supplemented with 250 mg l-1 cefotaxime sodium for 12 days. The transformed calli were selected on screening medium supplemented with 250 mg l-1 cefotaxime sodium and 2.0 mg l-1 hygromycin and further confirmed by PCR amplification of the GUS gene and histochemical GUS assay. Based on the optimal protocol, the induction and transformation efficiency of calli reached a maximum of 91.07%. The establishment of a genetic transformation system for C. pilosula calli lays the foundation for the functional analysis of genes related to bioactive components through genetic engineering technology.

Response of Bioactive Metabolite and Biosynthesis Related Genes to Methyl Jasmonate Elicitation in Codonopsis pilosula.

Bioactive metabolites in Codonopsis pilosula are of particular interest as an immunostimulant. Methyl jasmonate (MeJA) plays an important role in the elicitation of metabolite biosynthesis. Here, we explored the response of metabolites to MeJA elicitation in C. pilosula adventitious roots and multiple shoots. The results showed that the biomass, polysaccharide, and lobetyolin content of adventitious roots exhibited the highest increases with 100 µmol·L-1 MeJA at the 16th day of subculture, whereas the atractylenolide III (a terpenoid) content increased extremely with 50 µmol·L-1 MeJA treatment at the 7th day of subculture. In addition, the biomass and lobetyolin content significantly increased at the 4th day after treatment. Similarly, the polysaccharide and lobetyolin content increased in multiple shoots. Further identification of different metabolites responding to MeJA by ¹H-NMR showed an extremely significant increase of the lobetyolinin level, which coincided with lobetyolin. Accordingly, the precursor, fatty acids, showed a highly significant decrease in their levels. Furthermore, a significant increase in ß-d-fructose-butanol glycoside was detected, which was accompanied by a decrease in the sucrose level. Accordingly, the enzyme genes responsible for terpenoid and carbohydrate biosynthesis, CpUGPase, and CpPMK, were up regulated. In conclusion, MeJA promoted culture growth and accelerated bioactive metabolite accumulation by regulating the expression of the metabolite biosynthesis related genes, CpUGPase and CpPMK in C. pilosula.

[Study on antioxidant activity of flavonoids from leaves of Psidium guajava].

The present study is to study the chemical constituents from ethanol extract of Psidium guajava leaves. The constituents were separated and purified by silica gel column chromaiographios over, macroporous resin D-101, Sephadex LH-20, and ODS. Six flavonoids compounds were isolated and identified as quercetin(1), quercetin-3-O-α-D-arabinopyranoside(2), quercetin-3-O-α-D-ribopyranoside(3), quercetin-3-O-ß-D-galactopyranoside(4), quercetin-3-O-α-D-glucopyranoside(5), and quercetin-3-O-α-D-xylpyranoside(6). The antioxidant effects of six flavonoids was evaluated by scavenging ability of DPPH, superoxide anion, ABTS·âº, and reducing effect of Fe³âº as well as total antioxidant capacity(FRAP). Vitamin C was used as positive control. The results indicated that six flavonoids exhibited significant antioxidant effects.

Protective effects of hyperoside against H2O2-induced apoptosis in human umbilical vein endothelial cells.

The vascular endothelium is important in the physiological homeostasis of blood vessels. Increasing evidence demonstrates that oxidative stress­induced endothelial damage is involved in the pathogenesis of several cardiovascular diseases, including atherosclerosis. Hyperoside, one of major active components from Apocynum venetum L. (Luo­Bu­Ma), which is a traditional Chinese herbal medicine commonly used for the prevention of cardiovascular diseases, exhibits diverse bioactivities, including anti­inflammatory and antioxidant effects. In the present study, the protective effects of hyperoside against hydrogen peroxide (H2O2)­induced apoptosis of human umbilical vein endothelial cells (HUVECs) were investigated. The results demonstrated that hyperoside significantly prevented the loss of cell viability, the increase of endothelial Ca2+ content and apoptosis in H2O2­induced HUVECs. Additionally, reverse transcription-polymerase chain reaction and western blot analysis revealed that hyperoside significantly decreased the mRNA expression levels of B­cell lymphoma (Bcl)­2 associated X protein (Bax), cleaved caspase­3 and phosphorylated­p38, while increasing the mRNA expression of Bcl­2 in H2O2­induced HUVECs. The present findings suggested that hyperoside has protective effects against H2O2­induced apoptosis in HUVECs and serves a key role in the prevention of cardiovascular diseases.

iTRAQ-based quantitative proteomic analysis of the anti-apoptotic effect of hyperin, which is mediated by Mcl-1 and Bid, in H2O2-injured EA.hy926 cells.

Endothelial injury has been implicated in the pathogenesis of many cardiovascular diseases, including thrombotic disorders. Hyperin (quercetin-3-O-galactoside), a flavonoid compound and major bioactive component of the medicinal herb Apocynum venetum L., is commonly used to prevent endothelium dysfunction. However, its mode of action remains unclear. To the best of our knowledge, we have for the first time investigated the protective effect hyperin exerts against H2O2-induced injury in human endothelium-derived EA.hy926 cells using isobaric tags for relative and absolute quantitation (iTRAQ)­based quantitative proteomic analysis. The results showed that H2O2 exposure induced alterations in the expression of 250 proteins in the cells. We noted that the expression of 52 proteins associated with processes such as cell apoptosis, cell cycle and cytoskeleton organization, was restored by hyperin treatment. Of the proteins differentially regulated following H2O2 stress, the anti-apoptotic protein, myeloid cell leukemia-1 (Mcl-1), and the pro-apoptotic protein, BH3-interacting domain death agonist (Bid), exhibited marked changes in expression. Hyperin increased Mcl-1 expression and decreased that of Bid in a dose-dependent manner. In addition, flow cytometric analysis and western blot analysis of the apoptosis-related proteins, truncated BID (tBid), cleaved caspase-3, cleaved caspase-9, Fas, FasL and caspase-8, demonstrated that the rate of apoptosis and the pro-apoptotic protein levels were decreased by hyperin pre­treatment. In the present study we demonstrate that hyperin effectively prevents H2O2­induced cell injury by regulating the Mcl­1­ and Bid-mediated anti­apoptotic mechanism, suggesting that hyperin is a potential candidate for use in the treatment of thrombotic diseases.

