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HMN-214_SDS_MedChemExpress

Inhibitors, Agonists, Screening LibrariesSafety Data Sheet Revision Date:May-24-2017Print Date:May-24-20171. PRODUCT AND COMPANY IDENTIFICATION1.1 Product identifierProduct name :HMN-214Catalog No. :HY-12045CAS No. :173529-46-91.2 Relevant identified uses of the substance or mixture and uses advised againstIdentified uses :Laboratory chemicals, manufacture of substances.1.3 Details of the supplier of the safety data sheetCompany:MedChemExpress USATel:609-228-6898Fax:609-228-5909E-mail:sales@1.4 Emergency telephone numberEmergency Phone #:609-228-68982. HAZARDS IDENTIFICATION2.1 Classification of the substance or mixtureGHS Classification in accordance with 29 CFR 1910 (OSHA HCS)Acute toxicity, Oral (Category 4), H302Acute aquatic toxicity (Category 1), H400Chronic aquatic toxicity (Category 1), H4102.2 GHS Label elements, including precautionary statementsPictogramSignal word WarningHazard statement(s)H302 Harmful if swallowed.H410 Very toxic to aquatic life with long lasting effects.Precautionary statement(s)P264 Wash skin thoroughly after handling.P270 Do not eat, drink or smoke when using this product.P273 Avoid release to the environment.P301 + P312 IF SWALLOWED: Call a POISON CENTER or doctor/ physician if you feel unwell.P330 Rinse mouth.P391 Collect spillage.P501 Dispose of contents/ container to an approved waste disposal plant.2.3 Other hazardsNone.3. COMPOSITION/INFORMATION ON INGREDIENTS3.1 SubstancesSynonyms:IVX⁻214; HMN214; HMN 214; IVX 214; IVX⁻214; IVX214Formula:C22H20N2O5SMolecular Weight:424.47CAS No. :173529-46-94. FIRST AID MEASURES4.1 Description of first aid measuresEye contactRemove any contact lenses, locate eye-wash station, and flush eyes immediately with large amounts of water. Separate eyelids with fingers to ensure adequate flushing. Promptly call a physician.Skin contactRinse skin thoroughly with large amounts of water. Remove contaminated clothing and shoes and call a physician.InhalationImmediately relocate self or casualty to fresh air. If breathing is difficult, give cardiopulmonary resuscitation (CPR). Avoid mouth-to-mouth resuscitation.IngestionWash out mouth with water; Do NOT induce vomiting; call a physician.4.2 Most important symptoms and effects, both acute and delayedThe most important known symptoms and effects are described in the labelling (see section 2.2).4.3 Indication of any immediate medical attention and special treatment neededTreat symptomatically.5. FIRE FIGHTING MEASURES5.1 Extinguishing mediaSuitable extinguishing mediaUse water spray, dry chemical, foam, and carbon dioxide fire extinguisher.5.2 Special hazards arising from the substance or mixtureDuring combustion, may emit irritant fumes.5.3 Advice for firefightersWear self-contained breathing apparatus and protective clothing.6. ACCIDENTAL RELEASE MEASURES6.1 Personal precautions, protective equipment and emergency proceduresUse full personal protective equipment. Avoid breathing vapors, mist, dust or gas. Ensure adequate ventilation. Evacuate personnel to safe areas.Refer to protective measures listed in sections 8.6.2 Environmental precautionsTry to prevent further leakage or spillage. Keep the product away from drains or water courses.6.3 Methods and materials for containment and cleaning upAbsorb solutions with finely-powdered liquid-binding material (diatomite, universal binders); Decontaminate surfaces and equipment by scrubbing with alcohol; Dispose of contaminated material according to Section 13.7. HANDLING AND STORAGE7.1 Precautions for safe handlingAvoid inhalation, contact with eyes and skin. Avoid dust and aerosol formation. Use only in areas with appropriate exhaust ventilation.7.2 Conditions for safe storage, including any incompatibilitiesKeep container tightly sealed in cool, well-ventilated area. Keep away from direct sunlight and sources of ignition.Recommended storage temperature:Powder-20°C 3 years4°C 2 yearsIn solvent-80°C 6 months-20°C 1 monthShipping at room temperature if less than 2 weeks.7.3 Specific end use(s)No data available.8. EXPOSURE CONTROLS/PERSONAL PROTECTION8.1 Control parametersComponents with workplace control parametersThis product contains no substances with occupational exposure limit values.8.2 Exposure controlsEngineering controlsEnsure adequate ventilation. Provide accessible safety shower and eye wash station.Personal protective equipmentEye protection Safety goggles with side-shields.Hand protection Protective gloves.Skin and body protection Impervious clothing.Respiratory protection Suitable respirator.Environmental exposure controls Keep the product away from drains, water courses or the soil. Cleanspillages in a safe way as soon as possible.9. PHYSICAL AND CHEMICAL PROPERTIES9.1 Information on basic physical and chemical propertiesAppearance White to off-white (Solid)Odor No data availableOdor threshold No data availablepH No data availableMelting/freezing point No data availableBoiling point/range No data availableFlash point No data availableEvaporation rate No data availableFlammability (solid, gas)No data availableUpper/lower flammability or explosive limits No data availableVapor pressure No data availableVapor density No data availableRelative density No data availableWater Solubility No data availablePartition coefficient No data availableAuto-ignition temperature No data availableDecomposition temperature No data availableViscosity No data availableExplosive properties No data availableOxidizing properties No data available9.2 Other safety informationNo data available.10. STABILITY AND REACTIVITY10.1 ReactivityNo data available.10.2 Chemical stabilityStable under recommended storage conditions.10.3 Possibility of hazardous reactionsNo data available.10.4 Conditions to avoidNo data available.10.5 Incompatible materialsStrong acids/alkalis, strong oxidising/reducing agents.10.6 Hazardous decomposition productsUnder fire conditions, may decompose and emit toxic fumes.Other decomposition products - no data available.11.TOXICOLOGICAL INFORMATION11.1 Information on toxicological effectsAcute toxicityClassified based on available data. For more details, see section 2Skin corrosion/irritationClassified based on available data. For more details, see section 2Serious eye damage/irritationClassified based on available data. For more details, see section 2Respiratory or skin sensitizationClassified based on available data. For more details, see section 2Germ cell mutagenicityClassified based on available data. For more details, see section 2CarcinogenicityIARC: No component of this product present at a level equal to or greater than 0.1% is identified as probable, possible or confirmed human carcinogen by IARC.ACGIH: No component of this product present at a level equal to or greater than 0.1% is identified as a potential or confirmed carcinogen by ACGIH.NTP: No component of this product present at a level equal to or greater than 0.1% is identified as a anticipated or confirmed carcinogen by NTP.OSHA: No component of this product present at a level equal to or greater than 0.1% is identified as a potential or confirmed carcinogen by OSHA.Reproductive toxicityClassified based on available data. For more details, see section 2Specific target organ toxicity - single exposureClassified based on available data. For more details, see section 2Specific target organ toxicity - repeated exposureClassified based on available data. For more details, see section 2Aspiration hazardClassified based on available data. For more details, see section 212. ECOLOGICAL INFORMATION12.1 ToxicityNo data available.12.2 Persistence and degradabilityNo data available.12.3 Bioaccumlative potentialNo data available.12.4 Mobility in soilNo data available.12.5 Results of PBT and vPvB assessmentPBT/vPvB assessment unavailable as chemical safety assessment not required or not conducted.12.6 Other adverse effectsNo data available.13. DISPOSAL CONSIDERATIONS13.1 Waste treatment methodsProductDispose substance in accordance with prevailing country, federal, state and local regulations.Contaminated packagingConduct recycling or disposal in accordance with prevailing country, federal, state and local regulations.14. TRANSPORT INFORMATIONDOT (US)This substance is considered to be non-hazardous for transport.IMDGUN number: 3077Class: 9Packing group: IIIEMS-No: F-A, S-FProper shipping name: ENVIRONMENTALLY HAZARDOUS SUBSTANCE, SOLID, N.O.S.Marine pollutant: Marine pollutantIATAUN number: 3077Class: 9Packing group: IIIProper shipping name: Environmentally hazardous substance, solid, n.o.s.15. REGULATORY INFORMATIONSARA 302 Components:No chemicals in this material are subject to the reporting requirements of SARA Title III, Section 302.SARA 313 Components:This material does not contain any chemical components with known CAS numbers that exceed the threshold (De Minimis) reporting levels established by SARA Title III, Section 313.SARA 311/312 Hazards:No SARA Hazards.Massachusetts Right To Know Components:No components are subject to the Massachusetts Right to Know Act.Pennsylvania Right To Know Components:No components are subject to the Pennsylvania Right to Know Act.New Jersey Right To Know Components:No components are subject to the New Jersey Right to Know Act.California Prop. 65 Components:This product does not contain any chemicals known to State of California to cause cancer, birth defects, or anyother reproductive harm.16. OTHER INFORMATIONCopyright 2017 MedChemExpress. The above information is correct to the best of our present knowledge but does not purport to be all inclusive and should be used only as a guide. The product is for research use only and for experienced personnel. It must only be handled by suitably qualified experienced scientists in appropriately equipped and authorized facilities. The burden of safe use of this material rests entirely with the user. MedChemExpress disclaims all liability for any damage resulting from handling or from contact with this product.Caution: Product has not been fully validated for medical applications. For research use only.Tel: 609-228-6898 Fax: 609-228-5909 E-mail: tech@Address: 1 Deer Park Dr, Suite Q, Monmouth Junction, NJ 08852, USA。

