Hydroquinone (1,4-dihydroxybenzene, HQ) has been the gold standard for treating hyperpigmentation for more than 50 years and has been successfully used to treat melanosis. The compound can be found in wheat, tea, berries, beer and coffee, but is detoxified within the liver into inert compounds [6,47]. Hydroquinone is a phenolic compound and depigmenting agent that mainly exerts its effect on melanocytes with active tyrosinase. As HQ dependent melanogenic inhibition requires the presence of active tyrosinase, it is therefore not useful in altering the color of melanin that is previously present in the dermis and epidermis [48].

The structural similarity between HQ and melanogenic precursors enables HQ’s interaction with tyrosinase. This interaction mediates HQ’s inhibition of tyrosinase by binding histidines or copper at the active site of the enzyme. Additionally, HQ induced generation of reactive oxygen species and quinones leads to the oxidative damage of membrane lipids and proteins such as tyrosinase. Hydroquinone is also thought to inhibit pigmentation by depleting glutathione, modifying melanosome formation or reducing DNA and RNA synthesis with concomitant melanosome degradation and melanocyte destruction [10,20,49,50].

Traditional hydroquinone formulations contain other constituents that promote a synergistic effect. A popular formulation is comprised of hydroquinone and a corticosteroid to reduce inflammation, along with tretinoin, shown to reduce the atrophy associated with the corticosteroid and remove pigmentation by increasing keratinocyte turnover and the penetration of hydroquinone [50,51].

Due to the risks of side effects such as exogenous ochronosis and permanent depigmentation following long term use, hydroquinone has been banned by the European Committee (24^th Dir 2000/6/EC) and formulations have been withdrawn from cosmetics and are available only through prescription [10,20,50].

Other phenolic compounds have been evaluated for their depigmenting capabilities. In fact the chemical structures of several phenolic compounds have been investigated to delineate structure related tyrosinase inhibitory activity. It has been suggested that having a hydroxyl group para to an electron donator group is required for a compound to be an effective alternative substrate for tyrosinase [10]. Distinct structure-activity based analysis done by Ni-Komatsu et al. on quinolines, which contain a 4-substituted amino group with a tertiary amine side chain, shows significant inhibitory effect. Yet these quinolines, such as chloroquine, were not reported to influence the enzymatic activity of tyrosinase, but rather the intracellular trafficking of tyrosinase related proteins and lysosome associated membrane protein-1 (LAMP-1) [52].

The mono benzyl ether of hydroquinone (MBEH) is a related compound that is metabolized within the cell to form a quinone species that interacts with and results in permanent depigmentation, even at areas distant from the site of application. MBEH can destroy melanocytes and should not be used to treat post-inflammatory hyperpigmentation or melasma. MBEH therapy is appropriate for generalized depigmentation in the treatment of patients with vitiligo unresponsive to repigmentation therapy [10,20,48]. Proposed mechanisms of action for MBEH are both cytotoxicity to melanocytes as a result of free radical formation and competitive inhibition of tyrosinase activity [6].

Arbutin (hydroquinone-O-β-d-glucopyranoside), a derivative of hydroquinone, is a botanically derived compound found in cranberries, blueberries, wheat and pears [6,51]. Arbutin is used as an effective treatment of hyperpigmentary disorders, and displays less melanocyte cytotoxicity than hydroquinone. The compound inhibits melanogenesis by competitively and reversibly binding tyrosinase without influencing the mRNA transcription of tyrosinase. It also inhibits the maturation of melanosomes, possibly by its reported influence on DHICA polymerase activity and Pmel-17 protein. The mild effect of arbutin is attributed to the controlled release of hydroquinone as a result of in-vivo cleavage of the glycosidic bond. Higher concentrations of arbutin are more efficacious than lower concentrations, but may cause paradoxical hyperpigmentation [20,45,49,51,53]. Deoxyarbutin (dA), a synthetic form of arbutin synthesized without the hydroxyl moiety, provides a promising treatment for reducing skin hyperpigmentation [50]. dA shows reversible inhibition of tyrosinase activity with associated skin lightening in both a hairless guinea pig model system and in human skin. The reversibility of dA’s impact on skin pigmentation suggests that the compound does not permanently destroy melanocytes [20,54,55]. In addition to the reported efficacy, Hamed et al. have found that dA is less cytotoxic/cytostatic than HQ in treatment of cultured human melanocytes [56]. Chawla et al. have reported that dA and associated second-generation derivatives, dose-dependently inhibit tyrosinase hydroxylation and DOPAoxidase activity of tyrosinase. This may be attributed to the chemical structure of dA, as the deoxysugars may increase skin penetration and binding affinity for tyrosinase [20,54].

