Investigating the Impact of Collagen-Chitosan Derived from Scomberomorus Guttatus and Shrimp Skin on Second-Degree Burn in Rats Model.

BACKGROUND: The present study focused on burning as one of the main causes of mortality with detrimental economic and social effects in the world. The purpose of this study was to investigate the impact of collagen-chitosan gel extracted from Scomberomorus guttatus and shrimp skin in the treatment of second degree burn healing among rats. MATERIALS & METHOD: To fulfill the purpose of the study, chitosan and collagen were extracted respectively from shrimp and Scomberomorus guttatus skin waste by the acid-based method and were evaluated by using Pico Tag, SDS-PAGE. The burn wound healing efficiency of marine collagen-chitosan gel was examined in vivo using rats. Three different ratios of collagen and chitosan blend (Col-CH, 1:3, 1:1 and 3:1) were prepared to obtain the most effective Col-CH gel for burn wound healing and were compared to the animals treated with silver sulfadiazine ointment. Healing burn wound was studied by measuring wound surface area with Image J and histopathologic examination was carried out based on the mean of epithelialization, fibroblastic cells, acute and chronic inflammatory cells, angiogenesis, structure collagen and the amount of collagen on days 15 and 25 post-burn. RESULTS: The results of SDS-PAGE indicated that the extracted collagen was type I and it was composed of two α (α1 and α2) chains. Amino acid analysis showed a much higher glaycin content in extracted collagen which amounted to one-third of the total amino. The wound surface measurement showed a significant reduction in wound size in the group treated with Col-CH (3:1) compared to silver-sulfadiazine treated group on 15th and 25th days. Histopathological findings represented a high score in epithelialization, collagen, collagen structure, fibroblast cell and a decrease in inflammatory cells infiltration in Col-CH (3:1) treated group on 25th day. The most obvious finding of the present study is that chitosan-collagen gel (3:1) represented a better efficacy compared to sulfadiazine in burn wound healing on day 25 post-burn.

Significant Anticancer Activity of a Venom Fraction Derived from the Persian Gulf Sea Anemone, Stichodactyla haddoni.

Chemotherapy is still one of the main therapeutic regimens in cancer patients but its toxicity is a hard challenge for every patient yet. One of the available solutions is tracing for non-toxic anticancer agents from natural resources. Numerous proteins and peptides in the venom of sea anemones are potentially useful agents with pharmacological properties. Concerning to significance of this issue, the current study was aimed to finding a non-toxic anticancer fraction from the venom of the Persian Gulf sea anemone, Stichodactyla haddoni. Anticancer and hemolytic activity of crude venom was evaluated and followed by fractionation using RP-HPLC. Breast, Brain, and Colon cancer cell lines were selected to assessment of anticancer activity and toxicity. IC50 of crude venom on the abovementioned cancer cell lines was as 4.13, 6.58, and 31.54 µg, respectively. According to the results obtained by paired sample t-test and comparison of toxicity of the fractions in normal cell line, F10, designated as hadonin, was determined as the candidate anti-cancer fraction. The non-toxic dose of F10 was 20 ng in which showed respectively 66, 29, and 7 anticancer activities on breast, brain, and colon cancer cell lines. According to results, anticancer activity of hadonin is of high pharmaceutical value to follow its therapeutic potency in animal model. In conclusion, the venom of the Persian Gulf sea anemone contains a potential anticancer agent with reasonable activity at nanogram level against three kinds of cancer cells with no toxicity on normal cells.

Immobilization of platinum nanoparticles on the functionalized chitosan particles: an efficient catalyst for reduction of nitro compounds and tandem reductive Ugi reactions.

In this work, we reported a facile synthesis of Pt nanoparticles (NPs) on proline-functionalized cross-linked chitosan particles to catalyze the reduction of R-NO2 to R-NH2 in aqueous medium using sodium borohydride. Chitosan was functionalized with (3-bromopropyl)proline and cross-linked with glutaraldehyde for the synthesis of chitosan NPs. The platinum NPs was prepared on the chitosan NPs. This catalyst showed the good selectivity and activity for reduction of nitro compound as well as for tandem reduction of nitro groups in Ugi reaction.

