Protective Effect of Silibinin on Lipopolysaccharide-Induced Endotoxemia by Inhibiting Caspase-11-Dependent Cell Pyroptosis.

OBJECTIVE: To explore the protective effect and the underlying mechanism of silibinin (SIB), one of the active compounds from Silybum marianum (L.) Gaertn in endotoxemia. METHODS: Mouse peritoneal macrophage were isolated via intraperitoneally injection of BALB/c mice with thioglycolate medium. Cell viability was assessed using the cell counting kit-8, while cytotoxicity was determined through lactate dehydrogenase cytotoxicity assay. The protein expressions of interleukin (IL)-1 α, IL-1 ß, and IL-18 were determined by enzyme-linked immunosorbent assay. Intracellular lipopolysaccharide (LPS) levels were measured by employing both the limulus amoebocyte lysate assay and flow cytometry. Additionally, proximity ligation assay was employed for the LPS and caspase-11 interaction. Mice were divided into 4 groups: the control, LPS, high-dose-SIB (100 mg/kg), and low-dose-SIB (100 mg/kg) groups (n=8). Zebrafish were divided into 4 groups: the control, LPS, high-dose-SIB (200 εmol/L), and low-dose-SIB (100 εmol/L) groups (n=30 for survival experiment and n=10 for gene expression analysis). The expression of caspase-11, gasdermin D (GSDMD), and N-GSDMD was determined by Western blot and the expressions of caspy2, gsdmeb, and IL-1 ß were detected using quantitative real-time PCR. Histopathological observation was performed through hematoxylineosin staining, and protein levels in bronchoalveolar lavage fluid were quantified using the bicinchoninicacid protein assay. RESULTS: SIB noticeably decreased caspase-11 and GSDMD-mediated pyroptosis and suppressed the secretion of IL-1 α, IL-1 ß, and IL-18 induced by LPS (P<0.05). Moreover, SIB inhibited the translocation of LPS into the cytoplasm and the binding of caspase-11 and intracellular LPS (P<0.05). SIB also attenuated the expression of caspase-11 and N-terminal fragments of GSDMD, inhibited the relative cytokines, prolonged the survival time, and up-regulated the survival rate in the endotoxemia models (P<0.05). CONCLUSIONS: SIB can inhibit pyroptosis in the LPS-mediated endotoxemia model, at least in part, by inhibiting the caspase-11-mediated cleavage of GSDMD. Additionally, SIB inhibits the interaction of LPS and caspase-11 and inhibits the LPS-mediated up-regulation of caspase-11 expression, which relieves caspase-11-dependent cell pyroptosis and consequently attenuates LPS-mediated lethality.

Catalpol rescues LPS-induced cognitive impairment via inhibition of NF-Κb-regulated neuroinflammation and up-regulation of TrkB-mediated BDNF secretion in mice.

ETHNOPHARMACOLOGICAL RELEVANCE: Septic-associated encephalopathy (SAE) is a key manifestation of sepsis. Nevertheless, specific treatment for SAE is still lacking. Catalpol is an active component derived from Rehmanniae Radix, and has been demonstrated to be a potential neuroprotective agent. However, its effect on SAE still needs to be fully explored. AIM: To address the benefits of catalpol on post-sepsis cognitive deterioration and related mechanisms. MATERIALS AND METHODS: Novel object recognition test, temporal order task, histopathology, and immunochemistry were applied to address the benefits of catalpol on LPS-triggered post-sepsis cognitive decline in mice. Xuebijing injection (10 ml/kg) has been utilized as a positive control in the above animal studies. After treatment, the catalpol content in the hippocampus was determined using LC-MS/MS. Finally, the mechanisms of catalpol were further assessed in BV2 and PC12 cells in vitro using Western blot, RT-PCR, flow cytometry, molecular docking tests, thermal shift assay, transmission electron microscopy, and immunofluorescence analysis. RESULTS: Behavior tests showed that catalpol therapy could lessen the cognitive impairment induced by LPS damage. HE, Nissl, immunofluorescence, transmission electron microscopy, and Golgi staining further reflected that catalpol treatment could restore lymphocyte infiltration, blood-brain barrier (BBB) degradation, and the decreasing complexity of dendritic trees. According to LC-MS/MS analysis, catalpol had a 136 ng/mg concentration in the hippocampus. In vitro investigation showed that catalpol could inhibit microglia M1 polarization via blocking NF-κB phosphorylation, translocation and then reducing inflammatory cytokine release in BV2 microglia cells. Brain-derived neurotrophic factor (BDNF) release up-regulation and TrkB pathway activation were observed in the catalpol treatment group in vivo and in vitro. The effect of catalpol on enhancing BDNF expression was inhibited by the specific inhibitor of TrkB (GNF-5837) in PC12 cells. Further molecular docking tests showed that catalpol formed weak hydrophobic bonds with TrkB. Besides, thermal shift assay also reflected that catalpol incubation caused a considerable change in the melting temperature of the TrkB. CONCLUSION: Catalpol alleviates LPS-triggered post-sepsis cognitive impairment by reversing neuroinflammation via blocking the NF-κB pathway, up-regulating neurotrophic factors via the activation of TrkB pathway, and preserving BBB integrity.

