**3. The production of melanin**

#### **3.1. Tyrosinase**

Tyrosinase is responsible for the hydroxylation of monophenol and conversion of an *o*-di‐ phenol to the corresponding *o*-quinone which then undergoes several modifications to be‐ come melanin (Fig. 3). Tyrosinases are ubiquitously expressed by both plants and animals, and although they may vary in structure, they all contain copper as an important cofactor. Human tyrosinase is a transmembrane protein and is sorted into specialized organelles in melanocytes called melanosomes such that melanin is ultimately synthesized and stored within these melanosomes[17]. Tyrosinase is an excellent example of convergent evolution in that although both animals and plants express tyrosinase, and although tyrosinase essen‐ tially performs the same function in all of these organisms, the structure and enzymatic re‐ quirements of these proteins are diverse. Regardless, tyrosinase regulates both the type and amount of melanin synthesized in the specialized cells that contain it. In humans, tyrosinase is encoded by the *tyr* gene which when mutated causes albinism in humans[18]. In Caucasi‐ ans, tyr mutations can be identified in 56% of patients with autosomal recessive ocular albin‐ ism, characterized by reduced pigmentation of the eye[19].

#### **3.2. Microphthalmia-Associated Transcription Factor (MITF)**

Transcription of *tyr* is controlled by microphthalmia-associated transcription factor (MITF). MITF acts both as a transcription activator to promote expression of genes involved in mela‐ nogenesis within the cell cycle and as a transcriptional repressor of genes involved in inva‐ sion, making elevated MITF levels a possible biomarker for melanoma[20]. In humans, MITF regulates the expression of tyrosinase (TYR), tyrosinase-related protein-1 (TRP-1), and tyro‐ sinase-related protein-2 (TRP-2). A complex regulatory network precisely modulates MITF expression and activation and therefore controls tyrosinase and melanogenesis. Therefore, targeting only tyrosinase or only MITF is destined to be either an ineffectual or a temporary intervention. It is likely that combination therapy that targets MITF, tyrosinase and the other regulatory pathways is more likely to produce a measurable and prolonged effect on skin pigmentation.

One of the key pathways in the stimulation of melanocytes in response to UV radiation is the adenylate cyclase pathway. UV radiation mediates the synthesis and release of alphamelanocyte-stimulating hormone (α-MSH) which then promotes pigmentation by binding to the melanocortin 1 receptor (MC1R) on the surface of melanocytes. In people with the red-hair/fair-skin phenotype, the MC1R gene is non-signaling which leads to the increased risk for the development of melanoma. The production of cyclic adenosine monophosphate (cAMP) downstream of MC1R activates the receptors of MITF. MITF then participates in the conversion of tyrosine into melanin pigments by stimulating the transcription of melano‐ cyte-specific genes. Therapeutic agents that are able to inhibit MITF through the down regu‐ lation of MC1R activity are likely to be particularly effective in reducing melanocyte activity.

**Figure 3.** Melanin biosynthesis.

#### **3.3. Keratinocytes**

**3. The production of melanin**

ism, characterized by reduced pigmentation of the eye[19].

260 Using Old Solutions to New Problems - Natural Drug Discovery in the 21st Century

**3.2. Microphthalmia-Associated Transcription Factor (MITF)**

Tyrosinase is responsible for the hydroxylation of monophenol and conversion of an *o*-di‐ phenol to the corresponding *o*-quinone which then undergoes several modifications to be‐ come melanin (Fig. 3). Tyrosinases are ubiquitously expressed by both plants and animals, and although they may vary in structure, they all contain copper as an important cofactor. Human tyrosinase is a transmembrane protein and is sorted into specialized organelles in melanocytes called melanosomes such that melanin is ultimately synthesized and stored within these melanosomes[17]. Tyrosinase is an excellent example of convergent evolution in that although both animals and plants express tyrosinase, and although tyrosinase essen‐ tially performs the same function in all of these organisms, the structure and enzymatic re‐ quirements of these proteins are diverse. Regardless, tyrosinase regulates both the type and amount of melanin synthesized in the specialized cells that contain it. In humans, tyrosinase is encoded by the *tyr* gene which when mutated causes albinism in humans[18]. In Caucasi‐ ans, tyr mutations can be identified in 56% of patients with autosomal recessive ocular albin‐

Transcription of *tyr* is controlled by microphthalmia-associated transcription factor (MITF). MITF acts both as a transcription activator to promote expression of genes involved in mela‐ nogenesis within the cell cycle and as a transcriptional repressor of genes involved in inva‐ sion, making elevated MITF levels a possible biomarker for melanoma[20]. In humans, MITF regulates the expression of tyrosinase (TYR), tyrosinase-related protein-1 (TRP-1), and tyro‐ sinase-related protein-2 (TRP-2). A complex regulatory network precisely modulates MITF expression and activation and therefore controls tyrosinase and melanogenesis. Therefore, targeting only tyrosinase or only MITF is destined to be either an ineffectual or a temporary intervention. It is likely that combination therapy that targets MITF, tyrosinase and the other regulatory pathways is more likely to produce a measurable and prolonged effect on skin

