鸭血浆胆碱酯酶的分离纯化和性质的研究

将鸭血浆用0．2mol／L pH7．2磷酸盐缓冲溶液等体积稀释后，以硫酸铵为盐析剂进行二次盐析、再经DEAE-52离了交换层析和Sephadex G-200凝胶过滤层析对胆碱酯酶进行分离纯化，可获得电泳纯的胆碱酯酶，纯化倍数为278．5倍，酶活回收为17．8％。实验表明，该酶的最适温度为37℃，最适pH为7．8，有过量底物抑制现象。     

Use of cholinesterase activity as an indicator for the effects of combinations of organophosphorus pesticides in water from environmental sources

Organophosphorus pesticides (OPs) are commonly detected in agricultural products, animal-derived foodstuffs, and environmental samples. Until now, the focus of research has been to evaluate the adverse effect of a single OP. While each OP may be present at concentrations under recognized as “no observed adverse effect level (NOAEL)”, the combined effects of multiple OPs present at these low concentrations have not been sufficiently studied. Therefore, we developed an in vitro testing method to evaluate the toxicity of multiple OPs based on the degree of inhibition of cholinesterase (ChE) activity. This method requires only 10 min to complete and no specialized technology. We examined 15 OPs by this method and categorized them into three groups according to the degree of ChE inhibition. A relationship between the OPs’ chemical structures and the degree of ChE inhibition emerged with the moiety –P–O–CN– showing the strongest action. The degree of ChE inhibition increased with multiple OPs, and the degree of inhibition seemed to be additive. These results demonstrate that the combined toxicity of multiple OPs present in food or environmental samples is an easily determined and toxicologically relevant measure of overall toxicity of complex OPs mixtures. It is possible to apply this testing method as a monitoring technique in water quality management in order to control OPs. As a result, this method can play the role for the potential risk reduction to the ecosystem and may contribute to the preservation of the environment.     

Does mercury interact with the inhibitory effect of dichlorvos on Palaemon serratus (Crustacea: Decapoda) cholinesterase?

Mercury is one of the most hazardous metals that may contaminate estuarine ecosystems and induce toxic effects on wildlife organisms. It has been suggested that impairment of cholinesterase (ChE) activity may be involved in the resulting mercury toxicity. Following Palaemon serratus exposure to mercury chloride (HgCl₂), no effect on ChE activity was observed whatever the concentration used (to 37.5 µM) or the time of exposure (to 7 days). By contrast, following 24 h exposure to dichlorvos, an organophosphate insecticide with a well-characterised anti-ChE action, decrease of ChE activity was observed until 30 to 40% basal activity, which seems to be the minimum activity required for prawn survival. In addition, HgCl₂ does not affect dichlorvos toxicity and treatments with a mixture of both compounds can be interpreted as the sum of the two independent toxicities. Therefore, mercury and insecticide toxicities are independent and ChE activity from P. serratus eyes seems to be a reliable and sensitive biomarker for organophosphate insecticides even when organisms are simultaneously exposed to mercury.     