[Quality evaluation of Lysimachiae Herba from different habitats based on simultaneous determination of multiple bioactive constituents combined with multivariate statistical analysis].

A method based on ultra-high performance liquid chromatography coupled with triple quadrupole linear ion trap-tandem mass spectrometry(UHPLC-QTRAP-MS/MS) was developed for the simultaneous determination of 41 bioactive constituents of flavonoids, organic acids, nucleosides, and amino acids in Lysimachiae Herba. The content of multiple bioactive constituents was compared among the samples from different habitats. The chromatographic separation was performed in a Waters XBridge®C_(18) column(4.6 mm×100 mm, 3.5 µm) at 30 ℃. The gradient elution was performed with 0.4% methanol(A)-formic acid water(B) as the mobile phase at a flow rate of 0.8 mL·min~(-1), and the multiple-reaction monitoring(MRM) mode was adopted. According to the content of 41 constituents, hierarchical cluster analysis(HCA), orthogonal partial least squares discriminant analysis(OPLS-DA), and gray relational analysis(GRA) were perfomed to comprehensively evaluate the samples from different habitats. The results showed that the 41 constituents exhibited good linear relationship within the tested concentration ranges, with the correlation coefficients(r) greater than 0.999 4. The method featured good precision, repeatability, and stability with the relative standard deviations(RSDs) less than 5.0%. The average recoveries of the 41 constituents ranged from 98.06% to 101.9%, with the RSDs of 0.62%-4.6%. HCA and OPLS-DA separated 48 batches of Lysimachiae Herba samples from different habitats into three categories: the producing areas in Sichuan and Chongqing, the producing areas in Jiangsu, Zhejiang, and Jiangxi, and the producing areas in Guizhou. The content of 41 constituents varied among the Lysimachiae Herba samples from different habitats. The GRA results revealed that the Lysimachiae Herba sample from Nanchong City, Sichuan Province had the best comprehensive quality. The method developed in this study was accurate and reliable and thus can be used for comprehensive evaluation of Lysimachiae Herba quality and provide basic information for the selection of habitats.

Ethyl Lithospermate Reduces Lipopolysaccharide-Induced Inflammation through Inhibiting NF-κB and STAT3 Pathways in RAW 264.7 Cells and Zebrafish.

OBJECTIVE: To explore the anti-inflammatory effects of ethyl lithospermate in lipopolysaccharide (LPS)-stimulated RAW 264.7 murine-derived macrophages and zebrafish, and its underlying mechanisms. METHODS: 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazoliumbromide (MTT) assays were performed to investigate the toxicity of ethyl lithospermate at different concentrations (12.5-100 µ mol/L) in RAW 264.7 cells. The cells were stimulated with LPS (100 ng/mL) for 12 h to establish an inflammation model in vitro, the production of pro-inflammatory cytokines interleukin (IL)-6 and tumor necrosis factor α (TNF-α) were assessed by enzyme linked immunosorbent assay (ELISA). Western blot was used to ascertain the protein expressions of signal transducer and activator of transcription 3 (STAT3), nuclear factor kappa B (NF-κB) p65, phospho-STAT3 (p-STAT3, Tyr705), inhibitor of NF-κB (IκB) α, and phospho-I κB α (p-IκB α, Ser32), and confocal imaging was used to identify the nuclear translocation of NF-κB p65 and p-STAT3 (Tyr705). Additionally, the yolk sacs of zebrafish (3 days post fertilization) were injected with 2 nL LPS (0.5 mg/mL) to induce an inflammation model in vivo. Survival analysis, hematoxylin-eosin (HE) staining, observation of neutrophil migration, and quantitative real-time polymerase chain reaction (qRT-PCR) were used to further study the anti-inflammatory effects of ethyl lithospermate and its probable mechanisms in vivo. RESULTS: The non-toxic concentrations of ethyl lithospermate have been found to range from 12.5 to 100 µ mol/L. Ethyl lithospermate inhibited the release of IL-6 and TNF-α(P<0.05 or P<0.01), decreased IκBα degradation and phosphorylation (P<0.05) as well as the nuclear translocation of NF-κB p65 and p-STAT3 (Tyr705) in LPS-induced RAW 264.7 cells (P<0.01). Ethyl lithospermate also decreased inflammatory cells infiltration and neutrophil migration while increasing the survival rate of LPS-stimulated zebrafish (P<0.05 or P<0.01). In addition, ethyl lithospermate also inhibited the mRNA expression levels of of IL-6, TNF-α, IκBα, STAT3, and NF-κB in LPS-stimulated zebrafish (P<0.01). CONCLUSION: Ethyl lithospermate exerts anti-Inflammatory effected by inhibiting the NF-κB and STAT3 signal pathways in RAW 264.7 macrophages and zebrafish.

