Inhibiting Melanin Biosynthesis of Indonesian Medicinal Plant Extracts

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Title Inhibiting Melanin Biosynthesis of Indonesian Medicinal Plant_{Extracts( 本文(Fulltext) )}

Author(s) Andriyana Setyawati

Report No.(Doctoral Degree) 博士(農学) 甲第686号 Issue Date 2018-03-13 Type 博士論文 Version ETD URL http://hdl.handle.net/20.500.12099/75244 ※この資料の著作権は、各資料の著者・学協会・出版社等に帰属します。

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Inhibiting Melanin Biosynthesis of

Indonesian Medicinal Plant Extracts

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΢΢ϱχϋεΠ༂༽৪෼பड़෼͹ϟϧωϱਫ਼߻੔૏֒ͶؖͤΖݜڂ

)

2017

The United Graduate School of Agricultural Science, Gifu University Science of Biological Resources

(Gifu University)

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Inhibiting Melanin Biosynthesis of

Indonesian Medicinal Plant Extracts

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΢΢ϱχϋεΠ༂༽৪෼பड़෼͹ϟϧωϱਫ਼߻੔૏֒ͶؖͤΖݜڂ

)

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Summary

The potential use of traditional herbal medicines for development of new skin-care cosmetics has been encouraged. Many people now are attracted to the traditional herbal formulation to avoid unwanted side-effect. The pigment melanin in human skin is a product of the defense mechanism against ultraviolet light of the sun. The production of abnormal pigmentation, such as malesma, spots, post inflammatory and other forms of melanin hyperpigmentation can be a serious aesthetic problem. Melanin formation is enzymatically regulated in a human being. Melanin is the pigment in the human skin that is synthesized by tyrosinase from L-tyrosine. Commonly, skin whitening agents and depigmentation agents are commercially available such as kojic acid, arbutin, catechins, azelaic acid and so on, but some adverse effects of these compounds are irreversible. However, there are the natural products that provide a unique structural diversity and large number of the unexplored source of new drugs.

Indonesia which is straddling two major oceans, two great continents, across more than 17,000 islands and tropical climate has abundant medicinal plants and the rich source of the bioactive chemical. Moreover, it has created one of the most diverse ecosystems in the world. Indonesian cultures have been making use of the rich medicine by using the rich ecological system for a great variety of purposes. One of the traditional medication in Indonesia is called “jamu”. They have been adopting health care, beauty care, and physical maintenance regimens. A lot of medicinal plants are traditionally used as skin whitening agents. Then our research is focused on exploring the potential of

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ii Indonesian medicinal plants and its ingredients as whitening agents against skin hyperpigmentation.

This thesis is divided into three chapters. The first chapter reviewed the potential of medicinal plants as anti melanogenesis. Furthermore, it reviewed several papers for reporting the traditional medicinal plant extractives and the effective novel ingredients as anti-melanogenesis. Through this review, it was provided the list of potential medicinal plant which is possessing the potential of whitening agents. Chapter two and three provided the chemical and biological investigation on novel bioactive natural products as whitening agents.

The chapter one showed a number of the effects and ingredients in the medicinal plants which are distributed in the world and still unknown. It was mentioned that further investigation such as the toxicity assay, allergy assay, and other biological assay must be conducted to clarify their safety and clinical advantage to the human skin. According to the list of potential of medicinal plant as anti melanogenesis in the review, the investigation of novel bioactive for skin whitening agents is a challenge. Base on that reason, the exploration of active compound for whitening agents from the natural product are encouraged.

Furthermore, several potential medicinal plants from Indonesia were considering to be object of research on this project. Chapter two and three are provide the chemical and biological investigation novel bioactive natural product. The chemical investigation such as extraction of sample, isolation, purification and structure elucidation of active compound. Moreover, biological investigation performed by screening the extract and active compound into the melanoma cells.

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iii In chapter two, the methanol extract of Syzygium polyanthum leaf, an Indonesian medicinal plant, was evaluated for the inhibition of melanogenesis in B16 melanoma cells. Three novel compounds 1-(2,3,5-trihydroxy-4-methylphenyl)hexane-1-one (1), 1-(2,3,5-trihydroxy methylphenyl)octane-1-one (2), and (4E)-1-(2,3,5-trihydroxy-4-methylphenyl)decan-1-one (3) and one known compound 1-(2,3,5-trihydroxy-4-methylphenyl)decan-1-one (4) were isolated from the methanol extract. S. polyanthum leaf methanol extract decreased extracellular melanin formation more than 80% with high cell viability at 75–200 μg/ml. Compounds 1-4 were found to inhibit melanogenesis and tyrosinase activity. S. polyanthum leaf methanol extract and the isolated compounds were analyzed for their activity against tyrosinase. Compound 3 potently inhibited tyrosinase activity ((IC50; 83.98μM), particularly when L-tyrosine was the substrate. Compounds 2 and 3 significantly diminished extracellular melanin formation in B16 melanoma cells (>80%), with high cell viability.

Regarding to the results of chapter two, a novel bioactivity of anti melanogenesis and tyrosinase inhibitory were found by screening the activity compounds. In addition, further exploration of potential medical plants from Indonesia were continued by change the plant to the Zingiber purpureum rhizome, which is discussed in the chapter three.

In chapter three, the methanol extract of Zingiber purpureum rhizome was evaluated for the inhibition of melanogenesis and tyrosinase activity. Six known compounds A-F; (E)-4-(3,4-dimethoxyphenyl)-but-3-en-1-yl acetate (A), trans-3-(3’, 4’- Dimethoxyphenyl)-4-[(E)-3”’, 4”’-Dimethoxystryryl]Cyclohex-1-ene (B), cis-3-(3’, 4’- Dimethoxyphenyl)-4-[(E)-3”’,

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4”’-Dimethoxystryryl]Cyclohex-1-iv ene (C), (E)-4-(3,4-dimethoxyphenyl) but-3-en-1-ol (D), (E)-1-(3,4-dimethoxyphenyl)but-1-ene; (E), and 3,4-dimethoxylbenzaldehyde (F) were isolated from this plant. We found that Z. purpureum rhizome methanol extracts decrease extracellular melanin with low cytotoxicity in a dose-dependent manner. Furthermore, the result showed that compound B and C potently reduced the extracellular melanogenesis more than 50% at 32 PM without inhibiting tyrosinase with high cell viability. It is suggesting that they may inhibit the transportation of protein or release the melanosome or both, as well as inhibit melanin biosynthesis in B16 melanoma cells. While compounds A, D, E, and F showed no effect on the same level of compounds B and C.

In conclusion, the novel and known compounds from S. polyanthum leaf and Z. purpureum rhizome methanol extracts have potency for melanogenesis inhibitory activity. Novel compounds 1, 2, 3 and a known compound 4 were isolated from S. polyanthum leaf methanol extract. Compound 3 was most effective compound in the isolated compounds from S. polyanthum to inhibit melanogenesis in B16 melanoma cell, and it inhibited tyrosinase activity using L-tyrosine as the substrate. The known compounds A, B, C, D, E, and F were isolated from Z. purpureum rhizome methanol extract. Compounds B and C were found to suppress melanogenesis in the cells without tyrosinase inhibitory activity. These results suggested the potency of the plants and the ingredients as whitening agents.

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v ָҒ ࿨ช གྷࢭ ৿͢͏ηΫϱίΠԿ঻඾͹֋൅͹ͪΌ͹ఽ౹ద͵ਫ਼༂͗੷ۅదͶཤ ༽͠Ηͱ͘ͱ͏Ζɽ๮Ή͚͢͵͏෯ࡠ༽Νඈ͜ΖͪΌͶɾఽ౹ద͵ਫ਼༂͗ ଡ͚͹ਕʓͶ޹ΉΗͱ͏Ζɽਕؔ͹ൿේ͹৯ોͲ͍Ζϟϧωϱͺɾଢཇ͹ ࢷ֐તͶΓΖξϟʖζΝ๹ޜͤΖͺͪΔ͘Ν΍ͯਫ਼ର಼෾ࢢͲ͍Ζɽߜ൙ɾ ൙఼ɾԎ঳͵ʹͶΓΖҡ৙͵৯ો௞஥ͺɾ਄ࠃ͵ඔ༲৏͹໲ୌͳ͵Ζɽϟ ϧωϱܙ੔ͺਫ਼ର಼Ͳ߮ો͹ͺͪΔ͘ͶΓΕ੏ޜ͠Ηͱ͏Ζɽϟϧωϱͺɾ L-οϫεϱ͖Δ͹οϫεψʖκͶΓͮͱ߻੔͠ΗΖɽҲൢͶɾαΤζ࢐ɾ ΠϩϔοϱɾΩτΫϱɾΠκϧ΢ϱ࢐͵ʹ͹ൿේඔപࡐ͕Γ;୦৯ࡐ͗ࢤ ൤͠Ηͱ͏Ζ͗ɾ͞ΗΔ͹Կ߻෼ͺ͏͚Δ͖͹༙֒ࡠ༽Ν΍ͯɽ͖͢͢ɾ ಢಝ͹ߑଆదଡ༹੓Ν΍ͬɾາͫݜڂ͠Ηͱ͏͵͏ళષ༟པ෼࣯͹஦Ͷͺɾ ৿༂͹ϨʖχԿ߻෼Ͷ͵ΕಚΖՆ೵੓ΝඁΌͱ͏Ζ΍͹͗ଚࡑͤΖɽ ΢ϱχϋεΠͺ೦ଵـޫͲ 2 ͯ͹୉༺ɾ2 ୉୉཰ɾ17,000 Ґ৏͹ ౣʓ͖Δ͵Ζɽ͠ΔͶຌࠅͺ੊ֆ͹஦Ͳ΍࠹΍ଡ༹͵ਫ਼ସܧΝ੔͢ͱ͕Εɾ ਫ਼෼׈੓Կ߻෼͹๝෍͵ڛڇݱͳ͵ͮͱ͏Ζɽ΢ϱχϋεΠ͹ชԿͺ๝͖ ͵ਫ਼ସܧΝཤ༽͢ͱɾ͠Ή͡Ή͵໪ద͹ͪΌͶࣙષ༟པ͹ਫ਼༂Νਫ਼Ίड़͢ ͱͪ͘ɽ΢ϱχϋεΠ͹ఽ౹ద͵ਫ਼༂͹ 1 ͯ͗ʰζϡϞʱͳݼͻΗͱ͏Ζɽ

