Guided by the theory of "the kidney storing essence, governing the bones, and producing marrow", this study explored the mechanism of icariin(ICA) in regulating the osteogenic differentiation of rat bone mesenchymal stem cells(BMSCs) through caveolin-1(Cav1) via in vitro and in vivo experiments, aiming to provide a theoretical basis for the prevention and treatment of postmenopausal osteoporosis with traditional Chinese medicine(TCM). Primary cells were obtained from 4-week-old female SD rats using the whole bone marrow adherent method. Flow cytometry was used to detect the expression of surface markers CD29, CD90, CD11b, and CD45. The potential for osteogenic and adipogenic differentiation was assessed. The effect of ICA on cell viability was determined using the CCK-8 assay, and the impact of ICA on the formation of mineralized nodules was verified by alizarin red staining. A stable Cav1-silenced cell line was constructed using lentivirus. The effect of Cav1 silencing on osteogenic differentiation was observed via alizarin red staining. Western blot analysis was conducted to detect the expression of Cav1, Hippo/TAZ, and osteogenic markers such as Runt-related transcription factor 2(RUNX2) and alkaline phosphatase(ALP). The results showed that primary cells were successfully obtained using the whole bone marrow adherent method, positively expressing surface markers of rat BMSCs and possessing the potential for both osteogenic and adipogenic differentiation. The CCK-8 assay and alizarin red staining results indicated that 1×10~(-7) mol·L~(-1) was the optimal concentration of ICA for intervention in this experiment(P<0.05). During osteogenic induction, ICA inhibited Cav1 expression(P<0.05) while promoting TAZ expression(P<0.05). Alizarin red staining demonstrated that Cav1 silencing significantly promoted the osteogenic differentiation of BMSCs. After ICA intervention, TAZ expression was activated, and the expression of osteogenic markers ALP and RUNX2 was increased. In conclusion, Cav1 silencing significantly promotes the osteogenic differentiation of BMSCs, and ICA promotes this differentiation by inhibiting Cav1 and regulating the Hippo/TAZ signaling pathway.
Animals ; Mesenchymal Stem Cells/metabolism* ; Caveolin 1/genetics* ; Osteogenesis/drug effects* ; Rats, Sprague-Dawley ; Rats ; Cell Differentiation/drug effects* ; Female ; Signal Transduction/drug effects* ; Flavonoids/administration & dosage* ; Protein Serine-Threonine Kinases/genetics* ; Drugs, Chinese Herbal/pharmacology* ; Cells, Cultured ; Humans

A high performance liquid chromatography-tandem mass spectrometry(HPLC-MS/MS) method was established for simultaneous determination of seven characteristic components from the active fraction of Alpiniae Officinarum Rhizoma in rat plasma, including galangin, kaempferol, kaempferide, pinocembrin, 1,7-diphenyl-4-en-3-heptanone, 5-hydroxy-7-(4-hydroxy-3-methoxyphenyl)-1-phenyl-3-heptanone(DHPA), and 7-(4-hydroxy-3-methoxyphenyl)-1-phenyl-4-en-3-heptanone(DPHB). The new developed HPLC-MS/MS method was applied to study the pharmacokinetics of the 7 characteristic components in rats with Helicobacter pylori gastritis. A Waters Sunfire C_(18) column(2.1 mm×150 mm, 3.5 μm) was used. The acetonitrile-aqueous solution(containing 0.1% formic acid) was adopted as the mobile phase for gradient elution. Seven components and internal standard(chlorogenic acid) were separated within 12 min. Mass spectrometric detection was performed in multiple reaction monitoring(MRM) mode using electrospray ionization(ESI) source with fast switching between positive and negative ions. The method was verified by specificity, linearity, precision, accuracy, recovery, matrix effect, and stability and met the requirements of pharmacokinetic study on the 7 components in rat plasma. Pharmacokinetic results showed that the average peak time(T_(max)) of the 7 components was 0.31-2.19 h, their elimination half-life(t_(1/2)) was 5.26-16.65 h, and the average residence time(MRT) was 6.29-31.03 h after the oral administration of the active fraction of Alpiniae Officinarum Rhizoma to rats with H. pylori gastritis. The plasma exposure levels of galangin and DHPA were higher than those of the other components. The concentration-time curves of four detected flavonoids showed obvious double peaks. This study elucidated the pharmacokinetic characteristics of 7 characteristic components from the active fraction of Alpiniae Officinarum Rhizoma in rats with H. pylori gastritis, providing a scientific basis for the identification of the pharmacodynamic substances of Alpiniae Officinarum Rhizoma for treatment of H. pylori gastritis and the clinical application of Alpiniae Officinarum Rhizoma in the prevention and treatment of H. pylori gastritis.
Animals ; Rats ; Chromatography, High Pressure Liquid/methods* ; Tandem Mass Spectrometry/methods* ; Drugs, Chinese Herbal/administration & dosage* ; Male ; Helicobacter pylori/drug effects* ; Alpinia/chemistry* ; Rats, Sprague-Dawley ; Gastritis/metabolism* ; Helicobacter Infections/metabolism* ; Flavonoids/blood* ; Rhizome/chemistry* ; Liquid Chromatography-Mass Spectrometry

