Skin Improvement Effects of Ultrasound-Enzyme-Treated Collagen Peptide Extracts from Flatfish (Paralichthys olivaceus) Skin in an In Vitro Model.

Collagen is considered to be an intercellular adhesive that prevents tissue stretching or damage. It is widely utilized in cosmetic skin solutions, drug delivery, vitreous substitutions, 3D cell cultures, and surgery. In this study, we report the development of a green technology for manufacturing collagen peptides from flatfish skin using ultrasound and enzymatic treatment and a subsequent assessment on skin functionality. First, flatfish skin was extracted using ultrasound in distilled water (DW) for 6 h at 80 °C. Molecular weight analysis via high-performance liquid chromatography (HPLC) after treatment with industrial enzymes (alcalase, papain, protamex, and flavourzyme) showed that the smallest molecular weight (3.56 kDa) was achieved by adding papain (0.5% for 2 h). To determine functionality based on peptide molecular weight, two fractions of 1100 Da and 468 Da were obtained through separation using Sephadex™ G-10. We evaluated the effects of these peptides on protection against oxidative stress in human keratinocytes (HaCaT) cells, inhibition of MMP-1 expression in human dermal fibroblast (HDF) cells, reduction in melanin content, and the inhibition of tyrosinase enzyme activity in murine melanoma (B16F10) cells. These results demonstrate that the isolated low-molecular-weight peptides exhibit superior skin anti-oxidant, anti-wrinkle, and whitening properties.

Anti-Melanogenic Effects of a Polysaccharide Isolated from Undaria pinnatifida Sporophyll Extracts.

Undaria pinnatifida is a temperate brown alga known to exert free radical-scavenging and anti-inflammatory effects. In this study, we investigated the skin-whitening effects of U. pinnatifida sporophyll extracts (UPEs) in α-melanocyte-stimulating hormone (α-MSH)-stimulated B16F10 melanoma cells. The crude polysaccharide fraction (UPF) was obtained via ethanol precipitation. Four polysaccharide fractions (UPF1-4) were isolated and purified using ion-exchange column chromatography, and their anti-melanogenic activity was evaluated. UPF3 exhibited the highest anti-melanogenic activity, showing the highest sulfate (39.79%), fucose (143 µg/mg), and galactose (208 µg/mg) contents. UPF3 significantly inhibited intracellular tyrosinase activity in B16F10 cells. We also evaluated the melanogenic signaling pathway to determine the mechanism of action of UPF3 in melanongenesis. UPF3 reduced the expression of tyrosinase-related protein-1 (TRP-1), tyrosinase-related protein-2 (TRP-2), and tyrosinase, which play important roles in melanin production. Therefore, UPF3 has high potential for use in skin-whitening functional pharmaceuticals and cosmetics.

MLK3 is a novel target of dehydroglyasperin D for the reduction in UVB-induced COX-2 expression in vitro and in vivo.

Dehydroglyasperin D (DHGA-D), a compound present in licorice, has been found to exhibit anti-obesity, antioxidant and anti-aldose reductase effects. However, the direct molecular mechanism and molecular targets of DHGA-D during skin inflammation remain unknown. In the present study, we investigated the effect of DHGA-D on inflammation and its mechanism of action on UVB-induced skin inflammation in HaCaT human keratinocytes and SKH-1 hairless mice. DHGA-D treatment strongly suppressed UVB-induced COX-2 expression, PGE2 generation and AP-1 transactivity in HaCaT cells without affecting cell viability. DHGA-D also inhibited phosphorylation of the mitogen-activated protein kinase kinase (MKK) 3/6/p38, MAPK/Elk-1, MKK4/c-Jun N-terminal kinase (JNK) 1/2/c-Jun/mitogen, and stress-activated protein kinase (MSK), whereas phosphorylation of the mixed-lineage kinase (MLK) 3 remained unaffected. Kinase and co-precipitation assays with DHGA-D Sepharose 4B beads showed that DHGA-D significantly suppressed MLK3 activity through direct binding to MLK3. Knockdown of MLK3 suppressed COX-2 expression as well as phosphorylation of MKK4/p38 and MKK3/6/JNK1/2 in HaCaT cells. Furthermore, Western blot assay and immunohistochemistry results showed that DHGA-D pre-treatment significantly inhibits UVB-induced COX-2 expression in vivo. Taken together, these results indicate that DHGA-D may be a promising anti-inflammatory agent that mediates suppression of both COX-2 expression and the MLK3 signalling pathway through direct binding and inhibition of MLK3.

