Sulfated Galactans from Gracilaria fisheri with Supplementation of Octanoyl Promote Wound Healing Activity In Vitro and In Vivo.

Sulfated galactans (SG) isolated from Gracilaria fisheri is partially degraded (DSG), and subsequentially supplemented with octanoyl (DSGO) and sulfate (DSGS) groups. The molecular weights of DSG, DSGO, and DSGS are 7.87, 152.79, and 97.07 kDa, respectively. The modification is confirmed using FTIR and NMR, while in vitro wound healing activity is assessed using scratched wound fibroblasts. The results reveal that DSGO exhibits highest percentage of wound closure in scratched fibroblast L929 cells. Furthermore, DSGO is able to promote proliferation and accelerate migration of scratched fibroblasts, which correspond to the regulation of proteins and mRNA (Ki67, p-FAK, vimentin, and E-cadherin) determined by Western blotting and qPCR analysis. The superior wound healing activity of DSGO is also confirmed in excision wound of rats. The results demonstrate that DSGO significantly enhances the percentage of wound closure, re-epithelialization, and collagen arrangement, increases α-smoth muscle actin (α-SMA) and vimentin expression, and decreases that of tumor necrosis factor-α (TNF-α) at the wound site. The results suggest that degraded SG supplemented with medium-chain fatty acids of octanoyl group may pass through the membrane, subsequently activating the mediators associated with proliferation and migration of fibroblasts, which can potentially lead to the promotion of wound healing activity.

Bioencapsulation efficacy of sulfated galactans in adult Artemia salina for enhancing immunity in shrimp Litopenaeus vannamei.

Live food organisms like Artemia have been used for delivery of different substances such as nutrients, probiotics and immune-stimulants to aquatic animals. Previously, we reported that sulfated galactans (SG) from the red seaweed Gracilaria fisheri (G. fisheri) increased immune activity in shrimp. In the present study we further investigated the capacity and efficiency of bioencapsulation of SG in adult Artemia for delivery to tissues and potentially boosting the expression of immune genes in post larvae shrimp. SG were labelled with FITC (FITC-SG) for in vivo tracking in shrimp. Bioencapsulation of adult Artemia with FITC-SG (0-100 µg mL-1) was performed and the fluorescence intensity was detected in the gut lumen after enrichment periods of 30 min, 1 h, 2 h, 6 h and 24 h. The results showed the Artemia took up SG over time in a concentration-dependent manner. Shrimp were fed with the bioencapsulated Artemia (FITC-SG, 20 µg mL-1) and the shrimp were evaluated under a stereo-fluorescent microscope. At 24 h after administration, FITC-SG was located in gills and hepatopancreas and also bound with haemocytes. With daily SG administration, the genes IMD, IKKß were up-regulated (after 1 day) while genes dicer and proPO-I were up-regulated later (after 7 days). Moreover, continued monitoring of shrimp fed for 3 consecutive days only with SG at the dose of 0.5 mg g-1 BW showed increases in the expression of IMD, IKKß genes on day 1 and which gradually declined to normal levels on day 14, while the expression of dicer and proPO-I was increased on day 3 and remained high on day 14. These results demonstrate that bioencapsulation of SG in adult Artemia successfully delivers SG to shrimp tissues, which then bind with haemocytes and subsequently activate immune genes, and potentially increase immunity in shrimp. In addition, the present study suggests that a 3-consecutive-day regimen of SG supplemented in Artemia (0.5 mg g-1 BW) may boost and sustain the enhanced immune functions in post larvae shrimp.

Bioflocs substituted fishmeal feed stimulates immune response and protects shrimp from Vibrio parahaemolyticus infection.

