• Proceedings of the Plant Resources Society of Korea Conference
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• 2019.04a
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• pp.21-22
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• 2019

Melanin is a mixture of pigmented biopolymers synthesized by epidermal melanocytes that determine the skin, eye, and hair colors. Melanocytes produce two different kinds of melanin, eumelanin (dark brown/black insoluble pigments found in dark skin and dark hair and pheomelanin (lighter red/yellow). The biological role of melanin is to prevent skin damage by ultraviolet (UV) radiation. However, the overproduction or deficiency of melanin synthesis could lead to serious dermatological problems, which include melasma, melanoderma, lentigo, and vitiligo. Therefore, regulating melanin production is important to prevent the pigmentation disorders. Myanmar has a rich in natural resources. However, the chemical constituents of these natural resources in Myanmar have not been fully investigated. In the effort to search for compounds with anti-melanin deposition activity from Myanmar natural resources, five plants were collected in Myanmar. Extracts of these collected five plants were tested for anti-melanin deposition activity against a mouse melanoma cell line (B16-F10) induced with ${\alpha}$-melanocyte-stimulating hormone (${\alpha}$-MSH) and 3-isobutyl-1-methylxanthine (IBMX), and their anti-melanin deposition activities were compared with the positive control, arbutin. Among the tested extracts, the CHCl3 extracts of the Premna serratifolia (syn: P. integrifolia) wood showed anti-melanin deposition activities with IC50 values of $81.3{\mu}g/mL$. Hence, this study aims to identify secondary metabolites with anti-melanin deposition activity from P. serratifolia wood of Myanmar. P. serratifolia belongs to the Verbenaceae family and is widely distributed in near western sea coast from South Asia to South East Asia, which include India, Malaysia, Vietnam, Cambodia, and Sri Lanka. People in Tanintharyi region located in the southern part of Myanmar utilize the P. serratifolia, Sperethusa crenulata, Naringi crenulata, and Limonia acidissima as Thanaka, traditional cosmetics in Myanmar. Thanaka is applied in the form of paste onto skins to make it smooth and clear, as well as to prevent wrinkles, skin aging, excessive facial oil, pimples, blackheads, and whiteheads. However, the chemical constituents responsible for their cosmetic properties are yet to be identified. Moreover, the chemical constituents of P. serratifolia was almost uncharacterized. Investigation of the P. serratifolia chemical constituents is thus an attractive endeavor to discover new anti-melanin deposition active compounds. The investigation of the chemical constituents of the active CHCl3 extract of P. serratifolia led to isolation of four new lignoids, premnan A (1), premnan B (2), taungtangyiol C (3), and 7,9-dihydroxydolichanthin B (4), together with premnan C (5) (assumed to be an artifact), one natural newlignoid,(3R,4S)-4-(1,3-benzodioxol-5-ylcarbonyl)-3-[(R)-1-(1,3-benzo dioxol-5-yl)-1-hydroxy methyl]tetrahydro-2-furanone (6), and five known compounds (7-11)1,2). The structures of all isolated compounds were determined on the basis of their spectroscopic data and by comparison with the reported literatures. The absolute configurations of 1-3 and 5 were also determined by optical rotation and circular dichroism (CD) data analyses1). The anti-melanin deposition activities of all the isolated compounds were evaluated against B16-F10 cell line. 7,9-Dihydroxydolichanthin B (4) and ($2{\alpha},3{\alpha}$)-olean-12-en-28-oic acid (11) showed strong anti-melanin deposition activities with IC50 values of 18.4 and $11.2{\mu}M$, respectively, without cytotoxicity2). On the other hand, compounds 1-3, 5, and 7 showed melanogenesis enhancing activities1). To better understand their anti-melanin deposition mechanism, the effects of 4 and 11 on tyrosinase activities were investigated. The assay indicated that compounds 4 and 11 did not inhibit tyrosinase. Furthermore, we also examined the mRNA expression of microphthalmia-associated transcription factor (MITF), tyrosinase (TYR), tyrosinase-related protein-1 (TRP-1), and tyrosinase-related protein-2 (TRP-2). Compounds 4 and 11 down-regulated the expression of Tyr and Mitf mRNAs, respectively. Although the P. serratifolia wood has been used as traditional cosmetics in Myanmar for centuries, there are no scientific evidences to support its effectiveness as cosmetics. Investigation of the anti-melanin deposition activity of the chemical constituents of P. serratifolia thus provided insight into the effectiveness of the P. serratifolia wood as a cosmetic agent.

