Structural and functional characterization of a thermostable secretory phospholipase A2 from Sciscionella marina and its application in liposome biotransformation.

Secretory phospholipase A2 (sPLA2), which hydrolyzes the sn-2 acyl bond of lecithin in a Ca2+-dependent manner, is an important enzyme in the oil and oleochemical industries. However, most sPLA2s are not stable under process conditions. Therefore, a thermostable sPLA2 was investigated in this study. A marine bacterial sPLA2 isolated from Sciscionella marina (Sm-sPLA2) was catalytically active even after 5 h of incubation at high temperatures of up to 50°C, which is outstanding compared with a representative bacterial sPLA2 (i.e. sPLA2 from Streptomyces violaceoruber; Sv-sPLA2). Consistent with this, the melting temperature of Sm-sPLA2 was measured to be 7.7°C higher than that of Sv-sPLA2. Furthermore, Sm-sPLA2 exhibited an improved biotransformation performance compared with Sv-sPLA2 in the hydrolysis of soy lecithin to lysolecithin and free fatty acids at 50°C. Structural and mutagenesis studies revealed that the Trp41-mediated anchoring of a Ca2+-binding loop into the rest of the protein body is directly linked to the thermal stability of Sm-sPLA2. This finding provides a novel structural insight into the thermostability of sPLA2 and could be applied to create mutant proteins with enhanced industrial potential.

The Effects of Spirulina maxima Extract on Memory Improvement in Those with Mild Cognitive Impairment: A Randomized, Double-Blind, Placebo-Controlled Clinical Trial.

Spirulina maxima is a marine microalga that has been promoted worldwide as a super food. This study was conducted to evaluate its ability to improve memory in the older adults using Spirulina maxima 70% ethanol extract (SM70EE). This randomized, double-blind, placebo-controlled clinical trial comprised 80 volunteers recruited from Jeonbuk National University Hospital in Jeonju, Republic of Korea, who were randomly assigned to two groups. The participants received either 1 g/day of SM70EE or a placebo without otherwise changing their diet or physical activity. The participants were examined at baseline and after a 12-week interval to determine whether there were changes in their results for visual learning, visual working memory, and verbal learning tests from the Korean version of the Montreal Cognitive Assessment, brain-derived neurotrophic factor and beta-amyloid levels, and total antioxidant capacity. Compared to the placebo group, the treatment group showed a significant improvement in visual learning and visual working memory test results and enhanced vocabulary. SM70EE use was shown to improve memory, with no adverse effects. Its efficacy in alleviating Alzheimer's disease symptoms was verified for the first time through this clinical trial. SM70EE could play a role in the management of patients with dementia. This trial is registered with registration number of clinical research information service (CRIS: KCT0006161).

Spirulina maxima Derived Pectin Nanoparticles Enhance the Immunomodulation, Stress Tolerance, and Wound Healing in Zebrafish.

In this study, Spirulina maxima derived pectin nanoparticles (SmPNPs) were synthesized and multiple biological effects were investigated using in vitro and in vivo models. SmPNPs were not toxic to Raw 264.7 cells and zebrafish embryos up to 1 mg/mL and 200 µg/mL, respectively. SmPNPs upregulated Il 10, Cat, Sod 2, Def 1, Def 2, and Muc 1 in Raw 264.7 cells and tlr2, tlr4b, tlr5b, il1ß, tnfα, cxcl8a, cxcl18b, ccl34a.4, ccl34b.4, muc5.1, muc5.2, muc5.3, hamp, cstd, hsp70, cat, and sod1 in the larvae and adult zebrafish, suggesting immunomodulatory activity. Exposure of larvae to SmPNPs followed by challenge with pathogenic bacterium Aeromonas hydrophila resulted a two-fold reduction of reactive oxygen species, indicating reduced oxidative stress compared to that in the control group. The cumulative percent survival of larvae exposed to SmPNPs (50 µg/mL) and adults fed diet supplemented with SmPNPs (4%) was 53.3% and 76.7%, respectively. Topical application of SmPNPs on adult zebrafish showed a higher wound healing percentage (48.9%) compared to that in the vehicle treated group (38.8%). Upregulated wound healing markers (tgfß1, timp2b, mmp9, tnfα, il1ß,ccl34a.4, and ccl34b.4), enhanced wound closure, and restored pigmentation indicated wound healing properties of SmPNPs. Overall, results uncover the multiple bioactivities of SmPNPs, which could be a promising biocompatible candidate for broad range of aquatic and human therapies.

Spirulina maxima derived marine pectin promotes the in vitro and in vivo regeneration and wound healing in zebrafish.