Mequinol (hydroquinone monomethyl ether, 4-hydroxyanisole, para-hydroxymethoxybenzene), another derivative of hydroquinone, is enzymatically oxidized by tyrosinase to produce melanocytotoxic quinones. The formation of quinones results in pigment cell destruction and skin depigmentation [10,57]. The combination of 0.01% tretinoin with mequinol has been reported to inhibit melanin production and has been shown to be effective and safe in the treatment of solar lentigenes and related hyperpigmentation [48,50,58,59].

N-Acetyl-4-S-cysteaminylphenol (NCAP) is a phenolic thioether that has been used in the treatment of epidermal hyperpigmentation such as melasma and also in anti-melanoma studies. NCAP is structurally similar to tyrosine and acts specifically in melanin-synthesizing cells as an alternative tyrosinase substrate [48,50,60]. Ferguson et al. suggests that NCAP may undergo oxidation by tyrosinase to form a reactive o-quinone that is capable of alkylating thiol groups of essential enzymes, which may interfere with cell growth and proliferation [61]. In a 12 patient study of the efficacy of NCAP completed by Jimbow, the author describes an 8% complete loss and 66% marked improvement in visible changes of melanoderma after 2 to 4 weeks of topical application. The author attributes this depigmentation to a decrease in the number of functional melanocytes and the number of melanosomes transferred to keratinocytes. NCAP is suggested to be a depigmenting agent that is less irritating and more stable than HQ, with specificity for melanin-synthesizing cells [60].

Kojic acid (5-hydroxy-2-hydroxymethyl-4H-pyran-4-one, KA) is a naturally occurring hydrophilic fungal metabolite obtained from species of Acetobacter, Aspergillus and Penicillium [48,49]. Kojic acid is believed to inactivate tyrosinase by chelating copper atoms as well as suppressing the tautomerization of dopachrome to DHICA. [50] Although KA is a popular treatment for melasma, it is associated with sensitization, contact dermatitis and erythema [51]. A distinct, more stable derivative of kojic acid synthesized by Kim et al., 5-[(3-aminopropyl)phosphinooxy]-2-(hydroxymethyl)-4H-pyran-4-one (Kojyl-APPA), showed increased skin penetration and pigment lightening efficacy in melanoma and normal human melanocytes [20,62].

Azelaic acid (1,7-heptanedicarboxyilic acid, AZA) is a naturally occurring non-toxic straight chain, saturated dicarboxylic acid derived from Pityrosporum ovale [57,63,64]. AZA appears to selectively influence the mechanism of hyperactive and abnormal melanocytes, but minimally influences normal skin pigmentation, freckles, nevi and senile lentigenes [6,10,65]. AZA’s antiproliferative and cytotoxic effect may be mediated by the inhibition of DNA synthesis and mitochondrial oxidoreductase activity. The compound is also able to bind amino and carboxyl groups and may prevent the interaction of tyrosine in the active site of tyrosinase and thus function as a competitive inhibitor. Although not all authors are in agreement with the therapeutic efficacy of AZA, it has been reported to be effective in treatment of melasma and acne [10,65]. A 24 week multicenter, controlled, double blind clinical trial of 329 women completed by Baliña et al. compared the efficacy of a 20% AZA cream to a 4% HQ cream in treating melasma. The authors reported no significant difference between the results where ~65% of the patients treated with AZA were reported to achieve good to excellent results compared to ~73% of HQ treated patients [66]. 20% azelaic acid seems to be well tolerated in treated patients with no systemic side effects, but some local cutaneous irritation, a burning sensation, mild erythema, scaling and pruritus that subsided 2 to 4 weeks post treatment [65,67].

The methyl ester of gentisic acid (2,5-dihydroxybenzoic acid, MG) is a natural derivative of Gentianas root with the capacity to inhibit tyrosinase. MG can act as a pro-drug that releases HQ which subsequently inhibits tyrosinase. Yet, methyl gentisate is less cytotoxic and mutagenic than HQ [10,20,50].

