Development of Immediate Release Tablets Containing Calcium Lactate Synthetized from Black Sea Mussel Shells

Nowadays, the use of marine by-products as precursor materials has gained great interest in the extraction and production of chemical compounds with suitable properties and possible pharmaceutical applications. The present paper presents the development of a new immediate release tablet containing calcium lactate obtained from Black Sea mussel shells. Compared with other calcium salts, calcium lactate has good solubility and bioavailability. In the pharmaceutical preparations, calcium lactate was extensively utilized as a calcium source for preventing and treating calcium deficiencies. The physical and chemical characteristics of synthesized calcium lactate were evaluated using Fourier Transform Infrared Spectroscopy, X-ray diffraction analysis and thermal analysis. Further, the various pharmacotechnical properties of the calcium lactate obtained from mussel shells were determined in comparison with an industrial used direct compressible Calcium lactate DC (PURACAL^®). The obtained results suggest that mussel shell by-products are suitable for the development of chemical compounds with potential applications in the pharmaceutical domain.     

From Natural Xanthones to Synthetic C-1 Aminated 3,4-Dioxygenated Xanthones as Optimized Antifouling Agents

Biofouling, which occurs when certain marine species attach and accumulate in artificial submerged structures, represents a serious economic and environmental issue worldwide. The discovery of new non-toxic and eco-friendly antifouling systems to control or prevent biofouling is, therefore, a practical and urgent need. In this work, the antifouling activity of a series of 24 xanthones, with chemical similarities to natural products, was exploited. Nine (1, 2, 4, 6, 8, 16, 19, 21, and 23) of the tested xanthones presented highly significant anti-settlement responses at 50 μM against the settlement of mussel Mytilus galloprovincialis larvae and low toxicity to this macrofouling species. Xanthones 21 and 23 emerged as the most effective larval settlement inhibitors (EC₅₀ = 7.28 and 3.57 µM, respectively). Additionally, xanthone 23 exhibited a therapeutic ratio (LC₅₀/EC₅₀) > 15, as required by the US Navy program attesting its suitability as natural antifouling agents. From the nine tested xanthones, none of the compounds were found to significantly inhibit the growth of the marine biofilm-forming bacterial strains tested. Xanthones 4, 6, 8, 16, 19, 21, and 23 were found to be non-toxic to the marine non-target species Artemia salina (<10% mortality at 50 μM). Insights on the antifouling mode of action of the hit xanthones 21 and 23 suggest that these two compounds affected similar molecular targets and cellular processes in mussel larvae, including that related to mussel adhesion capacity. This work exposes for the first time the relevance of C-1 aminated xanthones with a 3,4-dioxygenated pattern of substitution as new non-toxic products to prevent marine biofouling.     

The role of gaping behaviour in habitat partitioning between coexisting intertidal mussels

Background  

Environmental heterogeneity plays a major role in invasion and coexistence dynamics. Habitat segregation between introduced species and their native competitors is usually described in terms of different physiological and behavioural abilities. However little attention has been paid to the effects of behaviour in habitat partitioning among invertebrates, partially because their behavioural repertoires, especially marine benthic taxa, are extremely limited. This study investigates the effect of gaping behaviour on habitat segregation of the two dominant mussel species living in South Africa, the invasive Mytilus galloprovincialis and the indigenous Perna perna. These two species show partial habitat segregation on the south coast of South Africa, the lower and upper areas of the mussel zone are dominated by P. perna and M. galloprovincialis respectively, with overlap in the middle zone. During emergence, intertidal mussels will either keep the valves closed, minimizing water loss and undergoing anaerobic metabolism, or will periodically open the valves maintaining a more efficient aerobic metabolism but increasing the risk of desiccation.     

