After treatment with the samples, the cells were incubated for about 5?days. The cells were then harvested and 1?mL of 10?mM Tris-HCl (pH?7.4) containing 2% SDS and 20?mM DTT was added to the pellet containing the cells. was determined by measuring the testosterone levels in rat liver microsomes. Results CTL and TI showed potent anti-oxidative activity and anti-inflammatory activities. Especially, the cytokine Cyclosporin H production inhibitory activities of TI were found to be similar to the positive control, epigallocatechin gallate (EGCG). CTL and TI enhanced the CE formation and filaggrin mRNA expression levels and showed potent activities compared to that in the positive control, 1.5?mM Ca2+. In additionally, CTL and TI showed 5-reductase inhibitory activities in a dose-dependent manner. Conclusion The results showed that CTL and TI inhibit AV endogenous factors such as 5-reductase and inflammatory cytokines and impact exogenous factors such as developing skin barrier function (CE and filaggrin levels). Therefore, CTL and TI may be plant-derived agent, encouraging in the treatment of acne vulgaris. (CT), a deciduous broad-leaved arboreous tree, Cyclosporin H is usually a member of the genus and Betulaceae family, native to Korea, Japan, and China [30, 31]. The genus has been widely and traditionally used to treat bladder contamination, osteoporosis, and stress disorders [32]. According to the Coloured flora of Korea, leaves of CT are oval shaped and have a doubly serrate margin [33]. The bark of CT has been used as a material for furniture and bed logs [34]. In a previous study, CT has been confirmed to show biological activities including cytoprotective activities, suppression of tyrosinase expression, whitening activities, anti-wrinkle, anti-allergic activities, and neuroprotective activities [31, 34C36]. Despite many studies on skin diseases, there Cyclosporin H have been few experiments demonstrating the skin improvement effects of CT in AV. The purpose of this study was conducted to assess the skin improvement effects of CT leaf (CTL) extract and tellimagrandin I (TI), which was isolated from CTL, on AV. Methods Plant materials The leaves of were obtained from the Yeoju Eco Park, Yeoju, Republic of Korea, in July 2018. Plant materials were distinguished by Kim Sungsik, Ph.D. (Korea National Arboretum, Pocheon). Voucher specimens were placed at the herbarium of the College of Pharmacy, Chung-Ang University or college (CTLYZ-1806). General experimental procedures The column chromatography isolation was performed on a Sephadex LH-20 column (10C25?m; GE Healthcare Bio-Science AB, Uppsala, Sweden). Structural identification was by one-dimensional nuclear magnetic resonance (1D-NMR) including 1H-NMR (600?MHz) and 13C-NMR (125?MHz) (JEOL, Tokyo, Japan) at Chung-Ang University. Extraction, isolation and structure elucidation Leaves (1.2?kg) of CT were extracted for 72?h at room temperature (25?C) with 70% prethanol A (ethyl alcohol), after removing the solution under vacuum, the CTL extract (252?g) was obtained. The CTL extract (142?g) was dissolved in water, the water layer was filtered through Celite 545 (Duksan Pure Chemicals Co. Ltd., Seoul, Korea). Filtrate was concentrated and applied to Sephadex LH-20 column (25C100?m; Pharmacia, Uppsala, Sweden) and eluted with water (H2O)-methanol (MeOH) gradient system, eleven fractions were obtained. Repeated column chromatography of portion 10 (11.64?g) on Sephadex LH-20 with water-methanol gradient system to obtained tellimagrandin I (TI, 1.98?g). The structure of TI was recognized by analysis of 1H-NMR and 13C-NMR spectra and comparison with reference [31]. Chemical and reagents Dulbeccos Modified Eagle Medium (DMEM), trypsin, and fetal bovine serum (FBS) were purchased from Welgene (Gyeongsan, Republic of Korea). StreptomycinCpenicillin was purchased from Gibco (NY, USA). Calcium-free DMEM, superscript? IV first-strand synthesis system, and Desire taq Green PCR Mix were purchased from Thermo Fisher Scientific (MA, USA). TRIzol reagent was purchased from Invitrogen (CA, USA). Sodium dodecyl sulfate (SDS), dithiothreitol (DTT), agarose, lipopolysaccharide (LPS), ethyl ether, 1,1-diphenyl-2-picrylhydrazyl (DPPH), Griess reagent, NG-Methyl-l-arginine acetate salt (L-NMMA), and thiazolyl blue tetrazolium bromide (MTT) were obtained from Sigma Aldrich (St. Louis, USA). Reagent set B, cytokine IL-6 and IL-8 ELISA units utilized for immunoassay were purchased from BD Biosciences (NJ, USA). TI was acquired in a previous study [31]. Anti-oxidative activity Measurement of DPPH radical scavenging activityTo evaluate the radical scavenging activities of CTL extract and TI, DPPH assay was conducted. DPPH is bound with the hydrogen of anti-oxidants, because nitrogen in hydrazyl on DPPH has an unstable radical. DPPH radical scavenging activities were assessed by confirming the color change in DPPH accompanied with the reaction to anti-oxidants [37]. To assess anti-oxidant activities, samples dissolved in anhydrous ethyl alcohol were added (20?L) into a 96-well plate, followed by addition of 0.2?mM DPPH (180?L). No sample adding, 0.2?mM DPPH 200?L was made as the negative control. After gentle shaking for 15?min at room heat, optical density (OD) was measured at 517?nm using an ELISA reader (TECAN, Salzburg, Austria). The OD was utilized for calculation as follows: the rate of inhibition (%)?