Flavonoids are natural plant polyphenols found in leaves, bark and flowers. Some 4,000 members have been identified to date, all benzo-γ-pyran derivates comprised of phenolic and pyran rings. These polyphenolic compounds are known to have anti-inflammatory, antiviral, antioxidant and anticarcinogenic properties [20,50,63]. The flavonoids may also have ROS scavenging properties and the ability to chelate metals at the active site of metalloenzymes. Flavonoids may have hypopigmenting capabilities by directly inhibiting tyrosinase activity at distal portions of the melanogenic pathway [20]. Structure-function analysis of flavonoids suggests that flavonoids with an α-keto group show potent tyrosinase inhibition due to the similarity between the dihydroxyphenyl group of DOPA and the α-keto containing flavonoids [63]. Similar analysis completed by Kubo et al. suggests that flavonoids have the capability to chelate copper in tyrosinase’s active site as long as the 3-hydroxygroup is free [64,68]. A comprehensive review on the properties of plant polyphenols has been published by Kim et al. [63] Flavonoids that will be reviewed here include aloesin, hydroxystilbene derivates and licorice extracts.

Ultraviolet radiation influences the proliferation of melanocytes and the production and secretion of paracrine and autocrine factors that stimulate melanogenesis. UV radiation can also produce reactive oxygen species (ROS) in the skin that may induce melanogenesis, DNA damage, melanocyte proliferation and/or apoptosis [76,77]. The skin contains a number of antioxidants that can be depleted by UV exposure and cause oxidative damage. The application of topical antioxidants has the capacity to prevent oxidative damage to the skin [78]. Redox agents capable of scavenging ROS generated in the skin can inhibit second messengers that may stimulate melanogenesis. Redox agents can also influence skin pigmentation by interacting with copper at the active site of tyrosinase or with o-quinones to impede the oxidative polymerization of melanin intermediates [10,79]. Here we will review vitamin C, vitamin E and later we will review the vitamin B derivative niacinamide.

The first step of melanosome transfer from melanocytes to surrounding keratinocytes is successful melanocytic dendrite formation and extension towards surrounding keratinocytes. The extension of melanocytic dendrites requires the reorganization of the melanocyte cytoskeletal elements such as actin filaments and microtubules [88]. Small GTPases Rho, Rac and Cdc42 play a pivotal role in cell morphology and dendrite formation. Specifically, Rac stimulates membrane ruffling and lamellipodia formation, Rho activates dendrite retraction and Cdc42 mediates filopodia and peripheral actin microspike formation [88–90]. Ito et al. has shown that treatment of melanocyte and keratinocyte co-cultures with methylophiopogonanone B (5,7-dihydroxy-6,8-dimethyl-3-(4-methoxybenzyl)chroman-4-one, MOPB), an agent reported to activate Rho and induce microtubule disorganization and tubule depolymerization, appeared to reduce melanosome transfer [89,90]. The authors also showed that treatment with 1μM MOPB did not influence melanin synthesis or the expression of melanogenic enzymes. MOPB appeared to induce a reversible dendrite retraction and transfer inhibition without associated cytotoxicity (tested up to 72 hours) [89]. Centaureidin (5,7,3’-trihydroxy-3,6,4’-trimethoxyflavone), a flavonoid glucoside derived from yarrow, also reduces melanosomal transfer to keratinocytes. Centaureidin is believed to directly or indirectly activate Rho, leading to melanocyte dendrite retraction without influencing melanogenic enzyme expression or melanin synthesis [88,91]. More analysis is needed to confirm the applicability of MOPB and centaureidin as skin lightening agents.

Niacinamide (vitamin B3, nicotinamide, 3-pyridinecarboxamide) is a biologically active form of niacin found in many root vegetables as well as in yeast [70,92]. Physiologically, niacinamide functions as a precursor to the co-factors nicotinamide adenine dinucleotide (NAD) and nicotinamide adenine dinucleotide phosphate (NADP). Along with their reduced forms NADH and NADPH, these enzymes participate in numerous enzymatic reactions and also act as antioxidants [70,92]. Niacinamide has several proposed medicinal applications in the skin including anti-inflammation, prevention of photoimmunosuppression and increased intercellular lipid synthesis [93]. Niacinamide’s role as a co-enzyme precursor may explain the multiple roles it has in skin, but this is not clearly defined [82,94]. Topical niacinamide is described to have several benefits on aging skin including but not limited to improved barrier function, improved appearance of photoaged facial skin (including texture, hyperpigmentation, redness, fine lines and wrinkles) and reduced sebum production [92,94–97]. Additionally, niacinamide is believed to influence cutaneous pigmentation by down-regulating transfer of melanosomes from the melanocytes to the keratinocytes [20,57,92]. Studies completed by Hakozaki et al. suggest that niacinamide has no effect on tyrosinase activity, melanin synthesis or melanocyte number in a monolayer culture system. Alternatively, the authors found that niacinamide down-regulated the number of melanosomes transferred from melanocytes to keratinocytes by 35 to 68% in a co-culture model system [92]. The actual process by which niacinamide down-regulates melanosome transfer remains to be properly established.

