HPLC法测定葛根滴丸中葛根素的含量

目的：建立葛根滴丸中葛根素含量的测定方法。方法：以30％乙醇溶液超声溶解葛根滴丸中葛根素，采用高效液相色谱法（HPLC）外标法测定葛根素含量，其中固定相为Kromasil C18流动相为乙腈：水=10：90，UV检测波长为250nm，流速为1mL／min，柱温为室温。结果：葛根素溶液浓度范围在10—200μg／mL时与其吸收值呈良好线性关系（r=0.9998），平均回收率为100．76％，RSD=1．12％。结论：该法具有良好的准确性、精密性，方法简便，结果可靠。     

HPLC determination of five polyphenols in rat plasma after intravenous administration of hawthorn leaves extract and its application to pharmacokinetic study

A simple and specific HPLC-UV method was developed to simultaneously determine five active compounds including vitexin-4"-O-glucoside (VG), vitexin-2"-O-rhamnoside (VR), vitexin (VIT), rutin (RUT) and hyperoside (HP) in rat plasma after intravenous administrating the hawthorn leaves extract (HLE). With baicalin as internal standard (I.S.), sample pretreatment involved a one-step extraction with methanol of 0.2 ml plasma. The HPLC assay was carried out using a Phenomsil C18 analytical column with UV detection at 332 nm. The mobile phase consisted of methanol-acetonitrile-tetrahydrofuran-1% glacial acetic acid (6:1.5:18.5:74, v/v/v/v). The calibration curves were liner over the range of 2.030-500.5, 0.1513-75.64, 0.2507-12.54, 0.5128-25.64 and 0.4032-20.16 μg/ml for VG, VR, VIT, RUT and HP, respectively. The relative standard deviations (RSD) of the intra- and inter-day precisions for the analysis of the five analytes were between 1.0 and 8.9% with accuracies (relative error) below 8.2% for the analysis of the five analytes. The average extraction recoveries of five analytes were more than 82.67 ± 4.74%. The HPLC method herein described was fully validated and successfully applied to the pharmacokinetic studies after intravenous administration of HLE solution to rats over three doses.     

The antidepressant effect of ethanol extract of radix puerariae in mice exposed to cerebral ischemia reperfusion

In our pilot study, the depressive-like behaviors of mice exposed to cerebral ischemia reperfusion (CIR) were observed and the antidepressant effects of radix puerariae (RP; root of the Pueraria plant) extract in CIR mice were assessed because it was speculated that the neuronal damage caused by CIR played an important role in the development of poststroke depression (a common and severe complication after stroke) and the RP extract was reported to exhibit effect of neuronal protection from cerebral ischemia damage. Our studies above indicated that the RP extract markedly shortened the increased immobility time induced by CIR of male mice in the forced swimming test (FST) and tail suspension test (TST), indicating a possible antidepressant activity. Thus, the aim of the present study was to confirm the putative antidepressant effect of RP extract (75, 150, and 300 mg/kg, administered orally 24 h after the CIR) on reserpine-induced symptoms. To get further insight into the mode of antidepressant action of RP extract, biochemical examination was conducted concomitantly to examine possible involvement of the brain monoamine systems in the behavioral syndromes observed. In CIR mice, pronounced low levels of norepinephrine (NE) and 4-dihydroxyphenylacetic acid (DOPAC, a metabolite of dopamine) in the hippocampus or striatum were detected, which were reversed by RP extract, whereas no significant change of serotonin (5-HT) was detected in either CIR or RP extract-treated mice. The data suggested that the disturbance of NE and DA systems in hippocampus and striatum played more important roles in the development of depressive-like behavior of CIR mice than 5-HT system did, and RP extract ameliorated the abnormal symptoms caused by CIR, which may throw new lights on the treatment of poststroke depression.     

