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BACKGROUND:Several studies have found that the total flavonoids of rhizome drynariae can promote neovascularization in the induced membrane,improve the biological properties of the induced membrane,and accelerate bone remodeling in the induced membrane,but the related molecular mechanisms still need to be further explored. OBJECTIVE:To explore the effect of total flavonoids of rhizome drynariae on bone remodeling in rat femoral Masquelet-induced membrane model by regulating H-type blood vessels. METHODS:Thirty-six male Sprague-Dawley rats were stratified by body mass and then randomly divided into blank group,model group and traditional Chinese medicine group,with 12 rats in each group.A 4-mm femoral bone defect model was established in all the rats.Bone defects in the model group and traditional Chinese medicine group were filled with polymethylmethacrylate bone cement.At 6 weeks after modeling,the tail bone of the rats was implanted in the blank group,as well as in the other two groups after removal of bone cement.The traditional Chinese medicine group was given 157.5 mg/kg per day of total flavonoids of rhizome drynariae at 3 days after bone implantation,while the model and blank groups were given the same amount of saline by gavage until the 8th week after bone implantation.Bone graft samples were taken for relevant testing at 8 weeks after implantation. RESULTS AND CONCLUSION:X-ray films showed that in the blank group,the fracture line in the defect area was clear,and only a small amount of bone callus formed;in the model group,the bone defect area still existed,where discontinuous cortical bone was visible;in the traditional Chinese medicine group,the defect area was filled with newborn bone tissues,the bone marrow cavity and part of the cortical bone formed,and the fracture line disappeared.Micro-CT scans showed that the amount of new bone in the defect area was low in the blank group,the number of bone trabeculae in the defect area was significantly increased in the model group,and a large amount of new bone tissue was filled in the bone defect area in the traditional Chinese medicine group.Hematoxylin-eosin staining results showed that in the blank group,only a small amount of new bone formed in the defect area and the quality of osteogenesis was poor;in the model group,there was more new bone tissue in the defect area,but some fibrous connective tissues were interspersed within the bone tissue;and in the traditional Chinese medicine group,a large amount of new bone formed in the defect area and the quality of osteogenesis was the best.CD31/Emcn immunofluorescence double-labeling staining results showed that the number of H-type blood vessels in the newborn bone tissue in the bone defect area of the blank group was sparse and sparsely distributed;compared with the blank group,there were more H-type blood vessels in the bone tissue in the bone defect area of the model group,and the blood vessels were distributed in relatively regular strips;the number of H-type blood vessels in the bone defect area of the traditional Chinese medicine group was the highest and the blood vessels were densely distributed.To conclude,the total flavonoids of rhizoma drynariae can upregulate the expression of H-type blood vessels to enhance the angiogenic-osteogenic effect,improve the osteogenic efficiency of the rat femoral Masquelet induced membrane model,and promote bone remodeling.
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Objective To screen the active components of total flavonoid extracts of Sarcandra glabra to promote megakaryocyte differentiation.Methods(1)A model of megakaryocyte differentiation disorder was established by co-culturing human megakaryocytic leukaemia cells(Dami)with human bone marrow stromal cells(HS-5)as an evaluation system,and the experimental groupings were as follows:the Dami group(Dami),the control group(Dami+HS-5),and the PMA group[Dami+HS-5+5 ng·mL-1 foprolol 12-tetradecanoate 13-acetate(PMA)],and model group[Dami+HS-5+1%rabbit anti-rat platelet serum(APS)+5 ng·mL-1 PMA]were cultured for 48 hours.The expressions of megakaryocyte differentiation and maturation surface marker molecules,CD41a and CD61 were detected by flow cytometry.(2)Forty-nine SD male rats were randomly divided into blank plasma group,15-minute group,30-minute group,60-minute group,90-minute group,120-minute group,and 240-minute group,with 7 rats in each group.The rats in each administration group were gavaged with 1.26 g·kg-1 of total flavonoids extracts of Sarcandra glabra,and blood was collected at six set time points(15,30,60,90,120,240 minutes)for the preparation of time-dependent serum-containing plasma of total flavonoids extracts of Sarcandra glabra.(3)Ultra-high performance liquid chromatography-quadrupole tandem time-of-flight mass spectrometry(UHPLC-Q-TOF/MS)was used to analyze the plasma of the time-dependent serum-containing plasma of the total flavonoids extracts of Sarcandra glabra,and the peak area was used to construct a matrix(X-matrix)of the amount of chemical composition change over time in the time-dependent serum-containing plasma of the total flavonoids extracts of Sarcandra glabra.The collected time-dependent serum-containing plasma of the total flavonoids extracts of Sarcandra glabra at six different time points was used to intervene in the model of megakaryocyte differentiation and maturation disorder,and the expression of cell surface molecules CD41a and CD61 was detected by flow cytometry to construct the matrix of effect of time-dependent serum-containing plasma of the total flavonoids extracts of Sarcandra glabra(Y-matrix).(4)After the data of X and Y matrices were standardized,partial least squares(PLS)was used to calculate and analyze the quantitative and qualitative effect relationship,and variable importance for projection(VIP)>1 was used as the threshold to screen the effect components related to the changes of cell surface molecules CD41a and CD61,and chemical composition identification,as the potential effector components in the total flavonoid extracts of Sarcandra glabra were used to promote the differentiation of megakaryocytes,and finally the regression evaluation system was used to verify the efficacy of its medicinal effect.Results(1)Compared with the Dami group,the expression level of CD41a on the surface of Dami cells in the control group was significantly increased(P<0.05).Compared with the control group,the expression levels of CD41a and CD61 on the surface of Dami cells in the PMA group were significantly increased(P<0.01).Compared with the PMA group,the expression levels of CD41a and CD61 on the surface of Dami cells in the model group were significantly reduced(P<0.01).(2)Compared with the blank plasma group,the expression levels of the molecules CD41a and CD61 on the surface of Dami cells at each time point of 15,30,60,90,120,and 240 minutes were significantly increased(P<0.01),and the expression levels of CD41a and CD61 were both highest in the 30-minute group.The potential effective components with VIP value greater than 1 were screened out in the positive and negative ion mode,and 540.3638@12.25 and 559.2991@11.53 were selected for pharmacodynamic verification.559.2991@11.53 was identified as daucosterol(Dau),540.3638@12.25 was identified as rosmarinic acid 4-O-β-D-glucoside(Ros).After Ros and Dau intervened in the megakaryocyte differentiation and maturation disorder model respectively,the expression levels of CD41a and CD61 on the surface of Dami cells in the low-,medium-and high-dose groups(40,60 and 80 μg·mL-1)of Ros and Dau were significantly increased compared with the model group(P<0.05,P<0.01).Conclusion Ros and Dau may be the active components of the total flavonoids extracts of Sarcandra glabra to promote the differentiation of megakaryocytes.
