XMEs detected in whole skin. Protein identification was based on the presence of ≥2 different tryptic peptides in at least two donors.

<p>The proteins identified have been classified into functional groups as indicated. The corresponding NCBI numbers are indicated for each protein and for all members of groups of related proteins. The sub-cellular fraction in which each protein was principally detected is shown. The proportion of donor samples (skin n = 10, liver n = 5) in which each protein was identified is indicated. Fold difference was calculated by summing the intensity values of all detected peptides for a protein and comparing the values obtained for skin and liver. Where no peptides were detected, an intensity value equivalent to the limit of detection was used. Statistical significance was assessed using the Mann-Whitney U test.</p

Comparison of XME profiles from <i>in vitro</i> skin models and whole skin.

<p>The relative amount of each protein is represented by the number of different tryptic peptides specific to each protein or protein family that were detected. Details of the protein accession numbers and their subcellular location are shown in Table S1. Shading indicates different enzyme classes: oxidoreductase (black), hydrolase (magenta), transferase (red), antioxidant (green), and other (blue).</p

Stability of Epiderm-200 XME expression profiles in culture.

<p>Epiderm-200 cultures were derived from either donor 254 or 1188 and were maintained for up to 3 days. The relative amount of each protein is indicated by the number of different tryptic peptides specific to each protein or protein family that were detected with adjacent bars representing results from 0, 1, 2 and 3 days, respectively. Details of the protein accession numbers and their subcellular location are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0041721#pone.0041721.s003" target="_blank">Table S2</a>. Shading indicates different enzyme classes: oxidoreductase (black), transferase (red), antioxidant (green), and other (blue).</p

Analysis of CYP expression in skin by immunoblotting.

<p>Samples of human whole skin microsomal fraction (75 µg) prepared from 5 donors were separated by SDS-PAGE, transferred to nitrocellulose filters and the presence of CYP1A1, CYP1A2, CYP2E1 and CYP3A4 detected using antibodies specific to each form. The lane on the left hand side contained either 25 µg lymphoblast cell microsomes containing recombinant human CYP1A1 (∼2 pmol), or a sample of human liver loaded with 25, 35 or 5 µg microsomal fraction for detection of CYP1A2, CYP2E1, or CYP3A4, respectively. Immunoreactive bands were developed using goat anti-rabbit-horseradish peroxidase and ECL detection.</p

Flow diagram summarizing the methodology.

<p>Flow diagram summarizing the methodology.</p

Detection of CYP proteins in skin and liver microsomal fraction by LC-MS/MS.

<p>Samples of skin microsomal fraction were spiked with a range of quantities of either recombinant CYP1A1 (expressed in lymphoblast cells) or with human liver microsomal fraction that contains known amounts of CYP1A2, CYP2E1, CYP3A4, and CYP3A5. The normal proteomics workflow was followed to identify peptides corresponding to the CYP proteins. Limits of detection based on the use of at least 2 tryptic peptides were established and based on these values the minimum level detectable by this technique was calculated for skin and compared with the mean level measured in liver. From these values the minimum comparative level in skin was calculated. CYP1A1 was not detected in either skin or liver making any comparison redundant (n/a; not applicable).</p

6,7-Seco-<i>ent</i>-Kauranoids Derived from Oridonin as Potential Anticancer Agents

Structurally unique 6,7-seco-<i>ent</i>-kaurenes, which are widely distributed in the genus <i>Isodon</i>, have attracted considerable attention because of their antitumor activities. Previously, a convenient conversion of commercially available oridonin (<b>1</b>) to 6,7-seco-<i>ent</i>-kaurenes was developed. Herein, several novel spiro-lactone-type <i>ent</i>-kaurene derivatives bearing various substituents at the C-1 and C-14 positions were further designed and synthesized from the natural product oridonin. Moreover, a number of seven-membered C-ring-expanded 6,7-seco-<i>ent</i>-kaurenes were also identified for the first time. It was observed that most of the spiro-lactone-type <i>ent</i>-kaurenes tested markedly inhibited the proliferation of cancer cells, with an IC₅₀ value as low as 0.55 μM. An investigation on its mechanism of action showed that the representative compound <b>7b</b> affected the cell cycle and induced apoptosis at a low micromolar level in MCF-7 human breast cancer cells. Furthermore, compound <b>7b</b> inhibited liver tumor growth in an in vivo mouse model and exhibited no observable toxic effects. Collectively, the results warrant further preclinical investigations of these spiro-lactone-type <i>ent</i>-kaurenes as potential novel anticancer agents

