Adulteration of Ginkgo biloba products and a simple method to improve its detection

Extracts of ginkgo (Ginkgo biloba) leaf are widely available worldwide in herbal medicinal products, dietary supplements, botanicals and complementary medicines, and several pharmacopoeias contain monographs for ginkgo leaf, leaf extract and finished products. Being a high-value botanical commodity, ginkgo extracts may be the subject of economically motivated adulteration. We analysed eight ginkgo leaf retail products purchased in Australia and Denmark and found compelling evidence of adulteration with flavonol aglycones in three of these. The same three products also contained genistein, an isoflavone that does not occur in ginkgo leaf.Although the United States Pharmacopeia – National Formulary (USP-NF) and the British and European Pharmacopoeias stipulate a required range for flavonol glycosides in ginkgo extract, the prescribed assays quantify flavonol aglycones. This means that these pharmacopoeial methods are not capable of detecting adulteration of ginkgo extract with free flavonol aglycones.We propose a simple modification of the USP-NF method that addresses this problem: by assaying for flavonol aglycones pre and post hydrolysis the content of flavonol glycosides can be accurately estimated via a simple calculation. We also recommend a maximum limit be set for free flavonol aglycones in ginkgo extract.     

Investigation of biologically active components in ginkgo leaf products on the Japanese market

The rapid and highly separative ultra high-performance liquid chromatography (UHPLC)-UV method was adopted and validated to investigate the flavonol glycoside compositions in ginkgo leaf products on the Japanese market. The result indicates that certain products contained amounts of flavonol glycosides approximately equivalent to the medicinal product. Additionally, we examined the correlations between the total amount of flavonol glycosides and of terpene lactones in various ginkgo leaf products.     

Liquid chromatography-atmospheric pressure chemical ionisation/mass spectrometry: a rapid and selective method for the quantitative determination of ginkgolides and bilobalide in ginkgo leaf extracts and phytopharmaceuticals

In order to evaluate the composition of active constituents in phytopharmaceutical preparations, valid analytical methods are required. For the determination of the active terpene constituents of Ginkgo biloba (the ginkgolides and bilobalide), a liquid chromatography-mass spectrometry (LC-MS) method has been developed using atmospheric pressure chemical ionisation (APCI) in the negative ion mode. This detection mode was found to be much more sensitive and selective compared to UV; indeed the ginkgo terpene trilactones lack strong UV chromophores and flavonoids interfere with their UV detection. LC-APCI/MS detection allowed a considerable reduction in analysis time when compared to LC-UV, because LC resolution was only needed between the pair of isomers ginkgolide B and ginkgolide J. All compounds were selectively detected by single ion monitoring of their specific deprotonated molecules [M-H]-. The samples were directly injected without pre-purification, and a fast gradient was applied, reducing the total time of analysis to 14 min. With this method, the ginkgo terpene trilactones were detected on-line in the picogram range. Several commercial ginkgo preparations on the Swiss market were analysed, and the ginkgolide and bilobalide contents were evaluated using the method described.     

Hydrophilic interaction ultra-performance liquid chromatography coupled with triple-quadrupole tandem mass spectrometry for highly rapid and sensitive analysis of underivatized amino acids in functional foods

This work presented a new analytical methodology based on hydrophilic interaction ultra-performance liquid chromatography coupled with triple-quadrupole tandem mass spectrometry in multiple-reaction monitoring mode for analysis of 24 underivatized free amino acids (FAAs) in functional foods. The proposed method was first reported and validated by assessing the matrix effects, linearity, limit of detections and limit of quantifications, precision, repeatability, stability and recovery of all target compounds, and it was used to determine the nutritional substances of FAAs in ginkgo seeds and further elucidate the nutritional value of this functional food. The result showed that ginkgo seed turned out to be a good source of FAAs with high levels of several essential FAAs and to have a good nutritional value. Furthermore, the principal component analysis was performed to classify the ginkgo seed samples on the basis of 24 FAAs. As a result, the samples could be mainly clustered into three groups, which were similar to areas classification. Overall, the presented method would be useful for the investigation of amino acids in edible plants and agricultural products.     

