[Effect of heavy metal lead on anticoagulant activity of leech based on label-free quantitative proteomics].

Whitmania pigra is the most widely distributed species of leeches in the market. In this study, the effect of heavy metal lead pollution on the anticoagulant activity of Wh. pigra was studied and the potential mechanism was explored. Pb(NO_3)_2 was used to contaminate the breeding soil which was then used to rear Wh. pigra for 50 days(lead-contaminated group, LC group), and meanwhile the blank control group(CG group) was set. Proteins were extracted from the obtained leech samples, and the differentially expressed proteins between LC and CG groups were analyzed by label-free proteomics technology. In this study, a total of 152 differentially expressed proteins were screened out, of which 93 proteins were up-regulated and 59 proteins were down-regulated in LC group. Bioinformatics analysis showed that the biological processes enriched with the differentially expressed proteins were mainly vesicle-mediated transport and transport positive regulation; the enriched cell components were mainly endocytosis vesicles and apical plasma membrane; the enriched molecular functions mainly included carbohydrate binding. The differentially expressed proteins were enriched in 76 KEGG pathways, which mainly involved metabolic pathways, biosynthesis of secondary metabolites, and bacterial invasion of epithelial cells. In this study, two differentially expressed proteins with Antistasin domain were presumed, which provides reference for further exploring the regulatory mechanism and signal transduction underlying the effect of lead pollution on the anticoagulant activity of leech.

A study of the influence of lead pollution on the anticoagulant activity of Whitmania pigra based on pharmacodynamics and metabolomics research.

Whitmania pigra Whitman (leech, also called Shuizhi in China, abbreviated as SZ), which has been used as a traditional Chinese medicine in the treatment of blood stasis syndrome (BSS) for a long time, is vulnerable to lead pollution in aquaculture environments. SZ has good anticoagulant activity. However, there are few studies on the influence of lead pollution on it. Therefore, we carried out the following researches to explore the influence of lead pollution on the anticoagulant activity of SZ and its mechanism. Firstly, the acute blood stasis model of rats was established by subcutaneous injection of adrenaline hydrochloride and ice water bath. Then unpolluted SZ (UPS) and lead-polluted SZ (LPS) were extracted. Next, the blood stasis model rats were administrated by gavage and the rats in normal control (NC) group and blood stasis model (BM) group were given the same amount of normal saline. Finally, the blood of the rats was collected to detect the coagulation function and hemorheology indexes. The metabolomics of rat plasma was studied by ultra-high-performance liquid chromatography coupled with orbitrap mass spectrometry (UPLC-Orbitrap-MS) technology. Principal component analysis (PCA), orthogonal partial least squares discriminant analysis (OPLS-DA) and Hierarchical clustering analysis (HCA) were used to perform metabolomics analysis. MetPA analysis was used to search for related metabolic pathways. The results of coagulation function and hemorheology showed that lead pollution could decrease the anticoagulant activity of SZ. The OPLS-DA score plots indicated that the plasma metabolites of rats in LPS group were close to BM group, while UPS group tended to be close to NC group both in the positive and negative ion mode. Hierarchical cluster analysis (HCA) suggested that UPS group and NC group were clustered into a branch, while LPS group and BM group were clustered into a branch. To sum up, lead pollution will reduce the anticoagulant activity of SZ. And lead pollution reduces the anticoagulant activity of SZ probably by influencing the metabolic pathways such as sphingolipid metabolism, amino acid metabolism and energy metabolism in rats.

[Determination of caffeine content in Ginkgo Folium by ultra high performance liquid chromatography-triple quadrupole mass spectrometry].

In this experiment, an ultra-high performance liquid chromatographytandem triple quadrupole mass spectrometry was established for the determination of caffeine in commercially available Ginkgo Folium. The samples were extracted by ultrasonic method with methanol, and separated on Waters CORTECS T3 column(2.1 mm×100 mm, 2.7 µm), with mobile phase of 0.1% formic acid solution-0.1% formic acid acetonitrile solution for gradient elution, at flow rate of 0.3 mL·min~(-1); column temperature of 30 ℃, and injection volume of 2 µL. Mass spectrometry was conducted at ESI~+ multiple reaction monitoring(MRM) mode; quantitative analysis was conducted with external standard method. The results showed that in the range of 0.099 6-9.96 ng·mL~(-1), there was a good linear relationship between the mass concentration of caffeine and the peak area, R~2=0.999; the average recovery was 84.51%, with RSD of 6.2%. The results of precision, repeatability and stability showed that the RSD was 5.1%, 5.9%, 7.2%, respectively. The content range of caffeine in 10 batches of Ginkgo Folium was 1.52-60.86 µg·kg~(-1). In conclusion, this method is accurate, reliable and reproducible, which provides a reference for the safety study of Ginkgo Folium.