圣草酚通过调控c-Myc

网络出版时间：２０２２－０５－２８１５：０５　网络出版地址：ｈｔｔｐｓ：／／ｋｎｓ．ｃｎｋｉ．ｎｅｔ／ｋｃｍｓ／ｄｅｔａｉｌ／３４．１０６５．Ｒ．２０２２０５２６．１０１６．００９．ｈｔｍｌ圣草酚通过调控ｃＭｙｃ／ＡＲ轴对三阴性乳腺癌ＭＤＡＭＢ２３１细胞增殖和凋亡的影响刘汉成１，李慧明１，张　杰１，孟庆来１，丁　实２，马立辉１２０２２－０４－１７接收基金项目：河北省卫生健康委医学科学研究课题计划（编号：２０２１１３３１）作者单位：１承德医学院附属医院乳腺外科，承德　０６７０２０２承德医学院药理学教研室，承德　０６７０００作者简介：刘汉成，男，主治医师；马立辉，男，教授，硕士生导师，责任作者，Ｅｍａｉｌ：ｗｌｊｙｌ００８＠１６３．ｃｏｍ摘要　目的　探究圣草酚（Ｅｒｉ）对三阴性乳腺癌ＭＤＡＭＢ２３１细胞增殖和凋亡的影响及其可能作用机制。

方法　采用不同浓度Ｅｒｉ干预ＭＤＡＭＢ２３１细胞后，ＭＴＴ法检测细胞增殖水平；平板克隆实验检测细胞克隆形成能力；流式细胞术检测细胞凋亡水平；Ｗｅｓｔｅｒｎｂｌｏｔ检测细胞中活性半胱天冬酶３（Ｃｌｅａｖｅｄｃａｓｐａｓｅ３）、Ｂ淋巴细胞瘤２相关Ｘ（Ｂａｘ）、Ｂ淋巴细胞瘤２（Ｂｃｌ２）、细胞髓细胞瘤（ｃＭｙｃ）、雄激素受体（ＡＲ）蛋白表达水平；ＭＤＡＭＢ２３１细胞转染ｓｉｃＭｙｃ，再联合５０μｍｏｌ／ＬＥｒｉ处理，ＭＴＴ法检测细胞增殖水平；流式细胞术检测细胞凋亡水平；ｑＲＴＰＣＲ检测细胞中ｃＭｙｃｍＲＮＡ表达水平；Ｗｅｓｔｅｒｎｂｌｏｔ检测细胞中ｃＭｙｃ和ＡＲ蛋白表达水平。

结果　Ｅｒｉ可抑制ＭＤＡＭＢ２３１细胞增殖活性，并呈现浓度和时间依赖性。

不同浓度Ｅｒｉ处理可降低ＭＤＡＭＢ２３１细胞克隆形成能力，促进细胞凋亡，上调Ｃｌｅａｖｅｄｃａｓｐａｓｅ３、Ｂａｘ蛋白表达水平，下调Ｂｃｌ２、ｃＭｙｃ、ＡＲ蛋白表达水平。

ｃＭｙｃ基因沉默可抑制ＭＤＡＭＢ２３１细胞增殖活性，促进细胞凋亡，下调ｃＭｙｃ和ＡＲ蛋白表达水平。

分子生物学词汇(中英文对照表 )