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vi ͨΗΔͺɾϖϩηίΠɾϑϣʖτΡʖίΠɾ͕Γ;݊߃ң࣍ྏ๑ͳ͢ͱ༽ ͏ΔΗͱ͏Ζɽఽ౹దͶൿේͶ͕͜Ζඔപࡐͳ͢ͱ༂༽৪෼͹ଡ͚͗࢘༽ ͠Ηͱ͏Ζɽͨ͞ͲຌݜڂͲͺ΢ϱχϋεΠ͹༂༽৪෼ͳͨ͹੔෾Ͷ஭໪ ͢ɾൿේ৯ો௞஥ཊ੏ࡐ͹୵ࡩΝߨ͑͞ͳͳͪ͢ɽ ͞͹࿨ชͺ 3 ͯ͹হͲߑ੔͠Ηͱ͏Ζɽ࠹ॵ͹হͲͺɾϟϧωϱਫ਼੔૏ ֒༂ͳ͢ͱ͹৪෼͹Ն೵੓Ͷͯ͏ͱ͹֕གྷΝफ़΄ͪɽ͠ΔͶɾఽ౹ద͵ҫ༂৪෼ பड़෼͕Γ;༙ް͵৿و੔෾Νϟϧωϱਫ਼੔૏֒ࡐͳ͢ͱๅࠄͪ͢͏͚͖ͯ͹࿨ ชΝखΕ৏ͪ͝ɽ͞͹ϪϑϣʖΝ௪ͣͱɾඔപࡐͳ͢ͱ͹Ն೵੓Ν΍ͯ༂༽৪෼ ͹ϨηφΝࡠ੔ͪ͢ɽ୊ 2 হ͕Γ;୊ 3 হͺɾϟϧωϱਫ਼੔Νཊ੏ͤΖ৿͢͏ళ ષ෼༟པ੔෾͹୵ࡩΝߨͮͪɽ ୊ 1 হͲͺɾ੊ֆͶ෾ා͢ͱ͏Ζາஎ͹༂༽৪෼͹ଡ͚͹׈੓ͳ੔෾Ν ࣖͪ͢ɽϐφ͹ൿේ΃͹҈સ੓ͳྡজ৏͹ཤ఼Ν໎Δ͖ͶͤΖͪΌͶɾ͠Δ͵Ζ ಡ੓෾ੵɾΠϪϩάʖ෾ੵɾͨ͹ଠ͹ਫ਼෼ָద෾ੵ͹චགྷ੓Νफ़΄ͪɽϪϑϣʖ ஦͹ϟϧωϱਫ਼੔૏֒༂ͳ͢ͱ͹Ն೵੓Ν΍ͯ༂༽৪෼͹ϨηφͶΓΗͻɾൿේ ͹ඔപࡐ͹ͪΌ͹৿و׈੓෼࣯͹௒ࠬ͗՟ୌͳ͵ͮͱ͏Ζɽ͞͹ͪΌళષ෼͖Δ ͹ඔപࡐͶ͵ΕಚΖ׈੓Կ߻෼͹୵ࡩ͗๮ΉΗͱ͏Ζɽͨ͞Ͳϟϧωϱਫ਼੔૏֒ ׈੓͗غଶͲ͘Ζ΢ϱχϋεΠࢊ༂༽৪෼Ν͞͹ݜڂ͹ଲেͳͪ͢ɽ

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vii ୊ 2 হͲͺɾ΢ϱχϋεΠࢊ༂༽৪෼Ͳ͍Ζ S. polyanthum ཁ͹ϟνόʖ ϩபड़෼ΝɾB16 ϟϧόʖϜࡋ๖Ͷ͕͜Ζϟϧωϱܙ੔૏֒Ͷͯ͏ͱ඲Ճͪ͢ɽ 3 ͯ ͹ ৿ و Կ ߻ ෼ trihydroxy-4-methylphenyl)hexane-1-one (1), 1-(2,3,5-trihydroxy methylphenyl)octane-1-one (2), and (4E)-1-(2,3,5-1-(2,3,5-trihydroxy-4- (4E)-methylphenyl)decan-1-one (3) and one known compound 1-(2,3,5-trihydroxy-4-methylphenyl)decan-1-one (4)Νϟνόʖϩபड़෼͖Δୱ཯ͪ͢ɽ S. polyanthum ཁ ϟνόʖϩபड़෼ͺ 75-200 μg/ml Ͳ߶͏ࡋ๖ਫ਼ଚིͲࡋ๖֐ϟϧωϱܙ੔Ν 80ˍҐ৏ݰঙͦͪ͠ɽԿ߻෼ 1-4 ͺɾϟϧωϱܙ੔͕Γ;οϫεψʖκ׈੓Ν૏ ֒ͤΖ͞ͳΝݡड़ͪ͢ɽS.polyanthum ཁϟνόʖϩபड़෼͕Γ;ୱ཯ͪ͢੔෾͹ οϫεψʖκ׈੓ࢾݩΝߨͮͪɽԿ߻෼ 3 ͺɾಝͶ L-οϫεϱ͗خ࣯Ͳ͍Ζ৖ ߻ɾک͏οϫεψʖκ૏֒׈੓Νࣖͪ͢(IC50; 83.98μM)ɽԿ߻෼ 2 ͕Γ; 3 ͺɾ B16 ϟϧόʖϜࡋ๖Ͷ͕͏ͱ߶͏ࡋ๖ਫ਼ଚིͲ(> 80%) ࡋ๖֐ϟϧωϱܙ੔Ν༙ қͶݰঙͦͪ͠ɽ ୊ 3 হͲͺɾZingiber purpureum ࠞܬ͹ϟνόʖϩபड़෼ΝɾB16 ϟϧό ʖϜࡋ๖Ͷ͕͜Ζϟϧωϱܙ੔͕Γ;οϫεψʖκ׈੓͹૏֒Ͷͯ͏ͱ඲Ճͪ͢ɽ 6 ͯ͹عஎԿ߻෼ (E)-4-(3,4-dimethoxyphenyl)-but-3-en-1-yl acetate (A);

trans-3-(3’, 4’- Dimethoxyphenyl)-4-[(E)-3”’, 4”’-Dimethoxystryryl]Cyclohex-1-ene (B); cis-3-(3’, 4’- Dimethoxyphenyl)-4-[(E)-3”’, 4”’-Dimethoxystryryl]Cyclohex-1-ene (C); (E)-4-(3,4-dimethoxyphenyl) but-3-en-1-ol (D); (E)-1-(3,4-dimethoxyphenyl)but-1-ene (E); (F) 3,4-dimethoxylbenzaldehyde Νຌ৪෼͖Δ

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viii ୱ཯ಋఈͪ͢ɽZ. purpureum ࠞܬϟνόʖϩபड़෼ͺɾఁ͏ࡋ๖উ֒੓Ͳೳౕғ ଚదͶࡋ๖֐ϟϧωϱΝݰঙͦͪ͠ɽ͠ΔͶɾԿ߻෼ B ͕Γ; C ͗߶͏ࡋ๖ਫ਼ ଚིͲοϫεψʖκΝ૏֒ͤΖ͞ͳ͵͚ 32PM Ͳ 50ˍҐ৏ࡋ๖֐ϟϧωϱਫ਼੔ ׈੓ΝఁԾͦ͠Ζ͞ͳΝࣖͪ͢ɽ͞ΗΔ͹Կ߻෼ͺ B16 ϟϧόʖϜࡋ๖Ͷ͕͜ Ζϟϧωϱਫ਼߻੔Ν૏֒ͤΖͫ͜Ͳ͵͚ɾνϱϏέ࣯༎ૻΏϟϧόλʖϞ͹๎ड़ Ν૏֒͢ͱ͏ΖՆ೵੓͗ߡ͓ΔΗΖɽҲ๏Կ߻෼ AɾDɾEɾF ͺԿ߻෼ C Ώ B Άʹ͹ϟϧωϱਫ਼੔૏֒׈੓ͺࣖ͠͵͖ͮͪɽ ݃࿨ͳ͢ͱɾS.polyanthum ཁ͕Γ; Z.purpureum ࠞܬϟνόʖϩபड़෼Ͷ ༟པͤΖ৿و͕Γ;عஎ͹Կ߻෼ͺɾϟϧωϱਫ਼੔૏֒׈੓Ν༙ͤΖ͞ͳ͗໎Δ ͖ͳ͵ͮͪɽ ৿وԿ߻෼ 1, 2, 3 ͕Γ;عஎԿ߻෼ 4 ΝɾS.polyanthum ཁϟνόʖ ϩபड़෼͖Δୱ཯ͪ͢ɽ Կ߻෼ 3 ͺɾ࠹΍߶͏ϟϧωϱਫ਼੔૏֒׈੓Νࣖ͢ɾ L-οϫεϱΝخ࣯ͳͪ͢οϫεψʖκ׈੓Ν૏֒ͪ͢ɽԿ߻෼ AɾBɾCɾDɾE ͕Γ; F ΝɾZ. purpureum ࠞܬϟνόʖϩபड़෼͖Δୱ཯ͪ͢ɽ Կ߻෼ B ͕Γ ; C ͺοϫεψʖκΝ૏֒ͦͥͶ߶͏ϟϧωϱਫ਼੔૏֒׈੓Νࣖ͞ͳ͗ݡड़͠ Ηͪɽ͞ΗΔ͹݃Վͺຌ༂༽৪෼͕Γ;ͨΗΔ͹੔෾͗ඔപࡐͳ͢ͱ͹Ն೵੓Ν ඁΌͱ͏Ζ͞ͳΝࣖࠨͪ͢ɽ

