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Keywords:

  • Cinnamomum subavenium;
  • human skin cells;
  • molecular docking;
  • tyrosinase inhibitor;
  • zebrafish

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Acknowledgements
  9. References
  10. Supporting Information

Abstract:  Tyrosinase is known to be the first two and rate-limiting enzyme in the synthesis of melanin pigments responsible for colouring skin, hair and eyes. Tyrosinase inhibition is one major strategy used to treat hyperpigmentation. In human skin melanocytes, the cellular tyrosinase inhibition was examined by the conversion of l-tyrosine and oxidation of l-DOPA to dopaquinone. We evaluated the skin pigmentation inhibitor effects with both in vitro and in vivo systems to find skin-whitening agents without cytotoxic concerns. First, linderanolide B and subamolide A were isolated from the stems of Cinnamomum subavenium and exhibited mushroom tyrosinase inhibition. Then, these two herbal compounds were proved to have good pigmentation inhibitory abilities at low doses and demonstrated free cytotoxicities to normal human skin cells and zebrafish system. With molecular docking, in a virtual model of human tyrosinase, linderanolide B and subamolide A displayed metal-coordinating interactions with Cu2+ ions. The results obtained from biological assays showed that linderanolide B and subamolide A possessed anti-tyrosinase properties, which exhibited potential for application in medical cosmetology.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Acknowledgements
  9. References
  10. Supporting Information

In mammals, skin, hair and eyes, darkening is determined by the synthesis and distribution of melanin (1). In skin, it is a mixture of pigmented biopolymers that is synthesized in a unique organelle, the melanosome of melanocytes (2,3). Excessive biosynthesis of melanin induces various related pigment disorders, such as senile lentigo, melasma, freckles and pigmented acne scars, that are of particular concern to women as well as men (3,4). Their treatment usually involves the use of medicines or medicinal cosmetics containing de-pigmenting or skin-whitening components. Safe and effective regulators that act to minimize skin pigmentation abnormalities include natural and synthetic de-pigmenting agents. However, only a few are used as therapeutic agents, primarily because of various safety concerns and low whitening bioactivity. In melanogenesis, l-tyrosine is hydroxylated to dihydroxyphenylalanine (l-DOPA) and then l-DOPA is oxidized to DOPA-quinone with two initial steps (2,5). Pigment colouring hair, skin and eyes because of the key protein, tyrosinase, is recognized to be the first two and rate-limiting enzyme in the biosynthesis of melanins (1,6–8). Recently, much attention has been drawn to the application of tyrosinase inhibitors to medical treatments and cosmetic businesses. Therefore, in clinical usage, tyrosinase inhibitors are being taken for dermatological disorder treatments related to melanin hyperaccumulation, and are thus fundamental in cosmetics for de-pigmentation (9).

From previous studies, it was seen that the zebrafish melanocytes were easily visualized and uniformly dispensable on the exterior surface (10,11). Given that the zebrafish system has several other advantages such as, numerous quantities of embryos amongst vertebrates, inducible spawning by light, convenience in observing melanin development, as well as a rapid pigmentation process and high permeability to small molecules. In addition, it possesses epidermal melanocyte equivalents that have similar structural and functional characteristics as mammals (12). So it can be used in phenotype-based screening for whole-animal models on pigmentary inhibitors.

We identified novel inhibitors of tyrosinase from Cinnamomum subavenium Miq. (Lauraceae), which is a medium-sized evergreen tree, found in the central to southern regions of mainland China, Taiwan, Malaysia, Cambodia, Burma, and Indonesia (13). There are only a few publications concerning the chemical constituents from this species (14), in addition, no biological effects of C. subavenium were reported in the literatures before. The phytochemical and bioactive compounds of C. subavenium, linderanolide B and subamolide A, chosen as the target compounds for de-pigmentation on human epidermal melanocytes and zebrafish system, were investigated. This was the first attempt to demonstrate the bioactivities for medical cosmetology purposes of these two pure compounds.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Acknowledgements
  9. References
  10. Supporting Information

Reagents and materials

Dimethyl sulfoxide (DMSO), l-DOPA, α-melanocyte-stimulating hormone (α-MSH), 1-phenyl-2-thiourea (PTU) and l-tyrosine were purchased from Sigma-Aldrich Chemical Inc. (St Louis, MO, USA). 3-[4,5-dimethylthiazol-2-yl]-5-[3- carboxymethoxyphenyl]-2-[4-sulfophenyl]-2H-tetrazolium, inner salt (MTS) from Promega (Madison, WI). Foetal bovine serum (FBS) and Dulbecco’s modified Eagle’s medium (DMEM) were obtained from Gibco BRL (Gaithersburg, MD, USA). All buffers and other reagents were of the highest purity commercially available.