To enhance the therapeutic efficacy of the baicalin-berberine complex(BA-BBR) in the treatment of ulcerative colitis(UC), BA-BBR nanocrystal microspheres(BA-BBR NC MS) were prepared using the dropping method. The microspheres were characterized in terms of morphology, particle size, differential scanning calorimetry(DSC), and powder X-ray diffraction(XRD). The release profiles of BA and BBR from the microspheres were measured, and the drug release mechanism was investigated. A rat model of UC was induced by 5% dextran sodium sulfate(DSS) and treated continuously for 7 days to evaluate the therapeutic effects of different formulations. The results showed that the prepared BA-BBR MS and BA-BBR NC MS were uniform gel spheres with particle sizes of(1.77±0.16) mm and(1.67±0.08) mm, respectively. After drying, the gels collapsed inward and exhibited a rough surface. During the preparation process, the BA-BBR nanocrystals(BA-BBR NC) were uniformly encapsulated within the microspheres. The release profiles of the microspheres followed a first-order kinetic model, and the 12-hour cumulative release of BA and BBR from BA-BBR NC MS was higher than that from BA-BBR MS. Compared with BA-BBR, BA-BBR NC, and BA-BBR MS, BA-BBR NC MS further alleviated UC symptoms in rats, most significantly reducing the levels of TNF-α, IL-1β, IL-6, and MPO, while increasing the level of IL-4 in colon tissues. These results indicate that BA-BBR NC MS, based on a "nano-in-micro" design, can deliver BA-BBR to the intestine and exert significant therapeutic effects in a UC rat model, suggesting it as a promising new strategy for the treatment of UC.
Animals ; Colitis, Ulcerative/metabolism* ; Rats ; Nanoparticles/chemistry* ; Microspheres ; Male ; Berberine/administration & dosage* ; Flavonoids/administration & dosage* ; Rats, Sprague-Dawley ; Drugs, Chinese Herbal/administration & dosage* ; Humans ; Particle Size ; Tumor Necrosis Factor-alpha/immunology* ; Drug Liberation ; Drug Compounding

This study aims to compare the metabolic differences of baicalin and its analogues between Shuganning Injection and Scutellariae Radix extract. Twelve SD rats were randomly divided into a Shuganning Injection group and a Scutellariae Radix extract group, with 6 rats in each group. Their liver microsomes were incubated with the drugs, and then the samples were collected. Ultra performance liquid chromatography-quadrupole/electrostatic field orbitrap high resolution mass spectrometry(UPLC-Q-Exactive Orbitrap-MS) was used to analyze the prototype components and metabolites of the drugs in liver microsomes of each group. Another 12 SD rats were also divided into a Shuganning Injection group and a Scutellariae Radix extract group, with 6 rats in each group. The rats were administrated with 4.2 mL·kg~(-1) Shuganning Injection or Scutellariae Radix extract by tail vein injection. After 48 h, the rat urine, feces, and bile were collected, and UPLC-Q-Exactive Orbitrap-MS was used to analyze the prototype components and metabolites in each biological sample. The results showed that 5 prototype components and 8 metabolites of Shuganning Injection and Scutellariae Radix extract were identified in liver microsomes. A total of 5 prototype components were identified in rat urine, feces, and bile separately. Fifteen metabolites were identified in the urine, 9 metabolites in the feces, and 12 metabolites in the bile. The differences of metabolic pathways and number of metabolites of baicalin were compared between Shuganning Injection and Scutellariae Radix extract. For both Shuganning Injection and Scutellariae Radix extract, the metabolites of baicalin or baicalein in rat liver microsomes, urine, bile, and feces were mainly formed glucuronic acid conjugates, and there were a small amount of glucose conjugates and methylation products. Differences were found in the number and types of metabolites of baicalin in urine samples between Shuganning Injection and Scutellariae Radix extract, indicating that differences existed in metabolism between the two. This suggests that the other components in the formula lead to changes of metabolites in vivo.
Animals ; Rats ; Rats, Sprague-Dawley ; Microsomes, Liver/chemistry* ; Drugs, Chinese Herbal/administration & dosage* ; Feces/chemistry* ; Scutellaria baicalensis/chemistry* ; Male ; Bile/chemistry* ; Flavonoids/metabolism* ; Urine/chemistry* ; Chromatography, High Pressure Liquid ; Mass Spectrometry ; Plant Extracts

