Research on the Technology for Enzymatic Preparation of Scutellarein / 中药新药与临床药理

Objective Scutellaria baicalensis stems and leaves glucuronic hydrolase(sbsl GUS)was used to enzymatically hydrolyze scutellarin in Erigeron breviscapus(Vant.)Hand.Mazz.to prepare scutellarein,and the high-purity scutellarein was obtained through separation and purification.Methods Orthogonal experiments were used to optimize the process parameters for the extraction of Erigeron breviscapus(Vant.)Hand.Mazz..Using the rate of enzymatic hydrolysis conversion of scutellarin as the index,the amount of enzyme,pH,temperature,time and antioxidant were investigated,and the preparation process parameters of scutellarein were optimized.Ethanol extraction,activated carbon decolorization,and fractional crystallization were used to purify the crude extract.Results The extraction process was determined to be:segments of Erigeron breviscapus were decocted twice with 10 times water for 1 hour each time.The preparation process of scutellarein was as follows:the amount of sbsl GUS extract and Erigeron breviscapus decoction was 1∶10 based on crude drugs,0.5%sodium metabisulfite was added,pH value was about 6.0,the temperature was about 45℃,and the time was 20 hours.The crude extract of scutellarein with the content more than 60%was obtained.The crude extract was purified by fractional crystallization,refluxed with 80%ethanol,decolorized with activated carbon,concentrated and crystallized,and the scutellarein extract with content more than 85%was obtained.Conclusion sbsl GUS enzymatic hydrolysis technology,which was used to prepare scutellarein,is simple and feasible.This study provides a new way for the manufacture of scutellarein.

A Cocktail of Natural Compounds Holds Promise for New Immunotherapeutic Potential in Head and Neck Cancer / 中国结合医学杂志

OBJECTIVE@#To obtain detailed understanding on the gene regulation of natural compounds in altering prognosis of head and neck squamous cell carcinomas (HNSC).@*METHODS@#Gene expression data of HNSC samples and peripheral blood mononuclear cells (PBMCs) of HNSC patients were collected from Gene Expression Omnibus (GEO). Differential gene expression analysis of GEO datasets were achieved by the GEO2R tool. Common differentially expressed gerres (DEGs) were screened by comparing DEGs of HNSC with those of PBMCs. The combination was further analyzed for regulating pathways and biological processes that were affected.@*RESULTS@#Totally 110 DEGs were retrieved and identified to be involved in biological processes related to tumor regulation. Then 102 natural compounds were screened for a combination such that the expression of all 110 commonly DEGs was altered. A combination of salidroside, ginsenoside Rd, oridonin, britanin, and scutellarein was chosen. A multifaceted, multi-dimensional tumor regression was showed by altering autophagy, apoptosis, inhibiting cell proliferation, angiogenesis, metastasis and inflammatory cytokines production.@*CONCLUSIONS@#This study has helped develop a unique combination of natural compounds that will markedly reduce the propensity of development of drug resistance in tumors and immune evasion by tumors. The result is crucial to developing a combinatorial natural therapeutic cocktail with accentuated immunotherapeutic potential.

Protective effects of twin drug ST-11 against PC12 cell injury induced by oxygen-glucose deprivation/reperfusion and its mechanism / 中国药房

