Effect of Zinc Oxide Nanoparticle Addition to Polycaprolactone Periodontal Membranes on Antibacterial Activity and Cell Viability.

During the design of membranes for guided tissue regeneration (GTR) to treat periodontal diseases, infection of the exposed membranes and postoperative complications can be prevented by increasing bacterial resistance. This study evaluated the antibacterial activity of PCL/ZnO membranes and their effect on cell viability via addition of antibacterial zinc oxide (ZnO) nanoparticles to a biocompatible and biodegradable material such as polycaprolactone (PCL). Neat PCL membranes and PCL/ZnO membranes containing 0.5 wt.% and 5 wt.% ZnO were produced, and divided into PCL (0% ZnO), LZ (0.5 wt.% ZnO), and HZ (5 wt.% ZnO) groups, respectively. The surface characteristics of the membranes including morphological features and changes in composition were analyzed. Adhesion of bacteria, including Streptococcus mutans and Porphyromonas gingi-valis, was analyzed using a crystal violet assay. The proliferation of MC3T3-E1 osteoblasts was evaluated using a WST-8 assay. Significant differences were analyzed using the Kruskal-Wallis test (P < 0.05). The results of groups were compared using the Mann-Whitney test (P < 0.017). ZnO nanoparticles were dispersed in the PCL matrix of PCL/ZnO membranes. Compared with neat PCL membranes, their ability to form crystals decreased and their amorphous structure increased. The adhesion of S. mutans and P. gingivalis in the LZ and HZ groups containing ZnO was significantly decreased compared with that of the neat PCL membranes (P < 0.05). No significant differences were observed in the proliferation of MC3T3-E1 cells between the PCL/ZnO membranes and the neat PCL membranes both on days 2 and 5 of culture (P > 0.05). This study has demonstrated that the PCL membranes carrying the ZnO nanoparticles inhibited bacterial adhesion without affecting the viability of osteoblasts, suggesting the potential application of ZnO in GTR to increase antibacterial activity of membranes.

Synthesis of 5-Aminolevulinic Acid-Based ALACELL Possessing Inhibitory Effect against Cutibacterium acnes: Whitening Effect and Protective Effect of UVB-Irradiated Damage Cells.

Five-aminolevulinic acid (5-ALA)-photodynamic therapy combined with infrared radiation is an effective and safe therapy for facial acne. Although there are various available agents for treating acne, therapies for resistant or severe strains have been limited. The aim of this study was to investigate the inhibitory efficacy of ALACELL synthesized by combining 5-ALA with Y-G-G-F-L peptide against Staphylococcus aureus, Bacillus cereus, Escherichia coli, and Yersinia enterocolitica, as well as Cutibacterium acnes. Furthermore, other effects of ALACELL on human skin cells, melanin formation, intracellular tyrosinase activity, and Ultra Violet B (UVB)-irradiated cell death were measured by treatment of ALACELL in vitro. ALACELL particularly showed a growth inhibitory effect on C. acnes and no inhibitory effect on the four bacteria strains. ALACELL reduced melanin formation and intracellular tyrosinase activity by α-melanin cell-stimulating hormone (α-MSH) in B16F10 cells, with no cytotoxicity. ALACELL also improved cell viability in UVB-irradiated HaCaT cells. The results of the experiment show that ALACELL exhibits more efficacy than 5-ALA against antimicrobial activity, melanin formation, intracellular tyrosinase activity, and UVB-irradiated cell death. Therefore, ALACELL is recommended as a candidate for clinical application in the treatment of acne and skin aging and will be further investigated to study the mode of action and in actual situations.

Screening ginseng saponins in progenitor cells identifies 20(R)-ginsenoside Rh2 as an enhancer of skeletal and cardiac muscle regeneration.

