Camellia sinensis, Ilex paraguariensis, Salvia officinalis, Tilia cordata, Levisticum officinale, Aegopodium podagraria, Urtica dioica, Capsicum baccatum, Viscum album, and marine algae Porphyra Yezoensis for green synthesis of AgNPs. The antioxidant power of methanolic extracts was estimated at the beginning according to their free radical scavenging activity by the DPPH method and reducing power activity by CUPRAC and SNPAC (silver nanoparticle antioxidant capacity) assays. The results obtained by the CUPRAC and SNAPC methods exhibited excellent agreement (R2~0.9). The synthesized AgNPs were characterized by UV-vis spectroscopy, Fourier transform infrared spectroscopy (FTIR), scanning electron microscopy (SEM), energy dispersive X-ray analysis (EDX), dynamic light scattering (DLS) particle size, and zeta potential. The UV-vis absorption spectra showed a peak at 423 nm confirming the presence of AgNPs. The shapes of extract-mediated AgNPs were mainly spherical, spheroid, rod-shaped, agglomerated crystalline structures. The NPs exhibited a high negative zeta potential value in the range from −49.8 mV to −56.1 mV, proving the existence of electrostatic stabilization. FTIR measurements indicated peaks corresponding to different functional groups such as carboxylic acids, alcohol, phenol, esters, ethers, aldehydes, alkanes, and proteins, which were involved in the synthesis and stabilization of AgNPs. Among the examined extracts, green tea showed the highest activity in all antioxidant tests and enabled the synthesis of the smallest nanoparticles, namely 62.51, 61.19, and 53.55 nm, depending on storage times of 30 min, 24 h, and 72 h, respectively. In turn, the Capsicum baccatum extract was distinguished by the lowest zeta potential, decreasing with storage time from −66.0 up to −88.6 mM.Natural extracts are a rich source of biomolecules that are useful not only as antioxidant drugs or diet supplements but also as complex reagents for the biogenic synthesis of metallic nanoparticles. The natural product components can act as strong reducing and capping substrates guaranteeing the stability of formed NPs. The current work demonstrates the suitability of extracts of  Escherichia coli, Bacillus subtilis, Vibria cholera, Pseudomonas aeruginosa, Syphillis typhus, and Staphylococcus aureus. Apart from gold and silver nanoparticles, the literature also describes nanoparticles of zinc oxide (ZnONPs), selenium (SeNPs), copper oxide (CuONPs), copper (CuNPs), nickel oxide (NiONP), and iron (INPs), covering iron oxide (ION) nanoparticles, iron hydroxide (IOOH), and zero-valent iron (ZVI) nanoparticles [Nanoparticles (NPs) are of great interest in engineering, chemical, and biomedical sciences. These small particles, due to their high surface-to-volume ratio, have different properties compared to chemical species of the same composition. The differences concern, among other aspects, changes in thermal and electrical conductivity, catalytic activity, optical absorption, melting point, and antibacterial properties. Currently, nanoparticles belong to highly advanced and specialized biomedical products . There aarticles ,9,10,11.Chemically synthesized nanoparticles often require toxic reducing and stabilizing agents  . The proThe biosynthesis of nanoparticles can be achieved by reducing metal cations with inactivated tissue, living organisms, or extracts of plants, yeasts, algae, lichens, fungi, and bacteria. The biosynthesized nanoparticles are more stable and less toxic in comparison to those obtained from chemical production. The extract components possessing various bioactive components such as trace metal ions, vitamins, alkaloids, carotenoids, polyphenols, fats, carbohydrates, proteins, biological catalysts (enzymes), etc., play a vital role in nanoparticle formation as strong reducing agents, stabilizing agents, or precursor molecules for NP formation ,14,15.The synthesis of nanoparticles takes place in two stages. In the first stage, the metal ions are reduced, and then the agglomeration of colloidal nanoparticles occurs, ultimately forming oligomeric clusters. The main problems encountered in the biogenic synthesis of nanoparticles concern: achieving their appropriate shape, size, and monodispersity in the solution phase. Usually, to control the synthesis process, several factors have been concerned such as pH, temperature, and reaction or incubation time ,17,18,19Calligonum comosum roots and Azadirachta indica leaf extracts against LoVo, MDA-MB231, and HepG2 cells. The AgNPs synthesized using Morinda citrifolia root extract caused the HeLa cell lines’ total death [Alternanthera tenella [Dendrophthoe falcate, and Datura inoxia [Cannabis sativa leaf extracts to mediate the green synthesis of AgNPs and their antibacterial activity against several human pathogens  have been proven [Iris tuberosa leaf extract. They proved the in vitro antimicrobial properties of AgNPs against typical pathogen contaminants in cosmetics, i.e., Escherochia coli, Pseudomonas aeruginosa, Staphylococcus aureus, Candida albicans, and Aspergillus brasiliensis. Then, the biogenic-silver nanoparticles were applied as preservative agents in moisturizing cream. Ali et al. [Calotropis gigantean, which inhibited the growth of Candida albicans.Various studies have described not only the ability of natural extracts and microorganisms to form AgNPs but also their excellent antioxidant activity, which is higher compared to substrates. It is believed that this activity is due to the preferential sorption of extract components on the surface of nanoparticles ,21. Extr tenella , Dendropa inoxia  extractsn proven . An inten proven . The auti et al.  also desNP preparations owe their therapeutic effects to the synergistic effect of the ingredients as well as a unique bioavailability. Taking into account the variability of NPs obtained by biosynthesis and the richness of natural extracts, there are still opportunities for discovering and developing their new aspects.The examined natural extracts are well-known sources of specific biomolecules with antioxidant potential ,37,38,39Many in vitro tests are used to measure antioxidant activity that are based on the mechanism of hydrogen transfer (HAT) or electron transfer (ET) from the antioxidant to free radicals. Recently published reviews detail the usefulness and principles of these procedures ,43. MostThe electron transfer (ET) assays include the Folina–Ciocalteu (FC) test, phosphomolybdenum assay, ferrous oxidation-xylenol orange assay (FOX), iron antioxidant power reduction (FRAP), chromium reducing antioxidant capacity (CHROMAC), copper antioxidant capacity reduction test (CUPRAC), or Ce (IV)-based reducing capacity (CERAC). These are color tests; therefore, spectroscopic techniques can be used for measurement. The change in absorbance level is linearly correlated with the total antioxidant capacity of the sample. From this group of tests, the CUPRAC test, which is commercially available in a microplate version, was selected for testing.Nanotechnology tests are also classified as electron transfer tests . In our ● or cationic radicals such as DMPD+● and ABTS+● are scavenged with an antioxidant. Despite the fact that the DPPH assay is usually classified as a mixed-mode (ET/HAT) assay, there is some evidence that the ET mechanism dominates in non-aqueous solutions due to the ability of organic solvents to form strong hydrogen bonds with antioxidants [There are also tests that operate in mixed mode (HAT/SET) such as DPPH , or DMPD . In these tests, a stable free radical such as DPPHoxidants ,47. PrepThe CUPRAC Antioxidant Assay Kit was used to determine the total antioxidant capacity (TAC) of examined extracts. In the first step, calibration curves were constructed for the reference substances such as chlorogenic acid, ascorbic acid, and Trolox. The linear regression analysis parameters evaluated for the standard curves are summarized in Camellia sinensis and Ilex paraguariensis extracts, and the lowest for Porphyra Yezoensis, a representative of marine algae. It has been shown many times that the high reduction potential of copper is associated with the high level of phenolic compounds in the extracts, which justifies the above result. Such a relationship was confirmed in the studies conducted by GonÇalves et al. [Asphodeline anatolica extracts, showed that the reduction ability is higher in methanol than in aqueous extracts.The reduction of metal ions, e.g., Cu (II), is commonly used as a test to assess the antioxidant activity of natural extracts. The reduction ability is a measure of their electron donating properties. The obtained results were expressed as equivalents of reference substances. It is known that the higher the equivalents, the higher the reducing activity of the test sample. Generally, the highest activity in the CUPRAC test was obtained for s et al.  and Srins et al. . Moreoves et al. , on the Thus, the preparation of methanol extracts in order to enhance the reduction potential seems to be a reasonable choice. This can also inspire further research to expand the range of extraction solvents.The DPPH owing to a stable nitrogen radical was very early adapted to study the free-radical quenching abilities of different antioxidants . DespiteIn the first step, calibration curves were performed for solutions of reference substances: ascorbic acid, chlorogenic acid, and Trolox. Measurements were performed for six different concentrations of standard solutions containing DPPH at a constant concentration of 0.1 mM at 517 nm after 15 min storage in a dark place, considering the previously confirmed fast reaction kinetics between DPPH and the chosen antioxidant standards . The calThe antioxidant activity of methanolic extracts was tested under uniform conditions, taking into account the optimized DPPH concentration of 0.1 mM at 517 nM. The reaction time was set at 60 min assuming the differentiated extracts’ components with possible slow kinetics. The addition of an increasing volume of the extract caused a clear change of the color of DPPH from purple to yellow. In order to compare the antioxidant properties of the tested samples, the obtained % I values were converted into equivalents of reference substances, using the appropriate calibration equation. The results are collected in Camellia sinensis > Tilia cordata > Ilex paraguariensis > Salvia officinalis > Aegopodium podagraria > Levisticum officinale > Urtica dioica > Viscum album > Capsicum baccatum > Porphyra Yezoensis. The antioxidant capacity of extracts is mainly attributed to the redox potential of phenolic compounds, acting as reducing agents. As it was mentioned before, DPPH can accept an electron as well as a hydrogen radical, becoming a stable diamagnetic molecule.The DPPH scavenging activity of the examined extracts varied from poor  to very good scavengers (green tea). Generally, the order of extract activities was similar to that established by the CUPRAC assay. The free radical scavenging activity of extracts can be arranged in the following order: In 2006, Scampicchio