Purified bioactive components of marine algae have shown great pharmaceutical and biomedical potential, including wound healing activity. However, the activity of Spirulina maxima is the least documented with regard to wound healing potential. In the present study, we investigated the regenerative and wound healing activities of a Spirulina (Arthrospira) maxima based pectin (SmP) using in vitro human dermal fibroblasts (HDFs) and in vivo zebrafish model. SmP treated (12.5-50 µg/mL) HDFs showed increased cell proliferation by 20-40% compared to the untreated HDFs. Moreover, in vitro wound healing results in HDFs demonstrated that SmP decreased the open wound area % in concentration-dependent manner at 12.5 (32%) and 25 µg/mL (12%) compared to the control (44%). Further, zebrafish larvae displayed a greater fin regenerated area in the SmP exposed group at 25 (0.48 mm2) and 50 µg/mL (0.51 mm2), whereas the untreated group had the lowest regenerated area (0.40 mm2) at 3 days post amputation. However, fin regeneration was significantly (P < 0.001) higher only in the SmP treated group at 50 µg/mL. Furthermore, the open skin wound healing % in adult zebrafish was significantly higher (P < 0.05) after topical application (600 µg/fish) of SmP (46%) compared to the control (38%). Upregulation of genes such as tgfß1, timp2b, mmp9, tnf-α, and il-1ß, and chemokines such as cxcl18b, ccl34a.4, and ccl34b.4, in the muscle and kidney tissues of SmP treated fish compared to the respective control group was demonstrated using qRT-PCR. Histological analysis results further supported the rapid epidermal growth and tissue remodeling in SmP treated fish, suggesting that SmP exerts positive effects associated with wound healing. Therefore, SmP can be considered a potential regenerative and wound healing agent.

Marine Microalgae, Spirulina maxima-Derived Modified Pectin and Modified Pectin Nanoparticles Modulate the Gut Microbiota and Trigger Immune Responses in Mice.

This study evaluated the modulation of gut microbiota, immune responses, and gut morphometry in C57BL/6 mice, upon oral administration of S. maxima-derived modified pectin (SmP, 7.5 mg/mL) and pectin nanoparticles (SmPNPs; 7.5 mg/mL). Metagenomics analysis was conducted using fecal samples, and mice duodenum and jejunum were used for analyzing the immune response and gut morphometry, respectively. The results of metagenomics analysis revealed that the abundance of Bacteroidetes in the gut increased in response to both modified SmP and SmPNPs (75%) as compared with that in the control group (66%), while that of Firmicutes decreased in (20%) as compared with that in the control group (30%). The mRNA levels of mucin, antimicrobial peptide, and antiviral and gut permeability-related genes in the duodenum were significantly (p < 0.05) upregulated (> 2-fold) upon modified SmP and SmPNPs feeding. Protein level of intestinal alkaline phosphatase was increased (1.9-fold) in the duodenum of modified SmPNPs feeding, evidenced by significantly increased goblet cell density (0.5 ± 0.03 cells/1000 µm2) and villi height (352 ± 10 µm). Our results suggest that both modified SmP and SmPNPs have the potential to modulate gut microbial community, enhance the expression of immune related genes, and improve gut morphology.

Novel pectin isolated from Spirulina maxima enhances the disease resistance and immune responses in zebrafish against Edwardsiella piscicida and Aeromonas hydrophila.

In this study, we demonstrate the enhanced disease resistance and positive immunomodulation of novel pectin isolated from Spirulina maxima (SmP) in zebrafish model. Zebrafish larvae exposed to SmP had significantly (p < 0.05) higher cumulative percent survival (CPS) at 25 (44.0%) and 50 µg/mL (67.0%) against Edwardsiella piscicida compared to the control. However, upon Aeromonas hydrophila challenge, SmP exposed larvae at 50 µg/mL had slightly higher CPS (33.3%) compared to control group (26.7%). SmP supplemented zebrafish exhibited the higher CPS against E. piscicida (93.3%) and A. hydrophila (60.0%) during the early stage of post-infection (<18 hpi). qRT-PCR results demonstrated that exposing (larvae) and feeding (adults) of SmP, drive the modulation of a wide array of immune response genes. In SmP exposed larvae, up-regulation of the antimicrobial enzyme (lyz: 3.5-fold), mucin (muc5.1: 2.84, muc5.2: 2.11 and muc5.3: 2.40-fold), pro-inflammatory cytokines (il1ß: 1.79-fold) and anti-oxidants (cat: 2.87 and sod1: 1.82-fold) were identified. In SmP fed adult zebrafish (gut) showed >2-fold induced pro-inflammatory cytokine (il1ß) and chemokines (cxcl18b, ccl34a.4 and ccl34b.4). Overall results confirmed the positive modulation of innate immune responses in larval stage and it could be the main reason for developing disease resistance against E. piscicida and A. hydrophila. Thus, non-toxic, natural and biodegradable SmP could be considered as the potential immunomodulatory agent for sustainable aquaculture.

Photo-oxidative stress by ultraviolet-B radiation and antioxidative defense of eckstolonol in human keratinocytes.

