钩吻素子在大鼠肝微粒体中的酶促反应动力学研究

目的建立体外实验法研究钩吻素子在大鼠肝微粒体中的酶促反应动力学。方法以钩吻碱甲标准品为内标,采用液质连用(UPLC/MS)的方法测定钩吻素子在体外代谢30min后剩余钩吻素子的含量。UPLC采用BEH C18色谱柱(2.1×50mm,1.7μm),流动相为0.1%甲醇酸-0.1%甲醇水(20∶80),流速为0.3mL·min-1,进样量为1μL,柱温为40℃,MS采用正离子、多反应监测(MRM)模式监测,离子监控通道m/z307.1>69.76(钩吻素子),m/z 323.1>69.76(钩吻碱甲),毛细管电压2.8kV,锥孔电压35V,离子源温度110℃,脱溶剂温度300℃,锥孔气(N2)流速50L·h-1,脱溶剂气(N2)流速450L·h-1。用Linewrave-Burk作图分析数据,计算酶促动力学参数。结果钩吻素子在0.5～30uM范围内线性良好Y=0.1544X+0.0593,r=0.9939结论此方法简单、快速、可靠,适用于钩吻素子的体外代谢研究。     

瑶药风湿骨痛喷雾剂Ⅲ的质量标准研究

目的:建立瑶药风湿骨痛喷雾剂Ⅲ的质量标准。方法:对制剂中钩吻进行薄层色谱法定性鉴别;用高效液相色谱法测定制剂中钩吻素子。结果:在薄层色谱中能检出钩吻;钩吻素子在0.199 2～3.187μg范围内呈良好的线性关系(r=0.999 7)。平均回收率为98.58%,RSD为1.41%(n=6)。结论:所建立的方法稳定可靠,可有效控制该制剂的质量。     

HPLC测定覆盆子中没食子酸含量

目的 用高效液相色谱法测定覆盆子中没食子酸的含量.方法 采用Alltech C18柱(250 mm×4.6 mm,5μm),流动相为甲醇- 0.4%磷酸水溶液(10:90),流速1.0 mL·min-1,检测波长278 nm,柱温室温.结果 没食子酸在5.24～104.80 μg·mL-1浓度范围呈良好的线性关系,r=0.999 2,平均回收率为97.6%,RSD为1.9%.结论 本法稳定性、重现性好,可作为覆盆子药材质量控制的依据之一.     

云南松松针中槲皮素的含量测定

目的建立利用高效液相色谱法测定云南松松针中有效成分槲皮素的含量。方法采用Inert Sustain C18(4.6 mm×250 mm,5μm)色谱柱;以乙腈-0.3%磷酸水溶液(35∶65)为流动相;检测波长370 nm;流速1m L·min-1;柱温:40℃。结果槲皮素进样量在0.096~0.485μg范围内线性关系良好(r=0.999 8),样品的平均回收率为100.4%,RSD为1.72%(n=6)。结论本法操作简便、快速、分离效果好、稳定性高、重现性好,可用于松针中槲皮素的含量测定。     

HPLC同时测定补肾活血颗粒中松脂醇二葡萄糖苷、苦杏仁苷、桂皮醛的含量

目的采用HPLC波长切换法,建立同时测定补肾活血颗粒中松脂醇二葡萄糖苷、苦杏仁苷、桂皮醛3成分含量的分析方法。方法采用Zorbax Extend-C18柱(4.6 mm×150 mm,5μm);以乙腈-水为流动相梯度洗脱,松脂醇二葡萄糖苷、苦杏仁苷、桂皮醛的检测波长分别为227,210,290 nm;进行样品前处理工艺和方法学考察。结果松脂醇二葡萄糖苷、苦杏仁苷和桂皮醛均得到很好分离,浓度分别在0.426 0~2.88 6μg·m L?1,7.455~86.60μg·m L?1和0.440 6~13.22μg·m L?1内线性关系良好(r≥0.999);平均加样回收率分别为98.3%(RSD=1.6%),101.4%(RSD=1.1%)和100.0%(RSD=1.2%);精密度试验峰面积RSD均≤1.4%。结论该方法预处理简单,方法灵敏、准确、重复性好,可用于补肾活血颗粒及相关制剂的质量控制。     

