Chemical Composition of Artemisia Scoparia and Their Bioactivities.

Three undescribed sesquiterpenes (1-3), two enantiomeric pairs of monoterpenes (4a/4b-5a/5b), one alkyne (6), two known alkynes (7-8) and eight known coumarins (9-16) were isolated from the aerial parts extracts of Artemisia scoparia. The structures of these compounds were fully elucidated by their 1D and 2D NMR, HRESIMS spectral data analyses, and comparison with literature. The absolute configurations of compounds were determined by single-crystal X-ray crystallography (1), a comparison of experimental and calculated electronic circular dichroism (ECD) data (2-6). 15 showed moderate inhibitory activity with the NO release in LPS-induced RAW264.7 cells. 9-16 showed varying degrees of promoting melanogenesis and tyrosinase activity in B16 cells.

Neurog1-Derived Peptides RMNE1 and DualPep-Shine Penetrate the Skin and Inhibit Melanin Synthesis by Regulating MITF Transcription.

Anti-pigmentation peptides have been developed as alternative skin-lightening agents to replace conventional chemicals that have adverse effects on the skin. However, the maximum size of these peptides is often limited by their low skin and cell penetration. To address this issue, we used our intra-dermal delivery technology (IDDT) platform to identify peptides with hypo-pigmenting and high cell-penetrating activity. Using our cell-penetrating peptides (CPPs) from the IDDT platform, we identified RMNE1 and its derivative RMNE3, "DualPep-Shine", which showed levels of α-Melanocyte stimulating hormone (α-MSH)-induced melanin inhibition comparable to the conventional tyrosinase inhibitor, Kojic acid. In addition, DualPep-Shine was delivered into the nucleus and regulated the gene expression levels of melanogenic enzymes by inhibiting the promoter activity of microphthalmia-associated transcription factor-M (MITF-M). Using a 3D human skin model, we found that DualPep-Shine penetrated the lower region of the epidermis and reduced the melanin content in a dose-dependent manner. Furthermore, DualPep-Shine showed high safety with little immunogenicity, indicating its potential as a novel cosmeceutical ingredient and anti-pigmentation therapeutic agent.

Discovery and identification of potential anti-melanogenic active constituents of Bletilla striata by zebrafish model and molecular docking.

BACKGROUND: Bletilla striata is the main medicine of many skin whitening classic formulas in traditional Chinese medicine (TCM) and is widely used in cosmetic industry recently. However, its active ingredients are still unclear and its fibrous roots are not used effectively. The aim of the present study is to discover and identify its potential anti-melanogenic active constituents by zebrafish model and molecular docking. METHODS: The antioxidant activities were evaluated by 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity, 2,2'-azino-bis-(3-ethylbenthiazoline-6-sulphonic acid) (ABTS) radical scavenging activity and ferric reducing antioxidant power (FRAP) assay. The anti-melanogenic activity was assessed by tyrosinase inhibitory activity in vitro and melanin inhibitory in zebrafish. The chemical profiles were performed by ultra-high-performance liquid chromatography combined with quadrupole time-of-flight tandem mass spectrometry (UPLC-Q-TOF-MS/MS). Meanwhile, the potential anti-melanogenic active constituents were temporary identified by molecular docking. RESULTS: The 95% ethanol extract of B. striata fibrous roots (EFB) possessed the strongest DPPH, ABTS, FRAP and tyrosinase inhibitory activities, with IC50 5.94 mg/L, 11.69 mg/L, 6.92 mmol FeSO4/g, and 58.92 mg/L, respectively. In addition, EFB and 95% ethanol extract of B. striata tuber (ETB) significantly reduced the melanin synthesis of zebrafish embryos in a dose-dependent manner. 39 chemical compositions, including 24 stilbenoids were tentatively identified from EFB and ETB. Molecular docking indicated that there were 83 (including 60 stilbenoids) and 85 (including 70 stilbenoids) compounds exhibited stronger binding affinities toward tyrosinase and adenylate cyclase. CONCLUSION: The present findings supported the rationale for the use of EFB and ETB as natural skin-whitening agents in pharmaceutical and cosmetic industries.

7-desacetoxy-6,7-dehydrogedunin discovered by high-throughput screening system suppresses melanogenesis through ATP-P2X7 signaling inhibition.