Natural Benzo/Acetophenones as Leads for New Synthetic Acetophenone Hybrids Containing a 1,2,3-Triazole Ring as Potential Antifouling Agents

Marine biofouling is a natural process that represents major economic, environmental, and health concerns. Some booster biocides have been used in biofouling control, however, they were found to accumulate in environmental compartments, showing negative effects on marine organisms. Therefore, it is urgent to develop new eco-friendly alternatives. Phenyl ketones, such as benzophenones and acetophenones, have been described as modulators of several biological activities, including antifouling activity (AF). In this work, acetophenones were combined with other chemical substrates through a 1,2,3-triazole ring, a strategy commonly used in Medicinal Chemistry. In our approach, a library of 14 new acetophenone–triazole hybrids was obtained through the copper(I)-catalyzed alkyne-azide cycloaddition “click” reaction. All of the synthesized compounds were evaluated against the settlement of a representative macrofouling species, Mytilus galloprovincialis, as well as on biofilm-forming marine microorganisms, including bacteria and fungi. The growth of the microalgae Navicula sp. was also evaluated after exposure to the most promising compounds. While compounds 6a, 7a, and 9a caused significant inhibition of the settlement of mussel larvae, compounds 3b, 4b, and 7b were able to inhibit Roseobacter litoralis bacterial biofilm growth. Interestingly, acetophenone 7a displayed activity against both mussel larvae and the microalgae Navicula sp., suggesting a complementary action of this compound against macro- and microfouling species. The most potent compounds (6a, 7a, and 9a) also showed to be less toxic to the non-target species Artemia salina than the biocide Econea^®. Regarding both AF potency and ecotoxicity activity evaluation, acetophenones 7a and 9a were put forward in this work as promising eco-friendly AF agents.     

pH-Dependent Solution Structure and Activity of a Reduced Form of the Host-Defense Peptide Myticin C (Myt C) from the Mussel Mytilus galloprovincialis

Myticin C (Myt C) is a highly variable host-defense peptide (HDP) associated to the immune response in the mediterranean mussel (Mytilus galloprovincialis), which has shown to be active across species due to its strong antiviral activity against a fish rhabdovirus found in fish cells overexpressing this HDP. However, the potential antimicrobial properties of any synthetic analogue of Myt C has not yet been analysed. Thus, in this work we have synthesised the sequence of the mature peptide of Myt C variant c and analysed the structure activity relationships of its reduced (non-oxidized) form (red-MytCc). In contrast to results previously reported for oxidized isoforms of mussel myticins, red-MytCc was not active against bacteria at physiological pH and showed a moderate antiviral activity against the viral haemorrhagic septicaemia (VHS) rhabdovirus. However, its chemotactic properties remained active. Structure/function studies in neutral and acid environments by means of infrared spectroscopy indicated that the structure of red-MytCc is pH dependent, with acid media increasing its alpha-helical content. Furthermore, red-MytCc was able to efficiently aggregate artificial phospholipid membranes at low pH, as well as to inhibit the Escherichia coli growth, suggesting that this activity is attributable to its more structured form in an acidic environment. All together, these results highlight the dynamic and environmentally sensitive behavior of red-Myt C in solution, and provide important insights into Myt C structure/activity relationships and the requirements to exert its antimicrobial/immunomodulatory activities. On the other hand, the pH-dependent direct antimicrobial activity of Myt C suggests that this HDP may be a suitable template for the development of antimicrobial agents that would function selectively in specific pH environments, which are sorely needed in this “antibiotic-resistance era”.     

The Negative Relationship between Fouling Organisms and the Content of Eicosapentaenoic Acid and Docosahexaenoic Acid in Cultivated Pacific Oysters,Crassostrea gigas

Bivalves serve as an important aquaculture product, as they are the source of essential fatty acids, such as eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), in our diet. However, their cultivation in the wild can be affected by fouling organisms that, in turn, affect their EPA and DHA content. The effects of fouling organisms on the EPA and DHA contents of cultivated bivalves have not been well documented. We examined the effects of fouling organisms on the EPA and DHA contents and condition index of cultured oysters, Crassostrea gigas, in an aquaculture system. We sampled two-year-old oysters from five sites in Shizugawa Bay, Japan, in August 2014. Most of the fouling organisms were sponges, macroalgae, and Mytilus galloprovincialis. A significant negative relationship existed between the DHA content in C. gigas and the presence of sponges and macroalgae. A lower C. gigas EPA content corresponded to a higher M. galloprovincialis fouling mass and a lower C. gigas condition index. This can be explained by dietary competition between C. gigas and M. galloprovincialis for diatoms, which were the main producer of EPA in our study sites. Our findings indicate that fouling organisms likely reduce the EPA and DHA content in cultivated oysters. Therefore, our results suggest that the current efforts to remove fouling organisms from oyster clusters is an effective strategy to enhance the content of EPA and DHA in oysters.     

