Secondary metabolite credentials ofEvolvulus alsinoides by high performance thin layer chromatography(HPTLC)

Plants and plant-based products are the bases of many modern pharmaceuticals that are current in use today for various diseases.The aim of the study was to investigate the biochemical constituents and high performance thin layer chromatography（HPTLC） finger printing of the ethanolic extract of Evolvulus alsinoides.Phytochemical screening was done by standard procedures and HPTLC method was also established to analyze alkaloids,flavonoids and phenolic compounds from the ethanolic extract of Evolvulus alsinoides.Preliminary phytochemical screening showed that ethanol extracted more secondary metabolites than other solvents.HPTLC fingerprinting analysis showed the presence of various alkaloids,flavonoids and phenols（quercetin） in the ethanolic extract.It can be concluded that Evolvulus alsinoides may serve as a source of potent antioxidants that may be used in the prevention of various diseases such as cancer,diabetes and cardiovascular diseases due to the presence of phenolic compounds.HPTLC finger print of Evolvulus alsinoides may be useful in the differentiation of the species from adulterants and act as a biochemical marker for this medicinally important plant in the pharmaceutical industry and plant systematic studies.

Stability indicating high performance thin-layer chromatographic method for simultaneous estimation of pantoprazole sodium and itopride hydrochloride in combined dosage form

A specific, precise and stability indicating high-performance thin-layer chromatographic method for simultaneous estimation of pantoprazole sodium and itopride hydrochloride in pharmaceutical formulations was developed and validated. The method employed TLC aluminium plates precoated with silica gel 60F254 as the stationary phase. The solvent system consisted of methanol：water：ammonium acetate; 4.0：1.0：0.5 （v/v/v）. This system was found to give compact and dense spots for both itopride hydrochloride （Rf value of 0.55__＋0.02） and pantoprazole sodium （Rf value of 0.85＋0.04）. Densitometric analysis of both drugs was carried out in the reflectance- absorbance mode at 289 nm. The linear regression analysis data for the calibration plots showed a good linear relationship with R2=0.9988___0.0012 in the concentration range of 100--400 ng for pantoprazole sodium. Also, the linear regression analysis data for the calibration plots showed a good linear relationship with R2=0.9990_＋0.0008 in the concentration range of 200-1200 ng for itopride hydrochloride. The method was validated for specificity, precision, robustness and recovery. Statistical analysis proves that the method is repeatable and selective for the estimation of both the said drugs. As the method could effectively separate the drug from its degradation products, it can be employed as a stability indicating method.

Evaluation of quantitative variation of secondary metabolites in Bergenia ciliata(Haw.)using high performance thin layer chromatography

Dear Editor： Therapeutically active metabolite contents in a med- icinal plant vary in nature, which may impact on its therapeutic efficacy. Bergenia （Saxifragaceae） is an evergreen perennial herb widely distributed in Central and East Asia with about 30 species reported worldwide. It grows at a range of altitudes from the Khasia hills at 400 feet to the temperate Himalayas from Kashmir to Bhutan at 7,000-10,000 feet. Its distribution over a wide range of altitudinal zones makes it a good candidate for studying variations in its metabolic profiles under different climatic conditions. Bergenin （C-glycoside of 4-O-methyl gallic acid） has been identified as a potent active secondary metabolite in Bergenia and other therapeutically active constituents including, among others, gallic acid （3,4,5 trihydroxybenzoic acid）, （＋） catechin, and gallicin （Fig. 1）.

