Subcritical Water and Pressurised Ethanol Extractions for Maximum Recovery of Antioxidants from Orange Peel Herbal Dust with Evaluation of Its Pharmacological Potential Using In Silico and In Vitro Analysis

Abstract

This research explored the potential of pressurised liquid extraction techniques for valorising herbal orange peel dust (OPD) waste from the filter tea industry. A series of experiments were conducted, varying the temperature (120-220 °C) and solvent (water and 50% (v/v) ethanol), while pressure and time were kept constant. Afterward, the obtained extracts were analysed by LC-ESI-MS/MS for determining the chemical composition. The highest concentrations of the most dominant compounds, the antioxidants hesperidin (662.82 ± 22.11 mg/L) and naringin (62.37 ± 2.05 mg/L), were found at specific temperatures using subcritical water extraction. In silico studies indicated that these compounds could interact with sirtuin-1 and growth factor beta receptors, suggesting potential anti-ageing benefits for skin. In vitro experiments on rat hepatoma cells (H4IIE) revealed that OPD extracts had antitumor potential, inhibiting cell proliferation and altering cell morphology. These findings underscore the importance of temperature and extraction technique in obtaining antioxidant-rich extracts with pharmacological potential. The resulting extracts, obtained using green solvents, show promise for cosmetic applications, though further in vivo studies are needed to confirm their therapeutic efficacy.

Keywords: antitumor activity; orange peel herbal dust; pressurised ethanol extraction; pressurised liquid extraction; subcritical water extraction; waste valorisation.

Figure 1

Effect of different concentrations (1 µg/mL, 10 µg/mL, 50 µg/mL, 100 µg/mL, and 300 µg/mL) of SWE 3 (extract obtained with subcritical water extraction procedure) (A,B) and PEE 4 (extract obtained with the pressurised ethanol extraction procedure) (C,D) extracts on the viability of H4IIE cells, measured by MTT assay. Cells were incubated with extracts for 24 h (A,C) and 48 h (B,D). Treatment with 300 µg/mL of the extract obtained with the pressurised ethanol extraction procedure in both periods significantly decreased the tumour growth rate compared to the other experimental groups (C,D) and the control (D). Results are expressed as viable cells (% of control) ± standard deviation (SD). Differences between groups were analysed where * p < 0.05 and # p < 0.001.

Figure 2

Morphology of the H4IIE cell line exposed to various concentrations of SWE 3 (extract obtained with a subcritical water extraction procedure): (A,G) control (non-treated), (B,H) treated with 1 µg/mL, (C,I) treated with 10 µg/mL, (D,J) treated with 50 µg/mL, (E,K) treated with 100 µg/mL, (F,L) treated with 300 µg/mL. Cellular morphological changes were analysed with phase contrast in different periods (A–F) after 24 h and (G–L) after 48 h. There were no observable differences in cell morphology and structure at both periods compared to the control group of cells. All photographs were taken at 200× magnification. Scale bars represent 100 µm.

Figure 3

Morphology of the H4IIE cell line exposed to various concentrations of PEE 4 (extract obtained with the pressurised ethanol extraction procedure): (A,G) control (non-treated), (B,H) treated with 1 µg/mL, (C,I) treated with 10 µg/mL, (D,J) treated with 50 µg/mL, (E,K) treated with 100 µg/mL, (F,L) treated with 300 µg/mL. Cellular morphological changes were analysed with phase contrast in different periods: (A–F) after 24 h and (G–L) after 48 h. The extract obtained with the pressurised ethanol extraction procedure at 300 µg/mL concentration caused classic apoptotic cell death (rounded and smaller cells with nuclear disintegration and chromatin condensation). All photographs were taken at 200× magnification. Scale bars represent 100 µm.

Figure 4

Rat hepatoma cells H4IIE were seeded in six-well plates (2000 cells/well) for long-term survival assay. After 24 h, the culture medium was replaced with (A) fresh medium or medium containing (B) 1 µg/mL, (C) 10 µg/mL, (D) 50 µg/mL, (E) 100 µg/mL, and (F) 300 µg/mL of SWE 3 extract (obtained with a subcritical water extraction procedure). Then, the cells were incubated for another 24 h. The culture medium was changed every three days for two weeks. Cells were fixed with methanol and stained with crystal violet. A 300 µg/mL concentration exerted a strong inhibitory effect on cell survival. Scale bars represent 1 cm.

Figure 5

Rat hepatoma cells H4IIE were seeded in six-well plates (2000 cells/well) for long-term survival assay. After 24 h, the culture medium was replaced with (A) fresh medium, or medium containing (B) 1 µg/mL, (C) 10 µg/mL, (D) 50 µg/mL, (E) 100 µg/mL, and (F) 300 µg/mL of PEE 4 extract (obtained with the pressurised ethanol extraction procedure). Then, the cells were incubated for another 24 h. The culture medium was changed every three days for two weeks. Cells were fixed with methanol and stained with crystal violet. The higher concentrations of 100 µg/mL exerted a strong inhibitory effect on cell survival. Scale bars represent 1 cm.