多波长HPLC梯度洗脱法同时测定眠安宁合剂中7个有效成分含量

目的建立多波长高效液相梯度洗脱法同时测定眠安宁合剂中5-羟甲基糠醛、远志(口山)酮Ⅲ、白术内酯Ⅰ、白术内酯Ⅲ、丹酚酸B、丹参酮ⅡA、梓醇7个成分的含量。方法采用依利特Hypersil C18色谱柱(4.6 mm×250 mm,5μm);流速:0.7 m L·min?1;流动相A为甲醇-乙腈(1∶3),流动相B为0.1%磷酸水溶液,梯度洗脱;检测波长:5-羟甲基糠醛为284 nm,远志(口山)酮Ⅲ为320 nm,白术内酯Ⅰ和白术内酯Ⅲ为220 nm,丹酚酸B和丹参酮ⅡA为270 nm,梓醇为210 nm,柱温为25℃。结果 5-羟甲基糠醛、远志(口山)酮Ⅲ、白术内酯Ⅰ、白术内酯Ⅲ、丹酚酸B、丹参酮ⅡA和梓醇质量浓度分别在10.08~100.80μg·m L?1(r=0.999 8)、3.55~35.50μg·m L?1(r=0.999 3)、5.80~58.00μg·m L?1(r=0.999 1)、4.20~42.00μg·m L?1(r=0.999 9)、40.50~405.00μg·m L?1(r=1.000)、12.28~122.80μg·m L?1(r=0.999 5)、7.90~79.00μg·m L?1(r=0.999 9)内与其峰面积呈良好的线性关系;5-羟甲基糠醛、远志(口山)酮Ⅲ、白术内酯Ⅰ、白术内酯Ⅲ、丹酚酸B、丹参酮ⅡA和梓醇的平均加样回收率分别为98.56%,96.94%,99.61%,97.54%,100.04%,98.59%,99.81%,RSD分别为0.97%,1.73%,1.03%,1.35%,0.86%,1.70%,1.43%(n=6)。结论建立的方法准确可靠,可用于眠安宁合剂的含量控制。     

钩吻生物碱钩吻素子的提纯及其抗神经病理性疼痛的作用

神经病理性疼痛(NPP)的新型治疗药物以及治疗靶点亟待研究。中国钩吻为马钱科植物胡蔓藤的全草,盛产于闽、粤、桂等地。钩吻的主要有效成分为吲哚类生物碱,含有数十种生物碱单体,其中钩吻素子(koumine,KM)为国产钩吻生物碱单体中含量最高的一种有效成分。民间一直应用钩吻原植物治疗各类疼痛,尤其神经性疼痛与癌性疼痛。有报道,钩吻总碱具有显著的镇痛效果,但治疗指数小,有效量与中毒量较接近。本文拟从国产钩吻分离纯化获取钩吻素子,并报道其抗神经病理性疼痛作用及其机制。选取本地采集的钩吻根和茎,提取钩吻总生物碱;应用高速逆流色谱等技术分离纯化钩吻总碱获得钩吻素子单体,纯度达99%;经紫外光谱、红外光谱、核磁共振谱、质谱分析、元素分析以及熔点测定等确证其化学结构,钩吻素子分子式为C20H22N2O,分子量306.3。钩吻素子原料药对光、温度、湿度稳定,钩吻素子水溶液在pH值3～8以及温度在室温、40℃、60℃条件下稳定。在大小鼠坐骨神经慢性束缚损伤(CCI)模型和脊神经结扎模型上,以动物机械阈值、热阈值和自发痛行为为指标,首次观察到ig或sc钩吻素子具有显著的抗NPP作用,有效剂量远低于中毒剂量(小鼠sc钩吻素子LD50为99 mg·kg-1)。脊髓可能是钩吻素子抗NPP的重要作用部位;脊髓神经活性甾体3α,5α-四氢孕酮(3α,5α-THP)参与调控NPP,钩吻素子可显著提高CCI模型大鼠脊髓腰段内3α,5α-THP的水平;3α-羟化类固醇脱氢酶(3α-HSD)是催化3α,5α-THP合成的关键酶,钩吻素子可显著上调脊髓3α-HSD酶的表达。上述结果表明,钩吻素子具有显著的抗NPP作用;增强脊髓3α-HSD酶的表达,促进脊髓3α,5α-THP的生成,可能是其发挥抗NPP作用的机制之一。     