marked manuscript

Quality evaluation of Flos Lonicerae through a simultaneous determination of seven saponins by HPLC with ELSDXing-Yun Chai1, Song-Lin Li2, Ping Li1*1Key Laboratory of Modern Chinese Medicines and Department of Pharmacognosy, China Pharmaceutical University, Nanjing, 210009, People’s Republic of China2Institute of Nanjing Military Command for Drug Control, Nanjing, 210002, People’s Republic of China*Corresponding author: Ping LiKey Laboratory of Modern Chinese Medicines and Department of Pharmacognosy, China Pharmaceutical University, Nanjing 210009, People’s Republic of China.E-mail address: lipingli@Tel.: +86-25-8324-2299; 8539-1244; 135********Fax: +86-25-8532-2747AbstractA new HPLC coupled with evaporative light scattering detection (ELSD) method has been developed for the simultaneous quantitative determination of seven major saponins, namely macranthoidinB (1), macranthoidin A (2), dipsacoside B (3), hederagenin-28-O-β-D-glucopyranosyl(6→1)-O-β-D- glucopyranosyl ester (4), macranthoside B (5), macranthoside A (6), and hederagenin-3-O-α-L-arabinopyranosyl(2→1)-O-α-L-rhamnopyranoside (7)in Flos Lonicerae, a commonly used traditional Chinese medicine (TCM) herb.Simultaneous separation of these seven saponins was achieved on a C18 analytical column with a mixed mobile phase consisting of acetonitrile(A)-water(B)(29:71 v/v) acidified with 0.5% acetic acid. The elution was operated from keeping 29%A for 10min, then gradually to 54%B from 10 to 25 min on linear gradient, and then keep isocratic elution with 54%B from 25 to 30min.The drift tube temperature of ELSD was set at 106℃, and with the nitrogen flow-rate of 2.6 l/min. All calibration curves showed good linear regression (r2 0.9922) within test ranges. This method showed good reproducibility for the quantification of these seven saponins in Flos Lonicerae with intra- and inter-day variations of less than 3.0% and 6.0% respectively. The validated method was successfully applied to quantify seven saponins in five sources of Flos Lonicerae, which provides a new basis of overall assessment on quality of Flos Lonicerae.Keywords: HPLC-ELSD; Flos Lonicerae; Saponins; Quantification1. IntroductionFlos Lonicerae (Jinyinhua in Chinese), the dried buds of several species of the genus Lonicera (Caprifoliaceae), is a commonly used traditional Chinese medicine (TCM) herb. It has been used for centuries in TCM practice for the treatment of sores, carbuncles, furuncles, swelling and affections caused by exopathogenic wind-heat or epidemic febrile diseases at the early stage [1]. Though four species of Lonicera are documented as the sources of Flos Lonicerae in China Pharmacopeia (2000 edition), i.e. L. japonica, L. hypoglauca,L. daystyla and L. confusa, other species such as L. similes and L. macranthoides have also been used on the same purpose in some local areas in China [2]. So it is an important issue to comprehensively evaluate the different sources of Flos Lonicerae, so as to ensure the clinical efficacy of this Chinese herbal drug.Chemical and pharmacological investigations on Flos Lonicerae resulted in discovering several kinds of bioactive components, i.e. chlorogenic acid and its analogues, flavonoids, iridoid glucosides and triterpenoid saponins [3]. Previously, chlorogenic acid has been used as the chemical marker for the quality evaluation of Flos Lonicerae,owing to its antipyretic and antibiotic property as well as its high content in the herb. But this compound is not a characteristic component of Flos Lonicerae, as it has also been used as the chemical marker for other Chinese herbal drugs such as Flos Chrysanthemi and so on[4-5]. Moreover, chlorogenic acid alone could not be responsible for the overall pharmacological activities of Flos Lonicerae[6].On the other hand, many studies revealed that triterpenoidal saponins of Flos Lonicerae possess protection effects on hepatic injury caused by Acetaminophen, Cd, and CCl4, and conspicuous depressant effects on swelling of ear croton oil [7-11]. Therefore, saponins should also be considered as one of the markers for quality control of Flos Lonicerae. Consequently, determinations of all types of components such as chlorogenic acid, flavonoids, iridoid glucosides and triterpenoidal saponins in Flos Lonicerae could be a better strategy for the comprehensive quality evaluation of Flos Lonicerae.Recently an HPLC-ELSD method has been established in our laboratory for qualitative and quantitative determination of iridoid glucosides in Flos Lonicerae [12]. But no method was reported for the determination of triterpenoidal saponins in Flos Lonicera. As a series studies on the comprehensive evaluation of Flos Lonicera, we report here, for the first time, the development of an HPLC-ELSD method for simultaneous determination of seven triterpenoidal saponins in the Chinese herbal drug Flos Lonicerae, i.e.macranthoidin B (1), macranthoidin A (2), dipsacoside B (3), hederagenin-28-O-β-D-glucopyranosyl(6→1)-O-β-D- glucopyranosyl ester (4), macranthoside B (5), macranthoside A (6), and hederagenin-3-O-α-L-arabinopyranosyl(2→1)-O-α-L-rhamnopyranoside (7) (Fig. 1).2. Experimental2.1. Samples, chemicals and reagentsFive samples of Lonicera species,L. japonica from Mi county, HeNan province (LJ1999-07), L. hypoglauca from Jiujang county, JiangXi province (LH2001-06), L. similes from Fei county, ShanDong province (LS2001-07), L. confuse from Xupu county, HuNan province (LC2001-07), and L. macranthoides from Longhu county, HuNan province (LM2000-06) respectively, were collected in China. All samples were authenticated by Dr. Ping Li, professor of department of Pharmacognosy, China Pharmaceutical University, Nanjing, China. The voucher specimens were deposited in the department of Pharmacognosy, China Pharmaceutical University, Nanjing, China. Seven saponin reference compounds: macranthoidin B (1), macranthoidin A (2), dipsacoside B (3), hederagenin-28-O-β-D-glucopyranosyl(6→1)-O-β-D- glucopyranosyl ester (4), macranthoside B (5), macranthoside A (6), and hederagenin-3-O-α-L-arabinopyranosyl(2→1)-O-α-L-rhamnopyranoside (7) were isolated previously from the dried buds of L. confusa by repeated silica gel, sephadex LH-20 and Rp-18 silica gel column chromatography, their structures were elucidated by comparison of their spectral data (UV, IR, MS, 1H- NMR and 13C-NMR) with references [13-15]. The purity of these