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List of Figure

Figure 1. Melanin biosynthetic pathway ... 14 Figure 2. Effect of some quercetin derivatives on the expression of Tyrosinase, TRP-1, TRP-2, MITF, p-p38MAPK, and p38MAPK in B16 melanoma cells. .... 16 Figure 3. Syzygium polyanthum (Weight) Walp ... 35 Figure 4. Structure of isolated compound 1–4 of S. polyanthum leaf ... 41 Figure 5. HMBC correlation of compound 1-4... 44 Figure 6. Melanin production and cell viability of B16 melanoma cells after treating with methanol extracts of S. polyanthum. ... 45 Figure 7. Melanin production and cell viability of B16 melanoma cells in the Fraction 5 after treating with methanol extracts of S. polyanthum. ... 46 Figure 8. Melanin production and cell viability of B16 melanoma cells after treating with compound 1-4 of S. polyanthum methanol extracts... 46 Figure 9. Zinger purpureum rhizome ... 53 Figure 10. Melanin production and cell viability of B16 melanoma cells after treating with methanol extracts of Z. purpureum. ... 59 Figure 11. Structure of isolated compound A–F of Z. purpureum rhizome

methanol extracts ... 61 Figure 12. Melanin production and cell viability of B16 melanoma cells after treating with compound A-F of Z. purpureum rhizome methanol extracts ... 62

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List of Scheme

Scheme 1. Mechanism of skin pigmentation ... 12

Scheme 2. Typical hair follicle ... 13

Scheme 3. Transcriptional regulation of expression of melanogenic enzyme ... 15

Scheme 4. Chemical structure of some whitening agent cosmetic ... 15

Scheme 5. Chemical structure possessing future possibility as cosmetic agent ... 21

Scheme 6. Chemical structure of potential whitening agent discovery from folk medicinal plant. ... 24

Scheme 7. Chemical structure of folk medicinal from Bengkoang (Pachyrhizus erosus) for whitening agents and commercially used as cosmetic product ... 25

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List of Table

Table 1. Some skin-whitening agents know so far ... 18 Table 2. Review of published literature relating to selected medical plants use in Melanogenesis ... 28 Table 3. Tyrosinase inhibitory activity of MeOH extract and fractions of S.

polyanthum ... 47 Table 4. Inhibitory activities of compounds isolated from S. polyanthum ... 48 Table 5. Inhibitory activities of compounds isolated from Z. purpureum ... 63

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1

Preface

Skin colour is one of the most discernible ways in which human vary. This colour is largely determined by epidermal melanin, its principal function has traditionally been believed to be photoprotective because of skin sensitivity to ultra violet radiation (UVR) which is relate to constitutive pigmentation and tanning ability. UVR is divided into three wavebands. UVC (200-290 nm) is filtered from reaching the earth’s surface by upper atmospheric ozone, UVB (290-320 nm) is also attenuated and only constitutes up to 6% of terrestrial UVR, whilst UVA (320-400 nm) makes up the remainder. Moreover, the higher wavelengths are visible light (Halliday et al., 2008).

UVR can be directly absorbed by DNA, causing damage (photolesions), or by other chromophores resulting in the production of reactive oxygen species (ROS) that cause damage to DNA, lipids and other cellular components. The sun screen preparation has been developed rapidly since it was found that the ultraviolet ray causes several damages on skin; for examples, sunburn, cancer, abnormally pigmented skin, wrinkling and coarsening of the skin surface.

Melanin is the principal surface pigments which found in plants and animals. The melanin also apparent in the human skin or animals not normally exposed to the sun are often heavily pigmented. For example, the skin of the genitalia of humans, including newborns, contains higher concentrations of melanocytes and the melanin than on the arms and chest (Robins, 1991). Melanocytes also occur in the other tissues including the throat, nasal and auditory passages in numerous animal species (Hill, 1992).

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2 Melanin has several biological functions (Riley, 1992). One of these is to strengthen structures by cross-linking of proteins. Melanin supply mechanical strength and may protect the protein from degradation. In the human, melanin may play a role as a photoprotective pigment and a case has been made for it being genoprotective by acting as a photosensitizer of cells that have been exposed to radiation of sufficient energy to produce genetic damage (Riley, 1997). The loss of skin pigmentation resulting from disappearance of pigment cells because of application of chemical or because of immunological and genetic factors. In the skin, in human (in vertebrate in general), the biosynthesis in melanin involve a metabolic pathway. Traditionally, skin types have not been classified according to the degree of pigmentation but rather to skin’s erythema response and ability to become tan color (Fitzpatrick, 1988).

The skin has epidermal units that are responsible for melanin production and distribution, a process called melanogenesis. Melanogenesis is a complex process with different stages. When disturbed, it may determine different types of pigmentary disorder, which are classified as hypo or hyperpigmentation and which may occur with or without an altered number of melanocytes. Pigmentary disorder is the conditions that is extremely common in black (acne vulgaris, eczema, fungal infection, seborrheic dermatitis, alopecias and contact dermatitis), Hispanic (acne, eczema, photoaging, acrochordons, seborrheic dermatitis and psoriasis), and Asian (xerosis, pruritus, nummular dermatitis, dyshidrosis, atopic dermatitis, malesma, photodermatoses and vitiligo) (Halder and Nootheti, 2003).

Skin hypopigmentation may result from decreased number or faction of melanocytes, degree of melaninization of melanosomes, or decreases

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3 phagocytosis of melanosomes by keratinocytes (Bolognia and Pawelek, 1988). Vitiligo is an acquired disease characterized by destruction of functional melanocytes mainly in the skin, resulting in the appearances of well-circumscribed white macules and patches. Affecting 0.5% to 2% of the general population, and 25% to 30% childhood patients (Nicolaidou et al, 2012). Postinflammatory hypomelanosis is hypopigmentation usually gradually resolve. Many dermatoses, including psoriasis, seborrheic dermatitis, atopic dermatitis, sarcoidosis, mycoses fungoides, and lupus can lead to postinflammatory hypopigmentation (Nicolaidou and Katsambas, 2014).

Hyperpigmentary disorder is skin darkening caused by follows auto immune conditions, sun damage (UV radiation and ionizing radiation), drug reactions (chemicals), hormonal changes, genetic factors, medications, hormonal therapy or birth control pills resulting in the hyper secretion of melanin from melanocytes (Maeda and Fukuda, 1991; Jennifer et al, 2012). Malesma is a common acquired hypermelanosis that occurs exclusively on sun-eposed areas, mostly on the face and occasionally on the neck and forearms. Malesma is more common in women. Whilst men have been reported to represent 10% of case (Katsambas et al, 2003). Pigmented contact dermatitis (Riehl melanosis) is a pigmentary dermatosis characterized by brownish gray facial pigmentation that is more marked on the temples and forehead. Erythomelanosis follicularis faciei (EFF) is a rare disease characterized by hyperpigmentation, erytherma and follicular papules localized on the face and neck. EFF was initially described in Asian men and later in white men (Watt et al, 1981). Erythema dyschromicum perstans (EDP) or ashy dermatosis is an idiopathic, acquired, and chronic skin

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4 disorder characterized by hyperpigmented patches on the runk, face, and extremities. Most adult patients with EPD are of Hispanic origin (Schwartz, 2004). Poikiloderma of civatte is a combination of linier telangiectasia mottled hyperpigmentation/depigmentation, and superficialatropy in a reticular pattern. Most frequently in fair-skinned, middle-aged, or elderly women (Katoulis et al, 2005).

To avoid damage effects of ultraviolet, it is necessary to use sun screening preparations. These preparations contain compounds which have the activity to prevent ultraviolet rays while it is penetrating the skin. Skin whitening agent is in close relationship with melanin (Briganti et al, 2003). The ideal depigmenting compound should be potent, rapid and selective bleaching effect on hyperactivated melanocytes, carry no short-or long-term side-effects and lead to a permanent removal of undesired pigment, acting at one at one or more steps of the pigmentation process. To decrease hyperpigmentation or melanogenesis on skin, we need to reduce the formation of melanin. The formation of melanin in the human body is influenced or reduced by several mechanisms, including anti-oxidant, direct tyrosinase inhibition of migration from cell to cell and hormonal activities, etc (Prota and Thomson, 1976; Pawelek and Komer, 1982).