Isoliquiritigenin induces apoptosis and inhibits xenograft tumor growth of human lung cancer cells by targeting both wild type and L858R/T790M mutant EGFR.

Non-small-cell lung cancer (NSCLC) is associated with diverse genetic alterations including mutation of epidermal growth factor receptor (EGFR). Isoliquiritigenin (ILQ), a chalcone derivative, possesses anticancer activities. In the present study, we investigated the effects of ILQ on the growth of tyrosine kinase inhibitor (TKI)-sensitive and -resistant NSCLC cells and elucidated its underlying mechanisms. Treatment with ILQ inhibited growth and induced apoptosis in both TKI-sensitive and -resistant NSCLC cells. ILQ-induced apoptosis was associated with the cleavage of caspase-3 and poly-(ADP-ribose)-polymerase, increased expression of Bim, and reduced expression of Bcl-2. In vitro kinase assay results revealed that ILQ inhibited the catalytic activity of both wild type and double mutant (L858R/T790M) EGFR. Treatment with ILQ inhibited the anchorage-independent growth of NIH3T3 cells stably transfected with either wild type or double-mutant EGFR with or without EGF stimulation. ILQ also reduced the phosphorylation of Akt and ERK1/2 in both TKI-sensitive and -resistant NSCLC cells, and attenuated the kinase activity of Akt1 and ERK2 in vitro. ILQ directly interacted with both wild type and double-mutant EGFR in an ATP-competitive manner. A docking model study showed that ILQ formed two hydrogen bonds (Glu-762 and Met-793) with wild type EGFR and three hydrogen bonds (Lys-745, Met-793, and Asp-855) with mutant EGFR. ILQ attenuated the xenograft tumor growth of H1975 cells, which was associated with decreased expression of Ki-67 and diminished phosphorylation of Akt and ERK1/2. Taken together, ILQ suppresses NSCLC cell growth by directly targeting wild type or mutant EGFR.

Development of an ultrasonic system for industrial extraction of unheated sesame oil cake.

Sesame is a popular functional food in Asia. However, research on sesame seed oil cake compounds and their extraction methods is lacking. Ultrasound technology was applied to develop an efficient extraction method for this purpose. First, pilot-scale extraction from sesame oil cake was performed and optimized using response surface methodology. The extract obtained using optimized conditions (0% ethanol for 4 h at 20°C) showed the highest yield (45.1%) and inhibitory effect on reactive oxygen species (ROS; 55.1%). Compared to extracts obtained by conventional extraction methods, those obtained by ultrasound technology exhibited a higher extraction yield, greater antioxidant effect, and increased lignan content. Based on pilot-scale experiments, an industrial-scale ultrasonic extraction system was designed to extract a 2.1-ton solution at once. The extract contained sesaminol 1,2-diglucoside (4.6 mg/g) as the major component and showed 28.3% ROS inhibition activity. Our industrial ultrasound-assisted extraction method has potential application for other compounds.

Inhibitory effect of Saccharomyces cerevisiae extract obtained through ultrasound-assisted extraction on melanoma cells.