saponins were determined to be more than 98% by normalization of the peak areas detected by HPLC with ELSD, and showed very stable in methanol solution.HPLC-grade acetonitrile from Merck (Darmstadt, Germany), the deionized water from Robust (Guangzhou, China), were purchased. The other solvents, purchased from Nanjing Chemical Factory (Nanjing, China) were of analytical grade.2.2. Apparatus and chromatographic conditionsAglient1100 series HPLC apparatus was used. Chromatography was carried out on an Aglient Zorbax SB-C18 column（250 4.6mm, 5.0µm）at a column temperature of 25℃.A Rheodyne 7125i sampling valve (Cotati, USA) equipped with a sample loop of 20µl was used for sample injection. The analog signal from Alltech ELSD 2000 (Alltech, Deerfield, IL, USA)was transmitted to a HP Chemstation for processing through an Agilent 35900E (Agilent Technologies, USA).The optimum resolution was obtained by using a linear gradient elution. The mobile phase was composed of acetonitrile(A) and water(B) which acidified with 0.5% acetic acid. The elution was operated from keeping 29%A for 10min, then gradually to 54%B from 10 to 25 min in linear gradient, and back to the isocratic elution of 54%B from 25 to 30 min.The drift tube temperature for ELSD was set at 106℃and the nitrogen flow-rate was of 2.6 l/min. The chromatographic peaks were identified by comparing their retention time with that of each reference compound tried under the same chromatographic conditions with a series of mobile phases. In addition, spiking samples with the reference compounds further confirmed the identities of the peaks.2.3. Calibration curvesMethanol stock solutions containing seven analytes were prepared and diluted to appropriate concentration for the construction of calibration curves. Six concentrationof the seven analytes’ solution were injected in triplicate, and then the calibration curves were constructed by plotting the peak areas versus the concentration of each analyte. The results were demonstrated in Table1.2.4. Limits of detection and quantificationMethanol stock solution containing seven reference compounds were diluted to a series of appropriate concentrations with methanol, and an aliquot of the diluted solutions were injected into HPLC for analysis.The limits of detection (LOD) and quantification (LOQ) under the present chromatographic conditions were determined at a signal-to-noise ratio (S/N) of 3 and 10, respectively. LOD and LOQ for each compound were shown in Table1.2.5. Precision and accuracyIntra- and inter-day variations were chosen to determine the precision of the developed assay. Approximate 2.0g of the pulverized samples of L. macranthoides were weighted, extracted and analyzed as described in 2.6 Sample preparation section. For intra-day variability test, the samples were analyzed in triplicate for three times within one day, while for inter-day variability test, the samples were examined in triplicate for consecutive three days. Variations were expressed by the relative standard deviations. The results were given in Table 2.Recovery test was used to evaluate the accuracy of this method. Accurate amounts of seven saponins were added to approximate 1.0g of L. macranthoides,and then extracted and analyzed as described in 2.6 Sample preparation section. The average recoveries were counted by the formula: recovery (%) = (amount found –original amount)/ amount spiked ×100%, and RSD (%) = (SD/mean) ×100%. The results were given in Table 3.2.6. Sample preparationSamples of Flos Lonicerae were dried at 50℃until constant weight. Approximate 2.0g of the pulverized samples, accurately weighed, was extracted with 60% ethanol in a flask for 4h. The ethanol was evaporated to dryness with a rotary evaporator. Residue was dissolved in water, followed by defatting with 60ml of petroleum ether for 2 times, and then the water solution was evaporated, residue was dissolved with methanol into a 25ml flask. One ml of the methanol solution was drawn and transferred to a 5ml flask, diluted to the mark with methanol. The resultant solution was at last filtrated through a 0.45µm syringe filter (Type Millex-HA, Millipore, USA) and 20µl of the filtrate was injected to HPLC system. The contents of the analytes were determined from the corresponding calibration curves.3. Results and discussionsThe temperature of drift tube and the gas flow-rate are two most important adjustable parameters for ELSD, they play a prominent role to an analyte response. In ourprevious work [12], the temperature of drift tube was optimized at 90°C for the determination of iridoids. As the polarity of saponins are higher than that of iridoids, more water was used in the mobile phase for the separation of saponins, therefore the temperature for saponins determination was optimized systematically from 95°C to 110°C, the flow-rate from 2.2 to 3.0 l/min. Dipsacoside B was selected as the testing saponin for optimizing ELSD conditions, as it was contained in all samples. Eventually, the drift tube temperature of 106℃and a gas flow of 2.6 l/min were optimized to detect the analytes. And these two exact experimental parameters should be strictly controlled in the analytical procedure [16].All calibration curves showed good linear regression (r2 0.9922) within test ranges. Validation studies of this method proved that this assay has good reproducibility. As shown in Table 2, the overall intra- and inter-day variations are less than 6% for all seven analytes. As demonstrated in Table 3, the developed analytical method has good accuracy with the overall recovery of high than 96% for the analytes concerned. The limit of detection (S/N=3) and the limit of quantification (S/N=10) are less than 0.26μg and 0.88μg respectively (Table1), indicating that this HPLC-ELSD method is precise, accurate and se nsitive enough for the quantitative evaluation of major non- chromaphoric saponins in Flos Lonicerae.It has been reported that there are two major types of saponins in Flos Lonicerae, i.e. saponins with hederagenin as aglycone and saponins with oleanolic acid as the aglycone [17]. But hederagenin type saponins of the herb were reported to have distinct activities of liver protection and anti-inflammatory [7-11]. So we adoptedseven hederagenin type saponins as representative markers to establish a quality control method.The newly established HPLC-ELSD method was applied to analyze