OBJECTIVE To study the protective effects of ligustrazine-scutellarein twin drug ST-11 on rat adrenal medullary pheochromocytoma PC12 cell injury induced by oxygen-glucose deprivation/reperfusion （OGD/R） and its mechanism. METHODS PC12 cells were divided into blank group， model group， nimodipine group （positive control， 5 μmol/L） and different concentration groups of ST-11 （5， 10， 20 μmol/L）. After 24 hours of pre-administration intervention， all the other groups except the blank group were cultured in glucose-free DMEM culture medium containing 10 mmol/L Na2S2O4 for 4 hours with glucose deficiency and hypoxia. After 4 hours of glucose and oxygen re-introduction， the survival rate of cells in each group， the contents of lactate dehydrogenase （LDH）， catalase （CAT）， glutathione （GSH）， malondialdehyde （MDA） and superoxide dismutase （SOD） in cell supernatant， apoptosis rate， the levels of reactive oxygen species （ROS） and mitochondrial membrane potential （MMP）， the protein expressions of B-cell lymphoma 2 related X protein （Bax）， B-cell lymphoma 2 （Bcl-2） and caspase-3 were all detected in each group. RESULTS Compared with blank group， the cell survival rate， the contents of CAT， GSH and SOD in cell supernatant， MMP level， relative expression of Bcl-2 and Bcl-2/Bax ratio in model group decreased significantly （P＜0.05）， while the contents of LDH and MDA， ROS level， apoptosis rate， relative expressions of Bax and caspase-3 were significantly increased （P＜0.05）. Compared with model group， above indexes of ST-11 groups （except for the protein expression of caspase-3 in 5 μmol/L ST-11 group） were reversed signifi-cantly （P＜0.05）. CONCLUSIONS ST-11 has a certain protec-tive effect on OGD/R-injured PC12 cells， and its effects may be related to reduction of oxidative stress and inhibition of cell apoptosis.

Study on protective effects of twin drugs of tetramethylpyrazine-scutellarein on cerebral ischemia-reperfusion injury model rats and its mechanism / 中国药房

OBJECTIVE To study the protective effects of twin drugs of tetramethylpyrazine-scutellarein （TMSC4） on cerebral ischemia-reperfusion injury （CIRI） model rats and its mechanism. METHODS One hundred and five SD rats were randomly divided into sham operation group， model group， scutellarein group （0.7 mmol/kg）， tetramethylpyrazine group （0.7 mmol/kg）， and TMSC4 low-dose， medium-dose and high-dose groups （0.35， 0.7， 1.4 mmol/kg）， with 15 rats in each group. Sham operation group and model group were given constant volume of normal saline intragastrically， and other groups were given relevant drug intragastrically， once a day， for consecutive 14 d. Except for sham operation group， all other groups were treated to establish the CIRI model using the thread occlusion method. After 2 hours of ischemia and 22 hours of reperfusion， the brain index and brain water content of the rats were measured. Serum levels of interleukin 1β （IL-1β）， IL-6 and tumor necrosis factor α （TNF-α）， the levels of superoxide dismutase （SOD）， malondialdehyde （MDA）， glutathione peroxidase （GSH-Px） and catalase （CAT） in brain tissues， the situation of neuronal cell apoptosis， and the protein expressions of B-cell lymphoma 2 （Bcl-2）， Bcl-2-associated X protein （Bax） and cleaved-caspase-3 were evaluated. RESULTS Compared with sham operation group， the brain index， brain water content， the serum levels of IL-1β， IL-6 and TNF-α， the levels of MDA in brain tissues， the brain cell apoptosis and the protein expressions of Bax and cleaved-caspase-3 in model group were significantly increased （P＜0.05）； the levels of SOD， GSH- Px and CAT and the protein expression of Bcl-2 in brain tissues were significantly decreased （P＜0.05）. Compared with model group， the above indexes of rats were reversed significantly in administration groups （P＜0.05）， while the reverse effects of TMSC4 medium-dose and high-dose groups were significantly better than those of scutellarein group and tetramethylpyrazine group （P＜0.05）. CONCLUSIONS TMSC4 has a certain protective effect in CIRI model rats， the mechanism of which may be related to relieving inflammatory reaction and oxidative stress， inhibiting cell apoptosis.

