蓖麻子中蓖麻碱的提取分离与含量测定

目的:建立蓖麻子中毒性成分蓖麻碱的含量测定方法。方法:采用溶剂提取、硅胶柱层析和重结晶等提取分离手段得到蓖麻碱对照品;采用HPLC法测定蓖麻子中蓖麻碱含量,以依利特Hypersil ODS2(150 mm×4.6 mm,5μm)色谱柱为固定相,以水-乙腈-二乙胺(89∶11∶0.03)为流动相,流速0.8mL·min-1,检测波长307nm。结果:从400 g蓖麻子种皮中分离、纯化得到纯度大于98.5%的蓖麻碱对照品210 mg;蓖麻碱在0.1255~2.510 0μg范围内,进样量与峰面积之间有良好的线性关系(R2=1),平均回收率为99.9%;蓖麻子中蓖麻碱含量为0.1695%~0.2396%。结论:所设计的提取分离步骤得到的蓖麻碱纯度高,可作为含量测定用对照品;所建立的含量测定方法简便、快速、重复性好,可用于蓖麻子中毒性成分蓖麻碱的含量测定。     

儿科危重急症急救讲座 第五讲 植物性中毒(中)

蓖麻子中毒俗称大麻子,是蓖麻的成熟种子,性平、味甘、辛,有小毒。外用可拔腐、提脓。榨油内服,可作泻药。中毒多因生食蓖麻子而致。小儿吃生蓖麻子5～6g即可致死。一、毒理蓖麻子含有两种毒性成分:蓖麻碱与蓖麻毒素。①蓖麻碱是一种白色结晶的毒性生物碱,约占蓖麻子的0.2%。蓖     

野蓖麻幼苗中毒1例救治体会

随着人们的养生保健意识的增强,饮食习惯也发生了改变,多喜欢采食野菜尝鲜。野蓖麻又名曼陀罗等,我国各地都有生长且称呼也不一致。晚春时节植株幼苗小,叶宽卵形,边缘有规则波状浅裂,可能与荠菜、菠菜等可食用的蔬菜相混淆而误食中毒。全株均有毒性,其毒性物质为莨菪碱、东莨菪碱、阿托品和蓖麻毒素等成分,中毒性成分具有不容易被高温破坏等特点。另外,野蓖麻也是麻沸散、蒙汗药的主要成分,可能被别人投毒,应该引起社会广泛关注。一旦诊断野蓖麻中毒,除按常规洗胃、导泻、利尿等抢救外,尽快使用新斯的明、毛果芸香碱或水杨酸毒扁豆碱等治疗。我院2015年5月29日成功救治1例误食野蓖麻叶中毒病例,报道如下。     

蓖麻子药效成分分离纯化和药理作用研究概述

蓖麻子为大戟科植物蓖麻(Ricinus communis Linn.)的干燥成熟种子,又名红麻、草麻、八麻子、牛蓖等.性平,味甘、辛,具有消肿拔毒,泻下通滞等功能[1].我国蓖麻是1400多年前由印度引入,现在全国各地均有种植.现代研究表明蓖麻子主要活性成分包括蓖麻毒蛋白、蓖麻油和蓖麻碱等[2].蓖麻毒蛋白具有抗肿瘤、抗生育、引产、泻下和抗病毒等作用.为深入系统研究蓖麻子药用价值,现对其药效成分分离纯化和药理作用研究进展作以综述,为后续研究提供参考.     

蓖麻壳水提液的镇痛作用及其成分分析

目的:研究蓖麻壳水提液的镇痛作用和主要成分的 HPLC 及质谱联用技术分析。方法:以啮齿动物的疼痛模型热板试验和乙酸扭体试验对蓖麻壳的水提液的镇痛进行了评价并利用高效液相色谱/质谱联用技术对浸提液的主要成分进行了分析。结果:小鼠 IP 蓖麻壳水提液组比对照组的扭体次数显著性减少和热板耐受时间显著性增加并有镇痛抑制率和耐受力随剂量增加而显著性提高(P<0.001)。利用 HPLC 和 HPLC/MS 联用技术分析蓖麻壳水提液其主要成分为蓖麻碱。结论:蓖麻壳水提液的活性成分可以明显抑制化学性刺激和热刺激所致的小鼠痛反应,有可能是多模式镇痛,减弱中枢神经系统接收到的疼痛信号和抑制外周疼痛信号。     

