Uthai Sotanaphun

Antioxidative and neuroprotective activities of extracts from the fruit hull of mangosteen (Garcinia mangostana Linn.).

Objective: The aim of this study was to investigate the antioxidative and neuroprotective activities of various extracts from the fruit hull of mangosteen (Garcinia mangostana Linn., GM). Materials and Methods: Four extracts: water, 50% ethanol, 95% ethanol and ethyl acetate, were used. The antioxidative activity was evaluated using 2,2-diphenyl-1-picrylhydrazyl free-radical scavenging assay at extract concentrations of 1, 10, 50 and 100 µg/ml. Based on the free radical scavenging activity of the extracts, two (water and 50% ethanol) were selected for their protective activity in NG108-15 neuroblastoma cells against H2O2-induced oxidative stress and for cell viability using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Results: All extracts exhibited antioxidative activity. The water and 50% ethanol extracts showed high free-radical scavenging activity with IC50 values of 34.98 ± 2.24 and 30.76 ± 1.66 µg/ml, respectively. Both water and 50% ethanol extracts exhibited neuroprotective activity on NG108-15 cells. The highest activity was observed at the concentration of 50 µg/ml for both the water and 50% ethanol extracts. For cytotoxicity test, none of the extracts was toxic to the cells except at the high concentration of 100 µg/ml. Conclusions: These results suggest that the water and 50% ethanol extracts from the fruit hull of GM may be potent neuroprotectants.

Quantitative determination of sibutramine in adulterated herbal slimming formulations by TLC-image analysis method

A simple thin layer chromatographic (TLC)-image analysis method was developed for rapid determination and quantitation of sibutramine hydrochloride (SH) adulterated in herbal slimming products. Chromatographic separation of SH was achieved on a silica gel 60 F(254) TLC plate, using toluene-n-hexane-diethylamine (9:1:0.3, v/v/v) as the mobile phase and Dragendorff reagent as spot detection. Image analysis of the scanned TLC plate was performed to quantify the amount of SH. The polynomial regression data for the calibration plots showed good linear relationship in the concentration range of 1-6 μg/spot. The limits of detection and quantitation were 190 and 634 ng/spot, respectively. The method gave satisfactory specificity, precision, accuracy, robustness and was applied for determination of SH in herbal formulations. The contents of SH in adulterated samples determined by the TLC-image analysis and TLC-densitometry were also compared.

Application of Scion image software to the simultaneous determination of curcuminoids in turmeric (Curcuma longa)

INTRODUCTION Curcumin, desmethoxycurcumin and bisdesmethoxycurcumin are bioactive constituents of turmeric (Curcuma longa). Owing to their different potency, quality control of turmeric based on the content of each curcuminoid is more reliable than that based on total curcuminoids. However, to perform such an assay, high-cost instrument is needed. OBJECTIVE To develop a simple and low-cost method for the simultaneous quantification of three curcuminoids in turmeric using TLC and the public-domain software Scion Image. METHODOLOGY The image of a TLC chromatogram of turmeric extract was recorded using a digital scanner. The density of the TLC spot of each curcuminoid was analysed by the Scion Image software. The density value was transformed to concentration by comparison with the calibration curve of standard curcuminoids developed on the same TLC plate. RESULTS The polynomial regression data for all curcuminoids showed good linear relationship with R(2) > 0.99 in the concentration range of 0.375-6 microg/spot. The limits of detection and quantitation were 43-73 and 143-242 ng/spot, respectively. The method gave adequate precision, accuracy and recovery. The contents of each curcuminoid determined using this method were not significantly different from those determined using the TLC densitometric method. CONCLUSION TLC image analysis using Scion Image is shown to be a reliable method for the simultaneous analysis of the content of each curcuminoid in turmeric.

Weed growth inhibitors from Aspergillus fischeri TISTR 3272

Chloroform and ethyl acetate extracts of Aspergillus fischeri TISTR 3272 showed good growth inhibitory activity on Mimosa pigra and Echinochloa crus-galli. Bioassay-directed fractionation of the active extracts led to the isolation of five known compounds, (+)-terrein (1), (−)-6-hydroxymellein (2), two diketopiperazines (cyclo-(S-Pro-S-Leu) (3) and cyclo-(S-Pro-S-Val) (4)) and butyrolactone I (5). Compounds 2–5 were reported for the first time in this fungus. Their structural determinations were based on analyses of spectroscopic data and their weed growth inhibitory effects were assessed.

Quinone-methide triterpenoids from Glyptopetalum sclerocarpum

Nine novel quinone-methide triterpenoids: 20-hydroxytingenone, 20,22beta-dihydroxytingenone, 20,22beta-dihydroxy-20-epi-tingenone, 20,21alpha-dihydroxy-22-oxo-21-desoxotingenone, 20-hydroxy-22-oxotingenone, 20-hydroxy-22-oxo-20-epi-tingenone, 21alpha-hydroxy-20,22-dioxo-30(20-->21)abeo-21-desoxotingenone, 20-oxo-20,21-seco-tingen-21-oic acid and 20-oxo-21-nor-20,21-seco-tingen-22-al, were isolated from the stem bark of Glyptopetalum sclerocarpum. Their structures were determined on the basis of spectroscopic evidence.