银杏DNA提取及RAPD分析

采用SDS裂解液和苯酚/氯仿/异戊醇提取液从银杏叶中提取银杏总DNA,并进行DNA样品分光光度测定和琼脂糖凝胶电泳分析。通过引物筛选和反应参数优化,选用3个随机引物对DNA样品进行RAPD扩增,获得较为清晰并有一定多态性差异的扩增谱带,初步摸索出适合于以银杏叶为材料的DNA提取方法和RAPD扩增程序,为研究银杏遗传多态性及种质资源研究提供一种实用的分析方法。     

A capillary electrophoretic method for monitoring the presence of alpha-tubulin in nuclear preparations

A sensitive capillary electrophoretic method was developed to detect the presence of alpha-tubulin, a microtubular cytoskeletal component, in isolated nuclear preparations. These preparations are treated with anti-alpha-tubulin primary mouse antibodies and then stained with a fluorescently labeled anti-mouse IgG antibody. The stained preparation is then analyzed by capillary electrophoresis with laser-induced fluorescence detection, a technique that allows for sensitive detection of fluorescently labeled species. Using this method, it is feasible to count individual subcellular aggregates containing alpha-tubulin (SATs), estimate the number of alpha-tubulin molecules per SAT, determine the cumulative intensity of all SATs as an estimate of the relative level of alpha-tubulin in a preparation, and obtain their apparent electrophoretic mobility distribution. The method was validated by comparing SATs from untreated cells with those from colchicine-treated cells. Since colchicine is a microtubule-disrupting agent, treatment reduced the number of SATs per cell as well as the cumulative intensity of all SATs in a preparation. In contrast, the apparent electrophoretic mobility distribution was not influenced by colchicine treatment, suggesting that this parameter is not strongly dependent on the alpha-tubulin content. Given the zeptomolar sensitivity of laser-induced fluorescence detection and the widespread availability of antibodies, the approach used here represents an improvement in the detection of cytoskeletal impurities in subcellular fractions.     

银杏外种皮提取物对病原菌﹙Cylindrocladium colhounii﹚的抑制作用

采用菌丝生长抑制法,研究了银杏外种皮3种不同提取物对病原菌（Cylindrocladium colhounii）的抑制作用并测定了这些提取物的MIC/MBC。结果表明,在相同实验天数内,抑菌效果（最小抑菌率）由高到低依次为银杏外种皮乙醇提取物（37.4%）、石油醚提取物（23.7%）和新鲜外种皮汁液（18.4%）。当提取物的浓度提升到一定程度时,三者的抑菌率均可达到100%;三种提取物的MIC/MBC分别为：86.25/86.25,172.5/276,293.25/345 mg·mL^-1。可见银杏外种皮具有明显抑制该病原菌生长的作用,且抑菌成分在乙醇中的溶解度较大。这为进一步研究该抑菌成分乃至生物农药的开发奠定了基础。     

核酸层析法转基因快速检测体系的建立及应用

随着我国转基因研究及产业化进程提速,转基因检测的重要性日益凸显,因而快速、高效的分子检测新方法的研发具有重要的生产实践及科研意义。在转基因检测中,常规PCR技术具有检测范围广的特点,但较为费时费力,且对实验条件要求较高;而胶体金蛋白试纸法检测方便、快捷,但可检范围窄。基于此,建立了一套基于核酸层析法快速转基因检测的方法体系。将经液氨研磨后的样品通过一管法提取DNA后直接进行PCR扩增,再将PCR扩增产物滴加到胶体金检测卡上,通过直接观察胶体金卡条显色状况进行结果判别。此方法最终检验出玉米内参基因ZSSⅡB及Zein、大豆内参基因SPS、水稻内参基因Lectin,转基因元件启动子CaMV35S及终止子NOS,以及抗虫基因Cry1Ab/1Ac、抗除草剂基因Bar、Pat、CP4-EPSPS及选择标记基因NPTⅡ、Pmi等外源基因;并成功检出特异性转基因事件Mir604及Bt11。研究获得的核酸层析检测方法具有灵敏度高、省时省力且对检测条件要求低等优点,集PCR法与蛋白试纸检测法二者优势于一身,可广泛应用于转基因产品的精确快速检测,为我国转基因生物安全监管提供了良好的技术支撑。     