Optimization of extraction conditions of antioxidants from sunflower shells (Helianthus annuus L.) before and after enzymatic treatment

The effects of three independent variables: solvent polarity, temperature and extraction time on the antioxidant capacity, total phenolic content and phenolic acid composition in extracts obtained from sunflower shells before and after enzymatic treatment were studied. Response surface methodology based on three-level, three-variable Box-Behnken design was used for optimization of extraction parameters and evaluation of their effect on antioxidant capacity and total phenolic content in shell extracts.The average antioxidant capacities of extracts from sunflower shells without enzymatic treatment (368.1-1574.4 μmol TE/100 g) were higher than those for cellulolytic and pectolytic enzymes-treated shells (222.7-1419.0 and 270.7-1570.7 μmol TE/100 g, respectively). The content of total phenolic compounds ranged between 58.2-341.2 mg CGA/100 g, 26.7-277.3 mg CGA/100 g and 51.4-301.5 mg CGA/100 g for extracts obtained from shells without enzyme and treated with cellulolytic and pectolytic enzymes, respectively. Total phenolic content (TPC) in the studied shell extracts correlated significantly (p < 0.0001) positively with their antioxidant capacity determined by the ferric reducing antioxidant power (FRAP) method (r = 0.9275). Results of FRAP, TPC and phenolic acid composition in the studied shell extracts depend on the extraction conditions (solvent polarity, temperature, time), but they are independent on the addition of enzyme solutions. The antioxidant capacity and total phenolic content in the resulting extracts increased with a line in extraction temperature and solvent polarity.     

Tetrodotoxins (TTXs) and Vibrio alginolyticus in Mussels from Central Adriatic Sea (Italy): Are They Closely Related?

Tetrodotoxins (TTXs), potent neurotoxins, have become an increasing concern in Europe in recent decades, especially because of their presence in mollusks. The European Food Safety Authority published a Scientific Opinion setting a recommended threshold for TTX in mollusks of 44 µg equivalent kg⁻¹ and calling all member states to contribute to an effort to gather data in order to produce a more exhaustive risk assessment. The objective of this work was to assess TTX levels in wild and farmed mussels (Mytilus galloprovincialis) harvested in 2018–2019 along the coastal area of the Marche region in the Central Adriatic Sea (Italy). The presence of Vibrio spp. carrying the non-ribosomal peptide synthetase (NRPS) and polyketide synthase (PKS) genes, which are suspected to be involved in TTX biosynthesis, was also investigated. Out of 158 mussel samples analyzed by hydrophilic interaction liquid chromatography coupled with tandem mass spectrometry (HILIC-MS/MS), 11 (7%) contained the toxins at detectable levels (8–26 µg kg⁻¹) and 3 (2%) contained levels above the EFSA safety threshold (61–76 µg kg⁻¹). Contaminated mussels were all harvested from natural beds in spring or summer. Of the 2019 samples, 70% of them contained V. alginolyticus strains with the NRPS and/or PKS genes. None of the strains containing NRPS and/or PKS genes showed detectable levels of TTXs. TTXs in mussels are not yet a threat in the Marche region nor in Europe, but further investigations are surely needed.     

