Wogonoside alleviates high glucose-induced dysfunction of retinal microvascular endothelial cells and diabetic retinopathy in rats by up-regulating SIRT1 / 南方医科大学学报

OBJECTIVE@#To investigate the effects of wogonoside on high glucose-induced dysfunction of human retinal microvascular endothelial cells (hRMECs) and streptozotocin (STZ)-induced diabetic retinopathy in rats and explore the underlying molecular mechanism.@*METHODS@#HRMECs in routine culture were treated with 25 mmol/L mannitol or exposed to high glucose (30 mmol/L glucose) and treatment with 10, 20, 30, 40 μmol/L wogonoside. CCK-8 assay and Transwell assay were used to examine cell proliferation and migration, and the changes in tube formation and monolayer cell membrane permeability were tested. ROS, NO and GSH-ST kits were used to evaluate oxidative stress levels in the cells. The expressions of IL-1β and IL-6 in the cells were examined with qRT-PCR and ELISA, and the protein expressions of VEGF, HIF-1α and SIRT1 were detected using Western blotting. We also tested the effect of wogonoside on retinal injury and expressions of HIF-1α, ROS, VEGF, TNF-α, IL-1β, IL-6 and SIRT1 proteins in rat models of STZ-induced diabetic retinopathy.@*RESULTS@#High glucose exposure caused abnormal proliferation and migration, promoted angiogenesis, increased membrane permeability (P < 0.05), and induced inflammation and oxidative stress in hRMECs (P < 0.05). Wogonoside treatment concentration-dependently inhibited high glucose-induced changes in hRMECs. High glucose exposure significantly lowered the expression of SIRT1 in hRMECs, which was partially reversed by wogonoside (30 μmol/L) treatment; interference of SIRT1 obviously attenuated the inhibitory effects of wogonoside against high glucose-induced changes in proliferation, migration, angiogenesis, membrane permeability, inflammation and oxidative stress in hRMECs. In rat models of STZ-induced diabetic retinopathy, wogonoside effectively suppressed retinal thickening (P < 0.05), alleviated STZ-induced retinal injury, and increased the expression of SIRT1 in the retinal tissues (P < 0.001).@*CONCLUSION@#Wogonoside alleviates retinal damage caused by diabetic retinopathy by up-regulating SIRT1 expression.

Synergistic Effect of Baicalin and Wogonoside on NF-κB Signaling Pathway Based on Medium-efficiency Equation / 中国实验方剂学杂志

Objective:Quantitative analysis of anti-inflammatory synergistic pharmacodynamics mechanism of baicalin and wogonoside by medium efficiency principle. Method:inflammatory cell model was constructed by stimulating RAW264.7 cells by lipopolysaccharide （LPS） 100 μg·L-1 in vitro. The experiment was performed in the normal group， the model group， the andrographolide group （10 μmol·L-1）， the baicalin group （2.06，4.13，8.25，16.5，33，66，132 μmol·L-1） and the wogonoside group （2.94，5.88，11.75，23.5，47，94，188 μmol·L-1） and the baicalin-wogonoside combination group ［（2.06+2.94）（4.13+5.88）（8.25+11.75）（16.5+23.5）（33+47）（66+94）（132+188） μmol·L-1］. The levels of tumor necrosis factor-α （TNF-α） and interleukin-6 （IL-6） in the cell culture supernatants after drug intervention for 50 min and 4 h were detected by enzyme-linked immunosorbent assay （ELISA） method. The level of nitric oxide （NO） in the cell culture supernatant after drug intervention for 24 h were detected by Griess method. Western blot was used to detect the activation levels of phosphorylation of nuclear factor-κB p65（p-NF-κB p65） and inducible nitric oxide synthase（iNOS） in cells after drug intervention for 2 h and 12 h. The fa/fu-dose profile of each indicator was drawn to observe the increase or decrease of effect. Result:Compared with normal group， the expression of p-NF-кB p65， iNOS and cytokines including TNF-α， IL-6 and NO （P<0.05，P<0.01） in the model group were significantly up-regulated. Compared with the model group， each group at high doses could inhibit the phosphorylation of NF-кB p65 protein（P<0.05），the baicalin group and the combined group could down-regulate the expression of iNOS protein in a concentration-dependent manner（P<0.01） and the baicalin group had no obvious inhibitory effect. each administration group at high dose could significantly inhibit the production of NO（P<0.05），but each group had no inhibitory effect on IL-6 production. The baicalin group and the combined group could significantly Inhibit the production of TNF-α（P<0.05） and there was no significant difference between the baicalin group and the model group. At the experimental dose， the fa/fu-dose table showed that the fa/fu value of p-NF-кB p65 and IL-6 in the combined group was not greater than the baicalin group and the wogonoside group. The fa/fu value of iNOS， TNF-α and NO in the combined group is higher than the baicalin group and the wogonoside group. Conclusion:The baicalin and wogonoside have different effects on different targets in the NF-κB pathway. The wogonoside is the main pharmacological substance in this combination and the combination shows different degrees of synergy or antagonism effects on different targets.

