Progress 10/01/14 to 09/30/15

Outputs
Target Audience: Nothing Reported Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?The essential oil distillated from a common food spice in southern Asian countries was found to exhibit potent tyrosinase inhibitory activity in initial cell-free experiments. In addition, most of the phytochemicals were characterized as tyrosinase inhibitors in initial cell-free experiments. It cannot be stressed enough that cell-free studies alone prove the participation of the enzyme, but their further evaluation using murine B16-F10 melanoma cells was performed as a model organism. Their effects on melanogenesis using cultured B16-F10 melanoma cells are currently under experiment. Compounds that showed potential activity in cell-free and cellular experiment are going to test crop pest insects. How have the results been disseminated to communities of interest? Nothing Reported What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? In our continuing search for melanogenesis inhibitors from plants, we became aware that the essential oil obtained from Polygonum odoratum (Polygonaceae), commonly known as Vietnam coriander, was previously reported to inhibit the oxidation of both L-tyrosine and L-3,4-dihydroxyphenylalanine (L-DOPA) catalyzed by tyrosinase (EC 1.14.18.1). The 25 scent compounds were characterized in the essential oil by GC-MS analysis. Aldehyde compounds are noted to be the most abundant, followed by alcoholic compounds but to a much lesser extent. Alkanals, dodecanal (55.49%) and decanal (11.57%), were the two most abundant in the essential oil, followed by anisaldehyde (6.35%). Notably, the enzymatic activity weakens over time but it does not inactivate the enzyme. Their tyrosinase inhibitory activity was likely caused by nonspecific disruption of the tertiary structure of the protein (tyrosinase) acting as surfactants. This is consistent with the fact that non-ionic surfactants do not denature proteins. Tyrosinase was inhibited when the concentration of these alkanals in the reaction mixture exceeded a certain threshold and the inhibition of the enzyme occurred without oxygen. However, the inhibition occurs slowly in these alkanal surfactants. Tyrosinase is a key enzyme in melanin biosynthetic pathway and therefore its inhibitors are expected to suppress melanogenesis. Hence, the essential oil was tested for their effects in cultured murine B16-F10 melanoma cells. The essential oil did not suppress melanin production in the cultured cells but rather slightly enhanced it. On the other hand, the essential oil exhibited significant cell viability at the concentration of 100 µg/mL. Dodecanal and decanal are two major compounds in the essential oil and their IC50 values were obtained as 92 µM and 112.5 µM, respectively. Their cytotoxicity was in combination with their ability to disrupt the native membrane-associated function nonspecifically as surfactants, and in reaction with biologically important nucleophilic groups. However, the surfactant damage is a more likely essential role. The work was performed in collaboration with the Professor W. Chavasiri's group of Chulalongkorn University, Thailand.

Publications

• Type: Journal Articles Status: Published Year Published: 2015 Citation: Masuoka, N. Nihei, K. Yamagiwa, Y. Maeta, A. Kubo, I. 2015. Inhibitory effects of cardols and related compounds on superoxide anion generated by xanthine oxidase. Food Chem. 166:270-274.
• Type: Journal Articles Status: Published Year Published: 2015 Citation: Fujita, K. Chavasiri, W. Kubo, I. 2015. Anti-Salmonella activity of scent compounds of Polygonum odoratum. Phytother. Res. 29:1081-1087.
• Type: Journal Articles Status: Published Year Published: 2015 Citation: C�spedes, C. L. Alarc�n, J. Aqueveque, P. M. �vila, J. G. Seigler, D. S. Kubo, I. 2015. In the search for new secondary metabolites with biopesticidal properties. Israel J. Plant Sci. 65:1-15.
• Type: Journal Articles Status: Published Year Published: 2015 Citation: C�spedes, C. L. Aqueveque, P. M. �vila, J. G. Alarc�n, J. Kubo, I. 2015. New advances in chemical defenses of plants: researches in Calceolaceae. Phytochem. Res. 14:367-380.
• Type: Journal Articles Status: Published Year Published: 2015 Citation: Ohsaki, A. Hoya, T. Ozawa, M. Kishida, A. Komiyama, K. Kubo, I. 2015. Cytotoxicity of the alkaloid constituents from the seeds of Licaria puchury-major, Brazilian medicinal plant, in vincristine-resistant P388 clells. Kenkyu KiyoNihon Daigaku Bunrigakubu Shizen Kagaku, Kenkyusho. 50:295-298.

Progress 10/01/13 to 09/30/14

Outputs
Target Audience: Nothing Reported Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? The essential oil distillated from a common food spice in southern Asian countries was found to exhibit potent tyrosinase inhibitory activity in initial cell-free experiments. In addition, most of the phytochemicals were characterized as tyrosinase inhibitors in initial cell-free experiments. It cannot be stressed enough that cell-free studies alone prove the participation of the enzyme, but their further evaluation using murine B16-F10 melanoma cells was performed as a model organism. Their effects on melanogenesis using cultured B16-F10 melanoma cells are currently under experiment. Compounds that showed potential activity in cell-free and cellular experiment are going to test crop pest insects. How have the results been disseminated to communities of interest? Nothing Reported What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? In our continuing search for melanogenesis inhibitors from plants, we became aware that the essential oil obtained from Polygonum odoratum (Polygonaceae), exhibited potent in vitro cytotoxicity with respect to murine B16F10 melanoma cells. This spice plant was reclassified as Persicaria odorata (Lour.) Sojak, locally known as "Vietnam coriander" and used as a food spice in Vietnam. The work is performed in collaboration with the Professor W. Chavasiri's group of Chulalongkorn University, Thailand. The essential oil of P. odoratum was extracted by steam distillation directly from the fresh leaves which has distinct odor and tastes pungent. Twenty-five compounds were characterized from this essential oil by GC-MS. The essential oil yield was 1.84% + 0.036 (v/w) and its composition was established by 97.4% of its total peak area from the GC-MS. Notably, aldehyde compounds were found as the most abundant, followed by alcoholic compounds but to a much lesser extent. The total amount of aldehyde compounds accounts for more than 78% of the essential oil. Alkanals, dodecanal (55.49%) and decanal (11.57%), were the two most abundant in the essential oil. In addition to these two alkanals, undecanal (1.31%) and tetradecanal (0.34%) were characterized in minute amounts. In connection with aldehydes, two aromatic aldehydes; anisaldehyde (6.35%) and methyl vanillin (0.86%); an α,b-unsaturated alkenal, 2E-dodecenal (2.3%); and a cyclic sesquiterpene dialdehyde, polygodial (0.01%) were characterized. Alkanols; dodecanol (3.3%), decanol (1.13%), undecanol (0.56%); two cyclic sesquiterpenoid, drimenol (0.39%) and bisabolol (0.41%); and a cyclic monoterpenoid, neoiso-menthol (0.45%); are the second most abundant compounds but to a much lesser extent compared to aldehyde compounds. It should be noted, however, that chemical analysis of the essential oil of P. odoratum collected from different locations (Australia, Thailands and Vietnam) was previously reported. In these previous papers, dodecanal and decanal were described as the two most abundant scent compounds, but in different yield. Besides these two major alkanals, other compounds were fairly different. Since tyrosinase is a key enzyme in melanin biosynthetic pathway, effects of the essential oil on mushroom tyrosinase using L-DOPA was examined first. The enzyme activity was monitored by measuring dopachrome formation at 475 nm spectrophotometrically. The essential oil quickly inhibited the tyrosinase-generated dopachrome formation up to around first 15 minutes, and then slowed down thereafter until a nearly straight line was appeared.

