仿刺参共附生真菌Aspergillus terreus来源的聚酮类化合物的研究

海参是海洋中重要的食物和药物资源,含有丰富的活性物质。海参共附生微生物非常丰富。为了从海参共附生微生物获得具有强生物活性的物质,对山东芝罘岛海域的仿刺参来源的共附生真菌Aspergillus terreus利用分子生物学手段进行了种属鉴定,并利用固体基进行培养,对其次级代谢产物进行了研究。通过薄层层析、柱层析、高效液相色谱对其化学成分进行了分离和纯化,综合核磁波谱、质谱对其进行结构鉴定。从该菌中获得dihydrogeodin(1)、Territrems A(2)、Territrems B(3)、2,4-二羟基苯乙酮(4)、questin(5)和emodin(6)六种物质。利用人口腔上皮癌细胞株(KB)和多药耐药性的细胞株(KBv200)对1,5和6进行了细胞毒活性测试。本研究首次对化合物1进行了完整的谱图解析,并首次发现海洋环境来源的微生物可以代谢化合物dihydrogeodin(1),以及具有强抑制乙酰胆碱酶活性Territrems(3),而这些物质是海参本体不能够产生的,为开发具有预防和治疗帕金森病相关的功能食品提供了新来源。     

Immunohistochemical and histochemical findings favoring the occurrence of autocrine/paracrine as well as nerve-related cholinergic effects in chronic painful patellar tendon tendinosis

The pathogenesis of the pain in patellar tendon tendinosis ("jumper's knee") is unclear. We have recently presented new information about the sensory nervous system in the human patellar tendon, but there is very little information regarding the possible occurrence of a cholinergic system in this tendon. In the present study, specimens of pain-free normal tendons and chronically painful tendinosis tendons were examined by different immunohistochemical and histochemical methods. Antibodies against the M(2) receptor, choline acetyltransferase (ChAT), and vesicular acetylcholine transporter (VAChT) were applied, and staining for demonstration of activity of acetylcholinesterase (AChE) was also utilized. It was found that immunoreactions for the M(2) receptor could be detected intracellularly in both blood vessel cells and tenocytes, especially in tendinosis specimens. Furthermore, in the tendinosis specimens, some tenocytes were seen to exhibit immunoreaction for ChAT and VAChT. AChE reactions were seen in fine nerve fibers associated with small blood vessels in both the normal control tendons and the tendinosis tendons. The observations suggest that there is both a nerve related and a local cholinergic system in the human patellar tendon. As ChAT and VAChT immunoreactions were detected in tenocytes of tendinosis tendons, these cells might be a source of local acetylcholine (Ach) production. As both tenocytes and blood vessel cells were found to exhibit immunoreactions for the M(2) receptor, it is likely that both of these tissue cells may be influenced by ACh. Thus, in conclusion, there appears to be an upregulation of the cholinergic system, and an occurrence of autocrine/paracrine effects in this system, in the tendinosis patellar tendon.     

乙酰胆碱酯酶在尼龙网上的固定化研究

以尼龙网为载体,分别用硫酸二甲酯和三乙基肟四氟化硼作烷基化试剂使尼龙网活化,然后将尼龙网浸入一定浓度的乙酰胆碱酯酶溶液中,置于4°C条件下进行固定化,获得了较好的固定化效果,酶膜活性分别达到4.2 U/cm2和11.1 U/cm2,固定化效率分别达到24.2%和64.1%,重现性好,稳定性好,5个月后活性无明显下降。     

Anticholinesterase potential of flavonols from paper mulberry (Broussonetia papyrifera) and their kinetic studies

It is necessary to develop food additives to help treat chronic disorders like neurodegenerative diseases from medicinal plants. Ethanol extracts of paper mulberry were found to display significant inhibition against cholinesterases, enzymes that are strongly linked with Alzheimer’s disease (AD). The active components were identified as prenylated flavonols (2-4) that inhibited two related human cholinesterases in a dose-dependent manner, with IC₅₀’s ranging between 0.8 and 3.1 μM and between 0.5 and 24.7 μM against human acetylcholinesterase (hAChE) and butylcholinesterase (BChE), respectively. Prenyl groups within these flavonols were found to play a critical role for inhibition because the parent compound 1, quercetin, was inactive (IC₅₀ > 500 μM) towards the target enzymes. Flavonols (2-4) showed mixed inhibition kinetics as well as slow and time-dependent reversible inhibition toward hAChE. The affinity between protein and inhibitors was investigated using fluorescence quenching. The affinity constants (K_SA) of inhibitors increased in proportion to their inhibitory potencies.     

兔血清乙酰胆碱酯酶用于农药残留速测的最佳条件研究

研究了磷酸缓冲液pH值、反应时间、反应温度、底物、酶量对兔血清乙酰胆碱酯酶(AChE)活力测定的影响,结果表明,供试AChE活力最佳条件是:磷酸缓冲液(pH8.0)3.0ml,显色剂0.6%二硫双对硝基苯甲酸(DTNB)50μl,0.75%酶液(pH8.0)100μl,反应温度35℃,混匀稳定后加入底物1%碘化硫代乙酰胆碱(BTCI)50μl,反应180s后在412nm处测定光密度。在上述条件下,测定表明有机磷杀虫剂丙硫磷(Prothiofos)浓度在0.0125～0.4μg/ml,保育时间5～30min之间与AChE抑制率之间存在良好的相关性,可根据检测需要调整保育抑制时间。本试验结果将为应用兔血清AChE研制、改进有机磷和氨基甲酸酯类农药残留速测卡,实现农产品、食品农药残留快速检测提供重要参考。     

地鳖虫提取物抑制乙酰胆碱酯酶活性的研究

采用乙酰胆碱酯酶抑制剂活性筛选模型对地鳖虫不同提取物进行活性筛选。结果表明,地鳖虫浸提最佳溶剂为乙醇,其对AchE的抑制率为27.7%;地鳖虫醇提物萃取最佳溶剂为乙酸乙酯,其对AchE的抑制率为59.78%;地鳖虫萃取物硅胶柱层析最佳流动相为氯仿:甲醇=91:9,其对AchE的抑制率为59.51%。     

基于功能化多壁碳纳米管的乙酰胆碱酯酶生物传感器制备

将功能化多壁碳纳米管(FCNTs)结合壳聚糖(CHIT)和戊二醛(GTA)导电聚合物形成凝胶溶胶,将该凝胶溶胶悬滴在玻碳电极(GCE)表面制得FCNTs-CHIT-GTA/GCE电极,结合自制的乙酰胆碱酶膜生成一种反应灵敏、快速、稳定、用于检测有机磷农药的电流型生物传感器。结果表明：该传感器具有出峰电位低、峰流值高、电子传递速率高、阻抗值小等优点,在研究不同浓度敌敌畏对酶抑制率的关系时,得到的关系曲线线性良好,最低检出限为20ng/L。该传感器适用于有机磷农药残留跟踪检测。