seven analytes in five plant sources of Flos Lonicerae, i.e. L. japonica,L. hypoglauca,L. confusa,L. similes and L. macranthoides(Table 4). It was found that there were remarkable differences of seven saponins contents between different plant sources of Flos Lonicerae. All seven saponins analyzed could be detected in L. confusa and L. hypoglauca, while only dipsacoside B was detected in L. japonica. Among all seven saponins interested, only dipsacoside B was found in all five plant species of Flos Lonicerae analyzed, and this compound was determined as the major saponin with content of 53.7 mg/g in L. hypoglauca. On the other hand, macranthoidin B was found to be the major saponin with the content higher than 41.0mg/g in L. macranthoides,L. confusa, and L. similis, while the contents of other analytes were much lower.In our previous study [12], overall HPLC profiles of iridoid glucosides was used to qualitatively and quantitatively distinguish different origins of Flos Lonicerae. As shown in Fig.2, the chromatogram profiles of L. confusa, L. japonica and L. similes seem to be similar, resulting in the difficulty of clarifying the origins of Flos Lonicerae solely by HPLC profiles of saponins, in addition to the clear difference of the HPLC profiles of saponins from L. macranthoides and L. hypoglauca.Therefore, in addition to the conventional morphological and histological identification methods, the contents and the HPLC profiles of saponins and iridoids could also be used as accessory chemical evidence toclarify the botanical origin and comprehensive quality evaluation of Flos Lonicerae.4. ConclusionsThis is the first report on validation of an analytical method for qualification and quantification of saponins in Flos Lonicerae. This newly established HPLC-ELSD method can be used to simultaneously quantify seven saponins, i.e. macranthoidin B, macranthoidin A, dipsacoside B, hederagenin-28-O-β-D-glucopyranosyl(6→1)-O-β-D- glucopyranosyl ester, macranthoside B, macranthoside A, and hederagenin-3-O-α-L-arabinopyranosyl(2→1)-O-α-L-rhamnopyranoside in Flos Lonicerae. Together with the HPLC profiles of iridoids, the HPLC-ELSD profiles of saponins could also be used as an accessory chemical evidence to clarify the botanical origin and comprehensive quality evaluation of Flos Lonicerae.AcknowledgementsThis project is financially supported by Fund for Distinguished Chinese Young Scholars of the National Science Foundation of China (30325046) and the National High Tech Program（2003AA2Z2010）.[1]Ministry of Public Health of the People’s Republic of China, Pharmacopoeia ofthe People’s Republic of China, V ol.1, 2000, p. 177.[2]W. Shi, R.B. Shi, Y.R. Lu, Chin. Pharm. J., 34(1999) 724.[3]J.B. Xing, P. Li, D.L. Wen, Chin. Med. Mater., 26(2001) 457.[4]Y.Q. Zhang, L.C. Xu, L.P. Wang, J. Chin. Med. Mater., 21(1996) 204.[5] D. Zhang, Z.W. Li, Y. Jiang, J. Pharm. Anal., 16(1996) 83.[6]T.Z. Wang, Y.M. Li, Huaxiyaoxue Zazhi, 15(2000) 292.[7]J.ZH. Shi, G.T. Liu. Acta Pharm. Sin., 30(1995) 311.[8]Y. P. Liu, J. Liu, X.SH. Jia, et al. Acta Pharmacol. Sin., 13 (1992) 209.[9]Y. P. Liu, J. Liu, X.SH. Jia, et al. Acta Pharmacol. Sin., 13 (1992) 213.[10]J.ZH. Shi, L. Wan, X.F. Chen.ZhongYao YaoLi Yu LinChuang, 6 (1990) 33.[11]J. Liu, L. Xia, X.F. Chen. Acta Pharmacol. Sin., 9 (1988) 395[12]H.J. Li, P. Li, W.C. Ye, J. Chromatogr. A 1008(2003) 167-72.[13]Q. Mao, D. Cao, X.SH. Jia. Acta Pharm. Sin., 28(1993) 273.[14]H. Kizu, S. Hirabayashi, M. Suzuki, et al. Chem. Pharm. Bull., 33(1985) 3473.[15]S. Saito, S. Sumita, N. Tamura, et al. Chem Pharm Bull., 38(1990) 411.[16]Alltech ELSD 2000 Operating Manual, Alltech, 2001, p. 16. In Chinese.[17]J.B. Xing, P. Li, Chin. Med. Mater., 22(1999) 366.Fig. 1 Chemical structures of seven saponins from Lonicera confusa macranthoidin B (1), macranthoidin A (2), dipsacoside B (3), hederagenin-28-O-β-D-glucopyranosyl(6→1)-O-β-D- glucopyranosyl ester (4), macranthoside B (5), macranthoside A (6), and hederagenin-3-O-α-L-arabinopyranosyl(2→1)-O-α-L-rhamnopyranoside (7)Fig. 2Representative HPLC chromatograms of mixed standards and methanol extracts of Flos Lonicerae.Column: Agilent Zorbax SB-C18 column（250 4.6mm, 5.0µm）, temperature of 25℃; Detector: ELSD, drift tube temperature 106℃, nitrogen flow-rate 2.6 l/min.A: Mixed standards, B: L. confusa, C: L. japonica, D: L. macranthoides, E: L. hypoglauca, F: L. similes.Table 1 Calibration curves for seven saponinsAnalytes Calibration curve ar2Test range(μg)LOD(μg)LOQ(μg)1 y=6711.9x－377.6 0.9940 0.56–22.01 0.26 0.882 y=7812.6x－411.9 0.9922 0.54–21.63 0.26 0.843 y=6798.5x－299.0 0.9958 0.46–18.42 0.22 0.724 y=12805x－487.9 0.9961 0.38–15.66 0.10 0.345 y=4143.8x－88.62 0.9989 0.42–16.82 0.18 0.246 y=3946.8x－94.4 0.9977 0.40–16.02 0.16 0.207 y=4287.8x－95.2 0.9982 0.42–16.46 0.12 0.22a y: Peak area; x: concentration (mg/ml)Table 2 Reproducibility of the assayAnalyteIntra-day variability Inter-day variability Content (mg/g) Mean RSD (%) Content (mg/g) Mean RSD (%)1 46.1646.2846.2246.22 0.1346.2245.3647.4226.33 2.232 5.385.385.165.31 2.405.285.345.045.22 3.043 4.374.304.184.28 2.244.284.464.024.255.204 nd1)-- -- nd -- --5 1.761.801.821.79 1.701.801.681.841.77 4.706 1.281.241.221.252.451.241.341.201.26 5.727 tr2)-- -- tr -- -- 1): not detected; 2): trace. RSD (%) = (SD/Mean) ×100%Table 3 Recovery of the seven analytesAnalyteOriginal(mg) Spiked(mg)Found(mg)Recovery(%)Mean(%)RSD(%)1 23.0823.1423.1119.7122.8628.1042.7346.1351.0199.7100.699.399.8 0.722.692.672.582.082.913.164.735.515.7698.197.6100.698.8 1.632.172.152.091.732.182.623.884.404.6598.8103.297.799.9 2.94nd1)1.011.050.980.981.101.0297.0104.8104.1102.0 4.250.880.900.910.700.871.081.561.752.0197.197.7101.898.9 2.660.640.620.610.450.610.751.081.211.3397.796.796.096.8 0.97tr2)1.021.101.081.031.111.07100.9102.799.1100.9 1.81): not detected; 2): trace.a Recovery (%) = (Amount found –Original amount)/ Amount spiked ×100%, RSD (%) = (SD/Mean) ×100%Table 4 Contents of seven saponins in Lonicera spp.Content (mg/g)1 2 3 4 5 6 7 L. confusa45.65±0.32 5.13±0.08 4.45±0.11tr1) 2.04±0.04tr 1.81±0.03 L. japonica nd2)nd 3.44±0.09nd nd nd nd L. macranthoides46.22±0.06 5.31±0.13 4.28±0.10 tr 1.79±0.03 1.25±0.03 tr L. hypoglauca11.17±0.07 nq3)53.78±1.18nd 1.72±0.02 2.23±0.06 2.52±0.04 L. similes41.22±0.25 4.57±0.07 3.79±0.09nd 1.75±0.02tr nd 1): trace; 2): not detected.. 3) not quantified owing to the suspicious purity of the peak.。