The parametric measurementof physico-chemical parameter and three types of effective components in Jiedu-Zhixue Decoction / 国际中医中药杂志

Objective@#To set up a method to measure the content of baicaline, wogonoside, and paeonol in Jiedu-Zhixue Decoction extract and to measure their physico-chemical parameters.@*Methods@#By using HPLC analytical methods to measure the content of baicaline, wogonoside, and paeonol in Jiedu-Zhixue Decoction extract. The RP18 column was adopted, gradient eluted with mobile phase of acetonitrile-1% phosphoric acid water, flow rate was 1 ml/min, detection wavelength was 274 nm, and the column temperature was 30 ℃. The method quantitatively analyze the compound extracts of baicaline, wogonoside and paeonol were used.@*Results@#The method isrepeatable, stable with good recorey rates. The calibration curves of baicaline was in good linearity over the range of 0.191 2-1.147 2 μg. The calibration curves of wogonoside was in good linearity over the range of 0.041 5-0.207 4 μg. The calibration curves of paeonol was in good linearity over the range of 0.019 5-0.156 3 μg. There was no significant difference among the component of 3 different batches of Jiedu-Zhixue Decoction extract, and the pH value range was 5.240-5.753, relative density range was 0.846-0.889.@*Conclusions@#The method used in this stydy to measure the effective components of Jiedu-Zhixue Decoction extract was stable and feasible, which is suitable for quality control of Jiedu-Zhixue Decoction.

Simultaneous Determination of 6 Components in Chaihuang Tablets by HPLC-DAD / 中国药房

OBJECTIVE： To establish a method for simultaneous determination of 6 components in Chaihuang tablets， such as baicalin， wogonoside， baicalein， wogonin， saikosaponin a and saikosaponin d in Chaihuang tablets. METHODS： HPLC-DAD method was used to detect 3 batches of Chaihuang tablets from same manufacturers. The determination was performed on Agilent Eclipse XDB-C18 column with mobile phase consisted of acetonitrile-triethylamine phosphate aqueous solution （pH adjusted to 7.0， gradient elution） at flow rate of 1.0 mL/min. The detection wavelengths were set at 210 nm （saikosaponin a， saikosaponin d） and 277 nm （baicalin， wogonoside， baicalein， wogonin）. The column temperature was 30 ℃， and sample size was 5 μL. RESULTS： The linear ranges of baicalin， wogonoside， baicalein， wogonin， saikosaponin a and saikosaponin d were 0.379 5-7.590 4 μg，   0.082 96-1.659 2 μg， 0.039 39-0.787 8 μg， 0.040 72-0.814 4 μg， 0.040 45-0.809 0 μg， 0.038 63-0.772 6 μg （all r≥0.999 3）， respectively. The limits of detection were 0.008， 0.007， 0.005， 0.005， 0.020 and 0.018 μg/mL. The limits of quantitation were 0.025， 0.022， 0.015， 0.015， 0.060， 0.054 μg/mL. RSDs of precision， reproducibility and stability tests （48 h） were all lower than 1.5％ （n＝6）. Average recoveries were 98.46％， 97.06％， 100.90％， 96.13％， 96.91％， 96.57％ （RSD＜2.0％， n＝6）. CONCLUSIONS： Established method is simple， accurate and reproducible for 6 components in Chaihuang tablets， and can be used for quality control of the tablet.