Publications

• Type: Journal Articles Status: Published Year Published: 2014 Citation: Ha, T. J. Shimizu, K. Kubo, I. 2014. Lipoxygenase inhibitory activity of alkyl protocatechuates. Food Chem. 15:471-476 .
• Type: Journal Articles Status: Published Year Published: 2014 Citation: Kamatani, J. Iwadate, T. Tajima, R. Kimoto, H. Yamada, Y. Masuoka, N. Kubo, I. Nihei, K. 2014. Stereochemical investigation and total synthesis of inuloidin, a biologically active sesquiterpenoid from Heterotheca inuloides. Tetrahedron 70:3141-3145.
• Type: Journal Articles Status: Published Year Published: 2014 Citation: C�spedes, C. L. Salvazar, J. R. Armand-Castolo, A. Yamaguchi, L. �vila, J. G. Aqueveqie, P. Kubo, I. Alarc�n, J. 2014. Biopesticides from plants: Calceolaria integrifolia s.l. Environ. Res. 132:391-406.

Progress 01/01/13 to 09/30/13

Outputs
Target Audience: Scientists who have been working on melanoma related research and business. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Fullbright Foundation supported postdoctoral research associate from Kenya. UC Berkeley graduate student for her dissertation. Four undergraduate student working with graduate student to develop science. How have the results been disseminated to communities of interest? Published papers What do you plan to do during the next reporting period to accomplish the goals? Continuing current research topic and assisting graduate student dissertation.

Impacts
What was accomplished under these goals? Methyl 4-hydroxycinnamate (methyl 4-coumarate) was previously characterized as a tyrosinase inhibitor from Heterothica inuloides (Compositae). Tyrosinase is a key enzyme in melanin biosynthetic pathway and therefore its inhibitors are expected to inhibit melanin production. Effect of methyl 4-hydroxycinnamate was evaluated on B16 melanoma cells and was found to suppress melanogenesis in cultured B16 melanoma cells. Methyl 4-hydroxycinnamate exhibited cytotoxicity but this activity was restored in a dose dependent manner by ascorbic acid. The results suggest involvement of the intracellular oxidation mechanism since the neutral molecule, methyl 4-hydroxycinnamate, enters into the cells by passive diffusion across the membrane. On the other hand, methyl 4-methoxycinnamate, which lacks the oxidizable phenolic hydroxyl group, still exhibited comparable cytotoxicity to that of methyl 4-hydroxycinnamate. In addition, methyl 3-(4-methoxyphenyl) propionate did not show any noticeable cytotoxicity, indicating that the double bond in the enone moiety is an essential element in eliciting this activity. Thus, the enone moiety is known to react with biologically important nucleophilic groups as a Michael reaction acceptor. Antioxidant activity of methyl 4-hydroxycinnamate is known and the formation of antioxidant-related phenoxy radical mediates the cidal effect. Another potent tyrosinase inhibitor, 5-pentadecanylresorcinol was isolated from the cashew Anacardium occidentale (Anacardiaceae). The resorcinol moiety first quickly and reversibly competes with the binuclear active site as a substrate analogue, and then the hydrophobic pentadecanyl side chain slowly and irreversibly interacts with the hydrophobic domain in close proximity to the active site in the enzyme (cooperative inhibition). Notably, this hydrophobic interaction disappeared when the reaction mixture was stirred. Consequently, the inhibitory activity could be monitored by measuring by dopachrome formation spectrophotometrically (in static condition) but could not be monitored by measuring oxygen consumption (in stirring condition). The inactivation of the enzyme underwent without oxygen and could be explained by their specific structural feature of cardols depending on their alkyl chain lengths. Thus, the hydrophobic pentadecanyl group plays an important role to inactivate tyrosinase since 5-pentylresorcinol did not inactivate this enzyme. Compounds that showed potential activity in cell-free and cellular experiment are going to test crop pest insects.

