It has been an active approach to screen the active ingredients in traditional Chinese medicines (TCMs) according to the affinity property between small molecule compounds and biomaterials such as cells, bacteria and proteins. On the other hand, the biomaterials can be immobilized on a solid support before the screening procedure. The immobilization method not only can maintain the biological activities of biomaterials, but also have other advantages such as high efficiency, simple operation, easy to be continuous and automatic, etc. Carrier materials (solid supports) for the immobilization including silica gel, magnetic materials, hollow fiber, and the surface plasma resonance sensor chips have been used to immobilize biomaterials and successfully applied in the screening of active ingredients from TCMs. In this paper, applications of immobilization techniques in the screening of active components from TCMs were reviewed to provide a scientific reference to the future applications.

In this study, 1-(3-(4-chlorophenyl)-5-methylthio-1H-1, 2, 4-triazol-1-yl)-butan-1-one discovered previously in our lab was selected as a inhibitor of human dihydroorotate dehydrogenase (HsDHODH) for structural optimization. The co-crystal of HsDHODH with the hit was obtained and analyzed for guiding the subsequent structural optimization. As a result, a series of novel triazole derivatives were designed and synthesized as potent HsDHODH inhibitors. Among them, compound (3-(4-chlorophenyl)-5-ethylthio-1H-1, 2, 4-triazol-1-yl)-furan-2-yl-methanone displayed high potency in the inhibition of HsDHODH with an IC₅₀ value of 1.50 μmol·L^-1. Meanwhile, the structure-activity relationships were analyzed based on the biological data and the co-crystal structure. These results provide a valuable reference for optimization of 1H-1, 2, 4-triazole derivatives as HsDHODH inhibitors in the future.

Puerarin (PUE), an isoflavone with anti-inflammation, anti-oxidation and neuroprotection effects, has been widely applied to the treatment of cardiovascular diseases in clinics in China. In the current study, we reported that the active pharmaceutical ingredient (API) of marketed products was the PUE monohydrate (PUEMH). During its supersaturated dissolution, the PUE concentration quickly reached a plateau, followed by a gradually concentration decrease to another lower plateau. In order to explore the internal mechanism of above phenomenon, the solid residues after saturated dissolution test were characterized by powder X-ray diffraction (PXRD), thermal gravity analysis (TGA) and Karl Fisher titration (KFT). PXRD suggested that a novel PUE crystal different from PUEMH formed during its dissolution, the following TGA and KFT confirmed the generation of PUE dihydrate (PUEDH) with much lower solubility. Moreover, polyvinylpyrrolidones (PVPK12, PVPK30 and PVPK90) were added in the dissolution medium to investigate their potential inhibition effects on such crystal transformation during dissolution process. We observed that polymers could inhibit the transformation from PUEMH to PUEDH and result in much higher PUE concentration level than that in pure water.

The study aims to investigate the effective components of Semen Ziziphi Spinosae (SZR) in nourishing the heart and tranquilizing the mind. A method of ultra high liquid chromatography (UHPLC) coupled with Q Exactive high resolution mass spectrometry (HR-MS) was developed. Based on the UV spectra, retention time and MS spectra, 25 compounds of SZR extract were identified or tentatively characterized, including 12 flavonoids, 8 triterpenoids saponins, 2 fatty acid and 3 alakoids. The study illuminated the major chemical components. Twenty bioactive components were determined in rat urine after oral administration of SZR extract by "in vitro to in vivo" translation approach, including 16 prototype compounds and 4 metabolites. Spinosin, swertisin, jujuboside A and B were considered as the effective and active constituents in SZR of the sedative and hypnotic effects, which emodies characteristics of multiple components. It was beneficial exploration for searching the effective and active constituents of SZR in nourishing the heart and tranquilizing the mind.

This study was designed to explore the impact of depression on kidney-yang deficiency in rats. Rats were repeatedly injected with hydrocortisone for 21 days to establish the depression model with kidneyyang deficiency. Tolbutamide, chlorzoxazone, theophylline, midazolam, omeprazole and dextromethorphan were used as substrates of CYP2C6, CYP2E1, CYP1A2, CYP3A2, CYP2D1, and CYP2D2 to test the depression impact on drug metabolism. Plasma concentrations of six CYP450 were determined by LC-MS/MS and used as pharmacokinetic parameters. Consequently, metabolism of theophylline, chlorzoxazone and tolbutamide were accelerated significantly in the model relative to the control (P < 0.01), but dextromethorphan, omeprazole and midazolam did not exhibit a significant difference. The present study suggests that depression with kidneyyang deficiency had a strong induction of CYP2E1 and moderate induction of CYP1A2, CYP2C6 in the rat model.