Liang-Ge-San inhibits dengue virus serotype 2 infection by reducing caveolin1-induced cytoplasmic heat shock protein 70 translocation into the plasma membrane.

BACKGROUND: Dengue virus (DENV) is a major public health threat. However, there are no specific therapeutic drugs for DENV. Many Chinese heat-cleaning formulas, such as Liang-Ge-San (LGS), have been frequently used in the virus-induced diseases. The antiviral effect of LGS has not been reported yet. PURPOSE: In this study, the effect of LGS on the inhibition of dengue virus serotype 2 (DENV-2) was investigated and the relevant mechanism was explored. METHODS: High-performance liquid chromatography was applied to analyze the chemical characterization of LGS. The in vitro antiviral activities of LGS against DENV-2 were evaluated by time-of-drug-addition assay. The binding of heat shock protein 70 (Hsp70) and envelope (E) protein or caveolin1 (Cav1) were analyzed by immunofluorescence and immunoprecipitation assays. Then the role of Cav1 in the anti-DENV-2 effects of LGS was further examined. DENV-2 infected Institute of Cancer Research suckling mice (n = 10) and AG129 mice (n = 8) were used to examine the protective effects of LGS. RESULTS: It was found that geniposide, liquiritin, forsythenside A, forsythin, baicalin, baicalein, rhein, and emodin maybe the characteristic components of LGS. LGS inhibited the early stage of DENV-2 infection, decreased the expression levels of viral E and non-structural protein 1 (NS1) proteins. LGS also reduced E protein and Hsp70 binding and attenuated the translocation of Hsp70 from cytoplasm to the cell membrane. Moreover, LGS decreased the binding of Hsp70 to Cav1. Further study showed that the overexpression of Cav1 reversed LGS-mediated E protein and Hsp70 inhibition in the plasma membrane. In the in vivo study, LGS was highly effective in prolonging the survival time, reducing viral loads. CONCLUSION: This work demonstrates for the first time that LGS exerts anti-DENV-2 activity in vitro and in vivo. LGS decreases DENV-2-stimulated cytoplasmic Hsp70 translocation into the plasma membrane by Cav1 inhibition, thereby inhibiting the early stage of virus infection. These findings indicate that LGS may be a candidate for the treatment of DENV.

[Analysis and evaluation of bioactive constituents from different parts of Epimedium brevicornum].

A comprehensive analytical method based on ultra-fast liquid chromatography coupled with triple quadrupole/linear ion trap tandem mass spectrometry(UFLC-QTRAP-MS/MS) was established for simultaneous determination of the content of 45 bioactive constituents including flavonoids, alkaloids, amino acids, phenolic acids, and nucleosides in Epimedium brevicornum. The multiple bioactive constituents in leaves, petioles, stems and rhizomes of E. brevicornum were analyzed. The gradient elution was performed at 30 ℃ in an XBridge~® C_(18) column(4.6 mm×100 mm, 3.5 µm) with 0.4% formic acid aqueous solution-acetonitrile as the mobile phase at a flow rate of 0.8 mL·min~(-1). Single factor experiment and response surface methodology were employed to optimize the extraction conditions. Multivariate statistical analyses including systematic cluster analysis(SCA), principal component analysis(PCA), partial least squares discriminant analysis(PLS-DA), and one-way analysis of variance(One-way ANOVA) were carried out to classify the samples from different parts and identify different constituents. Grey relation analysis(GRA) and entropy weight-TOPSIS analysis were performed to build a multi-index comprehensive evaluation model for different parts of E. brevicornum. The results showed that there was a good relationship between the mass concentrations of 45 constituents and the corresponding peak areas, with the correlation coefficients(r) not less than 0.999 0. The precision, repeatability, and stability of the established method were good for all the target constituents in this study, with the relative standard deviations(RSDs) less than 5.0%(0.62%-4.9%) and the average recovery of 94.51%-105.7%. The above results indicated that the bioactive constituents varied in different parts of E. brevicornum, and the overall quality followed the trend of leaves > petioles > rhizomes > stems. This study verified the rationality of the Chinese Pharmacopoeia(2020 edition) stipulating that the medicinal part of E. brevicornum is the leaf. Moreover, our study indicated that the rhizome had the potential for medicinal development. The established method was accurate and reliable, which can be used to comprehensive evaluate and control the quality of E. brevicornum. This study provides data reference for clarifying the medicinal parts and rationally utilizing the resources of E. brevicornum.

