Effect Of Sucrier Banana Peel Extracts On Inhibition Of Melanogenesis Through The ERK Signaling Pathway
Apr 25, 2023
Abstract
Hyperpigmentation is a type of pigmentary disorder induced by overexpression of melanin content activated severe esthetic problems such as melasma, freckle, ephelides, lentigo, and other forms on human skin. Several whitening agents have restricted use because of their side effects or stability such as kojic acid, ascorbic acid, and hydroquinone can act as cytotoxic substances associated with dermatitis and skin cancer. To find a safe substance, this study aimed to find the ability of several components in Sucrier banana peel (SBP) extracts to inhibit the melanogenesis process through the p38 signaling pathway in B16F10 mouse melanoma cells. Tyrosinase activity and the cellular melanin content were dose-dependent manners decreasing after SBP treatment. Furthermore, SBP decreased the expression of melanogenesis relate proteins as a microphthalmia-associated transcription factor (MITF) and tyrosinase protein after 24 hours of incubation with α-melanocyte stimulating hormones (MSH) stimulating. The findings demonstrated that SBP contained an effective agent for hyperpigmentation inhibitors through p38 signaling pathways without any effect on the ERK pathway, and subsequently down-regulate MITF expression and tyrosinase enzyme family production.
According to relevant studies,cistanche is a common herb that is known as "the miracle herb that prolongs life". Its main component is cistanoside, which has various effects such as antioxidant, anti-inflammatory, and immune function promotion. The mechanism between cistanche and skin whitening lies in the antioxidant effect of cistanche glycosides. Melanin in human skin is produced by the oxidation of tyrosine catalyzed by tyrosinase, and the oxidation reaction requires the participation of oxygen, so the oxygen-free radicals in the body become an important factor affecting melanin production. Cistanche contains cistanoside, which is an antioxidant and can reduce the generation of free radicals in the body, thus inhibiting melanin production.
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Keywords: sucrier banana peel; phenolic compounds; tyrosinase; microphthalmia-associated transcription factor; melanogenesis
Introduction
Melanin is produced within the melanosome which is an important organelle inside the melanocyte cells. Melanocyte is located in the basal layer of the skin epidermis. After the production process, melanin is the pigment responsible for skin, hair, and eye coloration which functions as a naturalized antioxidant, detoxification agent, and powerful-cation chelator and also can act as universal protection against UV damage [1]. Abnormal melanin production can cause pigmentary disorders such as hypopigmentation and hyperpigmentation. Hypopigmentation causes vitiligo, albinism, and abnormal hair problems while hyperpigmentation causes freckles, melasma, age spots, and post-inflammatory hyperpigmentation. Genetic predisposition, hormonal changes especially estrogen also others such as liver disease, tumor, cancer, malnutrition, or irregular function of the pituitary gland are the intrinsic factors including extrinsic factors like UV exposure, and toxic substances are the major cause of hyperpigmentation [2].
Tyrosinase is a rate-limiting step enzyme that targets the process of both stimulating and inhibiting the melanin production process. Melanin stimulation is used as a tanning agent or hair depigmentation treatment. Tyrosinase expression is controlled by microphthalmia-associated transcription factor (MITF) which is a melanocyte-specific transcription factor that controlled melanocyte differentiation, proliferation, and survival [3, 4]. The responsibility of MITF to melanogenesis is to increase tyrosinase expression by binding to DNA in the structure of homodimer or hetero dimer with MiT protein, these binding site involving E-Box and M-Box flanking thymidine nucleotide which stimulates the continuous steps to melanogenic enzyme production such as tyrosinase-related protein 1 (TRP-1), tyrosinase-related protein-2 (TRP-2) and dopachrome tautomerase. Regulation of MITF starts from signal transduction after α-MSH binds to the MC1R receptor which stimulates mitogen-activated protein kinase (MARKs) which is serine/threonine kinase and also includes extracellular signaling-regulated kinase (ERK) and also p38 as well. Several studies found that melanin synthesis is controlled by several signaling pathways such as phosphotidylineasital-3-kinase (PI3K/AKT) can be suppressed by natural substances which contain polyphenol group through the ERK signaling pathway of B16F10 mouse melanoma cells [5,6].