Publications

• Type: Journal Articles Status: Published Year Published: 2013 Citation: PUBLICATIONS: 2013/01 TO 2013/09 Mu��oz, E., Avila, J., Alarc�n, J., Kubo, I., Werner, E. and C�spedes, C. L. 2013. Tyrosinase inhibitors from Calceolaria integrifolia s.l.: Calceolaria talcana aerial parts. J. Agric. Food Chem. 61:4336-4343. Murata, W., Tanaka, T., Fujita, K. and Kubo, I. 2013. Protective effects of ?-tocopherol and ascorbic acid against cardol-induced cell death and reactive oxygen species generation in Staphylococcus aureus. Planta Med. 79:768-774. Kubo, I. and C�spedes, C. L. 2013. Antifungal activity of alkanols: inhibition of growth of spoilage yeasts. Phytochem. Rev. 12:961-977. C�spedes, C. L., Mu��oz, E., Salvazar, J. R., Yamaguchi, L., Werner, E., Alarcon, J. and Kubo, I. 2013. Inhibition of cholinesterase activity by extracts, fractions and compounds from Calceolaria talcana and C. integrifolia (Calceolariaceae: Scrophlariaceae). Food Chem. Toxicol. 62:919-926. Masuoka, N., Shimizu, K. and Kubo, I. 2013. Antioxidant activity of anacardic acids. In Antioxidants and Biocidals from Latin American Plants (C. L. C�spedes, D. A. Sampietro and D. S. Seigler, Eds) Cabi Publishing, London, pp. 137-147. Kubo, I., Fujita, K. and Shimizu, K. 2013. Anti-Salmonella agents from the Brazilian medicinal plant Tanacetum balsamita and their applications. In Antioxidants and Biocidals from Latin American Plants (C. L. C�spedes, D. A. Sampietro and D. S. Seigler, Eds) Cabi Publishing, London, pp. 239-253.

Progress 01/01/12 to 12/31/12

Outputs
OUTPUTS: Two hydrolyzable tannins were isolated as fungal protease inhibitors first from the root of Fuchsia tetradactyla LINDL (Onagraceae) collected in the highlands of Guatemala. Their subsequent assays to test for their effects various oxidases found that both tannins did not inhibit the oxidation of L-3,4-dihydroxyphenylalanine (L-DOPA) catalyzed by mushroom tyrosinase (EC 1.14.18.1) but were rather easily oxidized as soon as both were mixed with the enzyme. The available oxygen in the reaction mixture was used for the oxidation of both tannins and L-DOPA, and the enzymatically generated intermediates produced complex mixtures, and thus both tannins suppressed the initial rate of pigmented products formation. These tannins were also found to exhibit cytotoxicity against murine B16-F10 melanoma cells. In addition, gallic acid (3,4,5-trihydroxybenzoic acid) was characterized in a large quantity from the same source, but it acted neither as a substrate nor an inhibitor. However, gallic acid was oxidized by a redox reaction to the corresponding o-quinone. Thus, the enzymatically oxidized tannins and dopaquinone oxidized gallic acid as redox cyclers. This redox reaction-generated o-quinone condensed with one another through a Michael type addition, yielding a relatively stable dibenzotropolone intermediate, purpurogallincarboxylic acid. PARTICIPANTS: see publications TARGET AUDIENCES: UC and ANR researchers Researchers at other institutions Public Graduate students Undergraduate students PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
Tannic acid was enzymatically oxidized as soon as it was mixed with the enzyme. The available oxygen in the reaction mixture was mainly used for the oxidation of tannic acid and the enzymatically generated oxidized intermediates oxidized gallic acid as a redox cycler. Tannic acid exhibited potent cytotoxicity against the melanoma cells with an IC50 of 7 μM and almost complete lethality was observed at 20 μM. Tannic acid did not inhibit tyrosinase activity but was quickly oxidized as a substrate and the enzymatically oxidized product act as a redox cycler. Melanogenesis was not suppressed in the cultured melanoma cells by tannic acid, but slightly enhanced it.

Publications

• Satooka, H. and Kubo, I. 2012. Resveratrol as a kcat type inhibitor for tyrosinase: Potentiated melanogenesis inhibitor. Bioorg. Med. Chem. 20:1090-1099.
• Kubo, I., Ha, T. J. and Shimizu, K. 2012. Lipoxygenase inhibitory activity of alkyl protocatechuate. Chapter 8, In Soybean. A Review-Book 3 (J. Board, Ed.), InTech, Croatia, pp. 183-197.
• Kubo, I., Shimizu, K. and Fujita, K. 2012. Naturally occurring antifungal agents and their modes of action. In Fungicides for Plant and Animal Diseases (D. Dhanasekaran, N. Thajuddin and A. Panneerselvam, Eds.), InTech, Croatia, pp. 55-80.
• Kubo, I., Shimizu, K. and Nihei, K. 2012. Manicoba, a quercetin rich amazonian dish: Antioxidative prospects. Chapter 6, In Quercetin: Dietary Sources, Functions and Health Benefits (T. Chikamatsu and Y. Hida, Eds.), Nova Science Publishers, Hauppauge, pp. 129-154.
• Masuoka, N., Matsuda, M., Kubo, I. 2012. Characterization of the antioxidant activity of flavonoids. Food Chem. 131:541-545.
• Satooka, H. and Kubo, I. 2012. Effects of thymol on B16-F10 melanoma cells. J. Agric. Food Chem. 60:2746-2752.
• Satooka, H., Isobe, T., Nitoda, T. and Kubo, I. 2012. Melanogenesis inhibitors from Rabdosia japonica. Phytomedicine 19:1016-1023.
• Masuoka, N., Nihei, K., Masuoka, T., Kuroda, K., Sasaki, K. and Kubo, I. 2012. The inhibition of uric acid formation catalyzed by xanthine oxidase. Properties of the alkyl caffeates and cardol. J. Food Res. (Canada) 1:251-262.
• Ha, T. J., Lee, M.-H., Kim, H.-T., Kwon, H. S., Baek, I.-Y., Kubo, I. and Jang, D. S. 2012. Slow-binding inhibition of soybean lipoxygenase-1 by luteolin. Arch. Pharm. Res. 35:1811-1816.