Lumicolchicine as a Tool in the Study of Plant Microtubules: Some Biological Effects of Sequential Products Formed During Phototransformations of Colchicine

Lumicolchicine has been used as a control in studies of theeffects of colchicine on microtubule-dependent cellular processes.Prolonged UV-phototransformation of colchicine yields a seriesof lumi-derivatives with very different UV-absorption spectraand biological effects. Intermediate products strongly inhibitthe growth of cress seedlings. However, both root and hypocotylrespond with very different sensitivity to colchicine and tointermediate lumicolchicines prepared as described in the literature.Complete phototransformation yields a derivative with no detectablebiological activity and, therefore, of no value in distinguishingtoxic from microtubulebased cellular responses to colchicine.     

Colchicine inhibits ionophore-induced formation of leukotriene B4 by human neutrophils: the role of microtubules

Neutrophils which ingest particles (serum-treated zymosan, monosodium urate crystals) or are exposed to calcium ionophore A23187 generate leukotriene B4 (LTB4). Earlier work has shown that cells exposed to colchicine before exposure to monosodium urate crystals produce less LTB4; the formation of 5-HETE is unaffected. To determine whether inhibition by colchicine of LTB4 generation was stimulus-specific and was mediated by microtubule integrity, the effects of colchicine (10 microM, 60 min) on the release of lipoxygenase products from neutrophils exposed to ionophore A23187 (10 microM, 5 min) were examined. In the presence of exogenous arachidonic acid (100 microM, 15 min), colchicine decreased LTB4 to 48% +/- 11.7 of control and 5-HETE to 60.5% +/- 5.7 of control (mean +/- SEM); 15-HETE was also decreased to 61% +/- 10.3 of control. In the absence of exogenous arachidonate, LTB4 was decreased to 22.2% +/- 11.7 of control and 5-HETE to 13% +/- 4.8 of control. Lumicolchicine did not significantly affect formation of 5-HETE or LTB4. However, vinblastine sulfate (20 microM, 60 min), another microtubule-disruptive agent, decreased the formation of both 5-lipoxygenase products. The effects of colchicine and vinblastine were not due to impairment of cell viability because the release of cytoplasmic lactic dehydrogenase was unaffected. Ultrastructural analysis of centriolar microtubules showed that decrements in microtubule numbers of colchicine- and vinblastine-treated cells paralleled decrements in 5-lipoxygenase products. These pharmacologic manipulations suggested that functional microtubules might be required for optimal lipoxygenase activity. Consequently, we prepared neutrophil-derived cytoplasts, devoid of an intact microtubule system. No significant decreases in the 5- or 15-lipoxygenase products were found when cytoplasts were exposed to colchicine in the presence of exogenous arachidonate and A23187. The data show that colchicine inhibits the formation of lipoxygenase products from neutrophils stimulated with A23187, most likely via its effect on microtubules, the integrity of which appears necessary for full expression of 5- and 15-lipoxygenases.     

Toxicity of colchicine towards a marine fish,Dicentrarchus labrax

1. Hematology and blood biochemistry parameters were examined in order to determine the metabolic or endocrine disturbances caused by colchicine intoxication in sea bass (Dicentrarchus labrax) aged 1 + . The detection of non-lethal effects of colchicine requires the use of a broad concentration range (6.25–1020 mg 1⁻¹).2. The 48-hr lc₅₀, was 1020 mg 1⁻¹, a value which is close to that previously reported in the reference fresh water fish Brachydanio rerio. Exposure to 50 and 125 mg ⁻¹ colchicine for 48 hr resulted in 100% mortality one week after treatment.3. Colchicine involves the concomitant increase of packed cell volume and hemoglobin concentration in whole blood, in the concentration range of 12.5–1020 mg 1⁻¹.4. The activity of erythrocyte antioxidant enzymes undergoes low amplitude variations after 48-hr exposure to colchicine, regardless of concentration. Two days after recovery, decreased SOD, and increased catalase and peroxidase activities were shown.     