BACKGROUND: Hyperpigmented skin disorders such as melasma and lentigo are common photoaging diseases that cause cosmetic problems. The pigmentation is usually exacerbated by ultraviolet (UV) radiation, and various factors and pathways are involved in UV-mediated melanogenesis. Adenosine 5'-triphosphate (ATP), a well-known molecular unit of intracellular energy, is also regarded as a mediator of UV-mediated melanogenesis via the P2X7 purinergic receptor. OBJECTIVE: To discover natural substances with an anti-melanogenic effect through inhibition of ATP-P2X7 axis by high-throughput screening (HTS). METHODS: Among natural compounds provided by the Korea Chemical Bank, chemical compounds with a P2X7 inhibiting effect were screened through an HTS system. Then the selected compounds were verified for their anti-melanogenic effect after treating primary human epidermal melanocytes (PHEMs) with and without ATP. The expression of MITF, tyrosinase, and PMEL/gp100 was analyzed by Western blot, and melanin content was measured as 405 nm absorbance. RESULTS: Among 962 natural compounds, 58 showed greater than 80% suppression of YO-PRO-1 fluorescence, representing P2X7 activity. Among them, considering cell viability, chemical stability, and availability, 7-desaxacetoxy-6,7-dehydrogedunin (7DG), a limonoid natural compound, was selected. The expression of MITF, tyrosinase, and PMEL/gp100; tyrosinase enzyme activity; and melanin content, which were increased by ATP treatment were abrogated by 7DG. Even when 7DG was treated in PHEMs without addition of ATP, tyrosinase expression and melanin content were significantly decreased. Hypopigmenting effect of 7DG was confirmed in ex vivo culture of human skins. CONCLUSIONS: 7DG has an anti-melanogenic effect through ATP-P2X7 pathway inhibition and could be a potential skin whitening material.

Platelet-Rich Plasma in Melasma-A Systematic Review.

BACKGROUND: Melasma is a common relapsing hyperpigmentation disorder, which is often difficult to treat. Platelet-rich plasma (PRP) is a novel modality often used to treat acne scars, androgenic alopecia, chronic wounds, and skin rejuvenation. Recently, it has had a promising role in the treatment of melasma. OBJECTIVE: To review the published evidence on the efficacy and safety of PRP in the treatment of melasma. MATERIALS AND METHODS: A systematic review was performed. A meta-analysis could not be performed because of methodological differences across studies and data heterogeneity. RESULTS: Seven studies were fulfilled and analyzed. Most studies used intradermal injections of PRP and have shown significant improvement in melasma. Microneedling mediated delivery of PRP has been tried in melasma with good results. A single study showed no additional benefit of PRP in patients treated with topical tranexamic acid. Another study showed no benefit of intense pulsed light in patients treated with intradermal PRP. CONCLUSION: Platelet-rich plasma inhibits the melanin synthesis through its various components acting through several mechanisms. It demonstrates a moderate grade of recommendation according to the Oxford Center for Evidence-Based Medicine 2011 standards.

Theoretical Exploring of a Molecular Mechanism for Melanin Inhibitory Activity of Calycosin in Zebrafish.

Tyrosinase is an oxidase that is the rate-limiting enzyme for controlling the production of melanin in the human body. Overproduction of melanin can lead to a variety of skin disorders. Calycosin is an isoflavone from Astragali Radix, which is a traditional Chinese medicine that exhibits several pharmacological activities including skin whitening. In our study, the inhibitory effect of calycosin on melanin production is confirmed in a zebrafish in vivo model by comparing with hydroquinone, kojic acid, and arbutin, known as tyrosinase inhibitors. Moreover, the inhibitory kinetics of calycosin on tyrosinase and their binding mechanisms are determined using molecular docking techniques, molecular dynamic simulations, and free energy analysis. The results indicate that calycosin has an obvious inhibitory effect on zebrafish pigmentation at the concentration of 7.5 µM, 15 µM, and 30 µM. The IC50 of calycosin is 30.35 µM, which is lower than hydroquinone (37.35 µM), kojic acid (6.51 × 103 µM), and arbutin (3.67 × 104 µM). Furthermore, all the results of molecular docking, molecular dynamics simulations, and free energy analysis suggest that calycosin can directly bind to the active site of tyrosinase with very good binding affinity. The study indicates that the combination of computer molecular modeling and zebrafish in vivo assay would be feasible in confirming the result of the in vitro test and illustrating the target-binding information.

