A common feature of the inflammatory response in patients who have actually contracted influenza is the generation of a number of volatile products of the alveolar and airway epithelium. These products include a number of volatile organic compounds (VOCs) and nitric oxide (NO). These may be used as biomarkers to detect the disease. A portable 3-sensor array microsystem-based tool that can potentially detect flu infection biomarkers is described here. Whether used in connection with in-vitro cell culture studies or as a single exhale breathalyzer, this device may be used to provide a rapid and non-invasive screening method for flu and other virus-based epidemics.

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.

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.

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.

Benolea^® (EFLA^®943) is a standardized dry olive leaf extract (DOLE) considered safe for food consumption and has demonstrated superior pharmaceutical benefits such as antioxidant, anti-obesity, and anti-hypertensive activities. However, there is no study on its effects on melanogenesis yet. Disruption in the sequence of steps in melanogenesis can lead to hypopigmentary disorders which occur due to reduced production or export of pigment melanin in the skin. There is a need for safe and nontoxic therapeutics for the treatment of hypopigmentation disorders. Herein, we studied the effects of DOLE over a concentration range of 10–200 µg/mL on melanin synthesis and melanin secretion in B16F10 mouse melanoma cells and MNT-1 human melanoma cells and validated our results in primary human melanocytes (obtained from lightly pigmented (LP) and moderately pigmented (MP) cells) as well as their cocultures with keratinocytes. The capacity of melanocytes to export melanosomes was also estimated indirectly by the quantitation of melanocyte dendrite lengths and numbers. Our results show that DOLE significantly enhanced levels of extracellular melanin in the absence of effects on intracellular melanin, demonstrating that this plant extract’s pro-melanogenic activity is primarily based on its capacity to augment melanin secretion and stimulate melanocyte dendricity. In summary, our preliminary results demonstrate that DOLE may hold promise as a pro-pigmenting agent for vitiligo therapy and gray hair treatment by its exclusive and novel mechanism of functioning as a dendrite elongator. Further studies to elucidate the mechanisms of action of the pro-melanogenic activity and effects of DOLE on melanosome export as well as the last steps of melanogenesis are warranted.

Previous studies have reported that estrogen hormone promotes melanogenesis while progesterone inhibits it. A selective estrogen receptor modulator (SERM), tamoxifen, has been shown to promote melanogenesis; however, to date, there have been no reports on the effects of a selective progesterone receptor modulator (SPRM) on melanogenesis. In the present study, we hypothesized that asoprisnil (AP), a SPRM, inhibits melanogenesis. AP was tested for cytotoxicity to B16F10 mouse melanoma cells for screening the nontoxic concentrations using MTS cytotoxicity assay. Extracellular and intracellular melanin levels were estimated at nontoxic concentrations of AP. To evaluate the direct effect of AP on tyrosinase enzyme, tyrosinase activity and copper chelating activities were measured. Next, the effects of AP on melanogenesis were tested in normal human melanocytes, neonatal, darkly pigmented (HEMn-DP). Our results demonstrate that AP was nontoxic at a concentration range of 10–50 μM in B16F10 cells; AP at 50 μM significantly suppressed extracellular melanin levels comparable to kojic acid at 500 μM, with no significant effect on intracellular melanin levels. The mechanism of melanogenesis inhibition was studied to assess if AP downregulated tyrosinase activity in cell lysates or in a cell-free system. However, AP was found to increase intracellular tyrosinase activity without any effect on tyrosinase enzyme activity or copper chelating activity in a cell-free system, indicating that AP inhibits melanogenesis by mechanisms other than direct effects on tyrosinase enzyme activity. The capacity of AP to inhibit melanosome export was further validated in HEMn-DP cells; AP significantly suppressed dendricity at concentrations of 20 and 30 μM in the absence of effects on melanin synthesis or intracellular tyrosinase activity. In addition, AP was nontoxic to human keratinocytes (HaCaT) at these concentrations, validating its safety for topical use. Taken together, our preliminary results demonstrate that AP might be repurposed as a candidate therapeutic for treatment of hyperpigmentation disorders via a unique mechanism, which encompasses a selective inhibition of melanosome export.