Biochemical, Hematological Effects and Complications of Pseudosynanceia Melanostigma Envenoming.

OBJECTIVES: Venomous fishes have different pharmacological effects and are useful. Among the venomous fish, stonefishes; especially Pseudosynanceia melanostigma has various pharmacological effects on the nervous, muscular and cardiovascular system of humans. In this study, toxicological characteristics, some blood effects, pharmacological and enzymatic properties of Pseudosynanceia melanostigma venom was investigated. METHODS: Crude venom purified by using gel filtration chromatography and the molecular weights of the venom and its fractions were estimated. The approximate LD values of this venom were determinedand the effects of LD50 dose on the blood of rabbits were studied. Hemolytic and Hemorrhagic activity of the venom sample was determined. In this case coagulation tests were performed. RESULTS: The LD50 of the Pseudosynanceia melanostigma crude venom was also determined to be 194.54 µg/mouse. The effect of two doses of LD50 showed a non-significant differences decrease in RBCs and MCV. In other cases, the results showed significant differences in WBC, Plt, Hb, MCH, MCHC and HCT; also it's showed a significant decrease. WBC count showed a significant increase with two doses of LD50 groups. The prothrombin time and partial prothrombin time were increased after venom treatment. As well as bleeding and clotting time were increased. According to the results, a minimum dose for Haemorrhagic effect 40 µg was obtained. CONCLUSION: Venom of Pseudosynanceia melanostigma has inhibitory effect on platelet aggregation that can be used to design and develop of anticoagulant drugs.

Ontogenic development of the digestive tract in larval and juvenile Vimba bream, Vimba vimba.

In this study we examined the ontogenic development of the digestive tract of Vimba bream (Vimba vimba, Family: Cyprinidae) during the first 60 days of life (hatching to 60 days after hatching [DAH]). Samples of developing Vimba bream were randomly selected at various stages of development: 1-8, 10, 15, 20, 25, 30, 40, 50, and 60 DAH. For the histological and histochemical studies on the development of the alimentary canal, tissue sections prepared from the sampled hatchlings were stained with hematoxylin-eosin and periodic acid-Schiff and observed under a light microscope. The histological structures of both the mouth and esophagus were fully developed at 5 and 7 DAH, respectively. Intestinal differentiation was observed at 2 DAH, while mucosal folds appeared on the intestinal bulb at 7 DAH. At 5 DAH, with the appearance of goblet cells in the epithelium of the mouth, pharynx, and esophagus, the larvae showed secretion activity in these organs. At 6 DAH, secretion was observed in the intestine; at this stage of development, the surface of the gastrointestinal tract was covered in a neutral mucous-like layer of polysaccharide. The histological observations indicate that the early development of the digestive tract in Vimba vimba enables larvae to efficiently ingest and digest exogenous feed very quickly after hatching.

Assessment of the Inhibitory Effects of Ficin-hydrolyzed Gelatin Derived from Squid (Uroteuthis duvauceli) on Breast Cancer Cell Lines and Animal Model.

Marine novel natural products have been applied for cancer therapy. Enzyme-digested gelatin hydrolysates have proven to serve as promising sources of potent biologically active peptides. Potential anti-breast cancer properties of the extracted Ficin-digesterd gelatin hydrolysate from Indian squid (Uroteuthis duvauceli) was extensively characterized by cellular and animal models. Gelatin was extracted from squid skin, hydrolyzed by Ficin, and characterized by standard physico-chemical methods. Ficin-digested gelatin hydrolysate was used at various doses of 0-0.1 mg/mL for assessment of MCF-7 and MDA-MB-231 breast cancer cells versus HUVEC normal cells. Cytotoxicity, phase-contrast morphological examination, apoptosis/necrosis, clonal-growth, cell-migration, Matrix-metalloproteinases (MMPs) zymography, and Western blotting were used for cellular assessments. For animal studies, breast tumor-induced BALB/c mice received hydrolyzed gelatin regimen, followed by tumor size/growth and immune-histochemical analyses. Significant inhibition of MCF-7 and MDA-MB-231 with no cytotoxicity on HUVEC cells were detected. Apoptosis was increased in cancer cells, as revealed by elevated ratio of cleaved caspase-3 and PARP. MMP-2 and MMP-9 activities in both cancer cells were diminished. In mice, gelatin hydrolysate prevented weight loss, decreased tumor size, induced p53, and down-regulated Ki67 levels. These findings suggest that Ficin-digested gelatin hydrolysate could be a beneficial candidate for novel breast cancer therapies.