葛根口服液的工艺研究及葛根素的测定

目的对以葛根素为功效／指标性成分的嘲艮液进行工艺研究及其含量测定。方法口服液考察提取和精制工艺,含测采用HPLC。结果水提提取率可达90％,正交试验优选最佳提取条件为：固液比为1：8,提取次数2次,每次提取时间为2ho醇沉、壳聚糖澄清效果优于明胶和ZTC1＋1。HPLC测定中前处理操作简便。结论葛根口服液水提、精制工艺可满足要求,稳定。     

High-performance liquid chromatographic determination and pharmacokinetic study of vitexin-2'-O-rhamnoside in rat plasma after intravenous administration

A simple and specific high-performance liquid chromatographic (HPLC) method was developed for the pharmacokinetic study of vitexin-2'-O-rhamnoside (VOR) in rat after intravenous administration. The plasma samples were deproteinized with methanol after addition of internal standard (i.s.) hesperidin. HPLC analysis was performed on a Diamonsil ODS C18 analytical column, using acetonitrile-0.3% formic acid (20:80, v/v) as the mobile phase with UV detection at 270 nm. The standard curve was linear over the range of 0.1070-21.41 microg/mL in rat plasma. The average extraction recovery of VOR was 97.9+/-3.1%, and the relative standard deviations (R.S.D.s) of the intra- and inter-day precisions were no more than 7.4 and 8.5%, respectively. The lower limit of quantification (LLOQ) was 0.1070 microg/mL. The AUC of VOR was proportional to the dose after intravenous administration of 15, 30, 60 and 120 mg/kg body weight, and the elimination half-life (t1/2beta), systemic clearance (Cl) and apparent volume of distribution (Vc) were not significantly different among the four doses, and all the results indicated that the pharmacokinetics of VOR in rat obeyed first-order kinetics.     

HPLC method for the determination of fluvoxamine in human plasma and urine for application to pharmacokinetic studies

A simple, specific and sensitive high-performance liquid chromatographic (HPLC) method has been developed for the assay of fluvoxamine in human plasma and urine. The method was based on reaction of fluvoxamine with 1,2-naphthoquinone-4-sulphonic acid sodium salt (NQS) forming orange colored product. The fluvoxamine-NQ derivative was separated by isocratic reversed-phase HPLC and detected at 450 nm. The chromatographic conditions were as follows: Phenomenex C(18) (250 mm x 4.6 mm i.d., 5 microm) column, mobile phase consisting of acetonitrile/water (80:20 v/v) at a flow rate of 1 ml/min. Tryptamine was selected as an internal standard. The assay was linear over the concentration range of 5-145 and 2-100 ng/ml for plasma and urine, respectively. The limits of detection (LOD) were 1.4 and 1 ng/ml for plasma and urine estimation at a signal-to-noise (S/N) ratio of 3. The limits of quantification (LOQ) were 5 and 2 ng/ml for plasma and urine, respectively. The extraction recoveries were found to be 96.66+/-0.69 and 96.73+/-2.17% for plasma and urine, respectively. The intra-day and inter-day standard deviations (S.D.) were less than 1. The method indicated good performance in terms of specificity, linearity, detection and quantification limits, precision and accuracy. This assay was demonstrated to be applicable for clinical pharmacokinetic studies.     

葛根素微乳灌胃后大鼠血浆中葛根素的测定

目的建立反相高效液相色谱法测定大鼠血浆中的葛根素,用于葛根素微乳在大鼠体内的生物利用度研究。方法取血浆样品,用乙腈沉淀蛋白,Phenomenex C18柱（250 mm×4.6 mm,5μm）分离,4-羟基苯甲醛为内标,乙腈-0.1%醋酸溶液（23∶77）为流动相,流速0.8 mL.min-1,紫外检测波长250 nm,柱温30℃,进样量20μL。结果葛根素的线性范围为0.10～10.00μg·mL-1（r=0.999 6）,定量下限为0.10μg.mL-1,提取回收率〉79%,方法回收率〉92%,日内、日间精密度RSD〈6%。结论本方法灵敏度好、准确性高,可用于大鼠血浆中葛根素浓度的测定以及葛根素微乳在大鼠体内的生物利用度研究。     

Validated HPLC method for determination of amlodipine in human plasma and its application to pharmacokinetic studies