Clinopodium chinense, a traditional folk medicinal herb, has been used to treat abnormal uterine bleeding(AUB) for many years. Saponins and flavonoids are the main active components in C. chinense. To study the pharmacokine-tics of multiple components from the total extract of C. chinense(TEC), we established a sensitive and rapid method of ultra-perfor-mance liquid chromatography coupled with tandem mass spectrometry(UPLC-MS/MS) for simultaneous determination of five compounds in the plasma of AUB rats. After validation, the AUB model was established with SD female rats which got pregnant on the same day by gavage with mifepristone(12.4 mg·kg~(-1)) and misoprostol(130 μg·kg~(-1)). The established method was applied to the detection of hesperidin, naringenin, apigenin, saikosaponin a, and buddlejasaponin Ⅳb in AUB rats after the administration of TEC. The pharmacokinetic parameters were calculated by DAS 2.0. The five compounds showed good linear relationship within the detection range. The specificity, accuracy, precision, recovery, matrix effect, and stability of the method all matched the requirements of biolo-gical sample detection. The above 5 compounds were detected in the plasma of AUB rats after the administration of TEC. The C_(max) va-lues of hesperidin, naringenin, apigenin, saikosaponin a, and clinoposide A were 701.6, 429.5, 860.7, 75.1, and 304.1 ng·mL~(-1), respectively. All the compounds owned short half-life and quick elimination rate in vivo, and the large apparent volume of distribution indicated that they were widely distributed in tissues. Being rapid, accurate, and sensitive, this method is suitable for the pharmacokinetic study of extracts of Chinese herbal medicines and provides a reference for the study of pharmacodynamic material basis of C. chinense in treating AUB.
Administration, Oral ; Animals ; Apigenin/analysis* ; Chromatography, High Pressure Liquid/methods* ; Chromatography, Liquid ; Drugs, Chinese Herbal/chemistry* ; Female ; Flavonoids/analysis* ; Hesperidin ; Lamiaceae ; Mifepristone ; Misoprostol ; Oleanolic Acid/analogs & derivatives* ; Plant Extracts/chemistry* ; Rats ; Saponins ; Tandem Mass Spectrometry/methods* ; Uterine Hemorrhage

Qing-Fei-Pai-Du decoction (QFPDD) is a Chinese medicine compound formula recommended for combating corona virus disease 2019 (COVID-19) by National Health Commission of the People's Republic of China. The latest clinical study showed that early treatment with QFPDD was associated with favorable outcomes for patient recovery, viral shedding, hospital stay, and course of the disease. However, the effective constituents of QFPDD remain unclear. In this study, an UHPLC-Q-Orbitrap HRMS based method was developed to identify the chemical constituents in QFPDD and the absorbed prototypes as well as the metabolites in mice serum and tissues following oral administration of QFPDD. A total of 405 chemicals, including 40 kinds of alkaloids, 162 kinds of flavonoids, 44 kinds of organic acids, 71 kinds of triterpene saponins and 88 kinds of other compounds in the water extract of QFPDD were tentatively identified via comparison with the retention times and MS/MS spectra of the standards or refereed by literature. With the help of the standards and in vitro metabolites, 195 chemical components (including 104 prototypes and 91 metabolites) were identified in mice serum after oral administration of QFPDD. In addition, 165, 177, 112, 120, 44, 53 constituents were identified in the lung, liver, heart, kidney, brain, and spleen of QFPDD-treated mice, respectively. These findings provided key information and guidance for further investigation on the pharmacologically active substances and clinical applications of QFPDD.
Administration, Oral ; Alkaloids/analysis* ; Animals ; COVID-19 ; Chromatography, High Pressure Liquid ; Drugs, Chinese Herbal/pharmacokinetics* ; Flavonoids/analysis* ; Mice ; SARS-CoV-2 ; Saponins/analysis* ; Triterpenes/analysis*