Licorice is one of the most common herbs in traditional Chinese medicine, and classified as top grade in Shen Nong Ben Cao Jing. There are three different original plants of licorice stipulated in Chinese Pharmacopeia, Glycyrrhiza uralensis Fisch., Glycyrrhiza glabra L., and Glycyrrhiza inflata Bat. However, previous investigation showed that the pharmacodynamic effects of the three licorices were quite different. It is very difficult to identify them by the classical identification methods. In order to establish a fast and effective identification method, we collected 240 licorice plants from 21 populations of 7 provinces, and amplified their ITS and psbA-trnH sequences. ITS sequences with a full length of 616 bp and psbA-trnH sequences with a full length of 389 bp were obtained separately. Using DNAMAN to analyze these sequences, 4 variable sites were found in ITS sequences and 2 ITS haplotypes were determined, and 3 variable sites were found in psbA-trnH sequences and 4 psbA-trnH haplotypes were determined. With the combination analysis of ITS and psbA-trnH sequences, the molecular identification method of original licorice was established. Using this method, 40 samples of licorice slices collected from 4 main herbal material markets in China were identified successfully. Furthermore, the contents of 2 triterpenes, 18α-glycyrrhizic acid and 18β-glycyrrhizic acid, and 4 flavonoids, liquiritin, isoliquiritin, liquiritigenin, and isoliquiritigenin in these licorice pieces were examined by HPLC and the results were analyzed using SPSS 21.0. This study provides a new method in identification of licorice, which may serve as a guideline for quality control of licorice slices.

A type Ⅱ ribosome inactivating protein (RIP) gene was cloned from Polyporus umbellatus sclerotia by RT-PCR method. The full open reading frame cDNA sequence of this gene was 873 bp in length and encoded a 290-aa protein with a molecular weight of 32.33 kDa and an isoelectric point of 5.58. Multiple sequence alignment revealed that the deduced amino acids possessed conserved domains of RICIN superfamily protein. A neighbor joining phylogenetic analysis suggests that PuRIP was closely related to RIP in Marasmius oreades. Real time PCR results showed that this gene expressed in all tested tissues of P. umbellatus. Meanwhile, the expression of this gene was significantly up-regulated in the part infected by Armillaria mellea. This result suggested that this PuRIP might played important role with potential biotic stress tolerance of P. umbellatus. Otherwise, we successfully constructed the pET15b-PuRIP plasmid, produced and purified the His-PuRIP fusion protein, which would provide the basic material for polyclonal antibody preparation and gene function research.

Daphnetin is quickly eliminated in rats after dosing, but the mechanism remains unclear. This study was aimed to investigate the in vitro metabolism of daphnetin using rat liver S9 fractions (RLS9). The metabolites formed in RLS9 were identified and the kinetic parameters for different metabolic pathways were determined. HPLC-DAD-MS analysis showed that daphnetin was biotransformed to six metabolites, which were identified as 7 or 8 mono-glucuronide and mono-sulfate, 8-methylate, and 7-suflo-8-methylate. Methylation and glucuronidation of daphnetin exhibited the Michaelis-Menten kinetic characteristics, whereas the substrate inhibition kinetic and the two-site kinetic were observed for 8-sulfate and 7-sulfate formations. Of the 3 conjugation pathways, the intrinsic clearance rate for sulfation was highest, followed by methylation and glucuronidation. By in vitro-in vivo extrapolation of the kinetic data measured in RLS9, the hepatic clearance were estimated to be 54.9 mL·min^-1·kg^-1 which is comparable to the system clearance (58.5 mL·min^-1·kg^-1) observed in rats. In conclusions, the liver might be the main site for daphnetin metabolism in rats. Sulfation, methylation and glucuronidation are important pathways of the hepatic metabolism of daphnetin in rats.

A pre-column derivatization method combined with UHPLC-MS/MS was developed for the simultaneous determination of salidroside and tyrosol in Beagle dog plasma. After protein precipitation by acetonitrile, the liquid supernatant was treated with dansyl chloride under dark conditions at 60℃ for 30 min, and then, the sample solution was extracted using methyl tertiary butyl ether. The multiple reaction monitoring in positive ion mode was used for MS detection of the tested analytes with the specific ion transitions of m/z 534.2→372.0 for salidroside derivative, m/z 372.0→171.0 for tyrosol derivative and m/z 506.0→171.0 for arbutin derivative. The chromatograph separation was achieved on an ACQUITY UPLC^® BEH C₁₈ column (100 mm×2.1 mm, 1.7 μm) with a gradient mobile phase consisting of acetonitrile (0.1% formic acid)-water (10% acetonitrile, 0.1% formic acid) for 9 min. The assay showed a good linearity over the range of 0.02/0.1-20/10 μmol·L^-1 with a lower limit of quantitation of 0.02 and 0.1 μmol·L^-1 for salidroside and tyrosol in dog plasma, respectively. The intra-and inter-day precisions were all less than 8.68%, and the accuracy was within ±11.4%. The established method with a high sensitivity, good specificity and reliability was appropriate for simultaneous determination of salidroside and tyrosol in dog plasma and successfully applied to a pharmacokinetic study after intragastric administration of salidroside to Beagle dogs.

Five cassane diterpenes were isolated from the 95% ethanol extract of the seeds of Caesalpinia bonduc (Leguminosea) by a combination of various chromatographic methods, including silica gel, Sephadex LH-20, and semi-preparative HPLC. On the basis of spectroscopic techniques, their structures were identified as 3β-acetoxy-cassa-12, 14(17), 15-trien-7β-ol (1), caesalmin C (2), caesall E (3), caesalpinin MJ (4), and 1-deacetylcaesalmin C (5). Among them, compound 1 is a new compound and 2, 4, 5 were isolated from the plant for the first time.