Dihydromyricetin Alleviates H9C2 Cell Apoptosis and Autophagy by Regulating CircHIPK3 Expression and PI3K/AKT/mTOR Pathway / 中国结合医学杂志

OBJECTIVE@#To investigate the effect and potential mechanism of dihydromyricetin (Dmy) on H9C2 cell proliferation, apoptosis, and autophagy.@*METHODS@#H9C2 cells were randomly divided into 7 groups, namely control, model, EV (empty pCDH-CMV-MCS-EF1-CopGFP-T2A-Puro vector), IV (circHIPK3 interference), Dmy (50 µ mol/L), Dmy+IV, and Dmy+EV groups. Cell proliferation and apoptosis were detected by cell counting kit-8 assay and flow cytometry, respectivley. Western blot was used to evaluate the levels of light chain 3 II/I (LC3II/I), phospho-phosphoinositide 3-kinase (p-PI3K), protein kinase B (p-AKT), and phospho-mammalian target of rapamycin (p-mTOR). The level of circHIPK3 was determined using reverse transcriptase polymerase chain reaction. Electron microscopy was used to observe autophagosomes in H9C2 cells.@*RESULTS@#Compared to H9C2 cells, the expression of circHIPK in H9C2 hypoxia model cells increased significantly (P<0.05). Compared to the control group, the cell apoptosis and autophagosomes increased, cell proliferation rate decreased significantly, and the expression of LC3 II/I significantly increased (all P<0.05). Compared to the model group, the rate of apoptosis and autophagosomes in IV, Dmy, and Dmy+IV group decreased, the cell proliferation rate increased, and the expression of LC3 II/I decreased significantly (all P<0.05). Compared to the control group, the expressions of p-PI3K, p-AKT, and p-mTOR in the model group significantly reduced (P<0.05), whereas after treatment with Dmy and sh-circHIPK3, the above situation was reversed (P<0.05).@*CONCLUSION@#Dmy plays a protective role in H9C2 cells by inhibiting circHIPK expression and cell apoptosis and autophagy, and the mechanism may be related to PI3K/AKT/mTOR pathway.

Dihydromyricetin mediates epithelial mesenchymal transformation and regulates the proliferation and apoptosis of esophageal squamous cell carcinoma cells / 中华肿瘤杂志

Objective: To study the effects of dihydromyricetin (DMY) on the proliferation, apoptosis and epithelial mesenchymal transition (EMT) of esophageal squamous cell carcinoma (ESCC) cell KYSE150 and KYSE410. Methods: KYSE150 and KYSE410 cells were treated with different concentrations of DMY (0, 25, 50, 100, 150, 200 μmol/L) for 24 hours. The median inhibition concentration (IC50) values of KYSE150 and KYSE410 were detected by cell counting kit-8 (CCK-8) method. Then 0.5‰ dimethyl sulfoxide (DMSO) was used as control group, dihydromyricetin (DMY), dihydromyricetin and transforming growth factor-β1 (DMY+ TGF-β1), transforming growth factor-β1 (TGF-β1) were used as experimental group. Cell proliferation and apoptosis rates were measured by clonal formation and flow cytometry. Transwell invasion and wound healing assay were used to detect cell invasion and migration. The protein expression levels of Caspase-3, Caspase-9, Bcl-2, Bax, Smad2/3, phosphorylation-Smad2/3 (p-Smad2/3) and Vimentin were detected by western blot. Results: The IC50 values of DMY on KYSE410 and KYSE150 cells were 100.51 and 101.27 μmol/L. The clone formation numbers of KYSE150 and KYSE410 in DMY group [(0.53±0.03) and (0.31±0.03)] were lower than those in DMSO group [(1.00±0.10) and (1.00±0.05), P<0.05]. The apoptosis rates of KYSE150 and KYSE410 cells in DMY group [(1.84±0.22)% and (2.80±0.07)%] were higher than those in DMSO group [(1.00±0.18)% and (1.00±0.07)%, P<0.05]. The invasion numbers of KYSE150 and KYSE410 cells in DMY group [(0.42±0.03) and (0.29±0.05)] were lower than those in DMSO group [(1.00±0.08) and (1.00±0.05), P<0.05]. The migration rates of KYSE150 and KYSE410 cells in DMY group [(0.65±0.14)% and (0.40±0.17)%] were lower than those in DMSO group [(1.00±0.10)% and (1.00±0.08)%, P<0.05]. The clone formation numbers of KYSE150 and KYSE410 in TGF-β1 group [(1.01±0.08) and (0.99±0.25)] were higher than those in DMY+ TGF-β1 group [(0.73±0.10) and (0.58±0.05), P<0.05]. The apoptosis rates of KYSE150 and KYSE410 cells in TGF-β1 group [(0.81±0.14)% and (1.18±0.10)%] were lower than those in DMY+ TGF-β1 group [(1.38±0.22)% and (1.85±0.04)%, P<0.05]. The invasion numbers of KYSE150 and KYSE410 cells in TGF-β1 group [(1.19±0.11) and (1.39±0.11)] were higher than those in DMY+ TGF-β1 group [(0.93±0.09) and (0.93±0.05), P<0.05]. The migration rates of KYSE150 and KYSE410 cells in TGF-β1 group [(1.87±0.19)% and (1.32±0.04)%] were higher than those in DMY+ TGF-β1 group [(0.86±0.16)% and (0.77±0.12)%, P<0.05]. The protein expression levels of Bax, Caspase-3 and Caspase-9 in KYSE150 and KYSE410 cells in DMY group were higher than those in DMSO group, while the protein expression level of Bcl-2 was lower than that in DMSO group (P<0.05). The protein expression levels of p-Smad2/3, Smad2/3 and Vimentin in KYSE150 and KYSE410 cells in DMY group were lower than those in DMSO group (P<0.05). The protein expression levels of Bax, Caspase-3 and Caspase-9 in KYSE150 and KYSE410 cells in TGF-β1 group were lower than those in DMY+ TGF-β1 group, and the protein expression level of Bcl-2 was higher than that in DMY+ TGF-β1 group (P<0.05). The protein expression levels of Bax, Caspase-3 and Caspase-9 in KYSE150 and KYSE410 cells in DMY+ TGF-β1 group were lower than those in DMY group, and the protein expression level of Bcl-2 was higher than that in DMY group (P<0.05). The protein expression levels of p-Smad2/3, Smad2/3 and Vimentin in KYSE150 and KYSE410 cells in TGF-β1 group were higher than those in DMY+ TGF-β1 group (P<0.05). Conclusion: DMY can inhibit the proliferation and EMT of ESCC mediated by TGF-β1 and promote cell apoptosis.