=?[1 – (sample OD/negative control.CTL and TI enhanced the CE formation and filaggrin mRNA expression levels and showed potent activities compared to that in the positive control, 1.5?mM Ca2+. 5-reductase inhibitory activity was determined by measuring the testosterone levels in rat liver microsomes. Results CTL and TI showed potent anti-oxidative activity and anti-inflammatory activities. Especially, the cytokine production inhibitory activities of TI were found to be similar to the positive control, epigallocatechin gallate (EGCG). CTL and TI enhanced the CE formation and filaggrin mRNA expression levels and showed potent activities compared to that in the positive control, 1.5?mM Ca2+. In additionally, CTL and TI showed 5-reductase inhibitory activities in a dose-dependent manner. Conclusion The results showed that CTL and TI inhibit AV endogenous factors such as 5-reductase and inflammatory cytokines and affect exogenous factors such as developing skin barrier function (CE and filaggrin levels). Therefore, CTL and TI may be plant-derived agent, promising in the treatment of acne vulgaris. (CT), a deciduous broad-leaved arboreous tree, is a member of the genus and Betulaceae family, native to Korea, Japan, and China [30, 31]. The genus has been widely and traditionally used to treat bladder infection, osteoporosis, and anxiety disorders [32]. According to the Coloured flora of Korea, leaves of CT are oval shaped and have a doubly serrate margin [33]. The bark of CT has been used as a material for furniture and bed logs [34]. In a previous study, CT has been confirmed to show biological activities including cytoprotective activities, suppression of tyrosinase expression, whitening activities, anti-wrinkle, anti-allergic activities, and neuroprotective activities [31, 34C36]. Despite many studies on skin diseases, there have been few experiments demonstrating the skin improvement effects of CT in AV. The purpose of this study was conducted to assess the skin improvement effects of CT leaf (CTL) extract and tellimagrandin I (TI), which was isolated from CTL, on AV. Methods Plant materials The leaves of were obtained from the Yeoju Eco Park, Yeoju, Republic of Korea, in July 2018. Plant materials were distinguished by Kim Sungsik, Ph.D. (Korea National Arboretum, Pocheon). Voucher specimens were placed at the herbarium of the College of Pharmacy, Chung-Ang University (CTLYZ-1806). General experimental procedures The column chromatography isolation was performed on a Sephadex LH-20 column (10C25?m; GE Healthcare Bio-Science AB, Uppsala, Sweden). Structural identification was by one-dimensional nuclear magnetic resonance (1D-NMR) including 1H-NMR (600?MHz) and 13C-NMR (125?MHz) (JEOL, Tokyo, Japan) at Chung-Ang University. Extraction, isolation and structure elucidation Leaves (1.2?kg) of CT were extracted for 72?h at room temperature (25?C) with 70% prethanol A (ethyl alcohol), after removing the solution under vacuum, the CTL extract (252?g) was obtained. The CTL extract (142?g) was dissolved in water, the water layer was filtered through Celite 545 (Duksan Pure Chemicals Co. Ltd., Seoul, Korea). Filtrate was concentrated and applied to Sephadex LH-20 column (25C100?m; Pharmacia, Uppsala, Sweden) and eluted with water (H2O)-methanol (MeOH) gradient system, eleven fractions were obtained. Repeated column chromatography of fraction 10 (11.64?g) on Sephadex LH-20 with water-methanol gradient system to obtained tellimagrandin I (TI, 1.98?g). The structure of TI was identified by analysis of 1H-NMR and 13C-NMR spectra and comparison with reference [31]. Chemical and reagents Dulbeccos Modified Eagle Medium (DMEM), trypsin, and fetal bovine serum (FBS) were purchased from Welgene (Gyeongsan, Republic of Korea). StreptomycinCpenicillin was purchased from Gibco (NY, USA). Calcium-free DMEM, superscript? IV first-strand synthesis system, and Dream taq Green PCR Mix were purchased from Thermo Fisher Scientific (MA, USA). TRIzol reagent was purchased from Invitrogen (CA, USA). Sodium dodecyl sulfate (SDS), dithiothreitol (DTT), agarose, lipopolysaccharide (LPS), ethyl ether, 1,1-diphenyl-2-picrylhydrazyl (DPPH), Griess reagent, NG-Methyl-l-arginine acetate salt (L-NMMA), and thiazolyl blue tetrazolium bromide (MTT) were obtained from Sigma Aldrich (St. Louis, USA). Reagent set B, cytokine IL-6 and IL-8 ELISA sets used for immunoassay were purchased from BD Biosciences (NJ, USA). TI was acquired in a previous study [31]. Anti-oxidative activity Measurement of DPPH radical scavenging activityTo evaluate the radical scavenging activities of CTL extract and TI, DPPH assay was conducted. DPPH is bound with the hydrogen of anti-oxidants, because nitrogen.Inhibition of NO synthesis was calculated as inhibition rate (%)?