蓖麻根提取物抑制肝癌细胞的作用探讨

目的探讨蓖麻根提取物对肝癌细胞的作用。方法用（3-c4,5-二甲基噻唑-2）-2,5-二苯基四氮唑溴盐（MTT）实验法,检测蓖麻根提取物对肝癌细胞的表达程度。结果蓖麻根提取物中蓖麻石油醚成分对肝癌表达抑制作用最强。结论蓖麻根提取物在体外有较强的抗肝癌细胞作用。     

蓖麻蛋白：具有多种治疗用途潜力的植物毒素

本文概述了蓖麻蛋白的化学特性、作用机理、毒性步骤和对细胞培养、动物及人类的毒性效应,并综述了近年对蓖麻蛋白实验治疗及临床方面的研究。参考文献98篇。     

Promotion of ATP and S-140 to ribosome inactivation with camphorin,cinnamomin,and other RNA N-glycosidases

目的：研究ＡＴＰ和Ｓ１４０对Ⅰ型和Ⅱ型核糖体失活蛋白（ＲＩＰ）失活核糖体的影响．方法：采用凝胶电泳分析特征ＲＩＰ作用片断（Ｒ片断），以及改进的二步法标记苯丙氨酸外源ｐｏｌｙ（Ｕ）翻译体系，定量检测外加因子对ＲＩＰ失活核糖体作用的影响．结果：ＡＴＰ和Ｓ１４０对所有用于检测的Ⅰ型和Ⅱ型ＲＩＰ失活核糖体的功能均表现出不同程度的促进作用．对于Ⅰ型ＲＩＰ中克木毒蛋白、天花粉毒蛋白、γ苦瓜子毒蛋白、丝瓜毒蛋白Ａ、丝瓜毒蛋白Ｓ和Ⅱ型ＲＩＰ中蓖麻毒蛋白、蓖麻毒蛋白Ａ链；辛纳毒蛋白、辛纳毒蛋白Ａ链，ＩＣ５０增强活性比率分别为３１０８，１５，１５１，４５，５１和４７，７，２６，１２．结论：首次发现ＡＴＰ和Ｓ１４０对Ⅱ型ＲＩＰ及其Ａ链具有活性增强作用．克木毒蛋白和辛纳毒蛋白在对ＡＴＰ和Ｓ１４０的辅助需求方面有显著区别     

天花粉蛋白的研究进展

天花粉蛋白(trichosanthin,TCS)是从葫芦科栝楼属植物栝楼的根块中提取出来的一种碱性蛋白,属于Ⅰ型核糖体失活蛋白(RibosomeInactivating Proteins,RIPs).它与双链RIPs-蓖麻毒蛋白(Ricin)的A链在一级结构上有56%的同源性.     

蓖麻毒蛋白A链与抗表皮生长因子受体单克隆抗体偶联物的抗肿瘤效应

目的：将蓖麻毒蛋白（ricin）A链（RTA）与表皮生长因子受体（epidermal growth factor receptor,EGFR）mab偶联形成免疫毒素（immunotoxin,IT）,并评价该偶联物的体外抗肿瘤作用。方法：从蓖麻籽中提纯RTA,采用N-琥珀酰胺-3-（2一吡啶二硫）丙酸酯[N—succinimidyl-3-（2-pyridyldithio）-pm—pionate,SPDP]为偶联剂制备ITEGFRmab．RTA,以SDS—PAGE检测RTA的提取及IT的偶联,以DAB显色法检测IT对人肝癌HepG2细胞的结合能力,以间接ELISA方法测定IT的免疫反应性,以CCK-8法测定IT的体外抗肿瘤作用。结果：RTA从蓖麻籽中提取后,与EGFRmab成功偶联。IT基本保留了EGFRmab对HepG2细胞的结合能力和免疫反应性,及对HepG2细胞和L02细胞表现了不同的体外细胞毒作用。结论：EGFRmab—RTA具有特异性抑制肝癌细胞增殖的作用,有望成为新型的抗肝癌药物。     

Quantification of ricinine in rat and human urine: a biomarker for ricin exposure

Ricin is a toxalbumin derived from the castor bean plant, Ricinus communis. Ricinine is an alkaloid (3-cyano-4-methoxy-N-methyl-2-pyridone) that shares a common plant source with ricin, and its presence in urine infers ricin exposure. A new quantification method for ricinine was developed that uses solid-phase extraction to prepare 1-mL urine samples (81% recovery) for a 5-min, isocratic high-performance liquid chromatography method, followed by electrospray ionization tandem mass spectrometry. Protonated molecular ions were selected in the multiple reaction monitoring mode and quantified by isotope dilution with (13)C(6)-labelled ricinine as the internal reference. Urine pools enriched with ricinine at two concentrations were characterized as quality control materials and then used to validate the method. The method limit of quantification was 0.083 ng/mL, even with a confirmation ion of low relative abundance. Ricinine was stable in human urine when heated at 90 degrees C for 1 h, and during storage at 25 degrees C and 5 degrees C for 3 weeks. The method was applied to an animal exposure study, a crude ricin preparation scheme, and a forensic analysis. These studies show that ricinine can be measured in rat urine at least 48 h after exposure. Ricinine is present in crude preparations of ricin, and it can be found in human urine after a lethal exposure to ricin.     