[Study on HPLC specific chromatograms of Lu Dangshen].

In this paper, the RP-HPLC specific chromatography was adopted, with DIKMA-C18 (4.6 mm x 250 mm, 5 µm) as the chromatographic column, with a gradient elution compose of acetonitrile and 0.1% phosphoric acid at flow rate of 0.8 mL · min(-1), the detection wavelength was 220 nm. The difference of the HPLC specific chromatograms between the Lu Dangshen and other different base sources and different producing area of Codonopsis Radix was compared, involved in the similarities and differences of the number and the relative peak area of characteristic peaks in the HPLC specific chromatograms. The HPLC specific chromatograms of Lu Dangshen was established and the relative retention times of seven peaks was determined, and the peaks of codonopyrrolidium B, syringin, lobetyolin, tangshenoside I and atractylenoide III were identified; The HPLC specific chromatograms of Lu Dangshen provided a method for scientific evaluation and effective control the quality of Lu Dangshen from Shanxi famous-region.

Protective effects of farrerol against hydrogen-peroxide-induced apoptosis in human endothelium-derived EA.hy926 cells.

Vascular endothelium plays an important role in the physiological homeostasis of blood vessels. Endothelial injury is considered to be implicated in the pathogenesis of many cardiovascular diseases, including atherosclerosis. Farrerol, a flavonoid considered to be the major bioactive component in a traditional Chinese herb, "Man-shan-hong", which is the dried leaves of Rhododendron dauricum L., displays many bioactive properties, including antibechic, antibacterial, anti-inflammatory, and the inhibition of vascular smooth muscle cell (VSMC) proliferation. In this study, the protective effects of farrerol on hydrogen peroxide (H2O2)-induced apoptosis in human endothelium-derived EA.hy926 cells were investigated. The results showed that farrerol significantly inhibited the loss of cell viability and enhanced superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) activities in H2O2-induced EA.hy926 cells. Meanwhile, farrerol inhibited H2O2-induced elevation in the levels of intracellular malondialdehyde and reactive oxygen species, as well as cell apoptosis. Furthermore, real time RT-PCR and Western blot analysis showed that farrerol significantly decreased the expression of Bax mRNA, Bax, cleaved caspase-3, and phosph-p38 MAPK, while increasing the exporession of Bcl-2 mRNA and Bcl-2 in H2O2-induced EA.hy926 cells. These results are the first demonstration that farrerol has protective effects against H2O2-induced apoptosis in EA.hy926 cells, and suggests that farrerol is a potential candidate for the intervention of endothelial-injury-associated cardiovascular diseases.

Novel NGF-potentiating limonoids from the fruits of Melia toosendan.

Four new limonoids (1-4), together with five known limonoids (5-9), were isolated from the fruits of Melia toosendan. Their structures were elucidated based on extensive spectroscopic analyses (1D- and 2D-NMR, HRESIMS, IR, [α](D)). The isolated compounds were evaluated for their neurite outgrowth-promoting activities. Compounds 2 and 6 significantly enhanced NGF-mediated neurite outgrowth in PC12 cells at concentrations ranging from 0.1 to 50.0 µM.

Isolation of a new glycoside from starfish, Asterias amurensis Lutken, and its stimulation activity on the proliferation of the UMR106 cell.

A new glycoside compound (1) was isolated from the starfish Asteria amurensis Lutken. The structure for compound 1 was identified as 1-O-{beta-D-quinovopyranosyl-(1-2)-beta-D-fucopyranosyl-(1-4)-[beta-D-fucopyranosyl(1-2)] beta-D-quinovopyranosyl}-butanol by extensive NMR experiments as well as chemical evidence. The effects of compound 1 on UMR106 cell proliferation were screened by MTT assay. The results indicate that compound 1 (0.01-100 microM) significantly promotes osteoblastic proliferation.

Two new steroidal compounds from starfish Asterias amurensis Lutken.

Two new sulfated steroidal compounds (1 and 2), along with three known steroidal saponins (3, 4, and 5) were isolated from the starfish Asterias amurensis Lutken. The structures of new compounds were elucidated as 3beta-O-sulfated-cholest-5-ene-7alpha-ol (1) and (E) 25-O-beta-d-xylopyranosyl-26, 27-dinor-24(S)-methyl-22-ene-15alpha-O-sulfated-5alpha-cholest-3beta,6alpha-ol (2) by extensive NMR experiments and chemical evidence. Their effects on UMR106 cell proliferation were screened by MTT method. The results indicated that compounds 2 and 2a (0.01-100 microM) significantly promoted the osteoblastic proliferation. The initial structure-activity relationship analysis suggests that the sugar moiety is the necessary group for the activity.