非洲加纳籽生物学特性及其化学成分（综述）

2013,42(1):86-90.Subtropical Plant Science非洲加纳籽生物学特性及其化学成分(综述) 谌迪1,2，林德钦2，郑志忠2,3，童庆宣2，林河通1，明艳林2（1.福建农林大学食品科学学院，福建福州350002；2.厦门华侨亚热带植物引种园药用植物与植物药研发中心，福建厦门361002；3.天医堂(厦门)生物工程有限公司，福建厦门361022）摘要：现代植物化学研究发现，非洲加纳籽的主要化学成分为5-羟基色氨酸；现代药理研究表明，该植物具有抗抑郁、抑制肥胖和抗氧化等各种药理活性。

本文系统介绍非洲加纳籽的生物学特性及化学成分与药理药效的国内外研究进展，为其引种驯化与深度开发提供理论依据。

关键词：非洲加纳籽；引种驯化；化学成分；5-羟基色氨酸Doi: 10.3969/j.issn.1009-7791.2013.01.019中图分类号：Q949.95 文献标识码：A 文章编号：1009-7791(2013)01-0086-05Biological Characteristics of Griffonia simplicifolia and its Chemical Composition CHEN Di1,2, LIN De-qin2, ZHENG Zhi-zhong2,3, TONG Qing-xuan2, LIN He-tong1, MING Yan-lin2 (1.College of Food Science, Fujian Agriculture and Forestry University, Fuzhou 350002, Fujian China; 2.Research Center for Medicinal Plants and Plant Medicine, Xiamen Overseas Chinese Subtropical Plant Introduction Garden, Xiamen 361002, Fujian China; 3.Enjoye C&G (Xiamen) Bioengineering Co., Ltd, Xiamen 361022, Fujian China)Abstract: The modern phytochemistry study found that 5-hydroxytryptophan was the main chemical composition of Griffonia simplicifolia, and modern pharmacology researches showed that it had some pharmacological activities including depression, inhibition of obesity, antioxidation and so on. This article systematically introduced the biological characteristics of Griffonia simplicifolia and some correlative researches about its chemical composition and pharmacological efficacy, which provides a theoretical basis for its introduction, domestication and further development.Key words: Griffonia simplicifolia; introduction and domestication; chemical composition;5-hydroxytryptophan非洲加纳籽（Griffonia simplicifolia）为云实亚科加纳籽属植物，原产于加纳、科特迪瓦和多哥等西非国家[1-2]。

基于TCGA数据库分析GTSE1基因在肺腺癌中的表达及意义

论著文章编号１００６－８１４７（２０21）０3-0274-05作者简介高周勇(1994-)，男，硕士在读，研究方向：肺癌蛋白质组学；通信作者：王广舜，E-mail ：*******************。

基于TCGA 数据库分析GTSE1基因在肺腺癌中的表达及意义高周勇，张文成，王广舜（天津医科大学宝坻临床医学院肿瘤科，天津301800）摘要目的：探究G2/S 期表达蛋白1（G2and S phase-expressed protein1，GTSE1）在肺腺癌中的表达情况以及对预后的影响。

方法：从癌症基因组图谱（TCGA ）数据库中下载肺腺癌患者表达谱数据以及临床信息资料，分析GTSE1表达水平与临床指标信息之间的相关性及对预后的影响，并用Cox 比例风险回归模型分析影响患者预后的因素。

采用免疫组化检测14例肺腺癌肿瘤组织和13例癌旁组织中GTSE1表达水平。

采用基因集富集分析方法（GSEA ）预测GTSE1在肺腺癌中可能参与并调控的相关通路。

结果：在TCGA 数据中，在肿瘤组织样本中GTSE1基因表达情况明显高于正常组织（t =18.165，P <0.05），且表达水平与肿瘤分级（字2=17.543，P =0.001）、T （字2=8.825，P =0.032）和N 分期（字2=12.138，P =0.001）之间有显著相关，GTSE1高表达患者的总生存期显著低于GTSE1低表达的患者（字2=22.663，P <0.05），单因素Cox 分析提示肿瘤分级（HR =1.56，95%CI ：1.33～1.83）、TNM 分期（T ：HR =1.60，95%CI ：1.31～1.95；N ：HR =1.70，95%CI ：1.40～2.06；M ：HR =1.83，95%CI ：1.03～3.25）和GTSE1表达水平（HR =2.32，95%CI ：1.62～3.30）可影响患者的总生存期（均P <0.05）。