Aging is associated with increased prevalence of skeletal and cardiac muscle disorders, such as sarcopenia and cardiac infarction. In this study, we constructed a compendium of purified ginsenoside compounds from Panax ginseng C.A. Meyer, which is a traditional Korean medicinal plant used to treat for muscle weakness. Skeletal muscle progenitor cell-based screening identified three compounds that enhance cell viability, of which 20(R)-ginsenoside Rh2 showed the most robust response. 20(R)-ginsenoside Rh2 increased viability in myoblasts and cardiomyocytes, but not fibroblasts or disease-related cells. The cellular mechanism was identified as downregulation of cyclin-dependent kinase inhibitor 1B (p27Kip1) via upregulation of Akt1/PKB phosphorylation at serine 473, with the orientation of the 20 carbon epimer being crucially important for biological activity. In zebrafish and mammalian models, 20(R)-ginsenoside Rh2 enhanced muscle cell proliferation and accelerated recovery from degeneration. Thus, we have identified 20(R)-ginsenoside Rh2 as a p27Kip1 inhibitor that may be developed as a natural therapeutic for muscle degeneration.

Anti-photoaging effect of skin cream manufactured with ziyuglycoside I isolated from Sanguisorba officinalis on ultraviolet B-induced hairless mice.

In this study, skin cream containing ziyuglycoside I isolated from Sanguisorba officinalis was manufactured and examined the protective effects of the skin cream against UVB-induced hairless mice. UVB-induced hairless mice were topically treated with the skin cream once a day for 5 weeks. Application of the skin cream did not exhibit side effect on body growth showing normal body weight and food efficiency in the mice. The skin cream treatment also was inhibited mRNA expression of interleukin (IL)-1ß, matrix metalloproteinase (MMP)-2, MMP-9, and MMP-2 protein expression in the mice. Furthermore, the skin cream treatment inhibits epidermal wrinkle formation, wrinkle depth, wrinkle thickness, and collagen degradation in UVB-induced hairless mice. Therefore, the skin cream was able to play a role in the attenuation of photoaging caused by UVB irradiation via downregulation of mRNA expression of inflammatory cytokine IL-1ß, MMP-2, MMP-9, and suppression of MMP-2 proteins expression.

Biotransformed Metabolites of the Hop Prenylflavanone Isoxanthohumol.

A metabolic conversion study on microbes is known as one of the most useful tools to predict the xenobiotic metabolism of organic compounds in mammalian systems. The microbial biotransformation of isoxanthohumol (1), a major hop prenylflavanone in beer, has resulted in the production of three diastereomeric pairs of oxygenated metabolites (2⁻7). The microbial metabolites of 1 were formed by epoxidation or hydroxylation of the prenyl group, and HPLC, NMR, and CD analyses revealed that all of the products were diastereomeric pairs composed of (2S)- and (2R)- isomers. The structures of these metabolic compounds were elucidated to be (2S,2"S)- and (2R,2"S)-4'-hydroxy-5-methoxy-7,8-(2,2-dimethyl-3-hydroxy-2,3-dihydro-4H-pyrano)-flavanones (2 and 3), (2S)- and (2R)-7,4'-dihydroxy-5-methoxy-8-(2,3-dihydroxy-3-methylbutyl)-flavanones (4 and 5) which were new oxygenated derivatives, along with (2R)- and (2S)-4'-hydroxy-5-methoxy-2"-(1-hydroxy-1-methylethyl)dihydrofuro[2,3-h]flavanones (6 and 7) on the basis of spectroscopic data. These results could contribute to understanding the metabolic fates of the major beer prenylflavanone isoxanthohumol that occur in mammalian system.

Suppression of influenza B virus replication by sakuranetin and mode of its action.