Dihydromyricetin improves cardiac insufficiency by inhibiting HMGB1 in diabetic rats / 南方医科大学学报

OBJECTIVE@#To investigate the effect of dihydromyricetin (DHM) on cardiac insufficiency in diabetic rats and explore the underlying mechanism.@*METHOD@#Twenty-four male SD rats were randomized equally into normal control group, type 2 diabetes (T2DM) group fed on a high-glucose and high-fat diet for 6 weeks with low-dose streptozotocin (STZ) injection, metformin (MET) group with daily intragastric administration of MET (150 mg/kg) for 8 weeks after T2DM modeling, and dihydromyricetin (DHM) group with daily intragastric administration of DHM (250 mg/kg) for 8 weeks after modeling. The levels of fasting blood glucose, low density lipoprotein (LDL-C), triglyceride (TG), total cholesterol (TC), high density lipoprotein (HDL-C) and glycosylated hemoglobin (HbA1c) of the rats were measured, and plasma levels of insulin and high mobility group protein-1 (HMGB1) were detected with ELISA. The cardiac function of the rats was assessed using color echocardiography, ECG was measured using a biological signal acquisition system, and myocardial pathology was observed with HE staining. The protein expressions of HMGB1, nuclear factor-κB (NF-κB) p65 and phospho-NF-κB p65 (p-NF-κB p65) in the myocardial tissue were detected using Western blotting.@*RESULTS@#Compared with the control group, the rats in T2DM group showed significant anomalies in cardiac function after modeling with significantly increased plasma HMGB1 level and expressions of HMGB1, NF-κB p65 and p-NF-κB p65 proteins in the myocardial tissue (P < 0.05 or 0.01). Treatment with DHM significantly improved the indexes of cardiac function of the diabetic rats (P < 0.05 or 0.01), decreased plasma HMGB1 level and down-regulated the protein expressions of HMGB1 and p-NF-κB p65 in the myocardial tissue (P < 0.05 or 0.01).@*CONCLUSION@#DHM treatment can improve cardiac function in diabetic rats possibly by down-regulation of HMGB1 and phospho-NF-κB p65 expressions in the myocardium.

Dihydromyricetin reduces lipid accumulation in LO2 cells via AMPK/mTOR-mediated lipophagy pathway and inhibits HepG2 cell proliferation in vitro / 南方医科大学学报

OBJECTIVE@#To explore the mechanism underlying the hepatoprotective effect of dihydromyricetin (DMY) against lipid accumulation in light of the lipophagy pathway and the inhibitory effect of DMY on HepG2 cell proliferation.@*METHODS@#LO2 cells were cultured in the presence of 10% FBS for 24 h and treated with 100 μg/mL DMY, or exposed to 50% FBS for 24 h followed by treatment with 50, 100, or 200 μg/mL DMY; the cells in recovery group were cultured in 50% FBS for 24 h and then in 10% FBS for another 24 h. Oil red O staining was used to observe the accumulation of lipid droplets in the cells, and the levels of TC, TG, and LDL and activities of AST, ALT and LDH were measured. The expression of LC3 protein was detected using Western blotting. AO staining and transmission electron microscopy were used to determine the numbers of autophagolysosomes and autophagosomes, respectively. The formation of autophagosomes was observed with MDC staining, and the mRNA expression levels of LC3, ATG7, AMPK, mTOR, p62 and Beclin1 were determined with q-PCR. Flow cytometry was performed to analyze the effect of 50, 100, and 200 μg/mL DMY on cell cycle and apoptosis of HepG2 cells; DNA integrity in the treated cells was examined with cell DNA fragmentation test.@*RESULTS@#DMY treatment and pretreatment obviously inhibited lipid accumulation and reduced the levels of TC, TG, LDL and enzyme activities of AST, ALT and LDH in LO2 cells (P < 0.05). In routinely cultured LO2 cells, DMY significantly promoted the formation of autophagosomes and autophagolysosomes and upregulated the expression of LC3 protein. DMY obviously attenuated high FBS-induced inhibition of autophagosome formation in LO2 cells, up- regulated the mRNA levels of LC3, ATG7, Beclin1 and AMPK, and downregulated p62 and mTOR mRNA levels (P < 0.05 or 0.01). In HepG2 cells, DMY caused obvious cell cycle arrest, inhibited cell proliferation, and induced late apoptosis and DNA fragmentation.@*CONCLUSION@#DMY reduces lipid accumulation in LO2 cells by regulating the AMPK/ mTOR-mediated lipophagy pathway and inhibits the proliferation of HepG2 by causing cell cycle arrest and promoting apoptosis.

