Black pepper (Piper nigrum; BP), known as the 'king of spices', imported from various countries is widely available in Saudi Arabian markets, as its demand as a food as well as a medicine for minor ailments is increasing in the country. However, there is a lack of appropriate information regarding these samples in terms of quality variation and standardization. We thus aimed to evaluate the quality and standardize the BP sample with respect to its physicochemical characters, active principle variation [i.e. piperine (PPN)], toxicity, and biological activity. The main focus is to validate whether any difference exists in the quality and quantity of active principle in these samples. For this purpose, physicochemical (chemical tests and ash values) and instrumental analyses [accelerated solvent extraction (ASE), ultra-high-pressure liquid chromatography (UHPLC)-diode array detector, infrared (IR), nuclear magnetic resonance (NMR), and inductively coupled plasma-MS (ICP-MS)] and biological evaluation {in vitro antioxidant activity [2,2-diphenyl-1-picrylhydrazyl and 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)] and cytotoxicity assay} were performed. An extract yield (g) with %recovery of 2.26 ± 4.24 (11.3) was obtained for the Vietnamese sample, using a fast and rapid method of extraction (ASE). These values were 1.22 ± 2.64 (6.1) and 0.75 ± 1.69 (3.75) for the Pakistani and Indian samples. Physicochemical tests revealed the presence of flavonoids and phenolic compounds in all samples; however, in the Vietnamese sample a low amount of total, acid-insoluble, and high water-soluble ash value was noted. IR and NMR was applied to further standardize the samples. Results of ICP-MS analysis showed a high amount of macronutrients and micronutrients in the samples tested while UHPLC analysis revealed a high amount of PPN (ng/mL) in the Pakistani sample (1,362,614.09); these values were 1,051,848.04 and 768,512.81 for the Vietnamese and Indian samples, respectively. In vitro antioxidant and cytotoxicity activities revealed higher potential for the Vietnamese sample. The samples were properly standardized and effectively differentiated in terms of quality and biological activities using a fast and reliable method, however it certainly does not mean that a particular geographical region is more better or productive in terms of herbal products.
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