This study aimed to find the effect of melanogenesis inhibition from Sucrier banana peel (SBP) extracts. SBP may contain several types of polyphenolic compounds that act as tyrosinase inhibitors such as catechin, procyanidin, ferulic acid, and gallic acid which are detected by the expression of ERK signaling pathways that affects the production of melanogenic enzymes such as TRP-1 and TRP-2 [7-10].
Materials and methods
Chemicals and Materials
Ripped (7 days after harvesting) Sucrier banana (Musa spp.) peels from Kampangpetch Province, Thailand. Washed banana peels with water then sun-baked until totally dried, ground by blender, and extracted by soaking with 60% methanol for 24 hours (MW24), centrifuged with 95% ethanol for 10 minutes at 4 ℃(E4), boiling in DI water at 60 ℃ for 20 minutes (W60) and boiling in 95% methanol at 60 ℃ for 20 minutes (M60). The next step was to decrease the volume with a rotary evaporator at 40 ℃ and to dry by freeze dryer Christ Alpha- 4 LD plus.
Cell culture
B16F10 mouse melanoma cells were cultivated in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum at 37 ℃ in an incubated with 5% CO2. After 24 hours of incubation, then changed the medium to serum-free media with different concentrations of Sucrier banana peel (SBP) extracts with α-MSH stimulation. Then cells were harvested by trypsinization for the next experiments.
Cell viability assay
Cytotoxicity of SBP extracts was measured by MTT assay. The cells (5 ×10 3) were seeded to 96 well plates and cultivated in DMEM for 24 hours at 37℃ in an incubated with 5% CO2 then changed the medium to serum-free media with different concentrations of SBP extracts for 48 hours. After treatment, discarded the medium and washed twice with cold PBS, 100 ul of MTT solution (5mg/ml) was added into each well, incubated at 37 ℃ for 4 hours then measured by the ELISA reader at 420 nm.
Melanin content assay
Harvested cells were lysed in cold lysis buffer (20 mM sodium phosphate pH 6.8, 1% tritonX-100, 1mM PMSF, and 1 mM EDTA) and then centrifuged 1,200 rpm at 4 ℃ for 10 minutes to separate supernatant while melanin pellets were dissolved in 200 ul 1N NaOH for 60 minutes at 80 ℃. ELISA reader was used for measuring the absorbance at 415 nm. and calculated compared to the protein content from bicinchoninic acid (BCA) protein content assay (Pierce Biotechnology, Packford, IL, U.S.A).
Western blot analysis
Supernatant that was separated during the progression of cell lysis was taken to fractionate on SDS-PAGE and then transferred to Hybond enhanced chemiluminescence nitrocellulose membrane (Amersham, Little Chalfont, UK) and probe with primary antibody against tyrosinase (Santa Cruz, Biotechnology, Santa Cruz, CA, U.S.A), MITF ( Merck Millipore Darmstadt, Germany), ERK (Merck Millipore Darmstadt, Germany), P38 ( Merck Darmstadt Millipore, Germany), and monoclonal antibodies against B-Actin (AC-15, Sigma Aldrich). From then on, incubate the membrane with Horseradish peroxidase-conjugated secondary antibody complex visualization and quantified the signals by ImageJ software.
Statistical analysis All information was analyzed by mean ± standard deviation (SD). The one-way ANOVA test is used to measure the differences between each set of information. A P-value less than 0.05 was considered statistically significant.
