Comparison of Cd, Cu, Se, and Zn Concentration in the Muscle and Hepatopancreas of Sepia pharaonis and Uroteuthis duvauceli in the North of Persian Gulf (Iran).

The objective of this research was to examine and contrast the levels of cadmium (Cd), copper (Cu), zinc (Zn), and selenium (Se) in the muscle and hepatopancreas tissues of two species, namely pharaoh cuttlefish (Sepia pharaonis) and Indian squid (Uroteuthis duvauceli), from the Persian Gulf. A total of thirty individuals of each species were gathered in January 2009 from the northern waters of the Persian Gulf. The metal concentrations were significantly higher in muscle tissue (p < 0.05) than in other tissues. S. pharaonis had higher metal concentrations than U. duvauceli. In the muscle and hepatopancreas samples of S. pharaonis, the highest mean concentrations were found to be for Zn (58.45 ± 0.96 µg/g dw) and Cu (1541.47 ± 192.15 µg/g dw), respectively. In U. duvauceli, the highest concentration of measured elements was seen for Zn in both muscle (36.52 ± 0.56 µg/g dw) and hepatopancreas (60.94 ± 2.65 µg/g dw). Se had the lowest concentration among the elements measured in both species. There was a negative and significant correlation between Cu and biometrical factors (total body length and weight) in both muscle and hepatopancreas samples of S. pharaonic and only in the muscle samples of U. duvauceli (p < 0.01, R2 = - 052; p < 0.01, R2 = - 0.055). However, there was a strong correlation between Zn and biometrical factors in hepatopancreas samples of both species. The comparison of metal concentrations with standards revealed that only Cd levels in S. pharaonis exceeded the ESFA and WHO standards, whereas other metals were below the standards.

Effects of ammonia toxicity on the histopathology, detoxification, oxidative stress, and immune response of the cuttlefish Sepia pharaonis and the mitigation of γ-aminobutyric acid.

In this study, γ-aminobutyric acid (GABA) was examined as an additional supplement to improve the ammonia stress resistance of S. pharaonis. Specifically, we added different doses of GABA (0, 20, 40, 60, 80, and 100 mg/kg) to food, cultivated S. pharaonis in regular seawater for 8 weeks and then in 8.40 mg/L ammonia seawater for 48 h and then investigated the accumulation of ammonia (the hepatic ammonia content), ammonia detoxification process (the urea content), antioxidant enzymes (superoxide dismutase (SOD) and catalase (CAT) enzyme activities), immune response (the serum haemolytic complement (C3) and lysozyme (LYZ) contents), membrane lipid peroxidation (malondialdehyde (MDA)) and histopathology of the liver. The results showed that ammonia poisoning could induce ammonia and MDA accumulation and subsequently lead to oxidative stress (decreases in SOD and CAT activities), immunosuppression (reductions in the haemolytic C3 and LYZ content), and histopathological injury in the liver. The application of GABA had a significant effect on alleviating the adverse effect of ammonia poisoning, and 80-100 mg/kg treatment exerted the best effect. This treatment significantly reduced the ammonia and MDA contents, significantly increased the urea content, increased the SOD, CAT, C3 and LYZ activities, reduced the MDA content, suppressed membrane lipid peroxidation, and significantly improved the histopathological injury to the liver. In summary, the results could provide a new method for mitigating liver damage, alleviating the physiological and metabolic disorders caused by ammonia stress in cuttlefish, and provide a theoretical basis for the application of GABA in alleviating ammonia poisoning.

Preventive Effects of Three Polysaccharides on the Oxidative Stress Induced by Acrylamide in a Saccharomyces cerevisiae Model.

