葛根素和大豆甙元抑制[3H]氟硝西泮和体外大鼠脑膜的结合

从中药葛根中提取到两种苯二氮艹卓受体活性化合物:葛根素和大豆甙元。两种化合物在体外可抑制[3H]氟硝西泮和大鼠脑细胞膜的结合,IC₅₀值分别为18.46μmol·L^-1和15.43μmol·L^-1。大豆甙元还可抑制[³H]哌唑嗪和α1-肾上腺素受体的结合(IC₅₀值为89μmol·L^-1)。两种化合物的GABA比分别为1.11和1.12,提示两种黄酮化合物是苯二氮艹卓受体的拮抗剂或部分激动剂。Scatchardplot分析显示:两种化合物对[³H]氟硝西泮与膜结合的抑制作用是通过竞争性与非竞争性混合机制而实现的。     

酶法-超声提取黄精总黄酮及其抗氧化活性研究

目的 研究酶法-超声提取黄精中总黄酮（total flavonoids from Polygonatum sibirici,TFPS）的最佳工艺及总黄酮的体外抗氧化活性。方法 以总黄酮含量为指标,采用单因素和正交试验对纤维素酶用量、乙醇浓度、液料比和超声时间进行考察,优化工艺条件并考察总黄酮对DPPH自由基、ABTS⁺自由基的清除能力和亚铁离子螯合活性。结果 最佳提取条件：纤维素酶用量为0.75%,乙醇浓度为40%,液料比为20 mL·g^-1,超声时间30 min,总黄酮含量为1.595%。体外抗氧化试验表明,黄精总黄酮对DPPH自由基、ABTS⁺自由基有很强的清除能力,IC₅₀分别为27.55,11.47 μg·mL^-1,对亚铁离子的螯合活性较强,IC₅₀为32.26 μg·mL^-1。结论 酶法-超声提取工艺具有提取效率高、稳定性及重复性好等优点,可用于黄精中总黄酮的提取,在该工艺下提取的总黄酮具有较强的抗氧化活性。     

乌骨藤中C21甾体苷诱导SACC-83和SACC-LM细胞凋亡的作用及其机制研究

目的 研究乌骨藤提取物C₂₁甾体苷对唾液腺腺样囊性癌（salivary adenoid cystic carcinomacv,SACC）低侵袭细胞株（salivary adenoid cystic carcinoma-83,SACC-83）和肺高转移细胞株（SACC-LM）增殖抑制和诱导凋亡的作用及其机制。方法 用不同浓度（5,10,20,40,60,80,100 μmol·L^-1） C₂₁甾体苷处理SACC-83和SACC-LM细胞48 h后,MTT法检测细胞活力,并计算药物的IC₂₀和IC₅₀;细胞克隆形成实验检测细胞增殖能力;流式细胞术检测SACC-83及SACC-LM细胞凋亡情况;实时荧光定量PCR和Western blot检测SACC-83和SACC-LM细胞Bcl-2、Bax、caspase 3的mRNA和蛋白的表达。结果 不同浓度的C₂₁甾体苷降低SACC-83和SACC-LM细胞活力,抑制细胞增殖,并且作用于SACC-83细胞C₂₁甾体苷的IC₂₀浓度为7.49 μmol·L^-1,IC₅₀浓度为38.34 μmol·L^-1;作用于SACC-LM细胞的C₂₁甾体苷IC₂₀浓度为9.30 μmol·L^-1,IC₅₀浓度为46.04 μmol·L^-1;细胞克隆集落形成明显减少。C₂₁甾体苷IC₂₀浓度分别促进SACC-83及SACC-LM细胞凋亡,且随着给药时间延长,凋亡率增加,具有显著性差异（P<0.05,P<0.01）。经7.49,9.30 μmol·L^-1 C₂₁甾体苷分别处理SACC-83及SACC-LM细胞后,Bcl-2的mRNA及蛋白水平显著降低（P<0.01）,而Bax、Caspase 3的mRNA和蛋白水平显著升高（P<0.05）。结论 乌骨藤C₂₁甾体苷抑制SACC-83及SACC-LM细胞增殖、促进凋亡,其作用机制可能与调控Bcl-2、Bax和Caspase 3表达有关。     

阿魏酸衍生物的合成及其抗肿瘤活性研究

目的 设计、合成了一系列含有喹唑啉结构的阿魏酸衍生物，并对其抗肿瘤活性进行研究。方法 基于拼接原理，将阿魏酸与喹唑啉进行结合，并对喹唑啉6位进行修饰得到目标化合物，采用CCK-8法测试目标化合物对肺癌细胞A549的抗肿瘤活性，利用分子对接技术对目标化合物与表皮生长因子受体的结合模式进行模拟。结果 目标化合物的结构经HRMS(ESI)、¹H-NMR进行确证；体外抗肿瘤活性结果表明化合物11d、11f、12d和12g的IC₅₀低于阳性药吉非替尼，11c的IC₅₀值与吉非替尼相当。结论 化合物11d(IC₅₀=3.09 μmol·L^-1)对肺癌细胞A549抗肿瘤活性较强，值得深入研究。     

