高效液相色谱法测定体外大鼠肠道菌液中大豆苷及其代谢物

目的 建立HPLC法测定体外大鼠肠道菌液中的大豆苷及其代谢物,研究大鼠肠道菌群对大豆苷的代谢情况。方法 采用大鼠离体粪便温孵法,将大豆苷与大鼠肠道菌群培养液进行厌氧温孵。温孵样品采用甲醇沉淀蛋白处理后进行HPLC法分析,色谱条件：色谱柱为Diamonsil C₁₈柱;流动相为0.2%乙酸水溶液与甲醇,梯度洗脱;检测波长为260 nm;柱温为25℃;进样量为10 μl。结果 大豆苷及其代谢物大豆苷元分别在98.0~490、57.6~288 μg/ml浓度范围内线性良好,高、中、低3个浓度的相对回收率在96.6%~114.0%之间,相对标准偏差小于9.9%,表明本法的准确度和精密度符合要求。大豆苷在肠道菌液中代谢生成大豆苷元,其消除半衰期为37.6 min。结论 所建立的HPLC法适合同时测定体外大鼠肠道菌液中的大豆苷及其代谢物,大豆苷在体外代谢的主要途径为糖苷键断裂转化成对应苷元。     

HPLC法测定刺五加不同部位刺五加苷B、E含量

目的：建立HPLC法同时测定刺五加苷B和刺五加苷E含量的方法，并对刺五加不同部位的刺五加苷B和刺五加苷E进行含量测定，为合理开发、利用刺五加资源提供理论依据。方法：采用Nucleosil C18色谱柱（4．6mm×150mm，5μm），以乙腈（A）-水（B）为流动相，梯度洗脱（0～8rain：12％B；8—20min：12％B→18％B；20—25min：18％B；25min：12％B），流速0．8mL·min-1，检测波长210nm。结果：刺五加苷B在茎干中含量最高，叶中最低．束0五加苷E在根中含量最高，花中含量最低。除叶以外，其他各部位中刺五加苷E的含量均低于刺五加苷B。结论：本方法简便，重复性好，可作为刺五加药材的质量控制方法，也为合理利用刺五加这一资源提供依据。     

高效液相色谱法同时测定五加中刺五加苷B、苷E的含量

目的：测定甘肃产五加中刺五加苷B、苷E的含量。方法：高效液相色谱法,ODSKromasal柱。水：乙晴（95：5）为流动相,检测波长222nm,柱温度25℃。结果：本文可同时测定枣五加苷B、苷E的含量。刺五加苷B、苷E分别在0．064－0．320μg/ml;0.074-0.370μg/ml范围内峰面积与浓度呈线性关系,平均回收率分别为102．5％,RDS=4.2%,95.5%,RSD=4.6%。结论：剂五加苷Ｂ、苷Ｅ在红毛五加中含量最高,茎皮中含量最高,刺五加苷Ｅ的含量高于苷Ｂ。     

离体大鼠肠道菌群对野漆树苷的代谢研究

摘 要 目的：研究大鼠肠道菌群对野漆树苷的体外代谢转化作用。方法: 将野漆树苷与离体大鼠肠道菌群的孵育液在37 ℃厌氧条件下孵育,不同时间点取样经乙酸乙酯萃取后,采用HPLC-MS/MS对代谢产物进行定性和定量分析。结果: 孵育4 h后野漆树苷已被肠道菌代谢完全,在代谢组的孵育液中发现了芹菜素和柚皮素两种代谢产物,以芹菜素为主,并且在4 h达到较高浓度,随后缓慢降解,24 h后消失。结论:在离体条件下,大鼠肠道菌群对野漆树苷具有显著的代谢转化作用,提示代谢产物为其生物活性成分。     

刺五加苷B抗运动性疲劳作用的实验研究

目的研究刺五加苷B对大鼠运动性疲劳的作用。方法通过大鼠的递增强度跑台训练,建立运动疲劳模型,并测定跑台力竭时间以及血清中的BUN、CK、LDH、BUN、MDA、肌糖元、肝糖原、血糖等反映机体代谢和能量供应的指标。结果刺五加苷B能明显提高运动后大鼠体内肝糖原、肌糖原等指标,有效降低BUN、ALT、AST、LDH等指标。结论大鼠服用刺五加苷B有利于能量物质的储存和积累,加速某些导致疲劳代谢物质的清除,维持细胞的正常生理功能,延缓疲劳的发生,加快疲劳的恢复。     