Progress 01/01/11 to 12/31/11

Outputs
OUTPUTS: Methyl coumarate (methyl 4-hydroxycinnamate) isolated from the fresh flower of Trixis michuacana var longifolia (D. Dow) C. Anderson (Compositae), locally known as "Hierberina Salud" in Mexico, was found to significantly suppress the melanin formation in cultured murine B16-F10 melanoma cells, whereas coumaric acid (4-hydroxycinnamic acid) did not show this anti-melanogenesis activity. Methyl coumarate exhibited weak cytotoxicity. This citotoxicity was reversed by the addition of vitamin C dose-dependently but not by BHA, indicating the intracellular oxidation mechanism. Neutral molecule methyl coumarate enters into the cells by passive diffusion across the membrane. Once inside the cells, methyl coumarate is oxidized to the corresponding toxic quinone methide intermediate which binds with various cellular materials. Methyl cinnamate also exhibited cytotoxicity comparable to methyl coumarate but methyl 3-phenylpropionate did not possess this activity, indicating that the double bond in the enone moiety is an essential element in eliciting the activity. Both methyl coumarate and methyl cinnamate exhibited cytotoxicity but in different ways. Based on continuing structure-melanine production inhibitory activity relationship study, presence of phenolic hydroxyl group of thymol or carvacrol was found as an essential element in eliciting the inhibitory activity. Further experiments suggest that both monoterpenoids did not show any involvement on the enzymatic reactions. Thus, thymol is not a "tyrosinase" inhibitor. The formation of dopachrome from dopaquinone is extremely rapid and colorless. Therefore, alternative model of this reaction is required to test whether thymol affects this non-enzymatic process. Based on the new results, thymol quenched the redox reaction between L-DOPA and p-benzoquinone. Based on data obtained, thymol inhibits the redox reaction in melanin synthetic pathway. This inhibition of melanin formation is likely due to radical scavenging activity of thymol. The electron donating isopropyl and methyl groups in ortho position contribute to stability of phenoxy radical and their antioxidative activities. However, structural hindrance due to methyl or isopropyl group adjacent to hydroxyl group prevents for tyrosinase to recognize thymol as a monophenol. Thus, thymol is considered as a unique melanin synthesis inhibitor, but not an enzyme inhibitor. As described above, the antimelanogenic activity of thymol is linked with its antioxidant effect; in fact, melanin formation inhibitor, thymol, can help human health from oxidative damage as well as the prevention of undesirable browning. Radical-scavenging effect of thymol depletes an active electron in melanin synthesis, but antioxidant-related phenoxy radicals are newly formed. Hence, antioxidants are also known as a double-edged sword. In the case of thymol, it has been reported useful antioxidant properties without prooxidant effects. PARTICIPANTS: Not relevant to this project. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Not relevant to this project.

Impacts
In our continuing efforts to search for melanin synthesis inhibitors without affecting cell growth, two classes of chemicals have been found. The possibility that the observed inhibition of cellular melanin synthesis was due to their ability to chelate copper in the enzyme is very likely. The results obtained may indicate a more appropriate process to search for tyrosinase inhibitors and melanogenesis regulators. Methyl cinnamate is found naturally in a variety of plants, including in fruits, like strawberry, and some culinary spices, such as Sichuan pepper and some varieties of basil. Melanin production inhibitors isolated from regularly consumed foods and beverages, as well as their processed products, may be more effective than synthetic products.

Publications

• Kubo, I., Nitoda, T., Tocoli, F. E. and Green, I. R. 2011. Multifunctional cytotoxic agents from Anacardium occidentale. Phytother. Res. 25, 38-45.
• Satooka, H. and Kubo, I. 2011. Effects of thymol on mushroom tyrosinase-catalyzed melanin formation. J. Agric. Food Chem. 16, 8908-8914.
• Yutani, M., Hashimoto, Y., Ogita, A., Kubo, I., Tanaka, T. and Fujita, K. 2011. Morphological changes of filamentous fungus Mucor mucedo and inhibition of chitin synthase activity induced by anethole. Phytother. Res. 25, 1707-1713.
• Kubo, I., Lee, S. H. and Shimizu, K. 2011 Combination effect of miconazole with polygodial against Candida albicans. Open J. Med. Microbiol. 1, 7-11.

Progress 10/01/05 to 09/30/10

Outputs
OUTPUTS: Tyrosinase (EC 1.14.18.1), also known as polyphenol oxidase (PPO), is a copper containing mixed-function oxidase widely distributed in microorganisms, animals and plants. Tyrosinase is a key enzyme in the melanin synthetic pathway and therefore tyrosinase inhibitors are expected to inhibit melanin production. Our overall project objective is to develop ecologically friendly and environmentally acceptable insect control agents from botanical sources. Emphasis has now been placed on melanin production control agents based on natural products. Despite significant advances in recent decades in the techniques used for control insects continues to pose serious problems. The purpose of our AES project is to develop alternative insecticides by disrupting insect PO activity since tyrosinase is a key enzyme in insect cuticle sclerotization and melaninzation. However, we have also recognized that many phytochemicals exhibit potent cytotoxicity against murine B16-F10 melanoma cells which has been used as a model. By now, we have recognized that the inhibition of tyrosinase activity was not correlated with that of cellular tyrosinase and melanin production in cultured melanocytes. The results obtained may provide not only more scientifically sound and environmentally acceptable insect control agents but also information to understand melanin related diseases on a molecular basis. The inhibition of tyrosinase activity was previously described not correlative well with that of melanogenesis in cultured melanocytes. Thus, melanogenesis inhibition is not simply to inhibit tyrosinase but involves more complex biochemical reactions of which there is a lack of information and needs to be addressed. The proposed project will be performed mainly by two graduate students her at UC Berkeley. In addition, Professor Warinthon Chevasiri, Department of Chemistry, Chulalongkorn University, Bangkok, Thailand provides us plant crude extracts. The collaboration work has recently reached a stage where Dr. Chavasiri's group performs preliminary bioassay in conjunction with this lab. Two students will come from Thailand to Berkeley to learn and perform additional bioassay using melanoma cells and will be funded by their own institute. One of our former postdocs from Faculty of Agriculture, Kyushu University, Professor Kuniyoshi Shimizu, is now one of the world leaders in melanin science after working on this project for two years.In addition, an undergraduate student who has been involved melanin study and presented data he collected at a department student symposium. Lastly, the research has been presented at various conferences and symposiums such as the ACS and AAA annual meetings. PARTICIPANTS: Nothing significant to report during this reporting period. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
Many compounds have been tested to attempt to inhibit Tyrosinase in B16-F-10 melanoma cells. Tyrosinase inhibition was not correlative with that of melanogenesis in cultured melanocytes of murine B16-F-10 melanoma cells. When the same melanoma cells were cultured with 4-hydroxyanisole, melanogenesis was suppressed, but 4-hydroxyanisole exhibited potent cytotoxicity against the same melanoma cells and therefore the observed melanogenesis inhibition was due mainly to its melanocytotoxic effect. 5-Alk(en)ylresorcinols, commonly known as cardols isolated from various edible plants such as mango and pistachio, have been found to inactivate tyrosinase. The enzyme first reversibly binds cardols as a substrate analogue and then undergoes a slow irreversible course to inactivation. The pentadeca(en)yl side chain in cardols is an essential element to elicit the tyrosinase inactivation activity, presumably by interacting with the hydrophobic domain close to the binuclear copper active site. In a preliminary assay, one of the cardols tested has been found to slightly suppress melanogenesis in cultured murine B16-F10 melanoma cells up to 5 μM without affecting cell growth. Melanogenesis does not simply inhibit tyrosinase but involves more complex biochemical reactions and needs to be evaluated by direct usage of melanoma cells. However, some tyrosinase inhibitors characterized from plants suppressed melanin formation in cultured melanoma cells. 4-Methoxysalicylaldehyde isolated from various plants was found to inhibit the oxidation of L-tyrosine catalyzed by tyrosinase without itself being oxidized. The experiment was extended to test for the effects of this salicylaldehyde derivative on melanogenesis in cultured murine B16-F10 melanoma cells. Notably, melanogenesis was significantly suppressed in a dose-dependent manner without affecting cell growth up to 25 micro Moles. Similarly, chamaecin (4-isopropylsalicylaldehyde) was found to suppress melanogenesis without affecting cell growth up to 400 μM when the same melanoma cells were cultured. Both compounds possess a salicylaldehyde moiety and suppress melanin production in cultured B16 melanoma cells as a copper chelator. Hence, salicylaldehyde moiety is likely an essential element to elicit melanogenesis inhibitory activity. Based on the salicylaldehyde scaffold concept, various salicylaldehyde derivatives can be synthesized and their evaluation is in preparation. Based on the salicylaldehyde scaffold concept, various salicylaldehyde derivatives can be synthesized and their evaluation is in progress. Similarly, various resorcinol derivatives can be synthesized and tested for their effects on murine B16-F10 melanoma cells. When considered separately the head and tail moieties cardols suggests that optimization of melanogenesis inhibitory activity is achievable through a systematic synthetic approach. Based on the resorcinol scaffold concept, various resorcinol derivatives can be designed and synthesized for their further evaluation. All of these results will contribute to future testing on insects to prevent melanogensis by providing a framework of previously tested compounds.

