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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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Summary

Artemisia pallens L. (Compositae) is used in Indian traditional medicine to treat diabetes mellitus, jaundice, hysteria, body pain, and bacterial and fungal infections. A major cause of a variety of diseases is oxidative stress which is reduced by antioxidants such as polyphenols. These secondary metabolites are generally ubiquitous in plants and extensively used in the pharmaceutical, cosmetic, and food industries. In this study a simple and sensitive HPLC-UV-MS-MS-based method was developed for separation, identification, and quantification of polyphenols, for example gallic, protocatechuic, chlorogenic, caffeic, and ferulic acids, rutin, quercetin, and kaempferol. Amounts of polyphenols detected in 50% methanol-water extracts of the plant varied from 0.005% (kaempferol) to 0.24% (protocatechuic acid). Separation of the polyphenols was achieved on a reversed-phase C18 with a mobile phase prepared from 1% aqueous with acetic acid and acetonitrile at a flow rate of 0.6 mL min−1. The phenolic compounds were detected by UV absorption at 254 nm. The method was validated for linearity, accuracy, precision, LOD, LOQ, specificity, selectivity, and compound stability. Results from intra and inter-day validation (n = 6) showed the method was efficient and rapid. The optimized method was applied to extracts of A. pallens for identification and quantification of the polyphenols. The reference standards and their presence in A. pallens were confirmed by mass spectrometry.

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