Chemical and biological investigation for the search of novel bioactive natural products involves the extraction, isolation, purification and structure elucidation which can be challenging. Natural products (secondary metabolites) have been the most successful source of potential drug. However, their recent implementation in drug discovery and development efforts have somewhat demonstrated a decline in interest. Nevertheless, natural products continue to

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5 provide unique structural diversity in comparison to standard combinatorial chemistry, which presents opportunities for discovering mainly novel low molecular weight lead compounds. Since less than 10% of the world’s biodiversity has been evaluated for potential biological activity, many more useful natural lead compounds await discovery with the challenge being how to access this natural chemical diversity. Natural products have been used since ancient time and folklore for the treatment of many diseases and illnesses. Classical natural product chemistry methodologies enabled a vast array of bioactive secondary metabolites from terrestrial and marine sources to be discovered. Natural product is the most successful source of potential drug leads (Misra and Tiwari, 2011; Butler, 2004).

Traditional herbal medicines provide an interesting, largely unexplored source for development of potential new drugs. The potential use of traditional herbal medicines for development of new skin-care cosmetics has been emphasized recently (Kiken and Cohen, 2002). Traditional medicines are defined by the World Health Organization (WHO, 1978) as the sum total of knowledge or practices whether explicable or inexplicable, used in diagnosing, preventing or eliminating a physical, mental or social disease which may rely exclusively on past experience or observations handed down from generation to generation, verbally or in writing. It also comprises therapeutic practices that have been in existence often for hundreds of modern scientific medicines and are still in use today without any documented evidence of adverse effects.

Plants have an advantage based on their long-term use by humans (often hundreds or thousands of years). One might expect any bioactive compounds

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6 obtained from such plants to have low human toxicity. Obviously, some of these plants may be toxic. However, the goals of using plants as sources of medicinal agents are a) to isolate bioactive compounds for direct use as drugs, e.g., digoxin, digitoxin, morphine, reserpine, taxol, vinblastine, vincristine; b) to produce bioactive compounds of novel or known structures as lead compounds for semisynthesis to produce patentable entities of higher activity and/or lower toxicity, e.g., metformin, nabilone, oxycodone (and other narcotic analgesics), taxotere, and teniposide; c) to use agents as pharmacologic tools, e.g., lysergic acid diethylamide, mescaline and yohimbine; d) to use the whole plant or part of it as a herbal remedy, e.g., cranberry, echinacea, feverfew, garlic and ginkgo giloba (Fabricant and Fransworth, 2001).

Indonesia is one of the very few countries in the world that are endowed with rich biodiversity. Indonesian tropical forests, contain 28,000 plant species, convers about 143 million hectares. The use of plants and other natural resources for medicinal purpose in Indonesia dates back to prehistoric time. An existing proof can be found in the stone relief at the famous Borobudur temple dating around AD 800 showed the kalpataruh leaf, taken from mythological tree that never dies, and other ingredients are pounded to make a mixture for women’s health and beauty care (Agil, 2006).

Indonesian traditional medicine known as jamu is all medicine of Indonesian natural resources including plants, animals, minerals. Originally, jamu is a term used for Javanese traditional medicine, but now becomes popular as general term of Indonesian traditional medicine even modern health care has been

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7 developed but in the areas some families prefer to use only traditional medication, whilst others use it as a supplement to modern medication.

In order to find skin whitening agent from natural sources, our research is focus on exploration the potential Indonesian medicinal plants and its ingredients as whitening agents against skin hyperpigmentation.

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8

Chapter 1. Potential of Medicinal Plants Extractives as Anti

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9 1.1. Introduction

Skin darkening is one of the serious aesthetic problems in the human being. Melasma, post inflammatory melanoderma, solar lentigo, and freckles are skin diseases caused by the accumulation of melanin (Ahn et al., 2006; Unver et al., 2006; Altaei, 2012). The factors triggering these disorders are ultraviolet light, chronic inflammation, and rubbing of the skin as well as the abnormal _{ߙ-melanocyte stimulating hormone} (ߙ-MSH) release (Im et al., 2002 and Kang et al., 2002). Depigmentation is occurred by the loss of melanogenic enzymes in melanocytes (Mishima et al., 1972). The skin pigmentation relies on the melanin biosynthesis in melanocytes which is controlled by the melanogenic enzymes such as tyrosinase, tyrosinase related protein (TRP)-1, and 2 (Hearing, 1999).

Skin whitening agents are usually used to treat the skin pigmentation. It should be noted that safety is a priority consideration for its practical use in human. It has standard concentration which is set in each country for the addable concentration of active ingredients contained in the whitening agent, since it has different problems such as ochronosis, irritation and allergy. The adverse effect of whitening agent is dependent on the dose concentrations (Mahe et al., 2003) or frequent in use that it may induce skin tumorigenicity (Cheng et al, 2006; Burdock et al., 2001; Higa et al., 2002). Hydroquinone (HQ) allowed up to 2% as a cosmetic ingredient in European Union at 1984 by the Commission Directive 84/415/EEC (Fifth Commission Directive, 1984). Resorcinol (RS), an isomer of HQ is not permitted in either the European Union and United

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10 States (Sakuma et al., 1999). Finding effectives and safe skin-whitening substances have been encouraged to prevent human hyperpigmentation. Moreover, the potential safety has been taken into consideration.

The utilization of medicinal herbs as cosmetic materials is based on the ancestor experience which is usually called traditional cosmetics or traditional herbal formulation. Recently, the potential use of traditional medicines for the development of new skin care cosmetics has been emphasized (Kiken and Cohen, 2002). Several researchers have searched various plants for finding the novel compounds which utilized for curing skin diseases including over melanin production in the human being. Various active compounds have been found from the medicinal plants for hyperpigmentation as a skin whitening agent. This review reports the potential of traditional medicinal plant extractives and the effective novel ingredients as anti-melanogenesis. Through this review, the author provides the list of potential medicinal plant which is possessing future potential of whitening agents.

1.2. Research objectives

The effects as cosmetic materials are based on the ancestor experience which usually called traditional cosmetics or traditional herbal formulation. Recently, the potential use of traditional medicines for the development of new skin care cosmetics has been emphasized (Kiken and Cohen, 2002).

Several researchers have searched various plants for finding the novel compounds which utilized for curing skin diseases including over

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11 melanin production in the human being. Various active compounds have been found from the medicinal plants for hyperpigmentation as a skin whitening agent. This review reports the potential of traditional medicinal plant extractives and the effective novel ingredients as anti-melanogenesis.

1.3. Melanin Biosynthesis

Skin and hair coloration is caused by melanin pigment. Melanin production is regulated by the melanocytes distributed in the skin, hair follicles, and pigment epithelium in the retina (Costin and Hearing, 2007). It plays important role in photo-protection (Riley, 2003). Melanin in the human skin is important as a defense of human skin against the damage of UV irradiation due to the ability to absorb (Archambault et al., 1995; Chakraborty et al., 1996; Todd et al., 1993; Lukiewicz, 1972; Hengshan, 2006). Overproduction of melanin contents in human body causes hyperpigmentation in the epidermis that induce several kinds of skin problems (Ahn, 2006; Unver, 2006). In contrast, the deficiency of the melanin production caused skin aging or induces gray hair. The skin or hair pigmentation process are initiated after being stimulated keratinocytes in the skin surface by UV irradiation (Yoshida et al., 2000). Then the keratinocytes releasing messengers (histamine, ߙ-melanocytes-stimulating hormone (_{ߙ-MSH) and prostaglandin) and bind with the} receptors on the melanocyte. Melanin is biosynthesized in the melanosome then transported and accumulated in the keratinocytes, then cornification causes the skin pigmentation (Fig. 1).

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12 Correspondingly, hair pigmentation arises due to melanin released to the outside of the melanocyte and accumulated in the hair matrix. Melanocyte locates on the hair bulb to produce pigment of the hair shaft (Slominski et al., 2005; Slominski et al., 1993) (Fig. 2) (Yamauchi and Mitsunaga., 2016). In the late anagen, melanocytes begin to shut down melanogenesis and proceed to regression phase called catagen. Deficiency of melanin biosynthesis in the hair bulb on anagen is induced by a stress or an aging caused in gray hair (Tobin et al., 1998; Slominski et al., 1994; Tobin et al., 1999; Guo et al., 2012). Melanogenesis is a well-known physiological response of human skin induced by ultraviolet light and other sources. Melanin biosynthesis is formed by the melanogenic enzymes in melanocytes.

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13

Hair bulb Melanocyte

Hair shaft

Yamauchi et al., Letters in Drug Design and Discovery (2016)

Scheme 2. Typical hair follicle

1.4. Melanogenic enzymes

Tyrosinase, a major enzyme, catalyzes rate limiting reaction to synthesize melanin through the oxidation of L-tyrosine to L-DOPA (3,4-dihydroxyphenylalanine), following the oxidation of DOPA to L-DOPA quinone. The oxidation of L-L-DOPA quinone results in the formation of melanin (Olivares et al., 2009). Melanin is distinguished into two types that are blackish brown eumelanin and reddish yellow pheomelanin formed by the conjugation of cysteine or glutathione (Tsatmali et al., 2002; Slominski et al., 2004). The human tyrosine related proteins (TRP)-1 and TRP-2 are the key enzymes to biosynthesize eumelanin. Furthermore, cysteine or glutamine is involved in the formation of pheomelanin. The pathways are illustrated in Fig. 1.