Liquid-Liquid Chromatography Separation of Guaiane-Type Sesquiterpene Lactones from Ferula penninervis Regel & Schmalh. and Evaluation of Their In Vitro Cytotoxic and Melanin Inhibitory Potential.

Ferula penninervis Regel & Schmalh. is a perennial plant used in Kazakh traditional folk medicine to treat epilepsy, neurosis, rheumatism, gastroduodenal ulcers, dyspepsia, wounds, abscesses or tumors. The aim of this work was to isolate series of sesquiterpene lactones from a crude methanolic root extract and investigate their in vitro cytotoxic potential against androgen-dependent prostate cancer LNCaP and epithelial prostate PNT2 cells, as well as to evaluate their melanin production inhibitory effects in murine melanoma B16F10 cells stimulated with α-melanocyte-stimulating hormone (αMSH). Two new (penninervin P and penninervin Q) and five known (olgin, laferin, olgoferin, oferin and daucoguainolactone F) guaiane-type sesquiterpene lactones were isolated with the use of a simple and fast liquid-liquid chromatography method. Olgin and laferin showed the most promising cytotoxic effects in LNCaP cells (IC50 of 31.03 and 23.26 µg/mL, respectively). Additionally, olgin, laferin, olgoferin, and oferin (10 µg/mL) potently impaired melanin release (40.67-65.48% of αMSH + cells) without influencing the viability of B16F10 cells. In summary, our findings might indicate that guaiane-type sesquiterpene lactones from F. penninervis could be regarded as promising candidates for further research in discovering new therapeutic agents with anti-prostate cancer and skin depigmentation properties.

Salicylic acid in ginseng root alleviates skin hyperpigmentation disorders by inhibiting melanogenesis and melanosome transport.

Abnormal melanogenesis and melanosome transport can cause skin pigmentation disorders that are often treated using ginseng-based formulation. We previously found that phenolic acid compounds in ginseng root could inhibit melanin production and as a skin-whitening agents. However, mechanisms of action underlying effects of ginseng phenolic acid monomers on melanogenesis remain unclear. This study was conducted to investigate effects of salicylic acid, a main ginseng root phenolic acid component, on melanogenesis and melanosome functions in melanocytes of zebrafish and other species. Salicylic acid exhibited no cytotoxicity and reduced melanin levels and tyrosinase activity in B16F10 murine melanoma cells and normal human epidermal melanocytes regardless of prior cell stimulation with α-melanocyte stimulating hormone. Additionally, salicylic acid treatment reduced expression of melanogenic enzymes tyrosinase, tyrosinase-related protein 1 and tyrosinase-related protein 2, while reducing expression of their master transcriptional regulator, microphthalmia-associated transcription factor. Moreover, reduced phosphorylation of cAMP response-element binding protein was observed due to reduced cAMP levels resulting from salicylic acid inhibition of upstream signal regulators (adenylyl cyclase and protein kinase A). Furthermore, salicylic acid treatment suppressed expression of transport complex-associated proteins melanophilin and myosin Va in two UVB-treated melanocytic cell lines, suppressed phagocytosis of fluorescent microspheres by UVB-stimulated human keratinocytes (HaCaT), inhibited protease-activated receptor 2 activation by reducing both Ca2+ release and activation of phosphoinositide 3 kinase/AKT and mitogen-activated protein kinases and induced anti-melanogenic effects in zebrafish. Collectively, these results indicate that salicylic acid within ginseng root can inhibit melanocyte melanogenesis and melanin transport, while also suppressing keratinocyte phagocytic function.

The Pathogenesis and Management of Acne-Induced Post-inflammatory Hyperpigmentation.

Acne vulgaris is a common inflammatory disease. Among patients with darker skin phototypes (Fitzpatrick III-VI), the inflammatory processes of acne stimulate excess melanogenesis and abnormal melanin deposition, leading to pigmentary sequelae known as post-inflammatory hyperpigmentation and post-inflammatory erythema in all skin tones, although post-inflammatory hyperpigmentation is more common in darker skin and post-inflammatory erythema in lighter skin. These pigmentary alterations can be long lasting and are often more distressing to patients than the active acne lesions. This article discusses what is known about acne-related pigmentation, much of which is extrapolated from general study of nonspecific pigment deposition. Because dyspigmentation poses both a significant clinical concern to patients and a therapeutic challenge to clinicians, we formed a working group consisting of pigmentary experts with the aim of increasing awareness and education of acne-related pigmentary sequelae.