山胡椒内生真菌Trichoderma sp. SHJN1和Perenniporia sp. SHJG1的抗肿瘤活性成分研究

目的 从民族药山胡椒内生真菌Trichoderma sp.SHJN1和Perenniporia sp.SHJG1的代谢物中寻找活性先导化合物。方法 采用正相硅胶、反相硅胶、Sephadex LH-20凝胶及制备型HPLC等对Trichoderma sp.SHJN1和Perenniporia sp.SHJG1发酵物进行分离纯化，再通过NMR、ESI-MS等鉴定化合物结构，同时采用人乳腺癌细胞（MCF-7）和人肺癌细胞（A549）对这些化合物的抗肿瘤活性进行初步评价。结果 从2株内生真菌次级代谢产物中共分离鉴定了12个化合物：alantrypinone （1）、oryzalactam （2）、phomoindene A （3）、cis-gregatin B （4）、huaspenone B （5）、stigmasta-7，22-dien-3β，5α，6α-triol （6）、ergosterol （7）、1-deoxy-2-demethylviridiol （8）、viridiol （9）、trichodermamides A （10）、chromone （11）、对-羟基苯乙酸（12）。抗肿瘤活性评价结果显示，化合物3 抑制MCF-7细胞增殖活性IC₅₀为（62.9±1.02）μmol·L^-1[顺铂（cisplatin，DDP） IC₅₀为（30.1±1.67）μmol·L^-1]；化合物8 和9 抑制A549细胞增殖活性的IC₅₀分别为（34.6±1.57）μmol·L^-1和（44.9±1.74）μmol·L^-1[DDP IC₅₀为（20.6±1.42）μmol·L^-1]。结论 化合物3 ，8 和9 具有潜在抗肿瘤活性。     

基于UDP-葡萄糖醛酸转移酶1A1抑制探讨二蒽酮的潜在肝毒性

目的 基于胆红素代谢酶UDP-葡萄糖醛酸转移酶1A1（UGT1A1）靶点评价何首乌中二蒽酮类成分的潜在肝毒性。方法 采用Discovery Studio 2.5软件的From Receptor Cavities模块对UGT1A1酶蛋白空腔进行自动识别，将反式-大黄素-大黄素二蒽酮（trans-EMD）、顺式-大黄素-大黄素二蒽酮（cis-EMD）与UGT1A1酶蛋白进行对接，确定待测单体与酶蛋白的作用方式以及连接的紧密程度；应用大鼠肝微粒体孵育体系，加入底物胆红素对照品溶液，评价trans-EMD、cis-EMD（0.037、0.110、0.330、0.990、2.970 μg·mL^-1）对UGT1A1酶的作用，同时启动Ⅰ、Ⅱ相代谢，以表观抑制常数（K_i）为评价指标；CCK-8法检测trans-EMD、cis-EMD（0.04、0.10、0.30、1.00、3.00 μg·mL^-1）作用24 h对HepaRG细胞的毒性作用；实时荧光定量PCR（qRT-PCR）实验检测trans-EMD、cis-EMD（0.04、0.30、3.00 μg·mL^-1）作用24 h对HepaRG细胞UGT1A1 mRNA水平的影响。结果 分子对接实验显示trans-EMD、cis-EMD可与UGT1A1结合于site F，2个化合物10或10''位不同氢键构型可引起化合物空间构型的改变，影响其与UGT1A1的结合强弱；体外酶抑制实验表明trans-EMD、cis-EMD对UGT1A1酶均表现出竞争型抑制作用，抑制作用较强；细胞毒性实验表明trans-EMD（IC₅₀为1.333 μg·mL^-1）和cis-EMD（IC₅₀为1.715 μg·mL^-1）均表现出较明显的HepaRG细胞毒性，IC₅₀值较小；与对照组比较，trans-EMD和cis-EMD 0.30、3.00 μg·mL^-1均可显著下调UGT1A1 mRNA表达水平（P＜0.05），且作用存在浓度相关性。结论 具有大黄素（10→10''）大黄素或大黄素（10→10''）大黄素母核结构的二蒽酮化合物是一类具有潜在肝毒性的化合物，其毒性作用可能与抑制胆红素代谢酶UGT1A1相关。     