Toxicity and Potential Pharmacological Activities in the Persian Gulf Venomous Sea Anemone, Stichodactyla haddoni.

Numerous proteins and peptides in venomous marine animals are potentially active molecules with pharmacological properties. Particular condition of the Persian Gulf as a closed ecosystem is a good opportunity to study of biological activities and toxicity of venomous animals. In this study, Stichodactyla haddoni (S. haddoni), a sea anemone, selected to tracing for possible pharmaceutical agents and toxicological characterization. Analgesic, edematogenic, dermonecrotic, LD50, phospholipase, and proteolytic activities of the venom were estimated. LD50 was recorded at 675 µg by intraperitoneal injection. Analgesic activitiy of crude venom on Balb/c mice at both 100 and 150 µg were dose dependent as a linear trend. Three folds increase of activity was seen at both 100 and 150 µg after 240 min comparing to activity of morphine at 200 µg. The crude venom at amount of 0.23 µg produced 50% hemolysis. The highest edematogenic activity was seen on Balb/c mice just two hours after injection for both 168 µg (157%) and 335 µg (247%). The crude venom at 675 µg made 4 mm inflammation area on rabbit skin after 3 h but the amount of 1000 µg induced 8 mm necrosis area. Potent analgesic activity of the venom was seen below its toxic dose that was very greater than the other sea anemones in the other geographical areas. The results indicate that a persistent edematogenic activity could be happened after envenomation. Instant potent edematogenic and rapid dermonecrotic activity were significant phenomena. HD50 at 0.23 µg indicates that a very potent hemolytic agent exists in the venom. The results would also be of high value to better management of envenomation. This study confirmed the great value of further studies on the Persian Gulf S. haddoni venom.

Description of histopathological changes induced by the venom of the Persian Gulf Lionfish (Pterois russelli) in a mouse model of multiorgan toxicity.

Pterois russelli is a venomous fish belongs to Scorpaenidae family. Envenomation by the Persian Gulf lionfish is associated with local pain, marked inflammation and local heat. The present study was aimed to document the histopathological changes in liver, heart, lung, kidney and alterations in release of critical enzymes such as LDH, CK. AST, ALT and ALP induced by the administration of various doses of P. russelli venom in a mouse model. LD50 of venom was determined by intravenous injection in Balb/c mice. Histopathological alterations of lung, liver, heart and kidney following injection of one LD50, 1/2 and 1/3 LD50 doses of the venom were evaluated. Simultaneously, release of LDH, CK, AST, ALT and ALP were measured in serum following administration of 1/2 and1/3 LD50 doses of the venom too. LD50 was calculated as 10.5 mg/kg. The level of all enzymes were increased after 3 h and significantly raised after 24 h and rapidly reduced after 48 h. Histological studies showed that one LD50 and 1/2 LD50 doses of the venom induced significant histological alterations in the lungs, liver, heart and kidneys including congestion, hemorrhage, necrosis, apoptosis, edema, and infiltration of inflammatory cells. The results indicate that the venom of P. russelli has nephrotoxic, hepatotoxic, cardiotoxic and pneumotoxic effects in mouse model. Among four examined vital organs, the highest critical events were seen in liver. The findings are useful to give new insight in the fish's venom studies. Gathering the data resulted from this study together will be directed us toward a good aspect concerning the toxicity of potential therapeutic molecules in the venom of lionfish.

The first report on coagulation and phospholipase A2 activities of Persian Gulf lionfish, Pterois russelli, an Iranian venomous fish.

