The current study explored the hepatotoxicity among closed-ring genipin, open-ring tautomer of genipin and gardenia blue that generated from genipin and amino acid reaction using HepaRG cells to identify the material basis of genipin-induced hepatotoxicity in vitro. The effects of temperature, pH value and different kinds of amino acids on the chemical structure tautomerism between closed-ring and open-ring tautomer of genipin and the production of gardenia blue were investigated firstly, which aimed to explicit the conditions that could distinguish the closed-ring genipin and its open-ring tautomer, and the conditions generating gardenia blue, which were applied to prepare different kinds of gardenia blue; the CCK-8 kit was employed to analyze the hepatotoxicity of closed-ring genipin, open-ring tautomer of genipin and gardenia blue. From the results, it was found that, the structure transformation from close-ring to open-ring of genipin could be inhibited under the condition with acid environment; being essential groups to generate gardenia blue, the primary amino group and the open-ring tautomer of genipin reacting to generate the dihydropyridine ring was probably the key structure of gardenia blue; the structure characteristics existed apparent distinction at the reactive temperature of 37 ℃ and 80 ℃; compared to the culture condition with pH = 7.4, the concentration of genipin with close-ring in culture medium was significantly increased at pH = 5, but the cell viability did not decreased; the cell toxicity of gardenia blue was apparently lower than open-ring tautomer of genipin, and even some kinds of gardenia blue showed growth promoting effect on HepaRG cells. Here, it was suggested potentially that open-ring tautomer of genipin be the important material basis to induce hepatotoxicity, which could provide a cue and lay a foundation for the elucidation of the underlying mechanism of genipin-induced hepatotoxicity.

In this study, the ovarian surgery (ovariectomy, OVX) was used to establish the osteoporosis mice model of primary menstruation, in order to evaluate the protective effects and mechanisms of Zhibai Dihuang decotion on postmenopausal osteoporosis (PMOP). The animal experimental protocol has been reviewed and approved by Laboratory Animal Ethics Committee of Jinan University (number: 20210315-03), in compliance with the Institutional Animal Care Guidelines. C57BL/6 mice were divided into five groups, including Sham group, OVX group, low (32 g·kg^-1·day^-1) and high dose (64 g·kg^-1·day^-1) of Zhibai Dihuang decotion groups, positive drug group (alendronate, 9.9 mg·kg^-1·q3d). After modeling, mice were given medication intervention for 8 weeks, and then femoral and tibial tissues were taken to detect indicators such as bone microstructure, bone resorption, and oxidative stress. The experimental results showed that after Zhibai Dihuang decotion administration, the bone microstructure damage caused by OVX surgery was alleviated, and the relevant parameters bone mineral density (BMD), bone volume/total volume (BV/TV), trabecular number (Tb. N) and connectivity density (Conn. D) both significantly increased. At the same time, the number of TRAP positive osteoclasts decreased significantly, and the levels of proteins and genes related to osteoclast differentiation decreased, indicating that Zhibai Dihuang decoction could inhibit the increased activity of osteoclast caused by OVX. Afterwards, network pharmacology was used to construct the active compound action target network of Zhibai Dihuang decotion, and it was found that the target genes of its active ingredients were closely related to the oxidative stress pathway. Finally, the detection results of oxidative stress levels in bone tissues showed that after treatment with Zhibai Dihuang decotion, the levels of oxidative stress products 4-hydroxynonenal (4-HNE) and malondialdehyde (MDA) in bone tissues of mice significantly decreased, while the levels of antioxidant stress substance L-glutathione (GSH) increased. These above results indicated that Zhibai Dihuang decotion can regulate the level of oxidative stress in the body and inhibit osteoclast activity, which played a therapeutic role in PMOP, as well as provided theoretical basis for the prevention and treatment of PMOP with traditional Chinese medicine.