et al.  describe2 > 0.9). The limits of detection (LODs) for standards in the SNPAC assay were from 1.51 to 5.85 µM.The exemplary spectra recorded for different dilutions of the chlorogenic acid standard are presented in Camellia sinensis extract. As it can be seen, the increase in the volume of the extract in the test sample resulted in an increase in absorbance even beyond the measurable range. The visible wide absorption band in the UV-vis spectrum in the range of ~400–450 nm is characteristic of silver nanoparticles. One of the observed effects of the plasmon resonance of the formed nanostructures was an intense color that changes from straw yellow to dark brown. As the volume of the extract increases from 100 to 700 µL, an additional slight absorption band in the red light range appeared between 650 and 700 nm. Thus, in the case of larger volumes of the extract, i.e., 500 and 700 µL, the possibility of the agglomeration of nanoparticles should be considered. Such an effect can also be related to the possible formation of rod-shaped nanoparticles. In this case, two absorption bands appear on the spectrum: one caused by electron oscillations along the structure, and the other related to vibrations at its ends [To estimate the antioxidant activity of examined extracts, different volumes of extracts were added to the SNPs solution. its ends . If the The increasing absorbance value confirmed the ongoing intensive bioreduction process along with the increasing concentration of the extract. Photographs of the obtained spherical nanoparticles made by SEM microscopy show that the nanoparticles are “suspended” in a gelatinous organic substance  acting aCamellia sinensis, Ilex paraguariensis, Salvia officinalis, Tilia cordata, Levisticum officinale, Aegopodium podagraria, Urtica dioica, Capsicum baccatum, Viscum album, and marine algae.The antioxidant activity of the remaining extracts was measured utilizing appropriate volumes  to allow spectrophotometric reading. The measurement results were used to determine the equivalents of the reference substances on the basis of the calibration equations . CompariThe results of the reference substance equivalents obtained for the individual extracts differ significantly from one another. Nevertheless, the TAC equivalents found by the microdilution CUPRAC method in 96-well plates correlate well with those of the SNAPC. The correlations obtained are described by the following equations generated separately for the individual standards of chlorogenic acid (CAeq.), ascorbic acid (AAeq), and Trolox (Txeq):The highest equivalent capacities in the SNPAC method were observed for green tea and Yerba mate. The huge antioxidant potency of these extracts is caused by the catechin flavonoids, known as “tea antioxidants”, owing to the presence of multiple OH functional groups in their structures. The detection limits (LOD) for the reference substances , determined by the above methods, differed significantly from each other. In general, the most favorable values, and thus the smallest ones, were found for all reference substances in the DPPH free radical scavenging method (0.8498–1.8644 µM). An order or even two orders of magnitude of higher values were obtained for the CUPRAC method (65.48–115.23 µM). In turn, the LOD values obtained by the SNPAC method range from 1.5141 µM to 5.8548 µM.96h − Abs30min) can be arranged in the following order: Camellia sinensis (3.36), Salvia officinalis (1.75), Tilia cordata (0.95), Aegopodium podagraria (0.92), Urtica dioica (0.62), Capsicum baccatum (0.58), Levisticum officinale (0.50), Viscum album (0.03), Ilex paraguariensis (0.01), and Porphyra Yezoensis (0.0). The current results are in agreement with the literature [Boswellia ovaliofoliolata, Shorea tumbuggaia, and Svensonia hyderobodensia. Bala et al. [Aspergillus fumigates by surface plasmon resonance at 450 nm. Sriramulu [Spectrophotometric spectra of the synthesized nanoparticles were measured after 30 min, 24 h, 72 h, and 96 h of their storage in a dark place. The absorbance at 423 nm, which is characteristic of silver nanoparticles, increases with the storage time. The increase in absorbance was also visible to the naked eye as a darkening of the solution. The greatest increase in absorbance occurs after 96 h. The observed increase , bands C=C from aromatic rings and/or carboxylic acids, N-H from proteins (1730–1560 cm−1), and C-H from plant metabolites (2980–2770 cm−1). These results are in agreement with the investigation of Balaji Venkatesan et al. [Rosa damascene. Manikandan et al. [R. indica extract, indicated that the band at 1624 cm−1 shifted to a higher wavelength, confirming the formation of AgNPs.Additionally, the comparative spectra of pure extracts and extracts with synthesized AgNPs were obtained for chosen samples: 3-n et al.  who redun et al. , who redAegopodium podagraria  technique was applied with the aim to visualize the shape of silver nanoparticles. The representative SEM images including various forms of extract-mediated silver nanoparticles are presented in dagraria a; (ii) ricinalis b; and , indicating the sorption of the charged extract components, which stabilize the NPs and counteract aggregation. It should be emphasized that the NPs that were synthesized with Capsicum baccatum extract showed the most negative zeta potential. In this case, the zeta potential becomes increasingly negative over time, decreasing from −66.0 mV to −88.6 mM. Recent studies concerning the biosynthesis of AgNPs with Capsicum extract indicated that the proteins which have amine groups are responsible for the reduction during the formation of silver nanoparticles. It was proven that the secondary structure of the proteins is able to change after reaction with silver ions [Analyzing the effect of the storage time on the size and zeta potential of extract-mediated nanoparticles, the following trends could be observed : as timever ions .The biogenic synthesis of metallic nanoparticles with the use of extracts of natural plants, bacteria, fungi, lichens, etc., is widely described in the literature . The syn2~0.9). A similar correlation between the AgNP-based method and the cupric ion-reducing applied for the evaluation of the total antioxidant activity of polyphenols was reported previously by Özyürek et al. [2 = 0.8761. Azat Akbal et al. [Punica granatum, Cydonia oblonga, Castanea sativa, Ficus carica, Juglans cinerea, Morus nigra, and Morus alba aqueous leaf extracts.In order to assess the potential of the extracts to reduce silver ions, their antioxidant activity was determined using three independent methods , where electron transfer is the dominant mechanism and key for green synthesis. The results of the antioxidant activity were presented in the form of the equivalents of the reference substances of ascorbic acid, chlorogenic acid, and Trolox. The methods’ sensitivity can be arranged according to LOD values, estimated by the 3 σ procedure, in the following order: DPPH (0.8498–1.8644 µM) > SNAPC (1.5141–5.8548 µM) > CUPRAC (65.48–115.23 µM). A very good correlation was confirmed between the results estimated by the CUPRAC and SNPAC methods -epigallocatechin-3-gallate, (–)-epigallocatechin, (–)-epicatechin-3-gallate, and (–)-epicatechin, owing to the presence of multiple OH functional groups in their structures. As the research conducted so far shows, it is the hydroxyl groups, especially those located in the ortho and para positions, that are involved in the synthesis of metal nanoparticles [The obtained results showed that methanol extracts with a confirmed, differentiated antioxidant potential ensured the efficient synthesis of AgNPs. The mean size of the obtained nanoparticles ranged from 99.56 to 119.68 nm, changing with time from 30 min to 72 h. Green tea extract deserves special attention, as it showed the highest antioxidant activity in all tests and enabled the synthesis of the smallest nanoparticles, namely 62.51, 61.19, and 53.55 nm, depending on storage time. This observation confirms earlier findings that the size of the AgNPs formed may vary depending on the type of reducing agents and stabilizers of the reducing reaction . In genearticles ,72. Litvarticles ,74.Tilia cordata extract-mediated NPs increased by almost 2.5 times in size within 3 days after synthesis, whereas the smallest NPs obtained with the Camellia sinensis extract decreased by about 15%. The process is similar to the maturation of crystalline sediments when their size is normalized, as small forms disappear and larger ones expand. A similar effect was observed by Dwivedi et al. [The influence of elevated temperature on the kinetics of nanoparticle formation has already been investigated. It is known that at a higher temperature, even exceeding 100 °C, nucleation usually dominates over the growth of nanoparticles, which results in a reduction of their size ,76. In oi et al.  who perfCapsicum baccatum extract was distinguished by the lowest zeta potential. It is known that paprika extract is a source of phenolic compounds such as quercetin, capsaicinoids (mainly capsaicin), and polyphenolic compounds, including flavonoids , carotenoids , and vitamin E, and C [In turn, the E, and C ,79, whicThe increase in the volume of the extract caused an increase in the intensity of the SPR band, but in such conditions, an additional band appeared shifted towards longer wavelengths. The reasons for this may be different, but they are usually not favorable. Generally, absorbance at long wavelengths is enhanced due to developing shape anisotropy, the aggregation of small spherical NPs, or an increase in particle size ,80,81.All the chemicals were of analytic grade. 