Pterois russelli is a venomous fish belonging to scorpionidae family. Regarding to high significance value for tracing potential therapeutic molecules and special agents from venomous marine creatures, the present study was aimed to characterization of the Persian Gulf lionfish venom. Proteolytic, phospholipase, hemolytic, coagulation, edematogenic and dermonecrotic activities were determined for extracted venom. The LD50 of P. russelli venom was determined by intravenous injection in white Balb/c mice. Phospholipase A2 activity was recorded at 20 µg of total venom. Coagulation activity on human plasma was shown by Prothrombin Time (PT) and activated Partial Thromboplastin Time (APTT) assays and coagulation visualized after 7 and 14 s respectively for 60 µg of crude venom. LD50 was calculated as 10.5 mg/kg. SDS-PAGE revealed the presence of major and minor protein bands between 6 and 205 kDa. Different amounts of crude venom ranged from 1.87 to 30 µg showed proteolytic activity on casein. The highest edematic activity was detected at 20 µg. Our findings showed that the edematic activity was dose dependent and persisted for 48 h after injection. The crude venom did not induce dermonecrotic activity on rabbit skin and showed no hemolytic activity on human, mouse and rabbit erythrocytes. This is the first report for phospholipase A2 and coagulation activity in venomous fish and venomous marine animals respectively. Proteolytic activity of P. russelli venom is in accordance with the other genara of scorpionidae family. According to venom activity on intrinsic and extrinsic coagulation pathways, lionfish venom would be contained an interesting pharmaceutical agent. This study is pending to further characterization of phospholipase A2, coagulation, and protease activities and also in vivo activity on animal model of surface and internal bleeding.

A New, High Yield, Rapid, and Cost-Effective Protocol to Deprotection of Cysteine-Rich Conopeptide, Omega-Conotoxin MVIIA.

Almost all conopeptides purified from Conus venoms are cysteine-rich peptides. Among them, omega-conotoxin MVIIA, FDA approved peptide drug (Prialt(®)), selected as a cysteine-rich model that its protection from oxidation is critical during solid phase synthesis. Deprotection of cysteines is a crucial step after peptide synthesis. The current study aimed to set up a new highly efficient deprotection protocol for omega-conotoxin MVIIA. Deprotection was performed based on mercury acetate with significant major modification. The protocol accomplished based on the best molar ratio of peptide/mercury/2-ME that adjusted to 0.2 mm/3 mm/10 mm (50 µg/1 mg/10 µL). The yield and purity of omega-conotoxin MVIIA obtained at 93 and 95%, respectively. The total time of protocol shortened to 90 min instead of 6-20 h in routine methods. In this study, omega-conotoxin MVIIA was recovered in high yield and in the shortest time. Despite of other known protocols, molar ratio adjusted to minimum amount. In conclusion, this protocol would be suggested to cost-effective deprotection of thiol groups for similar cysteine-rich peptides.

Histopathological effects of atrazine on gills of Caspian kutum Rutilus frisii kutum fingerlings.

The use of chemical pesticides has increased environmental pollution and affects fishes as non-target organisms. To investigate the toxic effects of the widely used herbicide atrazine on Caspian kutum Rutilus frisii kutum fingerlings, fish were exposed to a sublethal concentration of half LC50 for 96 h. The main alterations visible in the gill tissue were detachment of the epithelium of the lamellae, necrosis, lamellar fusion, hyperplasia, club shaped lamellae, collapse of the lamellae, shrinkage and curling of the lamellae, and ultrastructural alterations such as necrosis of the apical microridges of the pavement cells. Results also showed that the gill ionocytes were fewer in number and larger in size in the atrazine-exposed fish. Atrazine appears to be highly toxic to Caspian kutum fingerlings even at a sublethal concentration (12.47 mg l(-1)) and acute exposure. This toxicity could affect gill respiration and ion regulation function of fingerlings by damaging tissue, pavement cells, and ionocytes.

Purification and characterization of pepsin-solubilized collagen from skin of sea cucumber Holothuria parva.

