Inhibitory effect of the extract of rhizome of Curcuma longa L in gelatinase activity and its effect on human skin.

Exposure to UV radiation to human skin up-regulates the synthesis of matrix metalloproteinase (MMP) family. Gelatinases are member of MMPs which have been suggested to play an important role in photoaging such as wrinkle formation. To inhibit gelatinase activity is regarded to be very important to keep healthy skin and to protect wrinkle formation. On the other hand, anti-photoaging agents are expected to be derived from natural resources, especially plants. Plant extracts having gelatinase-inhibitory effect that might be used as safe anti-photoaging ingredient were widely screened. An extract of rhizomes of Curcuma longa L. showed inhibitory effect of gelatinase activity. Curcuminoids and slight amount of compound, 6,11-dihydroxy-3-(4-hydroxy-3-mthoxyphenethyl)-7-[(E)-4-(4-hydroxy-3-methoxyphenyl)-2-oxo-3-butenyl]-10-methoxy-2-oxabicyclo[6.3.1.]dodeca-1(11),8(12),9-trien-5-yl (E)-3-(4-hydroxy-3-methoxyphenyl)-2-propenoate (curcuminoid D) were isolated as the gelatinase-inhibitory components from methanol extract of rhizomes. The structure of curcuminoid D was determined by means of spectral data including 1H- and 13C-NMR, and IR. Curcumin exerted the enhancing effect on deposition of basement membrane component at dermal-epidermal junction in skin equivalent model. Topical application of cream containing turmeric extract significantly improved facial skin elasticity and decreased the number of gelatinase-positive stratum corneum clusters in human facial skins. These results indicated that turmeric is an effective ingredient to improve skin condition and to prevent skin from photoaging by suppressing activation of gelatinase chronically caused by UV.

Quantification of activated and total caspase-14 with newly developed ELISA systems in normal and atopic skin.

BACKGROUND: Activation of caspase-14 occurs during terminal differentiation of keratinocytes and may play a role in filaggrin degradation. Therefore, down-regulation of caspase-14 may lead to impaired barrier function. OBJECTIVE: To compare the levels of active and total caspase-14 in healthy subjects in various age groups and in patients with atopic dermatitis (AD), using two enzyme-linked immunoassay (ELISA) systems. METHODS: We established four clones of monoclonal antibodies to caspase-14 and used clone 3 as the immobilizing antibody. A cleavage site-directed antibody, h14D146 [4] was used for specific quantification of active caspase-14 in extracts of tape-stripped corneocytes. Total caspase-14 was measured with a commercial antibody, H-99. RESULTS: The amount of caspase-14 remained constant (ca. 0.1% of extractable proteins) in healthy males from their twenties to their fifties. Caspase-14 was mostly in active form (71-94%) in these extracts. In contrast, caspase-14 level and active caspase-14 ratio were significantly decreased in females in their fifties and sixties. Contents of free amino acids were decreased in females in their sixties, and transepidermal water loss was increased in females in their forties and sixties. In patients with AD, active caspase-14 was markedly down-regulated compared to age-matched controls in both lesional (7.5%) and non-lesional skin (10.6%). Staining of active caspase-14 was considerably weaker in non-lesional skin and was hardly detectable in lesional skin with parakeratosis. CONCLUSION: Our new ELISA systems are effective tools to quantify activation of caspase-14. Our results indicate a role of caspase-14 in epidermal barrier function.

The evaluation of the amount of cis- and trans-urocanic acid in the stratum corneum by Raman spectroscopy.

