Retraction Note to: Biological and Biochemical Potential of Sea Snake Venom and Characterization of Phospholipase A2 and Anticoagulation Activity.

[This retracts the article DOI: 10.1007/s12291-015-0500-6.].

Evaluation of astaxanthin incorporated collagen film developed from the outer skin waste of squid Doryteuthis singhalensis for wound healing and tissue regenerative applications.

The present investigation was aimed to evaluate in vivo wound healing activity of astaxanthin incorporated collagen hydrogel film biomaterials extracted from the outer skin waste of squid Doryteuthis singhalensis, to releases antibiotic, delivering potentialities of excisional and incisional wound model in Wistar rats. These results suggested that the astaxanthin incorporated collagen film (ACF) and gentamicin incorporated collagen film (GCF) exhibited excellent wound healing activity (71%) in both full thickness excision and linear incision in rats. The in-vitro antioxidant abilities of extracted astaxanthin exhibited strongly significant 1,1­diphenyl­2­picrylhydrazyl (DPPH) radical scavenging activity. In addition, tensile strength, epithelialization, hydroxyproline content and protein content in ACF and GCF treated groups were significantly increased. Histopathological assessment revealed an increase in collagen content, fibroblasts, granulation, thickness of scar formation, effective neovascularization and faster epithelialization within the short duration after the treatment of ACF and GCF compared to the control groups. The structure of prepared ACF and GCF biomaterials were characterized by SEM, EDS, and XRD. The in vivo biological study of the collagen-based film releases the antibiotic substance. The composite of collagen based biomaterials displays a promising biocompatibility through the dermal wound healing process as well as an evidence of biodegradability. Thus, the marine-derived biomaterials gave a substantial pledge for the development of biodegradable materials in drug delivery and soft tissue regeneration process.

Formulation of alginate based hydrogel from brown seaweed, Turbinaria conoides for biomedical applications.

Sodium Alginate (SA) is an excellent carrier in various drug delivery systems. In this study, SA was synthesized from brown seaweed, Turbinaria conoides with a yield of 31.3 ± 0.86%. The analysis of physicochemical properties of extracted alginate (ALG) determined its purity. The structural confirmations of ALG were studied through FTIR, XRD and SEM analysis. Formulation of ALG with collagen (COL) as a wound healing microfilm showed potential anti-inflammatory properties (81.3 ± 1.77%) and sustained drug release. Likewise, the ALG microbead encapsulated with an anticancer drug, Tamoxifen indicated an in vitro sustained release in the range of 62 ± 0.70% - 91 ± 0.56%. The overall swelling behavior of both the hydrogels, microfilm and microbead provides new opportunities for development of natural ALG in this therapeutic era.

Isolation and Identification of Cytotoxic and Biological Active Toxin from the Puffer Fish Arothron stellatus.

This study is to investigate the biological, biochemical and cytotoxic effects of puffer fish (Arothron stellatus) toxin extracts under in-vitro condition. Extracted toxins from various organs of puffer fish were purified by using active charcoal column, and Bio-gel-P2 column chromatography. The lethality of toxin was tested in crabs, which consists of neurotoxic compounds. The degree of the brine shrimp lethality assay was found directly proportional to the concentration of the toxin extracts, which was well supported by hemolytic assay. The experimental results suggested that the gonad was found higher toxins than the liver and muscles. The mortality rate of brine shrimp nauplii was increased with the raise of concentrations of toxin level. Among the different doses and time dependent cytotoxic effect of human cervical carcinoma (HeLa) cells were showed 4.0 µg/mL of toxin, which was effectively inhibited cancer cell proliferation. HPLC and TLC analysis was revealed that the A. stellatus toxin contains tetrodotoxin (TTX), related compounds 4-epi TTX and anhydro-TTX. The present results suggested that the A. stellatus contain TTX as a major and anh-TTX as a minor toxin. It could be the potential candidate in the field of anticancer drug discovery against human cervical cancer cells. The present data is confirming that the puffer fish toxin as an interesting source of novel bioactive natural compounds with potent applications in pharmacology.

Biological and Biochemical Potential of Sea Snake Venom and Characterization of Phospholipase A2 and Anticoagulation Activity.