高效液相色谱波长切换法同时测定微达康膏中的梓醇、刺五加苷B、刺五加苷E、淫羊藿苷和宝藿苷Ⅰ

目的建立HPLC波长切换法同时测定微达康膏中的梓醇、刺五加苷B、刺五加苷E、淫羊藿苷和宝藿苷Ⅰ的方法。方法采用Waters Nova-pak C_(18)色谱柱,以乙腈(A)-0.2%磷酸溶液(B)为流动相,梯度洗脱,流速0.9 mL·min~(-1)。结果梓醇、刺五加苷B、刺五加苷E、淫羊藿苷和宝藿苷Ⅰ分别在4.53~90.60μg·mL~(-1)、6.18~123.60μg·mL~(-1)、3.92~78.40μg·mL~(-1)、7.51~150.20μg·mL~(-1)、7.14~142.80μg·mL~(-1)与峰面积呈良好的线性关系,相关系数分别为0.9999、0.9998、1.000、0.9996、0.9999;平均加样回收率及相应的RSD分别为98.5%(1.2%)、98.9%(0.77%)、99.1%(0.90%)、97.5%(1.8%)、97.1%(0.84%)。结论本方法快速、准确度高、专属性好,可用于微达康膏的质量控制。     

二苯乙烯苷在大鼠体内外的代谢研究

采用肝微粒体温孵和大肠杆菌培养等方法对二苯乙烯苷进行体外代谢研究.灌胃给予二苯乙烯苷后,对大鼠血液、胆汁、尿液、粪便、胃和小肠内容物进行体内代谢产物分析,并通过制备液相色谱从胆汁中获得主要代谢物的纯品,采用1HNMR、13CNMR及MS方法进行结构确证.结果表明,二苯乙烯苷在大鼠肝脏代谢为葡萄糖醛酸结合物,并经胆汁排泄,在肠道内菌或酶的作用下水解为二苯乙烯苷随粪便排出.     

安石榴苷大鼠体内肠代谢产物的鉴定分析

摘要：目的:研究安石榴苷在大鼠体内代谢转化的一般规律。方法:大鼠灌胃安石榴苷原料药后收集肠内容物和粪便样品,预处理后进高效液相色谱串联四级杆飞行时间质谱(HPLC-Q-TOF/MS)系统,负离子模式下扫描,结合相关文献推测可能的代谢产物。结果:在大鼠肠内容中鉴定出的代谢产物有尿石素D、尿石素C、尿石素A、二甲基-鞣花酸、尿石素B、硫酸化尿石素A-葡萄糖苷、甲基化尿石素A和尿石素C-二葡萄糖苷。在大鼠粪便中鉴定出的代谢产物有硫酸化尿石素A、尿石素A、二甲基-鞣花酸、尿石素C-二葡萄糖苷以及鞣花酸。安石榴苷在大鼠体内代谢的主要途径有甲基化、硫酸化、葡萄糖醛酸化等。结论:安石榴苷在肠道菌群作用下代谢为尿石素系列物。     

刺五加制剂用固相萃取后以高效液相色谱法测定刺五加苷B、苷E的含量

目的用高效液相色谱法研究刺五加制剂中2个主要活性成分刺五加苷B、苷E的含量测定方法.方法 Kromasil ODS柱, 水-乙腈梯度流动相, 流速0.8 mL·min-1, 测定波长刺五加苷B 206 nm,刺五加苷E 220 nm, 水杨酸作为内标, 选择了固相萃取条件.结果刺五加片中刺五加苷B、苷E的回收率范围分别是90.4%～96.8%和87.7%～93.3%;刺五加注射液中刺五加苷B、苷E的回收率范围分别是96.4%～99.8%和95.7%～98.5%.线性范围分别是4.45～22.25 μg·mL-1(r=0.999 8)和5.11～25.55 μg·mL-1(r=0.999 7).结论该方法节约了清洗色谱系统的时间, 提高了测定的灵敏度,提供了评价刺五加制剂质量的方法.     