Salvianolic acids from Salvia miltiorrhiza Bunge and their anti-inflammatory effects through the activation of α7nAchR signaling.

ETHNOPHARMACOLOGICAL RELEVANCE: Cardiovascular disease (CVD) is a serious disease with a high incidence rate and mortality. Inflammation is closely related to the occurrence of CVDs. As an essential medicine of promoting blood circulation and removing blood stasis in China, Salvia miltiorrhiza Bunge (Danshen) is widely used to treat CVDs due to its anti-inflammatory and cardiovascular protective effects. Salvianolic acids are the most abundant component in the water extract of S. miltiorrhiza, which has a significant effect on the treatment of CVDs. However, due to the complex composition of salvianolic acids, the active molecules and their underlying mechanisms have not been fully explored. AIM OF THIS STUDY: The present study aims to isolate and identify salvianolic acids from Danshen with anti-inflammatory activity and explore the potential mechanisms of isolates. METHODS: The structures of isolated salvianolic acids were elucidated by UV, IR, NMR, MS and electronic circular dichroism (ECD) calculations. Then anti-inflammatory activities of isolates were screened out by the zebrafish inflammation models. The most active compound was further used to explore the anti-inflammatory mechanisms on LPS-stimulated RAW 264.7 cells. The key inflammatory cytokines IL-6 and TNF-α were measured by enzyme-linked immunosorbent assay (ELISA). The protein expression levels of STAT3, p-STAT3 (Tyr705), NF-κB p65, IκBα, p-IκBα (Ser32) and α7nAchR were determined by Western blotting. The nuclear translocation of p-STAT3 (Tyr705) and NF-κB p65 was evaluated by immunofluorescence assays. Finally, the in vivo anti-inflammatory mechanisms were investigated by observation of neutrophil migration, H&E staining, survival analysis and quantitative PCR (Q-PCR) in LPS-microinjected zebrafish. RESULTS: Two new and four known compounds were isolated from Danshen. Among them, isosalvianolic acid A-1 (C1) and ethyl lithospermate (C5) inhibited neutrophil migrations in three zebrafish inflammation models and C1 with the best activities decreased the secretion of IL-6 and TNF-α and inhibited the expression level of p-IκBα (Ser32) in LPS stimulated RAW 264.7 cells. In addition, C1 also reduced the nuclear translocation of NF-κB p65 and p-STAT3 (Tyr705). Moreover, C1 significantly upregulated the protein expression of α7nAchR, and the knockdown of α7nAchR counteracted the effects of C1 on the production of IL-6 and TNF-α and the expression levels of p-STAT3 (Tyr705), NF-κB p65 and p-IκBα (Ser32). In vivo experiments, C1 decreased the migration and infiltration of inflammatory cells, increased the survival ratio and inhibited the mRNA level of IL-6, TNF-α, STAT3, NF-κB and IκBα in LPS-microinjected zebrafish. CONCLUSION: Two new and four known compounds were isolated from Danshen. Among them, C1 exerted anti-inflammatory activities by activating α7nAchR signaling and subsequently inhibiting STAT3 and NF-κB pathways. This study provided evidence for the clinical application of Danshen and contributed to the development of C1 as a novel in the treatment of cardiovascular disease.

N-Butanol Extract of Glycyrrhizae Radix et Rhizoma Inhibits Dengue Virus through Targeting Envelope Protein.

