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Keywords:

  • human tyrosinase inhibitor;
  • melanin;
  • p-coumaric acid;
  • Vaccinium bracteatum Thunberg

Synopsis

  1. Top of page
  2. Synopsis
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Acknowledgements
  7. References

Screening for tyrosinase (TYR) inhibitors potentially useful for control of skin pigmentation has been hampered by the limited availability of human TYR. To overcome this hurdle, we have established human embryonic kidney (HEK293)-TYR cells that constitutively express human TYR. In the current study, we assayed human TYR inhibition activities of 50 plant extracts using the lysates of transformed HEK293-TYR cells. The strongest inhibition of human TYR was shown by the extract of Vaccinium bracteatum Thunberg, followed by the extract of Morus bombycis Koidzumi. The former extract did not inhibit mushroom TYR activity whereas significant inhibition was observed with the latter extract, demonstrating the importance of using human TYR in the screening for human TYR inhibitors. Upon liquid-liquid partitioning of the extract from V. bracteatum, the active constituents were enriched in the ethyl acetate fraction, and the subsequent preparatory thin-layer chromatography identified p-coumaric acid (PCA) as the main active constituent. The hypo-pigmentation of PCA was verified in the MelanoDerm™ Skin Model. This study demonstrates that transformed HEK293-TYR cells could expedite the discovery of human TYR-specific inhibitors from natural sources which might be useful in the control of skin pigmentation.


Introduction

  1. Top of page
  2. Synopsis
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Acknowledgements
  7. References

Melanin is one of the major contributors to skin pigmentation and provides effective protection against harmful ultraviolet radiation [1, 2]. Melanin is synthesized through a series of oxidative reactions in a specialized organelle called melanosomes in epidermal melanocytes [3, 4]. The melanin-harbouring melanosomes are then transferred through dendritic tips to neighbouring keratinocytes in the epidermis [5].

Abnormal melanin deposition in the skin triggered by patho-physiological or environmental factors has great cosmetic relevance and has prompted research and development of natural and chemical agents that could interfere with melanin synthesis [6–8]. As an enzyme catalysing several reactions that constitute regulatory points of the melanogenic pathway, tyrosinase (TYR) has been a major target for the control of skin pigmentation [9, 10].

However, there has been a major problem associated with the use of mushroom TYR instead of the human enzyme in screening assays for hypo-pigmentation agents. The reason why mushroom TYR has been used for this purpose seems to be rather clear as human TYR is currently hardly available. Nonetheless, mushroom TYR is quite different from human TYR in terms of the amino acid sequence and substrate specificity [11–13]. Furthermore, previous studies have shown inconsistent effects of many kinds of compounds on the activity of TYR of mushroom, murine or human origin, demonstrating the biochemical differences between these enzymes [14, 15].

The lack of availability of human TYR and its high demand in screening assays has prompted us to develop a new source of human TYR by stable transfection of human embryonic kidney (HEK) 293 cells with a human TYR construct. As demonstrated in a previous study [16], the transformed cells, HEK293-TYR, proliferated rapidly and expressed the active form of human TYR constitutively and the cell-free extracts of these cells were very useful in the assay for TYR inhibition effects. Taking advantage of this approach, we attempted to discover specific and potent inhibitors of human TYR from natural sources. The extracts of fifty plants endemic to Jeju Island in the Republic of Korea were subjected to screening assays against human TYR. Screening assays were also run against mushroom TYR for comparative purpose.

Materials and methods

  1. Top of page
  2. Synopsis
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Acknowledgements
  7. References

Plant extracts

Samples of plant extracts used in the screening assays (Table I) were purchased from Jeju Biodiversity Research Institute of Jeju Technopark (http://jbri.jejutp.or.kr). Voucher specimens of the plants and other information regarding the plant extracts are available at this institute. The leaves, twigs or whole plants harvested in Jeju Island in the Republic of Korea were air-dried and extracted with 80% (v/v) aqueous ethanol at room temperature. The extracted solutions were evaporated under reduced pressure to obtain crude extracts.

