Effects of selected moraceae plants on tyrosinase enzyme and melanin content
Sukanya Dej-adisai1, Kedsaraporn Parndaeng1, Chatchai Wattanapiromsakul1, Wanlapa Nuankaew2, Tong Ho Kang2
1 Department of Pharmacognosy and Pharmaceutical Botany, Faculty of Pharmaceutical Sciences, Prince of Songkla University, Hat-Yai, Songkhla, Thailand
2 Department of Oriental Medicinal Biotechnology, Graduate School of Biotechnology, College of Life Sciences, Kyung Hee University, Gyeonggi-do, Republic of Korea
|Date of Submission||25-Jan-2019|
|Date of Decision||06-Mar-2019|
|Date of Web Publication||19-Sep-2019|
Department of Pharmacognosy and Pharmaceutical Botany, Faculty of Pharmaceutical Sciences, Prince of Songkla University, Hat-Yai, Songkhla 90112
Source of Support: None, Conflict of Interest: None
| Abstract|| |
Background: Hyperpigmentation is the one cause of skin disorder. The dark-colored skin causes from the increasing of melanin pigment production. It is synthesized by melanogenesis catalyzed by tyrosinase enzyme. Tyrosinase is one of the main causes of melanogenesis; thus, inhibition of the activity of tyrosinase can decrease melanogenesis. Hence, the potential tyrosinase inhibitor could be discovered from natural products. Objective: Discovery of tyrosinase inhibitor from natural products by focusing on Moraceae plants. Materials and Methods: Forty-eight Moraceae plant extracts were screened for antityrosinase and antibacterial activities; Streblus taxoides and Artocarpus chama were selected to study in B16F1 melanoma cell; intracellular antityrosinase activity and melanin content. Moreover, pigmentation inhibitory effect on the zebrafish of these samples was studied. Results: The extracts of S. taxoides and A. chama showed the potential activity against tyrosinase enzyme on both intracellular and extracellular enzymatic assays. Moreover, they suppressed pigmentation in zebrafish. Only ethyl acetate extract of these plants could show anti-bacterial activity. Conclusion: S. taxoides and A. chama are potential plants for further study of chemical constituents and biological activities especially the anti-tyrosinase activity of the isolated compound to find out the lead compound for whitening agent from natural product.
Keywords: Artocarpus charma, melanin content, moraceae, Streblus taxoides, tyrosinase inhibition
|How to cite this article:|
Dej-adisai S, Parndaeng K, Wattanapiromsakul C, Nuankaew W, Kang TH. Effects of selected moraceae plants on tyrosinase enzyme and melanin content. Phcog Mag 2019;15:708-14
|How to cite this URL:|
Dej-adisai S, Parndaeng K, Wattanapiromsakul C, Nuankaew W, Kang TH. Effects of selected moraceae plants on tyrosinase enzyme and melanin content. Phcog Mag [serial online] 2019 [cited 2021 Apr 23];15:708-14. Available from: http://www.phcog.com/text.asp?2019/15/65/708/267172
- Tyrosinase inhibition and anti-bacterial activity of 48 Moraceae plant extracts were reported
- Tyrosinase inhibition of Streblus taxoides woods and Artocarpus chama stem were reported for the first
- A. chama stem and S. taxoides wood extracts exhibited potential activity against tyrosinase enzyme both in enzymatic assay and intracellular assay
- A. chama stem and S. taxoides wood extracts showed the suppression of pigmentation in zebrafish.
Abbreviations used: DMSO: Dimethyl sulfoxide; OD: Optical density; DMEM: Dulbecco's modified eagle medium; SRB: Sulforhodamine B; TCA: Trichloroacetic acid; RIPA buffer: Radioimmunoprecipitation assay buffer; BSA: Bovine serum albumin; PBS: Phosphate-buffered saline; hpf: h postfertilization.
| Introduction|| |
The dark-colored skin causes from the increasing of melanin pigment production. This pigment shows brown and black colors. Melanin is distributed in the living organisms of the natural and has many different properties. Melanogenesis initiated from L-tyrosine hydroxylated to L-dihydroxyphenyl-alanine (L-Dopa), then oxidation of L-Dopa to its corresponding o-dopaquinone, catalyzed by tyrosinase enzyme. o-Dopaquinone can be divided into two different types of reaction to produce eumelanin and pheomelanin., Melanin plays an important role in preventing ultraviolet light-induced skin damage, but abnormal melanin (hyperpigmentation) or accumulation of an excessive level of melanin due to the overexpression of tyrosinase leads to skin disorders such as melasma, age spots, and sites of actinic damage. Tyrosinase is the key enzyme in melanogenesis, and then tyrosinase inhibition can decrease melanogenesis, that will be useful for the treatment of hyperpigmentation.
