No abstract

Curcumin, a bioactive from Curcuma longa, has been shown to possess anti-melanogenic activity previously; however, the effects of its hydrogenated metabolites (HMs)—Tetrahydrocurcumin (THC), Hexahydrocurcumin (HHC), and Octahydrocurcumin (OHC)—on melanogenesis have not been sufficiently explored. We have studied and compared three HMs (THC, HHC, and OHC) with the parent compound, curcumin (PC), on melanin synthesis in B16F10 mouse and MNT-1 human melanoma cells. Our results demonstrated that all the HMs were nontoxic over the concentration range 5–40 µM, while PC was nontoxic at 5 µM but induced toxicity at 20 and 40 µM in B16F10 cells. All three HMs enhanced melanin synthesis, while PC (5 µM) inhibited it. THC (40 µM) significantly stimulated melanin synthesis to a greater degree than HHC and OHC in both B16F10 and MNT-1 cells; the order of melanogenesis stimulation was THC = OHC > HHC in B16F10 mouse cells, while it was THC > HHC > OHC in MNT-1 cells. HMs stimulated melanogenesis by pathways not involving tyrosinase, as neither the intracellular tyrosinase activity nor the protein levels of tyrosinase were affected. In addition, mushroom tyrosinase activity, using L-Dihydroxyphenylalanine (L-DOPA) as the substrate, showed no direct effects of HMs. In summary, our results demonstrate that the HMs enhanced melanogenesis, which establishes that the hydrogenation of the heptadiene moiety of curcumin leads to a loss of its anti-melanogenic activity and instead results in the stimulation of melanogenesis. This stimulation is not further enhanced upon hydrogenation of the β-diketone, which was noted in MNT-1 cells, although the correlation to the number of keto groups differed in B16F10 cells where HHC was the weakest stimulator of melanogenesis. Collectively, THC with both keto groups intact is the best stimulator. Moreover, our results also validate that the electrophilicity of curcumin is necessary for its anti-melanogenic activity, as the non-electrophilic HMs did not inhibit melanogenesis. Furthermore, our results suggest that THC might hold promise as a stimulator of melanogenesis for treatment of hypopigmentation disorders and anti-graying therapies. Future studies to probe the molecular signaling mechanisms and test whether the pro-melanogenic activity of HMs is retained in primary human melanocytes are warranted.

Hyperpigmentation is a dermatological condition characterized by the overaccumulation and/or oversecretion of melanin pigment. The efficacy of curcumin as an anti-melanogenic therapeutic has been recognized, but the poor stability and solubility that have limited its use have inspired the synthesis of novel curcumin analogs. We have previously reported on comparisons of the anti-melanogenic activity of four novel chemically modified curcumin (CMC) analogs, CMC2.14, CMC2.5, CMC2.23 and CMC2.24, with that of parent curcumin (PC), using a B16F10 mouse melanoma cell model, and we have investigated mechanisms of inhibition. In the current study, we have extended our findings using normal human melanocytes from a darkly pigmented donor (HEMn-DP) and we have begun to study aspects of melanosome export to human keratinocytes. Our results showed that all the CMCs downregulated the protein levels of melanogenic paracrine mediators, endothelin-1 (ET-1) and adrenomedullin (ADM) in HaCaT cells and suppressed the phagocytosis of FluoSphere beads that are considered to be melanosome mimics. All the three CMCs were similarly potent (except CMC2.14, which was highly cytotoxic) in inhibiting melanin production; furthermore, they suppressed dendricity in HEMn-DP cells. CMC2.24 and CMC2.23 robustly suppressed cellular tyrosinase activity but did not alter tyrosinase protein levels, while CMC2.5 did not suppress tyrosinase activity but significantly downregulated tyrosinase protein levels, indicative of a distinctive mode of action for the two structurally related CMCs. Moreover, HEMn-DP cells treated with CMC2.24 or CMC2.23 partially recovered their suppressed tyrosinase activity after cessation of the treatment. All the three CMCs were nontoxic to human dermal fibroblasts while PC was highly cytotoxic. Our results provide a proof-of-principle for the novel use of the CMCs for skin depigmentation, since at low concentrations, ranging from 5 to 25 µM, the CMCs (CMC2.24, CMC2.23 and CMC2.5) were more potent anti-melanogenic agents than PC and tetrahydrocurcumin (THC), both of which were ineffective at melanogenesis at similar doses, as tested in HEMn-DP cells (with PC being highly toxic in dermal fibroblasts and keratinocytes). Further studies to evaluate the efficacy of CMCs in human skin tissue and in vivo studies are warranted.

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.