This study aimed to investigate in vitro the anti-influenza B/Lee/40 virus effect of sakuranetin and mode of its action. The sakuranetin exhibited potent antiviral activity against influenza B/Lee/40 virus, reducing the formation of a visible cytopathic effect, with a 50% inhibitory concentration (IC50 ) of 7.21 µg/ml and no cytotoxicity at a concentration of 100 µg/ml, and the derived therapeutic index (TI) was >13.87. Oseltamivir showed weak anti-influenza B/Lee/40 virus activity with IC50 of 80.74 µg/ml, 50% cytotoxicity concentration of >100 µg/ml, and TI of >1.24. Sakuranetin also showed effective inhibitory effects when added at the viral attachment, entry, and postentry steps. Moreover, sakuranetin effectively inactivated influenza B/Lee/40 virus infection in dose- and temperature-dependent manners. Sakuranetin indicated an inhibitory effect in viral RNA synthesis in the presence of 100 µg/ml of sakuranetin. Overall, this research revealed that sakuranetin could inhibit influenza B/Lee/40 virus replication and that sakuranetin may be involved in the virus attachment, entry, and postentry. Therefore, sakuranetin is a good candidate for a chemopreventive agent for influenza virus-related diseases.

Isolation and Characterization of Isofraxidin 7-O-(6'-O-p-Coumaroyl)-ß-glucopyranoside from Artemisia capillaris Thunberg: A Novel, Nontoxic Hyperpigmentation Agent That Is Effective In Vivo.

Abnormalities in skin pigmentation can produce disorders such as albinism or melasma. There is a research need to discover novel compounds that safely and effectively regulate pigmentation. To identify novel modulators of pigmentation, we attempted to purify compounds from a bioactive fraction of the Korean medicinal plant Artemisia capillaris Thunberg. The novel compound isofraxidin 7-O-(6'-O-p-coumaroyl)-ß-glucopyranoside (compound 1) was isolated and its pigmentation activity was characterized in mammalian melanocytes. Compound 1 stimulated melanin accumulation and increased tyrosinase activity, which regulates melanin synthesis. Moreover, compound 1 increased the expression of tyrosinase and the key melanogenesis regulator microphthalmia-associated transcription factor (MITF) in melanocytes. Compared to the parent compound, isofraxidin, compound 1 produced greater effects on these pigmentation parameters. To validate compound 1 as a novel hyperpigmentation agent in vivo, we utilized the zebrafish vertebrate model. Zebrafish treated with compound 1 showed higher melanogenesis and increased tyrosinase activity. Compound 1 treated embryos had no developmental defects and displayed normal cardiac function, indicating that this compound enhanced pigmentation without producing toxicity. In summary, our results describe the characterization of novel natural product compound 1 and its bioactivity as a pigmentation enhancer, demonstrating its potential as a therapeutic to treat hypopigmentation disorders.

Efficacy of a Complex of 5-Aminolevulinic Acid and Glycyl-Histidyl-Lysine Peptide on Hair Growth.

BACKGROUND: Pattern hair loss is a very common problem. Although effective therapeutics for the treatment of pattern hair loss have been used, novel therapeutic modalities are still required to enhance hair growth. OBJECTIVE: We investigated the efficacy and safety of a complex (ALAVAX) of 5-aminolevulinic acid (5-ALA) and glycyl-histidyl-lysine (GHK) peptide for the treatment of pattern hair loss. METHODS: Forty-five patients with male pattern hair loss were treated with ALAVAX 100 mg/ml (group A), ALAVAX 50 mg/ml (group B) or placebo (group C) once a day for 6 months. Total hair count, hair length, hair thickness, patient's assessment and adverse events were evaluated at month 1, 3, and 6. RESULTS: An increase in hair count for 6 months was 52.6 (p<0.05) in group A, 71.5 (p<0.05) in group B, and 9.6 in group C. The ratio of changes in hair count between group B (2.38) and group C (1.21) at 6 months showed a statistically significant difference (p<0.05). The proportion above good satisfaction was higher in group A (26.7%) than in the other groups (group B: 14.3%, group C: 7.1%). There was no statistically significant difference in hair length and hair thickness among 3 groups at 6 months. There was no adverse event in 3 groups. CONCLUSION: Our study showed that a complex of 5-ALA and GHK peptide may be considered as one of the complementary agents for the treatment of male pattern hair loss.