HPLC法测定多叶越南槐中红车轴草苷及高丽槐素的含量

目的:建立测定多叶越南槐药材中红车轴草苷及高丽槐素2个黄酮类成分含量的高效液相色谱法。方法:采用Agilent Eclipse XDB-C18色谱柱(4.6 mm×250 mm,5μm),柱温25℃,以乙腈-0.1%乙酸水溶液为流动相,梯度洗脱,流速1.0 m L·min-1,检测波长310 nm,进样量10μL,外标法定量。结果:红车轴草苷进样量在0.025 1~2.51μg范围内与峰面积呈良好的线性关系(r=0.999 0),平均回收率为97.0%(RSD=1.5%);高丽槐素进样量在0.025 2~2.52μg范围内与峰面积呈良好的线性关系(r=0.999 0),平均回收率为101.3%(RSD=2.2%)。结论:建立的含量测定方法符合方法验证要求。     

HPLC同时测定玉叶金花清热片中3种有效成分的含量

目的建立同时测定玉叶金花清热片中栀子苷、穿心莲内酯和脱水穿心莲内酯含量的HPLC。方法采用Agilent ZORBAX SB-C18色谱柱(4.6 mm×150 mm,5μm),流动相为甲醇-0.2%磷酸水溶液,梯度洗脱,流速为1.0 m L·min?1,检测波长:238 nm(0~10 min,栀子苷),225 nm(10~22 min,穿心莲内酯),254 nm(22~35 min)。结果栀子苷在0.166~2.981μg内线性关系良好(r=0.999 9),平均回收率为99.4%(RSD=1.2%);穿心莲内酯在0.146~2.621μg内线性关系良好(r=0.999 9,平均回收率为99.2%(RSD=1.3%);脱水穿心莲内酯在0.222~3.992μg内性关系良好(r=0.999 7),平均回收率为100.1%(RSD=1.4%)。结论该方法简便、快捷、准确,可用于该制剂的质量控制。     

高效液相色谱法测定药用辅料苯甲醇中苯甲酸和苯甲醛含量

目的建立HPLC法同时测定药用辅料苯甲醇中苯甲酸和苯甲醛的含量。方法采用Acclaim 120 C18柱(4.6 mm×250 mm,5μm),流动相为乙腈-0.02%甲酸(用氨水调至p H=4.5)(30:70),流速为1.0 m L·min-1,检测波长为230 nm,柱温为35℃,进样量为20μL。结果苯甲酸在0.500 6¹5.02μg·m L-1内与峰面积线性关系良好,平均回收率均>100%,RSD均<1.3%(n=3);苯甲醛在0.505 215.02μg·m L-1内与峰面积线性关系良好,平均回收率均>100%,RSD均<1.3%(n=3);苯甲醛在0.505 2¹5.16μg·m L-1内与峰面积的线性关系良好,平均回收率均>108%,RSD均<0.15%(n=3)。结论本方法准确度高、重现性好,可用于苯甲醇中苯甲酸和苯甲醛的含量测定。     

Efficacy of gut lavage,hemodialysis, and hemoperfusion in the therapy of paraquat or diquat intoxication