A rapid, simple and sensitive high-performance liquid chromatography (HPLC) method has been developed for quantification of amlodipine in plasma. The assay enables the measurement of amlodipine for therapeutic drug monitoring with a minimum detectable limit of 0.2 ng ml(-1). The method involves simple, one-step extraction procedure and analytical recovery was about 97%. The separation was performed on an analytical 125 x 4.6 mm i.d. Nucleosil C8 column. The wavelength was set at 239 nm. The mobile phase was a mixture of 0.01 M sodium dihydrogen phosphate buffer and acetonitrile (63:37, v/v) adjusted to pH 3.5 at a flow rate of 1.5 ml min(-1). The calibration curve was linear over the concentration range 0.5-16 ng ml(-1). The coefficients of variation for inter-day and intra-day assay were found to be less than 10%.     

葛根素对大鼠脑缺血再灌注损伤后Fas蛋白表达的影响

目的探讨葛根素对大鼠脑缺血再灌注损伤后Fas蛋白表达的影响。方法采用闭夹大脑中动脉后再通造成局部脑缺血再灌注模型,免疫组化SP法测定Fas表达情况,光镜观察脑组织Fas蛋白阳性细胞数和死亡细胞数。结果正常对照组Fas阳性细胞数及死亡细胞数明显少于再灌注组,再灌注葛根素组死亡细胞数和Fas阳性细胞数明显少于再灌注对照组(P均<0.01)。结论葛根素对大鼠急性脑缺血再灌注损伤具有保护作用。     

高效液相色谱法测定葛根总黄酮及制剂中葛根素的含量

以反相高效液相色谱法（ＲＰ－ＲＰＬＣ）测定葛根总黄酮提取物及制剂中葛根素的含量。检测波长２５０ｎｍ，流动相为甲醇－水（２５：７５），进样２０μｌ采用外标法。当葛根素浓度在０．５－１２μｇ／ｍｌ时，浓度与峰面积呈良好线性关系，ｒ＝０．９９９１。回收率及ＲＳＤ分别为１００．９％，１．２８％。     

CBN中葛根素、人参皂苷Rg1的高效液相色谱测定法及其在脑缺血再灌大鼠体内的药代动力学

ＣＢＮ(商品名 :金森脑泰 )是由我所独立研制成功的一个中药二类新药 ,是由葛根黄酮、三七皂苷等有效部分按一定比例配伍的复方中药注射剂 ,临床用于治疗缺血性脑卒中(脑血栓形成 )。药理实验表明 ,该药能治疗局部及全脑缺血所致的脑组织损伤 ,减少脑坏死面积 ;降低缺血脑组织中的ＭＤＡ生成 ,提高脑组织耐缺氧能力 ;明显增加小鼠脑膜血流量 ;直接保护神经元 ;对凝血过程的各个阶段有不同程度的抑制[1] 。为给ＣＢＮ的临床使用药提供借鉴 ,我们从葛根黄酮中选择葛根素 (ｐｕｅｒａｒｉｎ ,Ｐｕｒ) ,三七皂苷中选择Ｒｇ1作为指标成分 ,研究复…     

SPE-HPLC method for the determination and pharmacokinetic studies on paeoniflorin in rat serum after oral administration of traditional Chinese medicinal preparation Guan-Xin-Er-Hao decoction

A new HPLC method for the determination of paeoniflorin in rat serum with solid-phase extraction (SPE) for preconcentration is introduced. Paeoniflorin and an internal standard (pentoxifylline) were extracted from serum by means of SPE using cartridges with octadecyl chemically bound phase. The HPLC separation was then performed on a reversed-phase C(18) column using acetonitrile-water (18:82, v/v) as eluting solvent system, and UV detection at 230 nm to measure the analyte with a limit of quantitation about 10 ng ml(-1). The calibration curve for paeoniflorin was linear (r=0.9938) in the concentration range of 10-1200 ng ml(-1), both intra- and inter-day precision of the paeoniflorin were determined and their coefficience of variation did not exceed 10%. The validated method has been successfully applied for pharmacokinetic studies of paeoniflorin from rat serum after oral administration of Guan-Xin-Er-Hao decoction.     

