Zebrafish Model for Studying Dexamethasone-Induced Muscle Atrophy and Preventive Effect of Maca (Lepidium meyenii).

Loss of myofibers during muscle atrophy affects functional capacity and quality of life. Dexamethasone, an inducer of rapid atrophy of skeletal myofibers, has been studied as a glucocorticoid receptor in muscle atrophy or motor neurodegeneration. In this study, we examined dexamethasone-induced muscle atrophy using zebrafish (Danio rerio), a vertebrate model, and assessed whether administration of Lepidium meyenii (maca) as a dietary supplement can prevent muscle atrophy. Changes in skeletal myofibers in zebrafish were evaluated after exposure to dexamethasone for different periods and at different concentrations. Under optimized conditions, zebrafish pre-fed with maca for 3 days were exposed to 0.01% dexamethasone for 1 h/day for 7 days. Thereafter, myofiber loss, damaged muscle contractile proteins, and abnormal exploratory behavior due to the structural and functional impairment of skeletal muscle associated with muscle atrophy were investigated using hematoxylin-eosin, immunofluorescence staining, and behavioral analyses. Our findings suggest that dexamethasone induces muscle atrophy in zebrafish, inhibiting exploratory behavior by inducing myofiber loss, inhibiting muscle contraction, and causing changes in endurance and velocity. Thus, the zebrafish model can be used to screen pharmaceutical agents and to study muscle atrophy. Furthermore, maca is a potential dietary supplement to prevent muscle atrophy, as it protects muscle fibers.

Protective Effects of Fucoidan Isolated from Celluclast-Assisted Extract of Undaria pinnatifida Sporophylls against AAPH-Induced Oxidative Stress In Vitro and In Vivo Zebrafish Model.

Fucoidan is a fucose-enriched polysaccharide, obtained from brown algae, with demonstrated antioxidant properties. However, traditional extraction methods using water or chemical-based extraction methods have reduced yield and produced hazardous by-products. In this study, we isolated fucoidan at a high yield using enzyme-assisted extraction; the Celluclast enzyme assisted extract of Undaria pinnatifida sporophylls (FCUS). To examine the antioxidant properties of FCUS, oxidative stress was induced with 2,2'-azobis (2-methylpropionamidine) dihydrochloride (AAPH) in Vero cells and zebrafish model. FCUS was composed of 30.4% sulfate and 52.3% fucose. Pre-treatment of Vero cells with FCUS dose dependently inhibited AAPH-induced reactive oxygen species (ROS) production. Moreover, FCUS remarkably reduced cell death, ROS generation, and lipid peroxidation production in zebrafish larvae. Overall, these findings indicate that the sulfate-rich fucoidan of FCUS, obtained with an eco-friendly process, could be implemented as a beneficial antioxidant agent in the functional food industry.

Ishige okamurae Extract and Its Constituent Ishophloroglucin A Attenuated In Vitro and In Vivo High Glucose-Induced Angiogenesis.

Diabetes is associated with vascular complications, such as impaired wound healing and accelerated vascular growth. The different clinical manifestations, such as retinopathy and nephropathy, reveal the severity of enhanced vascular growth known as angiogenesis. This study was performed to evaluate the effects of an extract of Ishige okamurae (IO) and its constituent, Ishophloroglucin A (IPA) on high glucose-induced angiogenesis. A transgenic zebrafish (flk:EGFP) embryo model was used to evaluate vessel growth. The 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), gap closure, transwell, and Matrigel® assays were used to analyze the proliferation, migration, and capillary formation of EA.hy926 cells. Moreover, protein expression were determined using western blotting. IO extract and IPA suppressed vessel formation in the transgenic zebrafish (flk:EGFP) embryo. IPA attenuated cell proliferation, cell migration, and capillary-like structure formation in high glucose-treated human vascular endothelial cells. Further, IPA down regulated the expression of high glucose-induced vascular endothelial growth factor receptor 2 (VEGFR-2) and downstream signaling molecule cascade. Overall, the IO extract and IPA exhibited anti-angiogenic effects against high glucose-induced angiogenesis, suggesting their potential for use as therapeutic agents in diabetes-related angiogenesis.