This study is designed to isolate and purify a novel anti-clotting protein component from the venom of Enhydrina schistosa, and explore its biochemical and biological activities. The active protein was purified from the venom of E. schistosa by ion-exchange chromatography using DEAE-cellulose. The venom protein was tested by various parameters such as, proteolytic, haemolytic, phospholipase and anti-coagulant activities. 80 % purity was obtained in the final stage of purification and the purity level of venom was revealed as a single protein band of about 44 kDa in SDS-polyacrylamide electrophoresis under reducing conditions. The results showed that the Potent hemolytic activity was observed against cow, goat, chicken and human (A, B and O positive) erythrocytes. Furthermore, the clotting assays showed that the venom of E. schistosa significantly prolonged in activated partial thromboplastin time, thrombin time, and prothrombin time. Venomous enzymes which hydrolyzed casein and gelatin substrate were found in this venom protein. Gelatinolytic activity was optimal at pH 5-9 and (1)H NMR analysis of purified venom was the base line information for the structural determination. These results suggested that the E. schistosa venom holds good promise for the development of novel lead compounds for pharmacological applications in near future.

Isolation and characterization of biologically active venom protein from sea snake Enhydrina schistosa.

The present study is designed to investigate the isolation and characterization of biological and biochemical active venom protein from sea snake, Enhydrina schistosa. The highest purification peaks in ion-exchange chromatography on DEAE-cellulose column were obtained for fraction numbers 39-49 when eluted with 0.35-0.45 M NaCl. Eighty per cent purity was obtained in the final stage of purification, and a single protein band of about 44 kDa was visualized in SDS-polyacrylamide gel under reducing condition. Purified venom protein expressed as haemolytic, cytotoxicity and proteolytic activities with lethal concentration (LC50 ) at 2.0 µg/mL. Venom protein exhibits enzymatic activity and hydrolyzed casein and gelatin. Gelatinolytic activity was optimal at pH 5-9. In conclusion, the present results suggested that the sea snake venom might be feasible sources for biologically active substances. Thus, this low molecular weight component of the venom protein could be used in potentially serve biological and pharmaceutical aspects.

Screening of antiangiogenic potential of twenty two marine invertebrate extracts of phylum Mollusca from South East Coast of India.

OBJECTIVE: To evaluate the antiangiogenic potential of twenty two marine invertebrate species of Phylum Mollusca from south east coast of India. METHODS: Live specimens of molluscan species were collected and their methanolic extracts were evaluated for preliminary antiangiogenic activity using the in ovo chick chorio-allantoic membrane assay. The extracts were further evaluated for in vivo antiangiogenic activity using chemical cautery induced corneal neovascularization assay in rats and oxygen induced retinopathy assay in rat pups. RESULTS: In the chick chorio-allantoic membrane assay, four methanolic extracts of marine molluscan species viz. Meretrix meretrix, Meretrix casta, Telescopium telescopium and Bursa crumena methanolic extracts exhibited noticeable antiangiogenic activity at the tested concentration of 200 µg whereby they significantly inhibited the VEGF induced proliferation of new blood vessels. Among these four extracts, the methanolic extract of Meretrix casta exhibited relatively higher degree of antiangiogenic activity with an inhibitiory percentage (64.63%) of the VEGF induced neovascularization followed by the methanolic extracts of Telescopium telescopium (62.02%), Bursa crumena (60.48%) and Meretrix meretrix (47.01%). These four methanolic extracts were further evaluated for in vivo antiangiogenic activity whereby the methanolic extract of Telescopium telescopium exhibited most noticeable inhibition (42.58%) of the corneal neovascularization in rats in comparison to the sham treated group, and also exhibited most noticeable inhibition (31.31%) of the oxygen induced retinal neovascularization in rat pups in comparison to the hyperoxia group that was observed for considerable retinal neovascularization. CONCLUSIONS: The significant antiangiogenic activity evinced by the extract of Telescopium telescopium merits further investigation for ocular neovascular diseases.

Isolation and characterization of hyaluronic acid from marine organisms.

