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??OBJECTIVE To provide the basis of improving drug importation registration management system of our country by studying domestic and foreign drug importation management system adequately. METHODS Checked out and studied the laws associated with import drug of our country and United States with literature research and comparative analysis methods. RESULTS To reform the drug importation registration management system of our country, related problems should be analyzed. Also the recommendations should be put forward, including the registration of foreign drug establishment,unified registration procedures and adopted DMF system. CONCLUSION Based on national conditions, we must draw on the advantages of United States^?? system selectively, to improve our system, ensure the quality of import drug lifecycle and protect public health.     

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??OBJECTIVE To establish a UPLC-Q-TOF-MS method to characterize the metabolites of phillygenin in human liver microsomal incubation system for the first time. METHODS The chromatography separation was performed on a C₁₈ reversed phase LC column (Phenomenex Kinetex C₁₈, 2.1 mm??100 mm, 2.6 ??m). The mobile phase consisted of water-formic acid (100:0.1, V/V) and acetonitrile and a gradient elution program was adopted at the flow rate of 400 ??L??min^-1. The mass spectral analysis was performed in a positive electrospray ionization mode, and the turbo spray temperature was 550 ??. The full MS experiment was run with a scan range from m/z 100 to m/z 1 000. RESULTS The possible fragmentation pathways of phillygein were speculated in a positive electrospray ionization mode, and eight metabolites was identified in human liver microsomal incubation system. CONCLUSION The UPLC-Q-TOF-MS method is very convenient and efficient for detecting phillygein in human liver mirosomes. The developed method is suitable for the metabolism research of phillygein in human liver microsomes, which providing valuable reference for pharmacokinetic study of phillygenin.     

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??OBJECTIVE To abserve the antidepressant effect and mechanism of honokiol on acute and chronic stress mouse. METHODS Forced swimming model of acute stress (FST) and chronic stress mice model were used. The acute stress mouse were randomized into a control group, a fluoxetine group (3.3 mg??kg^-1), honokiol groups (2.5,5,10 mg??kg^-1). The chronic stress mouse were randomized into a blank group, a model group, a fluoxetine group(3.3 mg??kg^-1), honokiol groups (2.5,5,10 mg??kg^-1). Then, the immobility time of forced swimming, 5-hydroxytryptamine (5-HT) and 2,3- indole dioxygenase (IDO) contents in mouse brain tissue by Elisa Kit, and the expression of IDO mRNA in brain tissue used by quantitative real-time PCR were studied. RESULTS ??After acute stress, the immobility time of forced swimming in each treatment group was significantly shorter than that in the model group (P<0.05). The 5-HT content of fluoxetine and honokiol medium and high dose group was significantly higher than that of model group (P<0.05, P<0.01). The IDO content of honokiol high dose group was significantly higher than that of model group (P<0.05). ??After chronic stress, the immobility time of the model group were significantly higher than the blank group (P<0.01). The 5-HT content in brain tissue of the model group was significantly lower than that of the blank group (P<0.01), the IDO content in brain tissue and its expression level of mRNA increased comparing with the normal group (P<0.01). For each treatment group, the immobility time of forced swimming, IDO content and the expression level of IDO mRNA was significantly decreased, and the 5-HT content was significantly increased, comparing with the model group with significant difference (P<0.05). CONCLUSION Honokiol can relieve the depression behavior of mouse and have certain antidepressant effect. The main mechanism may be associated with the increase of 5-HT, reduction of the tryptophan pathway enzyme IDO content and its gene expression level.     

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??OBJECTIVE To investigate the effect of iridoids in Ajuga decumbens(ADI) on the metastasis of invasive breast cancer cells and related mechanisms. METHODS Several assays including cell-HUVEC adhesion assay were conducted, Transwell migration and Transwell invasion assay were conducted. In vivo antimetastatic effect of ADI was determined using experimental lung metastatic models induced by tail vain injection of 4T1-luc cells. Western blot analysis was employed to detect the expression of associated proteins of EMT and ERK1/2 MAPK signaling pathway. RESULTS The 50-200 mgL^-1 ADI could significantly inhibit the cell-HUVEC adhesion, migration and invasion of MDA-MB-231 cells. Administration with 200 mgkg ^-1 dose of ADI markedly inhibited the lung tumor nodules. Treatment with ADI resulted in markedly inhibition of phosphorylation of ERK1/2 and the reversal EMT of breast cancer cells, which increased the levels of epithelial biomarkers, such as E-cadherin and ZO-1, and reduced the levels of mesenchymal biomarkers, such as vimentin. CONCLUSION These results indicates that ADI can inhibit breast cancer cell invasion by suppressing the ERK1/2 MAPK pathway as well as reversing the epithelial-mesenchymal transition.     