Ricinus communis Intoxications in Human and Veterinary Medicine-A Summary of Real Cases

Accidental and intended Ricinus communis intoxications in humans and animals have been known for centuries but the causative agent remained elusive until 1888 when Stillmark attributed the toxicity to the lectin ricin. Ricinus communis is grown worldwide on an industrial scale for the production of castor oil. As by-product in castor oil production ricin is mass produced above 1 million tons per year. On the basis of its availability, toxicity, ease of preparation and the current lack of medical countermeasures, ricin has gained attention as potential biological warfare agent. The seeds also contain the less toxic, but highly homologous Ricinus communis agglutinin and the alkaloid ricinine, and especially the latter can be used to track intoxications. After oil extraction and detoxification, the defatted press cake is used as organic fertilizer and as low-value feed. In this context there have been sporadic reports from different countries describing animal intoxications after uptake of obviously insufficiently detoxified fertilizer. Observations in Germany over several years, however, have led us to speculate that the detoxification process is not always performed thoroughly and controlled, calling for international regulations which clearly state a ricin threshold in fertilizer. In this review we summarize knowledge on intended and unintended poisoning with ricin or castor seeds both in humans and animals, with a particular emphasis on intoxications due to improperly detoxified castor bean meal and forensic analysis.     

Surface plasmon resonance detection of ricin and horticultural ricin variants in environmental samples

A rapid, sensitive and robust immunoassay based on a commercial surface plasmon resonance (SPR) instrument (Biacore X) was developed for the detection of ricin in environmental samples. A total of 10 monoclonal antibodies were evaluated for their ability to recognise both a commercial ricin and horticultural ricin variants extracted from six different cultivars of Ricinus communis. Two suitable antibodies (7G12 and TFTA) were identified because of their strong affinity to all six ricin variants. The antibody 7G12 was used as the capture ligand in the SPR system. The assay was linear over a wide range of ricin concentrations (up to at least 750ng/ml) with a limit of detection of 0.5ng/ml. The assay was highly reproducible (coefficient of variation was less than 5%), and was able to detect all six ricin variants and environmental samples.     

Colloidal gold-based immunochromatographic assay for detection of ricin.

A rapid immunochromatographic assay was developed to detect ricin. The assay was based on the sandwich format using monoclonal antibodies (Mabs) of two distinct specificities. One anti-ricin B chain Mab (1G7) was immobilized to a defined detection zone on a porous nitrocellulose membrane, while the other anti-ricin A chain Mab (5E11) was conjugated to colloidal gold particles which served as a detection reagent. The ricin-containing sample was added to the membrane and allowed to react with Mab (5E11)-coated particles. The mixture was then passed along the porous membrane by capillary action past the Mab (1G7) in the detection zone, which will bind the particles that had ricin bound to their surface, giving a red color within this detection zone with an intensity proportional to ricin concentration. In the absence of ricin, no immunogold was bound to the solid-phase antibody. With this method, 50 ng/ml of ricin was detected in less than 10 min. The assay sensitivity can be increased by silver enhancement to 100 pg/ml.     

A Simple,Fast and Portable Method for Electrochemical Detection of Adenine Released by Ricin Enzymatic Activity

International authorities classify ricin toxin present in castor seed as a potential agent for use in bioterrorism. Therefore, the detection, identification, and characterization of ricin in various sample matrices are considered necessary actions for risk assessment during a suspected exposure. This study reports a portable electrochemical assay for detecting active ricin based on the adenine electro-oxidation released from herring sperm DNA substrate by its catalytic action. Also, kinetic parameters were calculated, and the values were K_m of 3.14 µM and K_cat 2107 min⁻¹. A linear response was found in optimized experimental conditions for ricin concentrations ranging from 8 to 120 ng/mL, and with a detection limit of 5.14 ng/mL. This proposed detection strategy emphasizes the possibility of field detection of active ricin in food matrices and can be applied to other endonucleolytic activities.     