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OBJECTIVE To study the inhibitory effect and mechanism of total flavonoids from Melicope pteleifolia (TF-MPL) on transplanted tumor of colorectal cancer in nude mice. METHODS The transplanted tumor model of colorectal cancer was induced by injecting 0.2 mL colorectal cancer cell LoVo subcutaneously via the right armpit of nude mice. After successful modeling, nude mice were randomly divided into model group, 5-fluorouracil group (positive control, 10 mg/kg), TF-MPL high- dose and low-dose groups (25, 12.5 mg/kg); a normal group (normal saline containing 0.3% carboxymethyl cellulose sodium) without modeling was additionally set up, with 6 mice in each group. Each group was intraperitoneally injected with the corresponding drug solution/solvent for 21 consecutive days. The inhibitory rate of the transplanted tumor, liver and spleen index, and the levels of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) in serum were detected after the last medication; the morphological changes of tumor tissue were observed; immunohistochemical staining was used to detect protein expressions of Toll- like receptor 4 (TLR4) and nuclear factor-κB subunit p65 (NF-κB p65) in tumor tissue of nude mice. Western blot assay was used to detect protein expressions of TLR4, myeloid differentiation factor 88 (MyD88), TNF receptor-associated factor 6 (TRAF6), interleukin-1 receptor-associated kinase 1 (IRAK-1), NF-κB p65 and caspase-3 in tumor tissue of nude mice. RESULTS Compared with the model group, TF-MPL high-dose group showed a significant decrease in tumor weight (inhibitory rate of 36.91%), liver and spleen index, serum levels of TNF-α and IL-6 and protein expressions of TLR4, MyD88, TRAF6,IRAK-1 and NF- κB p65 (P<0.05 or P<0.01); the expression of caspase-3 protein was increased significantly (P<0.05), and more tumor cell shrinkage and deformation, nuclear pyknosis and fragmentation were observed. CONCLUSIONS TF-MPL can significantly inhibit the growth of transplanted tumor of colorectal cancer in nude mice, the mechanism of which may be associated with reducing inflammatory response, inhibiting TLR4/MyD88/NF-κB signaling pathway, and promoting apoptosis in colorectal cancer cells.
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ObjectiveThe correlation of Pueraria lobata producing areas, climate factors, total flavonoids of P. lobata, polysaccharide content of P. lobata, and antioxidant activity of P.lobata for medicinal application was analyzed, and the relationship between climate factors and the formation of P. lobata quality was evaluated. MethodThe scavenging rates of 1,1-diphenyl-2-picrylhydrazyl radical 2,2-diphenyl-1-(2,4,6-trinitrophenyl)hydrazyl(DPPH) and 2, 2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)(ABTS) radicals by total flavonoids and polysaccharides of P. lobata were detected, and the correlation between the contents of each component and the information of producing areas and climate factors was analyzed. ResultThe ABTS+ scavenging rate by total flavonoids of P.lobata was negatively correlated with altitude (P<0.05) and positively correlated with annual sunshine hours (P<0.05). The altitude was positively correlated with the total flavonoid content, while the annual sunshine hours were negatively correlated with the total flavonoid content. There was a negative correlation between total flavonoid content and ABTS+ scavenging rate by total flavonoids. In other words, lower altitude and longer annual sunshine hours indicated lower total flavonoid content and higher ABTS+ scavenging rate by total flavonoids. The ABTS+ scavenging rate by polysaccharides of P. lobata was negatively correlated with the frost-free period (P<0.05) and the mean temperature in July (P<0.01). There was a positive correlation between the polysaccharide content of P. lobata and the frost-free period. The mean temperature in July was positively correlated with the polysaccharide content of P. lobata (P<0.05). The polysaccharide content of P. lobata was negatively correlated with the ABTS+ scavenging rate by polysaccharides of P. lobata. In other words, a shorter frost-free period in the producing area and lower mean temperature in July indicated lower polysaccharide content of P. lobata and higher ABTS+ scavenging rate by polysaccharides of P. lobata. The mean temperature in July was significantly correlated with the contents of total flavonoids and polysaccharides in P. lobata samples (P<0.05). The lower mean temperature in July was often accompanied by lower total flavonoid content of P. lobata, lower polysaccharide content of Pueraria lobata, and stronger antioxidant activity of P. lobata samples. ConclusionThe ability of P. lobata to remove ABTS+ is stronger than that of DPPH+. There is a significant correlation between climate factors, content, and antioxidant capacity in each producing area. Further research on the internal law of the formation of medicinal active components of P. lobata induced by core climate factors will provide a scientific basis for revealing the formation mechanism of genuine P. lobata and the subsequent control of P. lobata quality according to the environment of producing areas.