1,1-Diphenyl-2-picrylhydrazyl free radical (DPPH), ascorbic acid, and chlorogenic acid were purchased from Sigma-Aldrich . Methanol was obtained from Merck . Water purified by an ULTRAPURE Millipore Direct-Q 3UV-R  of the resistivity 18.2 MΩ cm was used to prepare all the aqueous solutions.The fresh plants Aegopodium podagraria, Urtica dioica, Salvia officinalis, Levisticum officinale, Tilia cordata, Capsicum baccatum, and Viscum album were harvested in the southeastern region of Poland in May 2020. The roasted sea algae (Porphyra Yezoensis) used to make nori, Camellia sinensis, and Ilex paraguariensis were purchased from a local market. The fresh plants were dried for about three months in a shady area. These samples were then ground into a powder and subjected to extraction. For extraction, 5 g of powder was added to 100 mL of methanol. The samples were left for 60 min in an ultrasonic bath  Bandelin Sonorex RK 103 H  at a temperature of 35 °C. The obtained extracts were centrifuged, and the supernatant was filtered through a syringe filter (0.7 µM or 0.45 µM) and stored in a refrigerator at 4 °C.570)sample represents the absorbance of the sample, (A570)blank is the absorbance of the medium blank, n stands for sample dilution factor, and Slope is related to linear calibration curve prepared for reference chemical.The CUPRAC method is based on testing the ability of antioxidants to reduce copper(II) ions to copper(I). Commercially available kits enable the performance of microscale spectrophotometric measurements with 96-well plates. The assay measures total antioxidant capacity (TAC). The detection is based on a colored complex formed between the resulting Cu(I) and a dye reagent. The color intensity is proportional to TAC of the sample. The absorbance measurements for standards  and extract samples in dilutions 1:1, 1:10, and 1:100 were performed in duplicate using Antioxidant Assay Kit-MAK334 purchased from Sigma-Aldrich Inc. . The 96-well microplate was mixed and incubated for 10 min at room temperature. The absorbance of extracts, reference chemicals, and blanks was measured at 570 nm by the use of a spectrophotometer Hybrid Multi-Mode Reader Synergy™ H1 . Calibration curves were constructed for the reference substances. Linearity in the range from 300 to 1000 μM was achieved. The TAC values of the examined extracts were calculated according to the following equation:control). Thus, the total concentration of DPPH was kept at a constant level of 0.1 mM. In the first step, calibration curves were performed for solutions of ascorbic acid, chlorogenic acid, and Trolox as reference antioxidants. Measurements were performed for six different concentration levels after 15 min storage in a dark place. The calibration curves were subjected to simple linear regression analysis. The DPPH radical scavenging activity of extracts (Abssample) was determined by measuring the absorbance of mixtures prepared by adding the different sample volumes to 1 mL of 1 mM methanolic solution of DPPH and made up to 10 mL with methanol. The measurements were performed after 1 h incubation in the dark at room temperature (20 °C). The DPPH radical scavenging activity of the samples was expressed as the percentage of inhibition (%I) calculated using the following equation:The stock solution of DPPH-R (1 mM) was prepared in methanol and stored in darkness at 4 °C in glass volumetric flasks. The working standard solutions were prepared daily by diluting the stock solution. The measurements were performed using spectrophotometer-Genesys 20 , which is able to measure absorbance in the range of 320–1100 nm. Spectrophotometric measurements were started with the selection of DPPH concentration so as to ensure absorbance on a level of 0.9 at 517 nm. The conducted experiments showed that this value corresponds to a concentration of 0.1 mM DPPH  of the examined extracts. The SNPAC measurements were performed using the procedure described by Özyürek et al. . The iniA measurement of 30 mL of 1 mM AgNO3 was heated for 15 min to approximately 90 °C. A total of 2 mL of the extract was then added dropwise with constant stirring using a magnetic stirrer. The finished solution was allowed to stand in the dark. The size of the silver nanoparticles and the zeta potential were measured with an analyzer  after 24 h, 72 h, and 96 h. UV-vis spectra were monitored as a function of the reaction time by a Genesys 20  spectrophotometer operated at a resolution of 1 nm.−1. For each material, 3 samples under the same conditions were examined. For each sample, 200 scans at a spectral resolution of 4 cm−1 were averaged. For a given material, the final average spectrum was then calculated. All spectral manipulation was carried out using Origin Pro 8.5 .Fourier transform infrared spectroscopy (FTIR) spectra were collected via a Nicolet 6700 FTIR spectrometer , with the Smart iTR attenuated total reflection  sampling accessory was used. Freeze dried samples were placed directly on ATR crystal and measured. The spectra were collected over the range of 4000–650 cmMorphological forms and chemical composition of AgNPs occurrence were characterized by scanning electron microscopy (SEM) using Quanta 250 FEG Scanning Electron Microscope by FEI  equipped with energy dispersive spectrometry (EDS). Prior to SEM analysis, samples in liquid form were dried. Residues were dissolved in ethanol and the solvent was evaporated in a vacuum dryer at 70 °C. Then, the samples were glued to carbon tape on an aluminum holder and sputtered with graphite.Salvia officinalis, Tilia cordata, Levisticum officinale, Aegopodium podagraria, Urtica dioica, Viscum album), vegetables (Capsicum baccatum), marine algae (Porphyra Yezoensis), and teas , can be useful for the synthesis of AgNPs. Most of them have never been used for this purpose. Their free radical scavenging activity and reducing power were confirmed by DPPH, CUPRAC, and SNPAC assays. The DLS measurements showed different hydrodynamic diameters (99.56 to 119.68 nm) and the zeta potentials (−49.8 mV to −56.1 mV) of the obtained nanoparticles. It should be emphasized that the longer storage of nanoparticles in the reaction mixture promotes the adsorption of biomolecules, increasing the negative surface charge.The current research proves that methanol extracts obtained from ten natural products, representatives of common herbs (Considering the fact that phytochemical synthesis leads to the formation of nanoparticles with different morphological features, undergoing modification by active ingredients such as carboxylic acids, alcohol, phenol, esters, ethers, aldehydes, alkanes, and proteins, differences in pharmacological activity can be expected. Thus, research on the biological activity of synthesized NPs will be continued on bacterial strains, cell lines, and animal models.


Gdue is a nutraceutical obtained from the association of two marine algae, Ascophyllum nodosum and Fucus vesiculosus, in addition to chromium picolinate, which could be useful for the treatment of dysglycemia, overweight, and the other components of the metabolic syndrome. The aim of the study was to assess the real-world effectiveness and safety of Gdue when administered to subjects with one or more components of the metabolic syndrome.  A longitudinal, retrospective, observational study, conducted among primary care physicians, nutritionists, and specialists from various disciplines. The impact of 180 days of administration of Gdue was assessed on body weight, waist circumference, fasting blood glucose, HbA1c, lipid profile, and blood pressure levels. The likelihood of experiencing a first major cardiovascular event over ten years was estimated using Italian risk charts. General linear models for repeated measures were applied to assess changes in the parameters of interest during the follow-up. Results are expressed as estimated marginal means with their 95% confidence interval.  Overall, 505 patients were enrolled by 282 physicians. After 6 months of treatment with Gdue, body weight was reduced on average by 7.3 kg , waist circumference by 7.5 cm , fasting blood glucose by 16.3 mg/dL , HbA1c by 0.55% , systolic and diastolic blood pressure by 7.1 mmHg  and 4.2 mmHg , respectively, LDL cholesterol by 18.2 mg/dL , and triglycerides by 39 mg/dL . HDL cholesterol was significantly increased by 2.9 mg/dL . The 10-year risk of cardiovascular events significantly decreased by 1.8%, corresponding to a relative risk reduction of 27.7%.  Our real-world study shows that 6 months of treatment with Gdue have an impact on all the components of the metabolic syndrome, thus offering the potential for decreasing the cardiovascular risk associated with metabolic syndrome. Metabolic syndrome has become a major health problem in most of the modern world and is characterized by abdominal obesity, insulin resistance, dysglycemia, hypertension, and hyperlipidemia , 2. The α-amylase and α-glucosidase enzymes in the intestine, causing a slowdown in the absorption and digestion of carbohydrates and demonstrating an important antihyperglycemic action in vivo, in particular on postprandial hyperglycemia [Two marine algae, Ascophyllum nodosum and Fucus vesiculosus, could be useful for the treatment of dysglycemia, overweight, and the other components of the metabolic syndrome –5. Ascopglycemia –17. In aglycemia . Furtherglycemia .Chromium is an essential trace element involved in the metabolism of carbohydrates, lipids, and proteins and is a necessary cofactor for many insulin functions, promoting the binding to its receptor in muscle cells, adipocytes, and hepatocytes and also promoting the phosphorylation of receptors –21.In streptozotocin-induced diabetic animals, administration of chromium picolinate decreased plasma glucose levels, normalized glycogen content in the liver, increased the activity of glycolytic enzymes , and suppressed the activity of gluconeogenic enzymes (glucose-6-phosphatase and phosphoenolpyruvate carboxykinase) in the liver . The inhβ-cell exhaustion.At the oral dose of 250-500 mg, Gdue reaches levels 25-50 times higher in intestinal fluids than in vitro inhibitory concentrations, completely, uncompetitively, and reversibly inhibiting carbohydrate-degrading enzymes. Preclinical studies have confirmed this mechanism of action , 8, respAnimal studies also suggest a positive effect of Ascophyllum nodosum and Fucus vesiculosus on liver steatosis, frequently associated with the metabolic syndrome .α levels, evaluated as endothelial damage markers [Open-label or double-blind controlled trials have shown how Gdue reduces the glycemic index of ingested foods, postprandial glycemic peaks, and the consequent insulin response , 9–11. W markers . Further markers , 11.Assessing whether the results of these clinical trials are applicable to the broader range of patient populations treated under routine clinical practice conditions and in different settings is of importance to determine the magnitude of effectiveness of Gdue use.The aim of the study was to assess the real-world effectiveness and safety of Gdue when administered to subjects with one or more components of the metabolic syndrome. The impact of 180 days of administration of Gdue was assessed on fasting blood glucose, HbA1c, body weight, waist circumference, lipid profile, and blood pressure levels.This is a longitudinal, retrospective, observational study, conducted among primary care physicians, nutritionists, and specialists from various disciplines.Male or female patients aged ≥18 yearsPresence of one or more components of the metabolic syndrome, according to the IDF definition , includiPatients treated with Gdue. The decision to prescribe Gdue was based on clinical judgment and was independent of study participationPatients were included if they fulfilled the following eligibility criteria:Gdue was generally administered at the recommended dose of 2-3 tablets per day at meals as integration of a dietary prescription.At baseline (start of Gdue) and after 90 days and 180 days of treatment, the following information was collected from clinical records on an ad hoc form: age, gender, body weight, waist circumference, fasting blood glucose, HbA1c, blood pressure, total cholesterol, HDL cholesterol, LDL cholesterol, and triglycerides. Information on comorbidities and concomitant treatments was also collected. Participating physicians were requested to report any adverse event registered during the follow-up.In a subgroup of 89 