Determination of methyl gallate in Bauhinia retusa extract by high-performance liquid and thin-layer chromatography

Two stability-indicating chromatographic methods are reported for the determination of methyl gallate in crude extracts of Bauhinia retusa. Separation by high performance thin layer chromatography was conducted on silica gel aluminum sheets using 9.5:0.5:0.2 (v/v/v) chloroform:methanol:acetic acid at 280 nm. The results from the 2–40 µg/band were used to prepare a linear calibration graph. The limits of detection and quantitation were 0.5 and 1.5 µg/band, respectively. The reverse phase high performance liquid chromatographic isolation of methyl gallate was performed at ambient temperature with an injection volume of 10 μL. The mobile phase consisted of 40:60 (v/v) methanol:0.1% ortho-phosphoric acid. The separation was performed at 1 mL/min using a detection wavelength of 280 nm. The calibration graph for methyl gallate was rectilinear from 0.02–40 µg/mL with limits of detection and quantitation of 0.004 and 0.010 µg/mL, respectively. For both methods, intra-day and inter-day precision were evaluated and the relative standard deviation was less than 2%, indicating good precision. The robustness was evaluated by making small and deliberate changes to appropriate parameters and the calculated relative standard deviation was less than 2%.The chromatographic methods were employed to determine methyl gallate in crude Bauhinia retusa extracts.     

茶黄效类高效薄层色谱指纹图谱研究

目的建立茶黄素类的高效薄层色谱(HpTLC)分离方法,应用聚酰胺薄板为分离材料,以不同溶剂,同一溶剂不同比例及不同预饱和时间分别对茶黄素类(TFs)的分离度进行研究,以前7个峰为考察对象,结果发现,展开剂为甲醇丙酮冰乙酸=853(体积比),预饱和时间为20min时分离效果最佳,Rf值分别为0.14、0.22、0.28、0.31、0.35、0.4、0.48.结论该方法是一种快速、有效、简便的TFs检测方法.     

Development and Validation of HPTLC Method for Estimation of Tacrolimus in Formulations

A new, simple, and rapid high-performance thin-layer chromatographic method with a derivatization procedure was developed and validated for quantitative determination of tacrolimus. Tacrolimus was chromatographed on silica gel 60 F₂₅₄ TLC plate using toluene–acetonitrile–glacial acetic acid (6:4:0.1, by volume) as mobile phase. Tacrolimus was visualized using a derivatization reagent containing anisaldehyde–sulfuric acid in absolute alcohol and quantified by densitometric analysis in the reflectance mode at 675 nm. The method was found to give compact spots for the drug (R_f?=?0.40?±?0.03). The linear regression analysis data for the calibration plots showed good linear relationship with r²?=?.9989 in the concentration range 100–800 ng/spot. The method was validated for precision, recovery, repeatability, and robustness as per the International Conference on Harmonization guidelines. The minimum detectable amount was found to be 28.90 ng, whereas the limit of quantitation was found to be 97.04 ng. Statistical analysis of the data showed that the method is precise, accurate, reproducible, and selective for the analysis of tacrolimus. The method was successfully employed for the estimation of equilibrium solubility and quantification of tacrolimus as a bulk drug and in commercially available capsules and in-house developed self-microemulsifying formulations.     

Multiplex PCR-based strategy to detect contamination with mycotoxigenic Fusarium species in rice and fingermillet collected from southern India