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OBJECTIVE To study the improvement effect of total flavonoids from Rosa multiflora root on vascular injury in rheumatoid arthritis (RA) model rats and its potential mechanism. METHODS Female Wistar rats were randomly divided into normal control group, model group, aspirin group (positive control, 30 mg/kg), low-dose and high-dose groups of total flavonoids from R. multiflora root (4.15, 8.30 g/kg, by crude drug), with 10 rats in each group. Except for the normal control group, the RA model was induced in other groups by collagen induction and high-fat diet. After 14 days of modeling, they were given corresponding drug solution/0.5% sodium carboxymethyl cellulose solution intragastrically, once a day, for 36 consecutive days. The total body score, arthritis index (AI) and swollen joint count (SJC) of the rats were evaluated regularly. After the last medication, serum levels of interleukin-6 (IL-6), intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule- 1 (VCAM-1) were determined. The pathological morphological changes in the vascular tissue of thoracic aorta were observed; the protein expression of Toll-like receptor 4 (TLR4) and the protein phosphorylation levels of Janus kinase 2 (JAK2) and signal transduction and activator of transcription 3 (STAT3) in vascular tissue of thoracic aorta were measured. RESULTS Compared with the normal control group, serum levels of IL-6, ICAM-1 and VCAM-1, protein expression of TLR4, and the protein phosphorylation levels of JAK2 and STAT3 in vascular tissue of thoracic aorta were increased significantly in model group (P< 0.01). The atherosclerotic plaque (atheroma), cholesterol crystal, lymphocyte infiltration and a small number of unbroken foam cell aggregation could be seen in the vascular tissue of thoracic aorta. Compared with the model group, total body score (except for the low-dose group), AI and SJC were decreased significantly in groups of total flavonoids from R. multiflora root on the 28th day (P<0.05 or P<0.01); total body score,AI and SJC were decreased significantly in low-dose group of total flavonoids from R. multiflora root on the 49th day (P<0.05 or P<0.01); the other quantitative indicators in serum and vascular tissue were significantly reversed in groups of total flavonoids from R. multiflora root (P<0.05 or P<0.01), and pathological damage of vascular tissue was significantly relieved. CONCLUSIONS Total flavonoids from R. multiflora root can significantly improve vascular injury in RA model rats, and its mechanism may be related to reducing the protein expression of TLR4 in vascular tissue and inhibiting the activation of IL-6/JAK2/ STAT3 signaling pathway.
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Bone defect has always been a major clinical challenge because of its great difficulty and long period of treatment. Drynariae Rhizoma is a commonly used medicine in osteology and traumatology of traditional Chinese medicine, and its active ingredients(mainly flavonoids) facilitate osteoblast differentiation of bone marrow mesenchymal stem cells, osteoclast proliferation, vascular-osteogenic coupling, and inhibit osteoclast activity to promote bone mineralization, and repair and reconstruction of bone defect. As a good substitute for bone regeneration drugs, the active constituents of Drynariae Rhizoma can be loaded on scaffold materials of tissue engineering, which greatly improves the bioavailability of the drug. Meanwhile, the sustained-release microspheres also solve some problems such as sudden drug release from the scaffolds, and the composite scaffolds with active ingredient of Drynariae Rhizoma prepared by them have good ossification activity and osteoinduction, with precise bone repair effects, which meet the diverse performance requirements of bone grafts and have a promising clinical application prospect.
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AIM:To observe how total flavonoids of Pterocarya hupehensis Skan(PHSTF)affects the migra-tion and invasion of human rheumatoid fibroblast-like synoviocyte line MH7A.METHODS:The MH7A cells were divided into control group(without any treatment),low-,medium-and high-dose(6.25,12.5 and 25 mg/L,respectively)PHSTF groups,phosphatidylinositol 3-kinase(PI3K)inhibitor 740Y-P(10 μmol/L)group,and 740Y-P(10 μmol/L)+high-dose(25 mg/L)PHSTF group.The viability of the MH7A cells was determined by CCK-8 assay,while the migration and inva-sion were assessed by scratch and Transwell assays.The protein levels of matrix metalloproteinase-2(MMP-2),MMP-9,PI3K,p-PI3K,AKT and p-AKT were detected by Western blot.RESULTS:Compared with control group,the cell via-bility in PHSTF treatment groups was reduced(P<0.05),the cell wound healing area was significantly decreased(P<0.01),migratory and invasive cells in the lower chamber were significantly reduced(P<0.01),and the protein expres-sion of MMP-2 and MMP-9 and the ratios of p-PI3K/PI3K and pAKT/AKT were decreased(P<0.01).Compared with high-dose PHSTF group,the addition of PI3K/AKT pathway agonist 740Y-P significantly increased the migration and invasion ability of MH7A cells(P<0.01)and elevated the protein expression of MMP-2 and MMP-9 and the ratios of p-PI3K/PI3K and pAKT/AKT(P<0.01)under the treatment with PHSTF.CONCLUSION:Total flavonoids of Pterocarya hupehensis Skan could inhibit the migration and invasion of MH7A cells by regulating the PI3K/AKT signaling pathway.