Results
Phenolic compound
The objective of this experiment was to measure the efficacy of the solvents used for extraction and also a method that can extract phenolic compounds as much as possible. Most phenolic compounds dissolve very well when extract with high polar solutions such as water, methanol (MeOH), ethanol (EtOH), acetone, ethylacetate, propanol, acetic acid, or their mixture in various portions [11]. Figure 1A shows that W60 can extract phenolic compounds as much as 16.24 µg/ml followed by M60, E4, and MW24 with total phenol 13.89, 9.21, and 8.38 µg/ml respectively. Cytotoxicity of SBP was experimented with by MTT reduction assay which measured the reduction environment from the mitochondrial function of living cells by measuring the formazan formation to access cytotoxic effects. The result found that SBP at indicated concentrations (up to 500 µg/ml) with 48 hours incubation. Cytotoxicity shows no significant effect after SBP treatment (Fig. 1B).
The effects of SBP on enzyme activity and melanin content in B16F10 cells
To test melanogenesis suppression of SBP, the first step was to test the capability of mushroom tyrosinase enzyme inhibition compared to kojic acid which is a commercial whitening agent. SBP (MW24) has higher efficacy than Kojic acid and other extractions at the same concentration (100-500 µg/ml). MW24 has the highest percentage of tyrosinase inhibition (66.39-77.05%) while kojic acid only has a percentage of tyrosinase inhibition at 44.68-59.93% as shown in Fig. 1C. From then on, MW24 with the highest percentage of tyrosinase inhibition was selected for anti-melanogenesis measurement by melanin production from B16F10 cells. The melanin content of B16F10 cells was reduced after SBP (MW24) treatment (Fig. 2A). The amount of melanin content of B16F10 cells decrease significantly after stimulated with α-MSH and treated with SBP (MW24) for 24 hours in a dose-dependent manner as shown in Fig. 2B.
Effects of SBP (M24) on the protein expression levels of tyrosinase and MITF
Western blot analysis was used for tyrosinase and MITF activity by measuring protein expression levels after being treated with SBP (MW24) for 24 hours. Regulation of melanogenic enzymes such as TRP-1, and TRP-2 resulted from the transcriptional effect of MITF and tyrosinase protein level. The results show that SBP (MW24) was capable of dose-dependent down-regulate both tyrosinase and MITF protein expression as shown in Fig 3.
Effect of SBP (M24) on melanogenesis inhibition through mitogen-activated protein kinase (MAPKs) signaling pathway
In melanogenesis, the MAPKs kinase family especially ERK and p38 are related directly to this procedure [12]. The ERK signaling will down-regulate while p38 signaling will up-regulate during the occurrence of melanin production. However, phosphorylation of ERK and p38 will have a reverse procedure of melanin production. The results showed a clear effect after SBP (MW24) treatment with α-MSH activated by immunoblotting to the MAPK signaling pathway. SBP (MW24) slightly decreased p38 protein expression and up-regulate phosphorylation of p38. This result showed the way to inhibit the melanogenesis process through the p38 pathway without any effect on ERK but phosphorylation of ERK protein level slightly decreased after α-MSH and SBP (MW24) treatment as shown in Fig. 4.
Discussion
Sucrier banana peels are agricultural waste that has an abundance of many active ingredient substances, especially in the group of phenolic compounds and carotenoids which are the secondary metabolite that plants create for self-defense mechanisms [7]. These substances can be found in leaves, bark, fruits, peels, and seeds of almost all plants. Phenolic compounds and carotenoids are phytochemical substances that have good properties for human health. The substances comprise anti-inflammatory, anti-viral, analgesic, antioxidant, anti-carcinogen, etc [13]. In general, phenolic compounds can be well dissolved in high-polar solvents. For the experiment, the extraction of MW24 should be able to extract the phenolic compounds the most because of the procedure of extraction from the Folin-Cicualteu assay. Low polar compounds were extracted first then follow by high polar substances depending on the final mixing ratio of solvent in the procedure. The results show that W60 can extract phenolic compounds the most, possibly due to 60 degrees Celsius temperature which could be the appropriate temperature to extract phenolic compounds [11]. It can also be assumed that SBP may contain high polar substances which dissolve well in water. When comparing the amount of total phenolic compounds as shown in Fig. 1A, W60, M60, E4, and MW24 demonstrated the possibility of substance solubility, significantly.