Saccharomyces cerevisiae was used as a model to explore the preventive effect of two marine polysaccharides separately derived from Sepia esculenta ink (SIP) and Laminaria japonica (FL) as well as one terrestrial polysaccharides from Eleocharis tuberosa peel (WCPP) on toxic injury induced by acrylamide (AA). The growth of yeast was evaluated by kinetics indexes including doubling time, lag phase and maximum proliferation density. Meanwhile, intracellular redox state was determined by contents of MDA and GSH, and SOD activity. The results showed that AA inhibited yeast growth and destroyed the antioxidant defense system. Supplement with polysaccharides, the oxidative damage of cells was alleviated. According to the growth recovery of yeast, FL and WCPP had similar degree of capacity against AA associated cytotoxicity, while SIP was 1.5~2 folds as strong as FL and WCPP. SIP and FL significantly reduced production of MDA by AA administration. Moreover, SIP, FL and WCPP increased SOD activity and repressed GSH depletion caused by AA.

[Analysis of arsenic speciation in Sepiae Endoconcha and research on its limit standard].

Sepiae Endoconcha is a common marine animal medicine,which generally contains high concentration of arsenic( As).The Chinese Pharmacopoeia( 2010 edition,part I) stipulated that the total As content of Sepiae Endoconcha should not exceed 2 mg·kg~(-1),while this limit was revised to 10 mg·kg~(-1) in the 2015 edition. So far,there is no research on the speciation of As in Sepiae Endoconcha,which made it hard to accurately evaluate its security risk. In this study,32 batches of Sepiae Endoconcha from different sources were collected. The safety risk assessment was carried out by determining the total As content and As speciation,inorganic As[As( Ⅲ),As( Ⅴ) ]and organic As( MMA,DMA,As C,As B) by HPLC-ICP-MS,and then the limit standard was discussed. The results showed that As B was the main form of As in Sepiae Endoconcha,followed by DMA and As( Ⅴ) ． Of the 32 batches of Sepiae Endoconcha,9 batches( accounting for 28%) were detected possessing i As. The maximum concentration of As( Ⅲ) was 103. 3 µg·kg~(-1),and the maximum concentration of As( Ⅴ) was 222. 4 µg·kg~(-1). According to the limit of i As in food,18. 75% of the samples exceeded the standard. The results indicate that there is no simple positive correlation between total As and As morphology in Sepiae Endoconcha. Besides,there is a risk in the total As limit,especially after the relaxation of the total As limit. The problem of high i As content caused by pollution and other factors is difficult to regulate. Since the toxicity of inorganic As is much higher than that of organic As,it is of great practical significance to establish inorganic As form limits in Sepiae Endoconcha.

Antimicrobial activity against Macrophomina phaseolina and the control of charcoal rot in soybeans using the homoeopathic drugs Sepia and Arsenicum album / Atividade antimicrobiana contra Macrophomina phaseolina e controle de podridão cinzenta da haste em soja pelos medicamentos homeopáticos Sepia e Arsenicum album

The aim of this study was to evaluate the control of charcoal rot in soybeans using Sepia and Arsenicum album homeopathic solutions in dynamizations of 6, 12, 24, 36 and 48CH (hahnemanian centesimal scale), in addition to verify the antimicrobial activity of these treatments against Macrophomina phaseolina. Additional treatments were also used, including distilled water and a hydroalcoholic solution of 30% ethanol. In vitro tests were performed to analyze the number of micro-sclerotia and the mycelial growth of the fungus. In vivo assays were used to evaluate the progress of the disease and the size of the lesions. For the area under the mycelial growth curve (AUMGC), A. album had no effect, however, Sepia reduced up to 32% of the fungal growth. For micro-sclerotia there was no statistical difference. For the area under the disease progress curve (AUDPC), both Sepia and A. album in 24CH dynamization reduced fungal growth up to 50% and 70% in the first and second assays, respectively, compared to control treatments. These results indicate the potential of these homeopathic drugs for controlling soybean charcoal rot.(AU)