Extraction and purification of linderanolide B and subamolide A

Linderanolide B and subamolide A were isolated from the stems of C. subavenium as described previously (13). (Fig. S1). In brief, the air-dried stems (8.0 kg) were extracted with methanol (80 l × 6) at room temperature, and the methanol extract (202.5 g) was obtained upon concentration under reduced pressure. The methanol extract, suspended in H2O (1 l), was partitioned with chloroform (2 l × 5) to give fractions soluble in chloroform (123.5 g) and H2O (74.1 g). The chloroform soluble fraction was subjected to chromatograph over silica gel (800 g, 70–230 mesh) using n-hexane/ethyl acetate/methanol mixtures as eluents to produce five fractions. Part of fraction 1 (7.46 g) was subjected to silica gel chromatography by eluting with n-hexane-ethyl acetate (30:1), enriched with ethyl acetate to furnish ten fractions (1). Fraction 1–3 (4.02 g) was subjected to silica gel chromatography, eluting with n-hexane-ethyl acetate (40:1) and enriched gradually with ethyl acetate, to obtain three fractions (1–3). Fraction 1-3-2 (4.11 g), eluted with n-hexane- ethyl acetate (40:1), was further separated using silica gel column chromatography and preparative thin layer chromatography (n-hexane-ethyl acetate (30:1) and gave linderanolide B (134 mg). Part of fraction 2 (9.31 g) was subjected to silica gel chromatography, by eluting with n-hexane-ethyl acetate (10:1), then enriched gradually with ethyl acetate to furnish five fractions (2-1-2-5). Fraction 2–4 (1.31 g) was subjected to silica gel chromatography, eluting with n-hexane-ethyl acetate (40:1), and enriched gradually with ethyl acetate to obtain four fractions (2-4-1-2-4-4). Fraction 2-4-2 (1.06 g), eluted with n-hexane-ethyl acetate (10:1), was further separated using silica gel column chromatography and preparative thin layer chromatography (n-hexane-ethyl acetate (30:1) and gave subamolides A (38 mg) (Fig. 1). The 1H NMR spectral data were consistent with the structure (1H NMR (400 MHz, CDCl3): subamolide A: δ 0.87 (3H, t, = 7.0 Hz), 1.27 (20H, br s), 1.49 (2H, m), 1.54 (3H, s), 2.74 (2H, m), 3.39 (3H, s), 4.39 (1H, d, = 1.2 Hz), 6.56 (1H, td, = 8.0, 1.6 Hz); linderanolide B: δ 0.88 (3H, t, = 7.0 Hz), 1.29 (20H, br s), 1.49 (2H, m), 2.73 (2H, m), 4.57 (1H, s), 4.71 (1H, s), 5.16 (1H, br s), 6.64 (1H, t, = 7.5 Hz).

image

Figure 1.  The structures of the two compounds—linderanolide B and subamolide A.

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Assay on mushroom tyrosinase activity

Tyrosinase activity inhibition was determined spectrophotometrically according to the method described previously (15,16), with minor modifications. Assays were conducted in a 96-well micro-plate, an ELSA plate reader being used to determine the absorbance at 490 nm (Molecular Devices). Kojic acid was used as a positive control on tyrosinase inhibitory assay. The testing substance was dissolved in aqueous DMSO, and incubated with l-tyrosine (2.5 mg/ml) in 50 mm phosphate buffer (pH 6.8). All samples were dissolved in DMSO, which did not affect tyrosinase activity when DMSO was <0.5% of the total volume. Then, 25 U/ml of mushroom tyrosinase in the same buffer was added, and the mixture was incubated at 37°C for 30 min (Table S1). Tyrosinase inhibitory activity was determined by the following equation:

  • image(1)

where A is the optical density (OD490) without testing substance; B is the OD490 without testing substance, but with tyrosinase; C is the OD490 with testing substance; and D is the OD490 with testing substance, but without tyrosinase. IC50 values of the inhibitors were determined by fitting the initial rates versus inhibitor concentrations using the following equation:

  • image(2)

In this equation, E(I) is the enzyme activity with inhibitor concentration I; E(0) is enzyme activity without inhibitor; I is the inhibitor concentration; and IC50 is the concentration of inhibitor which caused half reaction rate.