Periodontitis is an inflammatory autoimmune disease. Treatment should alleviate inflammation, regulate the immune reaction and promote periodontal tissue regeneration. Icariin is the main active ingredient of Epimedii Folium, and it is a promising compound for the enhancement of mesenchymal stem cell function, promotion of bone formation, inhibition of bone resorption, alleviation of inflammation and regulation of immunity. The study investigated the effect of icariin on periodontal tissue regeneration in a minipig model of periodontitis. The minipig model of periodontitis was established. Icariin was injected locally. The periodontal clinical assessment index, a computed tomography (CT) scan, histopathology and enzyme-linked immune sorbent assay (ELISA) were used to evaluate the effects of icariin. Quantitative analysis results 12 weeks post-injection demonstrated that probing depth, gingival recession, attachment loss and alveolar bone regeneration values were (3.72 ± 1.18) mm vs. (6.56 ± 1.47) mm, (1.67 ± 0.59) mm vs. (2.38 ± 0.61) mm, (5.56 ± 1.29) mm vs. (8.61 ± 1.72) mm, and (25.65 ± 5.13) mm vs. (9.48 ± 1.78) mm in the icariin group and 0.9% NaCl group, respectively. The clinical assessment, CT scan, and histopathology results demonstrated significant enhancement of periodontal tissue regeneration in the icariin group compared to the 0.9% NaCl group. The ELISA results suggested that the concentration of interleukin-1 beta (IL-1β) in the icariin group was downregulated compared to the 0.9% NaCl group, which indicates that local injection of icariin relieved local inflammation in a minipig model of periodontitis. Local injection of icariin promoted periodontal tissue regeneration and exerted anti-inflammatory and immunomodulatory function. These results support the application of icariin for the clinical treatment of periodontitis.
Animals ; Disease Models, Animal ; Enzyme-Linked Immunosorbent Assay ; Flavonoids ; administration & dosage ; pharmacology ; Gingival Crevicular Fluid ; chemistry ; Inflammation ; drug therapy ; Periodontitis ; diagnostic imaging ; drug therapy ; Swine ; Swine, Miniature ; Tomography, X-Ray Computed

Radix Scutellaria is widely applied to the treatment of diabetes mellitus in China. Its main bioactive constituents contain baicalin, wogonoside, oroxyloside, and their aglycones. To investigate the effect of type 2 diabetes mellitus on both pharmacokinetics and tissue distribution of these flavonoid compounds, the six flavonoids in plasma and tissues from the normal and type 2 diabetic rats after oral administration of Radix Scutellaria extract were simultaneously measured by ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method. The results showed that baicalin, wogonoside, and oroxyloside had higher C and AUC values (P < 0.05) in type 2 diabetic rats than that in normal rats and the tissue-distribution behaviors of the six flavonoid compounds in hearts, livers, spleens, lungs, kidneys, brains, pancreas, fat and muscle of the type 2 diabetic rats showed obviously differences from the normal rats (P < 0.05). In conclusion, the differences in the pharmacokinetics of oroxyloside and tissue distribution of the six flavanoids in Radix Scutellaria extract between diabetic and normal rats were found for the first time. The results from the present study provided a crucial basis for a better understanding of in vivo anti-diabetic mechanism of action of the six flavonoids from Radix Scutellaria.
Administration, Oral ; Animals ; Chromatography, High Pressure Liquid ; Diabetes Mellitus, Experimental ; drug therapy ; metabolism ; physiopathology ; Drugs, Chinese Herbal ; administration & dosage ; chemistry ; Flavonoids ; analysis ; chemistry ; pharmacokinetics ; Male ; Molecular Structure ; Plant Roots ; chemistry ; Rats ; Rats, Wistar ; Reproducibility of Results ; Scutellaria baicalensis ; chemistry ; Tandem Mass Spectrometry ; Tissue Distribution ; physiology

OBJECTIVETo evaluate the cytotoxic effects of ampelopsin sodium (Amp-Na) and carboplatin (CBP) used alone or in combination on human non-small cell lung cancer (NSCLC) cells SPC-A1 in vitro and its related mechanism.