Effects of ecological factors and gene expression of key enzymes on synthesis of major medicinal ingredients of Scutellaria baicalensis in autumn / 中草药

Objective:To study the dynamic changes of the pharmacodynamic components of Scutellaria baicalensis in the harvest period and the effects of ecological factors and key enzyme expression on it. Methods :The artificial cultivated annual S. baicalensis was studied and the expression of nine key enzyme genes (PAL, C4H, 4CL, CHS, CHI, FNS, F6H, UBGAT, and GUS) in the roots of S. baicalensis were determined by real-time quantitative PCR. The content of four main flavonoids (baicalin, wogonoside, baicalein, and wogonin) in the roots was determined by HPLC. The meteorological data of S. baicalensis were collected by the ecological meteorological station. SPSS statistical software and DPS statistical software were used for data analysis. Results: The content of flavonoids of four monomers of annual S. baicalensis decreased slowly in autumn, so the best harvest time of S. baicalensis was in early September. The results of grey correlation analysis showed that the ecological factors affecting the four flavonoids were SWC, Max Ta, RH, and PAR. The expression of C4H and UBGAT genes had an important effect on the accumulation of flavonoids in the roots of S. baicalensis in autumn. Maximum rainfall intensity may indirectly affect the accumulation of the pharmacodynamic components of S. baicalensis by affecting the gene expression of key enzymes. Conclusion: The dynamic changes of four main flavonoids of annual S. baicalensis in autumn and the expression of key enzyme genes of S. baicalensis annual are clarified, providing the theoretical basis for the clarification of the physiological and ecological mechanism of the biosynthesis of S. baicalensis and the improvement of the quality of S. baicalensis.

Research progress on pharmacological effects and mechanisms of flavonoids from Scutellaria baicalensis based on aging hypotheses / 中草药

Baicalin, baicalein, wogonoside, and wogonin are major flavonoids of Scutellaria baicalensis, which have been reported to possess various pharmacological effects such as anti-oxidation, immunomodulation, mitochondrial protection, telomerase inhibition, and anti-inflammatory activities. Recently, researches on their anti-aging activity have also gradually increased. Therefore, combining with kinds of aging hypotheses, e.g. the free radical aging hypothesis, immunosenescence aging hypothesis, spleen-kidney aging hypothesis of Chinese medicine, the mitochondrial aging hypothesis, the telomere hypothesis of cellular aging, inflamm-aging, we focus on the related pharmacological properties and mechanisms of these four flavonoids and make a review, aiming to provide a theoretical basis for the anti-aging research of flavonoids in S. baicalensis.

UPLC-Q-TOF/MS-based metabolic profiles of wogonoside by intestinal bacteria / 中草药

Objective To investigate the metabolic effects of wogonoside by human gut microbiota. Methods UPLC-Q-TOF/MS was used to analyze the metabolites of wogonoside. Additionally, 16 S rDNA molecular biology method was applied to the identification and composition analysis of intestinal bacteria. Results Wogonoside was mainly metabolized to wogonin and small quantities of demethylated metabolites. Most of Enterococcus and Escherchia had the metabolic abilities, among which a single bacterium Escherichia sp. 41 possessed the stronger metabolic activity. Conclusion This study has a good guiding significance for in vivo process and efficacy of wogonoside.

Quantitative determination of multiple characteristic components in Gegen Qinlian Pills by QAMS method / 中草药