Pyrogallol-Phloroglucinol-6,6'-Bieckol from Ecklonia cava Improved Blood Circulation in Diet-Induced Obese and Diet-Induced Hypertension Mouse Models.

Blood circulation disorders, such as hyperlipidemia and arteriosclerosis, are not easily cured by dietary supplements, but they can be mitigated. Although Ecklonia cava extract (ECE), as dietary supplements, are associated with improving the conditions, there are not many studies verifying the same. In this study, the beneficial effect of ECE and leaf of Ginkgo biloba extract (GBE), which is a well-known dietary supplement, were first confirmed in a diet induced-obese model. Afterwards, 4 phlorotannins were isolated from ECE, and their inhibitory effects on vascular cell dysfunction were validated. Pyrogallol-phloroglucinol-6,6-bieckol (PPB) was selected to be orally administered in two mice models: the diet induced obese model and diet induced hypertension model. After four weeks of administration, the blood pressure of all mice was measured, after which they were subsequently sacrificed. PPB was found to significantly improve blood circulation, including a reduction of adhesion molecule expression, endothelial cell (EC) death, excessive vascular smooth muscle cell (VSMC) proliferation and migration, blood pressure, and lipoprotein and cholesterol levels. Based on the excellent efficacy in diet-induced mouse models of obese and hypertension, our results demonstrate that PPB is a valuable active compound from among the phlorotannins that were isolated and it has the potential to be used in functional foods for improving the blood circulation.

Anti-Obesity and Anti-Diabetic Effects of Ishige okamurae.

Obesity is associated with several health complications and can lead to the development of metabolic syndrome. Some of its deleterious consequences are related to insulin resistance, which adversely affects blood glucose regulation. At present, there is a growing concern regarding healthy food consumption, owing to awareness about obesity. Seaweeds are well-known for their nutritional benefits. The brown alga Ishige okamurae (IO) has been studied as a dietary supplement and exhibits various biological activities in vitro and in vivo. The bioactive compounds isolated from IO extract are known to possess anti-obesity and anti-diabetic properties, elicited via the regulation of lipid metabolism and glucose homeostasis. This review focuses on IO extract and its bioactive compounds that exhibit therapeutic effects through several cellular mechanisms in obesity and diabetes. The information discussed in the present review may provide evidence to develop nutraceuticals from IO.

1-(5-Bromo-2-hydroxy-4-methoxyphenyl)ethanone [SE1] Inhibits MMP-9 Expression by Regulating NF-κB and MAPKs Signaling Pathways in HT1080 Human Fibrosarcoma Cells.

Hippocampus is a traditional medicine in China, which can be used for treating tumors, aging, fatigue, thrombosis, inflammation, hypertension, prostatic hyperplasia, and other diseases. 1-(5-Bromo-2-hydroxy-4-methoxyphenyl)ethanone [SE1] from seahorse (Hippocampus kuda Bleeler) has been shown to suppress proinflammatory responses. In the present study, SE1 potently inhibited gelatin digestion by MMP-9 induced by phorbol 12-myristate 13-acetate (PMA) and migration of human fibrosarcoma HT1080 cells in dose-dependent manner. Moreover, western blot analysis and immunofluorescence analysis have been studied on MAPKs (ERK1/2, p38 kinase and JNK) and NF-κB (p65 and IκB), which refer to the clear molecular mechanism. The results indicated that SE1 significantly suppressed the phosphorylation of mitogen-activated protein kinases (MAPK: p38 kinase and JNK) and NF-κB. Finally, molecular docking result showed SE1 interacts with TYR245 and HIS226 of MMP-9 by hydrogen bond and Pi-Pi bond to suppress MMP-9 activity. This data suggested that the SE1 may possess therapeutic and preventive potential for the treatment of MMP-9 related disorders.

