Abstract
Skin hyperpigmentation disorders arise due to excessive production of the macromolecular pigment melanin catalyzed by the enzyme tyrosinase. Recently, the therapeutic use of curcumin for inhibiting tyrosinase activity and production of melanin have been recognized, but poor stability and solubility have limited its use, which has inspired synthesis of curcumin analogs. Here, we investigated four novel chemically modified curcumin (CMC) derivatives (CMC2.14, CMC2.5, CMC2.23 and CMC2.24) and compared them to the parent compound curcumin (PC) for inhibition of in vitro tyrosinase activity using two substrates for monophenolase and diphenolase activities of the enzyme and for diminution of cellular melanogenesis. Enzyme kinetics were analyzed using Lineweaver-Burk and Dixon plots and nonlinear curve-fitting to determine the mechanism for tyrosinase inhibition. Copper chelating activity, using pyrocatechol violet dye indicator assay, and antioxidant activity, using a DPPH radical scavenging assay, were also conducted. Next, the capacity of these derivatives to inhibit tyrosinase-catalyzed melanogenesis was studied in B16F10 mouse melanoma cells and the mechanisms of inhibition were elucidated. Inhibition mechanisms were studied by measuring intracellular tyrosinase activity, cell-free and intracellular α-glucosidase enzyme activity, and effects on MITF protein level and cAMP maturation factor. Our results showed that CMC2.24 showed the greatest efficacy as a tyrosinase inhibitor of all the CMCs and was better than PC as well as a popular tyrosinase inhibitor-kojic acid. Both CMC2.24 and CMC2.23 inhibited tyrosinase enzyme activity by a mixed mode of inhibition with a predominant competitive mode. In addition, CMC2.24 as well as CMC2.23 showed a comparable robust efficacy in inhibiting melanogenesis in cultured melanocytes. Furthermore, after removal of CMC2.24 or CMC2.23 from the medium, we could demonstrate a partial recovery of the suppressed intracellular tyrosinase activity in the melanocytes. Our results provide a proof-of-principle for the novel use of the CMCs that shows them to be far superior to the parent compound, curcumin, for skin depigmentation.
Highlights
Melanocytes originate from neural crest-derived melanoblasts and have a key role in the synthesis of melanin pigment within organelles called melanosomes, which are secreted and transferred to keratinocytes [1]
Our results on the inhibition of monophenolase activity of tyrosinase showed that pure curcumin (PC) significantly inhibited monophenolase activity by 14.41%, 21.31%, and 22.10% at concentrations of 10, 20, and 25 μM, respectively
CMC2.24 inhibited the monophenolase activity by 32.08%, 34.66%, 34.84%, and 37.02% at 5, 10, 20, and 25 μM, respectively; this inhibition was significantly different from control as well as significantly greater than that of equivalent concentrations of the parent compound PC (Figure 1B)
Summary
Melanocytes originate from neural crest-derived melanoblasts and have a key role in the synthesis of melanin pigment within organelles called melanosomes, which are secreted and transferred to keratinocytes [1]. Biomolecules 2021, 11, 674 production of melanin in the skin can lead to hyperpigmentation and is associated with skin disorders such as melasma, post-inflammatory hyperpigmentation (PIH) and lentigo senilis (LS), resulting in significant psychosocial burden. 1.14.18.1) is a copper-containing membrane-bound glycoprotein that is the key rate-limiting enzyme in the synthesis of the macromolecular pigment, melanin; it catalyzes the conversion of L-tyrosine to L-3,4-dihydroxyphenylalanine (L-DOPA) and subsequent conversion to Dopaquinone. Tyrosinase contributes to excessive production of dopaquinone from dopamine, resulting in neurotoxicity and leading to neurodegenerative changes that have been linked to Parkinson’s disease [4,5,6]. A closely related soluble tyrosinase in plants causes browning of fruits and vegetables during post-harvest processing, leading to poor shelf-life [7,8,9]. The most popular commercial tyrosinase inhibitors, such as kojic acid (KA), hydroquinone and arbutin (glycosylated hydroquinone) exhibit serious sideeffects: KA causes pigmented contact dermatitis [12], hydroquinone is carcinogenic [13]
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