芹菜提取物中芹菜素的HPLC与HPTLC定量分析研究

通过采用高效液相色谱(HPLC)、紫外光谱和黄酮颜色特征反应对芹菜提取物主要成分芹菜素进行定性鉴定,同时建立定量测量芹菜素的高效薄层色谱(HPTLC)方法。结果表明:芹菜提取物的主要成分是黄酮醇-芹菜素,与芹菜素标准品具有相同的光谱和色谱性质;采用G60高效硅胶板,以氯仿-甲醇-水(18:2.3:0.35)为展开剂,芹菜素标样与试样有相同的Rf值为0.65;用最小二乘法作线性回归,芹菜素含量与斑点峰面积的标准曲线回归方程:y=15138x+7594.8,R2=0.993,线性范围:0.332～0.996μg/斑点。精密度实验RSD(n=5)=1.33%,平均回收率97.82%。

HPTLC法测定芹菜提取物中芹菜素的含量

本研究建立了测定芹菜素的高效薄层色谱（HPTLC）法。采用CAMAG全自动薄层色谱仪，高效硅胶G60薄层板，展开剂为氯仿-甲醇-水（18：2．3：0．35），测定波长为345nm，测定了芹菜提取物中芹菜素含量，在0．116～0．696μg／ml范围内呈良好的线性关系（R^2〉0．99）。该方法简单、快速，分离度好，灵敏度高，适用于芹菜提取物中芹菜素的测定。

茶叶中儿茶素薄层色谱分离所用展开剂的优化

室温条件下,以硅胶板为固定相,用5种展开剂(体积比9:9:2的甲苯-丙酮-甲酸、5.0:10.0:1.0:0.5的甲苯-甲酸乙酯-甲醇-甲酸、8.0:5.0:15.0:0.3的甲苯-三氯钾烷-丙酮-甲酸、12.5:7.5:1.8:0.6的三氯钾烷-丙酮-甲醇-水、12.5:7.5:1.8:0.6的三氯钾烷-丙酮-甲酸-水)对茶叶中的儿茶素进行薄层色谱分离,筛选出其中较优的展开剂后,进一步优化其配比,并用其分离绿茶和白茶提取物中的儿茶素以进行验证。结果表明,以体积比12.5:7.5:2.2的三氯甲烷-丙酮-甲酸为展开剂,茶叶内主要的4种儿茶素单体(EGCg、EGC、ECg、EC)可得到良好分离,绿茶与白茶提取物中的儿茶素能较好地得以分离。

高效薄层析进行农药吸附态光解的研究

研究了绿麦隆和消草醚２种除草剂在硅胶Ｇ高效薄层上的光稳定性，提出应用高效薄层析技术作为农药吸附态光解的一种试验方法。在高效薄层上直接点样，同一薄层上包括照光、黑暗对照和定量曲线样品，原始剂量４００—８００ｎｇ·１×５ｍｍ斑点－１，照光后直接展开和扫描定量测定，最低检测量可达２０ｎｇ·斑点－１。本方法无须对样品进行提取、净化等处理，具有快速高效、重复性好，且可直观光解产物等优点。

活血止痛制剂中三七的专属性鉴别方法研究

目的:建立活血止痛散和活血止痛胶囊中三七的专属性鉴别方法,并采用高分离度快速液相色谱-三重串联四极杆质谱(RRLC-QQQ/MS)对结果加以验证。方法:采用高效薄层色谱法研究三七与其混伪品人参、红参、三七茎叶的特征条带。以高效硅胶G薄层板为固定相,二氯甲烷-无水乙醇-水(70∶45∶6.5)展开,10%硫酸乙醇溶液显色,105℃加热至条带清晰。分别置紫外光灯(365 nm)和日光下检视。RRLC-QQQ/MS分析采用Agilent Rapid Resolution HT SB-C18 (2.1 mm×100 mm,1.8μm)色谱柱,以5 mmol·L-1醋酸铵溶液-乙腈为流动相梯度洗脱。离子化模式为ESI-,碎裂电压100 V。结果:三七可检出三七皂苷R1,未检出人参皂苷Rb3、人参皂苷Rf。人参和红参可检出人参皂苷Rb3和人参皂苷Rf,未检出三七皂苷R1。三七茎叶可检出人参皂苷Rb3,未检出三七皂苷R1或人参皂苷Rg1。以三七对照药材、三七皂苷R1(应检出)和人参皂苷Rb3(不得检出)为对照,可实现活血止痛制剂中三七的专属性鉴别和人参(红参)或三七茎叶的非法掺杂投料检查。6个厂家的93批样品均未发现人参、红参或三七茎叶冒充三七非法投料。RRLC-QQQ/MS与高效薄层分析结果一致。结论:该方法简便、快速、灵敏、准确,可为含三七中成药的质量控制提供参考。

