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Objectives: The current research delves into ethnopharmacology, specifically focusing on the influence of extraction solvent on parameters such as total phenol, flavonoid, coumarin, tannin, antioxidant, and antibacterial activity. This investigation spans different parts of Melilotus officinalis L. within two northern provinces of Iran. It is a holistic approach, exploring both traditional applications and the scientific impact of extraction methods on the plant’s bioactive properties. Materials and Methods: In various observational studies across different fields, ethnopharmacological data were acquired by collecting various parts of the M. officinalis plant (flowers, stem, and root) at different blooming stages from Charbagh Mountain (2340 msl) and Gorgan region (200 msl) between June and July 2016. "Ethanol and water extracts" of these plant parts were obtained through the maceration method. The quantification of "total phenol" (TP), "flavonoid" (TF), coumarin (CO), and tannin (TA) content in the extracts was conducted using spectrophotometry. The in vitro "antioxidant capacity" of the extracts was assessed through the DPPH free radical scavenging assay, the total antioxidant capacity (TAC) assay, and the reducing power assay. Additionally, the antibacterial activity of the extracts was examined against nine gram-positive and gram-negative bacteria using disc diffusion and minimum inhibitory concentration (MIC) assays. Results: Based on the results, the ethanol extract obtained from the flowers of M. officinalis collected at a high altitude (2340 meters) in Semnan province demonstrated the highest concentrations of total phenols (38.08 ± 0.13 mgGA/g), flavonoids (62.04 ± 0.01 mgQU/g), coumarins (19.32 ± 0.08 mL/g), and tannins (33.89 mg/g). Notably, coumarin and tannins were absent in the water extract. The ethanol extract from the flowers in Semnan province exhibited superior antioxidant activity, particularly in the DPPH method (IC50=10.61 ± 0.81 µg/mL), surpassing the efficacy of butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT). Moreover, the ethanol extract from high-altitude flowers displayed potent antimicrobial activity against gram-positive bacteria, specifically Staphylococcus aureus, S. epidermidis, and Bacillus cereus, with inhibition zone (IZ) values of 19.1 ± 0.01 mm, 15.8 ± 0.2 mm, and 14.4 ± 1.12 mm, respectively. The MIC values against these bacteria were 24.5 μg/mL, respectively. In summary, the ethanol extract exhibited greater effectiveness against gram-positive bacteria compared to gram-negative bacteria. Conclusions: Our results indicate a robust association among the extraction solvent, plant part, phytochemical composition, antibacterial efficacy, and antioxidant potential of M. officinalis L. Specifically, the ethanol extract derived from the flowers displayed the most significant antioxidant and antibacterial characteristics. This implies that the ethanol extract from the flowers of M. officinalis L. holds promise as a valuable reservoir of natural antioxidants, aligning with its traditional utilization in addressing various infections. |