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??OBJECTIVE To identify the metabolites of Cuscuta chinensis in the serum and feces of rats with estrogenic estrogen, and to provide the basis for elucidating the pharmacological basis of its efficacy. METHODS Using the HPLC-ESI-MS/MS technique, the molecular weight and molecular formula of the compounds were preliminarily estimated by the first-order mass spectrometry, and the chemical constituents of serum and feces were characterized by the retention time of the standard, the fragment ion information, the fragmentation law of mass spectrometry and the reference data. RESULTS Five prototypic components and 4 metabolites were identified in the serum. A total of 11 chemical constituents were identified in the feces after administration, including 5 prototype components and 6 metabolites. CONCLUSION Through the comparative analysis between serum and feces, the main metabolic pathways of Cuscuta chinensis compound are deduced, which provides reference for the basic research on the estrogenic substance of Cuscuta chinensis.     

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??OBJECTIVE To investigate the risk of magnolol interfering into propofol glucuronidation in human.METHODS This study was performed in pooled microsomes from human liver, intestine and kidney (HLM, HIM, HKM, respectively). Kinetic analyses were conducted to gain the inhibition potentials of magnolol against propofol glucuronidation in HLM, HIM, and HKM.RESULTS Magnolol can potently inhibit propofol glucuronidation in HLM and HKM, following mixed inhibition kinetics with K_i values of 0.1 and 0.2 ??mol??L^-1, respectively. Different from HLM and HKM, propofol glucuronidation in HIM was not affected by the presence magnolol. CONCLUSION Magnolol is hardly to depress systemic propofol glucuronidation due to lack of inhibition of the intestinal metabolic pathway.     

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??OBJECTIVE To investigate the mechanism of hyperthyroid rats treated by thiamazole tablets by the serum metabonomics technology. METHODS Hyperthyroid rats were modeled by subcutaneous injection of L-levothyroxine sodium 35 ??g??100 g^-1 for 7 d. Then rats of thiamazole-treated group were treated by intragastric administration of thiamazole tablets suspension 21 mg??kg^-1 for 21 d. The serum levels of model rats, control rats, thiamazole-treated rats were analyzed by using UPLC-QTOF-MS and UPLC-QTOF-MS was combined with two different modes of positive and negative ions as well as KEGG and HMDB. RESULTS Compared with normal rats, among hyperthyroid rats oxalacetic acid, dihydroxyacetone phosphate (DHAP), ethyl glucuronide, leukotriene E3, hydroxykynurenine, triglycerides (TG) and lysophospholipids were increased. Meanwhile, diacylglycerol (DG) and phosphatidylinositol (PI) were decreased. The 21 d later, the metabolism levels of oxalacetic acid, dihydroxyacetone phosphate, leukotriene E3, phospholipids and fatty acids in thiamazole-treated rats were close to the normality. CONCLUSION The RESULTS infer that tricarboxylic acid cycle of hyperthyroid rat is disturbedall of glucose metabolism, lipid metabolism and amino acid metabolism get into disorder. And these disorders are improved certainly by treating thiamazole. Meanwhile, this method provides an available reference for the establishment of the means of diagnosing hyperthyroidism based on metabonomics and the assessment of the therapeutic effect of thiamazole tablets.     