高效薄层层析法测定罗布麻烟中罗布麻浸膏的含量

利用高效薄层层析法,以罗布麻浸膏中的槲皮素为标准,测定罗布麻烟中罗布麻浸膏的含量。以甲苯:甲酸:乙酸乙酯:正己烷(2:0.2:1.5:0.2)作展开剂。可使得药烟中槲皮素与其它成分很好分离。分离后,用薄层扫描仪于波长410nm,参比波长750 nm扫描定量。本法需样品少,快速简便、灵敏准确。可适用于药烟生产厂家化验室检测使用。

高效薄层色谱法快速测定由药用植物内生真菌产生的紫杉醇(英文)

Taxol is an important anticancer drug used widely in the clinical field.In this study,some endophytic fungi were isolated from selected medicinal plants,and were screened for their potential in the production of taxol,using a rapid separation technique of high performance thin layer chromatography(HPTLC).Of the 20 screened fungi,only 13 fungal species produced taxol in the artificial culture medium.The results of HPTLC showed that the 13 fungal species had identical ultraviolet(UV) characteristics,positive reactivity with a spray reagent,yielding a blue spot,which turned to dark gray after 24 hours,and had Rf values identical to that of the authentic taxol.The amount of taxol was also quantified by comparing the peak area and the peak height of the fungal samples with those of authentic taxol.

高效薄层色谱-气相色谱-四级杆质谱法检测母乳中脂肪酸分布

母乳中肪酸组成对婴儿后天健康成长具有重要影响。大量测定和发现国人母乳中脂肪酸组成及分布信息不仅有助于制造更适合婴儿需要的母乳代用品,而且有助于指导通过母体补充的方式促进婴儿摄入具有功能特性的脂肪酸。样品经高效薄层色谱分离,甲酯化后,以SP2560柱(100 m×0.25 mm ID 0.20μm)分离,四级杆质谱定性定量,建立了高效薄层色谱-气相色谱-四级杆质谱(GC-MS)分析母乳中脂肪酸组成及分布的方法。四级杆质量扫描范围为50~500,扫描速率为20 000 amu/s。方法能够获得母乳中甘油三酯、甘油二酯、甘油磷脂中的脂肪酸组成及总脂肪酸分布,检出限符合方法学要求,为大批量调查国人母乳脂溶性成分的研究奠定技术基础。

烫王不留行有效成分及含量的测定

为了规范王不留行的炮制方法,并为新版《安徽省中药饮片炮制规范》的修订提供数据参考。采用薄层色谱法和高效液相色谱法对湖南和湖北共10批样品的有效成分和含量进行测定。经过测定,10批样品中均含有效成分王不留行黄酮苷,且含量均保持在1.7%以上,湖北产地的比湖南产地的含量偏高。本次实验得到的数据整体比较稳定,可以作为《安徽省中药饮片炮制规范》修订的参考依据,为后续烫王不留行的检测及使用提供依据。

In vitro Safety Evaluation and Anticlastogenic Effect of BacoMind^(TM) on Human Lymphocytes