BACKGROUND: At present, about half of the world's population is at risk of being infected with dengue virus (DENV). However, there are no specific drugs to prevent or treat DENV infection. Glycyrrhizae Radix et Rhizome, a well-known traditional Chinese medicine, performs multiple pharmacological activities, including exerting antiviral effects. The aim of this study was to investigate the anti-DENV effects of n-butanol extract from Glycyrrhizae Radix et Rhizome (GRE). METHODS: Compounds analysis of GRE was conducted via ultra-performance liquid chromatography/tandem mass spectrometry (UHPLC-MS/MS). The antiviral activities of GRE were determined by the CCK-8 assay, plaque assay, qRT-PCR, Western blotting, and the immunofluorescence assay. The DENV-infected suckling mice model was constructed to explore the antiviral effects of GRE in vivo. RESULTS: Four components in GRE were analyzed by UHPLC-MS/MS, including glycyrrhizic acid, glycyrrhetnic acid, liquiritigenin, and isoliquiritigenin. GRE inhibited the attachment process of the virus replication cycle and reduced the expression of the E protein in cell models. In the in vivo study, GRE significantly relieved clinical symptoms and prolong survival duration. GRE also significantly decreased viremia, reduced the viral load in multiple organs, and inhibited the release of pro-inflammatory cytokines in DENV-infected suckling mice. CONCLUSIONS: GRE exhibited significant inhibitory activities in the adsorption stage of the DENV-2 replication cycle by targeting the envelope protein. Thus, GRE might be a promising candidate for the treatment of DENV infection.

Discovery of anthraquinones as DPP-IV inhibitors: Structure-activity relationships and inhibitory mechanism.

Dipeptidyl peptidase IV (DPP-IV) is an integrated type II transmembrane protein that reduces endogenous insulin contents and increases plasma glucose levels by hydrolyzing glucagon-like peptide-1 (GLP-1). Inhibition of DPP-IV regulates and maintains glucose homeostasis, making it an attractive drug target for the treatment of diabetes II. Natural compounds have tremendous potential to regulate glucose metabolism. In this study, we examined the DPP-IV inhibitory activity of a series of natural anthraquinones and synthetic structural analogues on DPP-IV using fluorescence-based biochemical assays. The inhibitory efficiency differed among anthraquinone compounds with different structures. Alizarin (7), aloe emodin (11), emodin (13) emerged the outstanding inhibitory potential for DPP-IV with IC50 values lower than 5 µM. To clarifying the inhibitory mechanism, inhibitory kinetics were performed, which showed that alizarin red S (8) and 13 were effective non-competitive inhibitors of DPP-IV, while alizarin complexone (9), rhein (12), and anthraquinone-2-carboxylic acid (23) were mixed inhibitors. Emodin was determined as inhibitor with the strongest DPP-IV-binding affinity determined via molecular docking. Structure-activity relationship (SAR) demonstrated that hydroxyl group at C-1 and C-8 sites and hydroxyl, hydroxymethyl or carboxyl group at the C-2 or C-3 site were very essential for DPP-IV inhibition, replacement of hydroxyl group with amino group at C-1 could led to an increase of the inhibitory potential. Further fluorescence imaging showed that both compounds 7 and 13 significantly inhibited DPP-IV activity in RTPEC cells. Overall, the results indicated that anthraquinones would be a natural functional ingredient for inhibiting DPP-IV and provided new ideas for searching and developing potential antidiabetic compounds.

Postpartum dietary intake, depression and the concentration of docosahexaenoic acid in mature breast milk in Wuhan, China.

Background: Docosahexaenoic acid (DHA, C22:6) is an important fatty acid in breast milk and is essential for infantile growth and cognitive development. However, the factors that affect the DHA concentration in breast milk have not been completely clarified. Objective: This study aimed to characterize the composition of breast milk fatty acids and to identify maternal factors associated with breast milk DHA concentration in postpartum women in Wuhan, China. Methods: In this cross-sectional study, we analyzed milk fatty acids in 115 lactating women at 30-120 days postpartum using GC-MS. Maternal sociodemographic, health and other information were collected using a self-reported questionnaire. Maternal dietary intake information was collected through a 24-hour dietary recall method. Postpartum depression status was identified using the Edinburgh Postnatal Depression Scale (EPDS). Results: The mean DHA proportion in breast milk was 0.49%. The multivariate regression model showed that the milk DHA proportion was positively associated with maternal aquatic product intake (ß = 0.183, 95%CI: 0.052, 0.314) and DHA supplement use (ß = 0.146, 95%CI: 0.108, 0.185), and negatively associated with postpartum depression status (ß = -0.122, 95%CI: -0.243, -0.002) after adjustment for several maternal and infant factors. Conclusion: Increasing maternal aquatic product intake and DHA supplement use and improving postpartum depression status may increase DHA concentration in breast milk in lactating women.

Hybrid Therapeutic Device (CUHK-OA-M2) for Relieving Symptoms Induced by Knee Osteoarthritis.