用液相质谱法鉴定盐酸半附甲素在大鼠胆汁中I相和Ⅱ相代谢产物

目的：研究盐酸关附甲素在大鼠胆汁中的代谢产物。方法：建立了液相质谱和串联质谱法（LC－MS）对关附甲素及其代谢产物鉴定的方法。大鼠静脉注射盐酸关附甲素后采集胆汁，通过与对照化合物的色谱保留时间、分子离子峰、碎片离子峰和紫外图谱对照从而鉴定I相代谢物。胆汁经过葡萄糖醛酸酶或硫酸酯酶水解，鉴定其水解产物（苷元），从而确定Ⅱ相结合物，再通过LC－MS分离和确定分子离子峰，最后利用MS－MS寻找特征子离子和母离子的方法进行验证。结果：大鼠胆汁中存在I相代谢物关附壬素；Ⅱ相结合物经葡萄糖醛酸酶和硫酸酯酶酶解后，出现关附甲素和关附壬素；LC－MS检测发现胆汁中m/z 606和510两个准分子离子峰，推测分别为关附甲素葡萄糖醛酸苷和关附甲素硫酸酯；经MS－MS鉴定出m/z 606特征子离子m/z 177和m/z 430，进一步确证大鼠胆汁中存在关附甲素葡萄糖醛酸苷。结论：大鼠胆汁中存在I相代谢产物关附壬素，以及Ⅱ相结合物关附甲素葡萄糖醛酸和硫酸结合物、关附壬素葡萄糖醛酸和硫酸结合物。     

高效液相色谱法同时测定五加中剌五加苷B、苷E的含量

目的测定甘肃产五加中剌五加苷B、苷E的含量。方法高效液相色谱法,ODSKromasal柱。水乙晴(955)为流动相,检测波长222nm,柱温度25℃。结果本文可同时测定剌五加苷B、苷E的含量。剌五加苷B、苷E分别在0.064～0.320μg/ml;0.074～0.370μg/ml范围内峰面积与浓度呈线性关系,平均回收率分别为102.5%,RSD=4.2%,95.5%,RSD=4.6%。结论剌五加苷B、苷E在红毛五加中含量最高;茎皮中含量最高;剌五加苷E的含量高于苷B。     

Biotransformation of the xanthine oxidase inhibitor BOF-4272 and its metabolites in the liver and by the intestinal flora in rat

1. BOF-4272, (+/-)-8-(3-methoxy-4-phenylsulfinylphenyl) pyrazolo[1,5-a]-1,3,5-triazine-4(1H)-one), is a new drug intended for the treatment of hyperuricaemia. 2. This paper describes the detailed biotransformation of BOF-4272 andits metabolites in the liver and by the intestinal flora in rat. 3. Only the sulphoxide metabolite (M6) was detected in plasma in small amounts after the intravenous administration of M4 (hydroxy-BOF-4272) and in culture medium after the addition of M4. 4. Only M3 (the sulphide metabolite of M4) was detected in faeces, and its amount was ~50% of the administered dose within 1 day after the intravenous administration of M4. 5. These findings suggest that M4, which is excreted in the bile, is metabolized mainly to M3 (the corresponding sulphide of M4) by the intestinal flora in rat, whereas little M4 is metabolized to the sulphoxide (M6) in the rat liver. 6. M2, which is the demethylated form of BOF-4269, was detected in faeces after the oral administration of BOF-4272 to rat in which the common bile duct was cannulated, suggesting that BOF-4272 is metabolized to BOF-4269 and then to M2 by the intestinal flora. 7. These findings suggest that in rat the sulphoxide of BOF-4272 and its metabolites are demethylated and reduced by the intestinal flora, with other types of biotransformation occurring in the liver.     

Biotransformation of the xanthine oxidase inhibitor BOF-4272 and its metabolites in the liver and by the intestinal flora in rat

1. BOF-4272, (+/-)-8-(3-methoxy-4-phenylsulfinylphenyl) pyrazolo[1,5-a]-1,3,5-triazine-4(1H)-one), is a new drug intended for the treatment of hyperuricaemia. 2. This paper describes the detailed biotransformation of BOF-4272 and its metabolites in the liver and by the intestinal flora in rat. 3. Only the sulphoxide metabolite (M6) was detected in plasma in small amounts after the intravenous administration of M4 (hydroxy-BOF-4272) and in culture medium after the addition of M4. 4. Only M3 (the sulphide metabolite of M4) was detected in faeces, and its amount was approximately 50% of the administered dose within 1 day after the intravenous administration of M4. 5. These findings suggest that M4, which is excreted in the bile, is metabolized mainly to M3 (the corresponding sulphide of M4) by the intestinal flora in rat, whereas little M4 is metabolized to the sulphoxide (M6) in the rat liver. 6. M2, which is the demethylated form of BOF-4269, was detected in faeces after the oral administration of BOF-4272 to rat in which the common bile duct was cannulated, suggesting that BOF-4272 is metabolized to BOF-4269 and then to M2 by the intestinal flora. 7. These findings suggest that in rat the sulphoxide of BOF-4272 and its metabolites are demethylated and reduced by the intestinal flora, with other types of biotransformation occurring in the liver.     