Mechanism of scutellarein inhibiting foaming of macrophages by promoting cholesterol efflux pathway / 中国药理学通报

Aim To investigate the effects of scutellarein on the macrophage foam cell formation and cholesterol efflux, and the underlying mechanism. Methods THP-1 cells were differentiated with PMA, and the cell viability was detected by MTT assays. The effects of scutellarein on the cholesterol efflux and macrophage foam cell formation were evaluated by using NBD-la-beled cholesterol and the cholesterol detection kit. The effects of scutellarein on the activation of PPARγ-LXRα-ABCA1 signaling pathway were determined by molecular docking, ELISA, dual-luciferase reporter and Western blot. The effects of PPARγ knowdown on scutellarein-induced cholesterol efflux and inhibiting macrophage foaming were analyzed by siRNA interference. Results Scutellarein dose-dependently inhibited oxLDL-induced cholesterol accumulation, accelerated cholesterol efflux and significantly increased the protein expression of LXRα and ABCA1. At the same time, scutellarein could bind PPARγ and initiate its downstream LXRa-ABCAl signaling pathway. In addition, gene silencing of PPARγ not only significantly inhibited scutellarein-induced LXRα-ABCA1 signaling pathway and cholesterol efflux, but also reversed the inhibitory effect of scutellarein on macrophage foaming. Conclusions Scutellarein could promote the cholesterol efflux by activating PPARγ and initiating the downstream LXR-ABCA1 signaling pathway, thereby prevent the macrophage foam cell formation.

Anti-cerebral ischemia mechanisms of baicalein and genipin based on network pharmacology / 中草药

Objective: Using network pharmacology and molecular docking technology along with scutellarein (SE) as a reference, this study predicted the anti-cerebral ischemia targets of both baicalein (BE) and genipin (GE). It is hoped that these will provide a reference for clinical prevention and development of ischemic diseases. Methods: SE, BE and GE targets were predicted using TCMSP, Swiss Target Prediction, Stitch database search and literature mining methods. Targets related to cerebral ischemia diseases could be predicted by DisGeNET, CTD, NCBI Gene, OMIM, DrugBank and PharmGkb databases. Cytoscape 3.3.0 was used to construct the small molecule-target network. GO function enrichment and KEGG pathway analysis of SE, BE and GE specific anti-cerebral ischemia targets were analyzed with the DAVID database. Autodock Vina software was used for molecular docking, testing the binding energy of BE, GE and SE to targets of cerebral ischemia. The optimal target protein was selected according to the binding energy and inhibition concentration of receptor and ligand. Results: A total of 30 potential targets of SE, 59 potential targets of BE and 35 potential targets of GE were found. Common anti-cerebral ischemia targets of SE and BE were PIK3CG, CYP1A2, VEGFA, ALOX5 and PTGS2, while common anti-cerebral ischemia targets of SE and GE were PTGS2. Molecular docking results demonstrated that the binding energy and inhibitory concentration of receptor PTGS2 to the three drugs were relatively low. Enrichment of GO function showed that common targets of BE-SE were mainly distributed in cytoplasm, organelle membrane, endoplasmic reticulum and other elements. These elements had binding functions with metal ions and cations, catalytic and oxidoreductase activities, and they participated in cell lipid, carboxylic acid, oxygenic acid, organic acid metabolism and fatty acid synthesis. Results: of the KEGG analysis demonstrated that receptor PTGS2 mainly acted on the arachidonic acid metabolism pathway and the vascular endothelial growth factor signaling pathway. A combination of BE and GE functioned in the treatment of cerebral ischemia disease by inhibiting expression of PTGS2 (COX-2) and vascular endothelial growth factor protein, thus reducing brain injury caused by inflammatory factors and improving the permeability of the blood brain barrier (BBB). Conclusion: This study predicted potential targets of BE and GE compatibility in the treatment of cerebral ischemia diseases and preliminarily verified mechanisms of action in the treatment of cerebral ischemia diseases. It provides valuable data for further study into the mechanisms of BE and GE compatibility for the treatment of cerebral ischemia as well as developing a basis for the next synthesis of new derivatives.