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AIM:To investigate the impact of total flavonoids of Pterocarya hupehensis Skan(PHSTF)on the migration,invasion,and ferroptosis of non-small-cell lung cancer A549 cells.METHODS:The A549 cells were divided into control group,low-,medium-and high-dose(100,150 and 200 μg/mL)PHSTF groups,ferroptosis inhibitor liprox-statin-1(Lip-1)group,and high-dose PHSTF combined with Lip-1 group,each cultured in corresponding media.Cell via-bility was assessed using the CCK-8 assay,while cell migration and invasion abilities were determined through scratch and Transwell assays.Cell lipid peroxidation levels were measured using the glutathione(GSH)assay kit.RT-qPCR was em-ployed to assess the mRNA expression of solute carrier family 7 member 11(SLC7A11)and glutathione peroxidase 4(GPX4),while Western blot was utilized to examine the protein expression of SLC7A11,GPX4,Kelch-like epichlorohy-drin-associated protein-1(Keap-1),nuclear factor E2-related factor 2(Nrf2)and heme oxygenase-1(HO-1).RE-SULTS:Compared with control group,PHSTF significantly diminished the viability of A549 cells in a time-and dose-de-pendent manner(P<0.01),and the cell migration and invasion were also reduced(P<0.01),along with a significant de-crease in GSH level(P<0.01).Treatment with PHSTF inhibited the mRNA and protein expression levels of ferroptosis-re-lated proteins,including SLC7A11 and GPX4(P<0.01),suppressed the protein expression of Nrf2 and HO-1(P<0.01),and enhanced the expression of Keap-1(P<0.01).The Lip-1 partially restored the decrease in cell viability in-duced by PHSTF(P<0.01),significantly up-regulated the protein expression levels of SLC7A11,GPX4,Nrf2 and HO-1,and suppressed the protein expression of Keap-1(P<0.01).CONCLUSION:Total flavonoids of Pterocarya hupehen-sis Skan can inhibit the migration and invasion of non-small-cell lung cancer A549 cells,and induce the cell ferroptosis by regulating the Keap-1/Nrf2/HO-1 pathway.
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@#To investigate the influential mechanism of total flavonoids from Abelmoschus Manihot (HKZ) on cytochrome P450 (CYP450) isoforms in human liver microsomes and to verify its effect on the most significantly inhibited subtype CYP2C9 in rats.The inhibitory effects of HKZ on human CYP3A4, CYP2C9, CYP2C19, CYP2E1, CYP1A2 and CYP2D6 were evaluated through the cocktail method using ultra-performance liquid chromatography tandem mass spectrometry, then its inhibitory mechanism was investigated and kinetic parameters of enzyme inhibition were calculated By comparing the pharmacokinetic behaviors of tolbutamide after single or multiple administration of 200 mg/kg HKZ and equal dose of CMC-Na in rats, the effects of HKZ on CYP2C11 enzyme (CYP2C9 isoenzyme) was estimated.The results indicated the significant inhibitory effect of HKZ on CYP2C9 and CYP2E1 with IC50 of 3.22 and 8.64 μg/mL, respectively. Also, it showed certain inhibitory ability on other isoforms with IC50 of 20-30 μg/mL.As demonstrated, HKZ may not be a time-dependent inhibitor which competitively inhibited CYP2E1 and CYP2C9 with Ki of 3.84 and 6.33 μg/mL.In contrast, it showed noncompetitive inhibition on CYP3A4 mediated testosterone-6β-hydroxylation and midazolam-4-hydroxylation reaction with Ki of 7.37 and 3.32 μg/mL.It was also a noncompetitive inhibitor of CYP1A2, CYP2D6 and CYPC219 with Ki values of 8.66, 11.49 and 21.94 μg/mL. HKZ did not change the pharmacokinetic parameters of CYP2C11 probe substrate tolbutamide in rat, but it affected the AUC0-t, cmax of 4-hydroxytolubutamide (P < 0.05). Therefore, drug-drug interaction mediated by CYP450 should be considered in clinical study.
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OBJECTIVE To study pharmacodynamics and potential mechanism of Blumea balsamifera total flavonoids against acute myocardial infarction (AMI) model rats. METHODS AMI model of SD rats was established by ligating anterior descending branch of left coronary artery. Fifty model rats were randomly divided into model group (0.8% carboxymethyl cellulose solution), positive control group (Compound danshen tablet, 300 mg/kg), B. balsamifera total flavonoids low-dose, medium-dose and high- dose groups (3, 10, 30 mg/kg), with 10 rats in each group. Other 10 rats were included in sham operation group (0.8% carboxymethyl cellulose solution). After 1 day of surgery, they were given relevant medicine 3 mL/kg intragastrically, once a day, for 4 consecutive weeks. The changes of S-T segment were recorded before and after operation, after weekly intragastric administration. The hemodynamic indexes of rats were all determined, i.e. systolic blood pressure (SBP), diastolic blood pressure (DBP), mean arterial pressure (MAP), left ventricular systolic pressure (LVSP), left ventricular end diastolic blood pressure (LVEDP), maximal left ventricular pressure rising rate (+LVdp/dtmax), maximal left ventricular pressure decreasing rate (-LVdp/ dtmax). The levels of serum myocardial enzymes [lactate dehydrogenase (LDH), creatine kinase isoenzyme-MB (CK-MB)] and inflammatory factors [tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), IL-1β] were determined. The myocardial infarction rate of rats and the phosphorylation levels of phosphoinositide 3-kinase (PI3K) and protein kinase B (Akt) proteins in myocardial tissue were determined. RESULTS Compared with model group, S-T segments of electrocardiogram were all decreased significantly in administration groups (P<0.05). SBP, DBP, MAP, LVSP, +LVdp/dtmax, -LVdp/dtmax, and ratio of p-PI3KTyr607/ PI3K, p-AktThr308/Akt, p-Aktser473/Akt were increased significantly in B. balsamifera total flavonoids medium-dose and high-dose groups (P<0.05). The levels of LVEDP, serum myocardial enzymes and inflammatory factors, myocardial infarction rate were all decreased significantly (P<0.05). CONCLUSIONS balsamifera total flavonoids can improve cardiac function of AMI model rats, the mechanism of which may be associated with inhibiting the expression of inflammatory factor and activating PI3K/Akt signaling pathway.