Inhibition of melanogenesis by Aster yomena callus pellet extract in melanoma cells and patients with skin pigmentation.

Plant tissue culture holds immense potential for the production of secondary metabolites with various physiological functions. We recently established a plant tissue culture system capable of producing secondary metabolites from Aster yomena. This study aimed to uncover the mechanisms underlying the potential therapeutic effects of Aster yomena callus pellet extract (AYC-P-E) on photoaging-induced skin pigmentation. Excessive melanogenesis was induced in B16F10 melanoma cells using α-melanocyte stimulating hormone (α-MSH). The effects of AYC-P-E treatment on melanin biosynthesis inducers and melanin synthesis inhibition were assessed. Based on the results, a clinical study was conducted in subjects with skin pigmentation. AYC-P-E inhibited melanogenesis in α-MSH-treated B16F10 cells, accompanied by decreased mRNA and protein expression of melanin biosynthesis inducers, including cyclic AMP response element-binding protein (CREB), tyrosinase, microphthalmia-associated transcription factor (MITF), tyrosinase related protein-1 (TRP-1), and TRP-2. This anti-melanogenic effect was mediated by mitogen-activated protein kinase (MEK)/extracellular signal-regulated kinase (ERK) and protein kinase B (AKT) phosphorylation. Treatment of subjects with skin pigmentation with AYC-P-E-containing cream formulations resulted in 3.33%, 7.06%, and 8.68% improvement in the melanin levels at 2, 4, and 8 weeks, respectively. Our findings suggest that AYC-P-E inhibits excessive melanogenesis by activating MEK/ERK and AKT signaling, potentiating its cosmetic applications in hyperpigmentation treatment.

Novel Chemically Modified Curcumin (CMC) Analogs Exhibit Anti-Melanogenic Activity in Primary Human Melanocytes.

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.

Anti-Melanogenic Properties of Velutin and Its Analogs.

Velutin, one of the flavones contained in natural plants, has various beneficial activities, such as skin whitening, as well as anti-inflammatory, anti-allergic, antioxidant, and antimicrobial activities. However, the relationship between the structure of velutin and its anti-melanogenesis activity is not yet investigated. In this study, we obtained 12 velutin derivatives substituted at C5, C7, C3', and C4' of the flavone backbone with hydrogen, hydroxyl, and methoxy functionalities by chemical synthesis, to perform SAR analysis of velutin structural analogues. The SAR study revealed that the substitution of functional groups at C5, C7, C3', and C4' of the flavone backbone affects biological activities related to melanin synthesis. The coexistence of hydroxyl and methoxy at the C5 and C7 position is essential for inhibiting tyrosinase activity. However, 1,2-diol compounds substituted at C3' and C4' of flavone backbone induce apoptosis of melanoma cells. Further, substitution at C3' and C4' with methoxy or hydrogen is essential for inhibiting melanogenesis. Thus, this study would be helpful for the development of natural-derived functional materials to regulate melanin synthesis.

Novel 1,3,4-oxadiazole compounds inhibit the tyrosinase and melanin level: Synthesis, in-vitro, and in-silico studies.

In this research work, we have designed and synthesized some biologically useful of 1,3,4-Oxadiazoles. The structural interpretation of the synthesized compounds has been validated by using FT-IR, LC-MS, HRMS, 1H NMR and 13C NMR techniques. Moreover, the in-vitro mushroom tyrosinase inhibitory potential of the target compounds was assessed. The in-vitro study reveals that, all compounds demonstrate an excellent tyrosinase inhibitory activity. Especially, 2-(5-(2-methoxyphenyl)-1,3,4-oxadiazol-2-ylthio)-N-phenylacetamide (IC50 = 0.003 ± 0.00 µM) confirms much more significant potent inhibition activity compared with standard drug kojic acid (IC50 = 16.83 ± 1.16 µM). Subsequently, the most potent five oxadiazole compounds were screened for cytotoxicity study against B16F10 melanoma cells using an MTT assay method. The survival rate for the most potent compound was more pleasant than other compounds. Furthermore, the western blot results proved that the most potent compound considerably decreased the expression level of tyrosinase at 50 µM (P < 0.05). The molecular docking investigation exposed that the utmost potent compound displayed the significant interactions pattern within the active region of the tyrosinase enzyme and which might be responsible for the decent inhibitory activity towards the enzyme. A molecular dynamic simulation experiment was presented to recognize the residual backbone stability of protein structure.