• Journal of Life Science
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• v.29 no.3
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• pp.279-286
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• 2019

This study evaluated the anti-oxidant and whitening effects of a 70% ethanol extract of the Sambucus sieboldiana var. pendula (Nakai) (SS). At $1,000{\mu}g/ml$ concentration, the electron donating ability of this SS extract was found to be 86.21% and the ABTS+ radical scavenging ability was 97.9%. In terms of whitening activity, the tyrosinase inhibitory effect of the extract was 37%, also at $1,000{\mu}g/ml$ concentration. To explore the extractefftoxicity to B16F10 melanoma cells, a 3-[4,5-dimethyl-thiazol-2-yl]-2,5-diphenyl-tetrazoliumbromide assay was performed. Results showed 90% or more cells remained viable at $100{\mu}g/ml$ concentration. A Western blot of the SS extract was used to measure microphthalmia-associated transcription factor (MITF), tyrosinase-related protein-1 (TRP-1), tyrosinase relate protein-2 (TRP-2), and the tyrosinase protein expression inhibitory effect at 25, 50, $100{\mu}g/ml$ concentrations; ${\beta}-actin$ was used as a positive control. Consequently, the MITF, TRP-1, TRP-2, and the tyrosinase protein expression inhibitory effect were seen to decrease by 34.5%, 45.6%, 58.4%, and 79.6%, respectively, at $100{\mu}g/ml$ concentration. These were also then measured by reverse transcription-polymerase chain reaction at 25, 50, $100{\mu}g/ml$ concentrations with GAPDH as a positive control. As a result, the SS extract was seen to decrease MITF, TRP-1, TRP-2, and the tyrosinase protein expression inhibitory effect by 85.4%, 67.5%, 85.2%, 67.1%, respectively at the $100{\mu}g/ml$ concentration. We therefore confirmed the possibility of Sambucus sieboldiana var. pendula (Nakai) extract as a whitening material.

• Journal of Physiology & Pathology in Korean Medicine
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• v.21 no.5
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• pp.1243-1249
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• 2007

The aim of this study was to investigate the effect of ethanol extract of Fagopyrum esculentum on the melanogenesis. To determine whether ethanol extract of Fagopyrum esculentum suppress melanin synthesis in cellular level, B16F10 melanoma cells were cultured in the presence of different concentrations of Fagopyrum esculentum ethanol extract. In the present study, we examined the effects of Fagopyrum esculentum ethanol extract on cell proliferation, melanin contents, tyrosinase activity, expression of melanogenic enzyme proteins including tyrosinase, tyrosinase-related protein 1 (TRP-1) and tyrosinase-related protein 2 (TRP-2). Cell proliferation was slightly increased by treatment with ethanol extract of Fagopyrum esculentum $(25-200 {\mu}g/m{\ell}).$ The ethanol extract of Fagopyrum esculentum effectively suppressed melanin contents at a dose of $100 {\mu}g/m{\ell}).$ It was observed that the color of cell pellets was totally whitened compared with the control. The ethanol extract of Fagopyrum esculentum inhibited tyrosinase activity, regulate melanin biosynthesis as the key enzyme in melanogenesis. Using western blot analysis, the ethanol extract of Fagopyrum esculentum dose-dependently decreased tyrosinase and TRP-1 protein levels, and tyrosinase and TRP-1 were detected in similar manner. ${\alpha}-MSH$ leads to a stimulation of melanin synthesis through increase of tyrosinase activity, melanin contents and cytoplasmic dendricity. In this study, ethanol extract of Fagopyrum esculentum down-regulated the ${\alpha}-MSH$-induced tyrosinase activity, melanin contents and cytoplasmic dendricity. Regarding protein levels of the melanogenic enzymes, the amounts of tyrosinase and TRP-1 was increased after incubation with a-MSH. The treatment of ethanol extract of Fagopyrum esculentum decreased the ${\alpha}-MSH$-induced expression levels of tyrosinase and TRP-1. These results suggest that the ethanol extract of Fagopyrum esculentum exerts its depigmenting effects through the suppression of tyrosinase, TRP-1 and cytoplasmic dendricity. And it may be a potent depigmetation agent in hyperpigmentation condition.