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ObjectiveTo observe the effect of Cuscutae Semen total flavonoids combined with Tripterygium wilfordii polyglycoside tablets (TWPT) on ovarian germline stem cells of female physiological mice through neurogenic locus notch homolog (Notch) signaling pathway. MethodSixty female Kunming mice (5 weeks old) were randomly divided into normal group, Tripterygium wilfordii polyglycoside tablets low-, high-dose groups (13.65 mg·kg-1·d-1 and 27.3 mg·kg-1·d-1, 1 and 2 times clinical equivalent dose), Cuscutae Semen total flavonoids low- and high-dose groups (150 mg·kg-1·d-1 and 300 mg·kg-1·d-1), and combination group (13.65 mg·kg-1·d-1 TWPT and 150 mg·kg-1·d-1 Cuscutae Semen total flavonoids), with 10 in each group. After 3 weeks of continuous administration, the uterus/brain and ovarian/brain indexes were calculated, and the pathological changes of ovarian tissue were observed under light microscope. The content of estradiol in serum was determined by enzyme linked immunosorbent assay (ELISA). Immunofluorescence assay was performed to observe the expressions of germline stem cell markers in ovarian epithelium, including mouse vasa homologue (Mvh), octamer-binding transcription factor 4 (Oct4), tyrosine-protein kinase receptor (c-kit), Nanog, Notch signaling pathway molecules, neurogenic locus notch homolog protein 1 (Notch1), hes family BHLH transcription factor 1(Hes1), and jagged canonical Notch ligand 1 (JAG1). ResultCompared with the normal group, low and high doses of TWPT had no significant effect on the uterus/brain and ovary/brain indexes and the uterus and ovary morphologies of mice, while only the number of atretic follicles was increased (P<0.01). The expressions of ovarian germline stem cell markers and Notch signaling pathway molecules had a decreasing trend in TWPT low-dose group, while the expressions of Mvh, c-kit, and Nanog were down-regulated (P<0.05, P<0.01) and the expressions of Notch1 and Hes1 were also reduced (P<0.01) in TWPT high-dose group. However, the above indexes were increased in Cuscutae Semen total flavonoids low-dose group (P<0.05, P<0.01). Compared with the low does of TWPT group, the combination group had a decrease in the increased number of atretic follicles (P<0.01), an improvement in the down-regulated expressions of Mvh and Nanog (P<0.01), and an increase in the expressions of Notch1 and Hes1 (P<0.05, P<0.01). ConclusionOvarian germline stem cells are the source target of the reproductive toxicity of TWPT. Cuscutae Semen total flavonoids participate in the regulation of the germline stem cell pathways to alleviate the reproductive toxicity caused by TWPT, and its mechanism of action may be related to the Notch signaling pathway.
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OBJECTIVE To evaluate the quality of total flavonoids of Lamiophlomis rotata from different producing areas. METHODS Total flavonoids of S1-S15 batches of L. rotata from different producing areas were extracted by percolation and purified by polyamide column. The content of total flavonoids was determined by UV spectrophotometry, and its purity was calculated. HPLC fingerprint chromatograms and control fingerprint of total flavonoids from 15 batches of L. rotata were established with Similarity Evaluation System of Chromatographic Fingerprint of TCM (2012 version). Their similarities were analyzed. Cluster analysis, principal component analysis and orthogonal partial least squares-discriminant analysis were used to evaluate the quality of total flavonoids from 15 batches of L. rotata, and the main components which affected the quality of total flavonoids were analyzed. RESULTS The purities of total flavonoids from 15 batches of L. rotata were 55.82%-94.12%, with an average value of 77.72%; a total of five common peaks were identified in the fingerprint, and No. 3 peak was luteolin; the similarities between the fingerprint of each batch of samples and the control fingerprint were 0.925-1.000. By cluster analysis, S1 and S3-S9 were clustered into the first class, which were samples from Qinghai Province and Tibet Autonomous Region. S14 and S15 were clustered into the second class, which were samples from Yunnan Province. S10-S13 were clustered into the third class, which were all samples from Sichuan Province. S2 was clustered into the fourth class. The principal component analysis showed that the qualities of samples from the first and fourth classes were better; peaks 2, 3 and 5 were identified as the main components that caused the differences among different batches of samples by orthogonal partial least squares-discriminant analysis. CONCLUSIONS The qualities of total flavonoids of L. rotata from Qinghai Province and Tibet Autonomous Region are better.
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OBJECTIVE To screen the active ingredient with estrogenic effect from total flavonoids of Cuscutae Semen. METHODS The estrogenic effect of total flavonoids from 10 batches of Cuscutae Semen was evaluated with mouse uterus coefficient and endometrial thickness as evaluation indexes, establish its fingerprint and calibrate the common peak. Common peak and spectrum-effect relationship of the above two indicators were analyzed by bivariate relationship analysis and grey correlation analysis to screen active components with estrogenic effect. UPLC-Q-TOF-MS technology was used to characterize the active components. RESULTS The estrogenic effect of total flavonoids from 10 batches of Cuscutae Semen was good. Twenty-eight and thirty-three common peaks of total flavonoids in Cuscutae Semen were obtained in the positive and negative ion modes respectively. The constituents represented by peaks 7,10,12-16,26 in positive ion mode and peaks 2,5,8,9,12,16,19,22-26 in negative ion mode were highly correlated with the estrogenic effect of total flavonoids from Cuscutae Semen. Further identification showed that the active substances with estrogenic effect from the total flavonoids of Cuscutae Semen were 5,7,3′, 4′-tetramethoxyflavone, 6- O-(trans) p-coumarin-furanfructose-(2→1)-glucopyranoside, rutin, kaempferol-3,7-diglucoside, apigenin-7-O-glucoside, hyperoside, baicalin, quercitin, quercetin, apigenin, kaempferol, isorhamnetin, rhododendron, isoquercetin, kaempferol-3-furan arabinoside, 2,6-octadecanediacetylic acid. CONCLUSIONS A total of 16 chemical components with estrogenic effect are screened from total flavonoids of Cuscutae Semen in the study, which can provide reference for the development of phytoestrogens.