Objective BacoMind^TM （BM） is a standardized extract of Bacopa monnieri, which belongs to the family Scrophulariaceae and is a creeping annual plant found throughout the Indian subcontinent. It has been used by Ayurvedic medicinal practitioners in India for almost 3000 years and is classified as a medharasayana, a substance which improves memory and intellect. With the widespread traditional use as well as scientific validation of Bacopa monnieri for nootropic activity, a bioactive-rich unique phytochemical composition-BacoMind^TM was developed from B. monnieri for use as a cognition and memory enhancing agent. The present study aimed to investigate the in vitro toxicity of this formulation of BacoMind^TM on human lymphocytes and to rule out its possible contribution to mutagenicity. Methods In the present investigation the active ingredients present in BM were identified and quantified by high performance liquid chromatography （HPLC） and high performance thin-layer chromatography （HPTLC）. Antioxidant and anticlastogenic properties of BM were studied in vitro with and without metabolic activation. Doses of BM were chosen on the basis of mitotic index （MI） and cytokinesis-block proliferation index （CBPI）. Clastogenicity assays were performed at 31.2 μg/mL, 62.5 μg/mL, and 125 μg/mL, while the Salmonella reverse mutation assay （Ames test） was performed at doses of 61.72, 185.18, 555.55, 1666.67, and 5000.00 μg/plate. Results HPLC and HPTLC analysis of BM revealed the presence of bacoside A3. bacopaside Ⅰ, bacopaside Ⅱ, jujubogenin isomer of bacopasaponin C, bacosine, luteolin, apigenin, bacosine, and β-sitosterol D glucoside. BM demonstrated significant antioxidant activity. The number of chromosomal aberrations and the frequency of micronuclei induced by BM were not statistically significant up to a dose of 62.5 μg/mL. A subsequent dose of 125 μg/mL prior to metabolic activation induced mild clastogenicity, but it was found to be biologically insignificant as this effect was not seen post metabolic activation. BM also demonstrated a dose-dependent protection against the clastogens used in this study using the above tests for clastogenicity. Maximum protection was observed in presence of metabolic activation. Moreover, BM demonstrated no mutagenic effect on the tested strains, as observed in the Ames test. Conclusion BM protected human lymphocytes against various clastogens. BM also exhibited high antioxidant activity which might be responsible for the observed protective effects against the clastogens since the used clastogens are known to induce their clastogenic effects via production of oxidative radicals.

异丙肾上腺素在硅酸铋离子交换薄层上的选择性分离与测定（英文）

A simple and sensitive method for the separation and determination of isoproterenol from other doping drugs has been developed on thin layers of bismuth silicate,a synthetic inorganic ion exchanger as adsorbent in thin layer chromatography(TLC).A mixture of methanol and 0.1 mol/L formic acid(3∶7,v/v)was employed as the mobile phase.The development time was 32 min.The quantitative measurement were performed with a Camag TLC Scanner-3 at wavelength(λ)of 410 nm.The isoproterenol recovery in this procedure was 98.9%.The linear correlation coefficient was greater than 0.987 1 and the relative standard deviation(RSD)was less than 0.94.The limit of detection(LOD)and limit of quantification(LOQ)were 7.7×10-7mol/L and 3.85×10-6mol/L,respectively.This method has been applied in the determination of isoproterenol in dosage forms and in biological fluids.

基于糖谱法的半夏及其炮制品多糖结构特征分析及水/酶解前后抗氧化活性评价

目的:采用糖谱法,通过部分酸水解和特异性糖苷酶酶解结合高效薄层色谱法(HPTLC)和荧光辅助凝胶电泳法(PACE),分析半夏及其炮制品多糖的糖苷键结构特征,以及其水/酶解前后的抗氧化活性变化。方法:半夏及其炮制品多糖加入淀粉酶除淀粉后,超滤法除去3000 Da以下的小分子成分;精制后的多糖以酸水解和5种特异性糖苷酶酶解制备样品,采用HPTLC和PACE进行分析;以2,2′-联氮-双(3-乙基苯并噻唑啉-6-磺酸)二铵盐(ABTS)和2,2-联苯基-1-苦基肼基(DPPH)自由基清除实验评价多糖水/酶解前后抗氧化活性的变化。结果:通过HPTLC和PACE分析发现,半夏及其炮制品多糖能被β-半乳糖苷酶、β-甘露糖酶、纤维素酶和果胶酶水解,而几乎不能被葡聚糖酶水解,表明半夏及其炮制品的多糖含有β-吡喃半乳糖苷键、β-1,4-甘露糖苷键、β-1,4-葡萄糖苷键和α-1,4-半乳糖醛酸苷键。通过体外抗氧化实验发现,半夏及其炮制品多糖经酸水解和果胶酶酶解后清除ABTS自由基能力减弱,而经纤维素酶,β-半乳糖苷酶和β-甘露糖酶酶解后,清除ABTS自由基能力增强;而半夏及其炮制品多糖经不同水/酶解后,清除DPPH自由基能力均明显增强。结论:该研究通过糖谱法分析了半夏及其炮制品多糖的糖苷键结构特征,并明确了其与抗氧化活性之间的关系,有利于进一步阐明半夏及其炮制品相关功效的物质基础,并为中药多糖结构特征分析提供新的思路与方法。