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??OBJECTIVE To investigate the differences of the accumulation of the main secondary metabolites in the leaves of 11 kinds of Paris L. medicinal plants. METHODS The contents of the main secondary metabolites, polyphyllin??, ??, ??, and ?? in the leaves of 11 kinds of Paris L. medicinal plants were determined by UPLC, and the UPLC fingerprints were established. The accumulation of the main secondary metabolites was evaluated by one-way ANOVA and chromatographic analysis. RESULTS There was significant difference(P<0.01)in the contents of polyphyllin ??, ??, ??, and ?? in the leaves of 11 kinds of Paris L. medicinal plants, and polyphyllin ??, ??, ??, ?? were simutaneously detected only in var. yunnanensis-1, P. axialis, P. thibetica, P. forrestii and var. yunnanensis; there was significant difference in the UPLC chromatograms of 11 kinds of Paris L. medicinal plants, but the similarities among var. yunnanensis-1, P. axialis, P. thibetica, P. forrestii and var. yunnanensis all reached 0.902 with 16 common peaks, indicating smaller difference in their main secondary metabolites. CONCLUSION There is significant difference in the abilities of the 11 kinds of Paris L.medicinal plants to accumulate polyphyllin ??, ??, ??, and ?? , which may be the main reason that there are significant differences in the contents and classes of the major secondary metabolites of Paris L. roots.     

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??OBJECTIVE To study the flavonoid glycosides of Urena lobata. METHODS Compounds were isolated and purified using various column chromatographies such as D101 macroporous adsorption resin, silica gel, Sephadex LH-20, and prep HPLC. Their structures were identified on the basis of their physicochemical properties and various spectroscopic experiments, including HRESIMS, ¹H-NMR, ¹³C-NMR, HSQC, and HMBC. RESULTS Ten flavonoid glycosides were obtained from the n-BuOH extract of U. lobata including quercetin-3-O-??-D-glucopyranosyl-(1??2)-??-D-galactopyranoside(1), kaempferol-3-O-??-D-glucopyranosyl-(1??2)-??-D-glucopyranosyl-7-O-??-L-rhamnopyranoside(2), quercetin-3-O-??-D-apiofuranosyl-(1??2)-??-D-glucopyranosyl-7-O-??-L-rhamnopyranoside(3), kaempferol-4'-O-??-D-apiofuranosyl-3-O-??-D-glucopyranosyl-7-O-??-L-rhamnopyranoside(4), kaempferol-3-O-??-D-apiofuranosyl-(1??2)-??-D-glucopyranosyl-7-O-??-L-rhamnopyranoside(5), quercetin-3-O-??-D-glucopyranosyl-7-O-??-L-rhamnopyranoside(6), quercetin-3-O-??-D-glucopyranosyl-(1??2)-??-D-glucopyranoside(7), kaempferol-3-O-??-L-rhamnopyranosyl-(1??6)-??-D-glucopyranosyl-(1??2)-??-D-glucopyranoside(8), kaempferol-3-O-??-D-glucopyranosyl-(1??2)-[??-L-rhamnopyranosyl-(1??6)]-??-D-glucopyranoside(9) and kaempferol-3-O-??-D-glucopyranosyl-(1??2)-??-D-glucopyranoside(10). CONCLUSION Compounds 1-3 and 6-10 are firstly obtained from U. lobata.     

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??OBJECTIVE To optimize the extraction and purification process of eurycomanone (EN) from Eurycoma longifolia Jack. METHODS Using single factor test and orthogonal test to screen the best conditions of ethanol concentration, extraction time, amount of solvent and extraction times with the index of transfer rate of EN. Resin model was screened by static adsorption and desorption test, purification process of EN was investigated by single factor test and orthogonal test. RESULTS The optimal condition of the extracting was 20-fold water, 2 times with the first 2 and 1 h for the second time. Optimal purification process of HPD100 macroporous resin was 70% amount of saturated adsorption sample, 0.25 g??mL^-1 of sample liquid concentration, 3BV??h^-1 of sample flow rate, eluting using the 30% ethanol, 3 BV??h^-1 of elution velocity. CONCLUSION The optimized process is simple, stable and repeatable, which is suitable for industrial production.     