In this study, we investigated the effect of Cigu Xiaozhi formula on HSC-T6 activity in hypoxic microenvironment based on network pharmacology and computer-aided drug design, and predicted and verified its possible targets and related signaling pathways. The potential active components and targets of Cigu Xiaozhi formula were screened by searching Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform (TCMSP), Encyclopaedia of Traditional Chinese Medicine (ETCM) and Bioinformatics Analysis Tool for Molecular Mechanism of Traditional Chinese Medicine (BATMAN-TCM) databases, and the liver fibrosis related targets retrieved from Gene Cards and Pharm GK database were integrated to obtain the potential targets of Cigu Xiaozhi formula in the treatment of liver fibrosis. GO enrichment analysis and KEGG signaling pathway enrichment analysis were performed on Omic Share platform, and Cytoscape software was used to construct the "potential active ingredient-key target-pathway" network. The active components and target proteins were subjected to molecular docking analysis by Auto Dock software. According to the results of molecular dynamics simulation and binding free energy calculation, the top 5 active components with degree were scored. The active components stigmasterol and β-sitosterol were subjected to molecular docking. CoCl₂ was used to induce HSC-T6 cells to construct hypoxia model in vitro. The cell viability was detected by CCK-8 assay, and the optimal time and concentration of hypoxia model of HSC-T6 cells was determined to be 100 µmol·L^-1 CoCl₂ for 24 h. Under hypoxia condition, HSC-T6 cells were activated, the wound healing rate was significantly increased, and the fluorescence signal of activation marker protein α-smooth muscle actin (α-SMA) was significantly enhanced. However, 6% drug-containing serum could inhibit the activation of HSC-T6 cells, and the wound healing rate was significantly decreased, and the fluorescence signal of α-SMA was significantly weakened. Further studies showed that the expressions of hypoxia-inducible factor-1α (HIF-1α), α-SMA and key proteins of Hedgehog (Hh) signaling pathway in HSC-T6 cells were up-regulated under hypoxia, while the expressions of HIF-1α, α-SMA, Patched-1 (Ptch-1) and glioma related oncogene homology-1 (Gli-1) were down-regulated in 6% drug-containing serum group, the YC-1 group and the cyclopamine group. These results indicated that HIF-1α and Hh signaling pathways were involved in the activation of HSC-T6 cells, and the traditional Chinese medicine Cigu Xiaozhi formula could inhibit the activation of HSC-T6 cells, and the mechanism may be related to the inhibition of HIF-1α expression and the blocking of Hh signaling pathway. In conclusion, Cigu Xiaozhi formula can inhibit the activation of HSC-T6 cells by directly acting on HIF-1α and Hh signaling pathway, and exert an anti-hepatic fibrosis effect. The animal experimental protocol has been reviewed and approved by Laboratory Animal Ethics Committee of Gansu University of Chinese Medicine, in compliance with the Institutional Animal Care Guidelines.

The Ca²⁺-activated monovalent cation selective transient receptor potential melastatin 4 (TRPM4) channel expressed in pancreatic β-cells is implicated in the β-cell function and insulin secretion, but how pharmacological function of TRPM4 channel affects membrane excitability of β-cells remains largely unknown. Here, we report that pharmacological inhibition of TRPM4 by specific inhibitor 9-phenanthrol attenuates electrical activities of pancreatic β-cells. In whole-cell current clamp recordings, 9-phenanthrol results in inhibition of action potential frequency induced by tolbutamide of the INS-1 pancreatic β-cells in a dose-dependent manner with an IC₅₀ value of 14.99 ± 7.93 μmol·L^-1. Similarly, 9-phenanthrol also inhibited action potential firing in INS-1 cells stimulated by current injection. Further recordings of β-cells demonstrate the significant inhibitory effects on action potential peak and action potential amplitude by 9-phenanthrol. Taken together, our results show the involvement of TRPM4 channel function in pancreatic β-cells depolarization and action potential, it provides pharmacological experimental methods and theoretical support for the study of TRPM4 channel in pancreatic β-cells.

Eight compounds were isolated from the ethyl acetate fraction of the 80% aqueous ethanol extract of the roots and stems of Rubus pirifolius Smith by AB-8 macroporous resin, silica gel, ODS, Sephadex LH-20 column chromatography, and semi-preparative HPLC. Their structures were identified by spectral analysis such as 1D/2D NMR, MS, UV, IR and by comparison with literature information as rubussecotriterpene A (1), rubussecotriterpene B (2), cecropiacic acid (3), cecropiacic acid 3-methyl ester (4), alphitolic acid (5), betulinic acid (6), betulin (7), and obtusalin (8). Compounds 1 and 2 are new compounds, and compounds 3-8 were isolated from this plant for the first time.

To discover new structural hits, based on the important role of pyrazole ring and fragment of pyridinone carboxylic acid in drug design, novel title pyrazolo[3,4-b]pyridine-4-one-5-carboxylic acid derivatives (10a-10p) were designed and synthesized, the structures were confirmed by spectral data and elemental analyses. The antibacterial and antitumor activities were evaluated by the measured minimum inhibitory concentration (MIC) values against the tested four strains and half inhibitory concentration (IC₅₀) values against the tested four cancer cells, respectively. The results displayed markedly poor antibacterial activity and observably potent antitumor activity. In particularly, the title difluorophenyl (10d, 10e, 10f), pyridyl (10j), ethyl (10k) and cycloproyl (10l) compounds exhibited comparable activity against Capan-1 and A549 cells to that of the comparison doxorubicin. Thus, pyrazolo[3,4-b]pyridine-4-one-5-carboxylic acid derivatives as promising antitumor hits need to be developed.