Although the immune enhancing effect of yeast has been widely reported, studies specifically investigating its effects on skin cancer are lacking. Therefore, this study aimed to develop a yeast extract capable of inhibiting melanoma cells using ultrasound technology, which can lyse the cell walls allowing subsequent rapid yeast extraction. To compare the extraction efficiency across different extraction methods, the total yield, as well as total glucan, α-glucan, and ß-glucan yields were measured. Ultrasound-assisted extract of yeast (UAEY) was found to effectively inhibit melanoma cell growth and proliferation as well as the expression of cyclin D1 and c-myc, in vitro. Additionally, the extract reduced melanoma tumor volume and cyclin D1 levels in BALB/c nu/nu mice. The optimal extraction conditions were 0.2 M NaOH, 3 h, 70 °C, 20 kHz, and 800 W, resulting in an increased total extraction and ß-glucan yields of 73.6% and 7.1%, respectively, compared with that achieved using a conventional chemical (0.5 M NaOH) extraction method. Taken together, the results of this study suggest that UAEY may represent an effective anti-skin cancer agent.

Immune-enhancing effects of polysaccharide extract of by-products of Korean liquor fermented by Saccharomyces cerevisiae.

To increase the value of yeast-fermented Korean liquor by-products, we obtained crude polysaccharide (CPS) fractions via ultrasound-assisted extraction and stepwise-gradient ethanol precipitation and investigated their functionality. Nitric oxide production in RAW 264.7 cells was increased following treatment with the CPSs derived from extract. Analysis of the monosaccharide and amino acid composition of the CPS fractions using HPLC revealed that the polysaccharides were mainly composed of glucose (57.2%), mannose (22.6%), and galactose (17.6%), and no amino acids were detected. In addition, a higher concentration of ethanol solvent for fractionation yielded polysaccharides with lower molecular weights (<15 kDa). CPS 3 and 4 fractions increased the production of TNF-α (15 and 17-fold, respectively) and IL-6 (20 and 18-fold, respectively) and iNOS (65 and 35-fold, respectively) expression at concentration 12.5 µg/mL compared with levels in non-treated RAW 264.7 cells. Especially, CPS 4 at 200 and 400 µg/mL significantly increased the proliferation of mouse spleen cells by 126% and 153%, respectively. These results indicated that CPS 4 enhanced the proliferation of mouse spleen cells in vivo, indicating its immune-enhancing effects. Therefore, this research can contribute to the development of eco-friendly extraction techniques and immune-enhancing materials.

Silk peptide production from whole silkworm cocoon using ultrasound and enzymatic treatment and its suppression of solar ultraviolet-induced skin inflammation.

Silk fibroin, which is derived from sericin through degumming, is mainly used as a biomaterial. However, interest in functional verification and industrial applications of sericin has been growing for several years. We used ultrasonication to simplify the extraction process of the silk peptide under low salt conditions at 20 °C, instead of using the conventional conditions of high salt and temperature. The concentration of the silk peptide was measured to determine the optimized extraction time and solvent, which were 4 h and 0.1 N NaOH, respectively. The molecular weight of the enzyme-treated silk peptide was measured using SDS-PAGE and GPC. Silk peptide treated with papain after ultrasound had a molecular weight of less than 5 kDa, and the papain treated-silk peptide reduced solar ultraviolet-induced COX-2 expression through inhibition of ERK phosphorylation. This is the first study investigating simultaneous extraction of fibroin and sericin, which can be used for mass production of food materials.

Enhancement of the apoptotic effects of Arctii Fructus extracts on cancer cells by the enzymatic bioconversion of lignans.

The fruit of Arctium lappa L. (Arctii Fructus) is one of the most popularly used medicinal plant components in Asia. To enhance the functionality of Arctii Fructus extract, a bioconversion method was developed to produce arctigenin from arctiin. Treatment with ß-glucosidase increased the arctigenin content by >5 fold in Arctii Fructus extracts. The bioconversion products enhanced the apoptosis of cancer cells. The cell viabilities of gefitinib-resistant lung cancer HCC827 (HCC827GR) cells and colon cancer cells (DLD1) were decreased by 40% and 35%, respectively. The bioconversion products also decreased anchorage-independent growth of cancer cells. In addition, the increase of apoptosis in cancer cells by bioconversion was confirmed by the flow cytometry analysis. These results indicated that arctigenin exerts anticancer effects on lung and colon cancer cells and that Arctii Fructus can potentially function as a chemopreventive agent. In addition, bioconverted Arctii Fructus extract displayed higher anticancer activity than the same levels of purified arctigenin, indicating the advantage of consuming Arctii Fructus itself as a food or medicinal material.