• Journal of the Society of Cosmetic Scientists of Korea
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• v.43 no.1
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• pp.1-10
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• 2017

Cimicifuge dahurica (C. dahurica), Coptis chinensis (C. chinensis) and Phellodendri amurense (P. amurense) extracts has an detoxification effect and Magnol obovata bark (M. obovata) extracts has an antibacterial effect in oriental medicine. This study investigated the possibility of the extract mixture as a functional cosmetic ingredient by mixing C. dahurica, C. chinensis, P. amurense and M. obovata extracts. MTT assay was carried out for toxicity test and DPPH/ABTS experiments for antioxidant effects of the extract mixture. Results show that the extract mixture was safer and antioxidant effects in a dose-dependent manner than single extract of the mixture. The mixture effectively inhibited NO (nitric oxide) production, which indicate good efficacy for anti-inflammation. The mixture also protected UVB-induced cell damage and increased type 1 pro-collagen synthesis in fibroblast. In addition, it's treatment inhibited the melanin synthesis and tyrosinase activity by lowering expression of MITF, tyrosinase and TRPs in B16F10 melanoma cell. These results suggest that medicinal herbal extract mixture may be useful as a functional ingredient for anti-aging and skin whitening cosmetic formula.

• Nutrition Research and Practice
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• v.8 no.3
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• pp.257-266
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• 2014

BACKGROUND/OBJECTIVE: Licorice has been shown to possess cancer chemopreventive effects. However, glycyrrhizin, a major component in licorice, was found to interfere with steroid metabolism and cause edema and hypertension. The roasting process of licorice modifies the chemical composition and converts glycyrrhizin to glycyrrhetinic acid. The purpose of this study was to examine the anti-carcinogenic effects of the ethanol extract of roasted licorice (EERL) and to identify the active compound in EERL. MATERIALS/METHODS: Ethanol and aqueous extracts of roasted and un-roasted licorice were prepared. The active fraction was separated from the methylene chloride (MC)-soluble fraction of EERL and the structure of the purified compound was determined by nuclear magnetic resonance spectroscopy. The anti-carcinogenic effects of licorice extracts and licochalcone A was evaluated using a MTT assay, Western blot, flow cytometry, and two-stage skin carcinogenesis model. RESULTS: EERL was determined to be more potent and efficacious than the ethanol extract of un-roasted licorice in inhibiting the growth of DU145 and MLL prostate cancer cells, as well as HT-29 colon cancer cells. The aqueous extracts of un-roasted and roasted licorice showed minimal effects on cell growth. EERL potently inhibited growth of MCF-7 and MDA-MB-231 breast, B16-F10 melanoma, and A375 and A2058 skin cancer cells, whereas EERL slightly stimulated the growth of normal IEC-6 intestinal epithelial cells and CCD118SK fibroblasts. The MC-soluble fraction was more efficacious than EERL in inhibiting DU145 cell growth. Licochalcone A was isolated from the MC fraction and identified as the active compound of EERL. Both EERL and licochalcone A induced apoptosis of DU145 cells. EERL potently inhibited chemically-induced skin papilloma formation in mice. CONCLUSIONS: Non-polar compounds in EERL exert potent anti-carcinogenic effects, and that roasted rather than un-roasted licorice should be favored as a cancer preventive agent, whether being used as an additive to food or medicine preparations.