Publications

• Kubo, I., Shimizu, K., Ha, T. J., Masuoka, N. and Nihei, K. 2010. Multifunctional antioxidant activities of alkyl gallates. Open Bioact. Compd. J. Kubo, I., Ha, T. J. and Shimizu, K. 2010. Lipoxygenase inhibitory activity of 6-pentadecanylsalicylic acid without prooxidant effects. Nat. Prod. Commun. 5:85-90.
• Ha, T. J., Shimizu, K., Ogura, T. and Kubo, I. 2010. Inhibition mode of soybean lipoxygenase-1 by quercetin. Chem. Biodiv. 7:1893-1903.
• Kubo. I. 2010. Manicoba, quercetin rich Amazonian dish and lipoxygenase inhibitory action in VI Meeting of the Latin American Phytochemistry Society and the II Phytotherapics Conference of Mercosul, Belo Horizonte, Minas Gerais, pp 61-66.

Progress 01/01/09 to 12/31/09

Outputs
OUTPUTS: Tyrosinase, also known as polyphenol oxidase (PPO) and phenyloxidase (PO), is a copper containing mixed-function oxidase widely distributed in microorganisms, animals and plants. PPO is responsible for browning in plants and considered to be deleterious to the color quality of plant derived foods and beverages. This unfavorable darkening from enzymatic oxidation generally results in a loss of nutritional and economic values and has been of great concern. PO is a key enzyme in insect cuticle sclerotization and melaninzation and occupies several major roles in insect development and immunity. This mixfunction oxidase is a key enzyme in the melanin synthetic pathway and the oxidation of L-tyrosine catalyzed by this oxidase to melanin is important since melanin has many functions. Alterations in melannogenesis occur in many disease states. For example, melanoma specific anticarcinogenic activity is known to be linked with tyrosinase activity. Melanin synthesis inhibitors can be developed as alternative insecticides by disrupting insect PO activity. The tyrosinase inhibitors isolated from many sources have been monitored by dapachrome formation. As long as the enzyme activity was monitored by dapachrome formation, various compounds were characterized as tyrosinase inhibitors. However, this inhibitory activity was not present when the longer reaction time was observed. Hence, the compounds have been reevaluated by a more appropriate polarographic method. 5-Alk(en)ylresorcinols, commonly known as cardols isolated from various edible plants such as mango and pistachio, have been found to inactivate the enzyme. The inactivation of the enzyme by cardols does not require oxygen, indicating that inactivation of the enzyme by products of the reaction is not involved. This tyrosinase inactivation activity of cardols can be explained by their specific structural feature. The enzyme first reversibly binds cardols as a substrate analogue and then undergoes a slow irreversible course to inactivation. The pentadeca(en)yl side chain in cardols is an essential element to elicit the tyrosinase inactivation activity, presumably by interacting with the hydrophobic domain close to the binuclear copper active site. In a preliminary assay, one of the cardols tested has been found to slightly suppress melanogenesis in cultured murine B16-F10 melanoma cells up to 5 μM without affecting cell growth. When considered separately, the head and tail moieties cardols suggests that optimization of melanogenesis inhibitory activity is achievable through a systematic synthetic approach. Based on the head and tail concept, a series of alkyl 3,5-dihydroxybenzoates has been synthesized and their evaluation is in progress. Similarly, resveratol (3,5,4'-trihydroxystilbene) isolated from wine was found to suppress melanogenesis without affecting cell growth up to 400 μM when murine B16-F10 melanoma cells were cultured with this stilbene derivative. Interestingly, resveratrol was oxidized by tyrosinase as a substrate, indicating that inactivation of the enzyme was caused by oxidation products of the reaction. PARTICIPANTS: Nothing significant to report during this reporting period. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
The inhibition of tyrosinase activity is not correlated with that of melanogenesis in cultured melanocytes. Melanogenesis inhibition is not simply to inhibit tyrosinase but involves more complex biochemical reactions of which there is a lack of information and needs to be addressed. The results obtained may indicate a more appropriate process to search for tyrosinase inhibitors and melanogenesis regulators.