Fishmeal is the main source of protein in the shrimp feed industry and is normally derived from trash fish. As such, the production of fishmeal has an adverse effect on the marine environment by taking away small and juvenile fish, leading to depletion of marine species. There is a need for alternative sources of protein which will substitute fishmeal in the aquaculture industry. This study evaluated the components and nutritional efficacy of bioflocs, which were used to substitute fishmeal protein. The effect of bioflocs diets on growth performance, survival rate, and immune response in shrimp compared to normal fishmeal feed were determined. Bioflocs were harvested from the shrimp ponds (C:N ratio >12:1) at Shrimp Village, Chaiya district, Surat Thani, Thailand. The total protein in bioflocs was about 48% and the total lipid was about 5% (dried weight) and the percentages of essential amino acids (EAA) and fatty acids (EFA) in bioflocs were similar to those of fishmeal feed. Shrimp fed with the different dietary bioflocs feed regimens [% to replace fishmeal; 0% (B0), 25% (B25), 50% (B50), 75% (B75), and 100% (B100)] for 42 days revealed that all growth parameters were almost similar to those of the control shrimp (shrimp fed with normal fishmeal, B0) including final body weight, weight gain, specific growth rate, and feed conversion ratio. Remarkably, the survival rates, the levels of immune parameters, and expression of immune genes (proPO-I, PEN-4 and dicer) were significantly higher in bioflocs fed shrimp, especially in B25 and B50 shrimp. Moreover, B25 and B50 bioflocs fed shrimp showed notably increased survival rates following Vibrio parahaemolyticus (V. parahaemolyticus) infection. In conclusion, the present study demonstrates that shrimp survival and immunity are enhanced by biofiocs substituted fishmeal. Significantly, the bioflocs diets activated the immune response to prevent V. parahaemolyticus infection.

Ethanolic extract of red seaweed Gracilaria fisheri and furanone eradicate Vibrio harveyi and Vibrio parahaemolyticus biofilms and ameliorate the bacterial infection in shrimp.

Bacteria respond to host immunity for their proliferation and survival by cell-cell communications such as biofilm formation, bioluminescence, and secreting virulence factors. In the biofilm form, bacteria are more resistant to various antimicrobial treatments and withstand the host's immune system. The approaches of deciphering biofilm formation for treating bacterial infections are therefore highly desirable. Recently, we have reported that the ethanolic extract of the red seaweed Gracilaria fisheri (G. fisheri) enhanced immune activities and inhibited growth of the luminescent bacteria Vibrio harveyi in shrimp. We undertook the present research study in order to evaluate and compare the effectiveness of the ethanolic extract from G. fisheri and furanone, a known biofilm inhibitor, in inhibiting the formation of clinically important Vibrio biofilms. The results showed that sub-lethal concentrations of both the ethanolic extracts (5, 10 and 100 µg ml-1) and furanone (5 µM) inhibited biofilm formation by V. harveyi and Vibrio parahaemolyticus and also light production (luminescence) in V. harveyi. It is known that V. harveyi mediated light production via autoinducer AI-2 pathway, we further determined whether the inhibitory effect of the extract was involved the AI-2 signaling. The bioluminescence assay was conducted in an AI-2 deletion mutant V. harveyi. Supplementation of the AI-2 containing media with the extract or furanone impaired the light production in the mutant V. harveyi suggesting that the extract interfered AI-2 mediated light production similar to furanone. In vivo challenge study showed that the low concentrations (Sub MICs) of the ethanolic extract and furanone decreased bacterial adhesion and colonization in the surfaces of stomach lumen, down-regulated expression of a virulence factor, and protected shrimp against mortality from V. harveyi and V. parahaemolyticus infection. In conclusion, the present results suggest a potential application of the low concentrations of the ethanolic extract of G. fisheri as an efficient approach for treating biofilm-associated Vibrio diseases in aquacultures.

Assessment of the effects of sulfated polysaccharides extracted from the red seaweed Irish moss Chondrus crispus on the immune-stimulant activity in mussels Mytilus spp.