Design, Synthesis, and Biological Evaluation of Novel Hybrids Containing Dihydrochalcone as Tyrosinase Inhibitors to Treat Skin Hyperpigmentation

Excessive melanin deposition may lead to a series of skin disorders. The production of melanin is carried out by melanocytes, in which the enzyme tyrosinase performs a key role. In this work, we identified a series of novel tyrosinase inhibitor hybrids with a dihydrochalcone skeleton and resorcinol structure, which can inhibit tyrosinase activity and reduce the melanin content in the skin. Compound 11c possessed the most potent activity against tyrosinase, showing IC50 values at nanomolar concentration ranges, along with significant antioxidant activity and low cytotoxicity. Furthermore, in vitro permeation tests, supported by HPLC analysis and 3D OrbiSIMS imaging visualization, revealed the excellent permeation of 11c. More importantly, compound 11c reduced the melanin content on UV-induced skin pigmentation in a guinea pig model in vivo. These results suggest that compound 11c may serve as a promising potent tyrosinase inhibitor for the development of a potential therapy to treat skin hyperpigmentation

Design, Synthesis, and Biological Evaluation of Novel Hybrids Containing Dihydrochalcone as Tyrosinase Inhibitors to Treat Skin Hyperpigmentation

Excessive melanin deposition may lead to a series of skin disorders. The production of melanin is carried out by melanocytes, in which the enzyme tyrosinase performs a key role. In this work, we identified a series of novel tyrosinase inhibitor hybrids with a dihydrochalcone skeleton and resorcinol structure, which can inhibit tyrosinase activity and reduce the melanin content in the skin. Compound 11c possessed the most potent activity against tyrosinase, showing IC50 values at nanomolar concentration ranges, along with significant antioxidant activity and low cytotoxicity. Furthermore, in vitro permeation tests, supported by HPLC analysis and 3D OrbiSIMS imaging visualization, revealed the excellent permeation of 11c. More importantly, compound 11c reduced the melanin content on UV-induced skin pigmentation in a guinea pig model in vivo. These results suggest that compound 11c may serve as a promising potent tyrosinase inhibitor for the development of a potential therapy to treat skin hyperpigmentation

Antioxidant Properties of Novel Dimers Derived from Natural β‑Elemene through Inhibiting H₂O₂‑Induced Apoptosis

A series of novel β-elemene dimer derivatives were synthesized and evaluated for their antioxidant activities. The results indicated that most of the target compounds showed more potent cytoprotective effects than positive control vitamin E. In particular, dimer <b>D5</b> exhibited the strongest antioxidant activity, which was significantly superior to the active compound <b>D1</b> obtained in our previous study. Besides, <b>D5</b> did not produce obvious cytotoxicity in normal human umbilical vein endothelial cells (HUVECs) and increased the viability of HUVECs injured by H₂O₂ in a concentration-dependent manner. Further studies suggested that the cytoprotective action of <b>D5</b> might be mediated, at least in part, by increasing the intracellular superoxide dismutase activity and nitric oxide secretion as well as decreasing the intracellular malonyldialdehyde content and lactate dehydrogenase release. Furthermore, <b>D5</b> observably inhibited ROS generation and prevented H₂O₂-induced apoptosis in HUVECs possibly via inhibiting the activation of the MAPK signaling pathway