Human epidermal melanocyte, keratinocyte and dermal fibroblast cell cultures

Neonatal foreskin primary human epidermal melanocytes (HEMn-MP) were purchased from Cascade Biologics™, cultured in Medium 254 (M-254-500; Cascade Biologics, Portland, OR, USA) and supplemented with human melanocyte growth supplement (HMGS, cat.# S-002-5). The Medium 254 is a basal medium containing essential and non-essential amino acids, vitamins, organic compounds, trace minerals and inorganic salts. The human melanocyte growth supplement contains bovine pituitary extract, foetal bovine serum, bovine insulin, bovine transferrin, basic fibroblast growth factor, hydrocortisone, heparin and phorbol 12-myristate 13-acetate. Human keratinocytes were grown from foreskin primary culture, which was derived from Chung-Ho Memorial Hospital, Kaohsiung Medical University, Taiwan, and were kind gifts from Dr Ching-Ying Wu. Human keratinocytes were cultured in Keratinocyte-SFM (10724; GIBCO™), supplemented with Bovine Pituitary Extract (BPE, cat. #13028-014) and EGF Human Recombinant (cat.#10450-013). The medium and growth supplement for keratinocytes contain γ-epidermal growth factor, BPE, insulin, fibroblast growth factor and calcium (0.09 mm). The primary cultures of human skin fibroblasts were complimentary gifts from Dr Su-Shin Lee. All type cells were incubated at 37°C in a humidified incubator 5% CO2 atmosphere. To study melanin biogenesis, HEMn-MP were incubated in 24-well plates at a density of 105 cells per well.

Cell viability assay

To study the cytotoxicities of linderanolide B and subamolide A on human skin cells, we used MTS assay (17). The cells in 100 μl medium were exposed to 20 μl of CellTiter 96 AQueous One Solution (Promega, Cat.#: G3582), for 3 h according to the manufacturers’ instructions. Absorbance at 490 nm was recorded using an ELISA plate reader.

Assay on cellular tyrosinase activity

The tyrosinase activity was estimated by measuring the rate of dopachrome formation, based on the method described previously with minor modifications (18,19). HEMn-MP (105 per well) were placed in 24-well plates in 500 μl of medium containing various concentrations of testing samples and incubated for 2 days. The sample-treated cells were washed with phosphate-buffered saline (PBS) and lysed with 1% Triton X-100/PBS. The enzyme extract of cellular lysate was added to 10 μl of 10 mm l-tyrosine and 10 mm l-DOPA as substrates mixed in 0.1 m phosphate buffer (pH 6.8). This reaction was then incubated at 37°C for 3 h in a dark environment, and the absorbance at 490 nm was measured on a spectrophotometer.

Melanin quantification assay

Briefly, we followed the previous method with minor modifications (20). Cell pellets were dissolved in 1.0 N NaOH containing 10% DMSO and heated at 80°C for 1 h and suspensions were clarified by centrifugation for 10 min at 10 000 g. The amount of melanin was determined spectrophotometrically based on the absorbance at 475 nm (21–23).

Zebrafish (in vivo assay)

Synchronized embryos were collected and arrayed by pipette, according to the previous method (12), three embryos per well, in a 96-well plate containing 200 μL embryo medium. Test compounds were dissolved in 1% DMSO and then added to the embryo medium from 9 to 57 hpf (hours postfertilization, total 48 h exposure). The positive controls were 0.2 mm PTU and 20 mm arbutin. The effects on the pigmentation of zebrafish were observed under the stereomicroscope. Phenotype-based evaluations of the body pigmentation were then carried out at 57 hpf. For observation, embryos were dechorionated by forceps, anesthetized in tricaine methanesulfonate solution (Sigma-Aldrich), mounted in 1% methyl cellulose on a depression slide (Aquatic Eco-Systems, Apopka, FL, USA) and photographed under the stereomicroscope Z16 (Leica Microsystems, Ernst-Leitz-Strasse, Germany). The images were captured using a SPOT CCD Idea integrating camera (Diagnostic Instruments Inc., Sterling Heights, MI, USA). Afterwards, images were taken using the Scion Image alpha 4.0.3.2 software (Scion Co., Torrance, CA, USA) by a blinder observer. The pixel measurement analyzer program was then used to count the area of the zebrafish image pigmentation. The quantification of pigmentation data was expressed as a percentage change compared to the control group which was considered as 100%.