Seaweeds contain a number of health enhancing and antimicrobial bioactive compounds including sulfated polysaccharides (SP). In the present study, SP extracted from a European red seaweed Irish moss Chondrus crispus was chemically analyzed, SP content extracted and the immune-response effect on wild Irish mussels Mytilus spp. investigated for the first time. A high percent yield of SP was extracted from C. crispus and the immune-stimulant activity of SP was assessed in a laboratory trial with mussels exposed to three different treatments of low (10 µg mL-1), medium (20 µg mL-1) and high (50 µg mL-1) SP dose concentrations and a control mussel group with no exposure to SP. An initial mussel sample was processed prior to the trial commencing and mussels were subsequently sampled on Days 1, 2, 3, 4, 7, and 10 post SP exposure. Both cell, humoral and immune related gene responses including haemocyte cell viability, haemocyte counts, lysozyme activity and expression of immune related genes (defensin, mytimycin and lysozyme mRNA) were assessed. No mussel mortalities were observed in either the treated or non-treated groups. Mussels exposed with SP showed an increase in haemocyte cell viability and the total number of haemocytes compared to control mussels. Lysozyme activity was also higher in treated mussels. Additionally, up-regulated expression of defensin, mytimycin and lysozyme mRNA was observed in SP treated mussels shortly after exposure (on Days 1, 2, and 3) to SP. These results indicate that a high quality yield of SP can be readily extracted from C. crispus and more importantly based on the animal model used in this study, SP extracted from C. crispus can rapidly induce health enhancing activities in Mytilus spp. at a cellular, humoral and molecular level and with a prolonged effect up to ten days post treatment.

Sulfated galactans from the red seaweed Gracilaria fisheri exerts anti-migration effect on cholangiocarcinoma cells.

BACKGROUND: Seaweeds have a long history of use in Asian countries as functional foods, medicinal herbs, and the treatment of cancer. Polysaccharides from various seaweeds have shown anti-tumor activity. Cholangiocarcinoma (CCA), often with metastatic disease, is highly prevalent in Thailand as a consequence of liver fluke infection. Recently, we extracted sulfated galactans (SG) from Gracilaria fisheri (G. fisheri), a south east Asian seaweed, and found it exhibited anti-proliferation effect on CCA cells. PURPOSE: In the present study, we evaluated the anti-migration activity of SG on CCA cells and its underlined mechanism. METHODS: CCA cells were treated with SG alone or drugs targeting to epidermal growth factor (EGF) receptor (EGFR) or pretreated with SG prior to incubation with EGF. Anti-migration activity was determined using a scratch wound-healing assay and zymography. Immunofluorescence staining and western blotting were used to investigate EGFR signaling mediators. RESULTS: Under basal condition, SG reduced the migration rate of CCA, which was correlated with a decrease in the active-form of matrix metalloproteinases-9. SG decreased expression of phosphorylated focal adhesion kinase (FAK), but increased expression of E-cadherin to promote cells stasis. Moreover, phosphorylation of EGFR and extracellular signal-regulated kinases (ERK), known to stimulate growth of cancer cells, was blocked in a comparable way to EGFR inhibitors Cetuximab and Erlotinib. Pretreatment cells with SG attenuated EGF induced phosphorylation of EGFR, ERK and FAK. CONCLUSION: This study reveals that SG from G. fisheri retards migration of CCA cells, and its mechanism of inhibition is mediated, to some extent, by inhibitory effects on MAPK/ERK signal transduction pathway. Our findings suggest that there may be a therapeutic potential of SG in CCA treatment.

A sulfated galactans supplemented diet enhances the expression of immune genes and protects against Vibrio parahaemolyticus infection in shrimp.

A sulfated galactans (SG) supplemented diet was evaluated for the potential to stimulate immune activity in shrimp Penaeus vannamei (P. vannamei). Shrimp given the SG supplemented diet (0.5, 1 and 2% w/w) for 7 days showed enhanced expression of the downstream signaling mediator of lipopolysaccharide and ß-1,3-glucan binding protein (LGBP) and immune related genes including p-NF-κB, IMD, IKKß and IKKε, antimicrobial peptide PEN-4, proPO-I and II. Following immersion with Vibrio parahaemolyticus (V. parahaemolyticus) for 14 days, the shrimp given the SG supplemented diet (1 and 2% w/w) showed a decrease in bacterial colonies and bacterial toxin gene expression, compared to shrimp given a normal diet, and they reached 50% mortality at day 14. However, shrimp given the normal diet and challenged with the bacteria reached 100% mortality at day 6. SG-fed shrimp increased expression of immune genes related to LGBP signaling at day 1 after the bacterial immersion compared to control (no immersion), which later decreased to control levels. Shrimp on the normal diet also increased expression of immune related genes at day 1 after immersion which however decreased below control levels by day 3. Taken together, the results indicate the efficacy of the SG supplemented diet to enhance the immune activity in shrimp which could offer protection from V. parahaemolyticus infection.

