HPLC-ECD法测定大鼠脑微透析液中的多巴胺及其代谢产物

目的:建立应用常规高效液相-电化学法(HPLC-ECD)测定大鼠脑微透析液中多巴胺(DA)及其代谢产物3,4-二羟基苯乙酸(DOPAC)和高香草酸(HVA)的方法。方法:将探针插入大鼠右侧纹状体,在清醒自由活动状态下,用Ringer氏液以1.5μl.min-1的速度灌流,每20 min收集1管透析液,将其注入HPLC-ECD,考察本方法的专属性、线性范围、精密度和准确度等,并对其中所含的DA、DOPAC、HVA进行测定。结果:3种物质在12.5~250μg.L-1范围内线性关系良好,高、中、低3种浓度的回收率分别为98.33%、102.67%、92.33%,各物质的日内RSD值分别为3.3%、3.4%、2.5%;日间RSD值为4.2%、2.3%、5.6%。大鼠脑微透析液中的DOPAC及DA、HVA的含量分别为1.79±0.07、0.45±0.02、1.67±0.05μg.ml-1(n=6)。结论:本方法简便、准确、稳定,可用于应用脑微透析技术进行的DA相关疾病的基础研究。     

Cytoprotective role of taurine in a renal epithelial cell culture model

Taurine (TAU) is a sulfur-containing amino acid that has been shown to decrease during aging and is believed to be important for cytoprotection. A decrease in TAU could exacerbate the accumulation of free radical-induced damage that may lead to cell death during the aging process. We have shown previously that TAU directly inhibits dopamine (DA) and (-)-3-(3,4-dihydroxyphenyl)-L-alanine (L-dopa) oxidation. Experiments were conducted to establish a cytoprotective role for TAU. Porcine renal epithelial cells were treated for 1 hr with iron and catecholamines (L-dopa and DA) to produce cytotoxicity by a free radical and quinone mechanism in the absence and presence of 10 or 20mM TAU. Viability assays, protein, and DNA measurements were performed after a 24hr recovery period. In some experiments, cells were extracted immediately after the insult for DA and TAU content measurements using high performance liquid chromatography with electrochemical detection. Catecholamine-induced cytotoxicity caused a 50% loss in cell viability, and 10 or 20mM TAU provided significant protection from cytotoxicity and maintained the functional integrity of the cells. Photomicrographs showed attenuation in cell loss and swelling in the presence of TAU. Pretreatment with 1mM TAU followed by exposure to iron and L-dopa in the presence of 1mM TAU caused a moderate but non-significant increase in cell survival. These data conclusively show that TAU can play a cytoprotective role in the LLC-PK(1) cell culture model.     

促性腺激素释放激素类似物对大鼠胃肠道5-羟色胺分泌的影响

目的 研究GnRH对胃肠道内5-HT分泌的影响。方法胃腔直接注射GnRH类似物阿拉瑞林(A1arelin GnRH—A)以模拟外分泌产生的GnRH，并以免疫组织化学、高压液相色谱电化学检测(HPLC-ECD)的方法对阿拉瑞林刺激后胃及十二指肠内5-HT免疫反应细胞、血清中5-HT含量进行检测。结果 胃腔内注射GnRH—A后，大鼠胃及十二指肠内5-HT免疫反应阳性细胞密度显著增多，但血清中5-HT含量显著减少。结论 外分泌的OnRH对于5-HT的释放起明显抑制作用，但对5-HT的合成可能不产生影响。     

高效液相色谱-电化学检测法测定生物样品中单胺类神经递质及其代谢产物*

[目的]建立一种简单、快速、灵敏的测定生物样品中单胺类神经递质及其代谢产物的高效液相色谱方法.[方法]色谱柱采用Waters C18反相色谱柱(3.9 mm×150 mm,5 μm),流动相为水相(每500 mL中含辛烷磺酸钠1.25 g,磷酸二氢钾6.8 g,乙二胺四乙酸18 mg,pH值3.3)∶甲醇∶乙腈=78∶19∶3,流速为0.8 mL/min,柱温35 ℃;电化学检测器工作电极为玻璃碳电极,参比电极为银/氯化银(Ag/AgCl)电极,工作电压为0.7 V.[结果]5种物质保留时间分别为去甲肾上腺素3.6 min,5-羟吲哚乙酸4.5 min,高香草酸5.2 min,多巴胺6.2 min,5-羟色胺11.9 min;线性范围均为20-1 000 μg/L,最低检测限为4～8 μg/L.[结论]此方法测定生物样品中的单胺类神经递质及其代谢产物,简便,快速且灵敏度高.     

