Extraction of bioactive compounds from the leaves of Eucalyptus globulus using green solvents
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Eucalyptus globulus is a well-known source of phenolic compounds with many applications in the pharmaceutical, cosmetic, and food industries, being extensively found in Portuguese territory. Several studies have shown that the E. globulus extract contains important compounds for pharmacological and medicinal use with analgesic, antimicrobial, anti-inflammatory, antioxidant effects, among other applications. The use of its natural extracts has also been suggested as an alternative to synthetic drugs and additives, aiming to avoid possible undesirable side effects and prevent the development of resistance by microbiological organisms. The study of the matrix composition is essential to the development of new applications by obtaining bioactive compounds with high added value. In this work, the extraction of phenolic compounds from eucalyptus leaves was applied through heat-assisted and ultrasound-assisted techniques, using a green solvent (ethanol:water). Each extraction process was optimized using Response Surface Methodology (Traditional and Dynamic). The extraction time, solid/liquid ratio, the proportion of ethanol in the solvent, and temperature (or power, in the case of ultrasound-assisted extraction) were used as independent variables, and the extraction yield, phenolic acids, flavonoids, and total phenolic contents were the dependent variables. To support the development of new applications, bioactivity studies were carried out to evaluate the antioxidant, antimicrobial, anti-inflammatory and cytotoxicity activities of the extracts obtained under optimized conditions. Maximum antioxidant concentrations, between 5.95 to 6.85 μg/mL of dry extract, were determined by TBARS, expressed in terms of EC50. The antimicrobial activity was tested against several pathogens (Gram-positive and Gram-negative bacteria), demonstrating the ability to inhibit their growth. The highest antibacterial activity was obtained, with minimum inhibition concentration values of 0.6 mg/mL, for Yersinia enterocolitica and 0.3 mg/mL for Staphylococcus aureus. For the antifungal activity, Aspergillus fumigatus presented the highest sensibility. The anti-inflammatory activity was confirmed by inhibition of nitric oxide release in RAW 264.7 cells (murine macrophage). The extracts did not show cytotoxicity for the cell line tested (Vero cell line). The results obtained indicate that the extracts of E. globulus leaves have high bioactive capacity. Further studies should be conducted to verify the expected variability in the bioactive profile over time by collecting and analyzing periodically samples from the same region and under the same conditions.