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This study explores the effect of total flavonoids of Rhododendra simsii(TFR) on middle cerebral artery occlusion(MCAO)-induced cerebral injury in rats and oxygen-glucose deprivation/reoxygenation(OGD/R) injury in PC12 cells and the underlying mechanism. The MCAO method was used to induce focal ischemic cerebral injury in rats. Male SD rats were randomized into sham group, model group, and TFR group. After MCAO, TFR(60 mg·kg~(-1)) was administered for 3 days. The content of tumor necrosis factor-α(TNF-α), interleukin-1(IL-1), and interleukin-6(IL-6) in serum was detected by enzyme-linked immunosorbent assay(ELISA). The pathological changes of brain tissue and cerebral infarction were observed based on hematoxylin and eosin(HE) staining and 2,3,5-triphenyltetrazolium chloride(TTC) staining. RT-qPCR and Western blot were used to detect the mRNA and protein levels of calcium release-activated calcium channel modulator 1(ORAI1), stromal interaction molecule 1(STIM1), stromal intera-ction molecule 2(STIM2), protein kinase B(PKB), and cysteinyl aspartate specific proteinase 3(caspase-3) in brain tissues. The OGD/R method was employed to induce injury in PC12 cells. Cells were randomized into the normal group, model group, gene silencing group, TFR(30 μg·mL~(-1)) group, and TFR(30 μg·mL~(-1))+gene overexpression plasmid group. Intracellular Ca~(2+) concentration and apoptosis rate of PC12 cells were measured by laser scanning confocal microscopy and flow cytometry. The effect of STIM-ORAI-regulated store-operated calcium entry(SOCE) pathway on TFR was explored based on gene silencing and gene overexpression techniques. The results showed that TFR significantly alleviated the histopathological damage of brains in MCAO rats after 3 days of admini-stration, reduced the contents of TNF-α, IL-1, and IL-6 in the serum, down-regulated the expression of ORAI1, STIM1, STIM2, and caspase-3 genes, and up-regulated the expression of PKB gene in brain tissues of MCAO rats. TFR significantly decreased OGD/R induced Ca~(2+) overload and apoptosis in PC12 cells. However, it induced TFR-like effect by ORAI1, STIM1 and STIM2 genes silencing. However, overexpression of these genes significantly blocked the effect of TFR in reducing Ca~(2+) overload and apoptosis in PC12 cells. In summary, in the early stage of focal cerebral ischemia-reperfusion injury and OGD/R-induced injury in PC12 cells TFR attenuates ischemic brain injury by inhibiting the STIM-ORAI-regulated SOCE pathway and reducing Ca~(2+) overload and inflammatory factor expression, and apoptosis.
Subject(s)
Animals , Male , Rats , Apoptosis , Brain Ischemia/metabolism , Caspase 3 , Interleukin-1 , Interleukin-6 , Rats, Sprague-Dawley , Reperfusion Injury/metabolism , Tumor Necrosis Factor-alpha/genetics , Flavonoids/pharmacology , Rhododendron/chemistryABSTRACT
The present study observed the regulatory effect of total flavonoids of Ziziphora clinopodioides on autophagy and the phosphatidylinositol 3-kinase(PI3K)/protein kinase B(Akt)/mammalian target of rapamycin(mTOR) signaling pathways in ApoE~(-/-) mice and explored the mechanism of total flavonoids of Z. clinopodioides against atherosclerosis(AS). ApoE~(-/-) mice were fed on a high-fat diet for eight weeks to induce an AS model. The model mice were randomly divided into a model group, a positive control group, and low-, medium-and high-dose groups of total flavonoids of Z. clinopodioides, while C57BL/6J mice fed on a common diet were assigned to the blank group. The serum and aorta samples were collected after intragastric administration for 12 weeks, and the serum levels of total cholesterol(TC), triglyceride(TG), low density lipoprotein-cholesterol(LDL-C), and high density lipoprotein-cholesterol(HDL-C) were detected by an automatic biochemical analyzer. The serum expression levels of intercellular adhesion molecule-1(ICAM-1), vascular cell adhesion molecule-1(VCAM-1), matrix metalloproteinase-2(MMP-2), and matrix metalloprotei-nase-9(MMP-9) were detected by enzyme-linked immunosorbent assay(ELISA). Oil red O staining was used to observe the aortic plaque area in mice. Hematoxylin-eosin(HE) staining was used to observe the aortic plaque and pathological changes in mice. The expression of P62 and LC3 in the aorta was detected by the immunofluorescence method. The protein expression of LC3Ⅱ/Ⅰ, Beclin-1, P62, p-PI3K, p-Akt, and p-mTOR in the aorta of mice was detected by Western blot. The results showed that compared with the blank group, the serum levels of TC, TG, LDL-C, ICAM-1, VCAM-1, MMP-2 and MMP-9 in the model group were significantly increased(P<0.01 or P<0.05), the content of HDL-C was decreased(P<0.05), intra-aortic plaque area was enlarged(P<0.01), the expression of LC3 in the aorta was significantly down-regulated, P62 expression was up-regulated(P<0.01 or P<0.05), the expressions of LC3Ⅱ/Ⅰ and Beclin-1 in the aortic lysate were significantly down-regulated, and the expressions of p-PI3K, p-Akt, p-mTOR and P62 were significantly increased(P<0.01). The medium-and high-dose groups of total flavonoids of Z. clinopodioides could reduce the serum levels of TC, TG, LDL-C, ICAM-1, VCAM-1, MMP-2, and MMP-9 in AS model mice(P<0.01 or P<0.05), and increase the content of HDL-C(P<0.01 or P<0.05). The aortic plaque area of mice after middle and high doses of total flavonoids of Z. clinopodioides was significantly reduced(P<0.01), the content of foam cells decrease, and the narrowing of the lumen decreased. The total flavonoids of Z. clinopodioides significantly increased the expression of LC3 in the aorta and the expression of LC3Ⅱ/Ⅰ and Beclin-1 in the lysate, and decreased the expression of P62 in the aorta and the expression of p-PI3K, p-Akt, p-mTOR and P62 in the lysate(P<0.01 or P<0.05). The results showed that the total flavonoids of Z. clinopodioides could improve the content of blood lipids and inflammatory factors, and reduce the generation of foam cells and plaques in aortic tissue, and the mechanism may be related to the regulation of PI3K/Akt/mTOR signaling pathway.