Arctium lappa root extract containing L-arginine prevents TNF-α-induced early atherosclerosis in vitro and in vivo.

Atherosclerosis is a chronic inflammatory disease affecting the aorta and is a major cause of cardiovascular disease. Arctium lappa root is a plant widely used in traditional Chinese medicine (TCM), and Arctium lappa root extract (ALE) has been reported to exhibit anti-inflammatory capacity and to ameliorate endothelial dysfunction. Thus, we hypothesized that ALE would inhibit the early atherosclerotic stage. In this study, we evaluated the inhibitory effect of ALE on early arteriosclerosis and its mechanisms of action. ALE suppressed TNF-α-induced monocyte adhesion to the vascular endothelium by suppressing NF-κB signaling in HUVECs. In an acute mouse model of atherosclerosis, ALE suppressed TNF-α-induced monocyte infiltration of the vascular endothelium and the expression of genes encoding inflammatory cytokines including IL-1ß, IL-6, TNF-α, and MCP-1 in the mouse aorta. Moreover, inulin-type fructan and amino acids, especially L-aspartate and L-arginine (60.27 and 42.17 mg/g, respectively) were detected by NMR, MALDI-TOF MS, and HPLC analysis as the main components of ALE. Notably, L-arginine suppressed TNF-α-induced monocyte adhesion to HUVECs. Therefore, these results suggest that ALE may be a functional food for the suppression or prevention of early stages of atherosclerosis.

Irisin promotes C2C12 myoblast proliferation via ERK-dependent CCL7 upregulation.

Irisin is an exercise-induced myokine that has various physiological functions, such as roles in energy expenditure, glucose/lipid metabolism, and muscle development. In muscle development, myoblast proliferation is known to be a first step, and recent studies have reported that an increased irisin level is involved in the promotion of cell proliferation in various cell types, including myoblasts. However, the exact mechanism of action by which irisin promotes myoblast proliferation has not been reported. In this study, we aimed to determine the pro-proliferative effect of irisin on C2C12 myoblasts and its mechanism of action. Irisin induced C2C12 cell proliferation and upregulated the mRNA levels of markers of proliferation Pcna, Mki67, and Mcm2. Irisin increased extracellular signal-regulated kinase (ERK) phosphorylation, and U0126, an ERK pathway inhibitor, suppressed irisin-induced C2C12 cell proliferation. Transcriptomic and qRT-PCR analysis showed that Ccl2, Ccl7, Ccl8, and C3 are potential downstream regulators of ERK signaling that promote C2C12 cell proliferation. Knockdown of Ccl7 revealed that irisin upregulates chemokine (C-C motif) ligand 7 (CCL7) and subsequently promotes C2C12 cell proliferation. These results suggest that irisin promotes C2C12 myoblast proliferation via ERK-dependent CCL7 upregulation and may aid in understanding how irisin contributes to muscle development.

Ultrasonicated Lespedeza cuneata extract prevents TNF-α-induced early atherosclerosis in vitro and in vivo.