METHODSCytotoxic effects were assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays. The synergistic effects of the drugs were calculated with coefficient of drug interaction (CDI). Cell cycle was determined by flow cytometry (FCM). The levels of p53, p21, cyclinE, cyclinD1, and phosphorylated cyclin-dependent kinase-2 (p-CDK2) were evaluated by Western blot.

RESULTSAmp-Na (6.25-200 μg/mL) and CBP (3.13-100 μg/mL) alone exhibited prominent cytotoxic activity in a concentration-dependent manner on SPC-A1 cells with 50% inhibitive concentration values of 57.07±14.46 and 34.97±6.30 μg/mL, respectively. Drug combinations were associated with significantly higher cytotoxic effects than each drug alone (P<0.05 or 0.01). The CDI analysis confirmed the synergy of Amp-Na and CBP on inhibiting cancer cell viability across a wide concentration range (CDI <1). FCM and Western blot showed that synergistic cytotoxic effects of Amp-Na and CBP were related to Garrested which mainlym ediated by p 21 through the inhibition of CDK2 activity independent of the p53 tumor suppressor pathway.

CONCLUSIONSAmp-Na exhibits anticancer activities and enhances the antitumor activities of CBP through up-regulation of p21 and inhibition of CDK2 activity in human NSCLC cells SPC-A1. These results suggest that Amp-Na may be applied to enhance the anticancer action of CBP.

Antineoplastic Combined Chemotherapy Protocols ; pharmacology ; Carboplatin ; administration & dosage ; pharmacology ; Carcinoma, Non-Small-Cell Lung ; drug therapy ; pathology ; Cell Cycle ; drug effects ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Drug Synergism ; Flavonoids ; administration & dosage ; pharmacology ; Humans ; Lung Neoplasms ; drug therapy ; pathology

Asthma is characterized by chronic inflammation, goblet cell hyperplasia, the aberrant production of the Th2 cytokines, and eosinophil infiltration into the lungs. In this study, we examined the effects of baicalein, wogonin, and Scutellaria baicalensis ethanol extract on ovalbumin (OVA)-induced asthma by evaluating Th1/Th2 cytokine levels, histopathologic analysis, and compound 48/80-induced systemic anaphylaxis and mast cell activation, focusing on the histamine release from rat peritoneal mast cells. Baicalein, wogonin, and S. baicalensis ethanol extract also decreased the number of inflammatory cells especially eosinophils and downregulated peribronchial and perivascular inflammation in the lungs of mice challenged by OVA. Baicalein, wogonin, and S. baicalensis ethanol extract significantly reduced the levels of tumor necrosis factor α, interleukin (IL)-1β, IL-4, IL-5 and the production of OVA-specific IgE and IgG1, and upregulated the level of interferon-γ and OVA-specific IgG2a. In addition, oral administration of baicalein, wogonin, and S. baicalensis ethanol extract inhibited compound 48/80-induced systemic anaphylaxis and plasma histamine release in mice. Moreover, baicalein, wogonin, and S. baicalensis ethanol extract suppressed compound 48/80-induced mast cell degranulation and histamine release from rat peritoneal mast cells. Conclusively, baicalein and wogonin as major flavonoids of S. baicalensis may have therapeutic potential for allergic asthma through modulation of Th1/Th2 cytokine imbalance and histamine release from mast cells.
Administration, Oral ; Anaphylaxis* ; Animals ; Asthma ; Cytokines ; Eosinophils ; Ethanol* ; Flavonoids ; Goblet Cells ; Histamine Release* ; Histamine* ; Hyperplasia ; Immunoglobulin E ; Immunoglobulin G ; Inflammation* ; Interleukin-4 ; Interleukin-5 ; Interleukins ; Lung ; Mast Cells ; Mice ; Ovalbumin ; Ovum ; Plasma ; Rats ; Scutellaria baicalensis* ; Scutellaria* ; Tumor Necrosis Factor-alpha