O objetivo deste estudo foi avaliar o controle da podridão cinzenta da haste em soja, pulverizando soluções homeopáticas de Sepia e Arsenicum album, nas dinamizações 6, 12, 24, 36 e 48CH (centesimal hahnemaniana), bem como verificar a atividade antimicrobiana desses tratamentos contra Macrophomina phaseolina. Como tratamentos adicionais utilizou-se água destilada e solução hidroalcoólica a 30% de etanol. Foram realizados ensaios in vitro a fim de analisar o número de microescleródios e o crescimento micelial do fungo, e ensaios in vivo para acompanhar o progresso da doença e o tamanho da lesão. Para área abaixo da curva de crescimento micelial (AACCM), A. album não apresentou efeito, no entanto, Sepia reduziu em até 32% o crescimento. Para microescleródios não houve diferença estatística. Para área abaixo da curva de progresso da doença (AACPD), tanto Sepia quanto A. album na dinamização 24CH apresentaram redução de até 50% no primeiro ensaio e de até 70% no segundo ensaio quando comparados com a solução hidroalcoólica. Estes resultados indicam o potencial desses medicamentos homeopáticos no controle de podridão cinzenta da haste em soja.(AU)

Phenylboronic acid modified solid-phase extraction column: Preparation, characterization, and application to the analysis of amino acids in sepia capsule by removing the maltose.

Maltose, a common auxiliary material of pharmaceutical preparation, may disturb the analysis of total amino acids in sepia capsule by aldolization. Therefore, it is necessary to remove the maltose through a convenient method. In this work, a phenylboronic acid modified solid-phase extraction column has been synthesized and used to remove the maltose. The materials were synthesized by one step "thiol-ene" reaction and the parameters of the column such as absorption capacity, recovery, and absorption specificity have been investigated. The results showed the column (0.5 cm of length × 0.5 cm of inner diameter) can absorb 4.6 mg maltose with a linear absorption and absorption specificity. Then this technique was applied in the quantification of amino acids in sepia capsule. After the optimization of the method, four kinds of amino acids, which were the most abundant, were quantified by high-performance liquid chromatography with diode array detection. The amounts of the four kinds of amino acids are 1.5∼2 times more than that without the treatment of solid-phase extraction column, which almost overcomes the influence of the maltose. All the results indicate that the phenylboronic acid modified solid-phase extraction column can successfully help to accurately quantify the total amino acids in sepia capsule.

Impact of homeopathic remedies on the expression of lineage differentiation genes: an in vitro approach using embryonic stem cells.

BACKGROUND: Well-documented studies of the potential effects and safety of homeopathic medicines in pregnancy are required. In this study, specific genes were studied which could serve as biomarkers for specification of three lineages to predict the safety of homeopathic remedies using mouse embryonic stem (ES) cells. Thus, the present work was to study the effects of homeopathic remedies taken during pregnancy using ES cells as the model. METHODS: Mouse ES cells were exposed to 30C potency of Nux Vomica and Sepia, which are homeopathic medicines prescribed for the management of pregnancy related symptoms. Cytotoxicity studies were done using a modified Embryonic Stem cell test (EST). The expression levels of key genes and proteins were analyzed using real time polymerase chain reaction and immunocytochemistry, respectively. RESULTS: Homeopathic treatment led to modulations in the expression of certain lineage specific genes but this difference was not significant with respect to solvent control and showed normal differentiation as demonstrated by the expression of α/ß MHC and α-actinin proteins in the differentiated ES cells. CONCLUSIONS: Our study for the first time has shown the feasibility of using ES cells in the developmental toxicity testing of remedies. The results suggest that they are not associated with developmental toxicity.

Taurine depresses cardiac contractility and enhances systemic heart glucose utilization in the cuttlefish, Sepia officinalis.