Purification,characterization and toxicity profile of ricin isoforms from castor beans

The castor seed contains the toxin ricin, one of the most poisonous naturally occurring toxins. The whole of the plant is poisonous, however the seeds are considered the major source of ricin. Ricin exists in different forms in beans of different origin. We investigated the presence of ricin in different isoforms and elucidate some of their structural and biological features isolated from the castor seeds. The isoforms were sub fractionated into ricin I, II and III by chromatography. Their molecular weights lie between 60–65 kDa with difference in their relative electrophoretic mobility. An acidic native PAGE of ricin isoforms at pH 2.9 was performed. Ricin I, II and III are highly cytotoxic against Vero cell line with IC₅₀ values of 60, 30 and 8 ng/ml respectively. Difference in cytotoxicity of isoforms was confirmed through hemagglutination assay, ricin III caused high degree of hemolysis. The preliminary in vivo toxicity studies showed that ricin III is highly toxic. Immunological studies revealed that anti-ricin I and II antibodies are cross reactive with all the ricin variants, whereas the anti-ricin III antibody is highly specific. The present study shows that anti-ricin I and II antibodies can be used for detection of entire ricin isoforms.     

Simultaneous Detection of Ricin and Abrin DNA by Real-Time PCR (qPCR)

Ricin and abrin are two of the most potent plant toxins known and may be easily obtained in high yield from the seeds using rather simple technology. As a result, both toxins are potent and available toxins for criminal or terrorist acts. However, as the production of highly purified ricin or abrin requires sophisticated equipment and knowledge, it may be more likely that crude extracts would be used by non-governmental perpetrators. Remaining plant-specific nucleic acids in these extracts allow the application of a real-time PCR (qPCR) assay for the detection and identification of abrin or ricin genomic material. Therefore, we have developed a duplex real-time PCR assays for simultaneous detection of ricin and abrin DNA based on the OmniMix HS bead PCR reagent mixture. Novel primers and hybridization probes were designed for detection on a SmartCycler instrument by using 5′-nuclease technology. The assay was thoroughly optimized and validated in terms of analytical sensitivity. Evaluation of the assay sensitivity by probit analysis demonstrated a 95% probability of detection at 3 genomes per reaction for ricin DNA and 1.2 genomes per reaction for abrin DNA. The suitability of the assays was exemplified by detection of ricin and abrin contaminations in a food matrix.     

双抗体夹心酶联免疫法检测不同样品中的蓖麻毒素

目的应用酶联免疫吸附分析方法（ELISA）检测待测样品中的蓖麻毒素。方法用蛋白G亲和层析柱纯化蓖麻毒素单克隆抗体（4C13,3D74,5E4和5H6）,以3D74及辣根过氧化物酶（HRP）标记的4C13建立的双抗体夹心ELISA对含有蓖麻毒素的多种样品进行检测。结果抗蓖麻毒素的单克隆抗体经亲和层析纯化后具有较高的蛋白纯度,应用HRP标记的4C13与3D74建立双抗体夹心ELISA,对于溶解于磷酸缓冲液中的蓖麻毒素标准品的检测灵敏度可达2．5μg·L^-1;对于土壤、面粉、牛奶、咸菜汁、雪碧、可乐和腐乳汁．中的蓖麻毒素样品检测的灵敏度为2．5-5．0μg·L^-1;与磷酸缓冲液样品相比较,含有相同浓度蓖麻毒素的小鼠和人血清样品ELISA的阳性结果明显减弱．结论双抗体奕心酶联免疫法能够有效用于含有蓖麻毒素样品的检测分析。     

蓖麻碱的生物活性研究与应用开发前景

蓖麻碱是蓖麻中的主要毒素之一,具有一定的生物活性。在杀虫方面,对天幕毛虫、桃蚜和小菜蛾等3种害虫有不同程度的杀灭作用;在药理方面,蓖麻碱具有一定的肝保护作用和中枢神经兴奋作用,低剂量时具有改善记忆的效果,较大剂量时可作为工具药,用于制备癫痫动物模型来筛选抗惊厥药。     

Detection of ricin contamination in ground beef by electrochemiluminescence immunosorbent assay

Ricin is a highly toxic protein present in the seeds of Ricinus communis (castor), grown principally as a source of high quality industrial lubricant and as an ornamental. Because ricin has been used for intentional poisoning in the past and could be used to contaminate food, there is a need for analytical methodology to detect ricin in food matrices. A monoclonal antibody-based method was developed for detecting and quantifying ricin in ground beef, a complex, fatty matrix. The limit of detection was 0.5 ng/g for the electrochemiluminescence (ECL) method and 1.5 ng/g for enzyme-linked immunosorbent assay (ELISA). The detection of nanogram per gram quantities of ricin spiked into retail samples of ground beef provides approximately 10,000-fold greater sensitivity than required to detect a toxic dose of ricin (>1 mg) in a 100 g sample.