LC-MS/MS研究新型酪氨酸酶抑制剂UP302在大鼠肝微粒体中代谢的酶动力学

 目的 建立并验证测定大鼠肝微粒体孵育液中UP302含量的高效液相色谱-串联质谱法,研究UP302在大鼠肝微粒体中代谢的酶动力学。方法 经大鼠肝微粒体孵育的UP302样品,经色谱柱分离,电喷雾离子化负离子检测,扫描方式为选择反应监测,用于定量分析的离子反应分别为m/z 301.1→135.2（UP302）和m/z 252.9→132.0（内标大豆苷元）。考察不同孵育时间、底物浓度和微粒体浓度对UP302代谢的影响,确定最佳反应条件。结果 标准曲线线性范围为0.1～20 μmol·L-1,线性关系良好,日内日间准确度在86.42%～112.59%,日内日间精密度均小于9.09%,回收率在100.50%～109.91%之间。确定了酶动力学参数,最大反应速度V_max为196.08 μmol·L-1·min-1·g-1 ,米氏常数K_m为14.98 μmol·L-1,内在代谢清除率CL_int为13.09 mL-1·min-1·g-1。结论 该检测方法快速、专属、灵敏度高,可满足酶动力学检测要求。     

去甲异波尔定在大鼠肝微粒体中葡萄糖醛酸化代谢动力学研究

目的:研究去甲异波尔定在大鼠肝微粒体中的葡萄糖醛酸化酶促反应动力学。方法:优化去甲异波尔定与大鼠肝微粒体的反应体系,采用超高效液相色谱-质谱联用技术定量检测孵育体系中去甲波尔定代谢产物去甲异波尔定-9-O-α-葡萄糖醛酸苷的浓度,并应用Linewearve-Burk作图分析数据,计算酶促动力学常数。结果:去甲异波尔定-9-O-α-葡萄糖醛酸苷的酶促反应动力学参数Km40.7μmol.L-1,Vmax=909.1 pmol.(min.mg pro)-1,肝清除率CLint(Vmax/Km)=22.3μL.min-1.mg-1。结论:该方法简单、快速、可靠,适应于去甲异波尔定的葡萄糖醛酸化代谢研究;葡萄糖醛酸化是去甲异波尔定代谢的重要途径之一,提示葡萄糖醛酸转移酶的基因多态性及相关性的药物相互作用引起的去甲异波尔定活性和毒性作用的变化值得进一步关注。     

蛇床夫内酯在大鼠肝微粒体中的酶促反应动力学研究

 目的 研究蛇床夫内酯在大鼠肝微粒体中的酶促反应动力学。方法 以回芹内酯为内标,采用液质联用（LC/MS）的方法测定蛇床夫内酯在体外代谢中的代谢产物3″,8- 甲氧基异欧前胡内酯（M1）和5″- 羟基-8- 甲氧基异欧前胡内酯（M2）。用Linewrave-Burk作图分析数据,计算酶促动力学常数。结果 代谢物M1的V_max=1.152 5 μmol·(min·mg pro)-1, K_m=133.156 6 μmol·L-1,M2的V_max=1.630 8 μmol·(min·mg pro)-1, K_m=292.857 1 μmol·L-1。 结论 该方法简单、快速、可靠,适用于蛇床夫内酯的体外代谢研究。     

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??OBJECTIVE To study the enzymatic kinetics of TM-2 in rat, Beagle dog and human liver microsomes by LC-MS/MS. METHODS TM-2 was incubated with liver microsomal incubation system. LC-MS/MS method was established for quantitative analysis of TM-2 with cabazitaxel as internal standard. The enzyme kinetics parameters V_max and K_m was calculated by the GraphPad Prism 5.0 software. RESULTS A rapid and sensitive LC-MS/MS method was developed to study the enzyme kinetics of TM-2 in rat, Beagle dog and human liver microsome. The corresponding enzymatic kinetic parameters in rat, Beagle dog and human were as follows: V_maxvalues were 16.3, 354.6 and 154.8 nmol??min^-1??mg(protein)^-1, respectively; K_m values were 25.7, 313.8 and 89.4 ??mol??L^-1, respectively; CL_int values were 0.63, 1.13 and 1.73 mL??min^-1??mg(protein)^-1, respectively. CONCLUSION The result indicates that there are species differences in the activity of metabolic enzyme and the affinity of TM-2 to the metabolic enzyme. Enzyme kinetic parameters obtained of TM-2 provide important parameters for the further study.     