Molecular docking study of human tyrosinase

As no three-dimensional structures for human tyrosinase are available now, a theoretical homology model is retrieved for the docking study at present. To construct a human tyrosinase model with an active site in the likely ligand-bound protein conformation, a homology model was first simulated by computer docking with the substrate l-dopa and two copper ions, using molecular energy minimization and dynamic simulation. The crystallized structure of Octopus dofleini hemocyanin (1JS8) was downloaded from the Protein Data Bank (http://www.pdb.org). The active site was docked with inhibitors using the dock suite from Accelrys Discovery Studio 2.0 software (Accelrys, Inc., San Diego, CA, USA). All crystallographic water molecules, solvent molecules and ions were removed from the structure. The binding site was defined with the options of site opening = 5Å and grid resolution = 0.5 Å. Docking was performed with default values selected for Energy Grid Forcefield and ‘Minimizer’ chosen for Minimization Algorithm. The most preferable-choice orientation of protein-compound was presented. For docking result figures, PyMOL software was used to show the homology of human tyrosinase with final inhibitor blocking structure.

Statistical analysis

Results are presented as a mean value of the data obtained from triplicate experiments. Student’s t-test was used to determine the level of significance.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Acknowledgements
  9. References
  10. Supporting Information

Cytotoxicity of linderanolide B and subamolide A of HEMn-MP cells

Based on the above finding, we targeted linderanolide B and subamolide A from C. subavenium from a large number of plant component screenings of the mushroom tyrosinase assay (Fig S2). As a potent skin-lightening agent, the component should be harmless, without undesirable cytotoxic side effects. Considering the therapeutic or cosmetic usage of the two compounds, their effects on cell viabilities are significantly important. Thus, we did an assessment on the cytotoxic properties of both compounds on human skin cells, including epidermal keratinocytes, melanocytes and dermal fibroblasts.

An MTS assay was used to measure cell viability and investigate whether the inhibitors would induce cell death adversely. Testing samples were treated with various concentrations from 0.01 to 10 μm to verify the dose-dependent effects. Kojic acid and PTU, which are well-approved melanogenic inhibitors with obvious inhibitory effects at a high concentration of 100 μm, were used as positive controls in this current work. α-MSH, known to stimulate melanogenesis, was examined at 0.1 μm as the negative control group (24,25), and the effect on melanogenesis was compared with the testing samples. In Fig. 2, three kinds of normal human skin cells were exposed to high testing concentrations (10 μm) and exhibited viabilities of more than 50% after 48 h of treatment. This result revealed that these two compounds had little discernable toxic properties to human epidermal and dermal cells. The well-known tyrosinase inhibitors, such as kojic acid, PTU or arbutin, would induce tumorigenicity at high concentrations (26–28). We also observed cell death at treatments of 100 μm concentration. As the relative laws in each country has strictly limited on concentration doses, to use these agents as compositions in cosmetic products, we must abide by these regulations.

image

Figure 2.  Human normal skin cell viabilities of epidermal keratinocytes, dermal melanocytes and fibroblasts after treatment with various concentrations of linderanolide B and subamolide A. PTU and kojic acid at 100 μm as positive controls, and α-MSH at 0.1 μm as negative control. Human keratinocytes in black, melanocytes in light gray and fibroblasts in dark gray. (*< 0.01 and **< 0.001).