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14 Fig.1. Melanin biosynthetic pathway

The expression of melanogenic enzymes is stimulated through the intracellular signaling. When the human epidermis is exposed by UV radiation, a potent cyclic adenosine monophosphate (cAMP) activator and the cytokines bind to the MC1 receptor that activates cAMP-dependent protein kinase A (PKA) and other regulatory proteins (Busca and Ballotti, 2000). The cAMP response element-binding protein (CREB) is phosphorylated by PKA. Activated CREB induces Microphthalmia-associated transcription factor (MITF) transcription. MITF stimulates tyrosinase expression (Yasumoto et al., 1997). Also, it reported that MITF is regulator of melanocyte proliferation, differentiation and pigmentation (Tachibana, 2000; Costin and Hearing, 2007). MITF up-regulates the expressions of tyrosinase, tyrosinase-related protein (TRP)-1, and dopachrome tautomerase (Dct) that result in promoting melanin synthesis and skin pigmentation (Bertolotto et al., 1998a, b, c). Then

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15 MITF and the enzymes regarding to the expression of tyrosinase are known as the target to regulate melanognesis using ingredients in natural products (Scheme 3). Messenger p38 p-p38 MITF Tyrosinase TRP-1 TRP-2 ERK p-ERK Inhibition JNK p-JNK Inhibition Melanin Melanosome Melanocyte Activation Mitsunaga et al., J.W.S (2015)

Scheme 3. Transcriptional regulation of expression of melanogenic enzyme

Several studies reported that melanogenesis-modulating agents regulate the expression of tyrosinase, TRP-1, TRP-2, by regulating the expression of p38 MAPK, ERK, JNK, and MITF. Yamauchi et al. (2014) (Fig. 2) demonstrated that some quercetin deratives have effectivity on the expression of protein involved in melanin biosynthesis. Compound 3’, 4’, 7-O-trimetylquercetin is able to enhance the expression of tyrosinase, TRP-1, TRP-2, MITF, and p-p38 MAPK against B16 melanoma cell in dose dependent manner. It significantly stimulated the expression of MITF and p-p38 MAPK by increase the expression of tyrosinase, TRP-1, and TRP-2. Besides that, compound 3-O-methylquercetin indicated its effectivity to enhance the expression of tyrosinase, TRP-1, and TRP-2 by stimulating transcriptional factors that are yet to be indicated.

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16

A

O O CH₃ O OH O H OH OH Tyrosinase TRP1 TRP2 p-p38MAPK MITF p38MAPK GAPDH Control 3.1μM 6.3μM 12.5μM

B

Pr o te in e x pr es si o n (% )

C

O O CH₃ OH O CH3 O OH O CH₃ p-p38MAPK Control 3.1μM 6.3μM 12.5μM Tyrosinase TRP1 TRP2 MITF p38MAPK GAPDH

D

P rot ei n e xpr es si on (% )

Yamauchi et al., Bioorganic and Medicinal Chemistry (2014)

Figure 2. Effect of some quercetin derivatives on the expression of Tyrosinase, TRP-1, TRP-2, MITF, p-p38MAPK, and p38MAPK in B16 melanoma cells. (A) Representative blot of 3-O-methylquercetin. (B) Quantification of the ratio of protein expression in melanoma cells by 3-O-methylquercetin. (C) Representative blot of 3’, 4’, 7-O-trimetylquercetin. (D) Quantification of the ratio of protein expression in melanoma cells by 3’, 4’, 7-O-trimetylquercetin. The data show the means ±S.D. from three independent experiments. *≤0.05 and **p≤0.01 compared with control values.

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17 1.5. Natural resource for whitening agent

Traditional medicinal plants are a rich source of bioactive compounds. The skin-whitening agents derived from natural resources particularly plants have been known so far in cosmetic products (Table 1) (Scheme 4). A large number of skin-whitening agents such as kojic acid, arbutin, azelaic acid and aleosin originally from natural resources have been used in cosmetic products. 5-Hydroxy-2-(hydroxymethyl)-4H-pyran-4-one, known as kojic acid is a potent tyrosinase inhibitor that is produced by Aspergillus spp. and Penicillum spp. Lim (1999) revealed that kojic acid cures the melasma’s patient more than half. Arbutin, hydroquinone-O-β-D-glucopyranoside, is one of the most whitening agent used for cosmetic product. The arbutin analogs were isolated and identified from buds of Vaccinium dunalianum (Zhao et al., 2008). Azelaic acid (AZA) is obtained from Malassezia spp. AZA has been used to cure post-inflammatory hyperpigmentation and melasma (Bernal et al., 2000). Low et al., (1998) found that AZA exhibited decreasing hyperpigmentation in darker-skinned. Aleosin as skin-lightening agent is isolated from Aloe vera plant. Choi et al., (2002) revealed that aloesin shows tyrosinase inhibitory activity in human skin after UV radiation.

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18 T ab le 1. S om e s ki n-w hi te ni ng a ge nt s kno w n a s pra ct ic al us e s o fa r Ag en t So ur ce Me ch an is m of a cti on Adv anta ge Hyd ro qui non e m onob en zy l e the r (H MB E, m ono be nz one ) Hyd ro qui non e de ri va ti ve - Th e de pi gm en ta ti on th er ap y on e xte ns iv e vi tili go p ati en ts (K as ra ee e t a l, 2006) Hyd ro qui non e m ono m et hy l e the r (4 -hyd ro xy an is ol e, me qu in ol ) Hyd ro qui non e de ri va ti ve - R em ove r esi du al p igm en t of v er ti li go un iv er sa lis (N joo e t a l, 2000) A rbuti n Arc tost aphy los uv a-urs i (L .) In hi bi t th e o xid at io n o f L -ty ro si ne ( M ae da a nd F ukud a, 1996) C om pe titi ve white ni ng a ge nt ( H or i e t a l, 2004) A ze lai c aci d Ma la ss ez ia spp . In hi bi t t yro si ne T re at m en t o f a cn e ( D ute il a nd Or tonn e, 1992) Ko ji c a ci d Aspe rgillus sp p. In hi bi ts c at ec hol as e a ct iv it y of ty ro si na se in a non -c la ss ic al m anne r E xc el len t w hi ten in g a gen ts ( C ab an es e t al , 1994) Ma gn es iu m L -a sc or by l- 2-pho sp ha te / a sc or bi c ac id Citr us f ru it a nd le af y gr ee n ve ge ta bl e In hi bi t t yro si na se lig ht en in g in h ea lth y sk in ( R os e t a l, 1993) N ia cin am id e, V ita m in B3 R oot v ege ta ble , ye as t li m ita ti on m ela no so m e tr an sf er f rom m ela no cy te s t o ke ra tinoc yt es Sk in li gh te ni ng ( H ak oz ak i e t a l, 2002) E llag ic ac id P unic a granat um L. De cr ea si ng c oope r c onc en tr at io n a nd inh ib it t yr osi na se a ct iv it y Sk in li gh te ni ng ( S hi m og ak i e t a l, 2000) G la br idi n a nd l ic or ic e ex tr ac t G lycyw hi za g la bl a L. Sp ec if ic al ly d ecr ea si ng th e ty ro si na se ac ti vi ty In hi bit or y t yr osi na se a ct iv it y ( S hi n e t a l, 1998) Al eo si n Aloe v er a De pr es si on th e p igm en ta ti on v ia in hi bi t ty ro si na se a ct iv it y a fte r U V r ad ia ti on In hi bit ty ro si na se a cti vi ty ( C ho i e t a l, 2002) So y pr ot ei ns Gl yc ine ma x In fl ue nc e cy to sk el et al an d ce ll s ur fac e or ga ni za ti on Sk in li gh te ni ng ( Pa ine , 2001)

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19 O O H H yd ro qu ino ne m ono be nz yl e the r (H M B E, mo no be nz on e) O OH H yd ro qu ino ne m ono m et hy l e the r (4 -h yd ro xy an is ol e, me qu in ol) O O _OH OH O H O H OH Ar bu tin O O OH O H O H O H H O O H Al oe si n O O H O O H K oj ic ac id O H O O O H OH H OH Asc or bi c ac id N NH 2 O N iac in amid e O O O O OH OH OH OH El lag ic ac id O O HO H O G lab ri di n O H OH O O A ze laic ac id S ch em e 4 . C hemi cal s tr uc tu re o f s om e w hi ten in g a ge nt co sm eti c.

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20 Several studies of bioactive compound which is possessing future possibility as cosmetic agents have found (Scheme 5). Wang (2013) has demonstrated that linderanolide B and subamolide A isolated from Cinnamomum subavenium shows inhibitory activity on mushroom tyrosinase with low doses without cytotoxicity in normal human skin cells. (-)-N-formylanonaine isolated from the leaves of Michelia alba D.C. (Magnolianceae) is capable of inhibiting tyrosinase and reducing melanin activity in human epidermal melanocytes (HEMn) without cytotoxicity. The compound is also reported to inhibit tyrosinase activity due to chelation with two Cu2+_{ions that are the active site of tyrosinase (Wang et al., 2010).}

Sasa quelpaertensis containing p-coumaric acid inhibits melanin synthesis in human melanocytes stimulated by D-MSH (An et al., 2008).

8-Gingerol from Zingiber have identified to suppress intracellular tyrosinase activity and decrease the melanin contents. Besides, 8-Gingerol is reported to decrease intracellular reactive species (RS) and reactive oxygen species (ROS) level (Huang et al., 2013). Matsuda et al., (2009) exhibited that the constituents of Alpinia officinarum rhizome such as 5-hydroxy-1,7-diphenyl-3-heptanone, 7-(4”-hydroxy-3”- methoxyphenyl)-1-phenylhept-4-en-3-one, 5-hydroxy-7-(4” -hydroxy-3” -methoxyphenyl)-1-phenyl-3- heptanone, 3,5-dihydroxy-1,7-diphenylheptane, kaempferide, and galangin inhibit melanogenesis. Moreover, 7-(4”-hydroxy-3”-methoxyphenyl)-1-phenylhept-4-en-3-one, kaempferide, and galangin inhibit mRNA expression

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21 of tyrosinase, tyrosinase-related proteins-1, and -2, and reduces the protein level of MITF which is related to the expression of tyrosinase.