Antioxidant and Antimelanogenic Activities of Kimchi-Derived Limosilactobacillus fermentum JNU532 in B16F10 Melanoma Cells.

Melanin is a natural skin pigment produced by specialized cells called melanocytes via a multistage biochemical pathway known as melanogenesis, involving the oxidation and polymerization of tyrosine. Melanogenesis is initiated upon exposure to ultraviolet (UV) radiation, causing the skin to darken, which protects skin cells from UVB radiation damage. However, the abnormal accumulation of melanin may lead to the development of certain skin diseases, including skin cancer. In this study, the antioxidant and antimelanogenic activities of the cell-free supernatant (CFS) of twenty strains were evaluated. Based on the results of 60% 2,2-diphenyl-1-picrylhydrazyl scavenging activity, 21% 2,2'-azino-bis (3-ethylbenzthiazoline-6-sulfonic acid) scavenging capacity, and a 50% ascorbic acid equivalent ferric reducing antioxidant power value, Limosilactobacillus fermentum JNU532 was selected as the strain with the highest antioxidant potential. No cytotoxicity was observed in cells treated with the CFS of L. fermentum JNU532. Tyrosinase activity was reduced by 16.7% in CFStreated B16F10 cells (but not in the cell-free system), with >23.2% reduction in melanin content upon treatment with the L. fermentum JNU532-derived CFS. The inhibitory effect of the L. fermentum JNU532-derived CFS on B16F10 cell melanogenesis pathways was investigated using quantitative reverse transcription polymerase chain reaction and western blotting. The inhibitory effects of the L. fermentum JNU532-derived CFS were mediated by inhibiting the transcription of TYR, TRP-1, TRP-2, and MITF and the protein expression of TYR, TRP-1, TRP-2, and MITF. Therefore, L. fermentum JNU532 may be considered a potentially useful, natural depigmentation agent.

Platelet-rich plasma protects human melanocytes from oxidative stress and ameliorates melanogenesis induced by UVB irradiation.

To investigate the role of platelet-rich plasma (PRP) from different sources in alleviating oxidative stress and ameliorating melanogenesis in UVB-irradiated PIG1 cells, PIG1 cells were irradiated with 80 mJ/cm2 UVB prior to 1% PRP application and the following experiments were taken: the viability of UVB-irradiated PIG1 cells, cellular malondialdehyde (MDA) and reactive oxygen species (ROS) content, and activities of antioxidant enzymes. Western blotting was utilized to detect the expression level of proteins associated with melanin synthesis, apoptosis, and DNA lesions. We found that PRP intervention promoted cell proliferation, reduced MDA and ROS content, increased the activities of series of antioxidant enzymes, and alleviated DNA damages in UVB-damaged PIG1 cells. It is important to note that PRP treatment inhibited UVB-induced melanogenesis via the PI3K/Akt/GSK3ß signal pathway. Therefore, we suppose PRP treatment exerts a protective role through their antioxidation effect on UVB-damaged PIG1 cells and hinders melanogenesis induced by UVB irradiation.

A natural compound harmine decreases melanin synthesis through regulation of the DYRK1A/NFATC3 pathway.

BACKGROUND: Melanin plays important roles in determining human skin color and protecting human skin cells against harmful ultraviolet light. However, abnormal hyperpigmentation in some areas of the skin may become aesthetically unpleasing, resulting in the need for effective agents or methods to regulate undesirable hyperpigmentation. OBJECTIVE: We investigated the effect of harmine, a natural harmala alkaloid belonging to the beta-carboline family, on melanin synthesis and further explored the signaling pathways involved in its mechanism of action. METHODS: Human MNT-1 melanoma cells and human primary melanocytes were treated with harmine, chemical inhibitors, small interfering RNAs, or mammalian expression vectors. Cell viability, melanin content, and expression of various target molecules were assessed. RESULTS: Harmine decreased melanin synthesis and tyrosinase expression in human MNT-1 melanoma cells. Inhibition of DYRK1A, a harmine target, decreased melanin synthesis and tyrosinase expression. Further studies revealed that nuclear translocation of NFATC3, a potential DYRK1A substrate, was induced via the harmine/DYRK1A pathway and that NFATC3 knockdown increased melanin synthesis and tyrosinase expression. Suppression of melanin synthesis and tyrosinase expression via the harmine/DYRK1A pathway was significantly attenuated by NFATC3 knockdown. Furthermore, harmine also decreased melanin synthesis and tyrosinase expression through regulation of NFATC3 in human primary melanocytes. CONCLUSION: Our results indicate that harmine decreases melanin synthesis through regulation of the DYRK1A/NFATC3 pathway and suggest that the DYRK1A/NFATC3 pathway may be a potential target for the development of depigmenting agents.