Quantitation Overcoming Matrix Effects of Lipophilic Toxins in Mytilus galloprovincialis by Liquid Chromatography-Full Scan High Resolution Mass Spectrometry Analysis (LC-HR-MS)

The analysis of marine lipophilic toxins in shellfish products still represents a challenging task due to the complexity and diversity of the sample matrix. Liquid chromatography coupled with mass spectrometry (LC-MS) is the technique of choice for accurate quantitative measurements in complex samples. By combining unambiguous identification with the high selectivity of tandem MS, it provides the required high sensitivity and specificity. However, LC-MS is prone to matrix effects (ME) that need to be evaluated during the development and validation of methods. Furthermore, the large sample-to-sample variability, even between samples of the same species and geographic origin, needs a procedure to evaluate and control ME continuously. Here, we analyzed the toxins okadaic acid (OA), dinophysistoxins (DTX-1 and DTX-2), pectenotoxin (PTX-2), yessotoxin (YTX) and azaspiracid-1 (AZA-1). Samples were mussels (Mytilus galloprovincialis), both fresh and processed, and a toxin-free mussel reference material. We developed an accurate mass-extracted ion chromatogram (AM-XIC) based quantitation method using an Orbitrap instrument, evaluated the ME for different types and extracts of mussel samples, characterized the main compounds co-eluting with the targeted molecules and quantified toxins in samples by following a standard addition method (SAM). An AM-XIC based quantitation of lipophilic toxins in mussel samples using high resolution and accuracy full scan profiles (LC-HR-MS) is a good alternative to multi reaction monitoring (MRM) for instruments with HR capabilities. ME depend on the starting sample matrix and the sample preparation. ME are particularly strong for OA and related toxins, showing values below 50% for fresh mussel samples. Results for other toxins (AZA-1, YTX and PTX-2) are between 75% and 110%. ME in unknown matrices can be evaluated by comparing their full scan LC-HR-MS profiles with those of known samples with known ME. ME can be corrected by following SAM with AM-XIC quantitation if necessary.     

MytiLec,a Mussel R-Type Lectin,Interacts with Surface Glycan Gb3 on Burkitt’s Lymphoma Cells to Trigger Apoptosis through Multiple Pathways

MytiLec; a novel lectin isolated from the Mediterranean mussel (Mytilus galloprovincialis); shows strong binding affinity to globotriose (Gb3: Galα1-4Galβ1-4Glc). MytiLec revealed β-trefoil folding as also found in the ricin B-subunit type (R-type) lectin family, although the amino acid sequences were quite different. Classification of R-type lectin family members therefore needs to be based on conformation as well as on primary structure. MytiLec specifically killed Burkitt''s lymphoma Ramos cells, which express Gb3. Fluorescein-labeling assay revealed that MytiLec was incorporated inside the cells. MytiLec treatment of Ramos cells resulted in activation of both classical MAPK/ extracellular signal-regulated kinase and extracellular signal-regulated kinase (MEK-ERK) and stress-activated (p38 kinase and JNK) Mitogen-activated protein kinases (MAPK) pathways. In the cells, MytiLec treatment triggered expression of tumor necrosis factor (TNF)-α (a ligand of death receptor-dependent apoptosis) and activation of mitochondria-controlling caspase-9 (initiator caspase) and caspase-3 (activator caspase). Experiments using the specific MEK inhibitor U0126 showed that MytiLec-induced phosphorylation of the MEK-ERK pathway up-regulated expression of the cyclin-dependent kinase inhibitor p21, leading to cell cycle arrest and TNF-α production. Activation of caspase-3 by MytiLec appeared to be regulated by multiple different pathways. Our findings, taken together, indicate that the novel R-type lectin MytiLec initiates programmed cell death of Burkitt’s lymphoma cells through multiple pathways (MAPK cascade, death receptor signaling; caspase activation) based on interaction of the lectin with Gb3-containing glycosphingolipid-enriched microdomains on the cell surface.     

Growth,liver composition,and calcium balance of rats fed sorghum supplemented with two levels of beans

A 35-day experimental study consisting of 28-day growth and 7-day calcium (Ca) balance periods was conducted to assess the effects of fibre and N ratios on growth and Ca balance of rats. Growth and Ca balance of rats (45–80 g) fed mixtures of cooked (CS) and uncooked (RS) sorghum and dehulled (DB) and undehulled (UB) bean were studied. The diets contained 10% protein. Casein served as the control protein. The control group ate more food except for the group fed the CS:DU (60:40) diets (P<0.05) had higher values for all parameters tested than the test groups. There were increases in food and Ca intakes, fecal Ca, weight gain, protein efficiency ration (PER), liver weight and composition except for the low moisture value for the CS:DS (60:40) group when N ratios were changed from 80:20 to 60:40. These results appear to indicate that fiber and N ratio had significant effects on growth liver composition and calcium balance of the rats.     

