The medicinal plant Chromolaena odorata is traditionally used by people living in different communities of Nepal and the globe against diabetes, soft tissue wounds, skin infections, diarrhea, malaria, and several other infectious diseases. The present study focuses on the qualitative and quantitative phytochemical analyses and antioxidant, antidiabetic, antibacterial, and toxicity of the plant for assessing its pharmacological potential. The extracts of flowers, leaves, and stems were prepared using methanol and distilled water as the extracting solvents. Total phenolic content (TPC) and total flavonoid content (TFC) were estimated by using the Folin-Ciocalteu phenol reagent method and the aluminum chloride colorimetric method. Antioxidant and antidiabetic activities were assessed using the DPPH assay and α-glucosidase inhibition assay. A brine shrimp assay was performed to study the toxicity, and the antibacterial activity test was performed by the agar well diffusion method. Phytochemical analysis revealed the presence of phenols, flavonoids, quinones, terpenoids, and coumarins as secondary metabolites. The methanol extract of leaves and flowers displayed the highest phenolic and flavonoid content with 182.26 ± 1.99 mg GAE/g, 128.57 ± 7.62 mg QE/g and 172.65 ± 0.48 mg GAE/g, 121.74 ± 7.06 mg QE/g, respectively. The crude extracts showed the highest DPPH free radical scavenging activity with half maximal inhibitory concentration (IC₅₀) of 32.81 ± 5.26 µg/mL and 41.00 ± 1.10 µg/mL, respectively. The methanol extract of the leaves was found to be effective against bacterial strains such as K. pneumoniae (ZOI = 9.67 ± 0.32 mm), B. subtilis (ZOI = 15.00 ± 0 mm), and E. coli (7.3 ± 0.32 mm). The methanol extract of the flowers showed the most α-glucosidase inhibitory activity (IC₅₀ 227.63 ± 11.38 µg/mL), followed by the methanol extract of leaves (IC₅₀ 249.50 ± 0.97 µg/mL). The aqueous extract of the flowers showed the toxic effect with LC₅₀ 107.31 ± 49.04 µg/mL against the brine shrimp nauplii. In conclusion, C. odorata was found to be a rich source of plant secondary metabolites such as phenolics and flavonoids with potential effects against bacterial infection, diabetes, and oxidative stress in humans. The toxicity study showed that the aqueous extract of flowers possesses pharmacological activities. This study supports the traditional use of the plant against infectious diseases and diabetes and provides some scientific validation.

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