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Tyrosinase- and melanin-reducing activities of linderanolide B and subamolide A in HEMn-MP cells

Melanin plays an important role in determining mammalian skin colour. The melanogenesis pathway consists of the enzymatic l-tyrosine hydroxylation and the oxidation of l-DOPA to its corresponding dopaquinone. After the two tyrosinase-catalysed steps, additional multiple biosynthesis steps followed and yields melanin (29). Linderanolide B and subamolide A were investigated for human cellular tyrosinase inhibiting abilities and melanin content decreasing powers in HEMn-MP cells (Fig. 3). Both compounds demonstrated noticeable superior inhibition at a low dosage to tyrosinase activities and melanin contents. Kojic acid and PTU showed the same inhibition rate at higher concentrations (100 μm). With increasing concentrations of these two compounds, the human tyrosinase activities and melanin contents were decreased. In Fig. 3a,b, the melanin contents matched with the tyrosinase activities in the same dose-dependent tendencies upon the two compounds. As the figures indicate, the epidermal cellular melanin reductions might be because of the inhibition of human tyrosinase activities.

image

Figure 3.  (a) Tyrosinase activities of HEMn-MP cells after treatment with various concentrations of linderanolide B and subamolide A. (b) Melanin contents and the photographs at top side were pellet collection. PTU and kojic acid at 100 μm as positive controls, and α-MSH at 0.1 μm as negative control. (*< 0.01 and **< 0.001).

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Morphological changes of human epidermal melanocytes treated with linderanolide B and subamolide A

In Fig. 4, we observed HEMn-MP cell treatments with samples at various concentrations for more than 2 days. Here, we compared the cell morphologies of the control groups to the sample treatment groups under a phase contrast microscope. Kojic acid and PTU demonstrated melanin-reducing abilities, and the colour of cells after centrifugation became lighter (Fig. 3b). Otherwise, α-MSH stimulated the cellular melanin production and developed it into a darker colour than the others. We found that after being exposed to subamolide A or linderanolide B, the dendricity morphology of most of the human epidermal melanocytes changed to a bipolar, fibroblastic cell type, in a dose-dependent manner. While treated with higher dose handling, HEMn-MP cells showed less dendritic morphology and became almost circular in shape. At the same time, melanin productions apparently decreased. In Fig. 4, photographs of melanocytes treated with both compounds at 0.01–10 μm of concentrations separately were displayed. We discovered that as time goes by, the dendricity of both concentrations were reduced, and the phenomenon was much more obvious at 10 μm than at 0.01 μm. Simultaneously, melanin productions apparently decreased.

image

Figure 4.  After 48 hours of treatments of Linderanolide B or Subamolide A, the photographs of HEMn-MP cells were taken with a bright field microscope. Magnification is 200′ without staining. Red arrows showed the HEMn-MP cells with bipolar shape morphology.

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Zebrafish (in vivo assay)

In addition to the in vitro screening on human tyrosinase, we carried out the in vivo test on zebrafish (Fig. 5). Each test compound and concentration consists of about 5–9 embryos. With various dosages, we discovered suitable concentrations for de-pigmentation on the zebrafish exterior surface. We examined the inhibition effects on the pigmentation of zebrafish, arbutin, kojic acid and PTU, which are famous inhibitors, at 50 μm as control groups. When treated with low doses (1 and 5 μm) of linderanolide B and subamolide A, the pigmentation of zebrafish had no significant decrease. With 10 μm of these two compounds, the pigmentation level of zebrafish decreased about 30% and 25%, respectively. It was observed that both compounds showed de-pigmenting effects at 10 μm, a concentration much lower than the de-pigmenting concentration of PTU or arbutin, and showed no toxicity to zebrafish.

image

Figure 5.  The quantitated effects of linderanolide B and subamolide A on the pigmentation levels of the zebrafish system. Arbutin, kojic acid and PTU were at 50 μm as positive controls. (*< 0.01 and **< 0.001).

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Molecular docking study

Many tyrosinase inhibitors operate through chelating Cu2+ in the active site of this target enzyme. To explore the probable structure binding between linderanolide B/subamolide A and human tyrosinase, the efforts on molecular docking with the Cu2+ binding site were carried out. As no 3-D structures for human tyrosinase are available, for the present docking study, we retrieved a theoretical homology model. Through the virtual screening for the electron density near the active site, the structure of O. dofleini hemocyanin bound with Cu2+ was found to be six key histidines chelating the two Cu2+ (Fig. 6) (20,30). The docked molecules are then graded by the fitness function of GoldScore, a sum of internal torsional strain energy, van der Waals energy, internal ligand van der Waals and H-bond energy. The most preferable-choice orientation of protein-compound ranked by GoldScore can then be presented. Linderanolide B and subamolide A were docked into the active site, defining six histidine essential residues responsible for chelating with catalytic Cu2+. Each of these contains three His residue, named H180, H202, and H211 for one Cu2+, and H363, H367 and H390 for another. We observed that carbonyl of γ-lactone moiety of linderanolide B and subamolide A interacted to form metal-coordination interactions with two Cu2+. The oxygen atom may act as an electric sink to help the contact to copper ions. This coordination interaction was consistent with the essential binding interactions between the phenol substrates and monophenol or ortho-diphenol oxidases (31). Based on these docking models, we proposed that the melanin-reducing effects of linderanolide B and subamolide A on human epidermal melanocytes might be because of their binding with copper ions in the tyrosinase active site as indicated by the modelling program.

