The study was aimed to conduct phytochemical investigation and assess the in vitro antioxidant, cytotoxic, anti-thrombotic, anti-inflammatory and antibacterial properties of crude methanol extract of Dalbergia stipulacea leaf and its various extractives. All the fractions contain quinines, alkaloids, flavonoids, tannins, resins, glycosides, and flavonoids, while crude methanol, petroleum ether, and ethyl acetate fractions contain saponins and steroids in qualitative phytochemical analysis. In GC-MS/MS technique, a total of 44 phytoconstituents were identified and characterized, where 3-O-Methyl-D-glucose (22.80 %), 8,11,14-Docosatrienoic acid, methyl ester (19.86 %), pentadecanoic acid, methyl ester (14.17 %), 13-docosenamide (6.62 %), nonadecanoic acid, methyl ester (4.62 %), and phytol (3.04 %) were most prominent. In addition, some promising bioactive constituents, such as pantolactone, aromandendrene, D-allose, loliolide, neophytadiene, dibutyl phthalate, kolavenol, and squalene are present in the leaf of D. stipulacea plant species. Petroleum ether soluble fraction (PESF) of the plant had the highest phenolic content (27.34 mg of GAE/gm) and showed the most effective DPPH free radical scavenger with a half maximal inhibitory concentration (IC50) value of 3.87 μg/mL. The PESF also exhibited cytotoxicity with median lethal dose (LC50) value of 2.76 μg/mL compared to the standard medication vincristine sulfate (LC50 = 0.45 μg/mL) in brine shrimp lethality bioassay. Compared to streptokinase (63.34 %), aqueous soluble fraction (AQSF) displayed the highest percentage of clot lysis (38.61 %). The PESF exhibited the most significant suppression of heat-induced hemolysis (32.61 %) and hypotonic solution-induced hemolysis (33.86 %), respectively, revealing promising anti-inflammatory properties. In addition, the methanol extract and its fractions exerted promising antibacterial properties with notable zone of inhibition (6–25 mm) compared to ciprofloxacin (17–37 mm). The current evidence supports the traditional medicinal applications of the plant, particularly its ability to act as an antioxidant, cytotoxic, thrombolytic, anti-inflammatory, and antibacterial agent. However, further phytochemical isolation and in vivo screening is necessary to discover new drugs from the leaf of D. stipulacea plant species based on the current evidence.
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