Many problems from current whitening cosmetics have been reported such as dermatitis and skin irritation, melanocyte destruction, postinflammatory pigmentation, and sometimes, we found ochronosis, cytotoxicity, and skin cancer. The discovery of tyrosinase inhibitor from natural sources will be an alternative treatment. It might provide the lead compound of tyrosinase inhibitor and probably develop for whitening agent in cosmeceutical or medicine for hyperpigmentation.
Pathogenic bacteria are a major cause of human skin disease. Moreover, some micro-organisms can stimulate melanogenesis., Antibiotic is the choice for the treatment. However, the use of antibiotics may lead to drug resistance of many bacterial strains. Development of new antimicrobial compounds for resistant organisms is becoming critically important.
The bacterial infection is the one cause of heperpigmentation. Hence, maybe the synergistic effects of antimicrobial and antityrosinase activities from natural products will exhibit the decreasing of melanin pigment production.
Moraceae is the most interesting plant's family for biological especially, tyrosinase inhibition since many isolated compounds from Moraceae plants showed inhibitory effect against tyrosinase enzyme such as resveratrol and artocarpanone which were isolated from Artocarpus gomezianus and Artocarpus integer, respectively.,,,,,,,,,,
Hence, this study focused on Moraceae plants. Forty-eight Moraceae plants were selected for antityrosinase and antibacterial screening. Then, Streblus taxoides wood and Artocarpus chama stem extracts which showed the potential effects were selected for further study on biological activities such as both intracellular and extracellular antityrosinase and antibacterial activities.
| Materials and Methods|| |
Forty-eight Moraceae plant samples were collected from Rajjaprabha Dam, Surat Thani Province; Southern Literature Botanical Garden, Songkhla Province; Botanical Garden, Faculty of Pharmaceutical Sciences, Prince of Songkla University, Songkhla Province and Walailuk University, Nakhon Si Thammarat Province [Table 1]. All plant samples were identified by the botanists of each place. All sample specimens were kept in the Department of Pharmacognosy and Pharmaceutical Botany, Faculty of Pharmaceutical Sciences, Prince of Songkla University, Thailand. The voucher specimen numbers of two selected Moraceae plants, A. chama Buch.-Ham. and S. taxoides (Roth) Kurz were SKP 117 01 03 01 and SKP 117 19 20 01, respectively. These plants were selected for further biological studies.
|Table 1: Anti-tyrosinase activity of 48 Moraceae plant samples at 20 µg/mL|
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Preparation of plant extracts
Dried powder plant materials were extracted by maceration with absolute ethanol and extracted repeatedly at room temperature for 3 days (X3). The filtrates were pooled and evaporated under reduced pressure at temperature not exceeding 40°C by vacuum rotary evaporator to yield the ethanol extract.
The plants that showed the potential activities of antityrosinase and/or antibacterial activities and have not been reported were selected for further studies through this project.
The dried powder of selected plants was macerated with petroleum ether, ethyl acetate, and methanol at room temperature 3 days (X3, for each solvent separately) and boiled with H2O, respectively, to give petroleum ether, ethyl acetate, methanol, and H2O extracts.
Enzymetic antityrosinase activity assay
Antityrosinase activity was determined with the dopachrome method by using L-Dopa as the substrate. Dopachrome is one of the intermediate substrates in melanogenesis. The red color of dopachrome from the oxidation of L-Dopa can be detected by visible light at 492 nm.