白藜芦醇在人肝微粒体中代谢的酶动力学

目的研究白藜芦醇在体外人肝微粒体酶中的代谢以及葡糖醛酸转移酶(UGT)抑制剂对其代谢的影响。方法采用高效液相-质谱联用方法测定白藜芦醇在体外代谢系统中的代谢产物,并用Origin7.5软件分析数据,得到白藜芦醇的酶促反应动力学参数最大反应速率(Vm),米氏常数(Km),并计算UGT对白藜芦醇的内源性清除率(CLint)。通过抑制试验,观察不同浓度抑制剂对不同浓度白藜芦醇Ⅱ相代谢的影响。结果在体外代谢系统中,白藜芦醇生成两种单葡糖醛酸代谢产物M-1,M-2,初步推断其为白藜芦醇-4’,和3-葡萄糖醛酸化物。在肝微粒体中生成两种代谢产物的酶促反应的Vm和Km分别为M-1:(1.08±0.06)nmol.min-1.mg-1和(192.05±30.46)μmol.L-1;M-2:(2.20±0.10)nmol.min-1.mg-1和(34.82±6.95)μmol.L-1,白藜芦醇代谢为M-2的代谢速率明显高于M-1。UGT抑制剂姜黄素和槲皮素都能显著抑制白藜芦醇的代谢。结论代谢产物M-2的生成是白藜芦醇在肝微粒体中的主要消除途径。姜黄素、槲皮素在体外对白藜芦醇的Ⅱ相代谢有显著的抑制作用,对临床研究药物的相互作用有很大指导意义。     

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??OBJECTIVE To investigate the hepatic toxicity of 8 monomers in Polygonum multiflorum using a combination of UDP-glucuronic acid transferase 1A1(UGT1A1 enzyme). METHODS Bilirubin was used as the substrate for UGT1A1. Incubation method in RLM in vitro was adopted to test the apparent inhibition constants(K_i) of different components. Furthermore the structure-activity relationship between the 8 components and UGT1A1 was analyzed. RESULTS The inhibition effects on UGT1A1 enzyme of the 8 components were in the following sequence: emodin-8-O-glc??emodin??citreorosein??(+)-catechin??gallic acid??physcion??rhein??emodin-6-O-glc. Moreover, there was a structure-activity relationship, and it was presumed that the 6-position hydroxyl group is an active and necessary group. CONCLUSION The established method in vitro is stable and feasible. Experimental results shows that the enzyme inhibition has structural selectivity, which provides an experimental basis for predicting the enzyme inhibition activity of the analogues of components of Polygonum multiflorum.     

黄芩苷对大鼠和人肝微粒体CYP450酶的抑制作用

目的:观察黄芩苷对大鼠/人肝微粒体细胞色素P450(CYP450)酶活性的抑制作用,比较种属差异性,评价其发生药物相互作用的可能性。方法:通过采用体外肝微粒体温孵体系结合特异性探针底物的方法,结合应用LC-MS/MS检测各探针底物的代谢物的生成量,质谱检测条件为电喷雾离子化(ESI)源,选择性反应监测(SRM)扫描方式,色谱分离条件为ZORBAX Eclipse-plus C₁₈色谱柱(2.1 mm×100 mm,3.5 μm),流动相甲醇-0.1%甲酸水溶液梯度洗脱,流速0.2 mL·min^-1,计算半抑制浓度(IC₅₀)。结果:黄芩苷对大鼠肝微粒体CYP450酶7种亚型均无抑制作用,而对人肝微粒体CYP1A2,CYP2C19和CYP2E1有较弱的抑制作用,IC₅₀分别为39.72,40.91,32.83 μmol·L^-1。结论:黄芩苷对CYP450酶的抑制作用存在种属差异性,是人CYP1A2,CYP2C19,CYP2E1的弱抑制剂,临床静脉用药时,应注意可能因CYP450酶抑制引起的药物相互作用。     