Objective To develop a quantitative analysis multi-components by single marker (QAMS) method for the quantification of multiple characteristic components, namely, puerarin, daidzin, jatrorrhizine, palmatine, soybean glycol, wogonoside, wogonin, in Gegen Qinlian Pills (GQP) with puerarin as the internal reference. Methods Samples were analyzed by Waters ultra-high efficiency liquid chromatography (UPLC) system, equipped with a reverse phase Acquity BEH C18 chromatographic column, with the mobile phase of methanol-0.1% phosphoric acid solution at a flow rate of 0.3 mL/min. The column temperature and the detection wavelength set at 30 ℃ and 260 nm respectively. Based on puerarin as the internal reference, the relative correction factors (RCFs) with other six characteristic components were calculated, then compared with the external standard method. This result benefits to verify the accuracy and advantages of the established QAMS method. Results Under the linear range of determination, RCFs of daidzin, jatrorrhizine, palmatine, soybean glycol, wogonoside, wogonin with reference to puerarin, were 1.02, 1.07, 1.05, 1.32, 0.62, and 0.90 in GQP, respectively. And the repeatability was good in different experimental conditions. Moreover, there was no significant difference on the quantitative results of seven characteristic components, derived from between external standard method and QAMS method in the 10 batches of GQP samples. The range of seven characteristic components contents in the 10 batches of GQP samples, namely puerarin, daidzin, jatrorrhizine, palmatine, soybean glycol, wogonoside and wogonin, were 8.923-10.746 mg/g, 2.231-2.988 mg/g, 0.825-1.197 mg/g, 1.274-1.522 mg/g, 2.330-2.713 mg/g, 0.836-0.951 mg/g, and 0.901-1.092 mg/g, respectively. Conclusion In present study, a feasible, convenient and accurate QAMS method with puerarin as the internal reference was established. Therefore, it is suitable to quantify multiple characteristic components in GQP and provide a useful approach for the quality control of GQP.

Determination of Four Flavonoids in Shenyan Siwei Granules by Quantitative Analysis of Multi-components with Single Marker / 医药导报

Objective To establish an HPLC method to measure four flavonoids ( baicalin, wogonoside,baicalein and wogonin) in shenyan siwei granules by quantitative analysis of multi-components with single marker ( QAMS ) . Methods Agilent ZORBAX SB-C18 column(4. 6 mm × 250 mm,5 μm) was used. The mobile phase was composed with methanol and 0. 4% phosphoric acid solutionat at 1. 0 mL·min-1 flow rate with gradient elution. The detection wavelength was 278 nm. Baicalin was used as the internal reference substance. The relative correction factors ( RCF) between the baicalin and the other three flavonoids were established to detect the quantitation of baicalin and calculate the quantitation of the other three constituents. The external standard method was used for quantitating the four constituents, and the method was evaluated by comparing to the quantitative results between external standard method and QAMS method. Results The results of QAMS method had no significant difference with those of external standard method. Conclusion It is feasible and accurate to control the quality of shenyan siwei granules with QAMS.

Effects of 4 Flavonoids from Scutellaria Baicalensis on Different Cancer Cell Lines / 世界科学技术-中医药现代化

Wogonoside,wogonin,baicalin and baicalein are major chemical constituents of S.baicalensis.Baicalin,baicalein and wogonin have been reported previously to exert anti-cancer effects.The present study compared the anti-cancer effects of the four flavonoids individually towards four human cancer cell lines after 24-and 48-hour treatment based on cell viability assay.As a result,wogonin inhibited the growth of the four cell lines.The IC50 values after 48 hours of incubation were 97.9 μM for A549 lung adenocarcinoma cells,while 15.3 μM for HeLa cervical carcinoma cells,147 μM for HepG2 hepatocellular carcinoma cells and 104 μM for MCF-7 breast adenocarcinoma cells.In contrast,wogonoside,the glycoside of wogonin,showed no inhibitory effect against any one of the four cell lines.Baicalein inhibited the growth of HeLa cells,while baicalin inhibited the growth of HepG2 cells,both with higher IC50 values and less potent than wogonin.These findings established wogonin as the most active anti-cancer agent among the four major flavonoids of S.baicalensis.Since baicalin,the most abundant flavonoid in the herb,was found to enhance the growth of MCF-7 cells.Clinical applications of ingredients from S.baicalensis to the treatment of cancer should be carried out with purified compounds.