Taurine is the most abundant amino acid in the blood of the cuttlefish, Sepia officinalis, where levels can exceed 200 mmol L(-1). In mammals, intracellular taurine modulates cardiac Ca(2+) handling and carbohydrate metabolism at much lower concentrations but it is not clear if it exerts similar actions in cephalopods. Blood Ca(2+) levels are high in cephalopods and we hypothesized that taurine would depress cardiac Ca(2+) flux and modulate contractility in systemic and branchial hearts of cuttlefish. Heart performance was assessed with an in situ perfused systemic heart preparation and contractility was evaluated using isometrically contracting systemic and branchial heart muscle rings. Stroke volume, cardiac output, and Ca(2+) sensitivity were significantly lower in systemic hearts perfused with supplemental taurine (100 mmol L(-1)) than in controls. In muscle ring preparations, taurine impaired relaxation at high contraction frequencies, an effect abolished by supra-physiological Ca(2+) levels. Taurine did not affect oxygen consumption in non-contracting systemic heart muscle, but extracellular glucose utilization was twice that of control preparations. Collectively, our results suggest that extracellular taurine depresses cardiac Ca(2+) flux and potentiates glucose utilization in cuttlefish. Variations in taurine levels may represent an important mechanism for regulating cardiovascular function and metabolism in cephalopods.

Raman spectroscopy reveals variation in free OH groups and hydrogen bond strength in ultrahigh dilutions

Objective: To decipher the nature of water structure in two ultrahigh diluted (UHD) homeopathic drugs by Laser Raman Spectroscopy. Method: Two homeopathic drugs Calcarea carbonica (Calc.) and Sepia officinalis (Sep.) in 8cH, 202cH, and 1002cH and their diluent medium 90% ethanol in 8cH and 202cH were used in the present study. Laser Raman spectra of all the samples were obtained in the wave number region of 2400 – 4200 cm-1. The intensity ratio at vibration frequencies between 3200 and 3420 (R1) and that between 3620 and 3420 (R2) were calculated for each UHD of the samples. Results: The spectra show a marked difference in intensities in the stretching vibrations of CH and OH groups of all the samples. R1 values for three UHDs of Calc. and Sep. show negative and positive relationships, respectively. In the case of R2 values, the relationship in three UHDs is 8<202>1002 for Calc., and 8> 202 < 1002 for Sep. In the case of control (ethanol UHDs) both R1 and R2 show a negative relationship. Conclusion: R1 denotes a relative number of OH groups with strong and weak hydrogen bonds. R2 indicates the relative number of OH groups with broken and weak H-bonds. Therefore, the UHDs of the two drugs and the control are different from each other with respect to hydrogen bond strength of OH groups and the number of free OH groups or non-hydrogen bonded water molecules.(AU)

Raman spectroscopy reveals variation in free OH groups and hydrogen bond strength in ultrahigh dilutions

OBJECTIVE: To decipher the nature of water structure in two ultrahigh diluted (UHD) homeopathic drugs by Laser Raman Spectroscopy. METHOD: Two homeopathic drugs Calcarea carbonica (Calc.) and Sepia officinalis (Sep.) in 8cH, 202cH, and 1002cH and their diluent medium 90% ethanol in 8cH and 202cH were used in the present study. Laser Raman spectra of all the samples were obtained in the wave number region of 2400 – 4200 cm-1. The intensity ratio at vibration frequencies between 3200 and 3420 (R1) and that between 3620 and 3420 (R2) were calculated for each UHD of the samples. RESULTS: The spectra show a marked difference in intensities in the stretching vibrations of CH and OH groups of all the samples. R1 values for three UHDs of Calc. and Sep. show negative and positive relationships, respectively. In the case of R2 values, the relationship in three UHDs is 81002 for Calc., and 8> 202 < 1002 for Sep. In the case of control (ethanol UHDs) both R1 and R2 show a negative relationship. CONCLUSION: R1 denotes a relative number of OH groups with strong and weak hydrogen bonds. R2 indicates the relative number of OH groups with broken and weak H-bonds. Therefore, the UHDs of the two drugs and the control are different from each other with respect to hydrogen bond strength of OH groups and the number of free OH groups or non-hydrogen bonded water molecules.

Antifungal and antihepatotoxic effects of sepia ink extract against oxidative stress as a risk factor of invasive pulmonary aspergillosis in neutropenic mice.

