*1.1. Antioxidant Activity of Hesperidin*

Bioflavonoids have been known for a long time because of their noticeable antioxidant potential that mitigates the harmful impact of free radical and reactive oxygen species. Hydroxyl, alkoxyl, peroxyl, and peroxyl nitrite radicals induce ageing, tissue damage, and contribute to many disease developments like cancer, hypertension, atherosclerosis, amyloidosis, and senile dementia. Nevertheless, the cited free radicals are produced in everyday life and neutralized as well by enzymes [1]; with ageing and increased radical production, their nocive effects are enlarged and become far more potent. The radical scavenging property inherent to natural bioflavonoids is related to their structure and in HSD, hydroxyl groups at positions 3-- and 5- demonstrate mild antioxidant activities [15,16]. Hesperetin (HST) has an additional hydroxyl group (7-), thus showing a stronger antioxidant activity compared to HSD. Disaccharide in the HSD structure affects its capacity for electronic delocalization and decreases its antioxidant activity [6]. For example, HSD nano-loaded lipid carrier showed antioxidant effects in concentrations of 45 μM in 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay, and without any toxic effects on several cell lines [17].

#### *1.2. Hesperidin and Hesperetin Use in Cosmetics as Anti-Ageing Active Components*

Hesperidin and hesperetin are the foci of intensive research for topical application. For example, sulfonated, acetylated, or phosphorylated HSD derivatives are potent inhibitors of hyaluronidase. Moreover, HSD can also act on superoxide in electron transfer as well as proton transfer in vivo. HSD might act as a topical UV-protective agent, by protecting phosphatidylcholine liposomes from UV irradiation-induced peroxidation. The neohesperidin, for example, demonstrates the capacity to extend yeast's chronical lifespan, individually or in synergism with the HST, for 10 different aging factors, such as scavenging ROS effects, regulation of stress-related enzymes, and maintaining pH cellular value, favorable for life extension of yeas<sup>t</sup> cells [18]. Hesperidin was also proved as a potent anti-photoageing factor, through regulation of metalloproteinases MMP-9 via mitogen activation protein kinase (MAPK) signaling pathways. In the same study, Lee and colleagues [19] approved the positive effect of the hesperidin on wrinkle depth on a mouse dorsal skin model, and reduction in UVB-induced hydration changes and trans-epidermal water loss [20]. Different studies demonstrate accelerated cutaneous

diabetic or venous wounds healing and ameliorate skin epidermal barrier function already after 7 days of HSD application [20]. On the other side, HST was found to penetrate through the *stratum corneum* [5] and assays conducted in vitro showed that the presence of lecithin and d-limonene in formulations may aid towards faster hesperetin penetration into the skin [21]. Additionally, in vitro studies of the *stratum corneum* have demonstrated that flavonoids show differences in penetration capacities, which depend in a large part on the ingredients/vehicles present in the formulation. Therefore, the penetration rate of flavonoids, such as catechin, rutin, quercetin, and others, is influenced by moisturizing ingredients (glycerol, glycols, polyglycols, ethoxylated methyl glucoside, and urea) and by the type of cosmetic formulation (hydrogel, emulsion, microemulsion, and micellar system) [6]. For example, the water in oil microemulsion formulations significantly enhances quercetin skin penetration up to 12 h after application [22–24]. HSD-loaded nanostructured lipid vehicles show burst release in the beginning and further sustained bioflavonoid release from the lipid nanocarrier [17]. Despite their interesting and beneficial skin effects, bioflavonoids are very demanding for making effective formulations. In addition, their insolubility in water complicates their use in cosmetic products and influences greatly on their extraction from fruits and plants that is usually performed by the use of organic solvents.

Many different protocols are described and used for the extraction of hesperidin from various starting materials, including maceration [12,25–27], Soxhlet extraction [26,28], assisted extraction with ultrasound [25], high hydrostatic pressure and microwave assisted extraction [29,30], enzymatic process [14], and supercritical fluids extraction [31]. All the aforementioned extraction methods require the use of organic solvents, energy, and/or high-pressure, which makes those processes unfavorable for an ecologically clean way of treating orange peel. Herein, a new and green method, without using organic solvents and with low energy consumption, was implemented for the extraction of hesperidin from fresh orange bagasse. Hesperidin extracted in this manner is safe to use and biocompatible for topical application.
