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  • Author or Editor: Pankaj Nariya x
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DPPH scavenging assay is the widely used method for evaluation of in vitro anti-oxidant activity. Anovel, rapid, and precise online highperformance thin-layer chromatographic (HPTLC) method has been successfully developed for evaluation of DPPH reduction. In this paper, chemistry-based assays, like reduction or scavenging of DPPH with ascorbic acid, are performed and discussed. In its radical form, DPPH has strong absorption in the regions around 330 nm and 520 nm, but if it is reduced, there is no change in the first peak, but the intensity of the second peak (around 520 nm) decreases steadily with the increase in ascorbic acid concentration. The method has been combined with online planar chromatography (HPTLC) technique for rapid observation and quantitative estimation of reduction of DPPH by ascorbic acid. For scavenging of 660 ng to 1320 ng of DPPH, around 120 ng to 160 ng of ascorbic acid or its equivalent is the suitable concentration. The limit of detection (LOD) and limit of quantification (LOQ) of DPPH are 3.9 ng and 13 ng, respectively, while that of ascorbic acid are 4.5 ng and 15 ng, respectively. The focus is on the mechanisms involved rather than on the matrix. It can be used for rapid screening and evaluation of potential natural products for their anti-oxidant activity and will have wider applications.

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Piperine and gallic acid are of different chemical natures — piperine is an alkaloid, while gallic acid a phenolic compound. They are used as marker compounds in many plant-based formulations. A highperformance thin-layer chromatographic (HPTLC)-densitometric method has been developed and validated for the simultaneous quantification of piperine and gallic acid as such and in pharmaceutical dosage forms. Toluene-ethyl acetate (3:7) was used as mobile phase and scanning was done at 254 and 340 nm. The method was validated with respect to linearity, reproducibility, specificity, accuracy, precision, robustness, and ruggedness. Both compounds showed good linearities in the range of 250–1750 ng. LOD and LOQ for piperine were 9.98 and 33.29 ng, while for gallic acid 25 and 83.33 ng. Average % RSD values of precision for piperine and gallic acid were 0.46% and 0.72%, respectively. % Recovery was 96–103%. The method is accurate, reproducible, cost-effective, and can be used in routine analysis.

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A new method, involving the use of high-performance thin-layer chromatography, has been developed, which detects 10–20% (w/w) of adulterant tallow in cow ghee. The method consists of high-performance thin-layer chromatographic separation of both unsaponifiable and saponified portions using n-hexane-diethyl ether-glacial acetic acid (4:6:0.2, v/v) (for unsaponifiable portion) and n-hexane-diethyl ether-glacial acetic acid (6.5:3.5:0.2, v/v) (for saponifiable portion) as mobile phases. Beef tallow was mixed with cow ghee in various proportions (5 to 90%) to obtain admixtures of beef tallow with cow ghee. The analysis of the samples of cow ghee containing different proportions of beef tallow revealed that the addition of beef tallow to cow ghee affected the chromatographic profile; the effect increased with increasing proportions of beef tallow. Ghee adulterations with tallow at levels down to 20% are clearly seen visually in the chromatographic profile.

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The aim of this study was to isolate and identify three biologically active flavonoids, quercetin, kaempferol, and apigenin, from the hydrolyzed methanolic extract of three indigenous medicinal Cordia species, i.e., Cordia macleodii, Cordia dichotoma, and Cordia rothi barks using preparative thin-layer chromatography (TLC) and high-performance (HP)TLC methods. The isolation of flavonoid fraction was accomplished by preparative TLC method using reference standard. For achieving good separation, a mobile phase of toluene–ethyl acetate–GAA–formic acid (5:5:0.5:1) was used. The ultraviolet (UV)-based densitometry determination was carried out at 254 nm in reflection—absorption mode. The antioxidant compounds in the samples were screened through 2,2-diphenyl-1-picrylhydrazyl (DPPH) derivatization method. The method was partially validated in terms of linearity, specificity, and sensitivity. All three standards showed good linearity in the range of 2500–7500 ng with respect to area. Limit of detection (LOD) and limit of quantitation (LOQ) for quercetin were 9.4 and 28 ng; for apigenin, 30 and 92 ng; and for kaempferol, 57 and 173 ng. From the analysis, quercetin (0.21%) and kaempferol (0.19%) in C. rothi and apigenin (0.28%) in C. dichotoma were found to be the highest. The isolation and identification of these flavonoids have not yet been found in the literature and they are reported in this study for first time. From this, it is concluded that planar chromatography has a potential as a rapid and simple tool for the identification and quantification of phytochemicals in complex mixtures and marker-based samples.

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