Exploring the In Vitro Anti‐Inflammatory Effect of Citrus Fruit Hesperidin Supplementation

The aim of the present study was to assess the anti-inflammatory effect of hesperidin. The research was conducted by optimizing the hesperidin extraction process from citrus peel powder, followed by characterization and nutrition profiling of citrus peel hesperidin extract. Citrus peel was collected from the local market and dried in a hot air oven. Then it was ground in a grinder to make powder. Then, the powder was extracted first with petroleum ether and then with methanol inside a Soxhlet apparatus. Then, the methanolic extract was filtered and subjected to evaporation in a rotary evaporator. Hesperidin was precipitated and purified with the help of acetic acid and dimethylformamide. The anti-inflammatory and antioxidant perspective of hesperidin was assessed through NO₂ production assay, assay of inflammatory cytokines, DPPH, and FRAP, respectively. For instance, in the ferric reducing antioxidant power assay, for 10 μM of hesperidin, the mean reduction value of three different tubes was observed as 3.36 ± 0.197. With the rise of the quantity of hesperidin, that is, 50, 75, and 100 μM, the mean values were observed as 5.48 ± 0.279, 7.5 ± 0.259, and 10.050 ± 0.832, respectively. Similarly, for the 2, 2-diphenyl-1-picrylhydrazyl assay, the mean percentage reduction of the DPPH by hesperidin was 24 ± 0.5774, 35 ± 0.5774, 38 ± 0.57, and 40% ± 0.5% against the concentrations of 10, 25, 50, and 100 μM, respectively. As far as anti-inflammatory activity is concerned, in the NO2 (Nitrite ion) production assay, the mean production of NO2 by LPS induction was 6.3366 ± 0.1 mM. However, co-incubation of hesperidin with LPS in the concentrations of 10, 20, and 30 μM in the treatments has shown lesser productions of the NO2 as 4.8967 ± 0.5 μM, 3.6 ± 0.7 μM, and 2.8667 ± 0.5, respectively. Likewise, in the assay of IL-8, IL-1β and TNF-α production assay, the mean production rate of cytokines was 0.6 ± 0.21 ng/mL, 0.47 ± 0.012 ng/mL, and 0.3633 ± 0.045 ng/mL according to the exposure rate of hesperidin as 0.01 mg/mL, 0.1 mg/mL, and 20 μM of the positive control drug PD98059. With a similar exposure rate of hesperidin and the positive control drug, the mean rate of production of IL-1β was 0.0467 ± 0.079 ng/mL, 0.0367 ± 0.036 ng/mL, and 0.033 ± 0.021 ng/mL, respectively. The third cytokine TNF-α had also shown similar patterns of inhibition against the same dose rate of hesperidin and PD98059. The mean value for the production of TNF-α was 0.30 ± 0.18 ng/mL, 0.30 ± 0.091 mg/mL, and 0.1767 ± 0.084 ng/mL against the similar dose of hesperidin and PD98059. The inhibitory effect of hesperidin was more significant in IL-8 as compared to IL-1β and TNF-α. Conclusively, hesperidin did prove to be a significant antioxidant and anti-inflammatory agent.