The anti-melanogenic effects of ellagic acid through induction of autophagy in melanocytes and suppression of UVA-activated α-MSH pathways via Nrf2 activation in keratinocytes.

Ellagic acid (EA) is a natural phenol antioxidant in different fruits, vegetables, and nuts. As a copper iron chelator from the tyrosinase enzyme's active site, EA was reported to inhibit melanogenesis in melanocytes. Here, we demonstrated the anti-melanogenic mechanisms of EA through autophagy induction in melanoma B16F10 cells and the role of Nrf2 and UVA (3 J/cm2)-activated α-melanocyte stimulating hormone (α-MSH) pathways in keratinocyte HaCaT cells. In vitro data showed that EA suppressed the tyrosinase activity and melanogenesis by suppressing cAMP-mediated CREB and MITF signaling mechanisms in α-MSH-stimulated B16F10 cells. ERK, JNK, and AKT pathways were involved in this EA-regulated MITF downregulation. Notably, EA induced autophagy in B16F10 cells was evidenced from increased LC3-II accumulation, p62/SQSTM1 activation, ATG4B downregulation, acidic vesicular organelle (AVO) formation, PI3K/AKT/mTOR inhibition, and Beclin-1/Bcl-2 dysregulation. Interestingly, 3-MA (an autophagy inhibitor) pretreatment or LC3 silencing (siRNA transfection) of B16F10 cells significantly reduced EA-induced anti-melanogenic activity. Besides this, in UVA-irradiated keratinocyte HaCaT cells, EA suppressed ROS production and α-MSH generation. Moreover, EA mediated the activation and nuclear translocation of Nrf2, leading to antioxidant γ-GCLC, HO-1, and NQO-1 protein expression in HaCaT cells. However, Nrf2 knockdown has significantly impaired this effect, and there was an uncontrolled ROS generation following UVA irradiation. JNK, PKC, and ROS pathways were involved in the activation of Nrf2 in HaCaT cells. In vivo experiments using the zebrafish model confirmed that EA inhibited tyrosinase activity and endogenous pigmentation. In conclusion, ellagic acid is an effective skin-whitening agent and might be used as a topical applicant.

Anti-melanogenic activity of ellagitannin casuarictin in B16F10 mouse melanoma cells.

Ellagitannins such as casuarictin (CAS), isolated from clove extracts, have been shown to have superior benefits such as antioxidant and anti-inflammatory activity, but there have been no reports on their capacity to inhibit melanogenesis. Inhibition of melanogenesis by novel natural products has gained attention for cosmetic applications such as skin lightening. Here, we report the effects of CAS on melanogenesis in B16F10 mouse melanoma cells. Our results showed that CAS (30 µM) significantly inhibited intracellular melanogenesis while being nontoxic to B16F10 cells or to HaCaT cells at that concentration. CAS (30 µM) also inhibited intracellular tyrosinase activity as well as mushroom tyrosinase activity; possessed robust copper chelating ability comparable to that of 500 µM kojic acid; and downregulated MITF protein levels, all of which contribute to the inhibitory mechanisms underlying its anti-melanogenic activity. In summary, our results demonstrate that CAS might hold promise as a depigmenting agent for hyperpigmentation disorders.

The effect of isolates from Cassipourea flanaganii (Schinz) alston, a plant used as a skin lightning agent, on melanin production and tyrosinase inhibition.

