Central composite design expert-supported development and validation of HPTLC method: Relevance in quantitative evaluation of protopine in Fumaria indica

The current study was executed for method development, validation and to estimate the concentration of protopine in methanolic extract of Fumaria indica by high-performance thin-layer chromatography (HPTLC). Isolation of bioactive compounds was carried out using the mobile phase, toluene:ethyl acetate:diethyl amine (8:2.5:0.5 v/v/v), and detected at wavelength 290 nm. This method was validated for precision, specificity, linearity, limit of detection (LOD), limit of quantification (LOQ), etc. The calibration range was found to be 500–5000 ng/spot for the bioactive compound. Protopine was separated with an R_f value of 0.22 ± 0.03. The method was validated for linearity (r2 ≥ 0.996 ± 0.082), accuracy 98.75–102.12%), and RSD of precision (0.49–2.07) with a calibration curve range of 500.00–5000.00 ng/spot. The LOD and LOQ were found to be 83.92 ng/spot and 254.30 ng/spot., respectively. The Central Composite design expert was applied for the validation of robustness. Three independent variables such as the volume of toluene in solvent system, chamber saturation time and wavelength were investigated. The results indicated that a slight change in these variables had no significant effect on the peak response. This developed HPTLC method is simple, precise, robust, specific, rapid, and cost effective. It could be used for quality control study and quantification of protopine in the plant extract and different herbal formulations containing the plant species.     

Investigation of biological activity of Lamii albi flos extracts

Methanol, ethyl acetate and heptane extracts of Lamium album L. (Lamiaceae) were tested for the toxicity or ability to stimulate growth of human skin fibroblasts (HSF) in vitro. The biological effect depended on the HSF cells density and on the kind and concentration of extract that was used. At a density of 1x10(5) HSF cells/mL no cytotoxicity of extracts was observed during 24 h of incubation. During 24-72 h of incubation with a low (2x10(4) HSF cells/mL) density of cells, significant cytotoxicity was observed for methanol and ethyl acetate extracts at concentrations greater than 125 microg/mL. At concentration of 25 microg/mL the cells remained intact. However, when the cells were incubated with the heptane extract, there was observed relative high viability (>60%) of cells and significant, gradually increasing in time, cellular mitochondrial dehydrogenase activity, measured by MTT assay. Stimulation of human skin fibroblasts proliferation by the heptane extract of Lamii albi flos may indicate for its usefulness of wound healing in vivo and probability of perspectives its use in designing new medicinal plant preparations. In order to determine the biologically active compounds, the plant extracts were separated by high performance thin layer chromatography (HPTLC) on silica gel Si 60 F(254) combined with densitometry. Phenolic acids were investigated by high performance liquid chromatography (HPLC).     

用色谱和ESI-MS方法考察四物汤提取部位的化学成分

目的 :为了探讨四物汤补血作用的物质基础 ,制备 3种不同极性的四物汤提取部位 (C1,C2 ,C3) ,对这3种部位的化学成分特点进行考察。方法 :综合运用高效薄层色谱 (HPTLC)、高效液相色谱 (HPLC)、电喷雾离子化质谱 (ESI-MS) 3种方法对四物汤的C1,C2 ,C3部位及四味单药相应部位进行分析和比较。结果 :四物汤部位C1中含有种类丰富的成分 ,包括阿魏酸、芍药苷等 ,并且应含有藁本内酯 ,而糖类物质极少 ;C2的成分以芍药苷、单糖、二糖为主 ;C3的成分以单糖和二糖为主。结论 :综合运用色谱和直接输注ESI-MS ,可以获得关于方剂提取部位组成的丰富信息。不同方法的结果可以相互比较 ,相互印证 ,使所获得的信息比单一方法更全面更确切。本研究的结果对于探讨四物汤补血物质基础具有参考意义。     

高效薄层扫描法测定166种杜鹃中6种黄酮类成分的含量

在高效硅胶薄层板上以石油醚(bp60～90℃)—乙醚—甲酸(62:31:7)的上层(展开剂Ⅰ)分离杜鹃素、山奈素和槲皮素;扫描测定后再次展开,以氯仿—甲醇—水(7:3:1)的下层—甲酸(7:0.5)(展开剂Ⅱ)分离蓼属甙、槲皮甙干口金丝桃甙。用瑞士Camag 76510型单波长薄层扫描仪进行直线扫描,测定波长366nm,用美国Hewlett Packard 3390 A型积分仪测出峰面积。测定了166种杜鹃叶中上述6种黄酮类成分的含量。     

