**3. Discussion**

A new, water-based, eco-friendly extraction was performed to yield hesperidin (HSD, 1.2%) with approximate purity of 98%. This new method explored the use of calcium(II) and changes in the pH as to isolate HSD from orange peel. The use of calcium(II) enabled the complexation of pectin within the bagasse and release of HSD, which dissolved at high pH values. NaOH solution at pH ~11.5 deprotonated phenol HSD groups and promoted its better solubility. The next step counted on HSD protonation (HCl), which caused HSD precipitation on pH ~4.2. Pale yellow powder was obtained and characterized using different spectroscopic techniques. HSD is usually extracted with organic solvents [30,32] or by supercritical fluid extraction [31]. The organic solvent extraction for hesperidin extraction was performed by Cypriano and colleagues [37] in a 1.2% yield, following Ikan's method [12]. The method suggested in this work is energy- and organic solvents-free, and calcium(II) and pH-triggered. The use of calcium(II) facilitated complexation of pectin polysaccharide and at the same time, liberation of hesperidin from orange bagasse. The suggested process is longer but facilitates the extraction of hesperidin with greater purity compared to the Sigma-Aldrich analytical standard.

Observing the structure of HSD (Figure 1), it is evident that its B-ring is electron richer than the A-ring due to the contribution of the ortho methoxy group. The first site of oxidation in HSD is the 3--phenoxyl group in a one-electron reduction [8,38]. The pKa of HSD -OH groups is 8.9 and 11.2, respectively. However, the pKa of the hydroxyl radical in the 3- position drops to a value around 4–5. Due to this, HSD acts as antioxidant in the pH range of 7 to 10. Therefore, the mechanism of HSD oxidation involves the transfer of one electron and one proton. The reduction potential of the phenolate radical is strongly affected by substituents in the B-ring [8]. HSD has an electron-donating group (methoxy) attached to the B-ring, which elevates its reduction potential (E = 0.72 V) when compared to other flavonoids with catechol groups in the B-ring (E value from 0.5 to 0.7 V). Nevertheless, its reduction potential is still smaller than those from alkyl peroxyl radicals (E = 1.05 V) and superoxide radicals (E = 0.94 V), which confirms that HSD can scavenge and inactivate these harmful radical species in the organism [8,39–51]. HSD high chemical reactivity can prevent injuries caused by free radicals through direct interaction with radical oxygen species (ROS), or indirectly by inducing antioxidant enzymes activation, chelating of metal ions, reduction in α-tocopherol radicals, inhibition of oxidases, reduction in NO oxidative [4,38] stress, and increases in antioxidant effects of low molecular antioxidants [9,39].

Many other polyphenols, such as phenolic acid, flavonoids, coumarins, stilbenes, and lignans, are the most important polyphenols used in traditional and modern cosmetic and dermatologic products [3,52,53]. The antioxidant, anti-ageing, anti-inflammatory, antimicrobial, and anticancer properties of flavonoids are frequently deployed in skincare products. Their antiradical functions, as well as the ability to inhibit some enzymes, are some of the most important effects. Flavonoids have an influence on skin microcirculation and can be used as ingredients in creams for vascular, oily, and/or atypical skin [3]. Dermal bioavailability and antioxidant activity [54,55] of glycosides is beneath when compared to their aglycon forms, due to their low permeability [3]. One of the interesting physical-chemical properties of bioflavonoids is their ability to absorb ultraviolet radiation due to the presence of the conjugated double bonds in their structure. They may absorb UV radiations in the range of 550–300 and 285–240 nm, resulting from phenolic electron-donating substituents, as well as inter- and intramolecular hydrogen bonds and steric e ffects. The polyphenol concentrations used in cosmetics cannot replace conventional UV filters. Moreover, they can act as co-adjuvants in erythema and skin burns reduction caused by exposures to UVB radiation and IR rays of sunlight [3]. The anti-ageing properties of flavonoids are associated with their e ffect on the modulation of matrix metalloproteinases activities, which are dependent on the Zn2<sup>+</sup> ion and involved in connective tissue remodeling. Equally, by sequestering metal ions or the e ffects on the expression of endogenous protein tissue inhibitors of metalloproteinase (TIMPs) [9], flavonoids can act protectively. Flavonoids' skin whitening activity relates to their modulation of tyrosinase activity, through their chelating of calcium(II), in the formation of L-dopaquinone, which is a part of the melanogenesis process [56]. This ability is common to quercetin, cyanidin, and kaempferol, while their glycoside forms have not shown this capability [3]. Hyaluronic acid, which is an integral part of the dermis and blood vessel wall, is degraded by the activity of the enzyme hyaluronidase, which can be inhibited by the protective roles of flavonoids [3]. Additionally, phenolic compounds may have antimicrobial properties and assist in the preservation of cosmetic products against secondary infections [3].