Dihydromyricetin reverses Herceptin resistance by up-regulating miR-98-5p and inhibiting IGF1R/HER2 dimer formation in SKBR3 cells / 南方医科大学学报

OBJECTIVE@#To explore the effect of dihydromyricetin on the expression of miR-98-5p and its mechanism in the development of Herceptin resistance in SKBR3 cells.@*METHODS@#The expression of IGF2 and miR-98-5p and their interaction relationship were analyzed by bioinformatics analysis through TargetScan online databases. SKBR3 cells and drug-resistant SKBR3-R cells were cultured in cell experiments. Xenograft tumor mice were constructed by SKBR3 and SKBR3-R cells. Proteins were detected by western blotting and immunohistochemistry. Transfected cells were constructed by shRNA lentivirus vectors. RT-QPCR was used to detect RNA. Cell proliferation was detected by MTS method. Cell jnvasion was detected by Transwell assay. Luciferase reporting assays were used to verify RNA interactions. IGF-1R/HER2 heterodimer was determined by immunocoprecipitation.@*RESULTS@#The expression of IGF2, p-IGF1R, p-Akt and p-S6K in SKBR3-R cells were significantly higher than those in SKBR3 cells, while the expression of PTEN protein was lower in SKBR3-R cells (P < 0.05). IGF1R/HER2 heterodimer in SKBR3-R cells was significantly increased (P < 0.01).The expression of IGF2 and invasion ability were significantly reduced while transfected with miR-98-5p in SKBR3-R cells (P < 0.05), but the IGF2 mRNA were no difference in both cells (P > 0.05). The expression of miR-98-5p was up-regulated and IGF2 was decreased in drug-resistant xenograft tumor mice after feeding with dihydromyricetin, and the tumor became more sensitivity to Herceptin (P < 0.05).@*CONCLUSION@#Dihydromyricetin could induce the expression of miR-98-5p, which binds to IGF2 mRNA to reduce IGF2 expression, inhibit the IGF-1R/HER2 formation, thereby reversing cell resistance to Herceptin in SKBR3-R cells.

Recent advances in research on vine tea,a potential and functional herbal tea with dihydromyricetin and myricetin as major bioactive compounds / 药物分析学报

Vine tea has been used as an herbal tea by several ethnic minorities for hundreds of years in China.Flavonoids,a kind of indispensable component in a variety of nutraceutical,pharmaceutical and cosmetic applications,are identified to be the major metabolites and bioactive ingredients in vine tea.Interest-ingly,vine tea exhibits a wide range of significant bioactivities including anti-oxidant,anti-inflammatory,anti-tumor,antidiabetic,neuroprotective and other activities,but no toxicity.These bioactivities,to some extent,enrich the understanding about the role of vine tea in disease prevention and therapy.The health benefits of vine tea,particularly dihydromyricetin and myricetin,are widely investigated.However,there is currently no comprehensive review available on vine tea.Therefore,this report summarizes the most recent studies investigating bioactive constituents,pharmacological effects and possible mechanisms of vine tea,which will provide a better understanding about the health benefits and preclinical assessment of novel application of vine tea.

Therapeutic effects study on the contents of dihydromyricetin in ampelopsis grossedentata from different places and their compatibility with fructus arctiine / 药学实践杂志

Objective To study the content differences of Dihydromyricetin in Ampelopsis grossedentata from different origins, parts and processing techniques, and improve the therapeutic effects by optimizing the compatibility of Ampelopsis grossedentata with Fructus arctiine. Methods The HPLC separation of Dihydromyricetin was performed on an Agilent ZORBAX SB-C18 column with methanol, 0.05 % phosphoric acid (30∶70) as the mobile phase. The flow rate was 1 ml/min, the detection wavelength was 291 nm, and the column temperature was 25 °C. Compatibility efficacy verification was performed with the inflammation model caused by cotton ball implantation in rats and ear swelling in mice. The net granulomatosis in the rats with cotton ball implantation and the swelling rate of mouse ears were recorded. Results Dihydromyricetin had a good liner recovery between 0.019 9-0.318 mg/ml (r=0.999). The extracted recovery was in the range of 95.04 %-100.4 %. The sample was stable within 24 h. This method had good repeatability. The combination of optimized high-dose Ampelopsis grossedentata with Fructus arctiine resulted in significantly lower net granuloma in rats and ear swelling rate in mice compared to the blank control group. Conclusion This method is simple and accurate. The content of dihydromyricetin varies greatly with different origins, parts, and processing techniques. Among them, the natural sun-dried vine tea in Jiangkou County, Guizhou Province has the highest content. The combination use of Ampelopsis grossedentata and Fructus Arctiine can significantly alleviate the pharyngeal symptoms, reduce the degree of inflammation, and achieve the therapeutic effect of clearing pharynx.