几种国产药用紫草中萘醌色素的分析

从新疆软紫草石油醚提取物中分离出六个化合物。根据其理化常数、光谱数据(MS,IR,¹HNMR)鉴定为去氧紫草素(deoxyshikonin),β,β-二甲基丙烯酰阿卡宁(β,β-dimethylacryalkannin),乙酰紫草素(acetylshikonin),β-乙酰氧基异戊酰阿卡宁(β-acetoxyisovalerylalkannin),紫草素(shikonin),β-羟基异戊酰阿卡宁(β-hydroxyisovalerylalkannin)。用高效薄层层析扫描方法,分析了我国不同种类、不同产地的紫草根,均含萘醌类色素,但种间含量差异甚大。     

芍药甙类和丹皮酚类成分在芍药科植物中的存在

白芍、赤芍和牡丹皮均为芍药科Paeoniaceae常用中药,主要活性成分是芍药甙等单萜类化合物和丹皮酚类化合物。药理研究证明,这些成分具有抑制血小板凝聚、抗血栓形成、抑制中枢神经系统、扩张血管、抗炎、增强免疫系统功能等多种活性。本文研究了     

山荷叶中木脂素的薄层扫描测定

在高效硅胶薄层板上,用二氯甲烷-乙醚分离了山荷叶中6种苯基四氢萘丁内酯类木脂素:鬼臼酮、异苦鬼臼酮、苦鬼臼酮、苦鬼臼素、鬼臼毒素、4’-去甲基鬼臼毒素和2种苯基萘丁内酯:去氢鬼臼素、山荷叶素。用2.5%硫酸铈铵的20%硝酸溶液喷雾显色,氨气薰数分钟后,薄层扫描法测定,各木脂素的线性范围在0.48～2.52μg,并用此法测定了不同产地的山荷叶及相近属植物鬼臼和山荷叶中木脂素的含量,方法简便、快速、灵敏度高。     

A Reverse Phase HPLC-UV and HPTLC Methods for Determination of Plumbagin in Plumbago indica and Plumbago zeylanica

A reverse phase HPLC method with UV detection has been developed and validated in order to quantify plumbagin, the bioactive marker of the roots of P. indica and P. zeylanica. A quantitative HPTLC method was also developed using hexane: ethyl acetate (8:2) as the mobile phase. The plumbagin content in the roots were determined using both the methods. P. indica was found to contain significantly higher amount of plumbagin than P. zeylanica. The HPLC and HPTLC methods described here are simple, rapid, accurate and sensitive.     

Simultaneous estimation of andrographolide and wedelolactone in herbal formulations

Andrographolide and wedelolactone are active components of Andrographis paniculata and Eclipta alba, respectively. The extracts of these plants are used in many traditional hepatoprotective formulations. An attempt has been made to develop an accurate, precise and specific HPTLC method to quantify simultaneously both these chemical markers of diversified chemical structures in different dosage forms like tablet and syrup. Precoated silica 60F(254) plates with toluene:acetone:formic acid (9:6:1) as mobile phase and detection wavelength of 254 nm were used. The method was validated in terms of linearity, accuracy, precision and specificity. The calibration curve was found to be linear between 200 to 400 ng/spot for andrographolide and 100 to 200 ng/spot for wedelolactone. The limit of detection and the limit of quantification for andrographolide were 26.16 and 79.28 ng/spot, respectively and for wedelolactone 5.06 and 15.32 ng/spot, respectively.     

Flatworm models in pharmacological research: The importance of compound stability testing

Flatworms possess adult pluripotent stem cells, which make them extraordinary experimental model organisms to assess in vivo the undesirable effects of substances on stem cells. Currently, quality practices, implying evaluation of the stability of the test compound under the proposed experimental conditions, are uncommon in this research field. Nevertheless, performing a stability study during the rational design of in vivo assay protocols will result in more reliable assay results. To illustrate the influence of the stability of the test substance on the final experimental outcome, we performed a short-term International Conference on Harmonization (ICH)-based stability study of cyclophosphamide in the culture medium, to which a marine flatworm model Macrostomum lignano is exposed. Using a validated U(H)PLC method, it was demonstrated that the cyclophosphamide concentration in the culture medium at 20 °C is lowered to 80% of the initial concentration after 21 days. The multiwell plates, flatworms and diatoms, as well as light exposure, did not influence significantly the cyclophosphamide concentration in the medium. The results of the stability study have practical implications on the experimental set-up of the carcinogenicity assay like the frequency of medium renewal. This case study demonstrates the benefits of applying appropriate quality guidelines already during fundamental research increasing the credibility of the results.