Table I.   Plant extracts used in this study
No.Plant nameCatalogue no.Plant parts used for extractionYield of extract (% dry weight)
 1Aesculus turbinata Bl.JBRI-10506Leaves18.33
 2Ampelopsis brevipedunculata var. heterophylla (Thunb.) HaraJBRI-10552Whole plants17.32
 3Artemisia fukudo MakinoJBRI-10671Twigs11.26
 4Artemisia japonica Thunb.JBRI-10616Whole plants21.71
 5Artemisia montana (Nakai) Pamp.JBRI-10665Aerial parts12.98
 6Artemisia montana (Nakai) Pamp.JBRI-10666Whole plants5.38
 7Artemisia scoparia Waldst. Et KitamuraJBRI-10670Whole plants9.79
 8Atremisia capillaris Thunb.JBRI-10668Whole plants14.35
 9Caesalpinia decapetala var. japonica (S. et Z.) OhashiJBRI-10339Leaves21.54
10Callicarpa japonica ThunbJBRI-10444Leaves21.94
11Caragana sinica (Buchoz) RehderJBRI-10470Leaves12.77
12Castanea crenata S. et Z.JBRI-10569Leaves16.71
13Cayratia japonica (Thunb.) GagnepainJBRI-10648Leaves10.68
14Diospyros lotus L.JBRI-10571Fruit9.42
15Euonymus alatus (Thunb.) Sieb.JBRI-10316Leaves13.03
16Hedera rhombea (Miq.) BeanJBRI-10044Leaves20.17
17Hydrangea petiolaris S. et Z.JBRI-10296Leaves21.62
18Idesia polycarpa MaxJBRI-10464Leaves17.03
19Kadsura japonica (L.) DunalJBRI-10681Aerial parts10.07
20Kalopanax pictus (Thunb.) NakaiJBRI-10525Leaves21.28
21Lamium purpureum L.JBRI-10643Whole plants32.58
22Machilus thunbergii S. et Z.JBRI-10094Leaves13.97
23Magnolia kobus A. P. DC.JBRI-10462Leaves15.65
24Melandryum oldhamianum (Miq.) RohrbachJBRI-10073Aerial parts25.67
25Morus bombycis Koidz.JBRI-10473Leaves18.44
26Oenothera erythrosepala BorbasJBRI-10572Leaves15.72
27Oenothera odorata Jacq.JBRI-10545Whole plants14.39
28Platanus occidentalis L.JBRI-10531Leaves16.04
29Prunus maximowiczii RuprJBRI-10496Leaves18.12
30Prunus mume S. et Z.JBRI-10536Leaves21.75
31Prunus pendula for. Ascendens (Mak.) OhwiJBRI-10478Leaves20.80
32Prunus serrulata var. quelpaertensis UyekiJBRI-10480Leaves22.73
33Pyrrosia lingua (Thunb.) FarwellJBRI-10038Whole plants20.66
34Rhamnella frangulioides (Max) WeberbJBRI-10450Leaves24.78
35Rhododoendron weyrichii Max.JBRI-10439Leaves14.47
36Rhus succedanea L.JBRI-10561Leaves17.03
37Rubus buergeri Miq.JBRI-10634Leaves27.26
38Sambucus sieboldiana Bl.JBRI-10303Leaves26.26
39Sapium japonicum (Sieb. et Zucc.) Pax et HoffmannJBRI-10490Leaves27.12
40Sapium sebiferum (L.) ROXB.JBRI-10503Leaves26.09
41Sedum oryzifolium MakinoJBRI-10399Whole plants10.21
42Stauntonia hexaphylla (Thunb.) Decne.JBRI-10032Leaves28.32
43Styrax japonica S. et Z.JBRI-10454Leaves30.86
44Taxus cuspidata Sieb. et Zucc.JBRI-10021Leaves32.52
45Ternstroemia japonica ThunbJBRI-10468Leaves12.41
46Trifolium pratense L.JBRI-10509Leaves23.31
47Ulmus davidiana var. japonica (Rehder) NakaiJBRI-10484Leaves15.21
48Vaccinium bracteatum Thunb.JBRI-10685Aerial parts22.43
49Xylosma congestum (Lour.) Merr.JBRI-10481Leaves24.01
50Zelkova serrata MakinoJBRI-10498Leaves16.57