Ishophloroglucin A, a Novel Phlorotannin for Standardizing the Anti-α-Glucosidase Activity of Ishige okamurae.

Nutraceutical use of algae requires understanding of the diversity and significance of their active compositions for intended activities. Ishige okamurae (I. okamurae) extract is well-known to possess α-glucosidase inhibitory activity; however, studies are needed to investigate its active composition in order to standardize its α-glucosidase inhibitory activity. In this study, we observed the intensity of the dominant compounds of each I. okamurae extract harvested between 2016 and 2017, and the different potency of each I. okamurae extract against α-glucosidase. By comparing the anti-α-glucosidase ability of the dominant compounds, a novel Ishophloroglucin A with highest α-glucosidase inhibitory activity was identified and suggested for standardization of anti-α-glucosidase activity in I. okamurae extract. Additionally, a validated analytical method for measurement of Ishophloroglucin A for future standardization of I. okamurae extract was established in this study. We suggest using Ishophloroglucin A to standardize anti-α-glucosidase potency of I. okamurae and propose the significance of standardization based on their composition for effective use of algae as marine-derived nutraceuticals.

Protective Effect of Sulfated Polysaccharides from Celluclast-Assisted Extract of Hizikia fusiforme Against Ultraviolet B-Induced Skin Damage by Regulating NF-κB, AP-1, and MAPKs Signaling Pathways In Vitro in Human Dermal Fibroblasts.

Our previous study evaluated the antioxidant activities of sulfated polysaccharides from Celluclast-assisted extract of Hizikia fusiforme (HFPS) in vitro in Vero cells and in vivo in zebrafish. The results showed that HFPS possesses strong antioxidant activity and suggested the potential photo-protective activities of HFPS. Hence, in the present study, we investigated the protective effects of HFPS against ultraviolet (UV) B-induced skin damage in vitro in human dermal fibroblasts (HDF cells). The results indicate that HFPS significantly reduced intracellular reactive oxygen species (ROS) level and improved the viability of UVB-irradiated HDF cells in a dose-dependent manner. Furthermore, HFPS significantly inhibited intracellular collagenase and elastase activities, remarkably protected collagen synthesis, and reduced matrix metalloproteinases (MMPs) expression by regulating nuclear factor kappa B (NF-κB), activator protein 1 (AP-1), and mitogen-activated protein kinases (MAPKs) signaling pathways in UVB-irradiated HDF cells. These results suggest that HFPS possesses strong UV protective effect, and can be a potential ingredient in the pharmaceutical and cosmetic industries.

Cellular properties of the fermented microalgae Pavlova lutheri and its isolated active peptide in osteoblastic differentiation of MG­63 cells.

Fermented microalgae Pavlova lutheri (P. lutheri), the product of Hansenula polymorpha fermentation, exhibited an increase in alkaline phosphatase (ALP) activity in MG­63 osteoblastic cells when compared to that of non­fermented P. lutheri. Fractionation of the fermented P. lutheri resulted in identification of the active peptide [peptide of P. lutheri fermentation (PPLF)] with the sequence of EPQWFL. PPLF significantly increased ALP release from MG­63 cells and mineralization in a dose­dependent manner. In addition, the intracellular levels of ALP and osteocalcin (OCN) proteins were augmented by PPLF treatment. To identify the molecular mechanism underlying the effect of PPLF on osteoblastic differentiation, the phosphorylation levels of the mitogen­activated protein kinases, p38, extracellular signal­regulated kinases 1/2 and Jun, and nuclear factor (NF)­κB were determined following PPLF treatment and the differences in expression were analyzed using p38 and NF­κB selective inhibitors. These results concluded that PPLF from fermented P. lutheri induced osteoblastic differentiation by increasing ALP and OCN release in MG­63 cells via the p38/p65 signaling pathway, indicating that PPLF supplement may be effective for therapeutic application in the field of bone health.