patients, information on fasting insulin levels was also available at baseline and during the follow-up. In these patients, HOMA-IR was also calculated as an index of insulin resistance. Data collected were those available in medical records and prescribed according to routine clinical practice, and no laboratory test was performed specifically for the study.Descriptive data were summarized as mean and standard deviation (continuous variables) or counts and percentages .U-test for continuous variables and the chi-square test for categorical variables.Patient characteristics at baseline were compared by gender using the Mann-Whitney P value < 0.05 was considered for statistical significance.General linear models for repeated measures were applied to assess changes in the parameters of interest during the follow-up. Results are expressed as estimated marginal means with their 95% confidence interval (95% CI). Mean changes of each parameter were also estimated by tertile of the parameter at baseline. All tests are two-sided, and a The likelihood of experiencing a first major cardiovascular event  over the following ten years was estimated using the Progetto Cuore Italian risk charts .Statistical analyses were performed using the SPSS software ver. 23.0 .Overall, 505 patients were enrolled by 282 physicians (range 1-8). Among participating physicians, 65.5% were general practitioners, 10.9% were nutritionists, 8.1% were endocrinologists, and 15.5% were from other specialties . The characteristics of the patients enrolled by gender are reported in P = 0.03) and more likely to have hypertension . No other statistically significant differences emerged for all the other variables investigated.The proportion of patients with availability of information at baseline varied between 98.6% for body weight and 49.9% for total cholesterol; the availability of information relative to 180 days ranged from 88.9% for body weight to 41.8% for total cholesterol (supplementary table P < 0.0001).The analysis was also performed by gender  of the patients; moderate gastrointestinal side effects  were referred by 0.6% (N = 3) of the patients.Mild gastrointestinal side effects  were reported by 3.4%  [The beneficial effects of Gdue on waist circumference were also previously documented in a single-arm, 6-month study involving 50 overweight or obese patients , which s < 0.05) . In our  < 0.05) .P < 0.001), LDL cholesterol , and triglyceride levels  and a significant increase in HDL cholesterol levels . In our study, we found a statistically significant effect on all the parameters of the lipid profile, with an average reduction of 18.2 mg/dL for LDL cholesterol and 39.0 mg/dL for triglycerides, while HDL cholesterol was significantly increased by 5.2 mg/dL in men.The impact of Gdue therapy on lipid profile was assessed in a randomized, double-blind placebo-controlled trial involving 175 individuals with T2DM . In thisTo our knowledge, no previous studies have assessed the impact of Gdue therapy on blood pressure. We documented a significant reduction in systolic and diastolic blood pressure, evident after three months and even larger after six months, with an average reduction of 7.1 mmHg for systolic blood pressure and 4.2 mmHg for diastolic blood pressure. The effect on blood pressure can represent the consequence of weight loss; in fact, it is well established that weight loss is associated with a parallel decrease in systolic and diastolic blood pressure .Finally, a reduction vs. baseline in fasting insulin levels and HOMA index after 6 months of treatment with Gdue was described in the study by De Martin et al. , while aThe study has strengths and limitations. Among the strengths, the large number of patients and healthcare professionals involved provides a realistic picture of the effectiveness of Gdue under routine clinical practice conditions. Also, the study allowed to assess the impact of the treatment on a large array of different parameters of the metabolic syndrome.Among the limitations, it should be emphasized that follow-up data were not available for all patients. Also, BMI could not be calculated given the lack of information on height in most of the patients. However, it should also be noted that the degree of missingness was modest for some key variables such as FBG, body weight, and waist circumference. Furthermore, baseline characteristics of individuals with full data and those with missing data were largely superimposable, with the only exception of an older age and a higher prevalence of hypertension among those with all the information available. Another limitation is represented by the lack of a control group; in addition to the therapy with Gdue, additional aspects of care may have influenced the study results. In this respect, it is worth mentioning that study results were not affected when data were analyzed according to the dietary prescription made to the patients .In conclusion, our real-world study shows that 6 months of treatment with Gdue have an impact on all the components of the metabolic syndrome, thus offering the potential for decreasing the cardiovascular risk associated with metabolic syndrome. A significant reduction in the levels of key risk factors was documented even in patients in the lower tertile of the distribution of each variable, suggesting that also individuals with initial, mild elevation of FBG, blood pressure, or lipid parameters can benefit from this therapeutic approach.


Staphylococcus aureus ATCC 6538, Klebsiella pneumoniae ATCC 4352, Escherichia coli ATCC 25922, and Pseudomonas aeruginosa ATCC 27853) for one hour, a >99.99% removal rate was attained; when the filter was exposed to SARS-CoV-2 virus for one hour, it inactivated more than 99%. These beneficial properties minimize the risk of secondary infections, which are significantly more likely to occur when a conventional filter is replaced or removed.With the spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), disease prevention has become incredibly important. Consequently, mask and air-purifier use has increased. The filter is the core component of these items. However, most filter materials lack antimicrobial properties. Copper is a sustainable antimicrobial material. When copper is deposited onto the filter’s surface, the microorganisms that come into contact with it can be effectively inactivated. In this study, we used an oxygen ion beam with a controlled process temperature to treat filter surfaces with copper. This enabled a strong adhesion of at least 4 N/cm between the copper and the filter fibers without damaging them. Upon exposing the filter to bacteria ( Recently, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), first discovered in Wuhan, Hubei Province, China, has spread rapidly worldwide, raising widespread health concerns. The World Health Organization (WHO) declared the coronavirus disease of 2019 (COVID-19) a pandemic on 12 March 2020. Over two years after the outbreak started in December 2019, over 300 million people have been infected, over 5 million people have died worldwide, and the infections and mortality rates have continued to increase [In contrast, copper is a sustainable antimicrobial material proven to inactivate viruses even on the nanometer scale ,9,10,11.A key material used in masks and air purifier filters is the polymer membrane, which is an assembly of one-dimensional fibers. Air can pass through small pores in the membrane, but the fibers block droplets, bacteria, and dust. Nevertheless, while the polymer membrane can filter bacteria and viruses, the latter may remain on the membrane surface. This can cause secondary infections when the filters are replaced. Guo reported in 2020 that bacteria caught in high-efficiency particulate absorbing (HEPA) filters can survive longer than in dust. It was noted that bacteria in HEPA filters fill an ecological niche that may have been neglected in indoor environments .A solution to this problem is the application of a copper coating to antibacterial and antiviral filters. However, it is essential to adhere the copper to the surface of the filter firmly because if the copper detaches from the filter, a human can inhale the copper, which becomes a toxin when inside the body . In prevStaphylococcus aureus ATCC 6538, Klebsiella pneumoniae ATCC 4352, Escherichia coli ATCC 25922, and Pseudomonas aeruginosa ATCC 27853) and inactivated more than 99.8% of SARS-CoV-2.In this study, we observed that the ion beam surface treatment was very effective in improving the adhesion between the copper and the filter fibers. We found that the copper-coated filter inactivated more than 99.99% of four examples of bacteria   filter with an average diameter of 30 μm and a surface density of 70 g/m−5 torr of pressure, we proceeded with the subsequent process. First, oxygen gas was injected. Oxygen ion beams were generated from a linear ion beam source. The process was performed by varying the applied voltage, gas flow rate, and sample stage speed such that the total applied energy density was 1.45, 3.13, and 8.84 J/cm2. The latter three conditions were named Ion Beam 1, Ion Beam 2, and Ion Beam 3, respectively. Next, we measured the maximum temperature of the process by attaching labeled temperature-measuring tape  to the sample stage. After the ion treatments were performed, copper was deposited using a direct current magnetron sputtering system with a purity target of more than 99.99%. Argon gas (purity > 99.99%) was injected with 30 sccm to apply a working pressure of 1.0 mTorr, and the copper was deposited with a power density of 2.55 W/cm2 at a speed of 0.6 m/min in four separate runs. The thickness of the copper film was 30 nm. Detailed ion beam and sputtering process conditions are in Stopping and Range of Ions in Matter (SRIM) is software  that, as the name suggests, calculates the stopping and range of ions in matter ,22,23. T3, and the emitted ion energies were 180, 300, and 600 eV for Ion Beam 1, Ion Beam 2, and Ion Beam 3, respectively [16, 3.26 × 1016, and 4.60 × 1016 ions/cm2 for Ion Beam 1, Ion Beam 2, and Ion Beam 3, respectively.The density of the PET was 1.397 g/cmectively ,18. The TM 4910 Tape; 3M, St. Paul, MN, USA), illustrated in The test involving the T-peeling of tape  and field-emission scanning electron microscopy  were used to observe the surfaces of the filter and the detached tape.Staphylococcus aureus ATCC 6538, Klebsiella pneumoniae ATCC 4352, Escherichia coli ATCC 25922, and Pseudomonas aeruginosa ATCC 27853. The reduction rate was calculated usingA is the colony-forming unit (CFU) per mL of the control group, and B is the experimental group.The copper-coated filter’s antibacterial properties were evaluated, according to the KS K 0693:2016 test method, using the following bacteria: 6 plaque-forming units (PFU)/mL) were sprayed onto the filter at a flow rate of 320 μL/min for 300 s. For comparison, the same process was repeated using a filter that was not coated with copper. The filters were then immersed in 10 mL of DMEM for two minutes to separate the virus