Deep chemical identification of phytoecdysteroids in Achyranthes bidentata Blume by UHPLC coupled with linear ion trap-Orbitrap mass spectrometry and targeted isolation

Achyranthes bidentata Blume is widely used as a traditional Chinese medicine with the effects of nourishing the liver and kidneys and strengthening muscles and bones.In this work,a rapid and simple strategy was developed for characterizing phytoecdysteroids by ultra-high-performance liquid chromatography coupled with liner ion trap-Orbitrap mass spectrometry using electrospray ionization in the negative mode.As a result,47 phytoecdysteroids were unambiguously or tentatively characterized.Among them,seven known compounds were identified according to the reference standards along with molecular formula,retention time and fragmentation patterns,while others were mostly potential new compounds.Through targeted isolation,the structures of three new compounds were determined by NMR spectra,which were consistent with LC-MS characterization.The present study provides an efficient method to deeply characterize phytoecdysteroids.

Systematic Evaluation of Pharmacognostic Identification of Polygonum capitatum

[Objectives] To investigate the systematic evaluation of pharmacognostic identification of Polygonum capitatum . [Methods] 10 batches of P. capitatum cultivated in Guizhou were chosen for plant samples. Macroscopical identification was conducted on plant roots, stems, leaves, flowers and fruits. The P. capitatum powder was processed for physical and chemical distinction by FeCl 3 chromogenic reaction, hydrochloric acid magnesium powder reaction, AlCl 3 color development reaction and thin-layer chromatography.Microscope identification was carried out on the powder. Plant genome DNeasy Plant Kit was adopted for DNA molecular marker identification. [Results] The results showed that the stem of P. capitatum was tufted, the leaves were oval, 2 to 5 cm long, and 1 to 2 cm wide;the leaf apex was acute and cuneate at the base, the inflorescence was capitate, paired or solitary;the raceme was erect and nearly spherical, and the perianth was light red. Furthermore, for the chromogenic reaction of FeCl 3 ethanol extract of P. capitatum , appeared blue and turned to dark blue after long time storing at room temperature. For the reaction of hydrochloric acid magnesium powder, the alcohol extract of P. capitatum , exhibited deep red. In the color reaction of AlCl 3, the alcohol extract revealed yellow fluorescence under 360 nm UV lamp. Microscope identification of the powder displayed pollen grains, crystal sheath fibers, cellulose, vessels, starch grains, cork cells, and other characteristic fragments. In addition, DNA barcoding electrophoresis results showed that P. capitatum showed a clear and bright single band near 500 bp, and further sequencing results showed that the sequence differences were mainly concentrated in ITS1 and ITS2 region. [Conclusions] Systematic evaluation for the identification of P. capitatum is established, which combines with macroscopic identification, physicochemical identification, powder microscope identification, and DNA molecular identification. Finally, the original medicinal material is identified as P. capitatum Buch.-Ham. ex D. Don.

Separation and Preliminary Identification of Intestinal Probiotics of Laying Hens

[Objective] The paper was to isolate and identify probiotics in the intestine of laying hens. [Method] The intestinal probiotics in laying hens at peak period were isolated using conventional separation methods; the physical and chemical properties of target strains and in vitro antibacterial effects were measured. Moreover, the safety test of chicks was conducted. [Result] Four strains of lactobacillus were isolated from the jejunum of laying hens, including Lactobacillus plantarum, Bacillus acidophilus, L. delbrueckii subsp. Delbrueckii and L. delbrueckii subsp. Lactis, and their inhibition zone diameters were 18.30, 16.07, 11.45, 17.26 mm, respectively. One strain of Lactobacillus, L. brevis, was isolated from the cecum, with the inhibition zone diameter of 10.26 mm. Three strains of bacillus were isolated from the cecum, including Bacillus subtilis, B. cereus and B. licheniformis, and their inhibition zone diameters were 9.25, 8.46 and 8.37 mm, respectively. Daily drinking 2 billion units of viable bacteria was the safe dosage for chicks. [Conclusion] Eight strains of probiotics had certain inhibitory effect on Escherichia coli O;, and had no toxic side effects to chicks. Lactobacillus had strong antibacterial effect on E. coli O;, while the antibacterial effect of bacillus was relatively weak.

An novel identification method of the environmental risk sources for surfacewater pollution accidents in chemical industrial parks

The chemical industry is a major source of various pollution accidents. Improving the management level of risk sources for pollution accidents has become an urgentdemand for most industrialized countries. In pollution accidents, the released chemicals harm the receptors to some extentdepending on their sensitivity or susceptibility. Therefore, identifying the potential risk sources from such a large number of chemical enterprises has become pressingly urgent. Based on the simulation of thewhole accident process, a novel and expandable identification method for risk sources causingwater pollution accidents is presented. The newlydeveloped approach, by analyzing and stimulating thewhole process of a pollution accident between sources and receptors, can be applied to identify risk sources, especially on the nationwide scale. Three major types of losses, such as social, economic and ecological losses,were normalized, analyzed and used for overall consequence modeling. A specific case study area, located in a chemical industry park （CIP） along the Yangtze River in Jiangsu Province, China,was selected to test the potential of the identification method. The results showed that therewere four risk sources for pollution accidents in this CIP. Aniline leakage in the HS Chemical Plantwould lead to the most serious impact on the surroundingwater environment. This potential accidentwould severelydamage the ecosystem up to3.8 kmdownstream of Yangtze River, and lead to pollution over adistance stretching to 73.7 kmdownstream. The proposed method is easily extended to the nationwide identification of potential risk sources.

Identification and Preliminary Characterization of a New Chemical Affecting Glucosyltransferase Activities Involved in Plant Cell Wall Biosynthesis

Chemical genetics as a part of chemical genomics is a powerful and fast developing approach to dissect biological processes that may be difficult to characterize using conventional genetics because of gene redundancy or lethality and, in the case of polysaccharide biosynthesis, plant flexibility. Polysaccharide synthetic enzymes are located in two main compartments--the Golgi apparatus and plasma membrane-and can be studied in vitro using membrane fractions. Here, we first developed a high-throughput assay that allowed the screening of a library of chemicals with a potential effect on glycosyltransferase activities. Out of the 4800 chemicals screened for their effect on Golgi glucosyltransferases, 66 compounds from the primary screen had an effect on carbohydrate biosynthesis. Ten of these compounds were confirmed to inhibit glucose incorporation after a second screen. One compound exhibiting a strong inhibition effect （ID 6240780 named chemical A） was selected and further studied. It reversibly inhibits the transfer of glucose from UDP-glucose by Golgi membranes, but activates the plasma membrane-bound callose synthase. The inhibition effect is dependent on the chemical structure of the compound, which does not affect endomembrane morphology of the plant cells, but causes changes in cell wall composition. Chemical A represents a novel drug with a great potential for the study of the mechanisms of Golgi and plasma membrane-bound glucosyltransferases.