四级杆飞行时间串联质谱(QTofMS)辅助高效薄层色谱(HPTLC)鉴别乳香中乳香酸

目的建立薄层色谱法鉴别乳香中乳香酸类成分。方法样品经甲醇超声提取,C18固相萃取柱净化后,点样于硅胶GF254高效预制薄层板上,以甲苯-乙酸乙酯-庚烷-无水甲酸(8∶2∶1∶0.3)为展开剂展开,先置紫外灯(254 nm)下检视,再喷以10%硫酸乙醇溶液,并于105℃加热至条斑清晰,置紫外灯(365 nm)下检视。条斑借助QTofMS进行成分识别。结果紫外灯(254 nm)下,埃塞俄比亚乳香和索马里乳香的色谱均可见2个明显的暗条斑,紫外灯(365 nm)下,均可见8个荧光条斑,其特征图谱与其他常见树脂类药材(洋乳香、天然没药、胶质没药、安息香、松香和达玛树脂)区别明显。结论该法简便、专属、准确,适用于乳香的快速鉴别和质量控制。

花椒多糖抗阿尔茨海默病作用及其糖谱法结构特征分析

目的:建立阿尔茨海默病(AD)小鼠模型,评价花椒多糖对AD的治疗作用并采用糖谱法分析其结构特征。方法:采用腹腔注射D-半乳糖联合灌胃三氯化铝建立快速衰老的AD小鼠模型,通过Morris水迷宫实验评价小鼠的学习记忆能力,苏木素-伊红(HE)染色和尼氏染色观察小鼠脑组织病理学状况和神经元损伤情况。对花椒多糖进行酸水解和不同糖苷酶酶解后,采用高效薄层色谱法(HPTLC)和荧光辅助凝胶电泳法(PACE)分析水解产物的结构特征。HPTLC色谱条件为采用硅胶60高效薄层色谱板,点样量5μL,展开剂乙酸乙酯-冰乙酸-水(2∶2∶1),展开2次,苯胺-二苯胺-磷酸溶液显色,在105℃加热10 min后日光下检视。PACE检测条件为34%分离胶和8%浓缩胶,电泳缓冲液为0.1 mol·L^(-1)三羟甲基氨基甲烷(Tris)-硼酸缓冲液(pH 8.2),电泳在0℃环境下进行,上样量3~6μL,用15 mA恒流跑至样品达凝胶前端,紫外灯365 nm下检视。结果:Morris水迷宫实验结果表明,花椒多糖明显改善了AD模型小鼠的学习和记忆能力,逃避潜伏期缩短,穿梭平台次数和目标象限停留时间明显增加。组织病理学实验结果表明,花椒多糖能改善AD小鼠海马组织CA1、CA3区的病理情况和神经元的损伤,且尼氏小体数量明显增加。糖谱法分析结果表明,HPTLC和PACE分析结果基本一致,花椒多糖主要能被纤维素酶和果胶酶催化酶解成小分子糖类,表明其主要含有β-1,4-葡萄糖苷键和α-1,4-半乳糖醛酸苷键,能被葡聚糖酶、β-半乳糖苷酶和β-甘露糖酶少量酶解,表明其含有少量α-1,6-葡萄糖苷键、β-吡喃半乳糖苷键和β-1,4-甘露糖苷键。结论:花椒多糖对AD小鼠有明显治疗作用,其结构中主要含有β-1,4-葡萄糖苷键和α-1,4-半乳糖醛酸苷键,可为中药多糖的结构解析提供有益借鉴。