Isolation of 4,5-O-Dicaffeoylquinic Acid as a Pigmentation Inhibitor Occurring in Artemisia capillaris Thunberg and Its Validation In Vivo.

There is a continual need to develop novel and effective melanogenesis inhibitors for the prevention of hyperpigmentation disorders. The plant Artemisia capillaris Thunberg (Oriental Wormwood) was screened for antipigmentation activity using murine cultured cells (B16-F10 malignant melanocytes). Activity-based fractionation using HPLC and NMR analyses identified the compound 4,5-O-dicaffeoylquinic acid as an active component in this plant. 4,5-O-Dicaffeoylquinic acid significantly reduced melanin synthesis and tyrosinase activity in a dose-dependent manner in the melanocytes. In addition, 4,5-O-dicaffeoylquinic acid treatment reduced the expression of tyrosinase-related protein-1. Significantly, we could validate the antipigmentation activity of this compound in vivo, using a zebrafish model. Moreover, 4,5-O-dicaffeoylquinic acid did not show toxicity in this animal model. Our discovery of 4,5-O-dicaffeoylquinic acid as an inhibitor of pigmentation that is active in vivo shows that this compound can be developed as an active component for formulations to treat pigmentation disorders.

Delineation of the role of glycosylation in the cytotoxic properties of quercetin using novel assays in living vertebrates.

Quercetin is a plant-derived flavonoid and its cytotoxic properties have been widely reported. However, in nature, quercetin predominantly occurs as various glycosides. Thus far the cytotoxic activity of these glycosides has not been investigated to the same extent as quercetin, especially in animal models. In this study, the cytotoxic properties of quercetin (1), hyperoside (quercetin 3-O-galactoside, 2), isoquercitrin (quercetin 3-O-glucoside, 3), quercitrin (quercetin 3-O-rhamnoside, 4), and spiraeoside (quercetin 4'-O-glucoside, 5) were directly compared in vitro using assays of cancer cell viability. To further characterize the influence of glycosylation in vivo, a novel zebrafish-based assay was developed that allows the rapid and experimentally convenient visualization of glycoside cleavage in the digestive tract. This assay was correlated with a novel human tumor xenograft assay in the same animal model. The results showed that 3 is as effective as 1 at inhibiting cancer cell proliferation in vivo. Moreover, it was observed that 3 can be effectively deglycosylated in the digestive tract. Collectively, these results indicate that 3 is a very promising drug candidate for cancer therapy, because glycosylation confers advantageous pharmacological changes compared with the aglycone, 1. Importantly, the development of a novel and convenient fluorescence-based assay for monitoring deglycosylation in living vertebrates provides a valuable platform for determining the metabolic fate of naturally occurring glycosides.

Chemical targeting of GAPDH moonlighting function in cancer cells reveals its role in tubulin regulation.

Glycolytic enzymes are attractive anticancer targets. They also carry out numerous, nonglycolytic "moonlighting" functions in cells. In this study, we investigated the anticancer activity of the triazine small molecule, GAPDS, that targets the glycolytic enzyme glyceraldehyde 3-phosphate dehydrogenase (GAPDH). GAPDS showed greater toxicity against cancer cells compared to a known GAPDH enzyme inhibitor. GAPDS also selectively inhibited cell migration and invasion. Our analysis showed that GAPDS treatment reduced GAPDH levels in the cytoplasm, which would modulate the secondary, moonlighting functions of this enzyme. We then used GAPDS as a probe to demonstrate that a moonlighting function of GAPDH is tubulin regulation, which may explain its anti-invasive properties. We also observed that GAPDS has potent anticancer activity in vivo. Our study indicates that strategies to target the secondary functions of anticancer candidates may yield potent therapeutics and useful chemical probes.

Development of a highly visual, simple, and rapid test for the discovery of novel insulin mimetics in living vertebrates.

