Selective determination of formaldehyde by high-performance liquid chromatography with porous graphitic carbon column using N,N'-bis(9-anthrylmethyl)propane-1,3-diamine as derivatizing reagent.

Aromatic compounds containing two secondary amino groups were designed and prepared as new derivatizing reagents for aldehydes. One of them, N,N'-bis(9-anthrylmethyl)propane-1,3-diamine (APD), could achieve selective determination of formaldehyde (FA) on a porous graphitic carbon (PGC) column using xylenes, chlorobenzene, and 1-chloronaphthalene as mobile phases by high-performance liquid chromatography (HPLC). The APD-FA derivative was eluted from the PGC column, while the other APD-aldehyde derivatives remained on the column during the HPLC measurements. This specific elution was not observed using mobile phases such as acetonitrile, 1,4-dioxane, tetrahydrofuran, N,N-dimethylformamide, N,N-dimethylacetamide, N-methyl-2-pyrrolidone, chloroform, benzene, toluene, benzyl alcohol, 2-ethyl-1-hexanol, and pyridine. The APD-FA derivative had a six-membered ring of two tertiary amines identified using 1H NMR spectroscopy. When the π-π interaction of the solvent molecule of the mobile phase with PGC overcame that between the APD-FA derivative and PGC, the APD-FA derivative could be eluted from the column. The best resolution between the peak of the APD-FA derivative and that of free APD was observed when using o-xylene. The optimum derivatization and the HPLC conditions for selective HPLC determination of FA were to conduct the derivatization of FA by heating in an aqueous phase with APD in o-xylene at 100 °C. In this method, FA could be derivatized with APD at a mildly neutral pH of 6.7, unlike the low pH required for the derivatization of aldehydes with 2,4-dinitrophenylhydrazine (DNPH), which is commonly used for the derivatization of aldehydes. The detection and quantification limits of FA were 0.8 and 3.5 ng mL-1 in this HPLC method with fluorescent detection, respectively. This selective HPLC method could be applied to the determination of FA in various water samples. It was found that only APD among the derivatizing reagents containing two secondary diamines was useful for the selective determination of FA.

Suppression of melanin production by expression of HSP70.

Skin hyperpigmentation disorders due to abnormal melanin production induced by ultraviolet (UV) irradiation are both a clinical and cosmetic problem. UV irradiation stimulates melanin production in melanocytes by increasing intracellular cAMP. Expression of heat shock proteins (HSPs), especially HSP70, is induced by various stressors, including UV irradiation, to provide cellular resistance to such stressors. In this study we examined the effect of expression of HSP70 on melanin production both in vitro and in vivo. 3-Isobutyl-1-methylxanthine (IBMX), a cAMP-elevating agent, stimulated melanin production in cultured mouse melanoma cells, and this stimulation was suppressed in cells overexpressing HSP70. IBMX-dependent transcriptional activation of the tyrosinase gene was also suppressed in HSP70-overexpressing cells. Expression of microphthalmia-associated transcription factor (MITF), which positively regulates transcription of the tyrosinase gene, was up-regulated by IBMX; however, this up-regulation was not suppressed in HSP70-overexpressing cells. On the other hand, immunoprecipitation and immunostaining analyses revealed a physical interaction between and co-localization of MITF and HSP70, respectively. Furthermore, the transcription of tyrosinase gene in nuclear extract was inhibited by HSP70. In vivo, UV irradiation of wild-type mice increased the amount of melanin in the basal layer of the epidermis, and this increase was suppressed in transgenic mice expressing HSP70. This study provides the first evidence of an inhibitory effect of HSP70 on melanin production both in vitro and in vivo. This effect seems to be mediated by modulation of MITF activity through a direct interaction between HSP70 and MITF.

HSP70 inducers from Chinese herbs and their effect on melanin production.

Skin hyperpigmentation disorders as a result of abnormal melanin production induced by ultraviolet (UV) irradiation are both a clinical and a cosmetic problem. This melanin production is mediated by tyrosinase whose expression is positively regulated by microphthalmia-associated transcription factor (MITF). We recently found that expression of heat shock protein 70 (HSP70) inhibits melanin production. In this study, we searched for HSP70 inducers from Chinese herbs and selected an ethanol extract of Eupatorium lindleyanum (E. lindleyanum). Not only melanin production but also the activity and expression of tyrosinase were significantly suppressed in cells treated with E. lindleyanum extract as well as in HSP70-overexpressing cells. The expression of MITF was clearly suppressed in cells treated with E. lindleyanum extract but not in HSP70-overexpressing cells. These results suggest that E. lindleyanum extract suppresses the expression of tyrosinase and melanin production through both HSP70-dependent and HSP70-independent mechanisms.