Metabolomics-based study reveals the effect of lead (Pb) in the culture environment on Whitmania pigra.

Whitmania pigra, called Mahuang (MH) in Chinese, has been used as a traditional Chinese medicine for many years and is susceptible to Pb exposure in aquaculture environments. To understand the impact of Pb in the culture environment on MHs, we carried out a 50-day culture of MHs in environments with different levels of Pb pollution. Then, tissue samples of MHs reared in the different Pb-polluted environments were collected and analysed by UPLC-Q/TOF-MS. The results showed that the Pb residue in MHs increased with increasing Pb in the culture environment. There was no significant difference in MH Pb content (P < 0.05) between the low-Pb residue group (PbL) and the blank control group (BC), and those of the middle-Pb residue group (PbM) and the high-Pb residue group (PbH) were significantly different from that of the BC group. Metabolomics results showed significant changes in 24 metabolites in the PbL, PbM and PbH groups, some of which were dose-dependent. These metabolites were mainly lipids, nucleotides, and dipeptides, which are involved in metabolic pathways such as glycerophospholipid metabolism, sphingolipid metabolism, and nucleotide metabolism. Overall, the results proved that metabolomics can be an effective tool to understand the effects of Pb on the metabolic responses of MHs.

Anti-ageing and antioxidant effects of sulfate oligosaccharides from green algae Ulva lactuca and Enteromorpha prolifera in SAMP8 mice.

Oligosaccharides from green algae Ulva lactuca (ULO) and Enteromorpha prolifera (EPO) were used for investigation of anti-ageing effects and the underlying mechanism in SAMP8 mice. The structural properties of ULO and EPO were analyzed by fourier-transform infrared spectroscopy, gas chromatography-mass spectrometry, and agarose gel electrophoresis. These oligosaccharides enhanced the glutathione, superoxide dismutase, catalase, and telomerase levels and total antioxidant capicity, and decreased the levels of malondialdehyde and advanced glycation end products. After ULO and EPO treatment, the levels of inflammatory factors, including IFN-γ, TNF-α, and IL-6, decreased; the BDNF and ChAT levels increased; and hippocampal neurons were protected. Downregulation of the p53 and FOXO1 genes and upregulation of the Sirt1 gene indicated that ULO and EPO have potential therapeutic effects in the prevention of ageing in SAMP8 mice. By 16S rRNA gene high-throughput sequencing, the abundance of Desulfovibrio was discovered to be markedly different in mice treated with ULO and EPO. The abundances of Verrucomicrobiaceae, Odoribacteraceae, Mogibacteriaceae, Planococcaceae, and Coriobacteriaceae were positively correlated with age-related indicators. These results demonstrated that oligosaccharides from U. lactuca and E. prolifera are ideal candidate compounds that can be used in functional foods and pharmaceuticals to prevent ageing.

Anti-diabetic effect of oligosaccharides from seaweed Sargassum confusum via JNK-IRS1/PI3K signalling pathways and regulation of gut microbiota.

Brown seaweed Sargassum confusum (C. Agardh) has been used in traditional Chinese medicine to treat a variety of diseases. The aim of the present study was to evaluate the anti-diabetic effect of oligosaccharides from brown seaweed S. confusum (SCO). The anti-diabetic effect of SCO was evaluated in vivo using high-fat/high-sucrose fed hamsters. Molecular mechanisms of modulating gene expression of specific members of insulin signaling pathways were determined. The components of the intestinal microflora in diabetic animals were also analyzed by high-throughput 16S rRNA gene sequencing. And it was found that SCO had a sequence of sulfated anhydrogalactose and methyl sulfated galactoside units. Fasting blood glucose levels were significantly decreased after SCO administration. Histology showed that SCO could protect the cellular architecture of the liver. SCO could also significantly increase the relative abundance of Lactobacillus and Clostridium XIVa and decrease that of Allobaculum, Bacteroides and Clostridium IV. The active role of SCO in anti-diabetic effect was revealed by its regulation of insulin receptor substrate 1/phosphatidylinositol 3-kinase and c-Jun N-terminal kinase pathways. These results suggested that SCO might be used as a functional material to regulate gut microbiota in obese and diabetic individuals.