Dihydromyricetin inhibits proliferation and migration of gastric cancer cells through regulating Akt/STAT3 signaling pathways and HMGB1 expression / 南方医科大学学报

OBJECTIVE@#To investigate the inhibitory effects of dihydromyricetin on the proliferation and migration of gastric cancer BGC-823 cells and explore the molecular mechanisms.@*METHODS@#BGC-823 cells in routine culture were treated with different concentrations of dihydromyricetin (0, 40, 60, 80, 100, and 120 μg/mL) for 24 h, and the changes in cell viability were detected using CCK-8 assay; colony forming assay and Transwell assay were performed to assess the changes in colonyforming and migration abilities of the cells, respectively. The levels of MMP-2 and MMP-9 in the treated cells were determined using ELISA, and Western blotting was used to detect the expressions of E-cadherin, N-cadherin, cyclin D1, cyclin E1, HSP70 and HMGB1 and the phosphorylation levels of Akt and Stat3.@*RESULTS@#CCK-8 assay showed that dihydromyricetin treatment dose-dependently inhibited the viability of BGC-823 cells (@*CONCLUSIONS@#Dihydromyricetin inhibits the proliferation and migration of BGC-823 cells through suppressing the activation of Akt/stat3 signaling pathways and HMGB1 expression.

Preparation, characterization and pharmacokinetics of dihydromyricetin phospholipids complex loaded by nanostructured lipid carriers / 中草药

Objective: To prepare dihydromyricetin (DMY) phospholipids complex (DMY-PC) and its nanostructured lipid carriers (DMY-PC-NLC), and carry out in vitro and in vivo evaluation. Methods: DMY-PC was prepared by solvent evaporation method. High pressure homogenization method was used to prepare DMY-PC-NLC. Orthogonal test was employed to optimize the ratio of solid/liquid lipid, dose of lipids materials, dose of DMY-PC and the concentration of emulsifier of poloxamer. The lyophilized powder of DMY-PC-NLC was prepared with 5% of mannitol as protective agent. The comparation of in vitro release and pharmacokinetics between DMY-PC and DMY-PC-NLC was also studied. Results: DMY was in an amorphous state in DMY-PC. The results of 1HNMR showed that the structure of DMY was not changed. The optimized prescription of DMY-PC-NLC determined by orthogonal test was as follow: The ratio of solid/liquid lipid was 5:1, dose of lipids materials was 325 mg, dose of DMY-PC was 45 mg and the concentration of emulsifier of poloxamer was 0.9%. The average size, Zeta potential, entrapment efficiency and drug loading of DMY- PC-NLC was (197.25 ± 4.42) nm, (-18.2 ± 2.1) mV, (71.68 ± 1.36)% and (3.94 ± 0.24)%, respectively. The in vitro release model was accord with Weibull model and the equation was lnln(1-Mt/M∞)=0.700 1 lnt-1.954 1 (r = 0.971 4). The relative bioavailability of DMY-PC and DMY-PC-NLC were enhanced to 1.63 and 3.22 times compared to DMY, respectively. Conclusion: Compared with DMY-PC, the absorption was promoted by DMY-PC-NLC in further, and the bioavailability of DMY was enhanced effectively.

Effect of dihydromyricetin on the proliferation and migration ability in choriocarcinoma JEG-3 and JAR cells / 解剖学报

Objective To investigate the effect of dihydromyricetin (DM Y) on proliferation and migration in choriocarcinoma JEG-3 and JAR cells. Methods JEG-3 and JAR cells were treated with different concentrations (0 mg/L, 20 mg/L, 40 mg/L, 60 mg/L, 80 mg/L) of DMY for 24 hours and 48 hours, and the proliferation was analyzed by methylthio tetrazole (MTT) assay. The effect of DMY on migration was detected by wound healing (after 24 hours) and Transwcll assay (after treated JEG-3 for 36 hours and JAR for 24 hours). The expression of matrix metalloproteinase 2 (MMP-2) at mRNA and protein levels with different concentrations of DMY(0 mg/L, 40 mg/L, 60 mg/L, 80 mg/L) were detected by Real-time PCR (after 12 hours, 24 hours, 36 hours, 48 hours) and Western blotting (after treated 36 hours) respectively. Results The proliferation of JEG-3 and JAR ceils was inhibited significantly (/><0. 05) , after DMY treatment for 24 hours and 48 hours.DMY inhibited the migration of JEG-3 and JAR cells significantly (P<0. 05) with a dose-dependent manner. After JEG-3 and JAR Cells treated with DMY for 36 hours and 48 hours, the expression of MMP-2 mRNA decreased significantly (P<0. 05) , there were no significantly changes in DMY treatment with 12 hours and 24 hours. The expression level of MMP-2 protein was inhibited significantly after treatment with DMY for 36 hours (P<0. 05). Conclusion DMY might inhibit the proliferation in choriocarcinoma JEG-3 and JAR cells with a dose-dependent manner. The invasion and migration were inhibited by DMY through downregulation of MMP-2.