Fractionation of the extract from Vaccinium bracteatum Thunberg

The extract of V. bracteatum (800 mg) was suspended in 4 mL water and liquid-liquid partitioned twice with 4 mL methylene chloride (MC) and then, twice with 4 mL ethyl acetate (EtOAc). Combined MC layers, combined EtOAc layers and the aqueous layer were evaporated under reduced pressure to yield the MC fraction (165 mg), EtOAc fraction (46.3 mg) and the aqueous fraction (564 mg) (Fig. 2A).

The EtOAc fraction was subjected to preparatory thin-layer chromatography (TLC). The EtOAc fraction was dissolved in ethanol at 10% (w/v) and applied onto a TLC plate (Sigma-Aldrich, St. Louis, MO, U.S.A.) as a long streak (a total of 2 mg of the EtOAc fraction was applied). Reference compounds, p-coumaric acid (PCA) and quercetin, were dissolved in ethanol at 0.5% (w/v) and spotted at both ends of the TLC plate (5 μg per spot). Ascending TLC was run with a mixture of chloroform, methanol and acetic acid (90 : 10 : 1, v/v/v) as a mobile phase in an air-tight container. The bands and spots were viewed under a UV lamp. The chromatographic area on the TLC plate was divided into 12 horizontal sectors (Fig. 2C), and the adsorbent of each sector was scraped and eluted with 50 μL ethanol. With this method, a total of 12 sub-fractions of the EtOAc fraction were prepared.

HPLC analysis

Analytical HPLC was carried out using a Waters Alliance HPLC System (Waters, Milford, MA, U.S.A.) consisting of a Waters 2695 Separation Module and a Waters 2996 photodiode array detector. Stationary phase was 5 μm YMC-Pack Pro C18 column (4.6 × 250 mm) and mobile phase consisted of 30% methanol, 67% water and 3% formic acid (flow rate, 0.8 mL min−1).

Preparation of cell-free extracts from transformed HEK293-TYR cells

Human Embryonic Kidney 293 cells were purchased from American Type Culture Collection (Manassas, VA, U.S.A.) and transformed into HEK293-TYR cells that stably express human TYR, as previously described [16]. The transformed cells were cultured in the growth medium Dulbecco’s minimum Eagle’s medium (DMEM) containing 10% foetal bovine serum, 100 U mL−1 penicillin, 0.1 mg mL−1 streptomycin, 0.25 μg mL−1 amphotericin B and 1.0 mg mL−1 of G418 (Geneticin®; Invitrogen, Grand Island, CA, U.S.A.) with the medium renewed every 4 days. Cells were cultured at 37°C in a humidified atmosphere containing 5% CO2 and 95% air. The cells were homogenized in ice-cold lysis buffer (10 mmol L−1 Tris-Cl, pH 7.4, 120 mmol L−1 NaCl, 25 mmol L−1 KCl, 2.0 mmol L−1 EGTA, 1.0 mmol L−1 EDTA, and 0.5% Triton X-100 and protease inhibitor cocktail). The homogenates were then centrifuged at 13 000 g for 15 min at 4°C to obtain clear supernatants to be used as the cell-free extracts.