Hyaluronic acid (HA) being a viscous slippery substance is a multifunctional glue with immense therapeutic applications such as ophthalmic surgery, orthopedic surgery and rheumatology, drug delivery systems, pulmonary pathology, joint pathologies, and tissue engineering. Although HA has been isolated from terrestrial origin (human umbilical cord, rooster comb, bacterial sources, etc.) so far, the increasing interest on this polysaccharide significantly aroused the alternative search from marine sources since it is at the preliminary level. Enthrallingly, marine environments are considered more biologically diverse than terrestrial environments. Although numerous methods have been described for the extraction and purification of HA, the hitch on the isolation methods which greatly influences the yield as well as the molecular weight of the polymer still exists. Adaptation of suitable method is essential in this venture. Stimulated by the developed technology, to sketch the steps involved in isolation and analytical techniques for characterization of this polymer, a brief report on the concerned approach has been reviewed.

Biological activities of heparan sulfate.

Heparan sulfate was isolated from two bivalve mollusks such as Tridacna maxima and Perna viridis. The isolated heparin was quantified in crude as well as purified samples and they were estimated as 2.72 and 2.2g/kg (crude) and 260 and 248 mg/g (purified) in T. maxima and P. viridis, respectively. Both the bivalves showed the anticoagulant activity of the crude and purified sample as 20,128 USP units/kg and 7.4 USP units/mg, 39,000 USP units/kg and 75 USP units/mg, 9460 USP units/kg and 4.3 USP units/mg, and 13,392 USP units/kg and 54 USP units/mg correspondingly in T. maxima and P. viridis. The antiproliferative activity that was studied with pulmonary artery smooth muscle cells using RPMI media reported that the result is in a dose-dependent manner. Among the two clams, P. viridis showed more antiproliferative activity than that of T. maxima.

Screening of antiangiogenic potential of twenty two marine invertebrate extracts of phylum Mollusca from South East Coast of India

Objective: To evaluate the antiangiogenic potential of twenty two marine invertebrate species of Phylum Mollusca from south east coast of India.Methods:Live specimens of molluscan species were collected and their methanolic extracts were evaluated for preliminary antiangiogenic activity using the in ovo chick chorio-allantoic membrane assay. The extracts were further evaluated for in vivo antiangiogenic activity using chemical cautery induced corneal neovascularization assay in rats and oxygen induced retinopathy assay in rat pups.Results:In the chick chorio-allantoic membrane assay, four methanolic extracts of marine molluscan species viz. Meretrix meretrix, Meretrix casta, Telescopium telescopium and Bursacrumena methanolic extracts exhibited noticeable antiangiogenic activity at the tested concentration of 200 μg whereby they significantly inhibited the VEGF induced proliferation of new blood vessels. Among these four extracts, the methanolic extract of Meretrix casta exhibited relatively higher degree of antiangiogenic activity with an inhibitiory percentage (64.63%) of the VEGF induced neovascularization followed by the methanolic extracts of Telescopium telescopium (62.02%), Bursa crumena (60.48%) and Meretrix meretrix (47.01%). These four methanolic extracts were further evaluated for in vivo antiangiogenic activity whereby the methanolic extract of Telescopium telescopium exhibited most noticeable inhibition (42.58%) of the corneal neovascularization in rats in comparison to the sham treated group, and also exhibited most noticeable inhibition (31.31%) of the oxygen induced retinal neovascularization in rat pups in comparison to the hyperoxia group that was observed for considerable retinal neovascularization.Conclusions:The significant antiangiogenic activity evinced by the extract of Telescopium telescopium merits further investigation for ocular neovascular diseases.

Isolation and characterization of drug delivering potential of type-I collagen from eel fish Evenchelys macrura.

Acid soluble collagen (ASC) and pepsin-soluble collagen (PSC) from the outer skin waste of marine eel fish (Evenchelys macrura) were isolated and characterized. The prepared collagen was used to test its effective drug delivering potential in vitro condition. Present results were confirmed as collagen by different physico-chemical techniques like SDS-PAGE, HPLC, FTIR and SEM. Further amino acid analysis corroborates isolation of type I collagen. Both ASC and PSC comprising two different α-chains (α 1 and α 2) were characterized as type I and contained imino acid of 190-200 residues, respectively. The denaturation temperatures (T(d)) of ASC and PSC were 38.5 and 35.0 °C, respectively, which is promising as an advantage for biomedical application due to closeness in T(d) to mammalian collagen. Furthermore, the gel and film forming capability of collagen samples containing implant standard antibiotic was proved to be a suitable drug delivering system.