Clinical and in vitro investigations were carried out to test the efficacy of gut lavage, hemodialysis, and hemoperfusion in the treatment of poisoning with paraquat or diquat. In a patient suffering from diquat intoxication 130 times more diquat was removed by gut lavage 30 h after ingestion than was removed by complete aspiration of the gastric contents.Determination of in vitro clearances for paraquat and diquat by hemodialysis showed that, at serum concentrations of 1–2 ppm, such as are frequently encountered in poisoning in man, toxicologically relevant quantities of herbicide cannot be removed from the body. At a concentration of 20 ppm, on the other hand, hemodialysis proved to be effective, the clearance being 70 ml/min at a blood flow rate of 100 ml/min. The efficacy of hemoperfusion with coated activated charcoal was on the whole better. Especially at concentrations around 1–2 ppm, the clearance values for hemoperfusion were some 5–7 times higher than those for hemodialysis.In a patient suffering from paraquat poisoning, both hemodialysis as well as hemoperfusion were carried out. The in vitro results could be confirmed: At serum concentrations of paraquat less than 1 ppm no clearance could be obtained by hemodialysis while by hemoperfusion with activated charcoal quite high clearance values were measured and the serum level dropped down to zero.

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Zusammenfassung Klinische Untersuchungen und Laboratoriumsversuche wurden durchgeführt, um die Wirksamkeit von Darmspülung, Hämodialyse und Hämoperfusion bei Paraquat- und Deiquat-Vergiftungen zu prüfen.Bei einem Patienten wurde 30 Std nach Deiquat-Aufnahme durch Darmspülung 130mal mehr Deiquat entfernt als durch vollständige Aspiration des Mageninhaltes. In vitro-Versuche ergaben, daß bei Blutserumkonzentrationen von 1–2 ppm, die bei Vergiftungen oft gemessen werden, durch Hämodialyse keine toxikologisch relevanten Paraquat- oder Deiquat-Mengen entfernt werden können. Dagegen erwies sich die Hämodialyse bei 20 ppm und einer Blutumlaufgeschwindigkeit von 100 ml/min mit einer Clearance von 70 ml/min als wirksam. Die Hämoperfusion mit beschicheter Aktivkohle war in diesen Versuchen aber eindeutig überlegen, denn insbesondere bei Konzentrationen um 1–2 ppm waren die Clearance-Werte 5–7mal höher als bei der Hämodialyse.Die in vitro-Ergebnisse wurden bei einem Patienten mit einer Paraquat-Vergiftung bestätigt: Bei Konzentrationen unter 1 ppm war die Hämodialyse wirkungslos, während durch Hämoperfusion relativ hohe Clearance-Werte erreicht wurden, so daß der Serumspiegel rasch unter die Nachweisgrenze abfiel.

     

In vitro studies on sterically stabilized liposomes (SL) as enzyme carriers in organophosphorus (OP) antagonism

This study describes a new approach for organophosphorous (OP) antidotal treatment by encapsulating an OP hydrolyzing enzyme, OPA anhydrolase (OPAA), within sterically stabilized liposomes. The recombinant OPAA enzyme was derived from Alteromonas strain JD6. It has broad substrate specificity to a wide range of OP compounds: DFP and the nerve agents, soman and sarin. Liposomes encapsulating OPAA (SL)* were made by mechanical dispersion method. Hydrolysis of DFP by (SL)* was measured by following an increase of fluoride ion concentration using a fluoride ion selective electrode. OPAA entrapped in the carrier liposomes rapidly hydrolyze DFP, with the rate of DFP hydrolysis directly proportional to the amount of (SL)* added to the solution. Liposomal carriers containing no enzyme did not hydrolyze DFP. The reaction was linear and the rate of hydrolysis was first order in the substrate. This enzyme carrier system serves as a biodegradable protective environment for the recombinant OP-metabolizing enzyme, OPAA, resulting in prolongation of enzymatic concentration in the body. These studies suggest that the protection of OP intoxication can be strikingly enhanced by adding OPAA encapsulated within (SL)* to pralidoxime and atropine.     