Effect of scutellarein on acute pharyngitis in rats / 中国药科大学学报

@#In order to investigate the therapeutic effects of scutellarein on acute pharyngitis, 60 rats were randomly divided into five groups: blank group, model group, low-dose scutellarein group, high-dose scutellarein group and positive drug group. HE staining, blood-cell-analyzer, IL-6, IL-1β and TNF-α ELISA kit were used to study the effects of scutellarein on acute pharyngitis in pharyngeal tissue morphology, the counts of white blood cells and neutrophil and the serum concentrations of TNF-α, IL-1β and IL-6. Meanwhile, forty mice were randomly divided into four groups: blank group, low-dose scutellarein group, high-dose scutellarein group and positive drug group. Then, hot plate and writhing test of mice were carried out to study the analgesic effects of scutellarein. Results showed that, compared to the model group, scutellarein improved the physical status of acute pharyngitis rats, reduced the number of white blood cells significantly(P< 0. 05)and decreased the number of neutrophils and the levels of TNF-α, IL-1β and IL-6 in rats serum significantly(P< 0. 01). Meanwhile, scutellarein dramatically improved the pain threshold in hot plate test and decreased the number of writhing mice(P< 0. 01). It can be concluded that scutellarein can be used to treat acute pharyngitis with its anti-inflammatory and analgesic effect.

Assay and fingerprint chromatogram of scutellaria barbata formula granules by HPLC / 中国药学杂志

OBJECTIVE: To establish the HPLC methods for determining the contents of the active ingredients and fingerprint chromatogram of Scutellaria barbata formula granules. METHOD: The HPLC analysis was carried out on a Wondasil C18 column (4.6 mm×250 mm, 5μm) with mobile phase consisting of methanol-acetonitrile-0.1% phosphoric acid (12.5:15:72.5) at the flow rate of 1.0 mL·min-1. The column temperature was maintained at 30t. The detection wavelength was set at 335 nm to determine the contents of scutellarin and scutellarein and 320 nm to establish the fingerprint chromatogram of Scutellaria barbata formula granules, medical materials, and aqueous decoction. RESULTS: The linear ranges of scutellarin and scutellarein were 0.074 0-0.518 0 μg and 0.051 6-0.3612 μg, respectively. The average recoveries were 103.18% and 99.99%, respectively. Nine peaks in the fingerprint chromatogram of formula granules could be tracked in the aqueous decoction, and eight peaks in the fingerprint chromatogram could be tracked in the medical materials. CONCLUSION: The methods can provide more information for the quality control of Scutellaria barbata formula granules.

Chemical constituents from Scutellaria barbata / 中草药

Objective: To investigate the constituents in the aerial parts of Scutellaria barbata. Methods: The isolation and purification of the compounds were performed by AB-8 macroporous adsorption resin, silica gel, polyamide and Sephadex LH-20 column chromatography, and their structures were determined on physicochemical characters and spectroscopic data. Results: Fourteen compounds were separated and elucidated as hispidulin-7-O-β-D-methylgluzcuronide (1), apigenin (2), scutellarin (3), scutellarein (4), luteolin (5), scutellarein-7-O-β-D-glucuronide methyl ester (6), isoscutellarein-8-O-β-D-glucuronide-6″-methyl ester (7), apigenin-7-O-β-D-glucuronide-6″-methyl ester (8), 4'-hydroxywogonin (9), 4',5-dihydroxy-3',5',6,7-tetramethoxyflavone (10), isoscutellarein (11), 6-hydroxyluteolin (12), 5-hydroxy-6,7,3',4'-tetramethoxyflavone (13), salvigenin (14). Conclusion: Compound 7 is isolated from Lamiaceae for the first time, compounds 1 and 13 are for the first time isolated from the genus Scutellaria, and compounds 6 and 12 are for the first time obtained from S. barbata.

Pharmacokinetic study of scutellarin in healthy volunteers by enzymatic hydrolysis and LC-MS-MS analysis / 中国药理学通报