140 μL phosphate buffer (pH 6.8), 20 μL sample solution, and 20 μL tyrosinase solution (203.3 unit/mL) were mixed at 25°C for 10 min and then added with 20 μL of 0.85 mM L-Dopa. The visible absorption was measured at 492 nm. The solution was incubated at 25°C for 20 min. After incubation, the amount of dopachrome in the reaction was measured at 492 nm again. Tyrosinase inhibition (T) was calculated as this equation:
Tyrosinase inhibition (%) = (1 – [OD492 of sample/OD492 of control]) × 100
OD492: The difference of sample/control optical density (OD) before and after incubation at 492 nm.
Kojic acid and water extract of Artocarpus lakoocha wood were used as positive controls and Dimethyl sulfoxide (DMSO) was used as a negative control.
Murine melanoma B16-F1 cells (CLS-400122) were cultured in Dulbecco's Modified Eagle's medium containing 10% heat-inactivated fetal bovine serum at 37°C in a humidified atmosphere with 5% CO2. When cells reach 70%–80% confluence cell viability, cellular tyrosinase activity and melanin content were measured.,,,
Cell viability assay
Cell viability was determined by sulforhodamine B (SRB) assay. The cells were seeded in 96-well plate (5 × 103 cells/well). After incubation for 24 h, the cells were treated with test samples and 0.5% DMSO for negative control. After 48 h incubation, cells were fixed with 10% trichloroacetic acid and kept at 4°C, 1 h. After that, cells were strained with 0.45% SRB. Then, 10 mM Tris base was added on strained cells and then SRB color was dissolved by shaking. Optical densities were determined at 492 nm. The percent cell viability would be calculated.
Intracellular anti-tyrosinase activity and melanin content assays
The cells were seeded in 12 well plates (3 × 105 cells/well) and allowed to adhere at 37°C for 12 h. Cells were treated with test samples while control cells were treated with 0.5% DMSO. After 48 h incubation, cells were lysed with radioimmunoprecipitation assay buffer and centrifuged 14,000 rpm for 20 min (4°C) to separate the supernatant.
Intracellular antityrosinase activity
The supernatants were collected and the protein content was determined by the Bradford method using bovine serum albumin as standard. The supernatant of lysate cells and 2 mg/mL L-Dopa in phosphate-buffered saline were added to 96-well plate. The mixture was incubated at 25°C for 1 h, OD was determined at 492 nm. Tyrosinase would be calculated.
Cells pellet were dissolved with 1 M NaOH and incubated at 55°C for 1 h. Melanin concentration was calculated by comparison of the absorbance at 475 nm using a standard curve of synthetic melanin.
Pigmentation inhibitory activity on zebrafish
Zebrafish (Danio rerio) was used to determine the pigmentation inhibitory effect. The method was modified from Le et al. 2016. Briefly, embryos at 9 h postfertilization (hpf) were placed individually into 96-well plate filled with 100 μL/well 0.03% sea salt solution and each sample solution. 25 μM 1-phenyl-2-thiourea was used as positive group and 0.03% sea salt solution was used as normal group. The experiment used 20 embryos/group.
At 72 hpf, after embryos hatched, the larvae were put on glass slides and embedded using 2% low melting agarose. The dorsal view of the zebrafish fry was photo-captured, and the size of black spot in the head-dorsal region at 81 hpf was evaluated.
Antimicrobial activity assay
Micro-organisms: two types of bacteria were involved in this assay; aerobic bacteria were Staphylococcus aureus (ATTC 25923), methicillin-resistant S. aureus (MRSA) (DMST20654), and Staphylococcus epidermidis (TISTR 517), whereas anaerobic bacteria was Propionibacterium acnes (DMST 14916). Aerobic bacteria were cultured in Mueller-Hinton Agar for 18–24 h at 37°C. Anaerobic bacteria were cultured in Brain Heart Infusion Agar for 72 h at 37°C. The preliminary screening of antibacterial activity was assessed using agar disc diffusion method. The fractions of selected plants were determined for minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) in 96 well microplate by modified broth microdilution method., Oxacillin was used as positive controls except MRSA using vancomycin.