• Journal of the Society of Cosmetic Scientists of Korea
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• v.38 no.4
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• pp.277-287
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• 2012

To develop new therapeutic materials to prevent hair loss and enhance hair growth, we developed a medicinal herbal complex extract (MHCE) using 23 herbs traditionally used in oriental medicine. Medicinal Herbal complex extract was consist of Angelica gigas Nakai, Psoralea corylifolia Linne, Biota orientalis Endlicher, and Eclipta prostrata Linne, Rehmannia glutinosa Liboschitz var. purpurea Makino, Ligustrum lucidum Aiton, Polygonum multiflorum Thunberg, and Sesamum indicum Linne, Sophora angustifolia Sieboldet Zuccarini, Angelica dahurica Benthamet Hooker, and Leonurus sibiricus Linne, Salvia miltiorrhiza Bunge, Prunus persica Batsch, Commiphora molmol Engler, Chrysanthemum indicum Linne, Boswellia carterii Birdwood, Panax ginseng C. A. Meyer, Cnidium officinale Makino, Albizia julibrissin Durazzini, and Corydalis ternata Nakai that have traditionally been used for treating hair loss, preventing gray hair, anti-inflammation, and blood circulation in oriental medicine. In addition, we examined the hair growth effect of MHCE in vitro and in vivo. In vitro, we evaluated the effects of MHCE on cultured HFDPC, HaCaT cells, and murine embryonal fibroblasts (NIH3T3 cells). Also, we evaluated the ability of MHCE to prevent gray hair on murine melanoma cells (B16F1 cells). The hair growth-promoting effect of MHCE in vitro was also observed in vivo using C57BL/6 mice. Our results showed that MHCE significantly increased the proliferation of HFDPC (175 % proliferation at $50{\mu}g/mL$), HaCaT cells (133 % proliferation at $20{\mu}g/mL$), and NIH3T3 cells (120 % proliferation at $50{\mu}g/mL$). MHCE also showed consistent melanogenesis in B16F1 cells (154 % melanin synthesis at $50{\mu}g/mL$). Moreover, MHCE showed potential for hair growth stimulation in C57BL/6 mice experiments (98 % hair growth area on 4 weeks). These results indicate that MHCE may be a good candidate for promotion of hair growth.

• IMMUNE NETWORK
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• v.3 no.1
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• pp.53-60
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• 2003

Background: The role of macrophages in tumor angiogenesis is known to be the production of angiogenic cytokines and growth factors including TNF-${\alpha}$. Recently, macrophage also can produce the INF-${\gamma}$ that is being studied to be involved in angiogenic inhibition. Thus, the importance of macrophages in tumor angiogenesis is might being an angiogenic switch. Thus, the hypothesis tested here is that TNF-${\alpha}$ can modulate the INF-${\gamma}$ production in the macrophages from tumor environment as a part of tumor angiogenic switch. Methods: Macrophages in tumor environment were obtained from the peritoneal cavity of C57BL/6 mice injected with B16F10 melanoma cell line for 6 or 11 days. $Mac1^+$-macrophages were purified using magnetic bead ($MACs^{TM}$; Milteny Biotech, Germany) and cultured with various concentrations of TNF-${\alpha}$ for various time points at $37^{\circ}C$. The supernatants were analyzed for IFN-${\gamma}$ or VEGF by ELISA kit (Endogen, Woburn, MA). Results: Residential macrophages from the peritoneal cavity did not respond to LPS or TNF-${\alpha}$ to produce INF-${\gamma}$. However, the cells from tumor environment produced IFN-${\gamma}$ as well as VEGF and upregulated by the addition of LPS or TNF-${\alpha}$. RT-PCR analysis revealed the external TNF-${\alpha}$-induced IFN-${\gamma}$ gene expression in the macrophages from tumor environment. Conclusion: The overall data suggest that the macrophages in tumor environment might have an important role not only in angiogenic signal but also in anti-angiogenic signal by producing related cytokines. And TNF-${\alpha}$ might be a key cytokine in tumor angiogenic switch.

• Korean Journal of Plant Resources
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• v.34 no.1
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• pp.37-43
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• 2021

Angelica gigas Nakai (AGN) is a perennial plant belonging to the family Apiaceae. Its root has been utilized as a traditional medicine especially in Korea. This study was carried out to evaluate the potential use of extracts from AGN root parts as a cosmetic material. The dried AGN roots are divided into body (B), thick root (TkR), medium root(MR) and thin root (TnR) according to their diameter before cutting into medicine. B, TkR and MR of AGN are combined and used as medicinal herbs (MH). The extracts from AGN each root part (B, TkR, MR, TnR, MH) were used to test the effect on cell viability using MTS assay and to examine inhibitory effect on melanin accumulation in B16F10 melanoma cells. All extracts (50 - 200 ㎍/mL) from the each root part did not affect the cell viability. And inhibitory effect of all root extracts (200 ㎍/mL) on melanin accumulation was 12-19%. Especially, TnR showed similar inhibitory effect on melanin accumulation to MH. In addition, DPPH and ABTS free radical scavenging activity were higher in the TnR extract compared to MH. This study showed that the TnR extract exhibit high inhibitory effect on melanin accumulation and antioxidant activity compared to MH, suggesting that TnR extract has potential as a cosmetic ingredient.