Diabetes mellitus is a global epidemic with major impacts on human health and society. Drug discovery for diabetes can be facilitated by the development of a rapid, vertebrate-based screen for identifying new insulin mimetic compounds. Our study describes the first development of a zebrafish-based system based on direct monitoring of glucose flux and validated for identifying novel anti-diabetic drugs. Our system utilizes a fluorescent-tagged glucose probe in an experimentally convenient 96-well plate format. To validate our new system, we identified compounds that can induce glucose uptake via activity-guided fractionation of the inner shell from the Japanese Chestnut (Castanea crenata). The best performing compound, UP3.2, was identified as fraxidin and validated as a novel insulin mimetic using a mammalian adipocyte system. Additional screening using sets of saponin- and triazine-based compounds was undertaken to further validate this assay, which led to the discovery of triazine PP-II-A03 as a novel insulin mimetic. Moreover, we demonstrate that our zebrafish-based system allows concomitant toxicological analysis of anti-diabetic drug candidates. Thus, we have developed a rapid and inexpensive vertebrate model that can enhance diabetes drug discovery by preselecting hits from chemical library screens, before testing in relatively expensive rodent assays.

Acteoside improves survival in cecal ligation and puncture-induced septic mice via blocking of high mobility group box 1 release.

Acteoside, an active phenylethanoid glycoside, has been used traditionally as an anti-inflammatory agent. The molecular mechanism by which acteoside reduces inflammation was investigated in lipopolysaccharide (LPS)-induced Raw264.7 cells and in a mouse model of cecal ligation and puncture (CLP)-induced sepsis. In vitro, acteoside inhibits high mobility group box 1 (HMGB1) release and iNOS/NO production and induces heme oxygenase-1 (HO-1) expression in a concentration-dependent manner, while HO-1 siRNA antagonizes the inhibition of HMGB1 and NO. The effect of acteoside is inhibited by the p38 mitogen-activated protein kinase (MAPK) inhibitor SB203580 and Nfr2 siRNA, indicating that acteoside induces HO-1 via p38 MAPK and NF-E2-related factor 2 (Nrf2). In vivo, acteoside increases survival and decreases serum and lung HMGB1 levels in CLP-induced sepsis. Overall, these results that acteoside reduces HMGB1 release and may be beneficial for the treatment of sepsis.

Cytotoxic caffeic acid derivatives from the rhizomes of Cimicifuga heracleifolia.

Activity profiling of the n-BuOH extract from Cimicifuga heracleifolia rhizomes led to the identification of three cytotoxic caffeic acid derivatives, carboxymethyl isoferulate (2), cimicifugic acid A (3), and cimicifugic acid B (4) together with a series of structurally related inactive compounds. The extract was separated by time-based fractionation in a gradient HPLC condition, and cytotoxicity of each fraction was evaluated using HCT116 colon cancer cells in vitro. HPLChyphenated spectroscopy including LC/NMR and LC/PDA/MS provided structural information for phenolic compounds contained in the extract, and further preparative isolation of active compounds 2-4 was achieved by semi-preparative HPLC. Compounds 2-4 showed cytotoxic activity against cancer cells in a dose-dependent manner at the concentrations of 2.5-40 µM, and western blotting analysis showed that these compounds increased expression of cleaved poly ADP ribose polymerase (PARP), a critical apoptosis marker.

Variation in the total lengths of abutment/implant assemblies generated with a function of applied tightening torque in external and internal implant-abutment connection.