particles. The plaque assay was conducted in Vero76 cells  following the protocol described in a previous study [us study . The expThe surfaces of the filters were checked for changes to the filter fibers caused by the oxygen ion beam irradiation. Plateau–Rayleigh instabilities, in which a one-dimensional fluid is broken up into droplets due to the minimization of interfacial surface tension, can occur in a liquid column ,28,29. WThe SEM and energy-dispersive X-ray spectroscopy (EDS) mapping images of the bare filter and the copper-coated filter treated with Ion Beam 2 are shown in The strong adhesion between the filter fibers and the deposited copper film is essential because it can prevent copper nanoparticles  from entering the human body. The adhesion of the copper film deposited onto the filter fibers was evaluated using a T-peeling test with commercially available tape. This test confirmed that the peeling strengths of the filters treated with oxygen ion beams were higher than those of the bare filter, as shown in Staphylococcus aureus ATCC 6538, Klebsiella pneumoniae ATCC 4352, Escherichia coli ATCC 25922, and Pseudomonas aeruginosa ATCC 27853. The bare filter was used as the control sample. The antibacterial properties of the copper-coated filter are presented in The antibacterial properties of the copper-coated filter treated by Ion Beam 2 were investigated, according to the method described in The SARS-CoV-2 evaluation system, composed of a copper-coated filter using a cylindrical device, is shown in In summary, an ion beam treatment technique was used to strongly adhere copper to the fibers found in a filter, the core component in masks and air purifiers. After treating the filter surface with an oxygen ion beam, strong adhesion of at least 4.01 N/cm was achieved between the copper and the filter fibers. Furthermore, we found that the ion beam–surface treatment process should be performed below the glass transition temperature of filter material to prevent damaging the filter fibers. The copper-coated filter developed in this study demonstrated a capability to remove bacteria and viruses remaining on its surface. However, there is a high possibility that bacteria and viruses not caught in the filter cannot be removed, and studies on the possibility of changing the antibacterial and antiviral properties by accumulated dust, adsorbed moisture, and oxidized copper on the surface when the filter is used for a long period have not been conducted. Nevertheless, the use of this copper coating filter can minimize the risk of secondary infections that may occur during the replacement and disposal of filters compared to conventional. Thus, copper-coated filters should be able to effectively and sustainably prevent infections caused by not only SARS-CoV-2 but also unknown viruses that may occur in the future. Moreover, unlike coating copper on the finished mask product that we previously reported, depositing copper on the filter fiber is advantageous because it provides a wider range of personal hygiene supplies utilization . The cop


The aim of this study was to investigate factors responsible for the psychological performance in primary hyperparathyroidism (PHPT) patients.A group of 38 PHPT patients receiving questionnaires, including Beck Depression Inventory (BDI), State–Trait Anxiety Inventory (STAI), and 36-Item Short Form Survey (SF-36), was evaluated. The relationships between scores of questionnaires and clinical biomarkers were examined. Collinearity and linear regression model were applied to examine variables determining the scores of the questionnaire. In 192 PHPT patients, bivariate and partial correlation were used to analyze the relationships between serum concentrations of parathyroid hormone (PTH), calcium, osteocalcin (OCN), and cortisol.Among 38 patients receiving questionnaire tests, 50% (19/38) of the patients developed state anxiety, 60.5% (23/38) of the patients had the trait of developing anxiety. In addition, 18.4% (7/38) of the patients developed mild to severe depression. Serum cortisol at 8:00 was negatively and significantly correlated with social function  after controlling for age, sex, disease duration, serum PTH, calcium, phosphorus, and 25-hydroxyvitamin D [25(OH)D] concentration. OCN was significantly and negatively correlated with score of STAI-S . In the linear regression model for BDI score, variables with statistical significance were serum OCN  and cortisol at 0:00 . In 192 PHPT patients, the serum concentration of OCN  was positively correlated with PTH level. After controlling for age, sex, disease duration, serum 25(OH)D, phosphorus, and calcium concentration, the positive correlation between OCN and PTH was still statistically significant . The serum concentration of cortisol at 0:00 was significantly and positively correlated with serum calcium  in bivariate correlation analysis. After controlling for age, sex, disease duration, serum PTH, 25(OH)D, and phosphorus concentration, serum cortisol at 0:00 was still positively and significantly correlated with serum calcium .Serum levels of OCN and cortisol, rather than PTH and calcium, are associated with the development of anxiety and depression symptoms in PHPT patients. Primary hyperparathyroidism (PHPT) is a disorder of parathyroid hormone (PTH) hypersecretion by parathyroid gland(s) in patients with normal renal function, resulting in increased serum calcium concentration . While mIn fact, a number of hypotheses have been proposed with regard to the mechanism of depression and anxiety development. “Hypothalamus pituitary adrenal (HPA) axis disorder” hypothesis  was a clIn this study, both serum cortisol and OCN concentration changes and the correlation of psychological features with serum OCN and cortisol were analyzed in a group of PHPT patients.In this study, psychological questionnaires, which were not a mandatory requirement for every patient in our department, were tested from August 2020. Until December 2020, questionnaire information in 38 out of 52 patients in this period was obtained. The inclusion criteria included: i) elevated serum calcium level with inappropriately high serum PTH level and ii) with a complete record of serum PTH, OCN, and cortisol concentration. The exclusion criteria included: i) secondary and tertiary hyperparathyroidism, ii) multiple endocrine neoplasms, iii) malignancy, iv) chronic kidney disease (CKD) stages 4 and 5 or eGFR ≤30 ml/min, v) a history of head trauma or stroke, and vi) medications with glucocorticoid. In order to find out whether serum concentration of OCN and cortisol changed as PTH and calcium increased in PHPT patients, a total of 192 PHPT patients admitted to our department from January 2011 to December 2020, including the above 38 patients, were retrospectively evaluated, who met the above inclusion and none of the exclusion criteria. All the patients were managed by the standard protocol developed by our department. Due to the retrospective nature of this study, the written informed consent was waived, which was approved by the ethics committee of our hospital (2017-201).The age of onset was recorded according to the first identification of symptoms related to PHPT  or an elevation in serum calcium or PTH concentrations. The anthropometric information was also collected, including sex, age at the time of diagnosis, and body mass index (BMI).Blood samples were collected in the morning after 10 h of fasting. Fasting serum albumin, calcium, phosphorus, and creatinine levels were measured using an automatic biochemical analyzer . Serum level of PTH was measured by intact immunoradiometric assay . Serum level of 25-hydroxyvitamin D [25(OH)D] was measured by electrochemiluminescence immunoassay . Serum level of OCN was assayed by two methods: radioimmunoassay  and electrochemiluminescence  during different periods. Serum concentrations of Type I procollagen amino-terminal peptide (PINP) and Collagen I telopeptide-β (β-CTX) were examined through electrochemiluminescence method . Serum concentrations of cortisol  and 24-h urinary cortisol were assayed by chemical luminescence assay . Plasma adrenocorticotropic hormone (ACTH) level was tested by chemical luminescence assay . Area bone mineral densities (aBMDs) at the lumbar spines 1–4 (L1–L4) were measured by dual-energy X-ray absorptiometer .Testing was conducted preoperatively by a doctor who was not blinded to disease state, lasting approximately 0.5 h for each. Psychological tests included State–Trait Anxiety Inventory (STAI) , Beck DeThe STAI  measuresFor results in this study, continuous variables were expressed as means ± SD or median  according to their distributions. Categorical variables were summarized as group number/total number. The comparisons of continuous variables between groups were performed using t-test or one-way ANOVA for normally distributed variables; otherwise, nonparametric test. Categorical data were compared by chi-square test. As serum concentration of OCN was tested by two different kinds of methods, OCN was transformed into categorical variables according to its tertile values. Pearson correlation analysis (two-tailed) was used to investigate the relationship of PTH, calcium with cortisol, as well as the association between the serum PTH, calcium, OCN, cortisol levels, and parameters of questionnaires. Kendall’s tau-b was used to test the correlation between OCN and PTH or calcium, which were also transformed into categorical variables here. Partial correlation analysis (two-tailed) was used to examine the above associations when controlling for clinical features and biomedical markers. Linear regression analysis with backward mode was applied to examine determining variables of questionnaire score. In this part, age; sex; disease duration; serum PTH; calcium; phosphorus; 25(OH)D; OCN; cortisol of 8:00, 16:00, 0:00; and 24-h urinary free cortisol were considered. To get stable results, collinearity analysis was used to eliminate collinear variables in each questionnaire regression model. The cutoff value of the variance decomposition proportion for the diagnosis of multicollinearity is set to 0.3 in dimensions with condition index over 10 according to the work of Liao  and Kim Since only 38 out of 52 patients received psychologic evaluation, including BDI, STAI, and SF-36 questionnaire during August 2020 to December 2020, a sensitivity analysis was performed between patients who had taken the tests and those who had not. As shown in the For PHPT patients receiving questionnaire tests, the SF-36 MC items’ scores were 74.04 ± 23.32 for SF, 72.74 ± 18.71 for MH, 57.76 ± 22.17 for VI, and 66.67  for RE, respectively. The score of STAI-S was 35.43 ± 11.56, STAI-T 37.39 ± 10.34, and BDI was 5 . According to the reference , out of In addition, we assessed associations of PTH, calcium, OCN, and cortisol concentrations with psychological performance, as evaluated by questionnaires. As shown in As for the linear regression model of the BDI score, statistically significant variables were serum OCN  and cortisol at 0:00 , while