The symptoms of knee osteoarthritis (KOA) severely affect the life quality of the elderly population. Low-level laser therapy, heat therapy, and massage therapy are widely used as independent treatments for joint disorders. However, there are very limited reports of a combination of these therapies into an integrated device for KOA so far. This study aims to develop a novel hybrid therapeutic device that can meet various requirements for knee therapy. Our hybrid therapeutic device (CUHK-OA-M2) integrated with low-level laser therapy, heat therapy, and local massage therapy can effectively provide patients with KOA with relief from their clinical symptoms. A pilot test of 50 community-dwelling elderly volunteers with KOA was performed. Finally, 43 volunteers completed two treatment periods (30 days each) and two post-treatment periods (30 days each). The Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC) scores were collected and analyzed after each period. The outputs of the low-level laser, heating, and massage therapies significantly decreased the WOMAC scores in terms of pain, stiffness, function and total WOMAC after two treatment periods (p < 0.05). Although the score increased slightly after the post-treatment period, it was still lower than the baseline, indicating the treatment outcome could last for an extended period. Therefore, our CUHK-OA-M2 device, as an integrated multi-functional hybrid therapeutic device, is therapeutically significant for treating osteoarthritis symptoms on the knee joints of elderly subjects.

Serum Fat-Soluble Vitamin Levels of 6,082 Minors in Zhuzhou City.

Objective: To explore the nutritional status of serum fat-soluble vitamins such as vitamin A, 25-hydroxyvitamin D, and vitamin E of minors in the Zhuzhou area to provide a scientific basis for clinical guidance to supplement fat-soluble vitamins reasonably. Method: A total of 6,082 minors who underwent physical examination from January 2017 to February 2019 in the Children's Health Department of Zhuzhou Hospital affiliated with XiangYa School of Medicine of Central South University were selected as the subjects to measure the levels of serum fat-soluble vitamins A, D, and E. Results: (1) Their average levels of serum vitamin A, 25-hydroxyvitamin D, and vitamin E were (0.34 ± 0.08) mg/mL, (34.65 ± 10.24) ng/mL, and (10.11 ± 2.65) mg/mL, respectively. (2) Serum vitamin E showed a gender difference (P < 0.001). (3) The average levels of serum 25-hydroxyvitamin D and vitamin E in infancy, early childhood, preschool age, school age, and adolescence decreased gradually (P < 0.05). In contrast, the average level of serum vitamin A ranged between 0.32 mg/mL and 0.37 mg/mL. (4) The age was negatively correlated with serum 25-hydroxyvitamin D (r = -0.517, P < 0.001) and weakly negatively correlated with vitamin E (r = -0.366, P < 0.001), but weakly positively correlated with vitamin A (r = 0.269, P < 0.001). Conclusion: Minors from infancy to adolescence in Zhuzhou should strengthen their supplementation of fat-soluble vitamins.

Evaluation of the effects of solar withering on nonvolatile compounds in white tea through metabolomics and transcriptomics.

The mechanism through which solar withering (SW) affects the quality of white tea is unclear. To address this gap in the literature, in this study, we used metabolomics and transcriptomics to investigate the effect of SW on the quality of WT. WT that underwent SW was slightly more bitter and astringent than WT that underwent natural withering (control group). Specifically, SW considerably increased the concentration of astringent flavonoids and flavone glycosides in WT. This increase was mainly attributed to the upregulated expression of key genes in the shikimic acid, phenylpropanoid, and flavonoid biosynthesis pathways, such as shikimate kinase, chalcone synthase, and flavonol synthase. In addition, SW experienced considerable heat and light stress. The levels of glycerophosphatidylcholine and carbohydrates increased in response to the stress, which also affected the taste of WT. The results of this study indicate the mechanism through which SW affects the quality of WT.

Methyl Gallate Suppresses the Migration, Invasion, and Epithelial-Mesenchymal Transition of Hepatocellular Carcinoma Cells via the AMPK/NF-κB Signaling Pathway in vitro and in vivo.