BECODISC P 系列高纯度细胞化胶原浓缩过滤芯说明书

Technical Data Sheet Depth FiltrationBECODISC® P RangePremium Depth Filter Medium with High-Purity CelluloseBECODISC P stacked disc cartridges arecharacterized by unparalleled purity. The ion andendotoxin content is significantly lower than forconventional depth filter media.In Eaton’s innovative BECODISC P stacked disccartridge’s range, high-purity celluloses form aunique structure, which even for microbe removaldoes not require mineral components.The specific advantages of BECODISC P stacked disccartridges:-Minimum endotoxin contents. This ensures productsafety-Increased endotoxin retention-Without the addition of mineral components,therefore minimum ion content particularly ofcalcium, magnesium and aluminum ions-Very high chemical resistance and mechanicalstability-Rinsing volume reduced by up to 50%, resulting inreduced process costs- A Validation Guide is available upon requestIngredientsBECODISC P stacked disc cartridges are made only ofhigh-purity cellulose and wet strength agents.Areas of ApplicationBECODISC P stacked disc cartridges can be used for filtration of all liquid media. Application options range from coarse filtration to microbe removal.BECODISC P Stacked Disc Cartridges BECODISC P stacked disc cartridges are cationic. They are characterized by adsorption charge-related during filtration. Additionally, the depth filter medium has a very low content of soluble ions, especially of calcium, magnesium and aluminum. The chemical resistance and bursting strength is extremely high. BECODISC P stacked disc cartridges are suitable for applications involving mechanical separation of particles and adsorptive retention of negatively charged particles. Due to the minimum endotoxin contents and the increased endotoxin reduction the depth filter medium is ideal for pharmaceutical processes. Guide to Choosing the Right BECODISC P Stacked Disc CartridgeB171Microbial removal and increased endotoxin retention B271Microbial and endotoxin reductionB351Fine filtration, activated carbon removalB551Clarifying filtration, particle separationB581Coarse filtration, particle separationPhysical DataThis information is intended as a guideline for the selection of BECODISC stacked disc cartridges.The water throughput is a laboratory value characterizing the different BECOPAD ® P depth filter medium types. It is not the recommended flow rate. 2 2B171 BECOPADP 1700.2 – 0.4 0.15 (3.9) < 1 > 21.8 (150) 1.9 (77) < 0.025 B271 BECOPAD P 2700.5 – 0.7 0.15 (3.9) < 1 > 21.8 (150) 3.3 (135) < 0.025 B351 BECOPAD P 3500.7 – 1.0 0.15 (3.9) < 1 > 21.8 (150) 3.9 (160) < 0.025 B551 BECOPAD P 5502.0 –3.00.15 (3.9)< 1 > 21.8 (150) 14.0 (570) < 0.025 B581 BECOPAD P 5808.0 – 10.0 0.15 (3.9)< 1> 21.8 (150)87.6(3571)< 0.025*B = Polypropylene version (e.g. B171)** 100 kPa = 1 bar*** Endotoxin content analysis after rinsing with 0.61 gal/ft² (25 l/m²) of WFI (Water for Injection)Ordering Information1 Flat adapter/Double O-ring adapter |2 With cell spacer rail |3 Cannot be combined with double O-ring adapterExample: B17162SFPolypropylene stacked disc cartridge with BECOPAD P170 depth filter sheets, nominal retention range from 0.2 to 0.4 µm, 16 filter cells, 10.9 in (276 mm) high , 12", with silicone gaskets and flat adapter.BECODISC 12", Ø 11.6 in (295 mm) BECODISC 16", Ø 15.8 in (402 mm)Number of cells 16 14 91 9 5 16 14 91 9 5 Filter surface area [ft² (m²)]20.5 (1.9) 17.8 (1.65) 11.8 (1.1) 11.8 (1.1) 6.4 (0.59) 39.8 (3.7) 34.4 (3.2) 22.6 (2.1) 22.6 (2.1) 12.4 (1.15) Pre-coat volume [gal (l)]²- 0.9 (3.6) 2.1 (8.0)- -- 1.8 (7.0) 4.1 (15.4)- -Overall height flat adapter [in (mm)]10.9 (276) 10.9 (276) 10.9 (276) 7.7 (195) 4.4 (101) 10.9 (276) 10.9 (276) 10.9 (276) 7.7 (195) 4.4 (101) Overall height double O-ring adapter [in (mm)] 13.0 (329) 13.0 (329) 13.0 (329) 10.0 (248) -13.0 (329) 13.0 (329) 13.0 (329) 10.0 (248) -Cell spacer rail- - ✓ - -- - ✓ - -1 Special stacked disc cartridge configuration with cell spacer rails providing increased mechanical stability forholding filter cake | 2 Calculated values (BECO depth filter sheets with 0.16 in/4.0 mm thickness)171BECOPAD depth filter sheet 171 = P 170 271 = P 270 351 = P 350 551 = P 550 581 = P 580BDesignB = Polypropylene6Construction (overall height)16 = 16 filter cells(10.9/13.0 in) (276/329 mm) 4 = 14 filter cells(10.9/13.0 in) (276/329 mm) 7 = 9 filter cells 2(10.9/13.0 in) (276/329 mm) 9 = 9 filter cells(7.7/10.0 in) (195/248 mm) 5 = 5 filter cells 3(4.4 in) (101 mm)2Size2 = 12", ∅ 11.6 in(295 mm) 4 = 16", ∅ 15.8 in(402 mm)SGasket material E = EPDM F = FEP-coatedsilicone core S = Silicone V = FluoroelastomerFAdapterF = Flat adapter S = Double O-ringadapter Y = Flat adapter withgrounding deviseCompliance NoticeBECO depth filter sheets fulfill the requirements of Regulation (EC) 1935/2004 as well as the FDA Guideline 21 CFR §177.2260 test criteria. The polypropylene components comply with Regulation (EU) 10/2011. The polypropylene meets FDA requirements, 21 CFR § 177.1520. The sealing materials (silicone, EPDM) meet FDA requirements, 21 CFR § 177.2600. The depth filter sheet and the polypropylene components of the BECODISC P stacked disc cartridges meet the requirements of the USP Plastic Class VI – 70 °C test. For further details on individual components and materials see the declaration of conformity.Ion Concentration after Extraction with 40% EthanolCa < 50Mg < 25Fe < 5Al < 5* After rinsing with 0.61 gal/ft² (25 l/m²) of 40% EthanolRecommendations for Avoiding Damage BECODISC stacked disc cartridges can be used only in the specified flow direction. This applies to product filtering as well as sanitizing with hot water, and sterilizing with the stacked disc cartridges with saturated steam. In order to avoid damage to the filter cells, the system should be protected with a suitable non-return valve.Refer to the insert included with each BECODISC stacked disc cartridge carton for detailed application information.Depending on the filtered liquids, the operating temperature should not exceed 176 °F (80 °C). Please contact Eaton regarding filtration applications at higher temperatures.Intermediate PlatesIf more than two BECODISC stacked disc cartridges (12" or 16") with double O-ring adapters are stacked in the housing, install a central spindle for safety reasons. In the event, more than one 16" BECODISC stacked disc cartridge (flat adapter/double O-ring adapter) is used in the housing, Eaton recommends the installation of stainless steel intermediate plates between the BECODISC stacked disc cartridges. When silicone/FEP coated gaskets are used the stainless steel plates are mandatory. Sanitizing and Sterilizing (Optional)Sterilizing with Hot WaterThe hot water temperature should be 185°F (85 °C). A differential pressure of 21.8psi (150 kPa, 1.5 bar) must not be exceeded when sterilizing with hot water. Sterilization time: At least 30 minutes once a temperature of 185°F (85 °C) is reached at all filter openings. In the interest of energy conservation, the water may be circulated provided the specified temperatures are maintained.Sterilizing with SteamThe wetted BECODISC stacked disc cartridges can be sterilized with saturated steam up to a maximum temperature of 250 °F (121 °C) as follows:Steam quality: The steam must be free of foreignparticles and impurities. Temperature: Max. 250 °F (121 °C)(saturated steam)Duration: Approx. 20 minutes after steam exitsfrom all filter valvesRinsing: After sterilizing with 0.61 gal/ft²(25 l/m²) at 1.25 times the flow rateFilter Preparation and FiltrationUnless already completed after sterilization, rinse the stacked disc cartridges with 0.61 gal/ft² (25 l/m²) of water at 1.25 times the flow rate prior to the first filtration. Check the entire filter for leakage at maximum operating pressure.High-proof alcoholic solutions and products that cannot be rinsed with water should be circulated with the product. Discard the rinsing solution after rinsing. Differential PressureTerminate the filtration process once the maximum permitted differential p ressure of 43.5psi (300 kPa,3 bar) is reached. A higher differential pressure could damage the depth filter sheet material. For safety reasons, a differential pressure of 21.8psi (150 kPa, 1.5 bar) should not be exceeded in applications for separating microorganisms.SafetyWhen used and handled correctly, there are no known unfavorable effects associated with this product. Further safety information can be found in the relevant Material Safety Data Sheet, which can be downloaded from our website.DisposalDue to their composition, BECODISC stacked disc cartridges can be disposed of as harmless waste. Comply with relevant current regulations, depending on the filtered product.StorageBECODISC stacked disc cartridges must be stored in a dry, odor-free, and well ventilated place.Do not expose the BECODISC stacked disc cartridges to direct sunlight.BECODISC stacked disc cartridges are intended for immediate use and should be used within 36 months after production date.Quality Assurance According to DIN EN ISO 9001 The Quality Management System of Eaton Technologies GmbH has been certified according to DIN EN ISO 9001.This certification verifies that a fully functioning comprehensive Quality Assurance System covering product development, contract controls, choice of suppliers, receiving inspections, production, final inspection, inventory management, and shipment has been implemented.Extensive quality assurance measures incorporate adherence to technical function criteria and chemical purity and quality recognized as safe under the German legislation governing the production of foods and beverages.All information is given to the best of our knowledge. However, the validity of the information cannot be guaranteed for every application, working practice and operating condition. Misuse of the product will result in all warrantees being voided.Subject to change in the interest of technical progress.North America44 Apple StreetTinton Falls, NJ 07724Toll Free: 800 656-3344 (North America only)Tel: +1 732 212-4700Europe/Africa/Middle EastAuf der Heide 253947 Nettersheim, Germany Tel: +49 2486 809-0 Friedensstraße 4168804 Altlußheim, Germany Tel: +49 6205 2094-0An den Nahewiesen 2455450 Langenlonsheim, Germany Tel: +49 6704 204-0 ChinaNo. 3, Lane 280,Linhong RoadChangning District, 200335Shanghai, P.R. ChinaTel: +86 21 5200-0099Singapore100G Pasir Panjang Road #07-08Singapore 118523Tel: +65 6825-1668BrazilRua Clark, 2061 - Macuco13279-400 - Valinhos, BrazilTel: +55 11 3616-8400For more information, pleaseemail us at ********************or visit /filtration© 2018 Eaton. All rights reserved. All trademarks andregistered trademarks are the property of their respectiveowners. All information and recommendations appearing inthis brochure concerning the use of products describedherein are based on tests believed to be reliable. However,it is the user’s responsibility to determine the suitability forhis own use of such products. Since the actual use byothers is beyond our control, no guarantee, expressed orimplied, is made by Eaton as to the effects of such use orthe results to be obtained. Eaton assumes no liabilityarising out of the use by others of such products. Nor is theinformation herein to be construed as absolutely complete,since additional information may be necessary or desirablewhen particular or exceptional conditions or circumstancesexist or because of applicable laws or governmentregulations.EN1 A 2.8.210-2018。

