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  • Author or Editor: Sharad Srivastava x
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A simple, highly precise method has been established for the simultaneous determination of the bioactive molecules bergenin and gallic acid in three different species of Bergenia-B. ligulata (Wall) Eng., B. ciliata (Royle) Raizada, and B. stracheyi Engl. The assay combines separation and quantitative estimation of the analytes on silica gel 60GF 254 HPTLC plates with visualization under UV light and scanning at 260 nm. This study has enabled simultaneous analysis of bergenin and gallic acid in all three species.

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A validated online over-pressured layer chromatography (OPLC) method was developed for the rapid, cost-effective, and efficient separation of gallic acid. A reversed-phase (RP) ultraviolet (UV)–online OPLC analytical method was developed and validated as per the International Conference on Harmonization (ICH) guidelines. Methanolic extract of Annona muricata (fruit) was prepared by cold maceration. Separation of marker was achieved on RP-OPLC plates (5 × 20 cm) with isocratic solvent system of 0.1% acetic acid, water and methanol (30:70) at a flow rate of 0.15 mL min−1; detection was carried out at λmax 270 nm. The base peak of standard gallic acid was at 5.441 min with good linearity (r 2 > 0.999), precision, and accuracy. The limit of detection (LOD) (521.84 mg mL−1) and limit of quantification (LOQ) (1581.336 mg mL−1) values reflect that the method is sensitive with high peak purity; the recoveries of analyte were 99 to 103%. The achievement of the method was the early retention time of gallic acid which in turn increased the efficiency of the quantification of the targeted marker in a short duration of time for even larger number of samples in plant extract as well as biological fluids for pharmacokinetic studies. The application of the method can be extended in regulatory guidelines for the quality control of herbal drugs/products and formulations. The method is rapid and economical in terms of solvent consumption and, hence, can be preferred over other high-performance thin-layer chromatographic or liquid chromatographic methods.

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Commelina benghalensis (Commelinaceae) is widely used as traditional and folklore medicine in India. In the present study, a reverse-phase high-performance liquid chromatography—photodiode array detection (RP-HPLC—PDA) method was developed for the separation, identification, and quantification of bioactive phenolics. Antioxidant potential was also accessed to validate the presence of identified markers. Method was developed on C18 column with 1% formic acid (in water) and acetonitrile as solvent system, and data acquisitions were achieved at wavelength of 285 nm. The developed method was also validated for accuracy, precision, robustness, limit of detection and quantification (LOD and LOQ), repeatability, and recovery according to International Conference on Harmonization (ICH) guidelines. In this method, five phenolics, viz., protocatechuic acid (0.033%), vanillic acid (0.262%), ferulic acid (0.365%), apigenin (0.126%), and kaempferol (0.544%), were quantified in linearity range of 0.2–1.0 μg with correlation coefficient of more than 0.9949. Relative standard deviation (RSD) (%), LOD, LOQ, and recovery (%) are within the acceptable limit. Besides that, methanolic extract shows the inhibition (%) range from 24.45 to 68.75% at 0.02–0.12 mg mL−1. IC50 of extract was observed at 46.75 μg mL−1, suggesting the promising activity in methanol extract. Hence, the proposed method for simultaneous quantification of five bioactive phenolics in the tuber of C. benghalensis using HPLC–PDA detection under the specified conditions is specific and accurate, and validation proves its selectivity and reproducibility.

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Centella asiatica (L.) Urban (Apiaceae) possesses various healing effects and antioxidant properties. However, there has been very less focus on the investigation of chemotypic variations of C. asiatica found in different geographical zones of the country. In order to conserve C. asiatica, as it is an industrially valuable herb and overexploitation of this drug from wild is a common practice, different distinct accessions of C. asiatica from Nilgiri range (Deccan zones) of India were compared in relation to the levels of triterpenoid saponins. Physicochemical parameters were also evaluated in all the accessions. The metabolites investigated include madecassoside, asiaticoside, and its sapogenin, asiatic acid by high-performance thin-layer chromatography (HPTLC). CA-45 showed the highest content of asiaticoside, CA-51 showed the highest content of madecassoside, and CA-47 showed the highest content of asiatic acid among other accessions of Nilgiri range. It can be concluded that the geographical conditions (soil type and altitude) of these accessions are comparatively favorable for the production of higher levels of triterpenoid saponins in C. asiatica. The reported data will contribute to the establishment of knowledge about the triterpenoid saponin composition of different chemotypes of C. asiatica found in Nilgiri range of India in comparison to other geographical areas, and lays a foundation for the conservation and commercial cultivation of this plant.

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Ficus carica L. (Moraceae), the ‘fig tree’, is reported to help in the prevention of vein blockage. Its rich fiber content has a laxative effect and fig latex inhibits the growth of carcinoma cells. Despite the wide use in the Indian traditional system of medicine of, especially, the fruit as an antidiabetic drug, and pharmacological investigation of the leaves, very little investigation has been conducted on phytochemical properties of the plant. An HPTLC method has therefore been established for simultaneous quantification of four biomarkers — bergapten, psoralen, rutin, and chlorogenic acid — in different tissues of the plant. Levels of bergapten and psoralen were highest in the leaves and bark whereas amounts in the fruit were negligible. Levels of chlorogenic acid were highest in the fruit and the maximum concentrations of rutin were found in the leaves. It is therefore apparent that the part of the plant to be used as a drug should be decided on the basis of the activity desired.This HPTLC method can also be used for quality control and standardization of different parts of F. carica .

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JPC - Journal of Planar Chromatography - Modern TLC
Authors:
Pushpendra Kumar Shukla
,
Ankita Misra
,
Manish Kumar
,
Soundararajan Rajan
,
Pawan Kumar Agrawal
,
Ajay Kumar Singh Rawat
, and
Sharad Srivastava

Plant metabolite varies with season and geographic conditions. The present study is aimed at the identification of the potential chemotypes of Coleus forskohlii, available in the natural habitat of Nilgiri hills and adjoining area, in order to provide a basic lead for the industry concerning commercial exploitability, including the location-specific commercial cultivation of the plant. The effect of intra-specific variability in the forskolin content among the populations was estimated using high-performance thin-layer chromatography (HPTLC)—densitometric method. The roots of fourteen naturally occurring populations from the entire hill range were collected, covering the wide topography from foot hills up to the highest peak. The method developed for the quantification of forskolin was validated and found to be linear, specific, and accurate with precision and accuracy. The limit of detection (LOD) and limit of quantification (LOQ) were 1.04 and 3.16 ng spot−1. Precision studies (both inter-day and intra-day) were within the standard limit of relative standard deviation (RSD) (%) less than 3%. The quantification of forskolin within the population revealed that it varied from 0.0046 ± 0.0005 (NBC-36) to 1.156 ± 0.003% (NBC-46). The analysis of variance (ANOVA) suggested that there are significant differences in forskolin content among the populations. A positive correlation (Karl Pearson) was found between the altitude and the forskolin content. The cluster analysis of the population on forskolin content suspected the presence of two chemotypes. The study suggests the presence of chemotaxonomic variation among the populations which can be due to the change in phytogeographical factors.

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