Aquatic Insects and Trace Metals: Bioavailability,Bioaccumulation, and Toxicity

Abstract

The uptake of metals from food and water sources by insects is thought to be additive. For a given metal, the proportions taken up from water and food will depend both on the bioavailable concentration of the metal associated with each source and the mechanism and rate by which the metal enters the insect. Attempts to correlate insect trace metal concentrations with the trophic level of insects should be made with a knowledge of the feeding relationships of the individual taxa concerned. Pathways for the uptake of essential metals, such as copper and zinc, exist at the cellular level, and other nonessential metals, such as cadmium, also appear to enter via these routes. Within cells, trace metals can be bound to proteins or stored in granules. The internal distribution of metals among body tissues is very heterogeneous, and distribution patterns tend to be both metal and taxon specific. Trace metals associated with insects can be both bound on the surface of their chitinous exoskeleton and incorporated into body tissues. The quantities of trace meals accumulated by an individual reflect the net balance between the rate of metal influx from both dissolved and particulate sources and the rate of metal efflux from the organism. The toxicity of metals has been demonstrated at all levels of biological organization: cell, tissue, individual, population, and community. Much of the literature pertaining to the toxic effects of metals on aquatic insects is based on laboratory observations and, as such, it is difficult to extrapolate the data to insects in nature. The few experimental studies in nature suggest that trace metal contaminants can affect both the distribution and the abundance of aquatic insects. Insects have a largely unexploited potential as biomonitors of metal contamination in nature. A better understanding of the physico-chemical and biological mechanisms mediating trace metal bioavailability and exchange will facilitate the development of general predictive models relating trace metal concentrations in insects to those in their environment. Such models will facilitate the use of insects as contaminant biomonitors.     

Steroidal sapogenin contents in some domestic plants

In order to find out the values of the steroid resources for the future use. the compositions and contents of steroidal sapogenins from 13 domestic plants have been investigated. As a result,Dioscorea nipponica, D. quinqueloba andSmilax china were found to have large amount of diosgenin. And pennogenin inTrillium kamtschaticum andParis verticillata, yuccagenin inAllium fistulosum, hecogenin inAgave americana and neochlorogenin inSolanum nigum were appeared to be major steroidal sapogenins.     

Alzheimer’s disease – current trends in basic and clinical research. Highlights from the 16th ECNP

Advances in the molecular biological knowledge of neuronal nicotinic acetylcholine receptors (nAChRs) have led to a growing interest by the pharmaceutical industry in the development of novel compounds that selectively modulate nAChR function. The ability of (-)-nicotine, an activator of nAChRs, to enhance attentional aspects of cognition in animals and humans, to exert neuroprotective and anxiolytic-like effects, and presumably to mediate the negative correlation between smoking and Alzheimer's (and Parkinson's) Disease, has focused interest on the potential therapeutic utility of modulators of nAChR function for treatment of some of the deficits associated with these progressive, neurodegenerative conditions. Numerous compounds are known which activate nAChRs and which might serve as lead compounds toward the development of such agents. The pharmacologic diversity of neuronal nAChR subtypes suggests the possibility of developing selective compounds which would have more favourable side-effect profiles than existing agents. This broader class of agents, collectively called cholinergic channel modulators (ChCMs), is anticipated to encompass compounds which would have more favourable side-effect profiles than existing agents, which generally exhibit low selectivity. This selectivity may be achieved by preferentially activating some subtypes of nAChRs (i.e., Cholinergic Channel Activators, ChCAs) or inhibiting the function of other subtypes (Cholinergic Channel Inhibitors, ChCIs). An overview of the biology of nAChRs and the rationale for the use of ChCMs for the treatment of dementia related to neurodegenerative diseases are presented, followed by a discussion of lead compounds and compounds under consideration for clinical evaluation.