BACKGROUND: There is a great need for novel strategies to overcome the high mortality associated with invasive pulmonary aspergillosis (IPA) in immunocompromised patients. To evaluate the antifungal and antihepatotoxic potentials of Sepia ink extract, its effect on liver oxidative stress levels was analyzed against IPA in neutropenic mice using amphotercin B as a reference drug. MATERIALS AND METHODS: Eighty neutropenic infected mice were randomly assigned into four main groups. The 1(st) group was treated with saline, neutropenic infected (NI), the 2(nd) group was treated with ink extract (200 mg/kg) (IE) and the 3(rd) group was treated with amphotericin B (150 mg/kg) (AMB) and 4(th) group was treated with IE plus AMB. Treatment was started at 24 h after fungal inoculation (1×10(9) conidia/ml). RESULTS: The present study revealed good in vitro and in vivo antifungal activity of IE against A. fumigatus. IE significantly reduced hepatic fungal burden and returns liver function and histology to normal levels. Compared with the untreated infected group, mice in the IE, AMB, and IE+ AMB groups had increased glutathione reduced (GSH) and superoxide dismutase (SOD) and significantly reduced malondialdehyde (MDA) levels at 24 and 72 h after inoculation with A. fumigatus conidia. CONCLUSION: It is then concluded that in combination with antifungal therapy (AMB), IE treatment can reduce hepatic fungal burden, alleviate hepatic granulomatous lesions and oxidative stress associated with IPA in neutropenic mice.

Identification and analysis of an intracellular Cu/Zn superoxide dismutase from Sepiella maindroni under stress of Vibrio harveyi and Cd2+.

Superoxide dismutases (SODs) are ubiquitous family of metalloenzymes involved in protecting organisms from excess reactive oxygen species damage. In this paper, a novel intracellular Cu/ZnSOD from Sepiella maindroni (designated as SmSOD) was identified and characterized. The full-length cDNA sequence of SmSOD (GenBank accession No. KF908850) was 709 bp containing an open reading frame (ORF) of 459 bp, encoding 153 amino acid residues peptide with predicted pI/MW (6.02/15.75 kDa), a 131 bp-5'- and 116 bp-3'- untranslated region (UTR). BLASTn analysis and phylogenetic relationship strongly suggested that the sequence shared high similarity with known Cu/Zn SODs. Several highly conserved motifs, including two typical Cu/Zn SOD family domains, two conserved Cu-/Zn-binding sites (H-47, H-49, H-64, H-120 for Cu binding, and H-64, H-72, H-81, D-84 for Zn binding) and intracellular disulfide bond (C-58 and C-146), were also identified in SmSOD. Time-dependent mRNA expression of SmSOD in hepatopancreas was recorded by quantitative real-time RT-PCR after Vibrio harveyi injection and Cd(2+) exposure. The results indicated that SmSOD was an acute-phase protein involved in the immune responses against pathogens and biological indicator for metal contaminants in aquatic environment.

Relations between total mercury, methylmercury and selenium in five tissues of Sepia officinalis captured in the south Portuguese coast.

Mercury, methylmercury and selenium were determined in digestive gland, branchial hearts, mantle, kidney and gills of Sepia officinalis from two areas of the south Portuguese coast. To the best of our knowledge these are the first data on Hg, MeHg and Se in branchial hearts, kidney and gills of cuttlefish. Digestive gland, branchial hearts and kidney presented higher levels of Hg and Se than mantle and gills. Methylmercury was significantly higher in digestive gland, branchial hearts and mantle. The enhanced levels of Hg in digestive gland and branchial heart reinforce the elevated storage capacity of these two tissues. The percentage of MeHg varied from 6.1% in gills to 92% in mantle. Linear and positive MeHg-Hg relations were obtained for the five tissues, being the better relation and higher slope observed for mantle, followed by branchial hearts, digestive gland, kidney and gills. The Se:Hg molar ratios showed a surplus of Se in all tissues. Calculations based on the equimolarity of Se:Hg point that 95-99% of Se are not linked to Hg (Se free). The negligible quantity of Se associated with Hg suggests that the mechanism of MeHg demethylation was not triggered in none of the tissues, presumably because the threshold for MeHg toxicity was not achieved.