O H H H O H H O Linderanolide B O H O H H O H3CO Subamolide A O CH3 OH O CH3 5-hydroxy-7-(4''-hydroxy-3''-methoxyphenyl)-1-phenyl-3-heptanone O CH₃ OH O 7-(4''-hydroxy-3''-methoxyphenyl)-1-phenylhept-4-en-3-one OH CH3 3,5-dihydroxy-1,7-diphenylheptane CH2(CH2)5CH3 O OH O O H C H3 8-Gingerol O O CH3 O OH OH O H Kaempferide O OH O OH O H Galangin O OH 5-hydroxy-1,7-diphenyl-3-heptanone O O N H CHO (-)-N-formylanonaine OH O O H p-coumaric acid

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22 1.6. Potential of Indonesian natural medicinal plant for whitening agent

Natural medicinal treatments are used because it provides several beneficial effects such as anticancer, anti-inflammatory, and protection against UV. Indonesia is one of the world’s major country that possess the sources of useful plant materials and high number of indigenous medical plants. Most of the Indonesian people, particularly in outland areas use traditional herbal medicines known as jamu. Jamu is tribal language from Javanese expressing the traditional medicine from plants. Corresponding to the utilization as a medicine, it is distinguished into four categories: health care, beauty care (cosmetics), tonics, and bodily protection (Riswan and Roemantyo, 2002). The use of herbal ingredients is abundant in the practice of beauty care such as Indonesian traditional spa which is use of ground herbs in the form of powders and scrubs for body treatments. It has been involved into modern businesses which is distributed in the capital city of Indonesia, especially along Jawa and Bali Islands (Ministry of Trade Republic of Indonesia, 2009).

According to the abundance source medical plants, it is very encouraging to explore the potential of Indonesian natural source to maintain the health and to cure the disease. Zingiberaceae family such as Curcuma aeruginosa, C. aurantiaca, C. mangga, C. petiolate, C. purpurascens, C. soloensis, C. xanthorizae, C. domestica and C. zedora are the most frequently used as jamu. They are used for curing some illnesses such as appendicitis, asthma, itch, rheumatism, abdominalgia, anemia, hypertension,

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23 diarrhea, dysentery (Hatcher et al., 2008). Elfahmi et al., (2014) reported that Paeomiae alba, Polygalae tenuifolia, Rehmanniae prepareta, Carthami tinctorius, Leonuri heterophyclus, Angelicae sinensis, Concha ostrea gigas, Albizziae julibrissin have been used for regulating endocrine gland secretion and menstruation, promotes ovulation, and reduces menstrual clots.

Traditional herbal medicine gives an interesting and huge unexplored source for development of potential as new drugs and cosmetics. Several natural remedies have been used since centuries for caring skin conditions and dermatological disorders, including inflammation, phytotoxicity, psoriasis, atopic dermatitis, and alopecia aerate.

Several Indonesian plants that inhibit melanogenesis have been searched for effectivity as whitening agents Table 2 (Scheme 6). Bengkoang (Pachyrhizus erosus) has been use as a folk medicine for skin-lightening agents and commercially used in the cosmetic product. Lukitaningsih and Holzgrabe (2014) reported that constituent compounds in Bengkoang such as daidzein, daidzein-7-O-E-glucopyranose, 5-hydroxy-daidzein-7-O-E-glucopyranose, and 8,9-furanyl-pterocarpan-3-ol (Scheme 7) suppress tyrosinase activities.

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24 OH O O OH O H O H OH O O OH O H OCH 3 O OH OH OH OH O O H OH O H O H Ug in on J Ug in on K R ob in etin Re sv er at ro l OC H3 OH OC H3 O O O O OC H3 O O O H OH O OH O H O OH O H O H O H E uge no l Eu ge no l ac etate 7-me th ox yc ou mar in Q uer cet in -3’ -O-β-D -gl uc os ide O OH O H O H OH O OH O O O OH OH O H OH O O H OH O H O H O O O O O H O H OH OH OH O H OH O CH 3 CH 3 OC H3 O O Q uer cet in Q uer cet in 4 ’-O -β -D -gl uc op yr an os id e L eut in -7-O -β -g lu co si de Ele uth er in O O OH O O H O H O OH O O H O H O H O OH H H OH O H O H Br os im on 1 3-pr en yl lu te olin Gn et in C S ch em e 6. C he m ic al s truc ture of p ot ent ia l w hi te ni ng a ge nt d is cove ry f ro m fol k m ed ic in al p la nt .

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25 O O _O O H O H OH O O H

8,9-furanyl- pterocarpan-3-ol Daidzen

O OH O O OH OH OH OH OH O OH O O OH OH OH OH 5-hydroxy-daidzein-7-O-β-glucopyranose Daidzein-7-O-β-glucopyranose

Scheme 7. Chemical structure of folk medicinal from Bengkoang (Pachyrhizus erosus) for whitening agents and commercially used as cosmetic product

Alamanda cathartica is an ornamental plant in Indonesia containing glabridin as an active constituent to inhibit tyrosinase activity with high percentage inhibition (93%) (Yamauchi et al., 2011). Yokota et al (1998) revealed that glabridin from Licorice extracts is the main ingredient which affecting on skins by inhibit tyrosinase activity in the B16 melanoma cells and guinea pig skins. It was also detected that glabridin inhibited UVB-induced pigmentation and erythema in the skin guinea pigs. Allium cepa has constituents such as quercetin, quercetin -4’-O-glucoside, quercetin 4’-O-β-D-glucopyranoside, and quercetin-3’-O-β- D-glucoside which suppress melanin formation (Arung et al., 2011b, c, d).

A number of countries possess traditional herbal medicine for curing the disease. For instant, Korean traditional herbal medicines have been used

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26 to treat pimple, alleviate freckles, and melasma. Cuscuta japonica seed have found to inhibit induced melanin synthesis, decrease D-MSH-induced expression of MITF, TRPs, and reduce the level of phosphorylated p38 mitogen-activated protein kinase (MAPK) signaling through the down-regulation of D-MSH-induced cAMP (Jang et al., 2012).

Nardostachys chinensis has been used in melasma and lentigines disorder in Korea. Jang et al., (2011) found that Nardostachys chinensis inhibits melanogensis due to decreasing MITF, tyrosinase, TRP-1, dopachrome tautomerase (Dct), MITF, tyrosinase mRNA levels, and intracellular cAMP levels. It is also reported that it activates mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK) and phos-phatidylinositol 3-kinase (PI3K)/Akt expression that result in suppressing MITF expression.

Artocarpus communis is folk medicine to prevent skin disease as acne and dermatitis. Fu et al., (2014) demonstrated that the extract of Artocarpus communis decreases melanin contents and tyrosinase activity by inhibiting the expression of MITF and phosphorylated cAMP response element-binding protein (p-CREB). Oh et al., (2010) revealed that 1-O-Methyl-fructofuranose from Schisandra chinensis fruit inhibits both melanin synthesis and tyrosinase activity. It also reported to reduce the expression of melanogenic proteins including MITF and TRP-1. Moreover, it activates melanogenesis inhibitory proteins such as mitogen-activated protein kinase

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27 (MEK)/extracellular signal-regulated kinase (ERK) and Akt. An extract of brown seaweed (Sargassum polycystum) inhibits melanogenesis by inhibiting cellular tyrosinase activity and is known to treat skin related disorder (Chan et al., 2011).

A number of the effects and ingredients in the medicinal plant which is distributed in the world still unknown. Therefore, further investigation must be conducted to clarify their clinical advantage.

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28 Tab le 2 R ev iew o f p ub lis hed liter atu re r elat in g to s elected med ica l p la nts u se in M ela no ge ne si s No Pla nt N am e Pa rt Extr ac t C omp ou nd C omm en t Toxi c 1. P hy lla nt hus em blica ( mal ak a) fr ui t et hy l a cet ate ta ni n (ga la d a ci d a nd el eg at ) It in hi bi ts mel an og en es is an d de cr ea se s ty ro si na se ac ti vi ty w ith IC50 95.63 a nd 16.90 μ g/mL. (H in dr it ia ni e t a l., 2013) les s 2. H elm in th os ta ch y s z eyla nica roo ts 50% e th anol an d w ater m ixt ure U goni n J a nd U goni n K U gonin J a nd K de pre ss es ex tr ac el lu lar me la ni n p ro du ct io n to 75 a nd 19 % a t 12.5 μ M) . (Y am auc hi e t a l., 2015) no 3. D ur io ku tejen si s [B om bac ac ea e (H as sk ) Bec c] fr ui t n-he xa ne , et hy l a cet ate, et ha nol - Th e Et oA c ex tr act in hi bits m el ani n fo rm at io n b y 47% a t 200 μ g/ m L in B16 m el anom a c el ls w ith ou t c yt ot ox ic it y w hile d id n ot inhi bi t ty ro si na se a ct ivi ty . ( A ru ng et a l., 2015 a) no 4. In tia pa lem ba nica wo od m et ha nol Ro bi ne ti n Int si a pal em bani ca (M er ba u) ha s act iv it ies as m on op he no las e an d di ph enol as e i nhi bi to r of ty ro si na se on 10.4 μ g/ m L of I C50 an d an ti ox id an t acti vi ty . (Ba tuba ra , 2010) no 5. Allam anda ca th ar tica roo ts m et ha nol G la bri di n G lab ri di n i s th e ce nt er ac ti ve co m po un d of ty ro si na se inhi bi to ry a ct ivi ty b y 93% a t t he conc en tr at io n 19.3 μ M a nd 2.93 μ M of IC 50 . T he r es ul ts s ho w ed th at g lab ri di n h as act iv it y 1 0 times s tr on ger t ha n ko jic ac id . (Y am auc hi e t a l, 2011). no