高效液相色谱-电化学检测法测定黄芩中3种有效成分的含量

目的:建立同时测定黄芩中有效成分黄芩苷、黄芩素、汉黄芩素含量的HPLC-ECD检测法。方法:采用kromasil-C18色谱柱(4.6 mm×250 mm,5μm)进行分离,Waters2465电化学检测器进行检测,检测电位为+800 mVvs.ISAAC(in-situ silver/silver chloride),流动相为H₂O-THF-H₃PO₄(80∶10∶0.2)与CH₃OH等体积混合洗脱,流速1.0 mL.min^-1,柱温30℃,进样体积10μL。结果与结论:黄芩苷、黄芩素和汉黄芩素分别在下列范围内,有良好的线性:0.045 6～1.14μg(r=0.999 6),0.013～0.325μg(r=1.000 0),和0.009 5～0.047 5μg(r=0.999 9)。平均回收率(n=5)分别为97.9%(RSD 2.3%),97.2%(RSD 3.3%)和103.5%(RSD 3.5%)。测定了14个不同种源、9个不同产地黄芩中有效成分的含量,对黄芩品质进行了评价。     

8-Hydroxydeoxyguanosine levels in DNA of human breast cancers are not significantly different from those of non-cancerous breast tissues by the HPLC-ECD method

8-Hydroxydeoxyguanosine (oh⁸dG) is a promutagenic DNA lesion produced by oxygen radicals, and a high level of 8-hydroxyguanine in breast cancers was previously demonstrated by the gas chromatography-mass-spectrometry method. To confirm the previous observation, the oh⁸dG levels of DNA of 22 breast cancers and corresponding adjacent non-cancerous breast tissues were analyzed by high performance liquid chromatography-electrochemical detector (HPLC-ECD) system, and the correlation of the oh⁸dG levels in breast cancer DNAs with clinical and immunohistochemical parameters was examined. However, the levels of oh⁸dG in DNA of breast cancers are not significantly different from those of corresponding non-cancerous breast tissues (P = 0.084) by the HPLC-ECD method. Furthermore, the oh⁸dG levels in breast cancers were not associated with p53 and erbB-2 immunoreactions, with expression of estrogen and progesterone receptors, and with clinical stage and histological grade. Thus, in contrast to the previous data, the present study using the HPLC-ECD method does not indicate an increase of oh⁸dG levels in breast cancers.     

Antidepressant-like behavioral, neurochemical and neuroendocrine effects of naringenin in the mouse repeated tail suspension test

Our previous study demonstrated that the citrus bioflavonoid naringenin ameliorated behavioral alterations via the central serotonergic and noradrenergic systems in the tail suspension test (TST) induced mice. To better understand its pharmacological activity, mice were submitted to three 6min-TSTs one week apart (Day 1: test, Day 7: retest 1, Day 14: retest 2) followed by hippocampal glucocorticoid receptor (GR), monoamine neurotransmitters and serum corticosterone measurement. The results suggested that repeated TST detected the gradual increase in the efficacy of naringenin over time, additionally 1-day (20mg/kg), 7-day (10, 20mg/kg) and 14-day (5, 10, 20mg/kg) naringenin treatment markedly decreased the immobility time. Moreover, administration of naringenin for 14days (20mg/kg) increased hippocampal serotonin (5-HT), norepinephrine (NE) and GR levels, and reduced serum corticosterone levels in mice exposed to the repeated TST. Overall, the present study indicated that the re-exposure would facilitate the detection of the anti-immobility effects of antidepressant drugs in the mouse TST, and clearly demonstrated that the antidepressant-like effect of naringenin may be mediated by an interaction with neuroendocrine and neurochemical systems.     