常用中药单体及其制剂对CYP3A活性的体外抑制作用研究

摘 要 目的:研究常用中药单体及其制剂对CYP3A活性的抑制作用。方法: 在体外肝微粒体孵育体系中加入底物睾酮和不同浓度的中药单体或其制剂,以高效液相色谱法检测6β羟基睾酮的生成量,计算CYP3A酶活性。用GraphPad Prism v5.0软件,按照非线性回归计算出药物对CYP3A抑制作用的IC₅₀值。结果:不同中药单体及其制剂对CYP3A抑制作用的IC₅₀值如下：大黄酸IC₅₀值为36.74 μmol·L^-1;大黄素IC₅₀值为23.09 μmol·L^-1、芦荟大黄素IC₅₀值为23.91 μmol·L^-1,蜂胶IC₅₀值为60.3 μg·mL^-1;水飞蓟素胶囊IC₅₀值为24.5μg·mL^-1;丹参酮ⅡA磺酸钠注射液IC₅₀值为0.14%(v/v);五味子酯甲IC₅₀值为0.56 μmol·L^-1。结论: 大黄中的蒽醌、五味子酯甲、蜂胶、水飞蓟胶囊以及丹参酮ⅡA磺酸钠注射液在体外对鼠或人肝CYP3A酶具有抑制作用,临床上使用相关制剂时应密切关注其可能引起的药物相互作用。     

细胞色素P450介导的氯沙坦钾对瑞格列奈在大鼠肝微粒体中代谢的影响

目的 研究瑞格列奈在大鼠肝微粒体中的酶促反应动力学，并考察氯沙坦钾对其在大鼠肝微粒体中代谢的影响。方法 建立大鼠肝微粒体体外孵育体系对瑞格列奈的代谢进行研究；以洛伐他汀为内标，应用UPLC测定大鼠肝微粒体中瑞格列奈的浓度。采用底物减少法，通过GraphPad Prism 5.0软件计算瑞格列奈的酶促反应动力学常数V_max和K_m；分别以系列浓度氯沙坦钾（2.5~50μmol·L^-1）与瑞格列奈（44 μmol·L^-1）于37℃水浴中共同孵育，并测定肝微粒体中瑞格列奈的减少量，考察氯沙坦钾对瑞格列奈的抑制作用。结果 瑞格列奈在大鼠肝微粒体的最佳孵育时间为40 min，最佳蛋白质量浓度为1 mg·mL^-1；瑞格列奈酶促反应动力学参数V_max=47.29μmol·min^-1·（mg·protein）^-1，K_m=51.41 μmol·L^-1；氯沙坦钾对瑞格列奈在体外肝微粒体抑制作用的IC₅₀值为17.89 μmol·L^-1。结论 氯沙坦钾对瑞格列奈在大鼠肝微粒体中的代谢具有较强的抑制作用，两药联合应用可能发生相互作用，具有诱发低血糖的风险。     

衢枳壳不同组分体外降糖活性研究及4种黄酮组分含量分析

目的 研究衢枳壳不同极性组分体外降糖活性,并明确各组分中4种黄酮类化合物的含量。方法 采用系统溶剂法提取与分离衢枳壳中不同极性的黄酮组分,通过体外试验明确各组分对α-葡萄糖苷酶活性和HepG2细胞葡萄糖消耗的影响。采用Agilent Zorbax SB C₁₈色谱柱（250 mm×4.6 mm,5 μm）,以乙腈-0.02%磷酸水溶液（20︰80）为流动相,流速1.0 mL·min^-1,检测波长280 nm,柱温35℃,外标法测定衢枳壳各组分中芸香柚皮苷、柚皮苷、橙皮苷和新橙皮苷的含量。结果 衢枳壳各组分均具有α-葡萄糖苷酶抑制作用,抑制强弱顺序为正丁醇组分[IC₅₀：（0.033±0.010） mg·mL^-1] > 甲醇组分[IC₅₀：（0.092±0.006） mg·mL^-1] > 乙酸乙酯组分[IC₅₀：（0.170±0.014） mg·mL^-1] > 氯仿组分[IC₅₀：（0.509±0.070） mg·mL^-1];衢枳壳甲醇组分和正丁醇组分能够显著促进HepG2细胞葡萄糖的消耗,差异具有统计学意义（P<0.05）。含量测定方法经方法学认证,各项参数均符合要求。经分析发现：衢枳壳各组分中均含有芸香柚皮苷、柚皮苷、橙皮苷和新橙皮苷等黄酮类化合物,且正丁醇组分中各黄酮成分含量最高。结论 衢枳壳各组分均具有α-葡萄糖苷酶抑制作用,且随着各组分中黄酮类化合物含量增高作用增强。同时,含有较高黄酮类组分含量的衢枳壳组分具有促进HepG2细胞葡萄糖消耗的作用,如甲醇、正丁醇和乙酸乙酯组分。     