Anti-Inflammatory Activity and Mechanism of a Lipid Extract from Hard-Shelled Mussel (Mytilus Coruscus) on Chronic Arthritis in Rats

The present study was designed to investigate the anti-inflammatory activity and mechanism of a lipid extract from hard-shelled mussel (Mytilus coruscus) on adjuvant-induced (AIA) and collagen-induced arthritis (CIA) in rats. AIA and CIA rats that received hard-shelled mussel lipid extract (HMLE group) at a dose of 100 mg/kg demonstrated significantly lower paw swelling and arthritic index, but higher body weight gain than those which received olive oil (control group). Similar results were found in arthritic rats that received New Zealand green-lipped mussel lipid extract (GMLE) at the same dosage. The levels of leukotriene B₄ (LTB₄), prostaglandin E₂ (PGE₂), thromboxane B₂ (TXB₂) in the serum, and interleukin-1β (IL-1β), IL-6, interferon-γ (INF-γ), tumor necrosis factor-α (TNF-α) in the ankle joint synovial fluids of HMLE group rats were significantly lower than those of control group. However, the levels of IL-4 and IL-10 in HMLE group rats were significantly higher than those in the control group. Decreased mRNA expressions of matrix metalloproteinase 1 (MMP1) and MMP13, but increased tissue inhibitor of metalloproteinase 1 (TIMP1) were observed in the knee joint synovium tissues of HMLE group rats when compared with the control group. No hepatotoxicity was observed in both HMLE and GMLE group rats. The present results indicated that HMLE had a similarly strong anti-inflammatory activity as GMLE. Such a strong efficacy could result from the suppression of inflammatory mediators (LTB₄, PGE₂, TXB₂), pro-inflammatory cytokines (IL-1β, IL-6, INF-γ, TNF-α) and MMPs (MMP1, MMP13), and the promotion of anti-inflammatory cytokines (IL-4, IL-10) and TIMPs (TIMP1) productions.     

Mineral Profile and Variability in Vegetable Amaranth (Amaranthus tricolor)

Populations in North India depend on a number of vegetable crops of which Amaranthus spp. is the most important since it is the only crop available in the hot summer months when no other foliage crop grows in the field. However, reports on mineral composition of leaves are rare with absolutely no information on the qualitative improvement of foliage yield with special reference to minerals. Studies on correlation among the minerals as well as with yield and leaf attributes are also lacking. Hence, we report the proximate mineral composition in 30 strains of A. tricolor along with some suggestions for qualitative improvement of the foliage yield with reference to minerals. Our study showed that vegetable amaranth is a rich source of minerals like calcium (1.7±0.04 g/100 g), iron (1233.8±50.02 mg/kg), and zinc (791.7±28.98 mg/kg). The heritability estimates were high for most of the traits, with potassium and calcium showing high values, while comparatively lower values were recorded for magnesium and nickel. Nickel was the only mineral that showed positive correlation with all the minerals, as well as with leaf size and foliage yield. Zinc showed strong positive relationship with iron (0.66^**) and manganese (0.74^**), and was the only mineral exhibiting significant positive association with foliage yield. This study would be of use in enhancement of selected minerals in different regions according to local preferences and nutrient deficiency prevalent among the populations.     

Effects of 5 years of digestate application on biomass production and quality of cocksfoot (Dactylis glomerata L.)