Aim To establish a combined method ofβ-glucuronidase hydrolysis and LC-MS-MS analysis for the determination of scutellarein in human plasma, and investigate the pharmacokinetics of scutellarin prepara-tion in healthy male volunteers. Methods Plasma samples were prepared by enzymolysis with β-glucu-ronidase and protein precipitation with methanol. The analytes scutellarein and quercetin ( IS ) were separa-ted on an Agilent ZORBAX SB C18 column ( 2. 1 mm × 150 mm, 5 μm) with the mobile phases consisting of acetonitrile, methanol and water. Multiple reaction monitoring ( MRM) on MS was used to monitor precur-sor to produce ion transitions of m/z 285. 0→136. 8 for scutellarein and m/z 301. 1→120. 8 for IS. After method validation, this method was applied to deter-mine the plasma concentration of scutellarein in 12 male volunteers following single oral administration of 120 mg scutellarin preparation. Drug And Statistic soft-ware (1. 0) was used to process data and the pharma-cokinetic parameters were calculated. Results The assay was validated with linear range of 4 . 01-513. 38μg · L-1 for scutellarein. The intra- and inter-batch precisions ( RSD%) were within 7. 22%. The absolute recoveries were more than 84. 23%. The pharmacoki-netic parameters after a single dose were as follows:Cmax (μg · L-1 ): 159. 97 ± 58. 14; AUC(0-19) (μg · L-1·h):1151. 37 ±279. 80; AUC(0-∞)(μg·L-1· h):1194. 13 ± 264. 51; Tmax ( h):6. 33 ± 1. 67; T1/2 (h):2. 83 ± 0. 60. Conclusion The assay method is proved to be sensitive, accurate and convenient. It can be successfully applied to a pharmacokinetic study of scutellarin in healthy male volunteers.

Scutellarein Reduces Inflammatory Responses by Inhibiting Src Kinase Activity

Flavonoids are plant pigments that have been demonstrated to exert various pharmacological effects including anti-cancer, anti-diabetic, anti-atherosclerotic, anti-bacterial, and anti-inflammatory activities. However, the molecular mechanisms in terms of exact target proteins of flavonoids are not fully elucidated yet. In this study, we aimed to evaluate the anti-inflammatory mechanism of scutellarein (SCT), a flavonoid isolated from Erigeron breviscapus, Clerodendrum phlomidis and Oroxylum indicum Vent that have been traditionally used to treat various inflammatory diseases in China and Brazil. For this purpose, a nitric oxide (NO) assay, polymerase chain reaction (PCR), nuclear fractionation, immunoblot analysis, a kinase assay, and an overexpression strategy were employed. Scutellarein significantly inhibited NO production in a dose-dependent manner and reduced the mRNA expression levels of inducible NO synthase (iNOS) and tumor necrosis factor (TNF)-alpha in lipopolysaccharide (LPS)-activated RAW264.7 cells. In addition, SCT also dampened nuclear factor (NF)-kappaB-driven expression of a luciferase reporter gene upon transfection of a TIR-domain-containing adapter-inducing interferon-beta (TRIF) construct into Human embryonic kidney 293 (HEK 293) cells; similarly, NF-kappa B nuclear translocation was inhibited by SCT. Moreover, the phosphorylation levels of various upstream signaling enzymes involved in NF-kappaB activation were decreased by SCT treatment in LPS-treated RAW264.7 cells. Finally, SCT strongly inhibited Src kinase activity and also inhibited the autophosphorylation of overexpressed Src. Therefore, our data suggest that SCT can block the inflammatory response by directly inhibiting Src kinase activity linked to NF-kappaB activation.

Rapid pharmacokinetic characteristic evaluation of scutellarin derivatives in vitro / 中国药理学通报

Aim To study the pharmacokinetic char-acteristics of serial compounds that took the scutellarin and scutellarein as lead compounds by using the model of in vitro liver microsomes, and to screen compounds whose medicinal properties were superior to scutellarin and scutellarein. Methods The content of candidate compounds at different times by incubation system of rat liver microsome was determined using UPLC-MS/MS method. Candidate compounds that contained opti-mum T1/2 and CLint were screened. Enzyme kinetics and conversions of candidate compounds were com-pared with those of scutellarin and scutellarein. Re-sults The T1/2 and CLint were optimum of W11 com-pared with those of scutellarin and scutellarein; the Vmax, Km and CLint of compound W11 were (10.25 ±2.59 ) μmol · min-1 · g-1 , ( 4.64 ±0.24 ) μmol · L-1 and ( 2.29 ±0.23 ) L · min-1 · g-1; the Vmax , Km and CLint of scutellarin were (45.95±9.50) μmol · min-1 · g-1 , ( 10.19 ± 1.66 ) μmol · L-1 and (4.48±0.20) L·min-1 ·g-1; W11 might be me-tabolized into scutellarin and M1 ( a compound with mo-lecular weight of 577 after demethylating ) . Conclu-sion The pharmacokinetic properties of candidate compound W11 are better than those of scutellarin, and it could release scutellarin.