Simultaneous determination of baicalin, wogonoside, baicalein, wogonin, and oroxylin-A in Scutellaria baicalensis by UPLC / 中草药

Objective: To establish a UPLC method for the simultaneous determination of five constituents (baicalin, wogonoside, baicalein, wogonin, and oroxylin-A) in Scutellaria baicalensis. Methods: Five constituents in S. baicalensis were simultaneously determined by UPLC with Waters Acquity UPLC BEH C18 column (50 mm × 2.1 mm, 1.7 μm). With a gradient program of acetonitrile-0.1% formic acid at the flow rate of 0.6 mL/min, and the detection wavelength was set at 280 nm. Results: Baicalin, wogonoside, baicalein, wogonin, and oroxylin-A in S. baicalensis were clearly and respectively separated in 15 min. The relationship between the concentration and the peak areas of the five constituents in S. baicalensis was all linear. The RSD of recovery, precision, and reproducibility was all less than 3.0%. Conclusion: The method is rapid, simple, reliable, and accurate, and has been successfully used to the quantification of the five constituents in S. baicalensis.

Simultaneous determination of seven components in Fangfengtongsheng pills by HPLC / 中国药学杂志

OBJECTIVE: To develop an HPLC method for simultaneous determination of seven components in traditional Chinese medicines Fangfengtongsheng pills from different factories. METHODS: The chromatographic analyses were carried out on a kromasil C18 column(4.6 mm × 250 mm, 5 μm); and the mobile phase was methanol(A)-0.5 acetic acid at 1.0 mL · min-1 in gradient elution mode which was 15% A from 0 to 10 min, 15%-30% A from 10 to 15 min, 30%-40% A from 15 to 40 min, 40%-70% A from 40 to 80 min, and 70%-90% A from 80-120 min; the column temperature and detection wavelength were set at 25°C and 280 nm, respectively. RESULTS: Seven components were determined by HPLC, including baicalin, wogonoside, baicalein, wogonin, chrysin, emodin, and chrysophanol. The recoveries were 101.3%, 98.6%, 99.7%, 96.4%, 102.7%, 100.2% and 98.4%, respectively, and the linearity were good (r ≥ 0.9992) in the concentration ranges of 2.33-300.0, 0.407-120.0, 1.563-50.0, 0.625-40.0, 0.625-77.8, 1.563-100, and 1.625-120 μg · mL-1. CONCLUSION: Seven components are analyzed in Fangfengtongsheng pills by RP-HPLC with gradient elution, and the method has good specificity, reproducibility and high sensitivity, which can be applied to control the quality of Fangfengtongsheng pills.

Simultaneous determination of four flavones in Yinhuang preparations by QAMS / 中国药学杂志

OBJECTIVE: To establish a quality evaluation method, quantitative analysis of multi-components with a single-marker(QAMS), to simultaneously determine the contents of four flavones in Yinhuang preparations. METHODS: Baicalin was used as the internal reference substance. The relative correlation factors(RCF) of wogonoside, baicalein and wogonin to baicalin were calculated and established. The contents of these four flavones were determined by the external standard method and QAMS respectively. The QAMS method was validated through comparison of the results obtained by the two different methods. RESULTS: Within the linear ranges, the values of RCF determined at 274 nm of wogonoside, baicalein and wogonin to baicalin were 1.20, 1.62 and 1.68 respectively. The RCF showed good reproducibility within the range of 1.7%-4.8% for the three studied compounds. The two methods did not show significant difference in assay results. CONCLUSION: The QAMS method is feasible, credible, and can be used to determine multiple flavones in Yinhuang preparations.

High performance liquid chromatography in determination of baicalin and wogonoside contents in Xiaochaihu decoction / 第二军医大学学报

Objective: To establish an HPLC method for determination of baicalin and wogonoside contents in Xiaochaihu decoction. Methods: Decoction pieces were mixed and decocted with water. The chromatographic column was Agilent Zorbax XDB-C18 (150 mmX4.6 mm, 5 μm); the mobile phase was composed of 0.2% phosphate acid-water(A) and acetonitrile (B) with gradient elution at a flow rate of 1 ml/min. The detection wavelength was set at 275 nm, the temperature of column was 25°C, and the injection volume was 15 μL. Results: Baicalin and wogonoside were separated at baseline within 30 min with good linearity; the standard curves for baicalin and wogonoside were Y=44.16X-36.22 (r=0.999,9) and Y=52.08X-28.69(r= 0.999,9), respectively. The intra-day and inter-day precisions were both less than 1%, and the limits of qualification were 0.615,6 μg/ml for baicalin and 0.220,8 μg/ml for wogonoside. The recovery rates (n = 6) were 95.73% (RSD = 0.8%) for baicalin and 97.02% (RSD= 1.56%) for wogonoside. Conclusion: The method is simple, accurate, stable, and reliable in determining the contents of baicalin and wogonoside in Xiaochaihu decoction, and it can be used for the quality control of this preparation.