In vitro assay for TYR activity

The in vitro TYR assay was carried out in a 96-well microplate with the reaction mixture (200 μL) containing 100 mmol L−1 sodium phosphate buffer (pH 6.8), cell-free extracts of HEK293-TYR cells (200 μg protein mL−1) or mushroom TYR (100 unit mL−1) from Sigma-Aldrich, 0.5 mmol L−1 L-tyrosine, 1 μmol L−1 L-dihydroxyphenylalanine (DOPA) and a test sample or vehicle (ethanol, up to 4 μL). The constituted reaction mixture was incubated for 120 min (for human TYR) or 30 min (for mushroom TYR) at 37°C, and DOPA chrome formation was estimated at 490 nm by a BioRad Model 680 microplate reader (Bio-Rad Laboratories, Inc., Hercules, CA, U.S.A.).

Pigmentation assay in a reconstructed skin model

Hypopigmentation potency was assessed using a reconstituted human skin model, MelanoDerm™ (MEL-300-B) (MatTek Corporation, Ashland, MA, U.S.A.). The model consists of normal epidermal keratinocytes and melanocytes obtained from a highly pigmented donor, seeded at a 10 : 1 ratio on a nylon mesh basement membrane and cultured to form a multilayered fine structure similar to the human epidermis [17, 18]. The tissues were fed EPI-100-NMM-113 medium (MatTek Corporation) supplemented with 5 μg mL−1 gentamycin and 0.25 μg mL−1 amphotericin B, three times per week. The test materials were dissolved in 70% propylene glycol with a final concentration of 5 mmol L−1, and 25 μL of solution was topically applied every other day for 2 weeks [19]. At the end of the treatments, photographs of the tissues were taken under a phase-contrast microscope (Eclipse TS100; Nikon Instruments Inc., Melville, NY, U.S.A.). After removing the test materials by washing with PBS, cell viability and melanin content of the tissues were determined using procedures previously described [20]. Cell viability and melanin content are presented as the % of the vehicle control. The absorption spectra of the extracted melanin solution were recorded using a Shimadzu UV-1650PC spectrophotometer (Shimadzu Corporation, Kyoto, Japan).

Statistical analysis

Data were presented as means ± SE of three or more independent experiments. Significant differences among the groups were determined by a one-way ANOVA. Duncan’s multiple-range test was performed if differences were identified between the groups at < 0.05.

Results and discussion

  1. Top of page
  2. Synopsis
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Acknowledgements
  7. References

As a source of human TYR, transformed HEK293-TYR cells have many advantageous features. First of all, the cells express active forms of human TYR robustly as previously confirmed [16]. In contrast to HEMs that grew very slowly even with very expensive special culture media, HEK293-TYR cells proliferated rapidly in common culture media. HEK293-TYR cells, an immortalized cell line readily available from the commercial sources, can be conveniently transformed into HEK293-TYR cells. HEK293 cells only need to be transfected with a plasmid construct carrying the full coding sequence of the human TYR cDNA under a constitutively active promoter, followed by selection of stably transfected colonies. The established cell line can be maintained and expanded in the presence of the antibiotic agent, G418. Furthermore, the cell-free extracts of HEK293-TYR cells, without further purification, can be conveniently used in the screening of potential human TYR inhibitors [16].

The primary purpose of the present study was to select plant extracts that inhibit human TYR as such plant extracts might have potential to be cosmeceuticals for skin hypo-pigmentation. Fifty extracts derived from plants endemic to Jeju Island in the Republic of Korea were subjected to the primary screening assay using HEK293-TYR cell-free extracts. Although some of the plant extracts had previously been tested against mushroom TYR [21], the current study was the first to examine their effects on human TYR activity. In this assay, plant extracts were tested at 80 μg mL−1 compared to arbutin which provided a positive control. As shown in Fig. 1A, the strongest inhibition of human TYR was shown by the extract of V. bracteatum Thunberg (# 48), followed by the extract of Morus bombycis Koidzumi (#25). These plant extracts were as potent as arbutin, a well-known cosmeceutical used for the purpose of skin whitening [8].

image

Figure 1.  Effects of the plants extracts on the activities of human and mushroom TYRs in vitro. The activities of human TYR (A) and mushroom TYR (B) were determined with 0.5 mmol L−1 tyrosine and 1.0 μmol L−1 DOPA as the substrate, in the absence and presence of a plant extract or arbutin at 80 μg mL−1. Data are presented as the % of the uninhibited activity. *P < 0.05 vs. vehicle control; TYR, tyrosinase.