Methyl gallate (MG), a polyphenolic compound found in plants, is widely used in traditional Chinese medicine. MG is known to alleviate several cancer symptoms. However, most studies that have reported the antitumor effects of MG have done so at the cellular level, and the inhibitory effect and therapeutic mechanism of MG in hepatocellular carcinoma (HCC) have not been extensively explored in vivo. We aimed to understand the therapeutic mechanism of MG in HCC in vitro and in vivo. MTT and colony formation assays were used to determine the impact of MG on the proliferation of a human HCC cell line, BEL-7402; wound healing and transwell assays were used to quantify the migration and invasion of HCC cells. Western blotting was used to quantify the expression of the AMPK/NF-κB signaling pathway proteins. In vivo tumor growth was measured in a xenograft tumor nude mouse model treated with MG, and hematoxylin-eosin staining and immunohistochemistry (IHC) were used to visualize the histological changes in the tumor tissue. We found that MG showed anti-proliferative effects both in vitro and in vivo. MG downregulated the protein expression of AMPK, NF-κB, p-NF-κB, and vimentin and upregulated the expression of E-cadherin in a dose-dependent manner. Additionally, MG inhibited the migration and invasion of HCC cells by decreasing MMP9 and MMP2 expression and increasing TIMP-2 expression. These were consistent with the results of IHC in vivo. MG inhibited the proliferation, migration, and invasion of HCC cells. This effect potentially involves the regulation of the AMPK/NF-κB pathway, which in turn impacts epithelial-mesenchymal transition and MMP expression.

Expression Analysis of Lanosterol Synthase Gene in Dynamic Accumulation of Triterpenoids in Sanghuangporus baumii.

BACKGROUND: Sanghuangporus baumii is a traditional Chinese medicine with anti- cancer, anti-tumor, and anti-inflammatory effects. Triterpenoids are one of the main medicinal ingredients found in S. baumii. However, the dynamic changes of triterpenoids content and its molecular regulation mechanism are still unclear. OBJECTIVE: Some studies have shown that Lanosterol synthase ( LS) is a key enzyme involved in the mevalonate pathway (MVA pathway) to produce lanosterol, which is a precursor for synthesizing S. baumii triterpenoids. Therefore, the study of LS gene and expression characteristics can provide clues for the further study of triterpenoids synthesis. METHODS: The PCR, RACE PCR, RT-PCR, homologous recombination and prokaryotic expression technology were used to research the gene characteristic and dynamic changes of LS transcription level. RESULTS: The S. baumii LS sequence included a 5'-untranslated region (129 bp), a 3'-untranslated region (87 bp), and an open reading frame (2,229 bp) encoding 734 amino acids. The S. baumii LS protein was expressed in E. coli BL21 (DE3). The transcription start site of the S. baumii LS promoter sequence ranged from 1 740 bp to 1790 bp. The LS promoter contained 12 CAAT-boxes, 5 ABREs, 6 G-Boxes, 6 CGTCA-motifs, and so on. The LS transcription levels were the highest on day 11 in mycelia (1.6-fold), and the triterpenoids content also gradually increased. The transcription levels began to decrease on day 13, but the triterpenoids content still increased. CONCLUSION: The S. baumii LS was cloned and characterized to help to understand the mechanism of triterpenoids synthesis. In addition, we studied the relationship between LS transcription level and triterpenoid dynamic accumulation, and we found that they had a certain correlation.

Inhibition of drug-metabolizing enzymes by Jingyin granules: implications of herb-drug interactions in antiviral therapy.

Jingyin granules, a marketed antiviral herbal medicine, have been recommended for treating H1N1 influenza A virus infection and Coronavirus disease 2019 (COVID-19) in China. To fight viral diseases in a more efficient way, Jingyin granules are frequently co-administered in clinical settings with a variety of therapeutic agents, including antiviral drugs, anti-inflammatory drugs, and other Western medicines. However, it is unclear whether Jingyin granules modulate the pharmacokinetics of Western drugs or trigger clinically significant herb-drug interactions. This study aims to assess the inhibitory potency of the herbal extract of Jingyin granules (HEJG) against human drug-metabolizing enzymes and to clarify whether HEJG can modulate the pharmacokinetic profiles of Western drug(s) in vivo. The results clearly demonstrated that HEJG dose-dependently inhibited human CES1A, CES2A, CYPs1A, 2A6, 2C8, 2C9, 2D6, and 2E1; this herbal medicine also time- and NADPH-dependently inhibited human CYP2C19 and CYP3A. In vivo tests showed that HEJG significantly increased the plasma exposure of lopinavir (a CYP3A-substrate drug) by 2.43-fold and strongly prolonged its half-life by 1.91-fold when HEJG (3 g/kg) was co-administered with lopinavir to rats. Further investigation revealed licochalcone A, licochalcone B, licochalcone C and echinatin in Radix Glycyrrhizae, as well as quercetin and kaempferol in Folium Llicis Purpureae, to be time-dependent CYP3A inhibitors. Collectively, our findings reveal that HEJG modulates the pharmacokinetics of CYP substrate-drug(s) by inactivating CYP3A, providing key information for both clinicians and patients to use herb-drug combinations for antiviral therapy in a scientific and reasonable way.