Intestinal mast cell levels control severity of oral antigen-induced anaphylaxis in mice.

Food-triggered anaphylaxis can encompass a variety of symptoms that affect multiple organ systems and can be life threatening. The molecular distinction between non-life-threatening and life-threatening modes of such anaphylaxis has not yet been delineated. In this study, we sought to identify the specific immune functions that regulate the severity of oral antigen-induced anaphylaxis. We thus developed an experimental mouse model in which repeated oral challenge of ovalbumin-primed mice induced an FcεRI- and IgE-dependent oral antigen-triggered anaphylaxis that involved multiple organ systems. Strikingly, the severity of the systemic symptoms of anaphylaxis (eg, hypothermia) positively correlated with the levels of intestinal mast cells (r = -0.53; P < 0.009). In addition, transgenic mice with both increased intestinal and normal systemic levels of mast cells showed increased severity of both intestinal and extra-intestinal symptoms of IgE-mediated passive as well as oral antigen- and IgE-triggered anaphylaxis. In conclusion, these observations indicate that the density of intestinal mast cells controls the severity of oral antigen-induced anaphylaxis. Thus, an awareness of intestinal mast cell levels in patients with food allergies may aid in determining their susceptibility to life-threatening anaphylaxis and may eventually aid in the treatment of food-triggered anaphylaxis.

Cell type and gender-dependent differential regulation of the p202 and Aim2 proteins: implications for the regulation of innate immune responses in SLE.

Upon sensing cytosolic double-stranded DNA (dsDNA), the murine Aim2 (encoded by the Aim2 gene) protein forms an inflammasome and promotes the secretion of proinflammatory cytokines, such as IL-1ß and IL-18. In contrast, the p202 protein (encoded by the Ifi202 gene) does not form an inflammasome. Previously, we have reported that the interferon (IFN) and female sex hormone-induced increased nuclear levels of p202 protein in immune cells are associated with increased susceptibility to develop a lupus-like disease. However, signaling pathways that regulate the expression of Aim2 protein remain unknown. Here we report that the expression of Aim2 gene is induced in bone marrow-derived macrophages (BMDMs) by IFN-α treatment and the expression is, in part, STAT1-dependent. However, treatment of splenic T or B cells with IFN-α or their stimulation, which induced the expression of Ifi202 gene, did not induce the expression of Aim2 gene. Furthermore, treatment of cells with the male hormone androgen increased levels of Aim2 mRNA and protein. Moreover, treatment of murine macrophage cell lines (RAW264.7 and J774A.1) with IFN-α differentially induced the expression of Aim2 and p202 proteins and regulated their sub-cellular localization. Additionally, activation of Toll-like receptors (TLR3, 4, and 9) in BMDMs and cell lines also differentially regulated the expression of Aim2 and Ifi202 genes. Our observations demonstrate that cell type and gender-dependent factors differentially regulate the expression of the Aim2 and p202 proteins, thus, suggesting opposing roles for these two proteins in innate immune responses in lupus disease.

Increased susceptibility of 129SvEvBrd mice to IgE-Mast cell mediated anaphylaxis.

BACKGROUND: Experimental analyses have identified strain-dependent factors that regulate susceptibility to anaphylaxis in mice. We assessed the susceptibility of the widely used 129SvEvBrd (also known as 129S5) mouse strain to IgE/mast cell-mediated anaphylaxis as compared to BALB/c. Mice were subjected to passive and oral Ovalbumin [OVA]-induced active anaphylaxis. Tissue mast cell, plasma histamine, total IgE and OVA-specific IgE levels and susceptibility to histamine i.v infusion were assessed. Bone marrow mast cell (BMMC)s were examined for FcεRI, c-kit, degranulation efficiency, proliferation, apoptosis and cytokine profile. RESULTS: 129S5 mice had significantly increased susceptibility to passive and oral OVA-induced active anaphylaxis. Increased susceptibility to anaphylaxis was associated with increased homeostatic mast cell levels but not OVA-specific IgE or IgG1 levels. In vitro analyses of BMMCs revealed no difference in FcεRI and c-Kit expression, however, 129S5 BMMCs possessed greater proliferative capacity and reduced caspase-3-mediated apoptosis. IgE-BMMC degranulation assays demonstrated no difference in degranulation efficiency. Furthermore, 129S5 mice possessed increased sensitivity to histamine-induced hypothermia. CONCLUSIONS: We conclude that 129S5 mice have increased susceptibility to anaphylaxis as compared to BALB/c strain and their increased susceptibility was associated with altered mast cell proliferation and homeostatic tissue levels and responsiveness to histamine. Given the wide spread usage of the 129SvEvBrd strain of mice in experimental gene targeting methodology, these data have important implications for studying IgE-reactions in mouse systems.