AR-A014418_DataSheet_MedChemExpress

Inhibitors, Agonists, Screening Libraries Data SheetBIOLOGICAL ACTIVITY:AR–A014418 is a selective and effective GSK3β inhibitor with an IC 50 value of 104 nM, and has no significant inhibition on 26 other kinases.IC50 & Target: IC50: 104 nM (GSK3β)In Vitro: AR–A014418 inhibits tau phosphorylation at a GSK3–specific site (Ser–396) in 3T3 fibroblasts expressing human four–repeat tau protein with IC 50 of 2.7 μM, and protects cultured N2A cells from death induced by blocking PI3K/PKB pathway. In hippocampal slices, AR–A014418 inhibits neurodegeneration mediated by beta–amyloid peptide [1]. While in NGP and SH–5Y–SY cells,AR–A014418 reduces neuroendocrine markers and suppresses neuroblastoma cell growth [2].In Vivo: In ALS mouse model with the G93A mutant human SOD1, AR–A014418 (0–4 mg/kg, i.p.) delays the onset of symptoms,improves motor activity, slows down disease progression, and postpons the endpoint of the disease [3]. In addition, AR–A014418produces inhibition effect on acetic acid– and formalin–induced nociception in mice by modulating NMDA and metabotropic receptor signaling as well as TNF–α and IL–1β transmission in the spinal cord [4].PROTOCOL (Extracted from published papers and Only for reference)Kinase Assay:[1]The competition experiments are carried out in duplicate with 10 concentrations of the inhibitor inclear–bottomed microtiter plates. The biotinylated peptide substrate, biotin–AAEELDSRAGS(PO3H2)PQL, is added at a final concentration of 2 μM in an assay buffer containing 6 milliunits of recombinant human GSK3 (equal mix of both α and β), 12mM MOPS, pH 7.0, 0.3 mM EDTA, 0.01% β–mercaptoethanol, 0.004% Brij 35, 0.5% glycerol, and 0.5 μg of bovine serumalbumin/25 μL and preincubated for 10–15 min. The reaction is initiated by the addition of 0.04 μCi of [γ–33P]ATP and unlabeled ATP in 50 mM Mg(Ac)2 to a final concentration of 1 μM ATP and assay volume of 25 μL. Blank controls without peptide substrate are used.After incubation for 20 min at room temperature, each reaction is terminated by the addition of 25 μL of stop solution containing 5mM EDTA, 50 μM ATP, 0.1% Triton X–100, and 0.25 mg of streptavidin–coated SPA beads corresponding to appr 35 pmol of binding capacity. After 6 h the radioactivity is determined in a liquid scintillation counter. Inhibition curves are analyzed by non–linear regression using GraphPad Prism.Cell Assay: AR–A014418 is dissolved in DMSO.[1]Cell viability is assessed by calcein/propidium iodide uptake. Calcein AM is taken up and cleaved by esterases present within living cells, yielding yellowish–green fluorescence, whereas PI is only taken up by dead cells,which become orange–red fluorescent. In brief, N2A cells are cultured for 2 days in vitro and then treated with 50 μM LY–294002 in the presence of AR–A014418 or vehicle (DMSO) for 24 h. Subsequently, N2A cells are incubated for 30 min with 2 μM PI and 1 μM calcein–AM. The cultures are then rinsed three times with Hanks' buffered saline solution containing 2 mM CaCl 2, and the cells are visualized by fluorescence microscopy using a Zeiss Axiovert 135 microscope. Three fields (selected at random) are analyzed per well (appr 300 cells/field) in at least three different experiments. Cell death is expressed as percentage of PI–positive cells from the total number of cells. In every experiment, specific cell death is obtained after subtracting the number of dead cells present inProduct Name:AR–A014418Cat. No.:HY-10512CAS No.:487021-52-3Molecular Formula:C 12H 12N 4O 4S Molecular Weight:308.31Target:GSK–3; GSK–3Pathway:Stem Cell/Wnt; PI3K/Akt/mTOR Solubility:10 mM in DMSOvehicle–treated cultures.Animal Administration: AR–A014418 is formulated in normal saline.[3]First, to examine the effects of GSK–3 inhibition on the clinical symptoms, life span, and motor behavior function of ALS, 56 Tg mice are divided into four groups. In each group, 0.5 mL of normal saline is mixed with either 0 μg (control group), 1 μg (group A), 2 μg (group B) or 4 μg (group C) of AR–A014418 per gram of mouse, and injected intraperitoneally into 14 animals per group 5 days a week beginning 60 days after birth. The mice are sacrificed at the endpoint described below.References:[1]. Bhat R, Xue Y, Berg S, Structural insights and biological effects of glycogen synthase kinase 3–specific inhibitor AR–A014418. J Biol Chem. 2003 Nov 14; 278(46):45937–45.[2]. Carter YM, et al. Specific glycogen synthase kinase–3 inhibition reduces neuroendocrine markers and suppresses neuroblastoma cell growth. Cancer Biol Ther. 2014 May;15(5):510–5.[3]. Koh SH, et al. Inhibition of glycogen synthase kinase–3 suppresses the onset of symptoms and disease progression of G93A–SOD1 mouse model of ALS. Exp Neurol. 2007 Jun;205(2):336–46.[4]. Martins DF, et al. The antinociceptive effects of AR–A014418, a selective inhibitor of glycogen synthase kinase–3 beta, in mice. J Pain. 2011 Mar;12(3):315–22.Caution: Product has not been fully validated for medical applications. For research use only.Tel: 609-228-6898 Fax: 609-228-5909 E-mail: tech@Address: 1 Deer Park Dr, Suite Q, Monmouth Junction, NJ 08852, USA。

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