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The effects of the fifty plant extracts were also examined against mushroom TYR for comparative purposes (Fig. 1B). Surprisingly, the extract of V. bracteatum did not inhibit mushroom tyrosinase activity at all whereas a significant inhibition was observed with the extract of Morus bombycis that had been previously reported to inhibit mushroom TYR [22]. These results indicate that the extract of V. bracteatum, which inhibited human TYR most strongly, would not have been found if mushroom TYR had been used instead of the human enzyme in this assay. Together with the previous studies that have demonstrated the biochemical differences between mushroom and human TYRs [14, 15], the current observations demonstrate the importance of using human TYR in the screening assay for human skin hypo-pigmentation agents.

Vaccinium bracteatum Thunberg belongs to the genus Rhododendron and is grown worldwide and not restricted to Jeju Island. A literature search indicated that this plant contains various phenolic compounds including PCA and quercetin [23, 24]. The extracts derived from this plant have been described to have such physiological effects as antioxidant, anti-fatigue and anti-diabetic activities [23, 24]. But little is known regarding their effects on TYR activity or skin pigmentation. Therefore, the TYR-inhibiting active constituents of this plant were sought in the subsequent experiments. Among the aqueous, EtOAc and MC fractions obtained by a liquid-liquid partitioning (Fig. 2A), the EtOAc fraction was the most active against the human TYR (Fig. 2B). The EtOAc fraction was resolved through preparatory TLC in which PCA and quercetin were also developed as reference compounds (Fig. 2C). The resulting 12 sub-fractions of the EtOAc fraction were assayed against human TYR and the strongest inhibition was observed with sub-fractions 6–7 (Fig. 2D).

image

Figure 2.  Fractionation of the extract from Vaccinium bracteatum and analysis for human TYR-inhibiting activities. The crude extract of V. bracteatum was divided into the methylene chloride, EtOAc and H2O fractions (A) and each solvent-fraction was assayed against human TYR activity at 80 μg mL−1 (B). The EtOAc fraction was developed by preparatory thin-layer chromatography, with reference compounds (p-coumaric acid and quercetin), and the chromatographic area was divided into 12 horizontal sectors (C). Adsorbents of each sector were eluted with an equal volume of ethanol (50 μL) and an aliquot of each fraction (4 μL) was included in the reaction mixture (200 μL) to determine its effect against human TYR activity (D). Assay results are presented as the % of the uninhibited activity. *P < 0.05 vs. vehicle control. TYR, tyrosinase; EtOAc, ethyl acetate; MC, methylene chloride; PCA, p-coumaric acid.

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The main active compound of the EtOAc fraction was assumed to be PCA based on the similar mobility on the TLC (slight different mobility may be owing to overloading) and colour reaction with iodine vapour. This notion was verified by additional analysis using an HPLC system equipped with a photodiode array detector which provides the absorption spectra of resolved compounds. Typical HPLC profile of the EtOAc fraction and the absorption spectrum of a major peak marked with an arrow are shown in Fig. 3A. The major peak was attributed to PCA because its retention time and UV absorption spectrum were identical to those of authentic PCA. Co-elution of the sample and authentic PCA further verified this notion.