Pharmacokinetic studies of scutellarein and glucuronide scutellarin in rats / 中国药理学通报

Aims To establish a UPLC-MS/MS meth-od for the determination of plasma concentration of scutellarein and its metabolite and to study their phar-macokinetics in rat plasma. Methods The analysis was achieved by BEH C18 column with a mobile phase composed of 0 . 1 % formic acid in acetonitrile and 0 . 1% aqueous formic acid using step gradient elution. A TQD tandem mass spectrometry equipped with electros-pray ionization source was used as detector and opera-ted by multiple reaction monitoring( MRM) positive ion mode. After intravenous injection of scutellarein, the concentrations of scutellarein and its major metabolite glucuronide scutellarin in rat plasma were determined at different time points. The pharmacokinetic parame-ters were calculated by DAS 2. 0 software. Results Good linearity was achieved for scutellarein, the ex-traction recovery was between 80 . 5 % to 90 . 5 %, the precisions and accuracy were good. The result showed the pharmacokinetic profiles of scutellarein and glucu-ronide scutellarin both fit to the two-compartment mod-el. Conclusion The above mentioned method is spe-cific, rapid, sensitive and suitable for the pharmacoki-netic studies of scutellarein and its metabolite.

Study on sulfation metabolism of scutellarein in FVB/NCrIVr mice / 中国药学杂志

OBJECTIVE: To investigate the characteristics of sulfation of scutellarein in FVB/NCrIVr (FVB) mice. METHODS: FVB mouse intestinal perfusion model and incubation system with FVB mouse liver S9 fractions were adapted to conduct the study. HPLC-MS/MS and HPLC-UV were used to identify and quantify scutellarein and its metabolites in the samples. RESULTS: One sulfation metabolite and one glucuronidation metabolite were detected in the small intestinal perfusate. There was no significant difference between the excretion rates of sulfation metabolite and glucuronidation metabolite in small intestinal perfusate (P=0.435), while only sulfation metabolite of scutellarein could be detected in colon perfusate, scutellarein, sulfation metabolite and glucuronidation metabolite of scutellarein could all be detected in biliary samples, indicating an entero-hepatic circulation of scutellarein. In the liver S9 fractions, sulfation rate at 20 μmol·L-1 was sig nificantly higher than those at 10 and 40 μmol·L-1 (P<0.05). CONCLUSION: Sulfation was found to be the most important metabolism route in the intestinal disposition of scutellarein. There is probably a substrate inhibition effect in the sulfation of scutellarein in liver S9 fractions.

Inhibitory effect of 4'-methylether-scutellarein on human choriocarcinoma JAR cells and the possible mechanism / 中国肿瘤生物治疗杂志

Objective:To investigate the inhibitory effect of 4'-methylether-scutellarein(4-MS),an extract from Verbena officinalis,on human choriocarcinoma JAR cell line and the possible mechanism.Methods:JAR cells were exposed to 4'-methylether-scutellarein of different concentrations for 48 h.MTT assay was used to examine the anti-proliferative effect of 4'-methylether-scutellarein.Flow cytometry was used to investigate the apoptosis and the changes of cell cycle.AO/EB double staining was applied to discriminate the apoptotic cells from dead ones.The changes of Survivin,p38-MAPK and Caspase 3 mRNA expressions were detected by RT-PCR in JAR cells treated with 4-MS.Furthermore,Western blotting assay was used to determine Survivin protein expression,phosphorylation level of p38 and Caspase 3 in JAR cells before and after 4-MS treatment.Results:4-MS inhibited the proliferation of JAR cells in a dose- and time-dependent manner(P