One of the key pathways in the stimulation of melanocytes in response to UV radiation is the adenylate cyclase pathway. UV radiation mediates the synthesis and release of alphamelanocyte-stimulating hormone (α-MSH) which then promotes pigmentation by binding to the melanocortin 1 receptor (MC1R) on the surface of melanocytes. In people with the red-hair/fair-skin phenotype, the MC1R gene is non-signaling which leads to the increased risk for the development of melanoma. The production of cyclic adenosine monophosphate (cAMP) downstream of MC1R activates the receptors of MITF. MITF then participates in the conversion of tyrosine into melanin pigments by stimulating the transcription of melano‐ cyte-specific genes. Therapeutic agents that are able to inhibit MITF through the down regu‐ lation of MC1R activity are likely to be particularly effective in reducing melanocyte activity.

**3.1. Tyrosinase**

pigmentation.

Keratinocytes are an important structural cell of the epidermis and comprise the majority of the cells in this layer of the skin. Throughout their life cycle, keratinocytes can take on many forms. Keratinocytes are produced by mitosis in the most basal layer of the epidermis when they begin to produce a great deal of keratin. Eventually, older keratinocytes are pushed up to the upper layers by the rapidly dividing keratinocytes beneath them until they reach the stratum granulosum where they begin to drastically change their morphological and bio‐ chemical functions. In the stratum granulosum, keratinocytes lose their organelles and ac‐ quire a flattened appearance. Due to their production of keratin, these cells become strong and their tightly bound structure makes the outermost keratinocyte layer strong and rela‐ tively impermeable.

An important role of keratinocytes is the transport of melanin through the layers of the skin. Once melanin is produced by melanocytes, the pigments are packaged into melanosomes that are subsequently translocated along the dendrites and then captured and aggregated by keratinocytes. The mechanism of transfer has long been a mystery, however a recent study summarizes the transfer mechanism in three steps: (i) the melanocyte's dendrite attaches to the surface of the keratinocyte, (ii) a portion of the melanocyte's dendrite sheds in order to deposit the melanosomes on the surface of the keratinocyte, (ii) and finally, the keratinocyte engulfs the melanosome package[21].

#### **4. Natural ingredients that reduce pigmentation**

Since a number of naturally derived bioactive molecules have been found to reduce skin pigmentation, the cosmetic industry has focused on the development of these compounds for use in their products. These compounds inhibit the production of melanin at different stages of melanogenesis. There are several compounds that specifically inhibit tyrosinase. For example, terrein, a bioactive fungal metabolite agent isolated from the *Penicilllium* spe‐ cies, has been shown to strongly decrease the production of tyrosinase by down-regulating MITF through dual pathways[22]. One pathway of these pathways may involve the activa‐ tion of extracellular signal-regulated kinase (ERK) which has been reported to reduce mela‐ nin synthesis[23]. A second pathway recently identified may involve the ubiquitination and subsequent breakdown of tyrosinase itself[24].

Flavonoids, in particular, have received a great deal attention for their potential pigment-re‐ ducing activity because they are seen as "natural" and are associated with less side-effects than conventional medications and synthetically-designed compounds. Flavonoids are poly‐ phenolic compounds typically found in plants. These compounds are ideal candidates for cosmetic purposes because of their potent bio-activities and low toxicity. Aloesin, a com‐ pound isolated from aloe extracts, has been shown to successfully reduce tyrosinase *in vitro* by inhibiting the hydroxylase activities[25], [26]. Resveratrol, a compound found in red wine, appears to have an affinity for tyrosinase and, by an unknown mechanism, reduces tyrosinase activity and MITF expression[27] although some studies have questioned its abili‐ ty to reduce tyrosinase expression when used alone[28]. Flavonoids isolated from licorice are able to inhibit mushroom tyrosinase[29] and a skin lightening agent that uses licorice ex‐ tract in its formulation (comprised of many bioactive ingredients) has been shown to reduce pigmentation in patients[30]. There are many reports of flavonoids from various sources in‐ terfering with or reducing the activity of tyrosinase, and although they all show some effica‐ cy at reducing the biochemical pathways that lead to pigmentation, it is still unclear whether targeting these pathways in human skin would necessarily lead to reduced melanin forma‐ tion *in vivo*.

Melanin formation is a complex process and there are compounds that may inhibit melano‐ genesis without necessarily inhibiting tyrosinase function. Anti-oxidants such as fermented rice bran and vanillic acid can reduce expression of MITF, MC1R, and associated biochemi‐ cal pathways, without affecting tyrosinase activity,[31], [32] possibly due to their ability to impair peroxidase activity. Anti-oxidants, in general, may potentiate the MC1R pathway which itself is able to reduce downstream levels of hydrogen peroxide and promote nucleo‐ tide excision repair mechanisms in response to UV radiation[33]. The process of melanogen‐ esis involves cell-to-cell communication between keratinocytes and melanosomes and compound such as niacinamide, a bioactive form of niacin (vitamin B3), has been shown to reduce pigmentation both *in vitro* and *in vivo* by downregulating the amount of melano‐ somes transferred to keratinocytes[34].