Publications

• Kubo, I., Ha, T. J., Nitoda, T., Shimizu, K. and Kubo, A. 2008. Molecular design of acne control agents. Current Topics in Phytochemistry, 9:95-102.
• Ochi, M., Moriyama, K., Ohmae, K., Fukuyama, Y., Nihei, K. and Kubo, I. 2009. Sweet and bitter constituents of Wilbrandia species. Food Chem. 115:61-65.
• Kubo, I., Fujita, K. and Kubo, A. 2009. Naturally occurring anti-Salmonella agents and their modes of action. In Novel Therapeutic Agents from Plants: Progress and Future Perspectives (M. Rai and M. C. Carpinella, eds.) Science Publisher, Enfield, pp. 60-83.

Progress 01/01/08 to 12/31/08

Outputs
OUTPUTS: Constituents characterized from mycelium and sporocarp of American matsutake mushroom, Tricholoma magnivelare were tested for their effects on mushroom tyrosinase (EC 1.14.18.1) and murine B16-F10 melanoma cells. In a preliminary spectrophotometric assay, none of the compounds tested exhibited noticeable effects on the enzymic oxidation of L-3,4-dihydroxyphenylalanine (L-DOPA). However, methyl cinnamate significantly inhibited the tyrosinase activity when L-tyrosine was used as a substrate. In addition, the subsequent cellular assay, melanogenesis was significantly suppressed in melanocytes when murine B16-F10 melanoma cells were cultured with methyl cinnamate without affecting cell growth up to 50 μM. A rather rare naturally occurring chlorinated benzaldehyde derivative, 3,5-dichloro-4-methoxybenzaldehyde isolated from the same source, was found to show potent cytotoxicity with IC50 values of 22 μM and almost complete lethality was observed at 100 μM. Chlorine atom reduced tyrosinase inhibitory activity but enhanced cytotoxicity. Benzaldehyde derivatives usually inhibited the tyrosinase-catalyzed oxidation of L-DOPA (diphenolase activity) as long as the enzyme activity was monitored by measuring the steady-state oxidation of L-DOPA to dopachrome. This diphenolase inhibitory activity disappeared when a longer reaction time was observed. On the other hand, the same benzaldehydes significantly inhibited the tyrosinase-catalyzed oxidation of L-tyrosine (monophenolase activity) in different ways. The benzaldehyde derivatives exhibited antiproliferation activity against murine B16-F10 melanoma cell line in a dose-dependent manner as cross-linking agents, presumably by forming Schiff bases with primary amino groups in intracellular targets. The activity was significantly enhanced by introducing an additional hydroxyl group at the ortho-position of benzaldehyde, but the effect at the para-position was much lesser. Morphological observation showed that 4-hydroxybenzaldehyde induced B16-F10 melanoma cells to produce dendrite-like structure. In addition, flavor constituents characterized from the fresh leaves of Polygonum odoratum, locally known as "rau ram" and used as a food spice in Vietnam, were tested for their effects on murine B16F10 melanoma cells. None of them suppressed melanogenesis in cultured melanoma cells but rather enhanced it. Dodecanol exhibited cytotoxicity with IC50 of 60 μM and almost complete lethality was observed at 200 μM. In the case of 2E-alkenals, 2E-decenal was found to show cytotoxicity with an IC50 of 30 μM, followed by 2E-decenal with an IC50 of 50 μM. However, the cytotoxicity obtained were not the values of 2E-alkenals themselves but their adducts because they easily formed adducts in the medium (10%FBS-DMEM). PARTICIPANTS: Nothing significant to report during this reporting period. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
The results obtained may provide more scientifically sound and environmentally acceptable insect control agents. In addition, melanin has many functions in living systems and alterations in melanin synthesis occur in many disease states. Consequently, modulators of melanogenesis have assumed great importance for prevention of hypo- and hyper-pigmentary disorder for therapeutic purpose.

Publications

• Kubo, I., Fujita, K. and Nihei, K. 2008. Antimicrobial activity of anethole and related compounds from aniseed. J. Sci. Food Agric. 88:242-247.
• Nitoda, T., Isobe, T. and Kubo, I. 2008. Effects of phenolic compounds isolated from Rabdosia japonica on B16-F10 melanoma cells. Phytother. Res. 22:867-872.
• Kubo, I., Ha, T. J., Tsujimoto, K., Tocoli, F. E. and Green, I. R. 2008. Evaluation of lipoxygenase inhibitory activity of anacardic acids. Z. Naturforsch. C: J. Biosci. 63:539-546.
• Fujita, K., Fujita, T. and Kubo, I. 2008. Antifungal activity of alkanols against Zygosaccharomyces bailii and their effects on plasma membrane. Phytother. Res. 22:1349-1355.
• Kubo, I., Nihei, K., Satooka, H., Cespedes, C. L. and Calderon, J. S. 2008. Insect growth inhibitory activity and cytotoxicity of tannic acid from Galla Rhois. Biopestic. Int. 4:6-14.
• Cespedes, C. L., Alarcon, J., Avila, J. G. and Kubo, I. 2008. Antioxidant and biocide activities of selected Mexican and Chilean plants. in ACS Symposium Series 993, Functional Food and Health, pp. 277-306.
• Nitoda, T., Fan, M. D. and Kubo, I. 2008. Effects of cuminaldehyde on melanoma cells. Phytother. Res. 22:809-813.
• Green, I. R., Tocoli, F. E., Lee, S. H., Nihei, K. and Kubo, I. 2008. Design and evaluation of anacardic acid derivatives as anti-cavity agents. J. Eur. Med. Chem. 43:1315-1320.

