Insights on the Inhibitory Power of Flavonoids on Tyrosinase Activity: A Survey from 2016 to 2021

Tyrosinase is a multifunctional copper-containing oxidase enzyme that initiates melanin synthesis in humans. Excessive accumulation of melanin pigments or the overexpression of tyrosinase may result in skin-related disorders such as aging spots, wrinkles, melasma, freckles, lentigo, ephelides, nevus, browning and melanoma. Nature expresses itself through the plants as a source of phytochemicals with diverse biological properties. Among these bioactive compounds, flavonoids represent a huge natural class with different categories such as flavones, flavonols, isoflavones, flavan-3-ols, flavanones and chalcones that display antioxidant and tyrosinase inhibitor activities with a diversity of mechanistic approaches. In this review, we explore the role of novel or known flavonoids isolated from different plant species and their participation as tyrosinase inhibitors reported in the last five years from 2016 to 2021. We also discuss the mechanistic approaches through the different studies carried out on these compounds, including in vitro, in vivo and in silico computational research. Information was obtained from Google Scholar, PubMed, and Science Direct. We hope that the updated comprehensive data presented in this review will help researchers to develop new safe, efficacious, and effective drug or skin care products for the prevention of and/or protection against skin-aging disorders.


Introduction
Tyrosinase is a multifunctional copper-containing oxidase enzyme, involved in the initial steps of melanin production [1]. Melanin synthesis is regulated by the microphthalmiaassociated transcription factor (MITF) that is activated by multiple pathways such as cAMP-responsive element-binding protein (CREB), Wnt, glycogen synthase kinase 3β, and mitogen-activated protein kinases, and in turn modulates melanogenic enzyme expression such as tyrosinase [2]. This enzyme catalyzes the oxidation of L-tyrosine or L-3,4-dihydroxyphenylalanine (L-DOPA) to DOPA-quinone, which is the rate-limiting step of melanin synthesis [3].
Excessive production of melanin pigments resulted in different dermatological disorders such as skin aging spots, wrinkles, melasma, freckles, lentigo, ephelides, nevus and melanoma [4]. More seriously, tyrosinase catalyzes the oxidation of dopamine into quinone derivatives that initiate the progress of Parkinson disease [5]. These physiological disturbances are primarily ascribed to the excess accumulation of reactive oxygen species (ROS) or oxidative stress, which can affect the biological macromolecules and cellular functions, thus leading to aging-related disorders or hyperpigmentation [6]. Many patients suffer from these ailments, which have a damaging effect on their quality of life [7]. Furthermore,

