Eucalyptus grandis × Eucalyptus urophylla has been cultivated on a large scale in Malaysia for its timber values. Its leftover leaves are considered agricultural waste. This study aimed to optimize extraction yields and evaluate the chemical composition and bioactivities of leaf extracts from Eucalyptus hybrid produced using supercritical fluid extraction (SFE) with carbon dioxide. Leaves were collected from Eucalyptus hybrid trees aged 17 months to 31 months (group A) and 40 months to 50 months (group B). Optimization using response surface methodology (RSM) identified optimal extraction conditions (pressure, temperature, and time) as 29.7 MPa, 79.8 °C, and 116.2 min for A, and 30.0 MPa, 79.6 °C, and 108.6 min for B. Gas chromatography-mass spectrometry (GC-MS) showed that eucalyptol (18.82 %) and eucalyptin (16.46 %) were the dominant compounds in A and B, respectively. Incorporation of 10 % ethanol as co-solvent increased the SFE yield by approximately 10-fold from 0.475 % (w/w) (A) to 4.650 % (w/w) (A10) and 0.255 % (w/w) (B) to 2.640 % (w/w) (B10). All extracts demonstrated antimicrobial activity, with minimum inhibitory concentrations ranging from 0.02 mg/mL to 2.50 mg/mL against 10 bacterial strains, and 0.02 mg/mL to 1.25 mg/mL against seven fungal strains. The extracts were generally more effective against Gram-positive bacteria and exhibited variable antifungal activity depending on the fungal species. Notably, the ethanol-assisted SFE extract from B showed the strongest larvicidal activity against Aedes aegypti (LC50: 187.02 µg/mL) and Aedes albopictus (LC50: 294.48 µg/mL) after 48-hour exposure. These findings suggest that Eucalyptus hybrid leaves are a promising source of bioactive compounds with potential applications as disinfectants and mosquito larvicides.
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