PAR-2 belongs to a family of transmembrane G-protein coupled receptors (PAR1–PAR4) that are proteolytically activated by serine proteases. Specifically, the serine proteases (including trypsin or mast cell tryptase) cleave the extracellular amino terminal domain exposing a newly created N-terminus tethered ligand that undergoes a conformational change, binds and subsequently activates the receptors [98,99]. Within the epidermis, PAR-2 is expressed in keratinocytes, but not in melanocytes and is involved in the regulation of skin pigmentation through melanocyte-keratinocyte interactions [99–103]. Studies completed on keratinocyte PAR-2 indicate that it may influence melanosome incorporation and phagocytosis by keratinocytes and play a regulatory role in skin pigmentation. Therefore, modulation of PAR-2 activity augments or decreases melanosome transfer and in turn skin pigmentation [99,103–106]. PAR-2 activation can be achieved by synthetic peptides corresponding to the sequence of the N-terminal ligand. The peptide of mouse PAR-2 cleavage sequence SLIGRL is an equipotent activator of mouse and human PAR-2 receptor in comparison to the human tethered ligand SLIGKV [100]. Interestingly, PAR-2 expression and induction by ultraviolet irradiation is dependent on skin type, with a higher overall expression and induction in darker skin individuals [98,104,107]. Activation of PAR-2 enhances melanosome transfer, while inhibition of PAR-2 by serine protease inhibitors can result in reduced melanosomal transfer and distribution. Work published by Seiberg et al. and Paine et al. has shown that this inhibition leads to a dose-dependent lightening of skin pigmentation [103,104,106]. Work completed by Seiberg et al. examining the effect of serine protease inhibitor RWJ-50353 on epidermal equivalents shows an accumulation of melanosomes in melanocytes, with an increase in early stage melanosomes compared to untreated controls. They hypothesize that the keratinocytes inability to receive the presented melanosomes leads to an accumulation of melanosomes in the melanocytic dendrite and a concomitant negative feedback mechanism that slows pigment production. The authors additionally showed that Yucatan swine skin treated with RWJ-50353 for an 8 week period demonstrated a dose dependent, reversible skin lightening effect [103].

“Natural” therapies derived from soybeans have been explored for their safety and efficacy as depigmentation treatments. Two protein proteinase inhibitors have been isolated from soybean seeds Kunitz-type trypsin inhibitor (soybean trypsin inhibitor, STI) and the Bowman-Birk protease inhibitor (BBI). STI and BBI are found in the seeds of soybeans, but not in the other regions of the plant. STI inhibits trypsin proteolysis by forming a stable stoichiometric complex and BBI inhibits trypsin and chymotrypsin at separate reactive sites [106,108]. Work completed by Paine et al. has shown that soymilk and soybean extract reduces pigmentation in dark skinned Yucatan swine treated for an eight week period. The authors suggest that the soymilk inhibits PAR-2 activation in the skin and results in skin depigmentation. Moreover, the authors suggest that STI and BBI inhibit PAR-2 activation, causes a reduction in keratinocyte phagocytosis and a reduction in resulting skin pigmentation [106]. Soymilk also contains other constituents that may induce skin lightening such as trace amounts of free fatty acids and their acyl CoA esters that can inhibit trypsin and may participate in PAR-2 inhibition. Soybeans contain isoflavones, which are antioxidants that may reduce tyrosinase’s DOPA oxidase activity. In addition, soybeans contain phospholipids which the authors suggest may assist in the epidermal delivery of STI and BBI without the assistance of liposomes, when soy milk is used as a topical treatment [51,106].

