| Summary: | The increase in the use of herbal medicines has led to the implementation of more stern regulations in terms of quality variation and standardization. As medicinal plants are prone to quality variation acquired due to differences in geographical origin, collection, storage, and processing, it is essential to ensure the quality, efficacy, and biological activity of medicinal plants. This study aims to standardize the widely used fruit, i.e., <i>Prunus domestica</i> Linn., using evaluation techniques (microscopic, macroscopic, and physicochemical analyses), advanced instrumental (HPLC, HPTLC, and GC–MS for phytochemical, aflatoxins, pesticides, and heavy metals), biological, and toxicological techniques (microbial load and antioxidant activities). The results revealed a 6–8 cm fruit with smooth surface, delicious odor, and acidic taste (macroscopy), thin-walled epidermis devoid of cuticle and any kind of excrescences with the existence of xylem and phloem (microscopy), LOD (15.46 ± 2.24%), moisture content (13.27 ± 1.75%), the high extractive value of 24.71 ± 4.94% in water:methanol (1:1; <i>v/v</i>) and with ash values in the allowed limits (physicochemical properties), and the presence of numerous phytochemical classes such as alkaloids, flavonoids, carbohydrates, glycosides, saponins, etc. (phytochemical screening). Furthermore, no heavy metals (Pb, Hg, Cd, Ar), pesticides, ad microbial limits were detected beyond the permissible limits specified, as determined with AAS, GC–MS analysis, and microbial tests. The HPTLC was developed to characterize a complete phytochemical behavior for the components present in <i>P. domestica</i> fruit extract. The parameters utilized with the method used and the results observed for the prunus herein may render this method an effective tool for quality evaluation, standardization, and quality control of <i>P. domestica</i> fruit in research, industries, and market available food products of prunus.
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