The Role of Tyrosinase in the Pathway of Melanin Biosynthesis
Tyrosinase is bio-functional copper-containing enzyme implicated in multidisciplinary functions such as browning in plants, skin melanogenesis cascade in humans, the differentiation of the reproductive system, the host defense system in arthropods, and spore development in fungi [17]. Tyrosinase is mainly known as the key enzyme that regulates the quantity of melanin pigment formation in mammals [18]. Melanin is found in two major forms of pigments, namely eumelanin (black-brownish) and pheomelanin (redyellowish) [19]. The quantity and combination of these two forms can influence the colour of the skin, eyes, and hair. Furthermore, these pigments protect the human skin against harmful UV radiation and prevent DNA mutations and the progression of skin cancer. On other hand, the overproduction of melanin pigments could lead to hyperpigmentation diseases, such as lentigo, ephelides, melasma, freckles and nevus [20].
Tentative phytochemical research was conducted on the 50% methanol extract of Oroxylum indicum (Bignoniaceae) seeds using HPLC/TOF-MS, and then the identified compounds were screened for tyrosinase-binding affinity [29]. Among these compounds, two flavonoids, namely baicalein (5,6,7-trihydroxyflavone) (6) and oroxin A (baicalin-7glucoside) (7), exhibited the most potent inhibitory activities against the enzyme, with inhibition values of 64.90% and 59.70%, and IC 50 values of 0.29 and 0.50 mM. The molecular docking assays attributed these remarkable anti-tyrosinase properties to the structural similarities that make them fit on the same amino acid residues in the TYR catalytic pocket. Furthermore, the glycosylation of flavonoids could reduce their inhibitory activity.
Genkwanin (4 ,5-Dihydroxy-7-methoxyflavone) (15), previously isolated from Daphne gnidium stems, Alnus glutinosa seeds, and Asplenium normale leaves, was evaluated for its tyrosinase activity and its effect on melanin synthesis [34]. It demonstrated doseand time-dependent inhibition of tyrosinase activity in B16F10 melanoma cells. It also caused a significant dose-dependent reduction in melanin synthesis. Apigenin-7-O-β-Dglucopyranoside (16), the abundant flavone in different Thymus species, was evaluated at different concentrations for its tyrosinase activity and its influence on melanin synthesis [35]. It demonstrated significant stimulation of tyrosinase activity of B16F10 melanoma cells in a dose and time dependent manner. In addition, the flavone dose-dependently stimulated the synthesis of intracellular melanin. The up-regulation of the expression and the activity of the microphthalmia-associated transcription factor regulating the gene transcription of tyrosinase as well as activation of the cyclic AMP-protein kinase A pathway were the supposed underlying mechanisms. In a comparative study of the tyrosinaseinhibitory activities of fifty flavonoids, the antioxidant flavone swertiajaponin demonstrated the most potent inhibitory activity against mushroom tyrosinase, with an IC 50 value of 43.47 µM, comparable to the positive control kojic acid, with an IC 50 value of 41.26 µM [36]. Swertiajaponin (6-C-β-D-glucopyranosyl-7-O-methylluteolin) (17), abundant in Swertia japonica and Cymbopogon citratus, demonstrated significant inhibition of skin pigmentation in a human skin model in addition to the depression of melanin accumulation in αMSHor UVB-induced B16F10 cells. The interaction with the binding site of tyrosinase enzyme in addition to the reduction in tyrosinase protein levels via the inhibition of stress-mediated MAPK/MITF signaling is the suggested underlying mechanism behind the potent tyrosinase-inhibiting activity.
A comparative study was conducted for tyrosine inhibitory activities of structurally related flavones; norartocarpetin (5,7,2 ,4 -tetrahydroxyflavone) (18) and luteolin (3 ,4 ,5,7tetrahydroxy-flavone) (19) [37]. Despite the great similarity in the chemical structures of both flavones differing only in the position of a hydroxyl group, a vast difference was observed in their tyrosinase inhibitory activities. Norartocarpetin (18) demonstrated nearly 2200-fold stronger tyrosinase inhibitory activity than luteolin, with IC 50 values of 0.12 µM compared with luteolin (IC 50 = 266.67 µM). In addition, norartocarpetin demonstrated 570-fold stronger tyrosinase activity than kojic acid (IC 50 = 266.67 µM). The kinetic studies demonstrated the strong reversible competitive inhibition of tyrosine induced by norartocarpetin. However, luteolin (19) demonstrated weak, noncompetitive reversible inhibition. Computational docking simulations explained the reversible competitive inhibition induced by norartocarpetin by its ring B hydroxyl groups binding to Asn81 and His85 residues located in the catalytic pocket of tyrosinase. Meanwhile, the hydroxyl groups of the B ring of luteolin bind to residues Cys83 and Asn8. In another study, Park et al. [38] isolated fourteen flavonoids from the fruits of Juniperus chinensis and evaluated their inhibitory activity on mushroom tyrosinase. Among these compounds, hypolaetin-7-O-β-D-glucopyranoside (8-hydroxyluteolin-7-O-β-D-glucopyranoside) (20) and quercetin-7-O-α-L-rhamnopyranoside (21) were found to reduce tyrosinase activity at a concentration of 50 µM. They could effectively attenuate the cellular tyrosinase activity and melanogenesis in α-MSH plus IBMX-stimulated B16F10 melanoma cells. The chemical structures of the flavones with anti-tyrosinase activities are presented in Figure 2.
In a comparative study of the tyrosine inhibitory activity of nine flavonols and flavonol glycosides, quercetin-3-O-β-galactopyranoside (31) demonstrated the most potent significant inhibitory activity (IC 50 = 40.94 ± 0.78 µM) [43]. An in silico study supported the results demonstrating the crucial value of the hydroxyl group at the 7th position for competitive inhibition of tyrosinase enzyme. Moreover, the study supported the importance of hydroxyl substitution at C-3 and C-4 in binding with tyrosinase enzyme.