Pepsin-solubilized collagen (PSC) was extracted from the skin of sea cucumber Holothuria parva and was fractionally characterized. The PSC from H. parva skin consisted of three α1 chains (α1)3, in contrast to calf skin collagen type I with two α1 and one α2 chains (α1)2α2 with approximately 130 kDa each. The maximum transition (Tm) and denaturation temperature (Td) of PSC were determined to be 46.94 and 32.5 °C, respectively. The amino acid composition analysis revealed that glycine, proline, alanine, and hydroxyproline were the abundant amino acids available in extracted PSC. The results showed that the isolated collagen from H. parva has some similar characteristics to previously reported collagens used in food and pharmaceutical industries.

Alpha-amylase inhibitory activity and sterol composition of the marine algae, Sargassum glaucescens.

BACKGROUND: Sargassum species (phaeophyceae) are economically important brown algae in southern parts of Iran. Sargassum is mainly harvested as a row material in alginate production industries and is a source of plant foods or plant bio-stimulants even a component of animal foods. OBJECTIVE: In this study, Sargassum glaucescens, collected from the seashore of Chabahar, was employed for phytochemical and biological evaluations. MATERIALS AND METHODS: For that purpose, the dried algae was extracted by methanol and subjected to different chromatographic separation methods. RESULTS: Six sterols, fucosterol (1), 24(S)-hydroxy-24-vinylcholesterol (2), 24(R)-hydroxy-24-vinylcholesterol (3), stigmasterol (4), ß-sitosterol (5) and cholesterol (6) were identified by spectroscopic methods including (1)H-NMR, (13)C-NMR and mass spectroscopy. In vitro alpha-amylase inhibitory test was performed on the methanolic extract and the results revealed a potent inhibition (IC50 = 8.9 ± 2.4 mg/mL) of the enzyme compared to acarbose as a positive control. CONCLUSION: Various biological activities and distribution of sterols in Sargassum genus have been critically reviewed here. The results concluded that these algae are a good candidate for further anti-diabetic investigations in animals and human.

A cytotoxic hydroperoxy sterol from the brown alga, Nizamuddinia zanardinii.

BACKGROUND: The marine environment is a unique source of bioactive natural products, of which Nizamuddinia zanardinii is an important brown algae distributed in Oman Sea. Literature revealed that there is no report on phytochemistry and pharmacology of this valuable algae. METHODS: Bioguided fractionation of the methanolic extract of Nizamuddinia zanardinii, collected from Oman Sea, led to the isolation of a hydroperoxy sterol. Its structure was determined by analysis of the spectroscopic data as 24-hydroperoxy-24-vinyl cholesterol (HVC). In vitro cytotoxic activity of this compound was evaluated against HT29, MCF7, A549, HepG2 and MDBK cell lines. RESULTS: Although 24(R)-hydroproxy-24-vinylcholesterol has been previously reported from Sargassum and Padina species, it is the first report on the presence of this compound from N. zanardinii. This compound exhibited cytotoxicity in all cell lines (IC50, 3.62, 9.09, 17.96, 32.31 and 37.31 µg/mL respectively). HVC was also evaluated for apoptotic activity and demonstrated positive results in terminal deoxynucleotidyl transferase dUTP Nick End labeling (TUNEL) assay suggesting it a candidate for further apoptotic studies. CONCLUSIONS: Nizamuddinia zanardinii, a remarkable brown algae of Oman Sea, is a good source of hydroproxy sterols with promising cytotoxic on various cell lines particularly human colon adenocarcinoma.

The first report on some toxic effects of green scat, Scatophagus argus an Iranian Persian Gulf venomous fish.

Green scat namely as Scatophagus argus is a venomous aquarium fish belonging to Scatophagidae family. It can induce painful wounds in injured hand with partial paralysis to whom that touch the spines. Dorsal and ventral rough spines contain cells that produce venom with toxic activities. According to unpublished data collected from local hospitals in southern coastal region of Iran, S. argus is reported as a venomous fish. Envenomation induces clinical symptoms such as local pain, partial paralysis, erythema and itching. In the present study green scat (spotted scat) was collected from Persian Gulf coastal waters. SDS-PAGE indicated 12 distinct bands in the venom ranged between 7 and 250 kDa. The crude venom had hemolytic activity on human erythrocytes (1%) with an LC100 (Lytic Concentration) of about 1.7 µg. The crude venom can release 813 µg protein from 0.5% casein. Phospholipase C activity was recorded at 3.125 µg of total venom. Our findings showed that the edematic activity remained over 24 h after injection. The results demonstrated that crude venom extracted from Iranian coastal border has different toxic and enzymatic activities. This study is pending to further investigation on animal model regarding protein purification and in vivo studies.