Subject(s)
Animals , Mice , Apolipoproteins E , Atherosclerosis/genetics , Beclin-1 , Cholesterol, LDL , Intercellular Adhesion Molecule-1 , Matrix Metalloproteinase 2/genetics , Matrix Metalloproteinase 9/genetics , Mice, Inbred C57BL , Phosphatidylinositol 3-Kinases/metabolism , Plaque, Atherosclerotic , Proto-Oncogene Proteins c-akt/metabolism , TOR Serine-Threonine Kinases/genetics , Vascular Cell Adhesion Molecule-1/geneticsABSTRACT
This paper aimed to investigate the effect of total flavonoids of buckwheat flower and leaf on myocardial cell apoptosis and Wnt/β-catenin/peroxisome proliferator-activated receptor γ(PPARγ) pathway in arrhythmic rats. SD rats were randomly divided into a control group, a model group, a low-dose(20 mg·kg~(-1)) group of total flavonoids of buckwheat flower and leaf, a medium-dose(40 mg·kg~(-1)) group of total flavonoids of buckwheat flower and leaf, a high-dose(80 mg·kg~(-1)) group of total flavonoids of buckwheat flower and leaf, a propranolol hydrochloride(2 mg·kg~(-1)) group, with 12 rats in each group. Except the control group, rats in other groups were prepared as models of arrhythmia by sublingual injection of 1 mL·kg~(-1) of 0.002% aconitine. After grouping and intervention with drugs, the arrhythmia, myocardial cells apoptosis, myocardial tissue glutathione peroxidase(GSH-Px), catalase(CAT), malondialdehyde(MDA), serum interleukin-6(IL-6), prostaglandin E2(PGE2) levels, myocardial tissue apoptosis, and Wnt/β-catenin/PPARγ pathway-related protein expression of rats in each group were measured. As compared with the control group, the arrhythmia score, the number of ventricular premature beats, ventricular fibrillation duration, myocardial cell apoptosis rate, MDA levels in myocardial tissues, serum IL-6 and PGE2 levels, Bax in myocardial tissues, and Wnt1 and β-catenin protein expression levels increased significantly in the model group, whereas the GSH-Px and CAT levels, and Bcl-2 and PPARγ protein expression levels in myocardial tissues reduced significantly. As compared with the model group, the arrhythmia score, the number of ventricular premature beats, ventricular fibrillation duration, myocardial cell apoptosis rate, MDA leve in myocardial tissues, serum IL-6 and PGE2 levels, Bax in myocardial tissues, and Wnt1 and β-catenin protein expression levels reduced in the drug intervention groups, whereas the GSH-Px and CAT levels and Bcl-2 and PPARγ protein expression levels in myocardial tissues increased. The groups of total flavonoids of buckwheat flower and leaf were in a dose-dependent manner. There was no significant difference in the levels of each index in rats between the propranolol hydrochloride group and the high-dose group of total flavonoids of buckwheat flower and leaf. The total flavonoids of buckwheat flower and leaf inhibit the activation of Wnt/β-catenin pathway, up-regulate the expression of PPARγ, reduce oxidative stress and inflammatory damage in myocardial tissues of arrhythmic rats, reduce myocardial cell apoptosis, and improve the symptoms of arrhythmia in rats.
Subject(s)
Rats , Animals , PPAR gamma/metabolism , Fagopyrum/genetics , Rats, Sprague-Dawley , bcl-2-Associated X Protein , beta Catenin/metabolism , Interleukin-6 , Flavonoids/pharmacology , Propranolol/pharmacology , Ventricular Fibrillation , Dinoprostone , Wnt Signaling Pathway , Plant Leaves/metabolism , Flowers/metabolism , Apoptosis , Cardiac Complexes, PrematureABSTRACT
To study the effects of different drying methods on the quality of male flowers of Eucommia ulmoides(MFOEU), we treated fresh MFOEU samples with drying in the shade(DS), vacuum freeze drying(VFD), high-or low-temperature hot air drying(HTHAD, LTHAD), microwave drying(MD), and vacuum drying(VD), respectively. The color, total flavonoid content, total polysaccharide content, and main active components such as geniposide, geniposidic acid, rutin, chlorogenic acid, galuteolin, pinoresinol diglucoside, and aucubin in MFOEU were taken as the evaluation indicators. The quality of MFOEU was comprehensively evaluated by entropy weight method combined with color index method, partial least squares discriminant analysis and content clustering heat map. The experimental results showed that VFD and DS basically kept the original color of MFOEU. The MFOEU treated with MD had higher content of total polysaccharides, phenylpropanoids, lignans, and iridoids. The MFOEU treated with LTHAD had higher content of total flavonoids and that treated with VD had lower content of active components. According to the results of comprehensive evaluation, the quality of MFOEU dried with different methods followed the order of MD>HTHAD>VFD>LTHAD>DS>VD. Considering the color of MFOEU, the suitable drying methods were DS and VFD. Considering the color, active components, and economic benefits of MFOEU, MD was the suitable drying method. The results of this study are of a reference value for the determination of suitable methods for MFOEU processing in the producing areas.