HPLC-ECD法测定盐酸大观霉素有关物质

目的:建立HPLC-ECD法测定盐酸大观霉素有关物质。方法:应用高效液相色谱-电化学检测器,Waters XBridge C18色谱柱(4.6 mm×250 mm,5μm),以pH 3.2的草酸盐缓冲溶液-乙腈(1000∶105)为流动相,流速为1.0 mL.min-1;柱后溶液为21 g.L-1的氢氧化钠溶液,流速0.5 mL.min-1;ECD系统参数:极性为阳性,检波+0.12 V,氧化电位+0.70 V,还原电位-0.60 V,范围20μA。结果:盐酸大观霉素有关物质在2.25～45μg.mL-1范围内呈良好的线性关系,r=0.9999;Y轴截距在100%浓度响应值的7.3%处,在100%浓度的25%～500%的范围内,对任一点的响应值在此点理论值的97.7%至103.6%的范围内;平均回收率(n=9)为98.9%,RSD为2.9%。结论:该方法简便易行,准确度高,重复性好,可用于盐酸大观霉素及相关制剂产品的质量控制。     

松果菊苷对脑缺血大鼠纹状体细胞外液中单胺类神经递质的影响

目的研究松果菊苷(ECH)对局灶型脑缺血大鼠纹状体细胞外液中单胺类递质的影响,以探讨ECH对脑神经保护作用的可能机制。方法 SD大鼠随机分为对照组、模型组、ECH高、低剂量组和川芎嗪(CXQ)组。各组大鼠给予相应的药物或生理盐水腹腔注射,每天1次,连续7 d。给药第3天,脑纹状体埋置探针套管,末次给药1 h后,制作大鼠局灶性脑缺血模型(MCAO),造模后立刻进行微透析,将透析液注入高效液相-电化学检测器(HPLC-ECD)测定各组纹状体细胞外液中去甲肾上腺素(NE)、多巴胺(DA)、5-羟色胺(5-HT)、3,4-二羟苯乙酸(DOPAC)、高香草酸(HVA)、5-羟吲哚乙酸(HIAA)的含量。结果与对照组相比,模型组NE、DA、5-HT水平升高,其酸性代谢产物DOPAC,HVA,HIAA也随之升高。与模型组比较,ECH高、低剂量组(30、15 mg.kg-1.d-1)和CXQ组的6种物质的含量均有所降低。结论 ECH的神经保护作用可能与对抗脑缺血后单胺类神经递质的升高有关。     

HPLC-ECD法测定大鼠血浆中银杏叶黄酮成分及药代动力学研究

目的:建立大鼠灌胃银杏叶提取物后血浆中黄酮类成分的灵敏分析方法,并进行药代动力学研究。方法:血浆样品经葡萄糖醛酸苷水解酶水解后,采用高效液相色谱分离及电化学检测,同时分析大鼠血浆样品中槲皮素、异鼠李素和山柰酚。色谱柱为Agilent Zorbax Ec lipse XDB-C8(150 mm×4.6 mm,5μm),流动相为甲醇-异丙醇-磷酸盐缓冲液(磷酸二氢钠-磷酸,pH=2)(15?15?70),流速0.8 mL.m in-1,检测电压为200 mV,柱温30℃。结果:槲皮素、异鼠李素和山柰酚与其他内源性和药源性成分达到良好分离;最低检测浓度达到0.5 ng.mL-1;平均提取回收率分别为88.7%,86.2%,87.0%;准确度在92.7%~105.3%之间,日内精密度RSD小于6.6%,日间精密度RSD小于14.1%,样品分析时间为20 m in。大鼠灌胃给予银杏叶提取物10 mg.kg-1后,酶水解血浆中槲皮素、异鼠李素和山柰酚的半衰期分别为(3.53±1.88)h,(6.94±4.05)h,(3.97±2.30)h;3个黄酮类成分在大鼠血浆中都显示明显的二次达峰现象。结论:建立的检测方法能灵敏、特...