image

Figure 6.  (a) The proposed binding modes of linderanolide B. (b) The enlarged part was the active site of human tyrosinase docking. (c) The proposed binding modes of subamolide A and (d) the enlarged part.

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Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Acknowledgements
  9. References
  10. Supporting Information

Melanin is the most important pigment for skin colour in humans. Melanogenesis inhibitors are not only used in medical treatment for hyperpigmentation but usually used in cosmetic business applications (32,33). The aim of our study was to identify novel natural potent inhibitors of human tyrosinase. The inhibition of tyrosinase activity was described in numerous reports, most of which, however, used mushroom tyrosinase as the model. Mushroom tyrosinases have significant differences from human tyrosinase in catalysis mechanisms (34–36). We have known that the catalysis of the human tyrosinase and mushroom tyrosinase have diverse mechanism reactions. With l-DOPA as the substrate, melanin productions by the two types of enzymes have separate solubility properties, and l-DOPA has different stimulative effects upon mammalian enzyme activities and not to mushroom enzyme (37–39).

There are some well-known melanin synthesis inhibitors, including kojic acid, arbutin, PTU or hydroquinone, which are being utilized globally as cosmetics ingredients at present. However, these melanogenic inhibitors may induce human skin tumorigenicity at high concentration doses or frequent use (26–28). There are concentration standards for every country to use kojic acid, arbutin or other chemical ingredients as a composition in cosmetic additives. The inhibitory effects of linderanolide B and subamolide A on in vitro mushroom tyrosinase inhibition was measured (Data S1). Successfully, both compounds were discovered with the effects of reducing mushroom tyrosinase activity. In addition, our focus was on the effect of linderanolide B and subamolide A to the human skin cell and in vivo screening systems. In addition to the in vitro screening for mushrooms, the normal human skin cells were also carried out for a more physiologically relevant method and to elucidate the inhibition mechanisms of melanogenic inhibitors.

Our purpose was to find an effective and safe skin-whitening substance that can be used to prevent human hyperpigmentation. As melanogenic regulatory compounds are being developed, potential safety shall be taken into consideration, such as the sensitivity, allergy or toxicity tests, as most are not presently confirmed.

We showed the cell viability of human keratinocytes, melanocytes and fibroblasts treated with various concentrations of the two compounds for 48 h. Treatment at a low dose (0.01–1.0 μm) did not show significant cytotoxicity to human skin cells. Our natural compounds are taken from an edible herbal medicine source, and no significant decreases in human skin cell viabilities were observed. We believe that it can be used as skin-whitening agents with potential therapeutic usage to human skin cells or tissues (40,41).

In this human epidermal melanocyte system, we recognized that melanin production levels and tyrosinase activities were significantly decreased by the addition of linderanolide B and subamolide A in a dose-dependent manner. Decreased amounts of melanin were found in these cells when treated with both compounds at various doses (0.01–10 μm) for 2 days. As kojic acid and arbutin are weak skin-whitening agents, and high concentration usage would cause carcinogenic disease, two inhibitors were examined with strong whitening effects in low concentration and then compared to kojic acid and PTU.