非霍奇金淋巴瘤患者血清胆碱酯酶、乳酸脱氢酶及β2-微球蛋白的检测及其意义

目的 探讨非霍奇金淋巴瘤(NHL)患者血清胆碱酯酶(CHE)表达水平与患者临床特征及血清乳酸脱氢酶(LDH)、β2-微球蛋白(β2-MG)表达的相关性.方法 分别采用速率法、免疫法检测74例NHL患者血清CHE、LDH、β2-MG值,根据血清CHE均值5750 U/L分成低CHE水平组38例与高CHE水平组36例,比较两组患者的临床特征及LDH、β2-MG水平.结果 低CHE水平组与高CHE水平组血清CHE、LDH、β2-MG表达水平差异有统计学意义[(3714.2±1207.1)U/L、(435.7±36.4.4)U/L、(4.3±2.9)mg/L;(7898.2±1550.5)U/L、(247.4±134.8)U/L、(2.7±1.2)mg/L](t=10.510,P=0.000;t=2.969,P=0.005;t=3.043,P=0.004);两组年龄、病理类型、WBC、Plt差异无统计学意义(t=0.166,P=0.868;x2=0.751,P=0.386;t=1.626,P=0.111;t=1.987,P=0.056);两组性别、Ann Arbor分期有明显不同,低CHE水平组以男性(x2=5.432,P=0.020)、Ann Arbor Ⅲ～Ⅳ期(x2=9.394,P=0.024)患者为主.低CHE水平组血红蛋白含量[(97.6±25.8)g/L胝于高CHE水平组[(113.4±15.2)g/L],差异有统计学意义(t=3.230,P=0.002).结论 NHL患者CHE低活性与年龄、病理类型、WBC、Plt无关,与男性、Ann ArborⅢ/Ⅳ期、贫血、LDH、β2-MG高表达相关.     

拟胆碱药依酚氯铵的合成

探索拟胆碱药依酚氯铵的合成工艺。该工艺以间氨基酚为原料,经4步化学反应得到目标产物,总收率约为10％。     

Anticholinesterase and antioxidant effects of the ethanol extract, ethanol fractions and isolated flavonoids from Cistus laurifolius L. leaves

In the current study, the n-hexane, chloroform, ethyl acetate, water, and n-butanol fractions obtained from the main ethanol extract of Cistus laurifolius L. were evaluated for their cholinesterase inhibitory effects against acetyl- (AChE) and butyrylcholinesterase (BChE), at 50, 100, and 200 μg/ml, using an ELISA microplate reader. The antioxidative effect of the extract and fractions was also determined using 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity, ferrous ion-chelation capacity, and ferric-reducing antioxidant power (FRAP) test systems. Total phenol and flavonoid contents of the extract and fractions were calculated using Folin-Ciocalteau and aluminium chloride reagents. Three flavonoid derivatives; 3-O-methylquercetin (1), 3,7-O-dimethylquercetin (2), and 3,7-O-dimethylkaempferol (3) isolated from the CHCl₃ fraction were also tested in the same manner. Our experimental findings indicated that the ethanol extract exerted the highest AChE inhibition (80.07 ± 1.06% at 200 μg/ml). The ethyl acetate and n-butanol fractions displayed the best activity against DPPH and FRAP assays.     

Meserine对胆碱酯酶活性及东莨菪碱诱导痴呆小鼠学习记忆的影响

目的: 考察新型胆碱酯酶抑制剂Meserine对胆碱酯酶活性及东莨菪碱(Scopolamine)诱导的胆碱能障碍痴呆模型小鼠学习记忆的影响。方法: 选取小鼠脑匀浆、血浆、人源重组AChE(rHuAChE)为体外酶源,测定Meserine抑制AChE/BuChE的活性、选择性及酶动力学。通过鼻腔给药后检测脑部AChE活性和ACh浓度评价Meserine对小鼠脑内胆碱能系统的调节。选用避暗及水迷宫实验考察Meserine对痴呆模型小鼠学习记忆功能的影响。结果: Meserine对AChE和BuChE都具有较好的抑制活性,IC₅₀分别为(65.2±3.2) nmol/L和(86.7±4.9) nmol/L,并对rHuAChE呈现非竞争性抑制。经鼻给药Meserine可显著抑制脑内AChE活性、升高ACh水平,且二者变化的时程具有一致性,给药 15 min 后,AChE 抑制活性最强(26.9%),ACh浓度最高(1269.0 ng/g)。行为学实验结果显示,经鼻给药Meserine(10 μg/kg)能显著改善东莨菪碱诱导的痴呆模型小鼠的工作记忆及空间学习能力,较模型组具有统计学差异(P<0.01 vs 东莨菪碱组)。结论: 上述结果提示Meserine为强效非竞争性胆碱酯酶抑制剂,经鼻给药Meserine可通过调节脑内胆碱能系统有效改善东莨菪碱诱导的痴呆模型小鼠的学习记忆功能。     