| Results|| |
Enzymatic antityrosinase activity
Among 48 Moraceae plant samples [Table 1] investigated for antityrosinase activity, 12 samples showed antityrosinase activity at 20 μg/mL >30%, including Artocarpus altilis (branch), A. integer (branch), A. chama (wood), A. chama (stem), Ficus benghalensis (branch), F. benghalensis (wood), Ficus foveolata (wood), Ficus superba (leaf), Morus alba (branch), M. alba (leaf), Streblus ilicifolius (wood), S. taxoides (wood). The wood extracts of S. taxoides and S. ilicifolius showed the highest tyrosinase inhibition with 58.59% ±1.90% and 69.05% ±5.00%, respectively. However, chemical constituents and biological activities; anti-tyrosinase and antimicrobial activities were already reported for S. ilicifolius. Only, the extracts of S. taxoides and A. chama which showed the potential activity against tyrosinase enzyme were selected for further study and the following successive extraction was used.
The dried materials were extracted with petroleum ether, ethyl acetate, methanol and water, respectively. Percent antityrosinase activities of these crude extracts are shown in [Table 2]. The ethyl acetate and methanol extract of S. taxoides wood and A. chama stem showed the most potent effects against the tyrosinase enzyme.
|Table 2: Enzymatic anti-tyrosinase activity of petroleum ether, ethyl acetate, methanol and water extracts of Streblus taxoides wood and Artocarpus chama stem at 20 µg/mL|
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From the results of the enzymatic investigation, several extracts from S. taxoides wood and A. chama stem showed antityrosinase activity. Thus, the investigations were extended to cellular experiments. First, the extracts were determined for the inhibition of melanogenesis on cultured melanocytes. Then, the effect from the extracts on cell viability was measured. The results indicated that all sample extracts were not considerable cytotoxic in B16-F1 melanoma cells. Cell viability was still >80% at the concentration 100 μg/mL except ethyl acetate extracts of A. chama and S. taxoides, cell viability was >80% at the concentrations of 5 and 50 μg/mL, respectively.
Intracellular antityrosinase activity and melanin content
The effect of intracellular antityrosinase activity and melanin content on B16F1 melanoma cells were determined. The extracts were prepared at 100 μg/mL except ethyl acetate extract of A. chama was prepared at 5 μg/mL and ethyl acetate extract of S. taxoides was prepared at 50 μg/mL which followed the concentrations of cell viability results. After 48 h incubation with all sample extracts, the supernatant were measured antityrosinase activity, the results showed that the extracts from A. chama and S. taxoides exhibited antityrosinase activity, especially ethyl acetate extract from both plants which were prepared at lower concentration [Figure 1]. The ethyl acetate extract of A. chama showed 64.41% ±1.27% inhibition at 5 μg/mL, while the ethyl acetate extract of S. taxoides showed 54.37% ±1.55% inhibition at 50 μg/mL. Due to the inhibition of tyrosinase enzyme would result to reduce melanin content which results obtained as shown in [Figure 1].
|Figure 1: Intracellular anti-tyrosinase activity and melanin content. Arbutin, kojic acid = positive control. A1, A2, A3 and A4 = petroleum ether, ethyl acetate, methanol, water extracts of Artocarpus chama stem, respectively. S1, S2, S3 and S4 = petroleum ether, ethyl acetate, methanol and water extracts of Streblus taxoides wood, respectively. A1, A3, A4, S1, S3, S4 = 100 µg/mL, A2 = 5 ug/ml, S2 = 50 µg/mL. Data are expressed as mean ± standard deviation from three independent experiments. *P < 0.05, **P < 0.01 and ***P < 0.001 indicate a significant difference from control group|
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Pigmentation inhibitory activity on zebrafish
Zebrafish was used for screening of pigmentation inhibitory effect by measuring the size of black spot on zebrafish. The results showed that at concentration 200 μg/mL [Figure 2]a extracts could inhibit pigmentation while petroleum ether extracts of A. chama stem could stimulate pigmentation. However, ethyl acetate extract of A. chama stem, ethyl acetate, and methanol extracts of S. taxoides wood showed toxicity to zebrafish by the detection of coagulation of the embryo, nondetachment of the tail, lack of somite formation, and lack of heartbeat. After decrease, the concentration to 50 μg/mL, ethyl acetate and methanol extracts of S. taxoides wood suppressed the pigmentation on zebrafish [Figure 2]b.