=?[1 – (sample OD – blank OD)/(control OD – blank OD)]??100, and the IC50 values were defined as concentrations that inhibit 50% of NO production [38]. Measurement of inhibitory activity on cytokine productionDifferentiated HaCaT cells were inoculated into a 96-well plate and placed for 6?h at Cyclosporin H 37?C in 5% CO2. levels and showed potent activities compared to that in the positive control, 1.5?mM Ca2+. In additionally, Cyclosporin H CTL and TI showed 5-reductase inhibitory activities in a dose-dependent manner. Conclusion The results showed that CTL and TI inhibit AV endogenous factors such as 5-reductase and inflammatory cytokines and affect exogenous factors such as developing skin barrier function (CE and filaggrin levels). Therefore, CTL and TI may be plant-derived agent, promising in the treatment of acne vulgaris. (CT), a deciduous broad-leaved arboreous tree, is a member of the genus and Betulaceae family, native to Korea, Japan, and China [30, 31]. The genus has been widely and traditionally used to treat bladder infection, osteoporosis, and anxiety disorders [32]. According to the Coloured flora of Korea, leaves of CT are oval shaped and have a doubly serrate margin [33]. The bark of CT has been used as a material for furniture and bed logs [34]. In a previous study, CT has been confirmed to show biological activities including cytoprotective activities, suppression of tyrosinase expression, whitening activities, anti-wrinkle, anti-allergic activities, and neuroprotective activities [31, 34C36]. Despite many studies on skin diseases, there have been few experiments demonstrating the skin improvement effects of CT in AV. The purpose of this study was carried out to assess the pores and skin improvement effects of CT leaf (CTL) draw out and tellimagrandin I (TI), which was isolated from CTL, on AV. Methods Plant materials The leaves of were from the Yeoju Eco Park, Yeoju, Republic of Korea, in July 2018. Flower materials were distinguished by Kim Sungsik, Ph.D. (Korea National Arboretum, Pocheon). Voucher specimens were placed in the herbarium of the College of Pharmacy, Chung-Ang University or college (CTLYZ-1806). General experimental methods The column chromatography isolation was performed on a Sephadex LH-20 column (10C25?m; GE Healthcare Bio-Science Abdominal, Uppsala, Sweden). Structural recognition was by one-dimensional nuclear magnetic resonance (1D-NMR) including 1H-NMR (600?MHz) and 13C-NMR (125?MHz) (JEOL, Tokyo, Japan) at Chung-Ang University. Extraction, isolation and structure elucidation Leaves (1.2?kg) of CT were extracted for 72?h at space temperature (25?C) with 70% prethanol A (ethyl alcohol), after removing the perfect solution is under vacuum, the CTL draw out (252?g) was obtained. The CTL extract (142?g) was dissolved in water, the water coating was filtered through Celite 545 (Duksan Pure Chemicals Co. Ltd., Seoul, Korea). Filtrate was concentrated and applied to Sephadex LH-20 column (25C100?m; Pharmacia, Uppsala, Sweden) and eluted with water (H2O)-methanol (MeOH) gradient system, eleven fractions were acquired. Repeated column chromatography of portion 10 (11.64?g) about Sephadex LH-20 with water-methanol gradient system to obtained tellimagrandin I (TI, 1.98?g). The structure of TI was recognized by analysis of 1H-NMR and 13C-NMR spectra and assessment with research [31]. Chemical AKAP12 and reagents Dulbeccos Modified Eagle Medium (DMEM), trypsin, and fetal bovine serum (FBS) were purchased from Welgene (Gyeongsan, Republic of Korea). StreptomycinCpenicillin was purchased from Gibco (NY, USA). Calcium-free DMEM, superscript? IV first-strand synthesis system, and Desire taq Green PCR Blend were purchased from Thermo Fisher Scientific (MA, USA). TRIzol reagent was purchased from Invitrogen (CA, USA). Sodium dodecyl sulfate (SDS), dithiothreitol (DTT), agarose, lipopolysaccharide (LPS), ethyl ether, 1,1-diphenyl-2-picrylhydrazyl (DPPH), Griess reagent, NG-Methyl-l-arginine acetate salt (L-NMMA), and thiazolyl blue tetrazolium bromide (MTT) were from Sigma Aldrich (St. Louis, USA). Reagent arranged B, cytokine IL-6 and IL-8 ELISA units utilized for immunoassay were purchased from BD Biosciences (NJ, USA). TI was acquired in a earlier study [31]. Anti-oxidative activity Measurement of DPPH radical scavenging activityTo evaluate the radical scavenging activities of CTL draw out and TI, DPPH assay was carried out. DPPH is bound with the hydrogen of anti-oxidants, because nitrogen in hydrazyl on DPPH has an unstable radical. DPPH radical scavenging activities were assessed by confirming the color modify in DPPH accompanied with the reaction to anti-oxidants [37]. To assess anti-oxidant.