高效液相法测定松果菊苷在脑缺血大鼠血浆和脑组织中的浓度

<正>松果菊苷(echinacoside,ECH)是从新疆特色植物管花肉苁蓉中分离、纯化的一种苯乙醇苷类有效物质。研究表明[1-3],ECH具有较好的神经保护的作用。一种药物只有通过血脑屏障(blood brain barrier,BBB)并在中枢保持有效的浓度时,才能发挥药理作用。实验检测发现脑内含有原形ECH,推测ECH的中枢作用可能与其能透过BBB有关。用     

LC-MS determination and pharmacokinetic studies of ursolic acid in rat plasma after administration of the traditional chinese medicinal preparation Lu-Ying extract

Sambucus chinensis L. is a native perennial herb distributed throughout China. In traditional Chinese medicine (TCM), this herb is known as Lu-Ying. Ursolic acid is the major effective constituent of Lu-Ying. A rapid, sensitive, and accurate liquid chromatography-mass spectrometry (LC-MS) method for the determination of ursolic acid in rat plasma was developed and validated. Plasma samples taken from rats that had received Lu-Ying extract orally were acidified with acetic acid and then extracted with a mixture of hexane-dichloromethane-2-propanol (20:10:1, v/v/v). Separation of ursolic acid was accomplished on a C(18) column interfaced with a single quadrupole mass spectrometer. The mobile phase consisting of methanol and water (95:5, v/v) was delivered at a flow rate of 1.0 ml/min. Atmospheric pressure chemical ionization was operated in negative-ion mode. Using selected ion-monitoring mode, the deprotonated molecules [M-H](-) at m/z 455 and 469 were used to quantify ursolic acid and glycyrrhetic acid (internal standard), respectively. The assay was shown to be linear over the range of 10-1000 ng/ml (r> or =0.9960) with a lower limit of quantification of 10 ng/ml. The method was shown to be reproducible and reliable with intraday precision below 7.8%, interday precision below 8.1%, accuracy within +/-4.3%, and mean extraction recovery excess of 83.6%, which were all calculated from the blank plasma sample spiked with ursolic acid at three concentrations of 20, 200, and 800 ng/ml. The LC-MS method has been successfully applied to pharmacokinetic studies of ursolic acid after oral administration of Lu-Ying ethanolic extract (at a dose containing 80.32 mg/kg ursolic acid) to rats. The main pharmacokinetic parameters were: t(1/2), 4.3 h; K(e), 0.16 1/h; t(max), 1.0 h; C(max), 294.8 ng/ml; AUC(0-t) and AUC(0-infinity), 1007.1 ng.h/ml and 1175.3 ng.h/ml, respectively.     

鼠急性全脑缺血再灌注后葛根素对海马CA1区Bcl-2、Bax表达的影响

目的　观察全脑缺血再灌注后葛根素对神经细胞凋亡相关基因Bcl 2、Bax表达的影响。方法　采用免疫组织化学法、原位末端标记法检测大鼠全脑缺血再灌注不同时间内海马CA1区Bcl 2和Bax蛋白表达水平及凋亡细胞数的变化。结果　①脑缺血再灌注后 ,海马CA1区Bcl 2蛋白的表达随再灌注时间不同而变化 ,缺血 10min后再灌注 6h达高峰 ;葛根素治疗组Bcl 2蛋白的表达于相应的时间点明显增加 ;②Bax蛋白表达在再灌注 2 4h达高峰 ,葛根素治疗组Bax蛋白表达在相应时间点则下降 ;③神经细胞凋亡数随再灌注时间延长而增加 ,葛根素可减少神经细胞凋亡数。结论　在全脑缺血再灌注后细胞凋亡中 ,Bcl 2、Bax发挥重要作用 ;葛根素通过上调Bcl 2蛋白的表达 ,下调Bax蛋白的表达抑制细胞凋亡发挥神经保护作用     