Low drug permeability through human epidermis can be ameliorated using penetration enhancers (compounds such as surfactants, terpenes, lipophilic solvent, and fatty acids), which modify the permeability of the skin barrier reversibly. Rutin, catechin, epicatechin, and quercetin have a limited penetration. Quercetin shows poor permeability even in the presence of the enhancers, due to its absolute insolubility in water [35]. Several authors suggested that the permeability of this compound can be increased by uploading flavonoids in liposomes or other kinds of carrier systems. It can also be a ffected by the presence of promoters of permeability, such as glycerin and propylene glycol [3]. In the skin, HSD may significantly stimulate epidermal hyperplasia and improve epidermal permeability through epidermal proliferation, osteoblasts di fferentiation, and lipid secretion. Di fferent skin layers and skin proteins can respond di fferently to HSD treatment. Involucrin, present in the stratum spinosum, does not respond to HSD treatment. Filaggrin and loricrin (located in the stratum granulosum and stratum spinosum) expressions are increased. These beneficial e ffects that improve epidermal permeability and regulate filaggrin can be useful in the treatment of certain skin disorders, such as cutaneous inflammation and atopic dermatitis [52]. In vitro, HSD can inhibit the tyrosinase in melanocytes and reduce the process of melanogenesis, or influence melanocytes proliferation. Therefore, the possibility of a ffecting tyrosinase activity, and the practical application thereof in the field of anti-ageing preparations designed to prevent lentigo senilis and lentigo solaris makes HSD remarkably interesting for skin whitening [3]. Daily topical applications of HSD microemulsion have shown a significant skin whitening e ffect, reduction in trans-epidermal water loss, and inhibition of irritation e ffect after exposure to UV rays after four weeks. In their study, Kim et al. [56] showed that HSD had a depigmentation e ffect by blocking the melanophilin, a tripartite protein complex which is a response for transport of the melanosome into the melanocytes. This was proven by melanosome aggregation tests in cells. HSD did not inhibit melanin production in melanoma cells, but reduced skin pigmentation in the reconstruction of human epidermal skin [56]. HSD may also a ffect polyoxygenase, cyclooxygenase, hyaluronidase, collagenase, elastase, and tyrosinase; thus, it can contribute to the reduction in modifications in the skin's connective tissue remodeling [3]. Hyaluronidase plays a significant role in regulating the permeability of capillary walls and supporting tissues by causing the breakdown of hyaluronic acid and increasing the permeability of the tissue [5]. The HSD's ability to chelate multivalent metals such as iron(III), iron(II), copper(II), zinc(II), and manganese(II) is as relevant to the inhibition of enzymes, which contain metal ions in their reaction center or require metal cations cofactors, as to the inhibition of inflammatory processes and function of vascular vessels [3,15,57,58]. Chelation properties of HSD are particularly important for skin bleaching activities, as well as HSD

interaction with the tyrosinase during catalytic production of melanin. Tyrosinase has copper(II) in the active site, which is responsible for tyrosinase oxidation activities [9].