Denaturation and intermediates study of two sturgeon hemoglobins by n-dodecyl trimethylammonium bromide.

Varieties of hemoglobin (Hb) forms exist in fish, which are usually well adapted to the different ecological conditions or various habitats. In the current study, Hbs from two Sturgeon species of the Southern Caspian Sea Basin were purified and studied upon interaction with n-dodecyl trimethylammonium bromide (DTAB; as a cationic surfactant) by various methods including UV-visible absorption, dynamic light scattering (DLS), and ANS fluorescence spectrophotometry. The chemometric analysis of Hbs was investigated upon interaction with DTAB under titration, using UV-visible absorption spectra. The chemometric resolution techniques were used to determine the number of the components and mole fraction of the oxidized Hbs. These results provided the evidence for the existence of three different molecular components including native (N), intermediate (I) and denatured (D) in sturgeon Hbs. According to the distribution of intermediates, which were broadened in a range of DTAB concentration, the aggregation states, DLS experiments, and thermal stability (T(m) obtained by differential scanning calorimetry (DSC)), the Acipenser stellatus Hb was more stable compared to Acipenser persicus Hb. These results demonstrate a significant relationship between the stability of fish Hbs and the habitat depth requirements.

Biochemical characterization of hemoglobins from Caspian Sea sturgeons (Acipenser persicus and Acipenser stellatus).

Hemoglobin (Hb) variability is a commonly used index of phylogenetic differentiation and molecular adaptation in fish enabling them to adapt to different ecological conditions. In this study, the characteristics of Hbs from two Sturgeon species of the Southern Caspian Sea Basin were investigated. After extraction and separation of hemoglobin from whole blood, the polyacrylamide gel electrophoresis (SDS-PAGE), cellulose acetate electrophoresis, and isoelectric focusing (IEF) were used to confirm Hb variabilities in these fishes. We showed that although both species have variable Hbs with different isoelectric points, their dominant Hbs can be identified. Ion exchange on CM-cellulose chromatography was used for purification of the dominant Hbs from these fishes. The accuracy of the methods was confirmed by IEF and SDS-PAGE. Spectral studies using fluorescence spectrophotometery indicated that although the Hbs from these fishes had similar properties they exhibited clear differences with human Hb. A comparative study of Hbs alpha-helix secondary substructures was performed by circular dichroism spectropolarimetry (CD) analysis. UV-vis spectrophotometery was also utilized to measure oxygen affinity of Hbs by sodium dithionite. Oxygen affinities of these Hbs were compared using Hb-oxygen dissociation curves. Together, these results demonstrate a significant relationship between oxygen affinity of fish hemoglobins and environmental partial pressure of oxygen.

Morphological characterization and size of hemocytes in Anodonta cygnea.

The Anodont (Anodonta cygnea) constitutes one of the most important bivalves along of Anzali Lagoon. In last decade, Anodont have suffered a high degree of mortality. Description of the morphological characterization of hemocytes is a prerequisite to further exploring the causes of death in bivalves, therefore in this study; the circulating hemocytes of the A. cygnea in Anzali Lagoon were identified. At first, two types of hemocytes were recognized, granulocytes and agranulocytes, were identified based on the existence of cytoplasmic granules under light microscopy. The hemocytes were then stained and the granulocytes subclassified into eosinophilic and basophilic granulocytes and intermix. The eosinophilic granulocytes have distinct small and large granules. Agranulocytes could be subdivided into hyalinocytes and blast-like cells and another cell type, vesicular cells, was observed as unclassified cells. Results were compared with similar researches about other bivalves' observations.