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29 6. Sy zy gi um ar om aticu m m et ha nol a nd oi l E uge nol a nd Eu ge no l a cet ate E uge nol a nd eu ge nol a ce ta te exhi bi te d m el ani n i nhi bi ti on i n B16 m el ano m a ce lls b y 50 an d 80% a t 100 a nd 200 μ g/ m L re sp ec ti ve ly . ( A ru ng e t a l, 2011a ) les s 7. E upat or iu m tr ip lin er ve V ahl lea ve m et ha nol 7-met ho xy co umar in M eth an ol ex tr act o f E. tr ip lin er ve Va hl wa s d em on st ra te d fo r in hi bi to ry a cti vi tie s o n mel an in fo rm at ion i n B16 m el ano m a ce lls wi th I C50 1780 μ M a nd bot h ty ro si na se e nz ym e a cti vi ty L-ty ro si ne (I C 50 =2360 μ M) a nd L-DOPA ( IC 50 =2840 μ M ). ( A ru ng et a l, 2012) no 8. Alliu m c ep a dr ied s kin m et ha nol Q ue rc eti n an d Qu er ce ti n -4’ -O-gl uc os id e T he m et ha nol e xt ra ct s uppre ss ed m el ani n fo rm at ion i n B16 me la no ma ce lls b y 4 0-50% a t t he conc en tr at ion 50 a nd 100 μ g/ m L . Q ue rc eti n an d Q ue rc eti n-4’ -O -gl uc os id e de cr es ed t yr os ina se act iv it y w ith I C 50 26.5 an d 131 μ M , r es pe cti vely . ( A ru ng et al , 2011b) no 9. Sonne ra tia ca se ol ar is (R am bai S un gai ) lea ve E th anol (E tOH) lu te olin -7 -O -β -gl uc os id e lu te olin -7 -O -β -g lu co si de w as in hi bi to ry act iv it y o f m el an in fo rm at io n i n B16 m el ano m a w it h IC50 223.2 μ M . (A ru ng, 2015b) les s 10. Ar to ca rp us heter ophyl lus wo od m et ha nol Bro si m one I a nd 3 -pre nyl lu te ol in Bro si m one I a nd 3 -pre ny l lu te ol in w ere po te nt a s m el ani n inhi bi to ry act iv it y w it h IC50 0.8 a nd 56.7 μ M . (A run g e t a l, 2005; A ru ng les s

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30 et a l, 2006 ; A ru ng e t a l, 2007 ; A ru ng e t a l, 2010a ,b, c,d) 11. Alliu m c ep a dr ied s kin m et ha nol que rc et in 4’ -O - β -D -g lu cop yr an os id e Th e qu er cet in 4’ -O -β -D -gl uc op yr an os id e w as i nhi bi to ry of bo th ty ro si na se a cti vi ty L -ty ro si na se a nd L -DOPA wi th I C50 4.3 a nd 52.7 μ M . (A ru ng e t a l, 2011c ) no 12. W il lughbe ia co ri acea ba rk p art of aer ia l r oo t m et ha nol - Th e met ha no l ex tr acts w er e po ten t i nh ib ito ry mel an in fo rm at ion i n B16 m el ano m a ce lls by 92.5 % a t 100 μ g/mL a nd sc av en ge DPPH r ad ic al s. ( A ru ng et a l, 2009) les s 13. D endr opht hoe pe tandr a aer ia l r oo t m et ha nol - Th e met ha no l ex tr acts w er e po te nt i nhi bi to rs of ty ro si na se act iv it y a nd me la ni n f or m at io n in B16 m el anom a ce lls b y 95.9 % a t 100 μ g/mL, al so t o s ca ve ng e DPPH r ad ic al s. ( A ru ng et a l, 2009) les s 14. G loc hi di on phi lippc um aer ia l r oo t m et ha nol - Th e met ha no l ex tr acts w er e po ten ti al i nh ib ito ry mel an in fo rm at io n b y 71 % at 100 μ g/ mL in B16 m el ano m a c el ls ( A ru ng e t al , 2009) les s 15. Eleu th er in e pa lm ifo lia bul b m et ha nol - Th e met ha no l ex tr acts w er e inhi bi te d m el an in fo rm at ion b y 37. 9 % a t 100 μ g/mL in B 16 me la no ma ce lls , ex hi bited D P P H ra di ca l-sc av en gi ng a cti vi ty . les s

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31 (A ru ng e t a l, 2009) 16. Eu si de ro xylo n zw age ri seed m et ha nol - Th e met ha no l ex tr acts w er e inhi bi te d m el an in fo rm at ion b y 32.4 % a t 100 μ g/mL in B 16 m el an om a c el ls a nd DPPH ra di ca l-sc av en gi ng a cti vi ty . (A ru ng e t a l, 2009) les s 17. lans iu m do m es ticu m ba rk m et ha nol - It i nhi bi ts m el ani n fo rm at ion b y 13 % a t 100 μ g/mL in B 16 me la no ma ce lls . ( A ru ng et a l, 2009) les s 18. pa ss iflo ra fo etid a stem, f ru it m et ha nol - It i nhi bi ts m el ani n fo rm at ion b y 104.1 % a t 100 μ g/mL in B 16 me la no ma ce lls . ( A ru ng et a l, 2009) les s 19. Sol anum to rv um roo ts m et ha nol - It i nhi bi ts m el ani n fo rm at ion b y 98.6 % a t 100 μ g/mL in B16 me la no ma ce lls . ( A ru ng et a l, 2009) les s 20. Gn et um g ne mo n seed E th anol (E tOH) G ne ti n C a nd re sv er atr ol G ne ti n C an d re sv er at ro l w er e in hi bi to ry o f DOPA o xi da ti on b y 25.2 a nd 64.1 % 16 μ M. (Y an ag ih ar a e t a l, 2012) no 21. Alliu m c ep a dr ied s kin m et ha nol que rc et in -3’ -O -β - D -gl uc os id e It i nhi bi ts m el ani n fo rm at ion i n B16 m el ano m a c el ls a t 38.8 μ M of I C50 an d i nh ibi to ry bo th ty ro si na se act iv it y, L -t yr os ina se an d L -DOPA ( IC50 6.5 a nd 48.5 μ M re sp ec ti ve ly ). ( A ru ng e t a l, 2011d) no

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32 22. Eleu th er in e am er ic ana bul b m et ha nol na ph th oq ui no ne el eu th er in It i nhi bi ts m el ani n fo rm at ion i n B16 m el anom a ce lls b y 87 % a t 50 μ g/mL in B16 me la noma ce lls . (K us um a e t a l, 2010) les s 23. C us cu ta japoni ca seed w ater - It i nhi bi te d D-M S H -i nduc ed m el ani n s ynt he si s a nd ty ro si na se ac ti vi ty . (J an g e t a l, 2012). no 24. Nar dos ta ch ys ch in en si s seed w ater - It in hi bi ts me la ni n s yn th es is a nd ty ro si na se a ct iv it y >50% a t 0.1 – 2.5 g/ m l. (J an g e t a l, 2011). no 25. Ar to ca rp us co m m uni s he ar t-w ood met ha no lic - It d ecr eas es me la ni n c on te nt an d ty ro si na se a ct ivi ty at 39.50 μ g/mL o f I C 50 . (F u e t a l, 2014) no 26. Sar gas su m po lycys tu m seaw eed et ha nol 95% - It in hi bits ce llu lar t yr os in as e ac ti vi ty b y 87 % a t 100 μ g/mL. (Ch an e t a l, 2011). les s N ote : ( -) n ot i so lated t he co mp ou nd y et

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33

Chapter 2. Melanogenesis Inhibitory Activity of Components

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34 2.1. Introduction

Melanogenesis or melanin biosynthesis is a physiological process in which the human skin responds to ultraviolet light and other stimulants, resulting in the synthesis of melanin pigments (Cho et al, 2008). The excess accumulation of melanin causes hyperpigmentation and several skin diseases such as malaise, freckles, post-inflammatory, melanoderma, and solar lentigo (Sugamaran, 2002). Melanogenesis is regulated by enzymes such as tyrosinase, tyrosinase-related protein-1 (TRP-1), and tyrosinase-related protein-2 (TRP-2) (Kameyama et al, 1995). Tyrosinase is a rate-limiting enzyme that catalyzes the conversion of tyrosine to 3,4-dihydroxyphenylalanine (L-DOPA) and further oxidation to dopaquinone (Fitzpatrick et al, 1949). This reaction results in the production of eumelanin (blackish brown) and pheomelanin (red-orange). The inhibition of tyrosinase is the most common approach to achieving skin whitening (Solano et al, 2006). Currently, there are numerous pharmacological and cosmeceutical tyrosinase inhibitor or other melanogenic pathway targets (Briganti et al, 2003). Several whitening cosmetics are being developed and used widely, such as arbutin, kojic acid, hydroquinone, and hydroquinone derivatives (Komer and Pawelek, 1980). There is a need for novel, safe, and effective skin-whitening agents that possess melanogenesis inhibitory activity.