The morphological change photographs of human skin melanocytes treated with various concentrations of linderanolide B and subamolide A at different dosage levels under a phase contrast microscope. This implies that the loss of dendritic morphology may be related to the reduction in melanin production abilities. There were several reports concerning the relationships between the melanocyte morphologies, melanosome functions, melanin contents and tyrosinase activities of the epidermal cells (3). Human skin melanocytes have the bio-properties of regulation as sensors to receive the signals from UV or local environments (42,43). Skin melanins from melanocytes defend malignant melanocytes against noxious factors, including radio-, chemo- or photo- damages (44). In human epidermal cells, melanosomes are released from melanocyte dendrites and taken up by keratinocytes through endocytosis or phagocytosis (45). Human melanosomes assemble melanin at melanocyte dendrite ends to transport it to keratinocytes (46). Previous studies recognized that as melanocytes lose their characteristic dendritic structures and adopt fibroblastic bipolar forms, the intercellular contact between melanocytes and keratinocytes was significantly reduced, and resulted in a decrease in pigment transfer (47). Our study confirmed that there might be some connections among the dendritic morphology changes and the physiological properties of melanocytes. When exposed to higher dose inhibitor treatments, the HEMn-MP cells became almost round in shape with little dendricities and low melanin quantities. It deserves further investigation into comprehend its detailed physiological relationships.

When these skin-whitening agents are being further considered to be with potential cosmetic or therapeutic uses in human beings, the cytotoxic properties of two compounds are extremely important. In addition to the in vitro screening on normal human skin cell culture system, the in vivo zebrafish system was also carried out for a more physiologically relevant method and to elucidate the inhibition mechanisms of melanogenic inhibitors. Within this system, the compound toxicity could be determined simultaneously in a profitable vertebrate model owing to its having organ systems and gene sequences similar to humans (48). Linderanolide B and subamolide A showed a remarkable inhibitory potential on zebrafish pigmentation. Moreover, they suppress pigmentation production even at low doses without observable toxicity upon zebrafish. In other words, we observed that the zebrafish was alive after our treatments.

To understand the inhibitory mechanism of melanin synthesis, we simulated the model structure of the human tyrosinase active site. Using the human tyrosinase homology structure, the available pure compounds from C. subavenium were fitted to the active site. To construct a human tyrosinase model with a likely ligand-bound protein conformation on the active site, we first simulated a homology model by computer docking with Cu2+ and l-DOPA. Then, we performed the flexible-ligand docking using molecular energy minimization and dynamic simulation followed by docking studies with both compounds at the active site of human tyrosinase with the modelling program D.S. 2.0 (Accelrys, Inc., San Diego, CA, USA). The approving binding orientations provided with the lowest free binding energy of subamolide A and linderanolide B were estimated by theoretical modelling, respectively.

Conclusion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Acknowledgements
  9. References
  10. Supporting Information

From the previous descriptions and findings, the results uniquely reveal that both linderanolide B and subamolide A purified from the C. subavenium in human melanogenic inhibition have great potential. There is no previous biochemical characterization reference related to this species of plant in cosmetics or other applications. Both compounds could reduce 50% of human tyrosinase activities at a dose of 1 μm after 48 h of treatment and effectively inhibited the melanin production in HEMn-MP cells (40% reduction). Through computer docking, we discovered that the cellular melanin reduction may be because of blocking copper ions within the tyrosinase active site. We also computed the cytotoxicities of these two compounds with the in vivo zebrafish system, in preparation for further use in human medical dermatology. They did not illustrate significant toxicities to zebrafish. In conclusion, the data obtained from this study showed that linderanolide B and subamolide A were efficient and safe human tyrosinase inhibitors that can be produced in large quantities. We propose that linderanolide B and subamolide A are effective novel tyrosinase inhibitors to be considered as skin-lightening agents.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Acknowledgements
  9. References
  10. Supporting Information

This investigation was financially supported by National Science Council of Taiwan, ROC under grant nos. of NSC97-2221-E-037-002-, NSC98-2221-E-037-005- and NSC-97-2320-B-242-002-MY3; Ministry of Economic Affairs, 98-EC-17-A-10-S2-0066; as well as by the Kaohsiung Medical University under grant nos. of Q097002. The authors also would like to thank Yi-Ting Chou and Ya-Ling Yeh for the assistances and Min-Yii Julie Wang for English editing.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Acknowledgements
  9. References
  10. Supporting Information

Supporting Information

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Acknowledgements
  9. References
  10. Supporting Information

Figure S1. The purification flow chart and structures of the two compounds – Linderanolide B and Subamolide A.

Figure S2. Inhibitory effects of linderanolide B and subamolide A on mushroom tyrosinase.

Table S1. IC50 values of mushroom tyrosinase inhibition. Data were expressed as a mean value of three independent experiments. Kojic acid was used as a positive control of this assay.

Data S1. Extraction and purification of linderanolide B and subamolide A.

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