Optimization of Cellulase-assited Extraction Technology of the Steam-processed Scutellaria baicalensis by Orthogonal Experiment / 中国药房

OBJECTVIE:To optimize the cellulase-assisted extraction technology of steam-processed Scutellaria baicalensis. METHODS:The cellulase-assisted extraction technology of steam-processed S.baicalensis was optimized by orthogonal experiment with the extraction rates of baicalin and wogonoside as indexes and with cellulase concentration,extracting temperature,extracting time and pH value as factors. RESLUTS:The optimal cellulase-assisted extraction technology of steam-processed S.baicalensis was as follows:the concentration of cellulase was 15 U?g-1; the extracting temperature was 50 ℃; the extracting time was 7 h and the pH value was 4.8. Under the optimal conditions,the extraction rates of baicalin and wogonoside were 8.44% and 2.62%,respectively. CONCLUSION:The cellulase-assisted extraction technology of steam-processed S.baicalensis is superior to the traditional water-decoction method,and it proves to be an effective process for the extraction of baicalin and wogonoside from steam-processed S.baicalensis and by which the utilization rate of S.baicalensis can be improved.

Pharmacokinetic study on baicalin and wogonoside in diabetic rats in vivo after oral administrating Huanglian-Jiedu Decoction / 中草药

Objective To investigate the pharmacokinetic profiles of baicalin and wogonoside in diabe-tic rats in vivo,which are two major constituents in Huanglian-Jiedu Decoction(HJD).Methods The diabetic rats were induced by ip administration of streptozotocin(STZ).After the establishment of the method and the set-up of diabetic rats,the pharmacokinetic profiles of baicalin and wogonoside were investigated.Diabetic and normal rats were ig HJD extract,and then the blood samples were collected at the given time points.Contents of baicalin and wogonoside in diabetic and normal rat plasma were assayed by HPLC-UV method.The pharmacokinetic parameters(except Cmax and tmax) were analyzed by noncompartmental method.The area under the serum concentration-time curve(AUC0-t) was calculated using trapezoidal rule to the last point.Moreover,the presystemic metabolism of baicalin was investigated and compared to explore the pharmacokinetic difference by fermentation of baicalin in feces suspensions of normal and diabetic rats,respectively.Results The pharmacokinetic parameters of baicalin in normal and diabe-tic rats were:(4.50?1.92) and(7.5?1.0) h for tmax 2;(2.83?0.25) and(9.54?2.87) ?g/mL for Cmax1,(2.56?0.63) and(6.58?1.15) ?g/mL for Cmax2;(37.58?7.57) and(92.75?24.62) ?g?h/mL for AUC(0～24),(6.6?2.4) and(12.64?3.35) h for t1/2,respectively.And the pharmacokinetic parameters of wogonoside in normal and diabetic rats were:(5.5?1.0) and(8.00?1.63) h for tmax 2;(1.36?0.17) and(6.16?1.40) ?g/mL for Cmax1;(1.58?0.17) and(4.11?0.76) ?g/mL for Cmax2;(27.02?3.72) and(58.16?16.43) ?g?h/mL for AUC(0～24);(9.72?2.24) and(7.89?1.63) h for t1/2,respectively.The results indicated that Cmax1,Cmax2,AUC(0～24) of baicalin and wogonoside were significantly enhanced and t1/2 of baicalin was remarkably extended when compared with the normal rats.And the results also revealed that baicalin contained in HJD hydrolyzed more rapidly when incubated in the feces suspension of diabetic rats than in that of normal rats.Conclusion The results indicate that the pharmacokinetic difference of baicalin between diabetic and normal rats may result from the presystemic metabolism of baicalin.