头孢克洛血药浓度测定及其药代动力学研究

目的建立高效液相色谱法测定人血浆中头孢克洛浓度的方法。方法以头孢拉定为内标,采用DIS-COVERYC18色谱柱(4.6mm×250mm,5μm),以醋酸盐缓冲液-乙腈(83∶17)为流动相,流速:1mL/min;检测波长264nm。结果头孢克洛的线性范围为0.2～30μg/mL,回归方程为Y=0.0913X-0.0094,r=0.9996,最低检测浓度为0.1μg/mL,日内和日间RSD均小于7.5%。结论此法准确简便,适用于头孢克洛药代动力学的研究。     

AMS method validation for quantitation in pharmacokinetic studies with concomitant extravascular and intravenous administration

A technique has emerged in the past few years that has enabled a drug's intravenous pharmacokinetics to be readily obtained in humans without having to conduct extensive toxicology studies by this route of administration or expand protracted effort in formulation. The technique involves the intravenous administration of a low dose of (14)C-labelled drug (termed a tracer dose) concomitantly with a non-labelled extravascular dose given at therapeutically levels. Plasma samples collected over time are analysed to determine the total parent drug concentration by LC-MS (which essentially measures that arising from the oral dose) and by LC followed by accelerator mass spectrometry (AMS) to determine the (14)C-drug concentration (i.e., that arising from the intravenous dose). There are currently no published accounts of how the principles of bioanalytical validation might be applied to intravenous studies using AMS as an analytical technique. The authors describe the primary elements of AMS when used with LC separation and how this off-line technique differs from LC-MS. They then discuss how the principles of bioanalytical validation might be applied to determine selectivity, accuracy, precision and stability of methods involving LC followed by AMS analysis.     

离子交换纤维分离纯化葛根素的研究

目的:研究用离子交换纤维分离纯化葛根素的方法和工艺.方法:将葛根的浸提液在预处理过的纤维上吸附过瓣,再用70%乙醇洗脱,得纯化的葛根素.结果:最佳浸提葛根素的工艺参数:60%的乙醇浸提液pH=8,浸提温度60℃,提取时间3 h,浸提2次.得到的葛根素粗品收率为10.92%,纯度为4.56%.最佳分离纯化葛根素的工艺参数:在上柱药液pH=9,流速3 BV·h-1,药液浓度为1.703 mg·mL-1,体积为50 mL的条件下,解吸剂为70%乙醇和2 mmol·mL-1乙酸的混合液(体积比4:1)4 BV,洗脱流速6 BV·h-1.葛根素经离子交换纤维分离提纯后,产品纯度由4.56%提高到23.25%,产品总收率由10.92%降到2.89%.离子交换纤维对葛根素的吸附率为95.52%,解吸率为96.29%.结论:离子交换纤维分离纯化葛根素方法可行.     

simultaneous determination of mycophenolic acid and its metabolites by HPLC and pharmacokinetic studies in rat plasma and bile

In this study, we determined the pharmacokinetics of mycophenolic acid (MPA) and its metabolites mycophenolic acid glucuronide (MPAG) and acyl glucuronide (AcMPAG) in rat plasma and bile, using a newly developed HPLC method. Protein precipitation and liquid-liquid extraction were employed in sample preparation of plasma and bile, respectively. The HPLC methods included a gradient elution consisting of acetonitrile and phosphate buffer at a flow rate of 1.2 mL/min, with UV detection at 254 nm. The HPLC method was found to be sensitive and linear (r ² ≥ 0.9991, 1.0–128.0 and 0.25–32.0 mg/L for MPA; 1.0–128.0 and 0.5–64.0 mg/L for MPAG; 0.25–32.0 and 1.0–128.0 mg/L for AcMPAG in rat plasma and bile, respectively), precise (both the intra- and inter-day variability were ≤ 6.8%), and accurate (both the intra- and inter-day accuracy were between 92.2% and 105.4%). The average extraction efficiencies for MPA, MPAG and AcMPAG were 85.3%, 100.1%, and 94.7% in plasma, and 88.0%, 67.3%, and 68.3% in bile, respectively. The method was successfully employed for pharmacokinetic studies in plasma and bile after oral administration of mycophenolate mofetil (prodrug of MPA) in rats.