BACKGROUND: The genus Fusarium comprises a diverse group of fungi including several species that produce mycotoxins in food commodities. In the present study, a multiplex PCR was standardised for the group‐specific detection of fumonisin‐producing and trichothecene‐producing strains of Fusarium species. Primers for genus‐level recognition of Fusarium spp. were designed from the internal transcribed spacer regions 1 and 2 of rDNA. Primers for group‐specific detection were designed from the tri5 and tri6 genes involved in trichothecene biosynthesis and the fum1 and fum13 genes involved in fumonisin biosynthesis. RESULTS: Among the various genera and their strains tested, all the 85 confirmed Fusarium strains were positive for rDNA gene and the rest stayed negative. From among the Fusarium strains, 15 had amplification for trichothecene‐ and 20 for fumonisin‐encoding genes. All PCR positive trichothecene chemotypes of Fusarium species tested were positive for chemical analysis but in the case of fumonisins, of the 20 PCR positive cultures, only 13 showed positive for chemical analysis by HPTLC. CONCLUSION: The assay described here provided a rapid and reliable detection of trichothecene‐ and fumonisin‐producing Fusarium directly from natural food grains and the results were always comparable with a conventional HPTLC detection method. It can, therefore, be used by the food industry to monitor quality and safety. Copyright © 2011 Society of Chemical Industry     

Simultaneous Determination of Pentaerythritol Tetranitrate and 2,4,6‐Trinitrotoluene by High Performance Thin Layer Chromatography and Partial Least Squares Regression (PLSR) Method

High performance thin layer chromatography (HPTLC) has been used for the simultaneous determination of pentaerythritol tetranitrate (PETN) and 2,4,6‐trinitrotoluene (TNT). With this aim, the spots were developed on silica gel 60 F₂₅₄ layers with petroleum ether–acetone (2 : 1 v/v). Both PETN and TNT compounds were separated from other constituent materials, and were developed at the same speed, by this solvent system. Then ultraviolet (UV) spectra of these materials were recorded with TLC‐scanner3 of CAMAG Company, and partial least squares regression‐2 (PLSR‐2) method was applied for the calibration and quantitative determination of these materials. The figure of merit (FOM) of this method was determined, and the method was applied for the analysis of an artificial sample.     

Rapid Identification and Comparison of Compounds with Antioxidant Activity in Coreopsis tinctoria Herbal Tea by High‐Performance Thin‐Layer Chromatography Coupled with DPPH Bioautography and Densitometry

A simple and efficient method based on high‐performance thin‐layer chromatography coupled with 2,2‐diphenyl‐1‐picrylhydrazyl (DPPH) bioautography (HPTLC‐DPPH) was established for the screening and comparison of antioxidants in different parts of Coreopsis tinctoria herbal tea from different origins and other related herbal tea materials, which used Chrysanthemum morifolium cv. “Gongju” and “Hangju” in this study. Scanning densitometry after DPPH derivatization was applied for the determination of antioxidant capacities of isolated compounds in each sample. It is considered that ethanol extracts of C. tinctoria had stronger antioxidant activity and more characteristic bands than those of 2 compared samples, C. morifolium cv. “Gongju” and “Hangju.” Chemometric analysis results showed that the combination of hierarchical clustering analysis and principal component analysis based on determined antioxidant capacities could be used for the discrimination of different parts of C. tinctoria and C. morifolium. Results showed that 7 compounds made up the major contributions of antioxidant activity in C. tinctoria, including okanin, isookanin, marein, flavanomarein, 5,7,3′,5′‐tetrahydroxyflavanone‐7‐O‐glucoside, 3,5‐dicaffeoylquinic acid, and chlorogenic acid. Therefore, 7 compounds were identified as major antioxidant biomarkers for quality control of C. tinctoria. Results demonstrated that the established method could be applied for the identification of C. tinctoria, and were beneficial for the bioactivity‐based quality control of C. tinctoria.     