Seven compounds were isolated from fermentation extract of cave-derived Metarhizium anisopliae NHC-M3-2 by silica gel, semi-preparative HPLC and other chromatographic methods. Their structures were elucidated by UV, IR, MS and NMR methods as 2,3-dehydroindigotide G (1), (-)-regiolone (2), naphtho-γ-pyrone (3), indigotide G (4), indigotide B (5) destruxin A (6) and destruxin B (7). Compound 1 is a new glycoside naphthopyranone compound. The anti-hepatitis B virus (HBV) activity of these compounds was evaluated. The EC₅₀ and CC₅₀ of compound 3 against HBV were 4.5 μmol·L^-1 and 92.3 μmol·L^-1, respectively. This is the first report of the antiviral activity of compound 3.

An LC-MS method with natural isotope abundance correction and a ¹H NMR relative quantitative method were established to determine the deuterium incorporation of donafenib tosilate, a new deuterated drug molecule. First, the peak areas of isotopic impurities (non-deuterated and incompletely deuterated impurities) and deuterated drug were recorded through the single ion monitoring (SIM) mode of the established LC-MS method and then corrected in terms of the natural isotope abundance offered by ChemDraw soft, removing the nature isotope interference from ¹³C, ³⁷Cl, etc. The corrected areas were subsequently used to calculate mol% of isotopologues (D₀, D₁, D₂, D₃) and Atom% D, namely, deuterium incorporation. In addition, a ¹H qNMR experiment was conducted with the aromatic proton at δ 8.63 and the residual proton of isotopic impurities at δ 2.79 as quantitative peaks. The mixture of DMSO-d₆ and D₂O (10∶1) was employed as the solvent to change the spin-coupling between the residual proton and active hydrogen so that the residual proton could be measured as the single peak, and the sensitivity was greatly improved. The acquisition parameters were also optimized, and Atom% ¹H and the deuterium incorporation were then calculated. The two methods were applied to samples of three commercial batches, and the testing results were almost consistent. Both methods proved accurate, sensitive, fast and independent of standard substances and accurate weighing, which could be applied to the determination of the deuterium incorporation of donafenib tosilate and provide a reference for other deuterated drugs.

Sophorae Flavescentis Radix is the dried root of Sophora flavescens Ait. and Sophorae Tonkinensis Radix et Rhizoma is the dried root and rhizome of Sophora tonkinensis Gagnep. The two drugs are both from the same genus Sophora, having similar and different compositions and efficacies, however, their differences are not fully demonstrated in current standard. In this study, the high-performance thin-layer chromatography with multi-dimensional and multi-level features combined with electric spray mass spectrometry (HPTLC-ESI-MS) was used to discover and identify the characteristic zones in extracts of Sophorae Flavescentis Radix and Sophorae Tonkinensis Radix et Rhizoma, after optimizing the preparation method of the test solution and chromatographic parameters. As a result, 17 main characteristic zones were found on HPTLC chromatograms of Sophorae Flavescentis Radix and Sophorae Tonkinensis Radix et Rhizoma, among them, besides 3 known chemicals, another 12 unknown components were identified by HPTLC-ESI-MS, they are 1 alkaloid and 11 flavonoids. The identification results were verified by the reference standards partially and nuclear magnetic resonance spectra after guided-isolation. Finally, a unified HPTLC specific identification method with different markers was established to identify Sophorae Flavescentis Radix and Sophorae Tonkinensis Radix et Rhizoma simultaneously. Thanks to abundant chemical information provided when using diverse polarity mobile phases and derivatization reagents, the HPTLC technology offers a convenient strategy for discovery, quality evaluation, and identification of target chemicals when connecting with mass spectrometry.

Taking berberine (BBR) as an example, to study whether the supramolecular hydrogel formed by berberine and lotus root starch (Nelumbo nucifera Gaertn; LRS), a natural polysaccharide, affects the inhibition to Staphylococcus aureus and the ability of biofilm clearance. The chemical structure and rheological properties of BBR@LRS gel were characterized by infrared spectroscopy and rheometer. The in vitro release of supramolecular hydrogel was observed at pH = 1.2 and pH = 7.4. Broth dilution method and biofilm clearence experiment were used to observe the bacteriostasis and biofilm clearance respectively. Cytotoxicity test and in vitro hemolysis test were used to evaluate the biosafety preliminarily. The results showed that the LRS polysaccharide hydrogel could encapsulate BBR, and there was an interaction between them. The BBR@LRS gel had good rheological properties and biosafety, and played a role in solubility enhancement and slow release of BBR, which was stronger than BBR in inhibiting the growth of Staphylococcus aureus and clearing biofilm. This study provides reference for the effect of natural polysaccharide supramolecular hydrogels on biological functions of active components of traditional Chinese medicine.