Effect and mechanism of dihydromyricetin on cognitive dysfunction in model rats with Alzheimer's disease / 中华行为医学与脑科学杂志

Objective@#To explore the protective effect and mechanism of the dihydromyricetin (DHM) on cognitive dysfunction in Alzheimer’s disease (AD) rat model.@*Methods@#The AD model of rats was established by injecting Aβ1-42 oligomolymer into the hippocampus. According to the random number table, 30 successfully constructed AD model rats were divided into AD group, AD+ DHM1 group and AD+ DHM2 group, with 10 in each group.And the rats in the three groups were intraperitoneally injected with normal saline, 100 mg/kg DHM and 200 mg/kg DHM for 21 days, respectively.Another 10 rats with body mass matching were taken as the control group.Morris water maze was used to evaluate the spatial learning and memory ability of rats in each group, the expression of inflammatory cytokines were detected by Elisa, and the expressions of AMPK and SIRT1 proteins were detected by Western blot.@*Results@#Compared with the control group, the escape incubation period of rats in AD group was prolonged, and the difference was statistically significant (day 5 : (10.36±2.80)s, (22.40±2.98)s; t=-18.63, P<0.05). Compared with AD group, the escape latency of rats in AD+ DHM1 group and AD+ DHM2 group were shortened (day 5: AD+ DHM1 group (15.68±3.06) s, AD+ DHM2 group (18.85±3.22) s; t=10.65, 4.13, both P<0.05). Compared with AD group, rats in AD+ DHM1 group and AD+ DHM2 group had more crossing times ((1.87±0.76), (2.75±0.63) and (3.78±0.71); t=-6.86, -9.83, both P<0.05), and the target quadrant residence time were extended ((17.08±1.99) s, (16.33±4.33) s, (22.59±4.21) s; t= 28.5, 8.63, both P<0.05). Compared with the control group, the levels of IL-1β, IL-6 and TNF-α in the serum and hippocampus of the AD group were significantly increased (serum: t=4.98, 7.87, 5.43, all P<0.05; hippocampus: t=11.13, 30.50, 23.38, all P<0.05). Compared with the AD group, the levels of IL-1β, IL-6 and TNF-α in the serum and hippocampus of the AD+ DHM1 group and the AD+ DHM2 group were significantly decreased, the difference was statistically significant(serum: AD+ DHM1 group t=-4.13, -10.70, -9.22, AD+ DHM2 group t=-1.75, -3.63, -18.75, all P<0.05; hippocampus: AD+ DHM1 group t=-69.13, -15.13, -6.50, AD+ DHM2 group t=-10.25, -39.00, -8.00, all P<0.05). Compared with the control group, the expression of p-AMPK/AMPK protein and SIRT1 protein in the AD group were decreased.The expression of the two proteins in the AD+ DHM1 group and the AD+ DHM2 group were increased, comparing with those of AD group, and the difference was statistically significant(all P<0.05).@*Conclusion@#DHM exerts protective role in AD model rats, which may be related to the activation of AMPK/SIRT1 pathway and the inhibition of inflammatory response.

Effect and mechanism of dihydromyricetin on cognitive dysfunction in model rats with Alzheimer's disease / 中华行为医学与脑科学杂志

Objective To explore the protective effect and mechanism of the dihydromyricetin (DHM) on cognitive dysfunction in Alzheimer’s disease (AD) rat model. Methods The AD model of rats was established by injecting Aβ1-42 oligomolymer into the hippocampus. According to the random number table,30 successfully constructed AD model rats were divided into AD group,AD+DHM1 group and AD+DHM2 group,with 10 in each group. And the rats in the three groups were intraperitoneally injected with nor-mal saline,100 mg/kg DHM and 200 mg/kg DHM for 21 days,respectively. Another 10 rats with body mass matching were taken as the control group. Morris water maze was used to evaluate the spatial learning and memory ability of rats in each group,the expression of inflammatory cytokines were detected by Elisa,and the expressions of AMPK and SIRT1 proteins were detected by Western blot. Results Compared with the con-trol group,the escape incubation period of rats in AD group was prolonged,and the difference was statistically significant (day 5 :(10. 36±2. 80)s,(22. 40±2. 98)s;t=-18. 63,P<0. 05). Compared with AD group,the escape latency of rats in AD+DHM1 group and AD+DHM2 group were shortened (day 5:AD+DHM1 group (15. 68±3. 06) s,AD+DHM2 group (18. 85±3. 22) s; t=10. 65,4. 13,both P<0. 05). Compared with AD group,rats in AD+DHM1 group and AD+DHM2 group had more crossing times ((1. 87± 0. 76),( 2. 75± 0. 63) and (3. 78±0. 71);t=-6. 86,-9. 83,both P<0. 05),and the target quadrant residence time were ex-tended ((17. 08±1. 99) s,(16. 33±4. 33) s,(22. 59±4. 21) s;t= 28. 5,8. 63,both P<0. 05). Compared with the control group,the levels of IL-1β,IL-6 and TNF-α in the serum and hippocampus of the AD group were significantly increased (serum: t=4. 98, 7. 87, 5. 43, all P<0. 05; hippocampus: t=11. 13, 30. 50, 23. 38,all P<0. 05). Compared with the AD group,the levels of IL-1β,IL-6 and TNF-α in the serum and hippocampus of the AD+DHM1 group and the AD+DHM2 group were significantly decreased,the difference was statistically significant ( serum: AD+DHM1 group t=-4. 13,-10. 70,-9. 22, AD+DHM2 group t=-1. 75,-3. 63,-18. 75,all P<0. 05;hippocampus:AD+DHM1 group t=-69. 13,-15. 13,-6. 50,AD+DHM2 group t=-10. 25,-39. 00,-8. 00,all P<0. 05). Compared with the control group,the expression of p-AMPK/AMPK protein and SIRT1 protein in the AD group were decreased. The expression of the two pro-teins in the AD+DHM1 group and the AD+DHM2 group were increased,comparing with those of AD group, and the difference was statistically significant(all P<0. 05). Conclusion DHM exerts protective role in AD model rats,which may be related to the activation of AMPK/SIRT1 pathway and the inhibition of inflammato-ry response.

