Characterization of Anti-Ice Nucleation Activity of the Extract from Coffee Refuse.

　The supercooling-facilitating (SCF) activities, that is, the anti-ice nucleation activity of the hot water extracts from five types of processed food refuse was examined. The extract with the highest activity among five hot water extracts was coffee refuse, showing 1.50℃ of SCF activity at a final concentration of 0.1 mg/ml. From the hot water extract of coffee refuse, the coffee refuse extract containing various polyphenols was prepared by the ultrafiltration (less than MWCO 10,000), a solvent fractionation of ethyl acetate. The yield of coffee refuse extract was 0.9% (w/w) from dried coffee refuse. The SCF activity of the coffee refuse extract at a final concentration of 1.0 mg/ml was 4.2℃. HPLC analysis of the coffee refuse extract showed that caffeine and chlorogenic acid, which are major components of coffee, could be found at 173 and 62.3 µg/ml, respectively. However, the SCF activities of both compounds (0.70 and 1.06℃) at a final concentration of 0.1 mg/ml were lower than those of ferulic acid and coumaric acid, respectively at 3.40 and 2.35℃. This is the first report to our knowledge on the SCF activity of caffeine. The SCF activity of caffeine at a final concentration of 1.0 mg/ml was 2.3℃. The specificity of caffeine against various ice nuclei containing calcium oxalate, 9-fluorenon, and ice nucleating bacteria was examined. Caffeine at a final concentration of 1.0 mg/ml could inhibit the ice nucleation activity of calcium oxalate, and Pseudomonas fluorescens KUIN-1 at the same level that of as silver iodide. From these results, it was suggested that the extract could be able to be applied to the field to control the frost damage of the vegetables and that the harvested vegetables might be stored unfrozen even at 0℃ or less.

The Importance of 11α-OH, 15-oxo, and 16-en Moieties of 11α-Hydroxy-15-oxo-kaur-16-en-19-oic Acid in Its Inhibitory Activity on Melanogenesis.

Cosmetic industries have an interest in exploring and developing materials that have the potential to regulate melanin synthesis in human skin. Although melanin protects the skin from ultraviolet irradiation, excess melanin can be undesirable, particularly on the face where spots or freckles are associated with an appearance of aging. In this study, we found that ent-11α-hydroxy-15-oxo-kaur-16-en-19-oic acid (11α-OH KA) in Pteris dispar Kunze strongly inhibited melanin synthesis by suppressing tyrosinase gene expression. The melanogenic transcription factor microphthalmia-associated transcription factor (MITF) is required for this suppression. However, 11α-OH KA did not modulate the expression level or activity of MITF. Structure-activity relationship analyses suggested that the 11α-OH, 15-oxo, and 16-en moieties of 11α-OH KA are essential for the suppression of melanin synthesis. On the other hand, the 19-COOH moiety is important for preventing cellular toxicity associated with 11α-OH KA and its related compounds. These results suggest that 11α-OH KA is an attractive target for potential use in the production of cosmetic items.

Callicarpa longissima extract, carnosol-rich, potently inhibits melanogenesis in B16F10 melanoma cells.

Cosmetic industries focus on developing materials and resources that regulate skin pigmentation. Melanin, the major pigment in human skin, protects the skin against damage from ultraviolet light. An ethanolic extract of the leaves of Callicarpa longissima inhibits melanin production in B16F10 mouse melanoma cells by suppressing microphthalmia-associated transcription factor (MITF) gene expression. Following purification and analysis using liquid chromatography-mass spectrometry (LC-MS), NMR, and biochemical assays, carnosol was determined to be responsible for the major inhibitory effect of the C. longissima extract on melanin production. Carnosol is an oxidative product of carnosic acid, whose presence in the extract was also confirmed by an authentic reference. The carnosol and carnosic acid content in the extract was approximately 16% (w/w). These results suggest that C. longissima is a novel, useful, and attractive source of skin-whitening agents.

Induction of melanogenesis by 4'-O-methylated flavonoids in B16F10 melanoma cells.

Agents to control melanogenesis are in demand for the development of cosmetics to improve pigmentation disorders of skin and hair. In this study, we examined and evaluated the effects of flavonoids on melanogenesis in the melanogenic cells model, murine B16F10 melanoma cells. In the course of this study, we found that incubation of the cells in a medium containing 10 µM of the 4'-O-methylated flavonoids, diosmetin (4'-O-methylluteolin), acacetin (4'-O-methylapigenin) or kaempferide (4'-O-methylkaempferol), increased the melanin contents of the cells 3- to 7-fold higher than the control cells. The concentration-dependence test revealed that 20 µM acacetin showed the highest effect, up to 33-fold higher than the vehicle. On the other hand, the corresponding 4'-OH-type flavonoids, luteolin, apigenin and kaempferol, had a significantly smaller effect. Furthermore, by evaluating the melanogenic proteins, we found that the cells treated with 4'-O-methylated flavonoids showed higher tyrosinase activity, as well as upregulation of tyrosinase expression, preceded by activation of cAMP response element binding protein (CREB) and extracellular signal-regulated kinases types 1 and 2 (ERK1/2). These results indicate that the 4'-O-methyl group of flavonoids plays an important role in the induction of melanogenesis by activating its major signal transduction pathway through the upregulation of phospho-CREB in murine B16F10 melanoma cells.

Type II antifreeze protein from a mid-latitude freshwater fish, Japanese smelt (Hypomesus nipponensis).

A lot of reports of antifreeze protein (AFP) from fish have been published, but no report has mentioned of commercialized mid-latitude fresh water fish which producing AFP in its body fluid. We found that the AFP in the body fluid of Japanese smelt (Hypomesus nipponensis) from mid-latitude fresh water was purified and characterized. The N-terminal amino acid sequence of the Japanese smelt AFP was 75.0% identical to Type II AFP from herring. Results of EDTA treatment and ruthenium red staining suggested that the Japanese smelt AFP had at least one Ca2+-binding domain. Interestingly, the antifreeze activity of the Japanese smelt AFP did not completely disappear when Ca2+ ions were removed. The molecular mass of the Japanese smelt AFP was calculated to be 16,756.8 by the TOF-mass analysis. The Open reading flame of the gene coding for the Japanese smelt AFP was 444 bp long and was 85.0% identical with the entire herring AFP gene. The cDNA and amino acid sequence of the Japanese smelt AFP were the same length as those of herring AFP.