超临界流体萃取——高效液相色谱法测定百合中秋水仙碱

分别用超临界二氧化碳流体和有机溶剂萃取百合中的秋水仙碱,然后用高效液相色谱法直接测定萃取物中秋水仙碱的含量,从而测得百合中秋水仙碱的含量。超临界流体萃取的条件是：用乙醇作提携剂,萃取压力为18MPa,萃取温度为40℃,高效液相色谱测定条件为：ODS柱,甲醇：磷酸二氢钾溶液作流动相,检测波长为220nm,此法快速,简便,准确,可应用于秋水仙碱原料,制剂及其它植物中秋水仙碱含量的测定。     

Extraction,development and validation of HPLC-UV method for rapid and sensitive determination of colchicine from Colchicum autumnale L. Bulbs

Colchicum autumnale L. also known as the autumn crocus, contains colchicine alkaloid having antifungal properties. The tuber of this plant is rich in terms of colchicine. In this research an ultrasound-assisted extraction (UAE) method was optimized for the extraction of colchicine from Colchicum autumnale L. bulbs before high-performance liquid chromatography with UV detection (HPLC-UV). Optimization of various extraction parameters was performed using response surface methodology (RSM) to evaluate the maximum colchicine yield from Colchicum autumnale L. bulbs. The Box-Behnken design (BBD) and RSM were used to investigate the effect of three key parameters (extraction time (20–60?min), extraction temperature (40–80?°C) and ultrasound power (500–700?W) on extraction efficiency. The variance analysis suggested that the dependent response variable of yield of colchicine may be expressed by a quadratic polynomial model. The optimal theoretical extraction conditions were found to be an ultrasonication power of 602.4?W, an extraction time of 42?min and a temperature of 64?°C. Under these conditions, the optimum foreseen yield was 0.237%. The experimental colchicine yield obtained by following the optimized conditions was found to be 0.238%. These values are very well compatible with each other.     

Biomechanical measurement and analysis of colchicine-induced effects on cells by nanoindentation using an atomic force microscope

Colchicine is a drug commonly used for the treatment of gout, however, patients may sometimes encounter side-effects induced by taking colchicine, such as nausea, vomiting, diarrhea and kidney failure. In this regard, it is imperative to investigate the mechanism effects of colchicine on biological cells. In this paper, we present a method for the detection of mechanical properties of nephrocytes (VERO cells), hepatocytes (HL-7702 cells) and hepatoma cells (SMCC-7721 cells) in culture by atomic force microscope (AFM) to analyze the 0.1 μg/mL colchicine-induced effects on the nanoscale for two, four and six hours. Compared to the corresponding control cells, the biomechanical properties of the VERO and SMCC-7721 cells changed significantly and the HL-7702 cells did not considerably change after the treatment when considering the same time period. Based on biomechanical property analyses, the colchicine solution made the VERO and SMCC-7721 cells harder. We conclude that it is possible to reduce the division rate of the VERO cells and inhibit the metastasis of the SMCC-7721 cells. The method described here can be applied to study biomechanics of many other types of cells with different drugs. Therefore, this work provides an accurate and rapid method for drug screening and mechanical analysis of cells in medical research.     

The effect of colchicine on translocation of incorporated [3H] proline in Hymenolepis diminuta

Portions of adult Hymenolepis diminuta were exposed to a fixed concentration of colchicine (5 X 10(-4) M) in order to determine its effect upon incorporation of [3H] L-proline. Additional studies of the effect of colchicine upon tegumental morphology were performed. Autoradiographs showed a significant decrease in amount of incorporated label in the distal tegument of colchicine tissue and a heavy accumulation of label in the parenchyma. Radioassays indicated that the effect of colchicine on proline-incorporated protein was qualitative rather than quantitative suggesting that colchicine inhibits translocation in the tegument. It was hypothesized that microtubules within the internuncial processes facilitate movement of cell products from tegumentary cytons to the body surface.     