二茂铁杂环类化合物的合成及抗三阴性乳腺癌活性研究

目的 设计、合成杂环二茂铁衍生物,并研究其抗三阴性乳腺癌活性。方法 以二茂铁查耳酮为先导化合物,对其进行结构改造,合成了一系列含有杂环的二茂铁衍生物,并通过CCK8试剂盒测试化合物抗乳腺癌活性。结果 合成了28个二茂铁衍生物,其结构均通过¹H-NMR和MS加以确证。初步的生物活性测试结果表明,所合成的二茂铁衍生物对三阴性乳腺癌MDA-MB-231细胞有较强的选择性和抑制活性,其中咪唑杂环化合物抗肿瘤活性强于相应的吡唑类和嘧啶化合物。尤其是28a[IC₅₀=（1.6±0.23）μmol·L^-1]对MDA-MB-231的抑制活性分别是先导化合物3[IC₅₀=（10.7±1.41）μmol·L^-1]和他莫昔芬[IC₅₀=（13.7±1.17）μmol·L^-1]的6和10倍,同时这些二茂铁衍生物对正常乳腺上皮细胞MCF-10A均没有毒性。结论 本研究为开发具有抗三阴性乳腺癌活性的化合物提供了信息和依据。     

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.

     

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.     

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.     

2-(Acetoxyphenyl)-(Z)-styryl sulfides as cyclooxygenase inhibitors

2-(Acetoxyphenyl)-(Z)-styryl sulfides are described as selective cyclooxygenase-2 (COX-2) inhibitors, useful for treating inflammation and COX-2-mediated disorders including neoplasia. 2-(Acetoxyphenyl)-(Z)-styryl sulfide is claimed to be the most potent COX inhibitor in the series with a COX-2 selectivity ratio of 33. This compound is also claimed to be superior to celecoxib (Celebrex^®, Pfizer) in inhibiting cell growth of colorectal carcinoma cells. In this evaluation, the COX inhibitory activity of this compound is compared to that previously disclosed for diarylheterocycles and 2-(acetoxyphenyl)alkyl sulfides. The validity of the DLD-1 cell line in the growth inhibition studies is questioned based on recent literature reports indicating the lack of COX-2 expression in this cell line.     

Sleep-disordered breathing with chronic opioid use

Chronic opioid use for pain relief or as substitution therapy for illicit drug abuse is prevalent in our societies. In the US, retail distribution of methadone and oxycodone has increased by 824 and 660%, respectively, between 1997 and 2003. μ-Opioids depress respiration and deaths related to illicit and non illicit chronic opioid use are not uncommon. Since 2001 there has been an emerging literature that suggests that chronic opioid use is related to central sleep apnoea of both periodic and non-periodic breathing types, and occurs in ～ 30% of these subjects. The clinical significance of these sleep-related abnormalities are unknown. This review addresses the present knowledge of control of ventilation mechanisms during wakefulness and sleep, the effects of opioids on ventilatory control mechanisms, the sleep-disordered breathing found with chronic opioid use and a discussion regarding the future research directions in this area.     

Drug targets for cognitive enhancement in neuropsychiatric disorders

The investigation of novel drug targets for treating cognitive impairments associated with neurological and psychiatric disorders remains a primary focus of study in central nervous system (CNS) research. Many promising new therapies are progressing through preclinical and clinical development, and offer the potential of improved treatment options for neurodegenerative diseases such as Alzheimer's disease (AD) as well as other disorders that have not been particularly well treated to date like the cognitive impairments associated with schizophrenia (CIAS). Among targets under investigation, cholinergic receptors have received much attention with several nicotinic agonists (α7 and α4β2) actively in clinical trials for the treatment of AD, CIAS and attention deficit hyperactivity disorder (ADHD). Both glutamatergic and serotonergic (5-HT) agonists and antagonists have profound effects on neurotransmission and improve cognitive function in preclinical experiments with animals; some of these compounds are now in proof-of-concept studies in humans. Several histamine H3 receptor antagonists are in clinical development not only for cognitive enhancement, but also for the treatment of narcolepsy and cognitive deficits due to sleep deprivation because of their expression in brain sleep centers. Compounds that dampen inhibitory tone (e.g., GABA_A α5 inverse agonists) or elevate excitatory tone (e.g., glycine transporter inhibitors) offer novel approaches for treating diseases such as schizophrenia, AD and Down syndrome. In addition to cell surface receptors, intracellular drug targets such as the phosphodiesterases (PDEs) are known to impact signaling pathways that affect long-term memory formation and working memory. Overall, there is a genuine need to treat cognitive deficits associated with many neuropsychiatric conditions as well as an increasingly aging population.