Skin and cutaneous melanocytic lesion simulation in biomedical optics with multilayered phantoms.

The complex inner layered structure of skin influences the photon diffusion inside the cutaneous tissues and determines the reflectance spectra formation. Phantoms are very useful tools to understand the biophysical meaning of parameters involved in light propagation through the skin. To simulate the skin reflectance spectrum, we realized a multilayered skin-like phantom and a multilayered skin phantom with a melanoma-like phantom embedded inside. Materials used were Al(2)O(3) particles, melanin of sepia officinalis and a calibrator for haematology systems dispersed in transparent silicon. Components were optically characterized with indirect techniques. Reflectance phantom spectra were compared with average values of in vivo spectra acquired on a sample of 573 voluntary subjects and 132 pigmented lesions. The phantoms' reflectance spectra agreed with those measured in vivo, mimicking the optical behaviour of the human skin. Further, the phantoms were optically stable and easily manageable, and represented a valid resource in spectra formation comprehension, in diagnostic laser applications and simulation model implementation, such as the Monte Carlo code for non-homogeneous media.

Critical temperatures in the cephalopod Sepia officinalis investigated using in vivo 31P NMR spectroscopy.

The present study was designed to test the hypothesis of an oxygen limitation defining thermal tolerance in the European cuttlefish (Sepia officinalis). Mantle muscle organ metabolic status and pHi were monitored using in vivo 31P NMR spectroscopy, while mantle muscle performance was determined by recording mantle cavity pressure oscillations during ventilation and spontaneous exercise. Under control conditions (15 degrees C), changes in muscle phospho-L-arginine (PLA) and inorganic phosphate (Pi) levels could be linearly related to frequently occurring, high-pressure mantle contractions with pressure amplitudes (MMPA) of >0.2 kPa. Accordingly, mainly MMPA of >2 kPa affected muscle PLA reserves, indicating that contractions with MMPA of <2 kPa only involve the thin layers of aerobic circular mantle musculature. On average, no more than 20% of muscle PLA was depleted during spontaneous exercise under control conditions. Subjecting animals to acute thermal change at an average rate of 1 deg. h-1 led to significant Pi accumulation (equivalent to PLA breakdown) and decrements in the free energy of ATP hydrolysis (dG/dzeta) at both ends of the temperature window, starting at mean critical temperatures (Tc) of 7.0 and 26.8 degrees C, respectively. Frequent groups of high-pressure mantle contractions could not (in the warm) or only partially (in the cold) be related to net PLA breakdown in mantle muscle, indicating an oxygen limitation of routine metabolism rather than exercise-related phosphagen use. We hypothesize that it is mainly the constantly working radial mantle muscles that become progressively devoid of oxygen. Estimates of very low dG/dzeta values (-44 kJ mol-1) in this compartment, along with correlated stagnating ventilation pressures in the warm, support this hypothesis. In conclusion, we found evidence for an oxygen limitation of thermal tolerance in the cuttlefish Sepia officinalis, as indicated by a progressive transition of routine mantle metabolism to an anaerobic mode of energy production.

Aspergillus niger as a new allergic agent associated with bindis and its efficacy against homeopathic drugs.

Aspergillus was found as a dominant fungi to associate with brands of bindis. Among three potencies of four homeopathic drugs, Lycopodium 1M, Sulphur 1M, and Sepia 30 showed maximum inhibition zone of Aspergillus niger in inhibition zone technique. In poison food technique, Sepia 30M, Tellurium 30M, Sulphur 1M and Lycopodium 200 showed maximum percentage inhibition against A. niger