Quality Evaluation of Taxilli Herba from Different Hosts Based on Simultaneous Determination of Multiple Bioactive Constituents Combined with Multivariate Statistical Analysis.

Taxilli Herba (TAXH) is an important traditional Chinese medicine with a long history, dating from the Eastern Han Dynasty to the present times. However, the active constituents in it that parasitize different hosts vary, affecting its clinical efficacy. Given the complexity of the host origins, evaluating the quality of TAXH is critical to ensure the safety and effectiveness of clinical medication. In the present study, a quantitative method based on ultra-fast liquid chromatography tandem triple quadrupole mass spectrometry (UFLC-QTRAP-MS/MS) was established, which simultaneously determined the content of 33 active constituents, including 12 flavonoids, 4 organic acids, 12 amino acids, and 5 nucleosides in 45 samples. Orthogonal partial least squares discriminant analysis (OPLS-DA) was employed to classify and distinguish between TAXH and its adulterants, Tolypanthi Herba (TOLH). A hierarchical clustering analysis (HCA) was conducted combined with a heatmap to visually observe the distribution regularity of 33 constituents in each sample. Furthermore, gray relational analysis (GRA) was applied to evaluate the quality of samples to get the optimal host. The results demonstrated that TAXH excelled TOLH in quality as a whole. The quality of TAXH parasitizing Morus alba was also better, while those that were parasitic on Cinnamomum camphora and Glyptostrobus pensilis had relatively poor quality. This study may provide comprehensive information that is necessary for quality control and supply a scientific basis for further exploring the quality formation mechanism of TAXH.

Carboxylesterase inhibitors from clinically available medicines and their impact on drug metabolism.

Mammalian carboxylesterases (CES), the key members of the serine hydrolase superfamily, hydrolyze a wide range of endogenous substances and xenobiotics bearing ester or amide bond(s). In humans, most of identified CES are segregated into the CES1A and CES2A subfamilies. Strong inhibition on human CES (including hCES1A and hCES2A) may modulate pharmacokinetic profiles of CES-substrate drugs, thereby changing the pharmacological and toxicological responses of these drugs. This review covered recent advances in discovery of hCES inhibitors from clinically available medications, as well as their impact on CES-associated drug metabolism. Three comprehensive lists of hCES inhibitors deriving from clinically available medications including therapeutic drugs, pharmaceutical excipients and herbal medicines, alongside with their inhibition potentials and inhibition parameters, are summarized. Furthermore, the potential risks of hCES inhibitors to trigger drug/herb-drug interactions (DDIs/HDIs) and future concerns in this field are highlighted. Potent hCES inhibitors may trigger clinically relevant DDIs/HDIs, especially when these inhibitors are co-administrated with CES substrate-drugs with very narrow therapeutic windows. All data and knowledge presented here provide key information for the clinicians to assess the risks of clinically available hCES inhibitors on drug metabolism. In future, more practical and highly specific substrates for hCES1A/hCES2A should be developed and used for studies on CES-mediated DDIs/HDIs both in vitro and in vivo.

[Simultaneous determination of multiple bioactive constituents in Abelmoschi Corolla by UFLC-QTRAP-MS/MS].

A comprehensive analytical method based on ultra-fast liquid chromatography coupled with triple quadrupole/linear ion trap tandem mass spectrometry(UFLC-QTRAP-MS/MS) was established for simultaneous determination of the content of 38 active components in Abelmoschi Corolla, including flavonoids, organic acids, nucleosides and amino acids, so as to investigate the effects of different harvesting and processing methods on multi-active components in Abelmoschi Corolla. The chromatographic separation was performed on a XBridg®C_(18) column(4.6 mm×100 mm, 3.5 µm) with(0.1% formic acid water) methanol-acetonitrile(1∶1) as the mobile phase for gradient elution at 30 ℃. The flow rate was 0.5 mL·min~(-1). The components were detected in a multiple-reaction monitoring(MRM) mode. The gray relational analysis(GRA) was used to comprehensively evaluate the multiple active components of Abelmoschi Corolla at different harvesting times and drying temperatures. The results showed that 38 components had a good linearity with correlation coefficients all above 0.999 0. The method featured a good precision, repeatability and stability with the relative stan-dard deviations(RSDs) of less than 5.0%. Recoveries ranged from 98.06% to 104.4% with RSD between 0.22% and 4.9%. The results of GRA indicated that a better quality in the samples collected on September 9 th. Samples dried at 90 ℃ had a better quality. The established method is accurate and reliable, and can be used to assess the internal quality of Abelmoschi Corolla. This study can provide basic materials for determining appropriate harvesting time and processing method of Abelmoschi Corolla.