Simultaneous Determination of 7 Anti-rheumatic Active Ingredients in Mongolian Medicine Sendeng- 4 Decoction Powder by HPLC-DAD / 中国药房

OBJECTIVE： To establish a method for simultaneous determination of 7 kinds of anti-rheumatic active ingredients in Mongolian medicine Sendeng-4 decoction powder， such as catechin， jasminoidin， dihydromyricetin， texifolin， rutin， myricetin and quercetin. METHODS： HPLC-DAD method was adopted. The determination was performed on Sil Green C18 column with mobile phase consisted of acetonitrile-0.1％ phosphoric acid solution （gradient elution） at the flow rate of 1 mL/min. The detection wavelength was set at 238 nm， and column temperature was 30 ℃. The sample size was 10 μL. RESULTS： The linear range of catechin， jasminoidin， dihydromyricetin， texifolin， rutin， myricetin and quercetin were 8.590-2 290， 10.56-3 901， 13.00-3 958， 8.815-564.2， 4.030-257.8， 8.130-750.5， 7.075-454.2 μg/mL （r≥0.999 1）， respectively； the limits of detection were 0.429 5，  0.264 0， 0.325 0， 0.220 4， 0.201 5， 0.203 2， 0.176 9 μg/mL， respectively； the limits of quantification were 1.030， 1.321， 1.302， 1.397， 1.637， 0.813 0， 0.707 5 μg/mL， respectively. RSDs of precision， stability （48 h） and repetition tests were all lower than 2.0％ （n＝6）. The average recoveries were 96.24％-99.28％ （RSD＝1.03％-1.63％， n＝6）. CONCLUSIONS： The established method is simple， accurate and reproducible， and can be used for simultaneous determination of catechin， jasminoidin， dihydromyricetin， texifolin， rutin， myricetin and quercetin in Mongolian medicine Sendeng-4 decoction powder.

Method Validation of Dihydromyricetin in Anti-hangover preparation by High Performance Liquid Chromatography / Монголын Анагаах Ухаан

Background@#The high performance liquid chromatography (HPLC) method was developed for selective determination of dihydromyricetin in capsule formulation dietary supplement containing other components. Further, the proposed method was validated for linearity, precision (system precision, method precision, intermediate or inter-day precision), and accuracy, stability in analytical solution, system suitability and ruggedness. The developed method exhibited the best results in terms of the aforesaid validation parameters. The other components and additives did not interfere in their determinations. The method was found to be selective, simple, economical, accurate, reproducible, rapid and reliable for routine estimation purpose of dihydromyricetin in dietary supplement capsule.@*Goal @#The goal of this study was to develop the validation method of dihydromyricetin in the dietary supplement.@*Material and Methods @#The hangover preparation was produced by Technological section of Drug Research Institute. The standard dihydromyricetin was supplied from Sigma Aldrich Co. We used solvents for HPLC grade (methanol, acetonitrile). Chromatographic conditions: A gradient HPLC (Shimadzu LC20AD) with serial dual plunger pump; analytical column: Supelco inertsil С18 250 × 4.6 mm, particle size 5 μm; flow rate: 1 ml/min; column temperature: 350C, detection: UV 365 nm. Chromatographic procedure: 20 μl of the mixed standard preparation and assay (sample) preparation were separately injected into the chromatography, the chromatograms were recorded, and the responses for the major peaks were measured. The run time was approximately 10 minutes.@*Results @#The calibration curves for dihydromyricetin were made by plotting the peak area versus the concentration for each analyte using regression analysis. Each calibration curve was obtained using six levels of concentrations in the range 28-224 µg/mL. The linear correlation coefficient (r2 ) for all calibration curves was higher than 0.999 for all analytes. The LOD and LOQ for dihydromyricetin were in 11.29 µg/mL and 34.21 µg/mL, respectively. Accuracy and precision were assessed by analyzing five sets of samples, independently prepared at low, middle and high concentrations. The RSD values of both repeatability and intermediate precision were below 0.261% and 0.262%. The accuracy remaining between 101.65 to 104.7%. The resulting accuracy data were satisfactory for the quantitative analysis of dihydromyricetin in anti-hangover preparation. The results of summarized in Table 1, 2, 3. This article presents a simple, accurate, reproducible, and thoroughly validated HPLC-based method for qualitative and quantitative analysis of dihydromyricetin, as part of the quality assessment of products containing anti-hangover preparation.