不同来源乳香的高效薄层色谱数码轮廓指纹图谱分析及基原鉴别

目的:建立乳香药材的高效薄层色谱指纹图谱,并对不同来源乳香药材的指纹图谱进行比较分析。方法:样品经甲醇超声提取,点样于硅胶GF254高效预制薄层板上,以甲苯-乙酸乙酯-庚烷-无水甲酸(8∶2∶1∶0.3)为展开剂展开,先置紫外灯(254nm)下检视并成像,再喷以茴香醛试液,并于105℃加热至条斑清晰,日光下再次成像,图像经CHROMAP软件转换成数码轮廓图并生成共有模式后,进行相似度、聚类和主成分分析。结果:日光下,埃塞俄比亚乳香的指纹图谱由12个特征条斑组成,薄层色谱图像与数码轮廓图谱结合分析比较,可分为埃塞俄比亚乳香模式和索马里乳香模式,不同种的乳香可被鉴别并归类。结论:该法简便、专属、准确,适用于乳香的鉴别和质量控制。

高效薄层层析-病毒覆盖结合法比较细胞表面神经节苷脂与不同动物源性新城疫病毒结合特性

【目的】神经节苷脂是新城疫病毒入侵宿主细胞的受体,但不同动物源性的新城疫病毒利用受体的特性是否存在差异尚不明确。以鹅源新城疫病毒NA-1株和鸡源新城疫病毒F48E9株为研究对象,比较两株病毒受体结合特性的差异。【方法】提取鸡胚和鹅胚成纤维细胞新城疫病毒受体成分——神经节苷脂,用高效薄层层析法比较两种细胞所含神经节苷脂的类型和含量;通过高效薄层层析-病毒覆盖结合法比较两种病毒与不同细胞神经节苷脂的结合特性,最后通过病毒红细胞吸附抑制试验进一步验证神经节苷脂与病毒之间的相互作用关系。【结果】鸡胚和鹅胚成纤维细胞所含的神经节苷脂成分存在明显差异;高效薄层层析-病毒覆盖结合实验结果显示NA-1和F48E9与神经节苷脂的结合模式不同,NA-1主要与神经节苷脂GD1a结合,即包含双SAα2,3Gal末端的神经节苷脂,而F48E9可与更多类型的神经节苷脂结合,从单唾液酸到三唾液酸形式的神经节苷脂如GM1、GD1a、GD1b、GT1b等。【结论】鹅源新城疫病毒NA-1株和鸡源新城疫病毒F48E9株在入侵靶细胞时优先选择利用的受体不同。

高效薄层色谱测定湛江等鞭金藻培养过程脂质变化

【目的】研究湛江等鞭金藻(Isochrysis zhangjiangensis)由氮元素丰富至限制培养过程中脂质成分的变化。【方法】利用高效薄层色谱(HPTLC)分离分析微藻中脂质。【结果】随着培养基中营养物质的消耗,细胞逐渐处于胁迫状态,在这种状态下,细胞大量积累贮存脂质—甘油三脂(TAG),组成生物膜系统的单半乳糖甘油二脂(MGDG)、硫代异鼠李糖甘油二脂(SQDG)、磷脂酰甘油(PG)和磷脂酰胆碱(PC)含量降低,而游离脂肪酸(FFA)、甘油二脂(DAG)、双半乳糖甘油二脂(DGDG)、磷脂酰肌醇(PI)和磷脂酰乙醇胺(PE)则相对稳定。【结论】HPTLC可作为一种简便、可靠的微藻中脂质分离分析方法,为研究微藻油脂代谢途径以及甘油三脂(TAG)的调控积累提供有效手段。