Sulfated Galactans from Red Seaweed Gracilaria fisheri Target EGFR and Inhibit Cholangiocarcinoma Cell Proliferation.

Cholangiocarcinoma (CCA) is increasing in incidence worldwide and is resistant to chemotherapeutic agents, making treatment of CCA a major challenge. Previous studies reported that natural sulfated polysaccharides (SPs) disrupted growth factor receptor activation in cancer cells. The present study, therefore, aimed at investigating the antiproliferation effect of sulfated galactans (SG) isolated from the red seaweed Gracilaria fisheri (G. fisheri) on CCA cell lines. Direct binding activity of SG to CCA cells, epidermal growth factor (EGF) and epidermal growth factor receptor (EGFR) were determined. The effect of SG on proliferation of CCA cells was investigated. Cell cycle analyses and expression of signaling molecules associated with proliferation were also determined. The results demonstrated that SG bound directly to EGFR. SG inhibited proliferation of various CCA cell lines by inhibiting EGFR and extracellular signal-regulated kinases (ERK) phosphorylation, and inhibited EGF-induced increased cell proliferation. Cell cycle analyses showed that SG induced cell cycle arrest at the G0/G1 phase, down-regulated cell cycle genes and proteins (cyclin-D, cyclin-E, cdk-4, cdk-2), and up-regulated the tumor suppressor protein P53 and the cyclin-dependent kinase inhibitor P21. Taken together, these data demonstrate that SG from G. fisheri inhibited proliferation of CCA cells, and its mechanism of inhibition is mediated, to some extent, by inhibitory effects on EGFR activation and EGFR/ERK signaling pathway. SG presents a potential EGFR targeted molecule, which may be further clinically developed in a combination therapy for CCA treatment.

Green tea polyphenol epigallocatechin-3-gallate attenuates TNF-α-induced intercellular adhesion molecule-1 expression and monocyte adhesion to retinal pigment epithelial cells.

Epigallocatechin-3-gallate (EGCG) is a major polyphenol component of green tea (Camellia sinensis) and demonstrates anti-oxidant, anticancer and anti-inflammatory properties. EGCG has been shown to protect retinal pigment epithelium (RPE) against oxidative stress-induced cell death. The pathogenesis of diseases in the retina is usually initiated by local inflammation at the RPE cell layer, and inflammation is mostly associated with leukocyte migration and the secretion of pro-inflammatory cytokines. Whether EGCG can modulate the cytokine-induced inflammatory response of RPE, particularly leukocyte migration, has not been clearly elucidated, and was therefore the objective of this study. ARPE-19 cells were cultured with different concentrations of TNF-α in the presence or absence of EGCG to different time points. Intracellular reactive oxygen species (ROS) levels were determined. Intercellular adhesion molecule (ICAM)-1 and phosphor-NF-κB and IκB expression were determined by Western blot analysis. Phosphor-NF-κB nuclear translocation and monocyte-RPE adhesion were investigated using immunofluorescence confocal laser scanning microscopy. Scanning electron microscopy (SEM) was carried out to further determine the ultrastructure of monocyte-RPE adhesion. The results demonstrated that TNF-α modulated inflammatory effects in ARPE-19 by induction of ROS and up-regulation of ICAM-1 expression. Moreover, TNF-α-induced phosphor-NF-κB nuclear translocation, increased phosphor-NF-κB expression and IκB degradation, and increased the degree of monocyte-RPE adhesion. Pretreating the cells with EGCG ameliorated the inflammatory effects of TNF-α. The results indicated that EGCG significantly exerts anti-inflammatory effects in ARPE-19 cells, partly as a suppressor of TNF-α signaling and that the inhibition was mediated via the NF-κB pathway.

Sulfated galactans isolated from the red seaweed Gracilaria fisheri target the envelope proteins of white spot syndrome virus and protect against viral infection in shrimp haemocytes.