Subject(s)
Eucommiaceae/chemistry , Flowers/chemistry , Flavonoids/analysis , Rutin/analysis , Chlorogenic Acid/analysisABSTRACT
OBJECTIVE To comprehensively evaluate the quality of Eriobotrya japonica leaves from different producing areas. METHODS The contents of alcohol-soluble extracts were determined by hot-dipping method using 30 batches of E. japonica leaves from different producing areas as samples. The contents of total flavonoids and total triterpene acids were determined by ultraviolet spectrophotometry. The contents of five kinds of triterpenic acids (euscaphic acid,crataegolic acid,corosolic acid,oleanolic acid and ursolic acid) were determined by HPLC. The quality of E. japonica leaves from different producing areas was comprehensively evaluated by using entropy weight technique for order preference by similarity to an ideal solution (TOPSIS). The bivariate correlation analysis of E. japonica leaves was conducted by SPSS 22.0 software in terms of weight, comprehensive evaluation value, the content of alcohol-soluble extract, the contents of total flavonoids, total triterpene acids and five triterpenic acids. RESULTS The contents of alcohol-soluble extract in 30 batches of E. japonica leaves were (24.56±0.08)%-(34.85±0.13)%; the contents of total flavonoids were (4.69±0.11)-(14.23±0.27) mg/g; the contents of total triterpene acid were (27.58±0.59)- (63.95±1.27) mg/g; the contents of euscaphic acid, crataegolic acid, corosolic acid, oleanolic acid and ursolic acid were (0.728± 0.011)-(6.064±0.063), (0.526±0.013)-(3.245±0.022), (1.222±0.025)-(8.807±0.094), (0.856±0.021)-(2.931±0.075), (4.704±0.087)-(11.806±0.283) mg/g, respectively. The analysis result of entropy weight TOPSIS method showed that the top three samples with comprehensive evaluation values (No.Kjcx-5) were S14 (Huotian Town, Yunxiao County, Zhangzhou,Fujian), S19 (Qinnan District, Qinzhou, Guangxi) and S29 (Guoyang County, Bozhou, Anhui). Comprehensive evaluation 0596-2559522。E-mail:jxrcwxp@163.com of E. japonica leaves was positively correlated with the contents of five kinds of triterpenic acids, such as euscaphic acid, crataegolic acid, corosolic acid, oleanolic acid and ursolic acid (P<0.01). The weight of E. japonica leaves was positively correlated with the comprehensive evaluation value (P<0.01). CONCLUSIONS The qualities of E. japonica leaves from different producing areas are very different. Among them, the qualities of E. japonica leaves from Huotian Town, Yunxiao County, Zhangzhou of Fujian, Qinzhou Qinnan District of Guangxi, and Bozhou Guoyang County of Anhui are relatively better. The weight of E. japonica leaves is positively correlated with their quality.
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@#In order to guarantee the quality of traditional Chinese medicines (TCMs), the crystallization transformation of complex extracts of TCMs and the influence of solid form on their physicochemical properties were studied.The extract of total flavonoids from Pueraria lobata was taken as a model.Crystallization transformation happened when lofting under different conditions, and the intrinsic dissolution tests were carried out.It was found that humidity was the key factor to induce crystallization of total flavonoids from Pueraria lobata.The greater the wettability was, the more the crystallization was.The dissolution rate of total flavonoids from Pueraria lobata with the most crystallization amount significantly decreased by 96.51% compared to the sample without crystallization.After further simulating the preparation process of total flavonoids from Pueraria lobata, it was found that the wet granulation process with introduced water would also lead to crystallization and reduced dissolution rate.As for all crystallization samples, there was an inversely proportional relationship between the dissolution rates and the amount of crystallization.The risk of crystallization existed both in the storage and preparation process of TCM extracts.Crystallization would significantly affect the dissolution rate, and thus the quality of TCM products.In this study, the crystallization transformation of amorphous complex TCM extracts was discovered, and the effect of the crystallization transformation on its dissolution behavior was systematically studied, which provides a new research idea for assuring the quality of TCM products and promoting the improvement of TCM preparation level.
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OBJECTIVE To investigate the effect and mechanism of total flavonoids of Bidens pilosa L. (TFB) on lipopolysaccharide (LPS)-induced neuroinflammation in mice. METHODS Fifty C57BL/6 mice were randomly divided into normal control group, LPS group and TFB low-dose, medium-dose and high-dose groups, with 10 mice in each group. TFB low-dose, medium-dose and high-dose groups were given TFB solution intragastrically at 60, 120 and 240 mg/kg, and the normal control group and LPS group were given corresponding volume of normal saline, once a day, for consecutive 21 d. From the 15th day of administration, except for the normal control group, other groups were given LPS (400 μg/kg) intraperitoneally for 7 consecutive days to establish neuroinflammatory model. Brain tissues were taken under anesthesia 4 h after the final administration. The morphological changes of neuronal cells in mice were observed; the contents of nitric oxide (NO), tumor necrosis factor α (TNF- α), interleukin-1β (IL-1β), IL-6 and IL-10 were measured, and the expressions of inflammatory pathway-related proteins [inducible NO synthase (iNOS), cyclooxygenase-2 (COX-2), myeloid differentiation factor 88 (Myd88) and protein kinase C (PKC)] were measured in the brain tissues of mice. RESULTS Compared with the normal control group, the neuronal arrangement in the hippocampal region of the brain tissue of mice in the LPS group was sparsely disorganized, with a large number of neuronal fixations and shrunken nuclei; the contents of TNF-α, IL-1β, IL-6 and NO in the brain tissue were significantly increased, the contents of IL-10 were significantly decreased, and the relative expressions of iNOS, COX-2, Myd88 and PKC proteins were significantly increased (P<0.05). Compared with the LPS group, the neuronal pathological changes in the brain tissue of mice in the TFB low-dose, medium-dose and high-dose groups were 202014810) significantly improved, and the changes of the above indices in the brain tissue were significantly reversed (P<0.05) CONCLUSIONS TFB has an inhibitory effect on E-mail:pangxjun@163.com neuroinflammation, and its mechanism of action may be related to down-regulation of the expressions of inflammatory pathway-related proteins iNOS, COX-2, Myd88 and PKC, and reduction of inflammatory factors release.