variables excluded by collinearity analysis included age, serum PTH, 25(OH)D, calcium, cortisol at 8:00, and urinary free cortisol in 24 h. No significant variable was included in the equations of other questionnaires.In these 38 patients, we found that OCN was associated with PTH  and marginally associated with calcium , while cortisol was not related with these two parameters. In order to further test whether these findings could be replicated in a larger sample size, we further explored the relationships among OCN, cortisol, PTH, and calcium in 192 PHPT patients.The baseline characteristics of 192 PHPT patients in different PTH tertile groups were shown in When patients were grouped according to tertiles of serum calcium concentration, as shown in In addition, in the bivariate correlation model between serum PTH concentration and OCN, cortisol concentration showed that serum PTH was positively and significantly correlated with serum OCN . When controlling for age, sex, and disease duration, and additionally with phosphorus, calcium, and 25(OH)D, PTH was still positively and significantly correlated with serum OCN concentration . However, serum PTH concentration was not significantly correlated with serum cortisol of different time points and urinary cortisol excretion (p > 0.05) in the bivariate and partial correlation model.As to serum calcium level, it was positively and significantly correlated with serum OCN  and cortisol concentration at 0:00 . When controlling for age, sex, and duration, the correlation of serum calcium with OCN  and cortisol at 0:00  was still significantly positive. When serum 25(OH)D and phosphorus were adjusted, serum calcium was still significantly and positively correlated with serum cortisol at 0:00  and OCN . Even when serum PTH was further adjusted, the correlation between serum calcium and cortisol at 0:00 was still significantly positive , while the correlation between serum calcium and OCN  lost its significance.The findings from this study lie in two aspects. Firstly, the balance of serum OCN and cortisol was associated with the psychological performance in PHPT patients. Secondly, with the elevation of serum calcium and PTH, the concentration of serum cortisol and OCN increased.The effects of glucocorticoid –17, 19 ain vivo and in vitro , which is a metabolically active form of OCN at least in mouse studies . Third, To sum up, in this study, it was demonstrated that the serum levels of OCN and cortisol were independently associated with the development of psychological symptoms in PHPT patients. More basic and clinical studies are needed to test and verify this observation.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by Shanghai Ruijin Hospital. Written informed consent for participation was not required for this study in accordance with the national legislation and the institutional requirements.J-ML and L-HS conceived the project. S-MW carried out most of the information collection and data analysis and wrote the manuscript. YH and M-TZ collected part of patients’ clinical information. BT and H-YZ played a key role in maintaining and screening patients. All authors contributed to the article and approved the submitted version.This study was funded by the Yang Fan Project of Shanghai Science and Technology Commission (19YF1430000).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


During the first stage of an animal’s life, various physiological alterations with the concomitant development of different body organs occur. This period is also accompanied by different kinds of stressors, including, but not limited to, the stress of weaning, metabolic change, and peri-puberty changes in reproductive functions. Shiba goats, the main goat breed in Japan, are not commonly used as food animals. However, male Shiba goats largely contribute to reproductive and cardiology research activities, as well as being used for educational purposes for elementary school children. The physiological data regarding laboratory measurements in Shiba goats are lacking, especially at a young age. In this study, we investigated the age-related changes in hematology, plasma mineral concentrations, hormones, and oxidative stress markers during the first five months  in male Shiba goats.From a clinical point of view, the establishment of laboratory variables during the first few months of an animal’s life helps clinicians to make sure they base their medical decisions on laboratory values for the specific breed and age group. The present study aimed to investigate the monthly dynamics in some plasma elements, hematology, reproductive hormones, and oxidative stress marker profiles during the first five months of age  in male Shiba goat’s kids. Sixteen kids were investigated from the first to the fifth month (M1 to M5), and the data were presented as the statistical difference between them. Whole blood and plasma samples were collected monthly for analysis of basal hematology, plasma elements concentration , circulating hormones , and oxidative stress markers . The results showed age-related changes in the observed parameters. The fifth month recorded the lowest level of almost all investigated minerals, except for Cr. Plasma hormone levels revealed age-dependent increases in IGF-1 and testosterone, age-related decreases in T3 and T4, and non-significant changes in cortisol and FSH. Besides, the concentrations of inhibin and LH were significantly higher at M1–M3 compared with M4–M5. Plasma SOD, GPX, and CAT were increased with age. In conclusion, age-related changes and a distinction of age in months was found necessary to interpret the laboratory results, specifically in terms of age in months and the peri-puberty stage in young goats, which are important to follow up the age-specific diseases, reproductive status, and treatment follow-ups in this stage. The goat is one of the oldest domesticated animals and considered the greatest living species adaptable to the hot and dry regions of the world. Japanese Shiba goats are an efficient animal model in studying the pathophysiology of reproduction, cardiovascular, and hemodynamics of ruminants ,2,3,4. RThe health status and performance of an animal are affected by nutrient availability. It has been demonstrated that essential elements that are required for proper functions and homeostasis can have equally beneficial or detrimental effects depending on their balance . They acAssessment of mineral elements and their roles in oxidative stress are essential as a complementary tool in the evaluation of the nutritional and metabolic status of animals. Several studies have demonstrated the link between nutritional status, oxidative stress markers, and changes in organ and tissue contents of some mineral elements at normal and deficient levels concerning puberty onset, impaired reproductive efficiency, reproductive seasonality, growth rate, milk production, and mortality of goats and their kids ,15,16,17Dynamics in hematobiochemical parameters greatly contribute to the performance and health status of small ruminants during the early postnatal and the rapidly growing periods of life . Copioustravelmath.com, accessed on 4 November 2021). Local climatic conditions, including average environmental temperature, relative humidity (RH%), wind-speed, and temperature-humidity index (THI), concerning the sampling times, which correspond to the age of kids in months, were retrieved from the local Japan Meteorological Agency  and are presented in A total of 16 newborn miniature male Shiba goat’s kids obtained from 10 dams  were included in this study. All mothers became pregnant after natural mating without estrous synchronization and recorded no history of disease during pregnancy, abortion, dystocia, or post-parturient diseases. All kids were born in winter (February), and the investigation ended by June 2017. Kids were kept with their mothers under natural photoperiod and temperature in an adequately constructed goat barn belonging to the Laboratory of Veterinary Physiology, Tokyo University of Agriculture, Fuchu campus, Japan. The latitude and longitude of Fuchu city, where the study was conducted, are 35.67° N and 139.48° E, respectively . Licks of mineralized salt and water were provided ad libitum. Chemical analysis of the diet was done using standard methods and is illustrated in Special kid’s cages were provided to mothers and kids during the first month. Kids were allowed their mother’s milk The health status of animals was monitored throughout the study period, which lasted for five successive months, and the clinical data were recorded based on the clinical examination protocol of goats . Neither3EDTA) was used for hematological analysis using the automatic cell counter . To obtain plasma samples, five mL of whole blood in heparinized tubes  was collected and centrifuged at 3000 rpm for 15 min, and the clean non-hemolyzed plasma was collected and kept at −20 °C for analysis of elements, hormones, and oxidative stress markers. The reported hematological parameters include the following measurements: hemoglobin concentration (Hb), red blood cell count (RBC), hematocrit (HCT), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), total white blood cell count (WBC), red blood cell distribution width (RDW), platelet count (PLT), plateletcrit (PCT), mean platelet volume (MPV), and platelet distribution width (PDW). Modified Wright’s stain was used to perform the differential leukocytic count using a digital leukocytic calculator [Jugular venous blood samples were collected at the end of each month for five consecutive months. Two mL of whole blood in an ethylenediaminetetraacetic acid  and macroelements  were determined using the previously described dry ashing protocol . In brieA quality assurance procedure was performed following the previously described protocols ,31,32. TThe difference between wet weight and dry weight, as well as the water content of samples, was calculated. Mathematical calculation of the standard curve, limits of detection , and concentration of elements in PPM were performed using an excel worksheet. Serial dilution of the multi-element standard was used in the calculation of the standard curve of each element through the Trend function in the Microsoft Excel worksheet.To compare our results with element levels from previous studies, the obtained data (determined as PPM on a dry weight basis) were converted into wet weight basis (mg/L) using the water content values through the following equations :(1)WaterThe concentration of plasma hormones was determined in triplicate using a double-antibody radioimmunoassay system using I125-labeled radioligands. All procedures were done in the same laboratory at Tokyo University of Agriculture and Technology, Japan. Concentrations of plasma cortisol were determined using rabbit anti-cortisol (HAC-AA71-02RBP85), cortisol-3-CMOBSA (80-IC20) for iodination, and the reference standard of hydrocortisone (H-4001). Plasma concentrations of testosterone, cortisol, follicle-stimulating hormone (FSH), luteinizing hormone (LH), and