Evaluation of Antioxidant, Radical Scavenging Activity and Polyphenolics Profile in Solanum torvum L. Fruits

Solanum torvum fruit widely used in traditional medicine of India and also in food preparation. Three different extracts such as water (WE), methanol (ME), and ethanol (EE) were used to evaluate their antioxidant and radical scavenging activity by different methods. All the assays results were compared with well-known standard antioxidants. The IC(50) values of assays were determined. The total phenolic and flavonoids content were found to be maximum in water and ethanol extracts, respectively. The electron quenching ability of fruit extract was assayed by DPPH and reducing power assays succeeding order were ME > EE > WE, respectively. Inhibition of membrane damage, was assayed interns of oxidative hemolysis and lipid peroxidation assays, among all WE extract shows 58.00% and 68.55 5% percentage of inhibition with 0.9 and 0.8 correlations (r(2) ), respectively. Antioxidant and radical quenching efficiency were assayed by β-carotene bleaching and hydroxyl radical scavenging method and results were compared with vitamin C and catechin. The in vitro free radical quenching and antioxidant results were well correlated with in vitro DNA protection assay. As analyzed by HPTLC gallic acid content is high in WE (1394 ± 25.0) and ME (598 ± 54.0) whereas ferulic acid is high in EE (32 ± 5.94) μg/g, respectively. This study indicate that S. torvum fruit is an excellent source of natural antioxidant and could be an effective nutritional food supplement, which interns will have therapeutic applications. Practical Application: In siddha medicine on the traditional systems of India the, ripened fruits are used in the preparation of tonic named as a "sundaivattaral choornam" is used to improve the health and prevent several diseases. This study has given an experimental evidence that S. torvum fruit is an excellent source of natural antioxidants.     

Chemical and analytical screening of some edible mushrooms

Fractionation of extracts of the edible mushroom, Volvariella volvacea, led to the isolation of two heterocyclic carboxylic acids, namely pyridine-3-carboxylic acid [nicotinic acid, (5)] and pyrazole-3(5)-carboxylic acid (6) and the four steroidal metabolites ergosterol (1), 5-dihydroergosterol (2), ergosterol peroxide (3), cerevisterol (4). Significantly, compound (6) was identified for the first time, to our knowledge, in the mushroom kingdom and is of taxonomic significance. Compounds (2–4) were isolated for the first time from the Volvariella genus. In view of the structural similarity of compound (6) to pyrazole-3-carboxylic acids, which act as agonists for nicotinic acid receptors, the levels of compounds (5) and (6) were estimated for the first time using HPTLC in V. volvacea and two other edible mushrooms, namely Agaricus bisporus and Calocybe indica. Significant levels of compound (5) were found in C. indica, and compound (6) was found in abundance in A. bisporus. Correlations are suggested between the occurrence of these compounds in mushrooms and consumption as well as beneficial health effects of this food.     

Enzymatic acyl modification of phosphatidylcholine using immobilized lipase and phospholipase A2

Ethanol‐soluble (ES) lecithin mainly contains phosphatidylcholine (PC). The incorporation of caprylic acid into PC using immobilized phospholipase A₂ (PLA₂) and lipase was investigated. The Rhizomucor meihei lipase and the porcine pancreatic PLA₂ were immobilized on the hydrophobic resin Diaion HP‐20 and the modification was carried out in hexane as solvent. HPTLC with densitometer technique was successfully used for monitoring the production of structured phospholipids (PL) (ML‐type PC, MM‐type PC, and lysophosphatidylcholine; L: long‐chain fatty acid, M: medium‐chain fatty acid). The various parameters such as the effects of reaction temperature, enzyme loading, and the effect of molar proportion of substrate were studied in order to determine the optimum reaction conditions for the acidolysis reaction. The optimal operating conditions for the PLA₂‐catalyzed reaction were obtained as 50°C temperature, 50% (wt/wt of substrate) enzyme loading, and a 1:12 molar proportion of PC/caprylic acid. For the lipase‐catalyzed reaction, the optimized temperature was the same as for PLA₂, but the enzyme loading and molar proportion were slightly lower, i.e., 40 % w/w of substrate and 1:9 PC/caprylic acid, respectively. The effects of these parameters on the production of structured PL were compared. Under these optimal conditions, the ML‐type PC content was higher in the PLA₂‐catalyzed reaction, i.e., 45.29 mol%, and in the lipase‐catalyzed reaction it was 38.74 mol%.