Progress 01/01/07 to 12/31/07

Outputs
2-Hydroxy-4-methoxybenzaldehyde isolated from various plants significantly suppressed the oxidation of L-3,4-dihydroxyphenylalanine (L-DOPA) catalyzed by tyrosinase as a partial mixed type inhibitor without itself being oxidized. The mixed type inhibition comes from its ability to chelate copper in the active site and to form Schiff base adducts with a primary amino group in the enzyme. The same compound exhibited cytotoxicity against murine B16-F10 melanoma cells, but melanin production was suppressed in a dose-dependent manner without affecting cell growth. A related benzaldehyde, 3,5-dichloro-4-methoxybenzaldehyde isolated from the matsutake mushroom did not suppress melanogenesis in cultured melanoma cells but rather significantly enhanced it. Cardols, 5-pentadec(en)ylresorcinols isolated from the cashew, were found to inhibit the oxidation of L-tyrosine and L-DOPA catalyzed by tyrosinase without pro-oxidant effects. The hydrophobic pentadeca(en)yl side chain plays an important role to inhibit the enzyme activity. Thus, the resorcinol moiety first quickly competes with the binuclear active site as a substrate analogue, and then the hydrophobic alk(en)yl side chain slowly interacts with the hydrophobic domain close to the active site in the enzyme. Subsequently, cardols were found to slightly suppress melanogenesis in cultured murine B16-F10 melanoma cells without affecting cell growth. However, it exhibited potent cytotoxicity at higher concentrations, and this activity was found to come from their ability to disrupt the native membrane-associated function nonspecifically as surfactants. Since cardols are the derivatives of resorcinol with an alk(en)yl side chain, their activity was compared with that of resorcinol. Resorcinol exhibited weak effect on the tyrosinase catalyzed oxidation of L-DOPA to dopachrome but inhibited its further oxidation. Resorcinol did not suppress melanogenesis in cultured murine B16-F10 melanoma cells but rather slightly enhanced it. Simultaneously, resorcinol was noted to exhibit cytotoxicity in a dose-dependent manner and this activity was noted to come from partly its intracellular oxidation.

Impacts
Melanin has many functions in living systems and alterations in melanin synthesis occur in many disease states. The results obtained may provide not only more scientifically sound and environmentally acceptable insect control agents but also information to understand melanin related diseases.

Publications

• Nitoda, T., Fan, M. D. and Kubo, I. 2007. Anisaldehyde, a potent melanogenesis potenciator. Z. Naturforsch. C: J. Biosci. 62:143-149.
• Tamura, S., Nitoda, T. and Kubo, I. 2007. Effects of salicylic acid on mushroom tyrosinase and B16-F10 melanoma cells. Z. Naturforsch. C: J. Biosci. 62:227-233.
• Kubo, I., Nitoda, T. and Nihei, K. 2007. Effects of quercetin on mushroom tyrosinase and B16-F10 melanoma cells. Molecules 12:1045-1056.
• Ha, T. J. and Kubo, I. 2007. Slow-binding inhibition of soybean lipoxygenase-1 by dodecyl gallate. J. Agric. Food Chem. 55:446-451.
• Fujita, K., Fujita, T. and Kubo, I. 2007. Anethole, a potential antimicrobial synergist, converts a fungistatic dodecanol to a fungicidal agent. Phytother. Res. 21:47-51.
• Hayashi, A., Taniguchi, N., Tsujimoto, K. and Kubo, I. 2007. Mass spectrometric elucidation of phenolic oxidation process with Cu2+-adducts. J. Mass. Spectrom. Soc. Jpn. 55:7-13.
• Green, I. R., Tocoli, F. E., Lee, S. H., Nihei, K. and Kubo, I. 2007. Molecular design of anti-MRSA agents based on the anacardic acid scaffold. Bioorg. Med. Chem. 15:6236-6241.
• Tsujimoto, K., Hayashi, A., Ha, T. J. and Kubo, I. 2007. Mass spectrometric elucidation of anacardic acids and ferric ion chelation. Z. Naturforsch. C: J. Biosci. 62:710-716.
• Orozco, A., Ogura, T., Hirosawa, T., Garduno, R. and Kubo, I. 2007. In hydrolyzed cow's milk Helicobacter pylori becomes nonculturable and the growth of Salmonella typhi and Escherichia coli is inhibited. J. Food Sci. 72:M305-M309.
• Kubo, I., Nihei, K., Ha, T. J., Nitoda, T. and Masuoka, N. 2007. Antioxidants from arnica, a Mexican medicinal plant Heterotheca inuloides. In Recent Developments in Medicinal Plant Research (A. Capasso, ed.) Research Singpost, Kerala, pp. 193-216.

Progress 01/01/06 to 12/31/06

Outputs
Anisaldehyde was previously characterized as tyrosinase inhibitors in a cell free experiment. Tyrosinase is a key enzyme in the melanin synthetic pathway and therefore the effects on melanin formation in cultured melanocytes using murine melanoma cells were examined. It did not inhibit melanogenesis in cultured murine melanoma cells but enhanced it. This adverse effect of anisaldehyde was accompanied by melanocytotoxicity in a dose-dependent manner. The melanin content per cell was increased 5-fold indicating a 400% increase compared to control. Anisaldehyde was also examined against cultured human melanoma cells. The paper on this subject is in press now and a student who was involved this project was accepted by Cornel University Medical School. Chamaecin was previously found to be the most potent tyrosinase inhibitor among the tyrosinase inhibitors in cell-free experiment. However, it did not suppress noticeable melanogenesis in melanocytes. It exhibited potent cytotoxicity instead. On the other hand, quercetin was oxidized as a substrate to the corresponding o-quinone catalyzed by tyrosinase and subsequent isomerization to p-quinone methide type intermediate. This was followed by the addition of water on C-2 yielding a relatively stable intermediate, 2(hydroxybenzoyl)-2-hydroxybenzofuran-3(2H)-one. Melanogenesis was not suppressed in melanocytes but rather enhanced it when murine melanoma cells were cultured with quercetin. This adverse effect of quercetin was accompanied by melanocytotoxicity in a dose-dependent manner. The oxidized intermediate unlikely plays a role to elicit cytotoxicity. Rutin did not exhibit any activities observed with quercetin in cell free and cellular experiments. In addition, a student involved this project was accepted by UCLA's Dental School. Arbutin inhibited monophenolase activity of L-tyrosine and suppressed melanin production in cultured murine melanoma cells. This melanogenesis inhibitory activity was caused in part by intracellular oxidation of arbutin. The goal of the project is to develop alternative insecticides by disrupting insect phenoloxidase activity. This mixed-function oxidase is a key enzyme in insect cuticle sclerotization and melaninzation and occupies several major roles in insect development and immunity. Hence, the data expected was to inhibit melanin production without affecting cell growth. The benzaldehyde derivatives tested were found to affect cultured melanoma cells in different ways. The inhibition of tyrosinase activity was not correlated with that of cellular tyrosinase or with melanin production in cultured melanocytes.