Urocanic acid (UCA) is a major component in the epidermis which absorbs ultraviolet light (UV) and is isomerized from the trans to the cis form upon UV exposure. This study aimed to evaluate the amounts of cis- and trans-UCA in the stratum corneum (SC) in vivo using Raman spectroscopy. UV damage has previously been investigated by calculating the amount of trans-UCA by fitting Raman spectra. cis-UCA at pH 5.0 was also included in the fitting algorithm in this study. For validation, the SC was stripped from the subjects and the levels of cis- and trans-UCA were measured using high performance liquid chromatography (HPLC). Comparison between these two methods was performed at UV exposure and non-UV exposure sites on the volar forearm of 10 healthy Japanese subjects. Finally, the protective capacity of sunscreen was evaluated by Raman spectroscopy on the volar forearm of 18 healthy subjects. The amount of cis-UCA increased and the amount of trans-UCA decreased after UV exposure when measured by Raman spectroscopy. This was confirmed by HPLC analysis of the SC stripped from the same area of skin used in the Raman measurement. The protection against the increase in the cis-UCA and decrease in the trans-UCA amount after UV exposure by the application of sunscreen products was observed using Raman spectroscopy. The results show this method can be used to measure cis- and trans-UCA levels in vivo.

Neutral cysteine protease bleomycin hydrolase is essential for the breakdown of deiminated filaggrin into amino acids.

Filaggrin is a component of the cornified cell envelope and the precursor of free amino acids acting as a natural moisturizing factor in the stratum corneum. Deimination is critical for the degradation of filaggrin into free amino acids. In this study, we tried to identify the enzyme(s) responsible for the cleavage of deiminated filaggrin in vitro. First, we investigated citrulline aminopeptidase activity in the extract of newborn rat epidermis by double layer fluorescent zymography and detected strong activity at neutral pH. Monitoring the citrulline-releasing activity, we purified an enzyme of 280 kDa, comprised of six identical subunits of 48 kDa. The NH(2) terminus of representative tryptic peptides perfectly matched the sequence of rat bleomycin hydrolase (BH). The enzyme released various amino acids except Pro from beta-naphthylamide derivatives and hydrolyzed citrulline-beta-naphthylamide most effectively. Thus, to break down deiminated filaggrin, another protease would be required. Among proteases tested, calpain I degraded the deiminated filaggrin effectively into many peptides of different mass on the matrix-assisted laser desorption/ionization-time of flight mass spectrum. We confirmed that various amino acids including citrulline were released by BH from those peptides. On the other hand, caspase 14 degraded deiminated filaggrin into a few peptides of limited mass. Immunohistochemical analysis of normal human skin revealed co-localization of BH and filaggrin in the granular layer. Collectively, our results suggest that BH is essential for the synthesis of natural moisturizing factors and that calpain I would play a role as an upstream protease in the degradation of filaggrin.

Changes in environmental humidity affect the water-holding property of the stratum corneum and its free amino acid content, and the expression of filaggrin in the epidermis of hairless mice.

BACKGROUND: Seasonal changes affect the condition of skin and may trigger various cutaneous disorders. OBJECTIVE: To clarify the effects of the environmental humidity on the skin pathology, we studied the effects of the humidity on a water-holding function of the stratum corneum. METHODS: We evaluated the skin surface conductance, amino acid in the stratum corneum, and immunoreactivity of filaggrin of the epidermis of hairless mice kept in different environmental humidity. RESULTS: Skin surface conductance in the stratum corneum of hairless mice 3-7 days after transfer from a humid environment (>80% relative humidity) to a dry (<10% relative humidity) environment, was significantly lower than that of the mice transferred from a normal environment (relative humidity=40-70%) to a dry environment. The free amino acid content in the stratum corneum significantly decreased 24 h after we transferred the mice from a normal to a dry condition, then it recovered to the original level within 3 days, while the mice transferred from a humid to a dry condition showed a significantly lower amino acid content even 7 days after the transfer. No obvious change was observed in the relative composition of the major components of the free amino acids during the experiments. Immunoreactivity of filaggrin, which is the main precursor of free amino acids in the stratum corneum, also became faint in the epidermis of the mice transferred from a humid or normal to a dry environment. CONCLUSION: These results suggested that a drastic decrease in the environmental humidity reduced the total free amino acid generation and consequently induced skin surface dryness in the stratum corneum.