This study evaluated the use of ultrasonication to extract Lespedeza cuneata as a potential nutraceutical for preventing vascular inflammation. Ultrasonicated L. cuneata extract (ULCE) was prepared using 20% ethanol and 2 h of ultrasonication at room temperature, and its effects were investigated using relevant in vitro and in vivo models. ULCE suppressed tumor necrosis factor-alpha (TNF-α)-induced adhesion capacity, vascular cell adhesion protein 1 (VCAM-1) expression, and nuclear factor kappa-B (NF-κB) activity in human umbilical vein endothelial cells (HUVECs). ULCE also suppressed TNF-α-induced NF-κB signaling pathways and p65 translocation from the cytosol to the nucleus, as well as the mRNA expression of IL-1ß, IL-6, and TNF-α in HUVECs. Oral administration of ULCE suppressed TNF-α-induced monocyte infiltration into the intima and VCAM-1 expression, as well as the IL-1ß, IL-6, TNF-α, and monocyte chemoattractant protein-1 (MCP-1) mRNA expression in the main artery in mice. Among the compounds identified in the hydrolyzed ULCE, quercetin exhibited the strongest inhibitory effect against TNF-α-induced cell adhesion capacity. These results demonstrate that ULCE contains potent preventive factors against early atherosclerosis, which act by suppressing the NF-κB and VCAM-1 signaling axis.

Syringic acid prevents skin carcinogenesis via regulation of NoX and EGFR signaling.

Validation of nutraceutical and pharmaceutical targets is essential for the prediction of physiological and side effects. Epidemiologic evidence and molecular studies suggest that non-melanoma skin cancer is directly associated with excessive exposure to ultraviolet (UV) radiation. The aim of the present study was to evaluate the inhibitory effects of syringic acid on UVB-induced signaling and skin carcinogenesis, and determine the molecular targets. Treatment of human epidermal keratinocytes (HaCaT) cells with syringic acid resulted in the suppression of UVB-induced cyclooxygenase-2, matrix metalloproteinase-1, and prostaglandin E2 expression as well as activator protein-1 activity. Moreover, syringic acid inhibited the UVB-induced phosphorylation of mitogen-activated protein kinases and Akt signaling pathways as well as epidermal growth factor receptor (EGFR). Syringic acid treatment further inhibited intracellular reactive oxygen species and protein-tyrosine phosphatase-κ activity, a regulator of EGFR activation. Syringic acid and the antioxidant N-acetyl-l-cysteine inhibited UVB-induced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity. In vivo, pretreatment of mouse skin with syringic acid significantly suppressed UVB-induced skin tumor incidence in a dose-dependent manner. Overall, these results indicate that syringic acid exerts potent chemopreventive activity in skin carcinogenesis mainly by inhibition of the Nox/PTP-κ/EGFR axis. Syringic acid might serve as an effective chemopreventive and therapeutic agent against UVB-mediated skin cancer.

N-Benzyl-N-methyl-dodecan-1-amine, a novel compound from garlic, exerts anti-cancer effects on human A549 lung cancer cells overexpressing cancer upregulated gene (CUG)2.

Dietary garlic has been suggested to possess anticancer properties, and several attempts have been made to isolate the anticancer compounds. In this study, we efficiently synthesized N-benzyl-N-methyl-dodecan-1-amine (BMDA) by the reductive amination method. We evaluated the potential anticancer activities of BMDA against A549 lung cancer cells with cancer stem cell-like phenotypes due to the overexpression of cancer upregulated gene (CUG)2. N-Benzyl-N-methyl-dodecan-1-amine treatment sensitized A549 cells overexpressing CUG2 (A549-CUG2) to apoptosis and autophagy compared with those of the control cells. The treatment with BMDA also reduced tumor development in xenografted nude mice. Furthermore, BMDA inhibited cell migration, invasion, and sphere formation in A549-CUG2 cells, in which TGF-ß signaling is involved. Further analysis showed that BMDA hindered TGF-ß promoter activity, protein synthesis, and phosphorylation of Smad2, thus decreasing the expression of TGF-ß-targeted proteins, including Snail and Twist. N-Benzyl-N-methyl-dodecan-1-amine also decreased Twist expression in vivo. In addition, BMDA inhibited Akt-ERK activities, ß-catenin expression, and its transcriptional activity. These results suggest that BMDA can be a promising anticancer agent against cancer cells overexpressing CUG2.