Flavan-3-ols
The milk thistle (Silybum marianum) is well-known to be rich in flavonolignans with antioxidant, anti-inflammatory, antiviral and antifibrotic properties [56]. Among these compounds, silybin (47) was demonstrated to be the most potent one for tyrosinase inhibition, with an IC 50 value of 1.70 ± 0.07 µM, compared to kojic acid (IC 50 = 15.30 ± 0.50 µM) [57]. Based on kinetic studies, silybin (47) showed mixed type 1 inhibition of enzyme and a significant binding affinity with K i value of 0.7 µM. A study published by Chunhakant and Chaicharoenpong reported characterization of phytochemicals from Manilkara zapota (Sapotaceae) bark [58]. Among the isolated compounds, (+)-dihydrokaempferol (48) [59] studied the molecular docking of dihydromyricetin (49), a common natural flavonoid, on tyrosinase. This compound is fitted in the pocket of tyrosinase with hydrophobic interactions and hydrogen bonds, resulting in conformational changes of tyrosinase that hinder substrate binding. Consequently, it inhibited tyrosinase activity in a mixed-type manner with an IC 50 value of 3.66 ± 0.14 × 10 −5 mol/L, compared with kojic acid (IC 50 = 4.64 ± 0.37 × 10 −5 mol/L). In addition, the combination of dihydromyricetin (49) with vitamin D 3 displayed a synergistic effect on the enzyme inhibition. In Turkey, a group of researchers isolated a new catechin, namely (−)-8-chlorocatechin (50), from Quercus coccifera bark [60]. The isolated compound (50) expressed tyrosinase inhibition with IC 50 value of 4.05 ± 0.30 µg/mL, more potent even than kojic acid (IC 50 = 50.75 ± 0.25 µg/mL). Molecular modeling studies revealed the good fitting properties of the compound to the catalytic site of tyrosinase via 4-chromanone moiety with its hydroxyl groups. The chemical structures of the flavan-3-ols with anti-tyrosinase activities are presented in Figure 5.

Biflavonoids
Rhusflavanone (79) and mesuaferrone B (80) are major bioflavonoids, isolated from the methanolic extract of Mesua ferrea L. stamens with contents of 0.35 ± 0.04% and 0.55 ± 0.06%, respectively [75]. Both biflavonoids efficiently displayed inhibitory activities against mushroom tyrosinase, with IC 50 values of 10.6 and 10.3 µg/mL, respectively, compared with arbutin (IC 50 = 87.20 µg/mL). Furthermore, the dimerization of flavonoid units was found to contribute effectively in potent tyrosinase inhibitory activities, comparable to the original flavonoid monomers. The chemical structures of the biflavonoids with anti-tyrosinase activities are presented in Figure 9. We summarize the promising antityrosinase flavonoids, their sources, the type of the performed assays and their significance in Table 1. Additionally, we illustrated the different modes of actions reported for tested flavonoids in Table 2. As observed, flavonoids exhibited antityrosinase activity through different pathways such as competitive inhibition, non-competitive inhibition, mixed inhibition of tyrosinase, downregulation of MITF expression, and suppression of the cAMP-CREB signaling pathway.    Table 2. Summarized modes of tyrosinase inhibitory actions for flavonoids.

Mode of Action Compounds
Competitive inhibition of tyrosinase

Conclusions and Future Perspectives
In this review, reports from 2016-2021 on recently isolated flavonoids with tyrosinase inhibitor activity were summarized and critically analyzed. The review focused on the potential activities of different categories of flavonoids, including flavones, flavonols, isoflavones, flavan-3-ols, flavanones, prenylated flavonoids and biflavonoids in modulating the activity of tyrosinase enzyme. The studies varied between in vitro, in vivo and in silico computational assays. Moreover, the integration of inhibition kinetics and docking studies aided in the better understanding of structure-activity relationship (SAR) of flavonoids and tyrosinase inhibitory activity. It is worth mentioning that the number and the position of hydroxyl groups especially at ring B drastically affect the activity of different flavonoid classes via Cu 2+ chelate formation. In addition, the isoprene moiety and the dimerization of flavonoids contribute in the inhibitory activity of prenylated flavonoids and biflavonoids, respectively. Additionally, the mechanisms of tyrosinase activity inhibition for some promising flavonoids were discussed. Several inhibition mechanisms have been reported for the described inhibitors, pointing to the copper chelating and/or hydrophobic moieties as key structural requirements to achieve good inhibition properties. Some flavonoids were proved to suppress the expression of tyrosinase via the modulation of certain signaling pathways such as MITF, AMPK and MAPK proteins phosphorylation, CREB and p38.
Despite this extensive research, we observed that the studies addressing the safety profile the promising flavonoids are very scarce. Additionally, some studies are recommended to be extrapolated from in vitro assays to animal and clinical studies to assess the pharmacokinetics, topical permeation, bioavailability, dose issues, lifespan, anatomy and metabolic status. As the conventional whitening market products suffer from severe side effects such as dermatitis and skin cancer in long-term use, we highly recommend welldesigned long-term studies in the human subjects to investigate the potential toxic effects, biochemical and molecular mechanisms of the bioactive flavonoids. Interestingly, these studies will potentially push researchers to develop a novel drug for treating skin-aging ailments using medicinal chemistry approaches to synthesize more potent derivatives and study their detailed mechanisms of action.