羊耳菊提取物在大鼠粪便中的代谢产物分析

目的:研究羊耳菊提取物给药后在大鼠粪便中的代谢产物,为该药材的后续研究与开发提供参考。方法:采用超高效液相色谱-三重四级杆飞行时间质谱(UPLC-Q-TOF-MS/MS)检测大鼠口服羊耳菊提取物后粪便中的代谢产物,每次给药剂量100 g·kg~(-1)。使用RRHD Eclipse Plus C_(18)色谱柱(2.1 mm×100 mm,1.8μm),流动相0.1%甲酸水溶液-0.1%甲酸乙腈溶液梯度洗脱,选择电喷雾离子源,负离子模式进行扫描,准确质量测定采用甲酸钠校正标准液,采用Metabolite Detect(micr OTOF 2.3)等软件进行数据处理,鉴定代谢产物的化学结构。结果:在大鼠粪便中检测到原型M8(1,3-O-二咖啡酰基奎宁酸),M1~M4(二氢单咖啡酰基奎宁酸异构化产物),M5(二氢咖啡酸的硫酸酯化产物),M6~M7(单咖啡酰基奎宁酸的甲基化产物)等22个代谢产物。结论:羊耳菊提取物给药后,咖啡酰基奎宁酸类活性成分在大鼠粪便中的代谢途径以甲基化、还原反应为主。     

丹酚酸B大鼠体内代谢产物及途径分析

目的:研究分析丹酚酸B在大鼠体内的代谢产物及途径。方法:取SD大鼠12只,分为两组。用于收集空白血浆、胆汁、尿液样品的大鼠各2只;尾静脉注射丹酚酸B(100 mg·kg-1)后,用于收集血浆、胆汁、尿液中丹酚酸B代谢产物的大鼠各2只;采用超高效液相色谱串联四极杆飞行时间质谱(UPLC-Q-TOF)联用技术,对处理后的各样品进行检测,并对代谢产物进行定性与结构鉴定分析。结果:根据质谱信息,共检测到大鼠体内丹酚酸B相关代谢产物11个,其中血浆样品中检测到丹酚酸B酯键水解等2个代谢产物;胆汁样品中检测到丹参素、紫草酸和紫草酸的甲基化物等5个代谢产物;尿液样品中检测到丹酚酸B甲基化和硫酸化等4个代谢产物。结论:丹酚酸B可在大鼠体内代谢为丹参素等代谢产物;依据检测的代谢产物,推测丹酚酸B代谢反应位置主要在酯键和五元环上,代谢途径主要有酯键水解、分子内脱水、脱羧、羟化、甲基化和硫酸化等。     

HPLC测定大鼠肝微粒体中谷胱甘肽硫转移酶活性及体外动力学研究

 目的 建立一种测定1-氯-2,4-二硝基苯（CDNB）高效液相色谱分析方法,并以1-氯-2,4-二硝基苯为探针测定大鼠肝微粒体中谷胱甘肽硫转移酶（GST）活性并进行体外动力学分析。方法 色谱条件：Welch Materials Ultimate^TM XB C₁₈反相柱（4.6 mm×250 mm,5 μm）,流动相：乙腈-水（7∶3),流速0.8 mL·min^-1,柱温30 ℃,检测波长238 nm。实验方法：1-氯-2,4-二硝基苯与大鼠肝微粒体在37 ℃温孵7 min后,冰乙腈终止反应。反应液离心取上清液过滤后进行HPLC分析,通过Sigma Plot 软件作图求算V_max、K_m及代谢清除率（CL_int）值。结果 1-氯-2,4-二硝基苯的R_t=6.0 min,峰形良好,且无内源性干扰。最低检测限为1.0 μmol·L^-1,线性范围：2.5～100.0 μmol·L^-1。日内、日间精密度均小于10%。5 d内于室温及-20 ℃下较稳定。方法回收率为99.38%～108%。动力学分析表明,不同浓度1-氯-2,4-二硝基苯在0.02 mg·mL^-1蛋白浓度下孵育7 min,测得动力学参数：V_max为85.45 nmol·min^-1·（mg protein）^-1;K_m为15.09 μmol·L^-1; CL_lint为5.66 mL·min^-1·（mg protein）^-1。结论 该方法稳定可靠,灵敏度高,能准确快速测定谷胱甘肽硫转移酶活性,可用于其体外动力学研究。