1,8-cineole prevents UVB-induced skin carcinogenesis by targeting the aryl hydrocarbon receptor.

1,8-cineole is a natural monoterpene cyclic ether present in Eucalyptus, and has been reported to exhibit anti-inflammatory and antioxidant effects. However, the preventive effect of 1,8-cineole on skin carcinogenesis and the molecular mechanism of action responsible remains unknown. In the present study, we investigated the effect of 1,8-cineole on UVB-induced skin carcinogenesis. 1,8-cineole inhibited UVB-induced cyclooxygenase-2 (COX-2) protein and mRNA expression and prostaglandin E2 (PGE2) generation in HaCaT cells. 1,8-cineole also inhibited phosphorylation of extracellular signal-regulated kinase (ERK) 1/2, and phosphorylation of its upstream kinases, c-Src and epidermal growth factor receptor (EGFR). Quantitative real-time RT-PCR (qRT-PCR) and drug affinity responsive target stability (DARTS) assay results showed that 1,8-cineole suppressed UVB-induced expression of a target gene of the aryl hydrocarbon receptor (AhR), cyp1a1, and directly binds to AhR. Knockdown of AhR suppressed COX-2 expression as well as phosphorylation of ERK1/2 in HaCaT cells. Furthermore, topical treatment of 1,8-cineole on mouse skin delayed tumor incidence and reduced tumor numbers, while inhibiting COX-2 expression in vivo. Taken together, these results suggest that 1,8-cineole is a potent chemopreventive agent that inhibits UVB-induced COX-2 expression by targeting AhR to suppress UVB-induced skin carcinogenesis.

Protein Extraction from Porcine Myocardium Using Ultrasonication.

Porcine myocardium is regarded as a byproduct in slaughterhouses and is rarely used as a food source due to its unsuitability for processing and consumption. In this study, we sought to develop an efficient ultrasonication method to extract protein from porcine myocardium. Comparisons of protein yield using various ultrasonication conditions with porcine myocardium revealed that treatment with 0.2 M NaCl, with pH 8.0, at an extraction temperature of less than 40 °C and an amplitude of 60% to 80% was optimal, yielding an extraction rate of 90%. In addition, SDS-PAGE analysis showed that increasing the time interval for ultrasonication increased the presence of myosin heavy chain and actin protein content. Functional analysis of the physiological properties of the isolated proteins using an ATPase assay showed that Ca and Mg ATPase activity was virtually undetectable in the early stages of ultrasonic treatment and that the proteins denatured rapidly. An analysis of protein digestion also showed that the digestive capacity of proteins treated by ultrasonication methods was greater. These results demonstrate that the ultrasonication method is effective for high-yield protein extraction from cardiac myofibrils of porcine myocardium with low salt concentrations, low Ca and Mg ATPase activities, and high digestive capacities.

The role of sarcoplasmic protein in hydrostatic pressure-induced myofibrillar protein denaturation.

To observe the role of sarcoplasmic protein (SP) on myofibrillar protein (MP) denaturation under a hydrostatic pressure (HP), MP isolated from bovine muscle was treated with 300 MPa by increasing concentrations of SP (0, 0.8, 1.6, and 3.2 mg/ml) from bovine. SDS-PAGE patterns of soluble proteins in 0.1M NaCl (pH 7.4) indicated that a protein (about 100 kDa) from MP decreased with increasing concentrations of SP and that a 97 kDa protein from SP observed with 0.1 MPa was not observed with 300 MPa. SDS-PAGE patterns of soluble proteins in 0.6 M NaCl (pH 7.4) and Ca-ATPase activity showed that the denaturation of myosin heavy chain (MHC) was accelerated with increasing SP concentrations with the 300 MPa treatment. Thus, the addition of SP enhanced HP-induced denaturation of MHC and of a protein from MP of about 100 kDa.