Effects of dihydromyricetin on biological properties of Bloom syndrome helicase / 中国药理学通报

; Aim To explore the anti-tumor mechanism of dihydromyricetin (DMY), a kind of flavonoid compound with anti-inflammatory and anti-tumor effects, via studying the effect of DMY on biological activities of Bloom helicase. Methods The effect of DMY on the biological activities of BLM helicase was studied by ultraviolet spectrum (UV), circular dichroism (CD), fluorescence polarization and free phosphorus detection. Results The results of CD and UV showed that DMY could bind to a site of the BLM helicase. In the concentration of DMY in 0 ~ 25 μmol . L 1 range, DMY showed a positive correlation with the interference ability of BLM helicase secondary structure with the increase of concentration, while in the concentration of DMY in 25 ~ 75 μmol . L 1 range, DMY showed a negative correlation. Fluorescence polarization and free phosphorus detection experiments showed that DMY could bind to BLM helicase, thus inhibiting the helicase activity of BLM helicase. Conclusions DMY can competitively bind to the DNA binding site of BLM helicase and change the spatial structure of BLM helicase, inhibiting the binding of BLM helicase to DNA and the biological activity of BLM helicase accordingly.

Dihydromyricetin promoted browning of subscapular adipose tissues in obese mice fed with high-fat diet via activating AMPK-PGC1 a-Sirtl signaling pathway / 中国药理学通报

Aim To observe the effect of dihydromyricetin (DHM) on the browning of subscapular adipose tissues in the high-fat diet fed obese mice, and clarify the mechanism. Methods C57BL/6J mice were fed with normal and high-fat diet, and were treated with or without low dose (125 mg • kg"1 • d"1) or high dose (250 mg • kg"1 • d"1) DHM for 16W. The body mass of mice was measured every four weeks during the experiment. After 16 weeks, the mice were fasted overnight. Blood samples were taken for fasting blood glucose. The mice were then sacrificed and the body length measured. The Lee' s index was calculated. The size of the subscapular adipocytes was observed by HE staining. The mRNA and protein expression of uncoupling protein 1 (UCP1) , PR domain containing 16 (Prdml6) , adenylate-activated protein kinase (AMPK) , peroxisome proliferator-activated receptor gamma-assisted activator alpha (PGCla) and silencing signal regulator 1 (Sirtl) were assessed by real time quantitative PCR and Western blot assay. Results Compared with normal control group (ND group) , the body mass of mice in high-fat diet group (HFD group) significantly increased, suggesting that the obese mice model was successfully replicated. In addition , blood glucose, Lee' s index and the fat cell diameter of the subscapular adipose tissues in HFD group all significantly increased, while the mRNA and pro-tein expression of UCP1, Prdml6, AMPK, PGCla and Sirtl significantly decreased. DHM markedly re-versed the changes of the above indexes of HFD mice, but DHM did not significantly affect the above indexes of normal mice. Conclusions Dihydromyricetin promotes the browning of subscapular adipose tissues in mice fed with high-fat diet, which might be via activating AMPK-PGC1 a-Sirtl signaling pathway.

Preparation of long-circulating dihydromyricetinliposomes and its pharmacokinetics in rats / 中草药

Objective To prepare dihydromyricetin (DMY) long-circulating liposomes and evaluate in vitro release dynamics and in vivo pharmacokinetics in rats. Methods Film-ultrasonic method was used to prepare DMY liposomes by single factor experiment and orthogonal test to optimize the formulation and preparation of DMY liposomes. The particle size and zeta potential of liposomes were determined by laser particle size analyzer. The morphological examination of liposomes was performed by using transmission electron microscopy. The liposome release in vitro was studied using dialysis method. DMY concentration in rat plasma was determined by the established LC-MS/MS method. Results The optimal prescription was 75∶20∶5 for soybean phospholipid-cholesterol-mPEG 2000-DSPE, and 1∶12 for DMY-lipid (wt/wt) with the ultrasonic time of 20 min and loading temperature of 60 ℃ in pH 5.0 PBS buffer. Under the optimized conditions, DMY liposomes was sphere with mean particle size of (117.9 ± 5.5) nm and mean zeta potential of (−2.6 ± 1.7) mV, the encapsulation efficiency and drug-loading content was (54.7 ± 3.3) % and (4.3 ± 0.2) %, respectively. The in vitro accumulative release rate of 48 h was 86% in pH 1.2 and pH 6.8 dissolve medium. Compared with free DMY, the t1/2z and AUC0-∞ of DMY liposome were increased by 2.7-fold and 1.8-fold, respectively. Conclusion Compared with free DMY, DMY liposomes released gently and slowly in vitro, eliminated slowly in vivo, and had higher bioavailability of oral administration.