Phytochemical profile of a blend of black chokeberry and lemon juice with cholinesterase inhibitory effect and antioxidant potential

In this study, black chokeberry concentrate was added (5% w/v) to lemon juice, since previous reports suggested potential health benefits of this blend. The phytochemical composition, antioxidant capacity (scavenging of DPPH, superoxide and hydroxyl radicals, and hypochlorous acid), and inhibitory activity against cholinesterase of the new blend were determined and compared with those of lemon juice and chokeberry in citric acid (5%). The chokeberry concentrate, rich in cyanidin-glycosides, quercetin derivatives, and 3-O-caffeoylquinic acid, and lemon juice, possessing flavones, flavanones, quercetin derivates, and hydroxycinnamic acids, were characterised. The new drink showed a higher antioxidant effect than the chokeberry or lemon controls for all the tested methods, except for hypochlorous acid, in which lemon juice displayed higher activity. Both the lemon juice and chokeberry controls inhibited acetylcholinesterase and butyrylcholinesterase, and this effect was increased in the new mixtures. The results of the different radical scavenging assays indicate that the lemon–black chokeberry (5% w/v) mixture was more antioxidative than the respective controls separately. Moreover, their inhibition of cholinesterase is of interest regarding neurodegenerative disorders such as Alzheimer’s disease, Parkinson’s disease, or senile dementia.     

Risikoanalyse durch eine wirkungsbezogenen Analytik mit der instrumentellen Hochleistungs-Dünnschichtchromatographie in der Lebensmittelüberwachung

Zusammenfassung. Als wirkungsbezogene Analytik wird die Kopplung von biochemischen bzw. biologischen Testverfahren an chemisch/physikalische oder chromatographische Verfahren bezeichnet. So lassen sich mittels der Dünnschichtchromatographie die aufgetrennten Komponenten direkt auf dem Chromatogramm physikalisch-chemisch detektieren und quantifizieren. Durch die Kopplung von biochemischen (z.B. enzymatischen Hemmtests) oder biologischen Testverfahren k?nnen toxikologisch wirksame Substanzen in situ nachgewiesen werden. Mit diesen biologischen Testsystemen k?nnen – direkt auf dem Chromatogramm auf der Dünnschichtplatte – Fungizide, Antibiotika und Lumnineszenz-Hemmstoffen nachgewiesen werden; ein neues molekularbiologisches Testverfahren erm?glicht den qualitativen und quantitativen Nachweis von Hormonen. Mit biochemischen und biologischen Detektionsverfahren k?nnen Wirkstoffe in Lebensmittelproben sowie bei der Reinheitskontrolle und in der Metabolismusforschung von Chemikalien nachgewiesen werden. Au?erdem k?nnen die detektierten Wirkstoffe durch ihre Migrationsstrecke und ihr UV-Spektrum charakterisiert oder auch identifiziert werden. Pflanzliche Lebensmittel wurden mit der wirkungsbezogenen Analytik auf die Gegenwart von Pestiziden hin untersucht. Biochemische und biologische Detektionsverfahren auf dem Dünnschichtchromatogramm sind sehr selektiv und sensitiv und schlie?en damit die Lücke zwischen biologischen in vitro-Testverfahren und instrumenteller Analytik. Die Detektion von Wirkungs?quivalenten ist als Screening-Verfahren zun?chst unabh?ngig von Referenzsubstanzen. Neben verschiedenen Testverfahren wird ein Konzept zur Risikoanalyse und Risikobewertung vorgestellt, bei dem die wirkungsbezogene Analytik als Bindeglied zwischen Biotests und chemisch/physikalischen Analytik- und Identifizierungsverfahren fungiert. Eingegangen: 30. Juni 2006; angenommen 1. August 2006