大鼠和人肠道菌群对苯环喹溴铵代谢转化作用的研究

目的:研究大鼠和人肠道菌群对苯环喹溴铵(BCQB)代谢的影响。方法:采用体外肠道菌群代谢方法,将大鼠或人的粪便溶解于人工肠液中,搅拌混合后过滤,滤液加入1mg·mL-1的苯环喹溴铵溶液0.5mL,于37℃厌氧培养48h,采用高效液相色谱串联质谱法对大鼠或人肠内菌温孵液样品进行分离和定性分析,并设不加BCQB溶液的大鼠或人肠内菌温孵液为空白样品。结果:与空白样品比较,大鼠或人肠内菌温孵液样品中总离子流色谱中除BCQB峰外未见新增色谱峰,其质谱碎片信息显示只有BCQB的特征碎片离子,未检测到BCQB的代谢产物。结论:在离体条件下,BCQB不被大鼠和人的肠道菌群代谢。     

离体培养人肠道菌群对黄山药总皂苷的代谢研究

目的通过离体实验观察人肠道内细菌对黄山药总皂苷的代谢。方法离体培养人肠道内细菌，与黄山药总皂苷厌氧温孵，薄层色谱检测黄山药总皂苷的代谢，采用硅胶柱色谱对黄山药总皂苷肠道代谢产物进行分离和代谢转化产物的结构鉴定。结果代谢产物经IR、EI—MS、DEPT和NMR鉴定为，25（R）-螺甾-5-烯-3β，20（S）-二醇，此化合物为新的天然产物。结论体外模拟的人肠道菌群对黄山药总皂苷有代谢作用，其中转化出的新成分是不是起药理活性的真正成分，有待进一步确证。     

Identification of a novel dihydroxy metabolite of aflatoxin B1 produced in vitro and in vivo in rats and mice

HPLC analysis of bile obtained from rats given aflatoxin B1 (AFB) demonstrates the presence of numerous polar metabolites. The glutathione conjugate derived from AFB 8,9-epoxide and the glucuronide conjugate of aflatoxin P1 (AFP) have previously been identified as the two major polar metabolites. The most polar peak present in bile from AFB-treated rats is converted to a less polar peak upon incubation with beta-glucuronidase, which has a parent ion m/e of 314 amu. Treatment of this aglycon with diazomethane produced a product which cochromatographs with aflatoxin M1 (AFM). From these data it is concluded that the most polar peak in bile from AFB-treated rats is the glucuronide conjugate of 4,9a-dihydroxyaflatoxin B1. This dihydroxy AFB metabolite was produced in vitro in mouse microsomal incubations, and time-course studies of its production suggest that it is largely formed by 9a-hydroxylation of AFP, although some may be formed by 4-O-demethylation of AFM. Direct incubation of AFP and AFM with mouse microsomes confirmed that this metabolite can be formed from both AFP and AFM. An HPLC method is described which is capable of base line resolution of this novel dihydroxyaflatoxin metabolite and eight other hydroxylated metabolites of AFB, as well as the glutathione conjugate of AFB 8,9-epoxide.     

Mass spectrometric and NMR characterization of metabolites of roxifiban, a potent and selective antagonist of the platelet glycoprotein IIb/IIIa receptor

1. The methyl ester prodrug roxifiban is an orally active, potent and selective antagonist of the platelet glycoprotein GPIIb/IIIa receptor and is being developed for the prevention and treatment of arterial thrombosis. 2. Roxifiban was rapidly hydrolyzed to the zwitterion XV459 in vivo and by liver slices from the rat, mouse and human and by intestinal cores from dog. XV459 was metabolized to only a small extent in vitro and in vivo. 3. Studies with rat and dog given radiolabelled roxifiban showed limited oral absorption with the majority of the radiolabel being excreted in faeces. After i.v. doses of 14C-roxifiban, most of the radioactivity was recovered in the urine of rat whereas the dog excreted significant amounts of radioactivity in bile and urine. 4. XV459 could be metabolized extrahepatically by dog gut flora to produce an isoxazoline ring-opened metabolite. In vitro hepatic metabolism of XV459 was mainly by hydroxylation at the prochiral and chiral centres of the isoxazoline ring. These hydroxylated metabolites were not detected in the urine and plasma of human volunteers administered roxifiban. 5. Initial LC/MS identification of metabolites was achieved by dosing the rat with an equimolar mixture of d0:d4 roxifiban and detecting isotopic clusters of pseudomolecular ions. Unequivocal characterization of these metabolites was achieved by LC/MS, LC/NMR and high-field NMR techniques using synthetic standards of the metabolites. 6. The synthesis of one hydroxylated metabolite enabled the assignment of the correct stereochemistry of the substituted hydroxyl group on the isoxazoline ring.     