|Figure 2: Pigmentation inhibitory on Zebrafish. PTU (phenylthiourea) = positive control. A1, A2, A3 and A4 = petroleum ether, ethyl acetate, methanol, water extracts of Artocarpus chama stem, respectively. S1, S2, S3 and S4 = petroleum ether, ethyl acetate, methanol and water extracts of Streblus taxoides wood, respectively. (a) At concentration 200 µg/mL, (b) At concentration 50 µg/mL. Data are expressed as mean ± standard deviation from three independent experiments. *P < 0.05, **P < 0.01 and ***P < 0.001 indicate a significant difference from control group|
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Determination of antimicrobial activity
From the screening of antimicrobial activity, the ethanol extracts of 48 Moraceae plant samples displayed an inhibition zone against S. aureus, S. epidermidis, P. acnes, and MRSA as shown in the [Table 3]. Then, the extracts from selected Moraceae plants, S. taxoides and A. chama were determined of MIC and MBC (half-fold dilution; 15.625–2000 μg/ml as the results are shown in [Table 4]. The results showed that only the ethyl acetate extract of A. chama stem and S. taxoides wood against these microbes by exhibiting the MIC and MBC lower than 2000 μg/mL. Define what value, for each MIC and MBC, was considered as the extract had antimicrobial activity.
|Table 3: Anti-bacterial activity of 48 Moraceae plant samples at 2 mg/disc by agar disc diffusion method|
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|Table 4: Minimum inhibitory concentration and minimum bactericidal concentration of selected plant extracts (15.625-2000 µg/mL)|
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| Discussion|| |
A. chama stem and S. taxoides wood showed potential activity against tyrosinase enzyme both in enzymatic and intracellular assays. In addition, from in vivo study, they also showed the inhibition of melanogenesis by suppressing the pigmentation on zebrafish. Since, Moraceae is the most interesting plant family for biological study, especially, antityrosinase activity because the members of this family have been known to produce stilbenoids and flavonoids of various structural types.,, Structure-activity relationships with various flavonoids and stilbenes were demonstrated that 4-substituted resorcinol moiety was essential for showing the strong inhibitory activity against tyrosinase activity., The example of Moraceae plant, which showed the strong tyrosinase inhibition was Artocarpus lakoocha. Hence, water extract of the wood from this plant was used as positive control in the anti-tyrosinase assay. It exhibited the highest tyrosinase inhibition with >90%. The potent tyrosinase inhibition of A. lakoocha extract consorted with the previous reports as suggested that of oxyresveratrol (2,3', 4, 5'-tetrahydroxystilbene) seems to justify as the active component to show the high tyrosinase inhibition.,
S. taxoides wood and A. chama stem which demonstrated a capability to inhibit tyrosinase activity and they were described for the first. These plants could represent a potential source of new antityrosinase inhibitor. Further biological investigations on human melanocytes must be done to confirm these activities. Then, the toxicity on B16-F1 melanoma cells was evaluated with these samples. The results showed that they were non-toxic. However, only ethyl acetate extract of both plants showed the activity against bacteria. The isolation and the structural elucidation of the active constituents of these two selected plants will be useful to provide the lead compound in the development of skin-whitening agents.
| Conclusion|| |
S. taxoides and A. chama showed the potency of tyrosinase inhibition and reduction ability of melanin content without cytotoxicity. Then, they will be the are interested plants for further study of chemical constituents and biological activities, especially the antityrosinase activity of the isolated compound to find out the lead compound for whitening agent from natural product.
The authors would like to thank Department of Pharmacognosy and Pharmaceutical Botany and the Pharmaceutical Laboratory Service Center, Faculty of Pharmaceutical Sciences, Prince of Songkla University for the laboratory space and equipment.
Financial support and sponsorship
This study was financially supported by the Plant Genetic Conservation Project under The Royal Initiative of Her Royal Highness Princess Maha Chakri Sirindhorn (RSPG project) and the Prince of Songkla University (PSU) Ph.D. scholarship (PSU/95000201/2557).
Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2]
[Table 1], [Table 2], [Table 3], [Table 4]