immunoreactive inhibin (INH) were measured as described by previous studies ,34,35,362O2 at 520 nm using a redox dye [2 > 0.99), and the color intensity at the specified wavelength was directly proportional to the concentration in the sample. The ∆OD was used to determine the activity of the target in the sample from the generated standard curve.Plasma activity of malondialdehyde (MDA), catalase (CAT), superoxide dismutase (SOD), and glutathione peroxidase (GPX) were measured using a commercial colorimetric test . MDA was measured according to the modified method of Nielsen et al. . MDA foredox dye . The GPXedox dye , which iedox dye , after mp < 0.05). Spearman’s correlation analysis between reported parameters was performed. All analyses were carried out using GraphPad Prism 8.0 . All data were graphed and presented as the median and interquartile range. The numerical values of reported data are presented in All data were categorized depending on the age of kids in months  and used for statistical analysis. The sample size was measured based on the outcomes and calculation performed with G*Power 3.1.9.2 software , assuminp > 0.05). The HCT concentration at M1–M3 was higher than M4 (p = 0.013). RDW at M1–M2 was higher than M4–M5 (p = 0.007). Moreover, the highest PLT count was observed at M3–M4 (p = 0.018); meanwhile, the greatest MPV and PDW were observed at M1. Concerning the differential leukocytic count, a significantly higher neutrophil concentration at M4 compared with M2, and a significant increase in the lymphocytic count at M2 and M3 compared with M1, was observed. Increased monocyte and eosinophil count at M1 compared with the preceding age groups was also observed . p < 0.0001). Plasma Fe concentration showed a regular pattern of reduction with the advance of kid’s age and its lowest level was observed at M5, which was significantly lower than M1 (p = 0.0001). In contrast, Cr revealed a regular pattern of increment with age advance, and a significant increase in Cr level from M3 to M5 compared with M1 was noticed (p < 0.05). The level of Se and Zn showed no significant change with age. Plasma Ca and Mg levels were significantly decreased at M5 compared with earlier months .Data for element concentrations are presented in p = 0.045); however, there were no significant changes from M2 to M5. Concentrations of inhibin and LH were significantly higher at pre-puberty age (M1–M3) compared with M4–M5. In the current study, the concentrations of inhibin in the plasma during the first 5 months of age were also correlated positively with the levels of FSH and negatively with the levels of testosterone. Highly significant decreases in an age-dependent manner were observed in the concentrations of T3 and T4 .p = 0.001). In contrast, the highest concentration of plasma CAT was observed at M2 and M3, which was higher than M1. SOD and MDA showed no significant differences (p > 0.05). p < 0.001). Fe was correlated with Cu and Zn . Cr was significantly correlated with Zn, Se, and Mg levels . Zn showed a positive correlation with Hb and HCT . Correlation results between plasma elements and obtained measurements, as well as the correlation between oxidative stress markers and other variables, are illustrated in p < 0.05). There was a negative trend correlation between Se level and T3 and T4. In addition, the level of Ca, Fe, Cu, and Zn showed a negative trend correlation with plasma cortisol (p = 0.06). Plasma Cu concentration was correlated with plasma T3 and T4 levels  and negatively correlated with FSH  and LH , respectively. GPX showed a reverse relationship with T3 and T4 . Plasma CAT, SOD, and GPX were positively correlated with BW . However, HCT, PDW, PLT, PDW, monocytes, eosinophils, and lymphocytes showed significant differences from the first to the fifth month of life. There is a shortage in age-specific hematobiochemical indices and reference intervals in young goat kids during the investigated stage. Our results are consistent with previous studies ,21,47, wThe MCV and MCHC in goats fluctuated mostly depending upon RBC, Hb, and HCT values. Variation in RBC count could be related to the oxygen-carrying capacity of the blood, which is higher in the first few months of life in goats compared with older ages, and a normal Hb concentration in young goats is maintained by a higher RBC count to compensate for lower MCV ,29. HighThe lymphocytic count was significantly increased from the second month and remained higher than at the first month of age, but the WBC remained unchanged. The lymphocytes percentage in yearling goats was higher than the proceeding age . Along wMulti-elements analysis is necessary to uncover elements interaction, toxicity, and antagonism. ICP-MS is commonly used for this purpose which provide massive information regarding multiple element concentrations through one-shot analysis of acid-digested plasma samples . Our resPlasma Se concentration was almost the same from M1–M3 and showed a slight reduction from M4 to M5. A reduction in Se bioavailability with the advance of age due to the development of ruminal microflora was pointed out , which rReproduction in mammals is orchestrated by complex neuroendocrine networks, starting from early postnatal life to the peri-pubertal age and integrating a wide range of internal and external signals for the release of sex hormones under the control of the hypothalamic-pituitary-gonadal axis (HPG) . The HPGThe oxidative stress markers in the current study revealed age-related changes. Even though these changes are minimal, the reported level of these markers was lowest at M1. In addition, GPX was significantly elevated with age advance, which matched the trend of plasma Se concentration until three months. Notably, in our study, MAD, SOD, and GPX were elevated from the third month, and at the fifth month, MDA, CAT, and SOD reduced, while GPX remained elevated. This may reflect the increased oxidative stress and lipid peroxidation after weaning toward puberty, depicting the importance of oxidative stress during the neonatal and peri-puberty stage. In the biological system, the balance between antioxidants, including but not limited to CAT, SOD, and GPX, and levels of oxidants (such as MDA) are important to scavenge the oxidative metabolites or to repair the consequent damage by oxidative stress in various tissues including hematopoietic and reproductive organs ,77,78. OStudy limitations: The small sample size in this study should be considered; however, the population of goats in Japan is limited, since goats are frequently used for research and educational purposes rather than being a source of human food . Our invThis study reported the values of basic hematology, plasma minerals, reproductive hormones, and oxidative and antioxidative markers in male goats from the first month toward the puberty age, which can support the laboratory interpretation of diseases and help in the evaluation of the efficiency of treatment, specifically in the terms of age in months or peri-pubertal changes.


ASICs are a family of ion channels involved in the perception of different types of stimuli. They are excitatory receptors for extracellular HThe ASICs, in mammals as in fish, control deviations from the physiological values of extracellular pH, and are involved in mechanoreception, nociception, or taste receptions. They are widely expressed in the central and peripheral nervous system. In this review, we summarized the data about the presence and localization of ASICs in different organs of zebrafish that represent one of the most used experimental models for the study of several diseases. In particular, we analyzed the data obtained by immunohistochemical and molecular biology techniques concerning the presence and expression of ASICs in the sensory organs, such as the olfactory rosette, lateral line, inner ear, taste buds, and in the gut and brain of zebrafish. During the last decade, different types of ion channels, including acid-sensing ionic channels (ASICs), calcium-activated potassium (SK1), voltage-gated potassium channels (KV2), and transient receptor potential (TRP) family members, have been identified and investigated in many vertebrate and invertebrate species. Encoded by several genes in all cells type, ion channels show a wide diversity in molecular structure, selectivity to ions, and mechanisms of operation. However, they share the general structural feature of a pore that provides a pathway for charged ions to cross the plasma membrane down their electrochemical gradient. The present chapter covers the intriguing acid-sensing ionic channels in different zebrafish organs, highlighting their structures and function.+ channels gated by extracellular H+ and are widely expressed in the mammalian central and peripheral nerve systems [asic1a subunit is widely expressed in the central and peripheral nervous systems and contributes to synaptic transmission [asic1b and asic3 are expressed in the peripheral nervous system where they would seem to be involved in pain perception [asic2a is extensively expressed in the brain and contributes to synaptic transmission [asic4 transcripts are abundantly distributed in the adult wild-type mouse brain [asic4 mRNA is expressed mainly in the pituitary gland while its expression in other parts of the brain is weak [Acid-sensing ion channels (ASICs) are Na systems . ASICs a systems ,3,4. Six systems . Functio systems ,6,7,8,9  systems ,11. ASIC systems ,13,14. Tsmission ,16. asicrception ,18,19. Trception ,19. asicse brain , but in  is weak . Moreove is weak . +/H+ exchangers and Cl−/HCO3− exchanger beside the electrogenic absorption via epithelial Na+ channels (ENaC) at the mucosal side of the intestinal epithelium, the Na+ uptake in the teleost was channelized by the ASIC family members [+ from environmental FW in exchange for H+ through vacuolar-type H+-ATPase (VHA) [+ uptake was proved [Fish are, phylogenetically, the closest vertebrate group to amphibians and gave rise to mammals. Living in aquatic ecosystems, fish have different life histories and ecological pressures casting their evolution compared to mammals. An examination of the research findings from fish and mammals pointed up the extent of evolutionary conservation of ASICs across these animal taxa. Indeed, the proton-mediated activation of ASICs was highlighted in teleosts, while in a jawless fish and cartilaginous fish, ASICs were not activated by protons . In addi members . ASIC, lse (VHA) . In zebrs proved .