Ultraviolet-B (UV-B) irradiation has been known to generate oxidative stress by increasing reactive oxygen species (ROS) in skin cells. Several naturally occurring antioxidant compounds isolated from marine algae are believed to protect against ROS. In this study, we assessed the antioxidative effect of eckstolonol isolated from Ecklonia cava against UV-B-induced ROS in human keratinocytes (HaCaTs). We investigated the effects of photo-oxidative stress by UV-B (50 mJ/cm(2)) and the antioxidative effects of eckstolonol using fluorometry, flow cytometry, microscopy, and cell viability and comet assays. UV-B irradiation decreased cell viability, which was restored in a dose-dependent manner with eckstolonol treatment (0, 5, 50, 100, and 200 µM). Moreover, eckstolonol reduced UV-B-induced ROS, lipid peroxidation, damaged DNA levels, and cell death. These antioxidative effects seem to be due to the enzymatic activities of catalase (CAT) and superoxide dismutase (SOD). Collectively, these results indicate that eckstolonol is capable of protecting keratinocytes from photo-oxidative stress.

Neuroprotective effect of phlorotannin isolated from Ishige okamurae against H2O2 -induced oxidative stress in murine hippocampal neuronal cells, HT22.

The present study is designed to investigate the neuroprotective effect of a kind of phlorotannins, diphlorethohydroxycarmalol (DPHC) isolated from Ishige okamurae against hydrogen peroxide (H(2)O(2))-induced oxidative stress in murine hippocampal neuronal cells, HT22. H(2)O(2) treatment induced neurotoxicity, whereas DPHC prevented cells from H(2)O(2)-induced damage then restoring cell viability was significantly increased. DPHC slightly reduced the expression of Bax induced by H(2)O(2) but recovered the expression of Bcl-xL as well as caspase-9 and -3 mediated PARP cleavage by H(2)O(2). Intracellular reactive oxygen species (ROS) and lipid peroxidation was overproduced as the result of the addition of H(2)O(2); however, these ROS generations and lipid peroxidation were effectively inhibited by addition of DPHC in a dose-dependent manner. Moreover, DPHC suppressed the elevation of H(2)O(2)-induced Ca(2+) release. These findings indicate that DPHC has neuroprotective effects against H(2)O(2)-induced damage in neuronal cells, and that an inhibitory effect on ROS production may contribute to the underlying mechanisms.

Inhibitory effect of diphlorethohydroxycarmalol on melanogenesis and its protective effect against UV-B radiation-induced cell damage.

In this study, potential inhibitory effect of 21 species of marine algae on melanogenesis was assessed via tyrosinase inhibitory effect. The Ishige okamurae extract tested herein evidenced profound tyrosinase inhibitory effect, compared to that exhibited by other marine algae extracts. Thus, I. okamurae was selected for use in further experiments, and was partitioned with different organic solvents. Profound tyrosinase inhibitory effect was detected in the ethyl acetate fraction, and the active compound was identified as the carmalol derivative, diphlorethohydroxycarmalol (DPHC), which evidenced higher levels of activity than that of commercial whitening agent. Intracellular reactive oxygen species (ROS) induced by ultraviolet (UV)-B radiation was reduced by the addition of DPHC and cell viability was dose-dependently increased. Moreover, DPHC demonstrated strong protective properties against UV-B radiation via damaged DNA tail length and morphological changes in fibroblast. Hence, these results indicate that DPHC isolated from I. okamurae has potential whitening effects and prominent protective effects on UV-B radiation-induced cell damages which might be used in pharmaceutical and cosmeceutical industries.

Transcriptional up-regulation of disk abalone selenium dependent glutathione peroxidase by H(2)O(2) oxidative stress and Vibrio alginolyticus bacterial infection.

Selenium dependent glutathione peroxidase (Se-GPx) belongs to the family of selenoprotein, which acts mainly as an antioxidant in the cellular defence system. We have identified Se-GPx full length cDNA from disk abalone (Haliotis discus discus) designated as AbSe-GPx. It has a characteristic codon at (223)TGA(225) that corresponds to selenocysteine (Sec) amino acid as U(75). The full length cDNA consists of 675 bp, an open reading frame encoding 225 amino acids. Sequence characterization revealed that AbSe-GPx contains a characteristic GPx signature motif 2 ((97)LGFPCNQF(104)), an active site motif ((183)WNFEKF(188)) and essential residues for the enzymatic function. Additionally, the eukaryotic selenocysteine insertion sequence (SECIS) is conserved in the 3' UTR. The AbSe-GPx amino acid sequence exhibited the highest level of identity (46%) with insect (Ixodes scapularis) GPx, and shares 41% with bivalve (Unio tumidus) Se-GPx. The RT-PCR analysis revealed that AbSe-GPx mRNA was expressed constitutively in gill, mantle, gonad, abductor muscle, digestive tract, and hemocytes in a tissue specific manner. AbSe-GPx mRNA expression was significantly up-regulated in gill and digestive tract tissues after H(2)O(2) injection and Vibrio alginolyticus infection. However, AbSe-GPx expression was not up-regulated after Aroclor 1,254 injection. These results indicate that AbSe-GPx mRNA is expressed at a basal level in abalone tissues, which can be up-regulated transcriptionally by H(2)O(2) oxidative stress and Vibrio alginolyticus infection. Therefore, AbSe-GPx may be involved in a protective role against H(2)O(2) oxidative stress and immune defence against bacterial infection.