Cellular recognition between melanocytes and keratinocytes is an important event involved in melanosome transfer [87]. Lectins and neoglycoproteins have been explored as candidates that are involved in this phenomenon, because of their influence in cellular processes including intracellular trafficking, endocytosis and cell-cell recognition [87,93]. Interestingly, Minwalla et al. have demonstrated a role for melanocyte and keratinocyte membrane glycosylated residues in the process of receptor-mediated endocytosis to facilitate melanosome transfer. The authors suggest that lectins and neoglycoproteins play an inhibitory role in this process [20,87]. Specifically, plasma membrane lectins and their glycoconjugates are thought to interrupt melanocyte and keratinocyte contact and interaction, by binding their specific plasma membrane receptors, inhibiting melanosome transfer [109]. This inhibition is reversible and is shown to be enhanced by the presence of niacinamide [93].

α-Hydroxyacids (AHA) are weak organic acids found in fruits, plants and milk sugars [111]. For centuries, α-hydroxyacids have been one of the most commonly utilized peeling agents used to treat dry skin, acne, actinic damage and to improve skin color/texture [51]. AHAs are also reported to effectively treat pigmentary lesions such as solar lentigenes, melasma and post inflammatory hyperpigmentation (PIH). At low concentrations AHAs promotes exfoliation by decreasing corneocyte cohesion and stimulating new growth in the basal layer, while at higher concentrations AHAs promote epidermolysis and dispersed basal layer melanin. The accelerated desquamation of the stratum corneum by AHAs is complemented by a direct inhibition of tyrosinase, without influencing mRNA or protein expression [51,110,111]. Lactic acid (LA) and glycolic acid (GA) are AHAs derived from sour milk and sugarcane juice, respectively [110]. In a study investigating the histological differences between Japanese subjects treated for 6 weeks with 40% AHA, either glycolic, lactic or acetic acids, Yamamoto et al. reported an increase in epidermal thickness, decreased melanin deposition and up-regulated collagen levels. The authors also suggest that the AHAs induced a remodeling of the epidermis with accelerated desquamation [112].

Salicylic Acid (SA) is a β-hydroxyacid found in willow bark and sweet birch. It is also a phytohormone, a plant product that acts similar to a hormone and regulates cell growth and differentiation. SA functions as a desquamating agent that penetrates and dissolves the intercellular matrix of the stratum corneum [50,111].

Unsaturated fatty acids including oleic acid (C18:1), linoleic acid (C18:2) or α-linolenic acid (C18:3) suppresses melanogenesis and tyrosinase activity, while saturated fatty acids such as palmitic acid (C16:0) or stearic acid (C18:0) increases it [113]. Linoleic acid reduces the activity of tyrosinase in melanocytes, while mRNA levels remain unchanged [51]. No evidence of change in TYRP-1 and TYRP-2 protein levels suggest that fatty acids selectively target tyrosinase. This may influence the enzyme’s degradation via a physiologic proteasome-dependent mechanism, altering the tyrosinase protein content in hyperactive melanocytes [10,114]. Linoleic acid also influences skin pigmentation by stimulating epidermal turnover and increased desquamation of melanin pigment from the epidermis [51]. Studies completed to assess the skin lightening capabilities of unsaturated fatty acids, linoleic acid or α-linoleic acid, on UV induced hyperpigmentation of brown guinea pig skin, showed an efficient lightening effect [115]. It is thought that the unsaturated bonds of these molecules can be easily peroxidized, which in combination with an increase in epidermal turnover, correlate with an inhibitory effect on melanogenesis in vivo [51,115].

Retinoids are a common treatment option used to ameliorate acne, photodamage and PIH. The mechanism of action likely involves the inhibition of tyrosinase, the dispersion of keratinocyte pigmented granules, reduction in pigment transfer and a reduction in corneocyte cohesion with an associated acceleration of epidermal turnover [20,51,116]. Tretinoin (all-trans retinoic acid) is a derivative of vitamin A that is thought to have an inhibitory effect on tyrosinase transcription [117]. Tretinoin is reported to be effective in treating melasma, with some associated side effects including erythema, peeling at the site of application and PIH [20]. Tretinoin is also used in combination in topical creams, such as a formulation proposed by Kligman and Willis containing 5% HQ, 0.1% tretinoin and 0.1% dexamethasone. Tretinoin in this formulation acts as a stimulant of epidermal turnover and pigment reduction via epidermopoieses, an antioxidant to reduce the oxidation of HQ and a mild irritant to enhance the epidermal penetration of HQ [110].