Fibrinogenolytic and anticoagulant activities in the tissue covering the stingers of marine stingrays Dasyatis sephen and Aetobatis narinari.

Stingray envenomation is one of the major problems in the marine and freshwater ecosystem. Accidents in human cause immediate, local and intense pain, erythema, edema, hemorrhage, tissue necrosis and secondary bacterial infection are also common. To determine the effect of two marine stingray species Dasyatis sephen and Aetobatis narinari venom extract on coagulation, fibrin(ogen)olytic, proteolytic activities. Plasma coagulation, Thrombin catalyzed fibrinocoagulation, Fibrin plate assay, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), substrate SDS-PAGE and thrombin like activity by using chromogenic substrate were used to determine the effect of venom on plasma coagulation, its fibrin(ogen)olytic and proteolytic activity. The results show the presence of fibrin(ogen)olytic, anticoagulant and gelatinolytic activity in both stingray venom extracts. D. sephen venom delays coagulation of citrated plasma more significantly than A. narinari upon using increasing concentration of the venom. The same results were obtained in the fibrinocoagulation assays. SDS-PAGE analysis of fibrinogen and fibrin after incubation with D. sephen and A. narinari venom show fibrin(ogen)olytic activity. Through SDS-PAGE analysis it is confirmed that the delaying in coagulation process by stingray venom is due to its fibrin(ogen)olytic activity and fibrinolytic activity also confirmed through fibrin plate assay. Zymogram analysis shows the presence of array of gelatinolytic and fibrinogenolytic enzymes above 43-276 kDa in the D. sephen and A. narinari venom respectively. Protease inhibitor studies show the serine and metallo proteases are responsible for these activities. From the results, fibrinogenolytic, proteolytic activity of the stingray venom is confirmed, but it has no thrombin like activity and these activities may aid in hemorrhages, tissue necrosis and secondary bacterial infections at the site of envenomation.

Preliminary investigation on antimicrobial and proteolytic property of the epidermal mucus secretion of marine stingrays

Objective:To determine the antibacterial, antifungal, minimum inhibitory concentration (MIC) and the protease activity of the stingray mucus Dasyatis sephen (D. sephen) and Himantura gerrardi (H. gerrardi). Methods: Antimicrobial activity of crude aqueous, acidic and organic mucus extract was evaluated by disc diffusion method against human pathogens, MIC of the active samples were determined by spectrophotometric method and the protease activity which is responsible for the antimicrobial activity was determined by using zymogram method. Results:The crude acidic extracts of both the species showed antibacterial activity against Salmonella typhi (S. typhi), Klebsiella pneumoniae, Streptococcus aureus, Escherichia coli (E. coli), Vibrio cholerae (V. cholerae) and the acidic extracts of both the species exhibit antifungal activity against all the tested pathogens. Remaining extracts didn't show any inhibitory activity. The acidic extracts of H. gerrardi is significantly active against S. typhi, E. coli, V. cholerae, Trichophyton mentagrophytes (T. mentagrophytes), Alternaria alternaria (A. alternaria), Trichophyton rubrum (T. rubrum), Candida tropicalis (C. tropicalis) at the minimum concentration of 16μg/mL, but the acidic extract of D. sephen required 32μg/mL of protein to inhibit S.typhi, E. coli, Aspergillus niger (A. niger), penicillium sp, T. mentagrophytes, A. alternaria. Both the D. sephen and H. gerrardi shows the proteolytic activity above the molecular mass of> 66 KDa. The characterization of protease class using inhibitors showed the presence of both serine and metallo protease in the the samples. Conclusions:Protease activity present in the sting ray mucus is one of the key factor responsible for the antimicrobial activity and the results proved the role of mucus in the innate immunity.