Sustained Simultaneous Delivery of Metronidazole and Doxycycline From Polycaprolactone Matrices Designed for Intravaginal Treatment of Pelvic Inflammatory Disease.

Poly(ɛ-caprolactone) (PCL) intravaginal matrices were produced for local delivery of a combination of antibacterials, by rapidly cooling a mixture of drug powders dispersed in PCL solution. Matrices loaded with different combinations of metronidazole (10%, 15%, and 20% w/w) and doxycycline (10% w/w) were evaluated in vitro for release behavior and antibacterial activity. Rapid "burst release" of 8%-15% of the doxycycline content and 31%-37% of the metronidazole content occurred within 24 h when matrices were immersed in simulated vaginal fluid at 37°C. The remaining drug was extracted gradually over 14 days to a maximum of 65%-73% for doxycycline and 62%-71% for metronidazole. High levels of antibacterial activity up to 89%-91% against Gardnerella vaginalis and 84%-92% against Neisseria gonorrhoeae were recorded in vitro for release media collected on day 14, compared to "nonformulated" metronidazole and doxycycline solutions. Based on the in vitro data, the minimum levels of doxycycline and metronidazole released from PCL matrices in the form of intravaginal rings into vaginal fluid in vivo were predicted to exceed the minimum inhibitory concentrations for N. gonorrhea (reported range 0.5-4.0 µg/mL) and G. vaginalis (reported range 2-12.8 µg/mL) respectively, which are 2 of the major causative agents for pelvic inflammatory disease.

In vitro cytotoxicity of Nicotiana gossei leaves, used in the Australian Aboriginal smokeless tobacco known as pituri or mingkulpa.

The Aboriginal population of Central Australia use endemic Nicotiana species to make a smokeless tobacco product known usually as pituri or mingkulpa. Nicotiana leaves are masticated with wood ash into a 'quid' that is chewed/sucked for absorption of nicotine. In addition to nicotine, smokeless tobacco products contain a spectrum of biologically active compounds that may contribute to effects on health. The objective of this study was to quantify nicotine, and related alkaloids and tobacco specific nitrosamines (TSNAs), in Nicotiana leaves used in pituri, and compare in vitro toxicity of pure nicotine with Nicotiana leaf extract at the same concentration of nicotine. An aqueous extract of dry leaves of Nicotiana gossei and a reference smokeless tobacco (CORESTA CRP2) were quantified for major pyridine alkaloids and TSNAs using HPLC-UV and LC-MS/MS. A range of extract concentrations and corresponding concentrations of nicotine standard were tested using an MTS assay to measure human lung epithelium cell (A549) survival. Cells treated for 24h with the maximum concentration of 1.5mg/ml of nicotine resulted in 77% viability. In contrast, extracts from N. gossei leaves and CRP2 containing a similar concentration of nicotine (1.3mg/ml) resulted in remarkably lower viability of 1.5 and 6%, respectively. Comparison of cytotoxicity of pure nicotine with that of the extracts revealed that nicotine was not the source of their cytotoxicity. Other biologically active compounds such as the known carcinogens NNK and NNN, derived from nicotine and nornicotine and found to be present in the smokeless tobacco extracts, may be responsible.

A reversed-phase HPLC-UV method developed and validated for simultaneous quantification of six alkaloids from Nicotiana spp.

A reversed-phase HPLC-UV method was developed, optimized, and validated for the separation and quantitation of six target alkaloids from leaves of Nicotiana species (nicotine, nornicotine, anatabine, anabasine, myosmine, and cotinine). A bidentate reversed-phase C18 column was used as stationary phase and an alkaline ammonium formate buffer and acetonitrile as mobile phase. The alkaloids were well separated in a short run time of 13min with mobile phase pH 10.5 and a small gradient of 9-13% acetonitrile, and detected using UV at 260nm. Peak parameters were acceptable for all six closely related alkaloids. The proposed method has enough linearity with correlation coefficient >0.999 within the investigated range for all tested alkaloids. Satisfactory precision was achieved for both intra- and inter-day assay, with RSD less than 2% for all alkaloid standards. Reproducibility was also within the acceptable range of RSD <2%. Limit of detection was 1.6µg/mL for nicotine and below 1µg/mL for all other alkaloids. The limit of quantification was 2.8 and 4.8µg/mL for nornicotine and nicotine respectively, and below 2µg/mL for all other alkaloids. The method was successfully applied for simultaneous analysis of alkaloids in leaves of Nicotiana benthamiana.