ETHNOPHARMACOLOGICAL RELEVANCE: The Zulu and Xhosa people of South Africa use the stem bark of Cassipourea flanaganii as a skin-lightning cosmetic. AIM OF THE STUDY: To isolate and identify compounds responsible for the skin lightning properties from the stem bark of Cassipourea flanaganii and to evaluate their cytotoxicity towards skin cells. MATERIALS AND METHODS: Extracts from the stem bark of Cassipourea flanaganii were isolated using chromatographic methods and structures were determined using NMR, IR and MS analysis. The tyrosinase inhibitory activity and the ability to inhibit the production of melanin were determined using human primary epidermal melanocyte cells. Cytoxicity was established using the same melanocytes and a neutral red assay. RESULTS: One previously undescribed compound, ent-atis-16-en-19-al (1) along with the known ent-atis-16-en-19-oic acid (2), ent-atis-16-en-19-ol (3), ent-kaur-16-en-19-oic acid (4), ent-kaur-16-en-19-al (5), ent-manoyl oxide (6), guinesine A (7), guinesine B (8), guinesine C (9), lichenxanthone (10), 2,4-dihydroxy-3,6-dimethyl benzoic acid methyl ester (11), lynoside (12), lupeol (13), ß-amyrin (14), docosyl ferulate (15), stigmasterol, sitosterol and sitosterol-O-glucoside were isolated in this investigation. An impure fraction containing compound 3 was acetylated to obtain 19-acetoxy-ent-atis-16-ene (3a). Compounds 10 and 11 are usually isolated from lichen, hence they are possible contaminants of lichen harvested with the bark. Compounds 1, 3a, 5-14 were not significantly cytotoxic to the primary epidermal melanocyte cells (P > 0.05) when compared to the negative and positive controls (DMSO, 0.1% and hydrogen peroxide, 30 wt% in water). Inhibition of tyrosinase was significantly greater with respect to the negative control (P < 0.001) for compounds 3a, 5-8 and 9-10 at 10 µM and for compounds 5-8 and 9-10 at 100 µM. Compared to hydroquinone (the positive control) at 10 µM, the level of inhibition was comparable or to that of compounds 3a, 5, 6, and 8-10 at 10 µM, with 9 and 10 showing a greater level of inhibition. Inhibition of melanin was both concentration and time dependent for all compounds tested with higher melanin content at 24 h compared to 48 h s and at 10 mM compared to100 mM at both time points; melanin content was significantly lower for hydroquinone at both time points and concentrations. CONCLUSIONS: Compounds 1, 5-14, isolated from Cassipourea flanaganii and the derivative 3a showed low cytotoxicity. All compounds had a clear time and concentration dependent effect on melanin content which did not appear to be dependent on their inhibition of tyrosinase.

Xanthones for melanogenesis inhibition: Molecular docking and QSAR studies to understand their anti-tyrosinase activity.

The human skin is constantly exposed to external factors that affect its integrity, UV radiation being one of the main stress factors. The repeated exposure to this radiation leads to increased production of Reactive Oxygen Species (ROS) which activate a series of processes involved in photoaging. Excessive UV exposure also exacerbates melanin production leading to a variety of pigmentation disorders. Xanthones are reported to exhibit properties that prevent deleterious effects of UV exposure and high levels of ROS in the organism, so in this work a wide library of xanthones with different patterns of substitution was synthesized and tested for their inhibitory activity against the skin enzymes tyrosinase, elastase, collagenase and hyaluronidase, many of which were evaluated for the first time. Most of the compounds were tyrosinase inhibitors, with the best one (xanthone 27) presenting an IC50 of 1.9 µM, which is approximately 6 times lower than the IC50 of the positive control kojic acid. Concerning the other enzymes, only one compound presented IC50 lower than 150 µM in elastase inhibition (xanthone 14 = 91.8 µM) and none in collagenase and hyaluronidase inhibition. A QSAR model for tyrosinase inhibitory activity was built using six molecular descriptors, with a partial negative surface area descriptor and the relative number of oxygen atoms being positively contributing to the tyrosinase inhibitory activity. Docking using AutoDock Vina shows that all the tested compounds have more affinity to mushroom tyrosinase than kojic acid. Docking results implied that the tyrosinase inhibitory mechanisms of xanthonic derivatives are attributed to an allosteric interaction. Taken together, these data suggest that xanthones might be useful scaffolds for the development of new and promising candidates for the treatment of pigmentation-related disorders and for skin whitening cosmetic products.