AIM: Settling (embedment relaxation), which is the main cause for screw loosening, is developed by microroughness between implant and abutment metal surface. The objective of this study was to evaluate and compare the relationship between the level of applied torque and the settling of abutments into implants in external and internal implant-abutment connection. MATERIAL AND METHODS: Five different implant-abutment connections were used (Ext, External butt joint + two-piece abutment; Int-H2, Internal hexagon + two-piece abutment; Int-H1, Internal hexagon + one-piece abutment; Int-O2, Internal octagon + two-piece abutment; Int-O1, Internal octagon + one-piece abutment). All abutments of each group were assembled and tightened with corresponding implants by a digital torque gauge. The total lengths of implant-abutment samples were measured at each torque (5, 10, 30 N cm and repeated 30 N cm with 10-min interval) by an electronic digital micrometer. The settling values were calculated by changes between the total lengths of implant-abutment samples. RESULTS: All groups developed settling with repeated tightening. The Int-H2 group showed markedly higher settling for all instances of tightening torque and the Ext group was the lowest. Statistically significant differences were found in settling values between the groups and statistically significant increases were observed within each group at different tightening torques (P<0.05). After the second tightening of 30 N cm, repeated tightening showed almost constant settling values. CONCLUSIONS: Results from the present study suggested that to minimize the settling effect, abutment screws should be retightened at least twice at 30 N cm torque at a 10-min interval in all laboratory and clinical procedures.

Evaluation of the accuracy of implant-level impression techniques for internal-connection implant prostheses in parallel and divergent models.

PURPOSE: This study evaluated the accuracy of 2 implant-level impression techniques (direct nonsplinted and splinted) for the fabrication of multi-unit internal-connection implant restorations in 2 simulated clinical settings (parallel and divergent) using a laboratory model. MATERIALS AND METHODS: A dental stone master model was fabricated with 2 pairs of implant replicas. One pair simulated a parallel clinical condition and the other an 8-degree-divergent condition. Ten stone casts were made from vinyl polysiloxane impressions of the master model for each impression technique. Half of the samples were created by a direct nonsplinted technique (square impression copings, custom tray), and the other half were made by a direct splinted technique (square impression copings splinted with autopolymerizing acrylic resin, custom tray). Four strain gauges were fixed on each metal framework to measure the degree of framework deformation for each stone cast in half-Wheatstone-bridge formations. Deformation readings were made twice in 4 directions (anterior, posterior, superior, and inferior). Deformation data were analyzed using repeated-measures analysis of variance at a .05 level of significance. RESULTS: No significant difference in deformation was found between the direct nonsplinted and splinted samples in either simulated clinical condition (P > .05). No significant difference in deformation was found between the techniques regardless of condition (P > .05). CONCLUSIONS: Within the limitations of this study, using a 2-implant model, the accuracy of implant-level impressions for internal-connection implant restorations was similar for the direct nonsplinted and splinted techniques in settings with divergence up to 8 degrees.

A cholesterol biosynthesis inhibitor from Rhizopus oryzae.

A bile acid derivative, methyl cholate (1), was isolated from EtOAc extract of the fungus Rhizopus oryzae as a cholesterol biosynthesis inhibitor. It showed moderate inhibitory activity on cholesterol biosynthesis in human Chang liver cells. Compound 1 exhibited inhibitory effect on the later step of cholesterol biosynthesis, indicating that its action mode is different from that of statins that act on the HMG-CoA reductase.

A novel iridoid from Boschniakia rossica.

A novel iridoid, (4R)-4-hydroxymethylboschnialactone (1), has been isolated from Boschniakia rossica, together with three previously known compounds, (24S)-3beta-hydroxy-24-ethylcholest-5-en-7-one (2), (24R)-3beta-hydroxy-24-ethylcholest-5-en-7-one (3) and methyl p-coumarate, using column chromatography. The structures of compounds were elucidated by spectroscopic methods.

Microbial metabolism of the environmental estrogen bisphenol A.

Preliminary microbial metabolism studies of bisphenol A (BPA) (1) on twenty six microorganisms have shown that Aspergillus fumigatus is capable of metabolizing BPA. Scale-up fermentation of 1 with A. fumigatus gave a metabolite (2) and its structure was established as bisphenol A-O-beta-D-glucopyranoside (BPAG) based on spectroscopic analyses.