Metabolism of vesatolimod in rat,dog, and human liver microsomes: Metabolic stability assessment,metabolite identification,and interspecies comparison

Vesatolimod (GS‐9620) is an agonist of toll‐like receptor (TLR7) 7, which has been developed as an anti‐hepatitis B virus (HBV) agent. The focus of the present study is on the metabolic stability evaluation and metabolite identification of GS‐9620 in rat, dog, and human liver microsomes, as well as interspecies comparison. The average observed in vitro T_1/2 values were 3.06, 13.06, and 15.56 minutes in rat, dog, and human liver microsomes, respectively. The findings suggested that GS‐9620 was rapidly metabolized in the presence of reductive nicotinamide adenine dinucleotide phosphate (NADPH) in rat liver microsomes (RLM), and moderately metabolized in dog liver microsomes (DLM) and human liver microsomes (HLM). Subsequently, the metabolites were characterized using an ultra‐high performance liquid chromatography coupled with linear ion trap orbitrap tandem mass spectrometer (UHPLC–LTQ–Orbitrap–MS) with dd‐MS² on‐line data acquisition mode. Under the current conditions, a total of 18 metabolites were detected and their identities were proposed by comparing their accurate masses, fragmental ions, and retention times with those of GS‐9620. Three metabolites (M2, M4, and M18) were authentically identified by using reference standards. In RLM, 16 metabolites were identified with M2 being the most abundant metabolite. M4, M5, and M9 were rat‐specific. In DLM, 12 minor metabolites were identified with a dog‐specific metabolite (M6). In HLM, GS‐9620 showed similar metabolic profiles to that in DLM, and 11 minor metabolites were detected with M12 being human‐specific. Based on the identified metabolites, the metabolic pathways of GS‐9620 were proposed, including hydroxylation, bis‐hydroxylation, dehydrogenation, and oxidative deamination.     

大鼠肠内菌转化连翘苷的代谢产物

应用液相色谱-串联质谱法研究肠内菌转化连翘苷的代谢产物.将连翘苷与大鼠肠内菌于体外厌氧温孵培养,在温孵液中检测到连翘苷及其3种代谢产物.放大制备了转化产率高的代谢物,依据1H NMR和ESI-MS数据推测代谢物结构并假设了连翘苷可能的代谢途径.     

Identification of in‐vivo and in‐vitro metabolites of palmatine by liquid chromatography–tandem mass spectrometry

Objectives Despite its important therapeutic value, the metabolism of palmatine is not yet clear. Our objective was to investigate its in‐vivo and in‐vitro metabolism. Methods Liquid chromatography–tandem electrospray ionization mass spectrometry (LC‐ESI/MSⁿ) was employed in this work. In‐vivo samples, including faeces, urine and plasma of rats, were collected after oral administration of palmatine (20 mg/kg) to rats. In‐vitro samples were prepared by incubating palmatine with intestinal flora and liver microsome of rats, respectively. All the samples were purified via a C₁₈ solid‐phase extraction procedure, then chromatographically separated by a reverse‐phase C₁₈ column with methanol–formic acid aqueous solution (pH 3.5, 70: 30 v/v) as mobile phase, and detected by an on‐line MSⁿ detector. The structure of each metabolite was elucidated by comparing its molecular weight, retention time and full‐scan MSⁿ spectra with those of the parent drug. Key findings The results revealed that 12 metabolites were present in rat faeces, 13 metabolites in rat urine, 7 metabolites in rat plasma, 10 metabolites in rat intestinal flora and 9 metabolites in rat liver microsomes. Except for six of the metabolites in rat urine, the other in‐vivo and in‐vitro metabolites were reported for the first time. Conclusions Seven new metabolites of palmatine (tri‐hydroxyl palmatine, di‐demethoxyl palmatine, tri‐demethyl palmatine, mono‐demethoxyl dehydrogen palmatine, di‐demethoxyl dehydrogen palmatine, mono‐demethyl dehydrogen palmatine, tri‐demethyl dehydrogen palmatine) were reported in this work.