Protective effect and mechanism of dihydromyricetin on atherosclerosis in ApoE-/-mice / 中成药

AIM To explore the protective effect and mechanism of dihydromyricetin (DHM) on atherosclerosis (AS) in ApoE-/-mice induced by high fat diet (HFD).METHODS Thirty healthy 6-week-old male ApoE-/-mice were randomly divided into model group,DHM group [50 mg/(kg · d)] and Atorvastatin group [5 mg/(kg · d),i.g.],and another 10 male C57BL/6J mice were taken for control group.The mice in the control and model group were administered with 0.5％ CMC-Na solution,while the other two groups were given DHM and atorvastatin suspension (0.2 mL/10 g).All mice went on with a 10-week HFD diet (0.3％ cholesterol,20％ fat),after which the orbit blood was procured for serum isolation and subsequent determination of levels of SOD,GSH-Px,CAT and MDA by the biochemical analyzer and test of lipid accumulation in the aotic root by oil red O.The detection of macrophages accumulation and Caspase-3 expression level in the aortic root were achieved by immunofluorescence,and the macrophages apoptosis by TUNEL assay.RESULTS Significant post-treatment reduction in levels of TG,TC,and remarkable amelioration of LDL-C and plaque area in the aortic root were observed in DHM group when compared with model group.Meanwhile,DHM contributed to marked decrease of MDA level and improvement in activities of CAT,GSH-Px and SOD,the obvious macrophages accumulation prevention in atherosclerotic plaque.Additionally,its significant inhibition on macrophage apoptosis in aortic root was verified by the TUNEL assay.CONCLUSION DHM reduces the lipid accumulation in HFD-induced ApoE-/-mice,mechanically a resultant of its inhibition on macrophage apoptosis.

Regulation of dihydromyricetin on immune function of spleen in the rats with rheumatoid arthritis / 上海交通大学学报（医学版）

Objective • To investigate the immunoregulatory effect of dihydromyricetin (DMY) on the spleens of rats with rheumatoid arthritis. Methods • Thirty Wistar rats were randomly divided into five groups, i.e., normal group, model group and DMY administration groups (5, 25, and 50 mg/kg). The type Ⅱ collagen-induced rheumatoid arthritis (CIA) model was established in model group and DMY administration groups. DMY administration groups were intraperitoneally injected with DMY every other day at set doses. After five weeks of administration, rats of each group were sacrificed, and the spleens were taken out to prepare tissue sections, and pathological changes were observed after hematoxylin-eosin staining. The spleen lymphocytes of model group were isolated, and the effect of DMY on the proliferation of spleen lymphocytes in inflammatory state was detected by cell-counting-kit 8. The percentages of different subsets of T lymphocytes were detected by flow cytometry. The expression of inflammatory factors in spleen lymphocytes was determined by PCR and ELISA. Results • Compared with model group, inflammation-related pathological changes in the spleens of DMY-administered groups were significantly relieved. DMY could inhibit the proliferation of spleen lymphocytes, reduce the percentage of CD3+CD4+ cells and slightly increase the percentage of CD3+CD8+ cells after the treatment of concanavalin (ConA) in vitro. The results of PCR and ELISA showed that DMY could inhibit the expression of proinflammatory cytokines and promote the expression of anti-inflammatory factors both in vitro and in vivo. Conclusion • DMY has immunomodulatory effects on immune-associated T lymphocytes in the spleens of CIA rats in vivo and in vitro, thereby inhibiting systemic inflammatory response.

Protective effects and mechanism of dihydromyricetin on non-alcoholic fatty liver disease in ApoE-/-mice / 中成药

AIM To explore the protective effects and mechanisms of dihydromyricetin (DHM) against non-alcoholic fatty liver disease (NAFLD) in ApoE-/-mice.METHODS The 40 male healthy six week-old ApoE-/mice were randomly divided into model group,DHM (50,100 mg/kg) group and Bicyclol group.Ten male C57 mice were used as the control group.The control and model groups were administrated with 0.5％ CMC-Na solution and the other three groups were given DHM [50,100 mg/(kg-d),i.g.] and Bicyclol [140 mg/(kg · d),i.g.].All mice were fed with high fat diet (0.3％ cholesterol,20％ fat) for 12 weeks.At the end of the experiment,the blood was collected from the orbit and the serum was separated for blood lipid detection.AST and ALT expression levels were detected by automatic biochemical analyzer.HE staining and oil red O monitored the liver injury and lipid accumulation.The oxidase enzymes were measured by the commercial kits.The hepatocyte apoptosis was determined by TUNEL assay,and the Bcl-2,Caspase-3 expression levels were detected by immunohistochemistry.The AMPK and phosphorylation of AMPK expression levels were determined by Western blot.RESULTS After the treatment,liver lipid accumulation and liver injury,TUNEL postive cells sharply increased compared with the control group.Meanwhile,the Caspase-3 expression level was significantly upregulated and the Bcl-2 was markedly downregulated in the model group.DHM [50,100 mg/(kg · d)] remarkably reduced AST and ALT levels,liver lipid accumulation and TUNEL positive cells and modulated oxidase enzymes expression level compared with the model group.Moreover,DHM obviously increased Bcl-2 expression and decreased Caspase-3 expression in liver tissue,and inhibited AMPK phosphorylation.CONCLUSION DHM reduces the lipid accumulation and protects liver injury from high fat diet induced NAFLD mice,the protective mechanism of DHM may be related to inhibiting AMPK phosphorylation and inhibiting hepatocyte apoptosis.