Optimization of hydrolysis conditions, isolation, and identification of neuroprotective peptides derived from seahorse Hippocampus trimaculatus.

Hippocampus trimaculatus is one of the most heavily traded seahorse species for traditional medicine purposes in many countries. In the present study, we showed neuroprotective effects of peptide derived from H. trimaculatus against amyloid-ß42 (Aß42) toxicity which are central to the pathogenesis of Alzheimer's diseases (AD). Firstly, H. trimaculatus was separately hydrolyzed by four different enzymes and tested for their protective effect on Aß42-induced neurotoxicity in differentiated PC12 cells. Pronase E hydrolysate exerted highest protection with cell viability value of 88.33 ± 3.33 %. Furthermore, we used response surface methodology to optimize pronase E hydrolysis conditions and found that temperature at 36.69 °C with the hydrolysis time 20.01 h, enzyme to substrate (E/S) ratio of 2.02 % and pH 7.34 were the most optimum conditions. Following several purification steps, H. trimaculatus-derived neuroprotective peptides (HTP-1) sequence was identified as Gly-Thr-Glu-Asp-Glu-Leu-Asp-Lys (906.4 Da). HTP-1 protected PC12 cells from Aß42-induced neuronal death with the cell viability value of 85.52 ± 2.22 % and up-regulated pro-survival gene (Bcl-2) expressions. These results suggest that HTP-1 has the potential to be used in treatment of neurodegenerative diseases, particularly AD. Identification, characterization, and synthesis of bioactive components derived from H. trimaculatus have the potential to replace or at least complement the use of seahorse as traditional medicine, which further may become an approach to minimize seahorse exploitation in traditional medicine.

Antioxidant peptides from protein hydrolysate of microalgae Navicula incerta and their protective effects in HepG2/CYP2E1 cells induced by ethanol.

Marine microalgae have been reported as valuable new sources of pharmacologically active compounds and there are now numerous commercial applications of microalgae. Hence, in this study we evaluated the protective effects of peptides purified from marine microalgae, Navicula incerta, against alcohol-induced damage in HepG2/CYP2E1 cells. To obtain bioactive peptides from microalgae, N. incerta was hydrolysed using various enzymes (alcalase, α-chymotrypsin, neutrase, papain, pepsin, pronase-E and trypsin), and the hydrolysates were evaluated for cytoprotective activity. Among them, papain-derived hydrolysate exhibited higher antioxidant activities than those of other enzymes. Therefore, papain hydrolysate was purified in order to obtain potent antihepatotoxic and antioxidative peptides. The amino acid sequences of the purified peptides were analysed as; NIPP-1 (Pro-Gly-Trp-Asn-Gln-Trp-Phe-Leu) with molecular mass 1 171 Da, and NIPP-2 (Val-Glu-Val-Leu-Pro-Pro-Ala-Glu-Leu) with molecular mass 1108 Da. Furthermore, this study demonstrated that NIPP-1 and NIPP-2 peptides inhibited ethanol-induced cytotoxicity in HepG2/CYP2E1 cells.

Sea cucumber, Stichopus japonicus ethyl acetate fraction modulates the lipopolysaccharide induced iNOS and COX-2 via MAPK signaling pathway in murine macrophages.