Several plants are used as traditional medicines in Indonesia called jamu which is distinguished into four categories based on use: healthcare, beauty care (cosmetic), tonic or beverages, and body protection (Risman and Roemantyo, 2002). Syzygium polyanthum (Weight) Walp (Figure 3) is a

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35 traditional medicine plant, belongs to the Myrtaceae family. It originated in Indonesia and is abundantly distributed there. S. polyanthum, also known as Daun Salam, has been widely used as culinary additive due to its flavor (Noorma et al, 1995; Har et al, 2012). It is also used in the treatment of diarrhea (Sumono and Wulan, 2008), diabetes (Widharna et al, 2015), hypertension (Ismail et al, 2013), Staphylococcus aureus infection (Grosvenor et al, 1995), Bursaphelenchus xylophilus infection (Mackeen et al, 1997), leukemia (Ali et al, 2000), Alternaria alternata and Colletotrichum capsici infection (Mohamed et al, 1996). However, the efficacy of S. polyanthum leaf extract in the treatment of other diseases needs to be investigated. In the present study, the effects of the methanol extract of S. polyanthum on melanogenesis were evaluated using B16 melanoma cells. To the best of our knowledge, this is the first study to investigate the effects of S. polyanthum leaf extract on melanogenesis.

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36 2.2. Material and Methods

2.2.1. Plant Material

Salam leaf (Syzygium polyanthum (Wight) Walp.) was collected from Bogor, West Java, Indonesia, and identified by Research Center for Biology, Indonesia Institute of Sciences, Cibinong, Jakarta. Voucher specimens (No BMK0089082016) were deposited in Tropical Biopharmaca Research Center, Bogor Agricultural University, Indonesia.

2.2.2. Extraction and Fractionation of S. polyanthum Leaf Powder

S. polyanthum leaf powder (500 g) was extracted with methanol (2.5 L), and the methanol extract (30.1 g) was separated with water and ethyl acetate. The aqueous layer was lyophilized (5.22 g) and the ethyl acetate layer was evaporated in the rotary evaporator (13.05 g). The ethyl acetate fraction (10.1 g) was separated via silica gel column chromatography (80 mm φ x 520 mm L) by stepwise elution from n-hexane to ethyl acetate (2:1 to 1:1 v/v) to obtain fractions 1–6 (0.20, 1.93, 0.92, 0.94, 0.69, 0.06 g, respectively). Further elution with ethyl acetate yielded fraction 7 (0.72 g) and with methanol yielded fraction 8 (4.73 g). The fraction 5 was separated using preparative HPLC [ODS-3 (20 mm φ x 250 mm L) (MeOH:0.05% TFA aq. = 60%:40%→100%:0% (40 min)].

2.2.3. Identification of Compounds

Compounds 1–4 from fraction 5 were identified by 1_H-NMR,13

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37 spectrometer) and MALDI-TOF-MS (Shimadzu Biotech Axima Resonance 2.9.1.20100121: Mode Negative, Low 100+, power: 120). Methanol-D4 (compounds 1-3) and acetone-D6 (compound 4) were used as the solvents for the NMR experiment. Compound 1: 1-(2,3,5-Trihydroxy-4-methylphenyl)hexane-1-one: λmax 252, 292 nm; 1H-NMR (METHANOL-D4, 600 MHz): δppm 5.87 (1H, s, H-6), 3.00 (2H, t, J = 7.6 Hz, H2-2´), 1.89 (3H, s, 4-CH3), 1.63 (2H, quint, J = 7.4 Hz, H2-3´), 1.33 (4H, m, H2-4´, H2-5´), 0.90 (3H, t, J = 7.2 Hz, H2-6´); 13_{C-NMR (METHANOL-D4, 150 MHz): δppm 206.2 (C-1´), 163.7 (C-3),} 162.4 (C-5), 160.0 (C-2), 103.8 (C-1), 102.2 (C-4), 93.4 (C-6), 43.6 (C-2´), 31.6 (C-4´), 24.8 (C-3´), 22.3 (C-5´), 13.0 (C-6´), 6.0 (4-CH3). The molecular

weight was determined to be 238 m/z by MALDI-TOF-MS (Shimadzu Biotech Axima Resonance 2.9.1.20100121: Mode negative, Low 100+, power: 120 % int. 936 mV Profile 1-14: Threshold Gradient). The molecular formula is C13H18O4. Compound 2: 1-(2,3,5-Trihydroxy-4-methylphenyl)octane-1-one: λmax 252, 288 nm; 1H-NMR (METHANOL-D4, 600 MHz): δppm 5.87 (1H, s, H-6), 3.00 (2H, t, J = 7.6 Hz, H2-2´), 1.89 (3H, s, 4-CH3), 1.62 (2H, quint, J = 7.6 Hz, H2-3´), 1.31 (8H, m, H2-4´, H2-5´, H2-6´, H2-7´), 0.88 (3H, t, J = 7.2 Hz, H2-8´); 13C-NMR (METHANOL-D4, 150 MHz): δppm 206.3 (C-1´), 163.6 (C-3), 162.4 (C-5), 160.0 (C-2), 103.8 (C-1), 102.2 (C-4), 93.5 (C-6), 43.6 (C-2´), 31.6 (C-6´), 29.3 (C-4´), 29.0 (C-5´), 25.1 (C-3´), 22.4 (C-7´), 13.1 (C-3´), 6.0 (4-CH3). The molecular weight was determined by

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38 2.9.1.20100121: Mode negative, Low 100+, power: 120 % int. 936 mV Profile 1-14: Threshold Gradient). The molecular formula is C15H22O4.

Compound 3: (4E)-1-(2,3,5-Trihydroxy-4-methylphenyl)dec-4-en-1-one: λmax 252, 292 nm; 1H-NMR (METHANOL-D4, 600 MHz): δppm 5.88

(1H, s, H-6), 5.36 (2H, m, H-4´, H-5´), 3.04 (2H, t, J = 7.2 Hz, H2-2´), 2.37 (2H, q, J = 7.3 Hz, H2-3´), 2.01 (2H, q, H2-6´), 1.89 (3H, s, 4- CH3), 1.27 (6H, m, H2-7´, H2-8´, H2-9´), 0.87 (3H, t, J = 7.2 Hz, H2-10´); 13C-NMR (METHANOL-D4, 150 MHz): δppm 205.3 (C-1´), 163.6 (C-3), 162.5 (C-5), 160.0 (C-2), 130.2 (C-5´), 128.4 (C-4´), 103.8 (C-1), 102.2 (C-4), 93.4 (C-6), 43.6 (C-2´), 31.3 (C-8´), 29.2 (C-7´), 26.7 (C-6´), 22.8 (C-3´), 22.3 (C-9´), 13.1 (C-10´), 6.0 (4- CH3). The molecular weight was determined to be 292

m/z by MALDI-TOF-MS (Shimadzu Biotech Axima Resonance 2.9.1.20100121: Mode negative, Low 100+, power: 120 % int. 936 mV Profile 1-14: Threshold Gradient). The molecular formula is C17H24O4.

Compound 4: 1-(2,3,5)-trihydroxy-4-methylphenyl)decan-1-one: λmax

252, 292 nm; 1H-NMR (ACETONE-D6, 600 MHz): δppm 6.04 (1H, s, H-6), 3.04 (2H, t, J = 7.6 Hz, H2-2´), 1.93 (3H, s, 4-CH3), 1.64 (2H, quint, J = 7.6 Hz, H2-3´), 1.27 (12H, m, H2-4´, H2-5´, H2-6´, H2-7´, H2-8´, H2-9´), 0.85 (3H, t, J = 7.6 Hz, H2-10´); 13C-NMR (ACETONE-D6, 150 MHz): δppm 205.8 1´), 164.3 3), 161.9 5), 159.5 2), 104.2 1), 102.6 4), 94.0 (C-6), 43.7 (C-2´), 31.8 (C-8´), 28.6~29.5 (C-4´-7´), 24.9 (C-3´), 22.5 (C-9´), 13.5 (C-10´), 6.6 (4-CH3). The molecular weight was determined to be 294

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39 2.9.1.20100121: Mode negative, Low 100+, power: 120 % int. 936 mV Profile 1-14: Threshold Gradient). The molecular formula is C17H26O4.

2.2.4. Cell Culture

Cells were cultured as previously described (Yamauchi et al, 2015) with minor modifications. Murine melanoma B16-F0 cells (DS Pharma Biomedical, Osaka, Japan) were grown in Dulbecco’s modified Eagle medium (DMEM) supplemented with 9% fetal bovine serum (FBS Biosera, Japan), 100,000 unit/L penicillin, and 100 mg/L streptomycin. The cells were cultured in a CO2 incubator with humidified atmosphere containing 5% CO2 in air at

37°C.

2.2.5. Determination of Melanin Content in B16 Melanoma Cells

The cellular melanin content was determined as previously described (Yamauchi et al, 2011; 2015; Arung et al, 2011) with minor modifications. In brief, the confluent culture of B16 melanoma cells was rinsed in phosphate-buffered saline (PBS) and removed using 0.25% trypsin/EDTA. The cells were added to a 24-well plate (0.5 × 105 cells/well) and allowed to adhere at 37°C for 24 h. After adding the samples with various concentration and arbutin was used as a positive control, furthermore cells were incubated for 72 h. Each experiment was repeated twice. The supernatant (extracellular melanin formation) was collected in an Eppendorf tube; 200 PL was transferred to a 96-well plate and the absorbance was determined at 510 nm. To determine intracellular melanin, the cells were washed with PBS, followed by lysis in 600 PL of 1N NaOH by heating for 30 min at 100qC to dissolve the