After the total amount of phenolic compound was known, we can proceed to pinpoint the type and amount of flavonoid and carotenoid substances. We found SBP (MW24) extracts contain ferulic acid as high as 906.62 mg/100g and also found a small amount of lutein and β-carotene which were categorized in the carotenoid group at 1.23 and 2.37 mg/100g respectively. SBD (MW24) still has a high percentage of tyrosinase inhibition. Therefore, SBP (MW 24) was selected for further study.
From cytotoxicity testing, no significant cytotoxic was found from SBP although the amount of usage concentration was up to 500 µg/ml. possibly because the small amount of carotenoid within extraction has the capability of protecting cells. Substances in this group have antioxidant properties that can terminate poison through Cytochrome P-450 especially β-carotene which has a stimulating role in immune functions by interacting with Reaction Oxygen Species (ROS). This will support the effectiveness of a phenolic compound that has ROS scavenging effect property to decrease inflammatory cytokine [14]. So, SBD (MW24) not only has no toxic effect, it has the role of cell protection as well.
Our study also found that SBP (MW24) has the capability of inhibiting the melanogenesis of B16F10 cells. The expression of tyrosinase and MITF were reduced after SBP (MW24) treatment in both assessed with mushroom tyrosinase and Western blot analysis in a dose-dependent manner. This test indicated that SBP (MW24) can suppress the creation of melanin by down-regulate p38 signaling pathway and up-regulate phosphorylation of p38 which activate MITF protein degradation and then effect to decrease the expression of melanogenic enzyme family such as tyrosinase. Suppression of p38 decreases cAMP Response Binding Protein (CREB) functions which is an important transcription factor that works with PAX3 and SOX10 that attaches to M-BOX of MITF gene to activate MITF protein expression that affects the melanin production process and cells survival activity [15]. In general, activating ERK leads to phosphorylation of MITF at serine 73 which causes ubiquitination and eventually degradation. Most natural compounds can inhibit melanogenesis through the stimulation process of the ERK signaling pathway, but SBP (MW24) was not significantly influenced the ERK signaling pathway [16].
Many research found that phenolic compounds and carotenoids can inhibit the production of melanin pigments because beta-carotene has the capability of absorbing UVB photons while also has the property of the lipid-soluble antioxidant and phenolic compound can activate Nrf2, blocking ROS receptors, decrease inflammatory cytokine production and induce y-GSC expression which support to our study but in the different way of melanogenesis inhibition [17]. Other than that ferulic acid found in SBP (MW24) was the main substance within the group of phenolic compounds confirmed by several studies that it has the property of anti-melanogenesis because it can modulate vascular endothelial growth factor (VEGF) expression, induces nitric oxide synthase, act as tumor suppressor gene and also relate to the procedure of melanin pigments production. In SBP (MW24) solution, ferulic acid can increase effectiveness when mixed with carotenoid or other phospholipids because the water solubility, lipid solubility, and bioavailability will be increased resulting to improve melanogenesis inhibition of B16F10 cells [18]. The implication of all the results concludes that SBP (MW24) could reduce melanogenesis effectively.
Abbreviations
MITF: microphthalmia-associated transcription factor; α-MSH: α-melanocytes stimulating hormone; MC1R: melanocortin-1 receptor; TRP1: tyrosinase-related protein 1; TRP2: tyrosinase-related protein 2; MAPK: mitogen-activated protein kinase.
Acknowledgments
This work was supported by grants from the Royal Golden Jubilee Ph.D.: RGJPHD. Thailand. (PH.D./0017/2558).
Author Contributions
R.H., K.T., K.S., and U.P. conceived and designed the experiments; R.H. C.H.L. and M.C. performed the experiments; R.H. and M.C. and C.H.L analyzed the data; R.H. contributed reagents/materials/analysis tools; R.H. and C. H. L.wrote the paper.
Competing Interests
The authors have declared that no competing interest exists.
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