• Proceedings of the Korean Society of Applied Pharmacology
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• 2007.11a
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• pp.79-92
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• 2007

Oxidative stress have known to be a risk factor for the degenerative processes and closely related to a lot of diseases. It is well established that antioxidants are good in protection and therapeutic means against oxidative damage. There is increasing interest in natural antioxidants and many natural antioxidants have been found and utilized as the possible protection for various diseases and skin aging. We have screened natural antioxidant agents for cosmeceuticals, nutraceuticals, and drugs as therapeutic and preventive means against oxidative stress, and have developed a number of novel antioxidants from various natural sources. A novel melanin synthesis inhibitor, Melanocin A, isolated from the metabolite of a fungal strain Eupenicillium shearii F80695 inhibited mushroom tyrosinase and melanin biosynthesis of B16 melanoma cells with $IC_{50}$ value of 9.0 nM and MIC value of $0.9\;{\mu}M$, respectively. Melanocin A also exhibited potent antioxidant activity by scavenging of DPPH and superoxide anion radicals. UV was found to increase the level of hydrogen peroxides and other reactive oxygen species (ROS) in skin tissues. This increase in ROS may not only alter the structure and function of many genes and proteins directly but may also modulate their expressions through signal transduction pathways and, ultimately, lead to skin damage. We investigated the effect of Melanocin A on UV-induced premature skin aging. Firstly, the effect of Melanocin A on UV-induced matrix metalloproteinase (MMP)-9 expression in an immortalized human keratinocyte cell line, HaCaT in vitro was investigated. Acute UV irradiation induced MMP-9 expression at both the mRNA and protein levels and Melanocin A suppressed this expression in a dose-dependent manner. We then investigated UV-induced skin changes in hairless mice in vivo by Melanocin A. Chronic exposure of hairless mouse dorsal skin to UV increased skin thickness and induced wrinkle formation and the gelatinase activities of MMP-2 and MMP-9. Moreover, Melanocin A significantly suppressed UV-induced morphologic skin changes and MMP-2 and MMP-9 expression. These results show that Melanocin A can prevent the harmful effects of UV that lead to skin aging. Therefore, we suggest that Melanocin A should be viewed as a potential therapeutic agent for preventing and/or treating premature skin aging. Terrein is a bioactive fungal metabolite isolated from Penicillium species. Terrein has a relatively simple structure and can be easily synthesized. However, the biologic effects of terrein are comparatively unknown. We found for the first time that terrein potently inhibit melanin production in melanocytes and has a strong hypopigmentary effect in a spontaneously immortalized mouse melanocyte cell line, Mel-Ab. Treatment of Mel-Ab cells with terrein (10-100 mM) for 4 days significantly reduced melanin levels in a dose-dependent manner. In addition, terrein at the same concentration also reduced tyrosinase activity. We then investigated whether terrein influences the extracellular signal-regulated protein kinase (ERK) pathway and the expression of microphthalmia-associated transcription factor (MITF), which is required for tyrosinase expression. Terrein was found to induce sustained ERK activation and MITF down-regulation, and luciferase assays showed that terrein inhibits MITF promoter activity in a dose-dependent manner. To elucidate the correlation between ERK pathway activation and a decreased MITF transcriptional level, PD98059, a specific inhibitor of the ERK pathway, was applied before terrain treatment and found to abrogate the terrein-induced MITF attenuation. Terrein also reduced the tyrosinase protein level for at least 72 h. These results suggest that terrain reduces melanin synthesis by reducing tyrosinase production via ERK activation, and that this is followed by MITF down-regulation.