The present study was aimed at evaluating an underlying mechanism of the antiviral activity of the sulfated galactans (SG) isolated from the red seaweed Gracilaria fisheri against white spot syndrome virus (WSSV) infection in haemocytes of the black tiger shrimp Penaeus monodon. Primary culture of haemocytes from Penaeus monodon was performed and inoculated with WSSV, after which the cytopathic effect (CPE), cell viability and viral load were determined. Haemocytes treated with WSSV-SG pre-mix showed decreased CPE, viral load and cell mortality from the viral infection. Solid-phase virus-binding assays revealed that SG bound to WSSV in a dose-related manner. Far Western blotting analysis indicated that SG bound to VP 26 and VP 28 proteins of WSSV. In contrast to the native SG, desulfated SG did not reduce CPE and cell mortality, and showed low binding activity with WSSV. The current study suggests that SG from Gracilaria fisheri elicits its anti-WSSV activity by binding to viral proteins that are important for the process of viral attachment to the host cells. It is anticipated that the sulfate groups of SG are important for viral binding.

Diarylheptanoid 7-(3,4 dihydroxyphenyl)-5-hydroxy-1-phenyl-(1E)-1-heptene from Curcuma comosa Roxb. protects retinal pigment epithelial cells against oxidative stress-induced cell death.

Chronic exposure to oxidative stress causes damage to retinal pigment epithelial cells which may lead to the development of age-related macular degeneration, the major cause of vision loss in humans. Anti-oxidants provide a natural defense against retinal cell damage. The present study was designed to evaluate the potential anti-oxidant activity and protective effect of two diarylheptanoids isolated from a medicinal herb Curcuma comosa; 7-(3,4 dihydroxyphenyl)-5-hydroxy-1-phenyl-(1E)-1-heptene (compound A), and 1,7-diphenyl-4(E),6(E)-heptadien-3-ol (compound B) against oxidative stress (H(2)O(2))-induced human retinal pigment epithelial (APRE-19) cell death. The 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay indicated that the anti-oxidant activity (IC(50)) of compound A was similar to that of vitamin C. Pre-treatment of ARPE-19 cells with 20 µM compound A for 4h afforded greater protection against the insult from 500 µM H(2)O(2), compared to a similar protection period for compound B. Compound A lowered H(2)O(2)-induced lipid peroxidation, malondialdehyde formation and intracellular reactive oxygen species. Furthermore, compound A ameliorated the H(2)O(2)-induced decrease in anti-oxidant enzyme activities and subsequent apoptotic cell death in ARPE-19 cells in a dose and time-dependent manner. These results suggest that compound A protects ARPE-19 cells against oxidative stress, in part, by enhancing several anti-oxidant defense mechanisms. Therefore, compound A may have therapeutic potential for diseases associated with oxidative stress, particularly degenerative retinal diseases.

Solvent extracts of the red seaweed Gracilaria fisheri prevent Vibrio harveyi infections in the black tiger shrimp Penaeus monodon.

Vibriosis is a common bacterial disease that can cause high mortality and morbidity in farmed shrimp. Since compounds from seaweed have been reported to have anti-bacterial and immunostimulant activity, this study was conducted to determine whether solvent extracts from the red seaweed Gracilaria fisheri might be a possible alternative for prevention and treatment of shrimp vibriosis caused by Vibrio harveyi. Seaweed extracts prepared using ethanol, methanol, chloroform and hexane were evaluated for anti-V. harveyi activity by the disc-diffusion method. The ethanol, methanol and chloroform extracts showed activity against a virulent strain of V. harveyi with potency (minimal inhibitory concentrations in the range of 90-190 µg ml(-1)) equivalent to the antibiotic norfloxacin. The ethanol extract was not toxic to the brine shrimp Artemia salina when it was fed to them for enrichment prior to their use, in turn, as feed for postlarvae of Penaeus monodon. Postlarvae fed with these enriched Artemia gave significantly lower mortality than control postlarvae after challenge with V. harveyi. In addition, P. monodon juveniles injected with the ethanol extract showed a significant increase in the total number of haemocytes and an increased proportion of semi-granulocytes and granulocytes when compared to control shrimp. The activities of phenoloxidase and superoxide dismutase were also increased, with an accompanying increase in superoxide anion production. When these juvenile shrimp were challenged with V. harveyi, mortality was markedly reduced compared to that of control shrimp. The results indicated that ethanol extracts of G. fisheri had immunostimulant and antimicrobial activity that could protect P. monodon against V. harveyi.