Anaerobic digestion of biomass produces biogas for combustion and also provides a residual digestate. Although sometimes regarded as a waste product, the nutrient‐rich chemical composition of digestate makes it a potential organic fertilizer for agriculture. The goal of this study was to evaluate the effectiveness of digestate as a fertilizer on the biomass yield and chemical composition of cocksfoot (Dactylis glomerata L.). In a 5‐year small‐plot field experiment digestate fertilization treatments supplying 90, 180, 270, 360 and 450 kg N ha^?1 were compared with untreated plots and plots fertilized with 180 kg N ha^?1 of mineral N fertilizer. Swards fertilized with digestate produced higher biomass yield compared with the control. The same rate of nitrogen fertilizer (180 kg N ha^?1) supplied as digestate and from mineral fertilizers gave similar results on biomass yield. Herbage in swards fertilized with digestate contained less nitrogen, but the C:N ratio was much more suitable for biogas production. Digestate fertilization resulted in higher concentrations of cellulose and hemicellulose in biomass and lower contents of the inhibitors of anaerobic digestion—sulphur, calcium, magnesium and phosphorus—compared with those of swards receiving mineral fertilizers.     

New approaches concerning the utilization of natural amendments in cadmium phytoremediation

Different vegetal biomass resources such as Picea abies bark, Castanea sativa chestnuts shell and Asclepias syriaca plant were considered to provide a source of natural bioactive compounds, which can be properly used in several directions such as: plant growth regulators and amendments in bioremediation. Having in mind a complex processing biomass technology, these raw materials are used in a first step to separate bioactive compounds by an aqueous extraction. The obtained extracts containing polyphenols were tested in germination tests to evaluate their influence on cadmium bioaccumulation in oat plant (Avena sativa). It was observed that the mentioned extracts modulated cadmium bioaccumulation, photosynthesizing pigments biosynthesis, plant growth and its development depending on metal ion concentration (5, 12.5, 25 mg/L), and extracts composition determined by vegetal raw material and their total polyphenols content (130, 122, 114 mg/L related to gallic acid). FTIR spectra and histo-anatomical cross section of the roots certify that P. abies bark and A. syriaca plant extracts amendments stimulated cadmium bioaccumulation, promoting the translocation of heavy metals to the aerial part of the plant, meanwhile chestnuts shell extract reduced the mobility of cadmium determining in situ inactivation of heavy metal ions.     

Synthesis of a new thiophosphorylated compound derived from cashew nut shell liquid and study of its antioxidant activity

This work describes the synthesis and study of the antioxidant activity in mineral oils of new thiophosphorylated compound derived from cashew nut shell liquid (CNSL), a by-product of the process of improvement of cashew nuts. This study was conducted through the thermal analysis observing the T_onset variations and the accelerated oxidation experiments observing the induction time variations. The results of thermogravimetric and acceleration oxidative experiments showed a significant improvement in the oxidative stability of the doped oils. These results suggest that this new compound acts efficiently in the stabilization of the naphthenic mineral oils.     

Fumigant Toxicity and Repellence Activity of Camphor Essential Oil from Cinnamonum camphora Siebold Against Solenopsis invicta Workers (Hymenoptera:Formicidae)

The red imported fire ant (RIFA) Solenopsis invicta Buren causes severe damage to humans and animals as well as the environment. Chemical treatment is the main strategy of RIFA management, which also is potentially toxic to the environment. Plant essential oils (EOs) are considered as potential substance that can be used to control insects. This study aimed to identify the chemical composition of camphor EO and investigate the insecticidal activity on RIFAs. The chemical composition of the EO was analyzed by gas chromatography/mass spectrometry and gas chromatography with flame ionization detection. Results revealed that 36.61% camphor and 30.05% cineole were the major components. The insecticidal activity of camphor EO was assessed against RIFA workers by conducting two different bioassays: fumigant toxicity and repellence. Fumigant toxicity assay results showed that the lethal dose (LC₅₀) of the EO at 24 h was 1.67 and 4.28 μg/ml for minor and major workers, respectively; knockdown time (KT₅₀) was 10.82 and 14.73 h. At 2.55 μg/ml, the highest average mortality of the ants was 84.89% after 72 h. Camphor EO exhibited fumigant toxicity against minor and major workers as indicated by the effects on attacking, feeding, and climbing behaviors. This EO was also strongly repellent to the two size workers of the colony as observed in their behavior against Tenebrio molitor treated with 5 µl EO. The fumigant toxicity and repellence of camphor EO against RIFA indicated that this substance could be a potential alternative for the development of eco-friendly products used to control pests.