Impacts
The results obtained may provide more scientifically sound and environmentally acceptable insect control agents.

Publications

• Kubo, I., Masuoka, N., Ha, T. J. and Tsujimoto, K. 2006. Antioxidant activity of anacardic acids. Food Chem. 99:555-562.
• Masuoka, N., Isobe, T. and Kubo, I. 2006. Antioxidants from Rabdosia japonica. Phytother. Res. 20:206-213.
• Kubo, I., Masuoka, N., Nihei K. and Burgheim, B. 2006. Manicoba, a quercetin-rich Amazonian dish. J. Food Composit. Anal. 19:579-588.
• Nihei, K., Asaka, Y., Mine, Y., Ymada, Y., Iigo, M., Yanagisawa, Y. and Kubo, I. 2006. Musidunin and musiduol, insect antifeedants from Croton jatrophoides. J. Nat. Prod. 69:975-977.
• Masuoka, N., Nihei, K. and Kubo, I. 2006. Effects of alkyl gallates on xanthine oxidase. Mol. Nutr. Food Res. 50:725-731.
• Hayano, F. and Kubo, I. 2006. Novel synergic combinations of aminoglycosides with natural potentiators. Nippon Bunri Daigaku Kankyo Kagaku Kenkyusho. 5:31-35.
• Kubo, I., Fujita, K., Nihei, K. and Kubo, A. 2006. Molecular design of anti-Salmonella agents based on natural products. In Lead Molecules from Natural Products: Discovery and New Trends Advances in Phytomedecine Vol. 2. (M. T. H. Khan and A. Ather, eds.). Elsevier, Amsterdam, pp. 353-372.
• Kubo. I. 2006. New concept to search for alternate insect control agents from plants. In Naturally Occurring Bioactive Compounds: Advances in Phytomedecine Vol. 2. (M. Rai, and M. C. Carpinella, eds.) Elsevier, Amsterdam, pp. 61-80.

Progress 01/01/05 to 12/31/05

Outputs
A series of benzaldehyde derivatives was previously characterized as tyrosinase inhibitors in a cell free experiment. Tyrosinase is a key enzyme in the melanin synthetic pathway and so the effects on melanin formation in cultured melanocytes using murine B16-F10 melanoma cells were examined. The goal of the project is to develop alternative insecticides by disrupting insect phenoloxidase activity. This mixed-function oxidase is a key enzyme in insect cuticle sclerotization and melaninzation and occupies several major roles in insect development and immunity. Hence, the data expected was to inhibit melanin production without affecting cell growth. The benzaldehyde derivatives tested were found to affect cultured melanoma cells in different ways. The inhibition of tyrosinase activity was not correlated with that of cellular tyrosinase or with melanin production in cultured melanocytes. Most of the benzaldehyde derivatives tested were found to exhibit potent cytotoxicity. For example, chamaecin (2-hydroxy-4-isopropylbenzaldehyde) was characterized as a potent tyrosinase inhibitor, exhibiting potent cytotoxicity while inhibiting melanin formation in the cultured melanocytes. On the other hand, anisaldehyde (4-methoxybenzaldehyde) did not inhibit melanogenesis in cultured melanoma cells but rather significantly enhanced it. This adverse effect of anisaldehyde was accompanied by melanocytotoxicity in a dose-dependent manner. The melanin content per cell was increased 5-fold indicating a 400% increase compared to control and morphological observation showed deposits of melanin-like granules. However, its structurally closely related analogue, 2-hydroxy-4-methoxybenzaldehyde, previously characterized as a potent tyrosinase inhibitor exhibited expected results. As melanoma cells were cultured with this benzaldehyde derivative, melanin production was suppressed without affecting cell growth in a dose-dependent manner. This activity was not noticeable with cultured human A375 melanoma cells.

Impacts
The results obtained may provide more scientifically sound and environmentally acceptable insect control agents.

Publications

• Nihei, K., Ying, B.-P., Murakami, H., Matsuda, N., Hashimoto, M. and Kubo, I. 2005. Pachyelasides A, B, C, and D; Novel molluscicidal triterpene saponins from Pachyelasma tessmannii. J. Agric. Food Chem. 53:608-613.
• Nihei, K., Asaka, Y., Mine, Y. and Kubo, I. 2005. Insect antifeedants from Croton jatrophoides: Structures of zumketol, zumsenin and zumsenol. J. Nat. Prod. 68:244-247.
• Kubo, I., Lee, S. H. and Ha, T. J. 2005. Combination effect of EDTA with polygodial on the growth of Saccharomyces cerevisiae. J. Agric. Food Chem. 53:1818-1822.
• Ha, T. J. and Kubo, I. 2005. Lipoxygenase inhibitory activity of anacardic acids. J. Agric. Food Chem. 53:4350-4354.
• Fujita, K. and Kubo, I. 2005. Naturally occurring antifungal agents against Zygosaccharomyces bailii and their synergism. J. Agric. Food Chem. 53:5187-5191.
• Ha, T. J., Tamura, S. and Kubo, I. 2005. Effects of mushroom tyrosinase on anisaldehyde. J. Agric. Food Chem. 53:7024-7028.
• Fujita, K. and Kubo, I. 2005. Multifunctional action of antifungal polygodial against Saccharomyces cerevisiae: Involvement of pyrrole formation on cell surface in antifungal action. Bioorg. Med. Chem. 13:6742-6747.
• Kubo, I., Fujita, K. and Nihei, K. 2005. Molecular design of antioxidative anti-Salmonella agents. Rev. Latinoamer. Quim. 33:60-75.
• Kubo, I., Fujita, K., Lee, S. H. and Ha, T. J. 2005. Antibacterial activity of polygodial. Phytother. Res. 19:1013-1017.