Pectins from Citrus maxima

Pectins from the fruit peel of Citrus maxima (Burm. f.) Merr. or pomelo (Rutaceae) were composed of two main groups: 1) water-soluble pectin (WSP) [high-methoxyl pectin, degree of methoxylation (DM) = 69.32-78.68%], and 2) oxalate-soluble pectin (OSP) (low-methoxyl pectin, DM = 21.01-55.41%). Variation of plant cultivar and storing time of the fruits after harvesting did not significantly influence the content, percentage galacturonic acid, DM and neutral-sugar proportion of both WSP and OSP. The contents of WSP and OSP were 8.12-10.87% and 4.89-8.62%, respectively, whereas percentage galacturonic acid of WSP and OSP were 68.31-79.29% and 48.99-74.02%, respectively. Comparison between albedo (inner layer of the peel) and flavedo (outer layer of the peel), the content and percentage galacturonic acid of pectins in albedo were higher. After storing the fruits for 30 days, the molecular weight of WSP in albedo increased from 22-25 KDa to 41-50 KDa. Infrared (IR) spectra confirmed DM difference between WSP and OSP, and suggested more consistency character of WSP among cultivars and storing times. The proportion of neutral sugars in pectins was not influenced by cultivar and storing time, but it was different between WSP and OSP, and between pectins from albedo and flavedo.

Geraniinic acid derivative from the leaves of Phyllanthus reticulatus

Chemical constituents as well as cytotoxic and insecticidal activity of the crude methanol extract from the leaves of Phyllanthus reticulatus Poir. (Euphorbiaceae) were investigated. (5R*,6R*)-4,6-Dimethoxycarbonyl-5-[2′,3′,4′-trihydroxy-6′-(methoxycarbonyl) phenyl]-5,6-dihydro-2H-pyran-2-one (1) along with 3,4,3′-tri-O-methylellagic acid, and methyl gallate were isolated from the dichloromethane extract. Determination of their structures was based on spectroscopic analysis. Compound 1 possessed a very weak insecticidal activity against Spodoptera frugiperda (Sf9) with an IC50 value of 27.27 μg/mL.

Synergistic antioxidant action of Phikud Navakot ameliorates hydrogen peroxide-induced stress in human endothelial cells

Background Phikud Navakot (PN), a combination of nine herbs, has been used traditionally in Thai medicinal formulas to relieve circulatory disorder. The present study aimed to compare the synergistic antioxidant efficacy and toxicity of the hydroethanolic and water extracts of PN at cellular level. Methods PN and its nine herbs were extracted with either 50% ethanol or water. All extracts were tested for in vitro antioxidant potential using standard antioxidant assays. Evaluation of cytotoxicity, genotoxicity, and intracellular reactive oxygen species were performed using human endothelial ECV304 cells. Results Antioxidant assays in cell-free systems showed that the hydroethanolic extract of PN scavenged superoxide, hydroxyl, nitric oxide radicals, and hydrogen peroxide more effectively than its water extract. Combination indices were calculated to show that the ingredients of the hydroethanolic extract acted synergistically to exhibit antioxidant activities against all tested radicals, whereas, in the case of water extract, this effect was observed only against 2,2-diphenyl-1-picrylhydrazyl, superoxide, and hydroxyl radicals. A cell-based assay also revealed that the hydroethanolic extract concentration-dependently attenuated hydrogen peroxide-induced stress more effectively than the water extract. At the antioxidant and cytotoxic concentrations of both extracts, no genotoxicity was found. Conclusion Our findings demonstrate that the synergistic antioxidant action of PN ameliorates endothelial stress, which may provide some clues for understanding the traditional use of PN for the treatment of circulatory disorder. Additionally, the selection of a suitable solvent for the extraction of PN herbal combination is essential for maximal efficacy and safety.

Comparison of In Vitro Binding of Bile Salt by Pectins from Various Sources

Binding of bile salts by dietary fiber is believed to promote their excretion and hence to reduce the serum cholesterol level in man and experimental animals. In this study, the binding efficiency of soluble pectin from various sources, i.e., apple, citrus and pomelo, was examined. Sodium deoxycholate and sodium cholate hydrate were used as a model to represent bile salt in human body. The binding efficiency was assayed by acid reaction, thin layer chromatography (TLC) and enzyme cycling method. The results demonstrated that enzyme cycling method was the most suitable for assaying the in-vitro binding of bile salts while the TLC was not very sensitive, i.e., low amount of bile salts cannot be detected by TLC. Excess pectin from binding test could also interfere the acid reaction method even though the centrifugation was used to remove the excess pectin. When the concentration of pectin was increased, the binding efficiency with sodium deoxycholate increased. However, at 1% w/w of pectin, the binding efficiency decreased. The exception is for pomelo pectin in which the binding efficiency increased when the pectin concentration increased. With sodium cholate hydrate, only slight difference in binding efficiency was observed for all types and concentrations of pectin. The results indicate that the ability to bind bile salts of pectin might be responsible for its hypocholesterolemic action observed in experimental animals and humans.

Stability-Indicating TLC-Image Analysis Method for Quantification of Ceftriaxone Sodium in Pharmaceutical Dosage Forms

A simple and rapid thin-layer chromatographic (TLC)-image analysis method has been established for stability-indicating studies and quantification of ceftriaxone sodium (CFX) in bulk and in pharmaceutical dosage forms. Chromatographic separation was achieved on RP-18 F254S plates with 15% (w/v) ammonium acetate buffer (pH 6.2)-methanol-acetonitrile 12:0.5:0.25 (v/v) as mobile phase. The method was validated in accordance with International Conference on Harmonization (ICH) guidelines. Polynomial regression data for the calibration plot were indicative of a good linear relationship between response and amount in the range 1–8 µg per spot. The limits of detection and quantitation were 228 and 759 ng per spot, respectively. The precision, accuracy, and robustness of the method were satisfactory. Good separation was achieved between CFX and the degradation products obtained under different stress conditions, suggesting the method is stability-indicating. The CFX content of dosage forms determined by use of TLC-image analysis, TLC-densitometry, and the official HPLC method was not significantly different. The proposed TLC method was found to enable reliable and convenient analysis of CFX and can be used as a stability- indicating assay.