The sea cucumber Stichopus japonicus is an important food and traditional medicine in Asian countries. However, ethyl acetate solvent fraction of S. japonicus (SCEA-F) is largely unknown for its anti-inflammatory activity and related molecular mechanisms. In this study, effect of SCEA-F on inflammation was investigated in LPS stimulated RAW264.7 cells. SCEA-F significantly inhibited the productions of NO and PGE(2) by inhibiting iNOS and COX-2 at their protein and gene levels. The production and the gene transcription of pro-inflammatory cytokines are also inhibited. The responsible molecular signaling for these inhibitory actions was found to be through suppression of the phosphorylation of MAPK molecules; ERK and p38 MAPK. These results indicate that SCEA-F inhibits LPS-induced inflammatory response via blocking of MAPK signaling pathway in murine macrophages, thus demonstrated its in vitro anti-inflammatory potential. Therefore it could be suggested that SCEA-F could be effectively used in functional food preparations.

Differentiation of human osteosarcoma cells by isolated phlorotannins is subtly linked to COX-2, iNOS, MMPs, and MAPK signaling: implication for chronic articular disease.

Arthritis is one of the most prevalent chronic inflammatory diseases, and it is characterized by structural and biochemical changes in major tissues of the joint, including degradation of the cartilage matrix, insufficient synthesis of extracellular matrix (ECM). Ecklonia cava (EC) is a member of the family of Laminariaceae, which is an edible marine brown alga with various bioactivities. In this study of the methanol extract of brown alga EC, the dieckol (1) and 1-(3',5'-dihydroxyphenoxy)-7-(2'',4'',6''-trihydroxyphenoxy) 2,4,9-trihydroxydibenzo-1,4,-dioxin (2) were isolated and characterized by NMR techniques with high yield. Phlorotannin derivatives (1, 2) promoted osteosarcoma differentiation by increasing alkaline phosphatase (ALP) activity, mineralization, total protein and collagen synthesis in human osteosarcoma cell (MG-63 cells), respectively. Furthermore, these phlorotannin derivatives (1, 2) inhibited mRNA gene and protein levels of matrix metalloproteinase (MMP-1, MMP-3, and MMP-13), iNOS and COX-2 in casein zymography, Western blot and reverse transcriptase-polymerase chain reaction (RT-PCR) assays. In addition, it was observed that the phlorotannins inhibited phosphorylation of JNK and p38 MAPK in human osteosarcoma cell. These results suggested the phlorotannin derivatives (1, 2) could promote cell differentiation, attenuate MMP-1, MMP-3, MMP-13 expressions, and inflammatory response via MAPK pathway in chronic articular diseases.

Chemical components and its antioxidant properties in vitro: an edible marine brown alga, Ecklonia cava.

Seven phlorotannins were isolated and characterized from an edible marine brown alga Ecklonia cava (EC), along with three common sterol derivatives (fucosterol, ergosterol, and cholesterol) according to the comprehensive spectral analysis of MS and NMR data. Compounds 5 (7-phloro eckol) and 7 (6,6'-bieckoll) of phlorotannin derivatives were obtained for the first time with the high yields. No reports of compound 3 (Fucodiphloroethol G) was published up to date. The antioxidant properties of all phlorotannins were assessed by total antioxidant activity in a linoleic acid model, free radicals scavenging assay using electron spin resonance spectrometry (ESR) technique, cellular reactive oxygen species (ROS) assay by DCFH-DA, membrane protein oxidation assay; measurement of cellular glutathione (GSH) level in RAW264.7 cell line, and myeloperoxidase (MPO) assay in HL-60 cell line. The results revealed that all phlorotannins had antioxidant properties in vitro, especially, compounds 7 (6,6'-bieckol), 6, and 3 showed the significant activities compared to the other phlorotannins. Furthermore, the structure-activity relationship (SAR) was discussed based on the structural differences of the tested phlorotannins which have polymeriged phloroglucinal units with diverse skeletons and linkages. It could be suggested that phlorotaninns from this genus would be more potential candidates for the development of unique natural antioxidants for further industrial applications as functional foods, cosmetics and pharmaceuticals. As well as our results makes it clear to understand the reason behind the use of EC as traditional folk herb for a long history.