A self-probing primer PCR method for detection of very short DNA fragments

A novel self-probing primer method that based on the fluorescence resonance energy transfer principle is designed to detect DNA fragments of approximately 40 bp. Four self-probing primer reaction systems were developed to target a maize endogenous reference gene (HMG), a soybean endogenous reference gene (Lectin), a rapeseed endogenous reference gene (CruA) and an exogenous gene 5-enolpyruvylshikimate-3-phosphate synthase (ctp2-cp4epsps). These four primer systems were confirmed to have a high level of inter-species specificity and good intra-species stability. The limit of detection was estimated to be 10 copies of haploid genomes for all four assays. The validation results demonstrated that the self-probing primer methods are able to quantify the DNA amount in the different samples with good sensitivity and precision. When highly processed food products were assayed, the self-probing primer method produced better results than the TaqMan probe method. Overall, the self-probing primer method is suitable for qualitative and quantitative detection of very short DNA targets in samples of different sources.     

六种作物内标准基因开发与检测研究进展

内标准基因（internal reference gene）是指能够区分物种的特异性、具有单一或恒定的低拷贝数、低变异的保守DNA序列。指定作物种类的内标准基因在转基因成分检测、物种及其产品判别等众多领域内作为鉴别其他物种的主要遗传标志,尤其是在转基因产品定量定性检测中,可用于检测样品中的物种来源以及转基因含量,对转基因产品定量定性检测具有重要意义。目前,已有多种作物的内标准基因被开发,其中玉米、油菜和水稻开发的内标准基因数量较多,但仍缺乏更为透彻的比较研究,导致选择与应用较为困难。基于此,根据国内外已有的研究成果,综述了6种检测或鉴定需求较大的作物（包括玉米、大豆、油菜、棉花、水稻和小麦）的内标准基因的研究进展,并对常见作物的内标准基因进行了系统的比较和研究,以期为植物源性、转基因成分及相关检测鉴定提供技术支撑。     

Effects of curcumin and Ginkgo biloba on matrix metalloproteinases gene expression and other biomarkers of inflammatory bowel disease

Treatment of inflammatory bowel disease (IBD) by synthetic active ingredients leads to many side effects. The objective of this study was to manage IBD using natural products as curcumin and Ginkgo biloba. Rats were divided into four groups (control, IBD, curcumin treated, and ginkgo treated). Inflammation was assessed by determination of myeloperoxidase, matrix metalloproteinases, metalloproteinase-1 inhibitor, nitric oxide, hydroxyproline, tumor necrosis factor-alpha, ceruloplasmin, and histopathological scoring. IBD induction significantly increased all measured parameters. Treated groups had significantly lower levels when compared with the IBD group. In conclusion, curcumin and ginkgo were effective in prevention and treatment of IBD.     

高效液相色谱法测定银杏叶提取物中黄酮甙含量

用高效液相色谱法测定银杏叶提取物经酸水解后黄酮甙元含量．采用Ｃ１８柱,甲醇：水：磷酸（５５：４４．５：０．５）为流动相,检测波长３７０ｎｍ,方法回收率９５．１％～１０３．２％,变异系数（ＣＶ）３．１７％,１０批样品含测平均为２５．４％．     

Improvement in Salmonella detection in milk and dairy products: comparison between the ISO method and the Oxoid SPRINT Salmonella test

The Oxoid SPRINT Salmonella test was compared with the ISO method (ISO 6579: 1993) for the detection of Salmonella in milk and dairy products. Samples were artificially contaminated, in some cases with sublethally injured salmonellas. Experiments with raw milk, soft cheese made from heat-treated milk (mould-ripened and with smear) and soft cheese with smear made from raw milk showed no significant differences between the SPRINT and ISO methods. With dried milk products and mould-ripened soft cheese made from raw milk the reference method gave significantly more positive results. The addition of ferrioxamine E to pre-enrichment (ISO) or pre-enrichment/enrichment broth (SPRINT test) did not improve Salmonella detection.