image

Figure 3.  Identification of PCA as a main active constituent of Vaccinium bracteatum that inhibits human TYR activity in vitro and confirmation of its hypo-pigmentation effect in the MelanoDerm™ Skin Model. A typical HPLC profile of the ethyl acetate fraction derived from V. bracteatum monitored at 309 nm is shown (A). The UV absorption spectrum (maximum absorbance at 309 nm) of a major peak (marked with an arrow) is shown in inset. This peak was attributed to PCA based on the chromatographic and spectroscopic evidence. The effects of PCA and the extract from V. bracteatum against human TYR activity were determined in vitro using the human embryonic kidney 293-TYR cell-free extracts (B). The potential hypo-pigmentation effect of PCA was examined in comparison with arbutin using the MelanoDerm™ Skin Model (C). Tissues were treated with 5 mmol L−1 PCA or arbutin or vehicle (70% propylene glycol) every other day for 2 weeks. Microscopic tissue images are shown. Cell viability and melanin content are presented as the % of the vehicle control. *P < 0.05 vs. vehicle control. Typical absorption spectra of the extracted melanin from the treated tissues are shown (D). TYR, tyrosinase; PCA, p-coumaric acid.

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The content of PCA in the crude extract of V. bracteatum was estimated to be 0.8% (w/w). In addition, enzyme assays indicated that PCA inhibited human TYR activity by 50% at 0.5 μg mL−1 which means that this compound is 100 times more active than the crude extract of V. bracteatum (Fig. 3B). These results suggest that PCA might be the main active constituent of V. bracteatum providing the human TYR-inhibiting effects. When tested in human epidermal melanocytes, V. bracteatum extract exhibited a higher toxicity than PCA and arbutin, and showed no significant anti-melanogenic effect at sub-toxic concentrations (data not shown). Thus, purification steps seemed to be essential for this extract to be used as a safe and effective cosmeceutical.

The potent inhibitory effects of PCA against human TYR activity, cellular melanin synthesis and skin pigmentation have previously been demonstrated in a number of studies [9, 15, 25, 26]. Therefore, it was of interest to examine its effect on the pigmentation of reconstructed skin models. The three-dimensional human skin model, MelanoDerm™, was used for this purpose. As shown in Fig. 3C, topical treatment of 5 mmol L−1 PCA for 2 weeks decreased the melanin content of the skin model without causing a loss in cell viability. Arbutin at 5 mmol L−1 had no significant effects on the melanin content and cell viability. Microscopic tissue images indicated that the number of highly pigmented cells decreased owing to the treatment with PCA. The typical absorption spectra of melanin extracted from the treated tissues are shown in Fig. 3D.

As an extension of the previous study that established HEK293-TYR cells as a human TYR enzyme source [16], the present study demonstrated the utility of the assay method using the HEK293-TYR cells in the screening of plants extracts. Many of the tested plant extracts were found to have different effects against human and mushroom TYRs. Some plant extracts inhibited human or mushroom TYR selectively and the other plant extracts inhibited both or none. These results suggest that plants’ extracts that specifically inhibit human TYR can be conveniently selected by the screening assay using HEK293-TYR cells and the comparative assay against mushroom TYR.

In conclusion, this study demonstrated a model case for the discovery of human TYR-specific inhibitors from natural sources. By comparing the effects of various plant extracts on both human and mushroom TYR activities, the extract of V. bracteatum was found to inhibit human TYR activity very potently and specifically without having an effect on the mushroom TYR activity. The potent and specific inhibition of human TYR by the extract from V. bracteatum could be attributed to its constituent, PCA, of which the hypo-pigmentation effect was verified in the MelanoDerm™ Skin Model. The current study also demonstrates why human TYR should be used in screening assays for skin hypo-pigmentation agents.

Acknowledgements

  1. Top of page
  2. Synopsis
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Acknowledgements
  7. References

This work was supported by a grant from the Regional Industry Technology Development Program of the Ministry of Knowledge Economy, Republic of Korea.

References

  1. Top of page
  2. Synopsis
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Acknowledgements
  7. References