+, Na+ selectivity, and the inhibiting effects of amiloride [zasic1.1, zasic2, and zasic4.1 are orthologs of mammalian asic1, asic2, and asic4, respectively, while zasic1.2 and zasic1.3 are paralogs of zasic1.1 and, finally, zasic4.2, is a paralog of zasic4.1  of teleosts and are considered the morpho-functional unit of the LLS. In zebrafish, the neuromasts are grouped into superficial and deep neuromasts. They consist of centrally located sensory cells, supporting and mantle cells ,37,38. Lasic mRNA and protein were detected in the taste buds of adult zebrafish. The anti-ASIC1 antibody identified, by Western blot analysis, a protein band with an estimated molecular weight of about 60 kDa, the anti-ASIC2 antibody detected a single protein band with an estimated molecular weight of about 64 kDa, and also the antibody against ASIC4 detected a single band with an estimated molecular weight of 64kDa [Taste buds are chemosensory organs devoted to evaluating food composition and detecting variations in the environmental chemical composition . They arof 64kDa . Westernof 64kDa ,49. asic2 mRNA and protein were observed. asic2 hybridization was observed in the luminal pole of the nonsensory epithelium, particularly, in the cilia basal bodies and ASIC2 immunoreactivity was found only in the cilia of the nonsensory cells. The immunoreaction for ASIC2 was never observed in the olfactory cells [The olfactory organ consists of numerous lamellae that insert into a midline raphe, thus forming an oval-shaped rosette. In each lamella, there are distributed sensory and nonsensory regions located separately. Three types of receptor cells were identified in the olfactory epithelium: two with cilia or microvilli in the apical border, the third is a different type of sensory cell, the crypt cells with submerged microvilli, and cilia ,51,52. Try cells . The zebrafish retina consists of the ganglion cell layer (GCL), inner plexiform layer (IPL), inner nuclear layer (INL), outer plexiform layer (OPL), and outer nuclear layer (ONL) . Seven dzasic1 mRNA was found in the whole eye of the adults [asic1 in zebrafish retina was analyzed using whole mount in situ hybridization on zebrafish larvae and immunostaining was detected on retina slices [zasic1 transcript was detected in 4–5 day post-hatch zebrafish larvae [asic2 and asic4.2 was affected by light and darkness [In zebrafish, similarly to mammals, e adults . The spea slices . The zash larvae . The immh larvae . In adulh larvae . ASIC2 wh larvae . ASIC4 wdarkness . +, Cl−, and Ca2+ ions, which occurs through the activity of particular gill cells, specialized ionocytes defined as mitochondria-rich cells. Four types of ionocytes called mitochondria-rich cells (MRCs) involved in Na+ uptake have been demonstrated in zebrafish gills: VHA-rich cells (HR cells), NKA-rich cells (NaR cells), cells expressing Na+/Cl− cotransporter (NCC cells), and K+-secreting cells (KS cells) [2+/TGF-b–activated kinase 1 (TAK1)/NF-kB, by means of pharmacological and genetic inhibition experiments [+ uptake demonstrated the role of ASIC4.2 in regulating Na+ uptake in zebrafish exposed to low and ultra-low-sodium media at gill level [The gills of teleosts are composed of gill arches that provide support for the primary lamellae considered the functional unit of the gill, and are interspersed with five gill slits called chambers . Their eS cells) . Furthereriments . The experiments . Furtherll level .As in mammals, the enteric nervous system of adult zebrafish is organized into two plexuses, the myenteric and submucosal. Structurally, the myenteric plexus consists mainly of enteric neurons, while the submucous plexus has few neurons and numerous nerve fibers . Specifizasic genes in zebrafish neurons was demonstrated through the in situ hybridization technique [zasic genes were different. Particularly, zasic1.1 was demonstrated within 30 h postfertilization (hpf) at anterior and posterior lateral line ganglia and optic sensory neurons and at 48 hpf, also in the trigeminal ganglia [zasic1.2 was evident, although with weak expression at 48 hpf, in the ventral thalamus, ventral midbrain, ventral cerebellum and in the dorsal thalamus, hypothalamus and telencephalon along the anterior commissure. zasic1. 2 at 48 hpf was present in the dorsal midbrain (dMb) and olfactory bulb, whereas from 96 hpf it was also expressed in the tectum. zasic1.3 was expressed at 30 hpf in the ganglia of the lateral line while between 30 and 72 hpf in the telencephalon it was expressed along the anterior commissure tract, in the ventral thalamus, ventral midbrain, and ventral cerebellum [zasic2 was observed at 30 hpf along the anterior commissure tract and at 48 hpf, also in the preoptic area, ventral thalamus, and ventral midbrain. At 72 and 96 hpf, it was present in the whole brain except for the dorsal forebrain and was also expressed in retinal ganglion cells. zasic4.1 at 48 hpf showed a similar pattern to that of zasic1.2. zasic4.1 was expressed in the dorsal midbrain and retinal ganglion cells. zasic4.2 as early as 24 and 30 hpf was expressed along the anterior commissure tract and in cells along the commissure tract. At 48 hpf, it was observable in the preoptic zone, posterior hypothalamus, ventral midbrain, cerebellum, and retinal ganglion cells [ASICs are widely expressed in the nervous system of zebrafish embryos and larvae. The expression of six echnique . In geneasic2 gene, behavioral responses to acidic taste stimuli remained unaffected [asics between mammals and zebrafish. Indeed, while asic4 was demonstrated in zebrafish, it was always absent in mouse and rat, asic1 and asic3 have always been absent in zebrafish while they were observed as RNAs in mice and rats. asic2 was observed in zebrafish and rats but not in mice [asic2 and asic4 in taste buds would also suggest in zebrafish a role as a receptor for acid taste or an involvement of ASICs in taste cells, taste or more functions in zebrafish.The presence of ASICs has been demonstrated in different organs of zebrafish, such as sensory organs, gills, gut, and brain. The expression of ASICs in the hair cells of LLS neuromasts and the neurosensory cells of the inner ear could support the hypothesis of an involvement in the transduction of mechanical stimuli from the aquatic environment similar to the role they play in mammalian inner ear cells. It is known that, functionally, ASICs control moderate deviations from the physiological values of extracellular pH. In the rat and mouse, ASIC1a and ASIC2a immunoreactivity was detected in small vestibular ganglion neurons and afferent fibers of the macula utricle and crista stroma, whereas ASIC2b, ASIC3, and ASIC4 were observed in vestibular ganglion neurons. It has also been demonstrated in these species that the acidification of the extracellular pH generates action potentials in vestibular neurons, confirming a function of ASICs in their excitability ,63,64. Raffected ,66,67. T in mice . These dasic1a, asic2, and asic3 in the mammalian retina has been demonstrated [asic1 is the most represented and is widely expressed in the retina and involved in retinal activity, particularly in cone function [asic1a, functionally expressed in retinal pigment epithelium (RPE) cells, might play an important role in the neuroprotection of RPE cells from oxidative stress [asics in zebrafish gills, at mRNA and protein levels, was demonstrated, and their involvement in Na+ regulation of adult zebrafish acclimated to low-Na+ medium was validated using pharmacological blockade of Na+ uptake. Moreover, the presence of alternative transport mechanisms was also hypothesized. The differences found between zebrafish larvae and adults in response to specific inhibitors of ASICs, could be explained by a difference in the ionic compositions and pH used in the two different studies or could suggest the use of different Na+ uptake mechanisms in the two stages examined, as previously demonstrated in rainbow trout [asic3 have been observed in gastrointestinal disorders associated with inflammation, while disruption of the asic2 gene results in altered emptying from the gastrointestinal tract [+ physiology, learning/memory, fear conditioning, and in pathological conditions such as brain ischemia, multiple sclerosis, epileptic seizures, depression-related behavior, anxiety disorders and malignant glioma [zasic genes were mainly expressed in the nervous system of embryos and larva, although the expression pattern of the six zasic genes, compared to mammals, was limited primarily to neurons and perhaps some glial cells. However, further studies are also needed in the adult zebrafish brain to demonstrate the exact role of ASICs in this experimental model. Therefore, such evidence on the involvement of these ion channels in both physiological and pathological processes in the central nervous system could suggest them as novel and effective therapeutic interventions for CNS diseases.The localization of ASIC2 in the nonsensory epithelium of the olfactory rosette suggests that it is not involved in olfaction. However, the presence of immunoreactivity for ASIC2 in the basal bodies of the cilia of nonsensory cells, notoriously involved in the perception and transduction of mechanical and chemical stimuli, would suggest a role for this ion channel in other types of stimuli from the aquatic environment, pH changes, or movement of water. The presence of nstrated ,70. Amonfunction . In mammfunction ,70. As pfunction ,73. Morefunction ,75. Finaow trout . Numerouow trout . Indeed,ow trout ,16 in thow trout ,16,77,78al tract . The rolal tract ,16. On tt glioma . In zebrIn zebrafish the pattern of the distribution of ASICs in different organs and tissues suggests their involvement in multiple physiological functions, including mechanosensation, hearing, temperature sensing, taste, vision, intestinal physiology and brain activity as previously demonstrated in mammals ,81,82. F


The year 2021 is the 100th anniversary of the confirmation of the neurotransmission phenomenon by Otto Loewi. Over the course of the hundred years, about 100 neurotransmitters belonging to many chemical groups have been discovered. In order to celebrate the 100th anniversary of the confirmation of neurotransmitters, we present an overview of the first two endogenous gaseous transmitters i.e., nitric oxide, and carbon monoxide, which are often termed as gasotransmitters. The year 2021 is the 100th anniversary of the confirmation of the neurotransmission phenomenon by Otto Loewi .Neurotransmitters are chemicals that enable communication, i.e., the flow of nerve impulses between nerve cells, or between nerve cells and muscles and glands. Recently, one can distinguish excitatory and inhibitory mediators, which are endo– or exogenous compounds that control the function of the entire organism. From a chemical point of view, neurotransmitters belong to many different structural groups including amino acids (such as glycine), peptides , monoamines , purine derivatives (such as adenosine), gases , and acetylcholine. From a medical point of view, disturbances in the concentration of neurotransmitters in the body result in the occurrence of mental disorders and diseases , which contribute to the occurrence of dementia (including Alzheimer’s disease) and others. For this reason, they are used in medicine , but they can also be a serious problem in non-medical use .NO and CO are the first two endogenous gaseous transmitters identified and are often termed as gasotransmitters . NO was NO is an important cellular signaling molecule that participates in diverse physiological functions in mammals, including vasodilation, smooth muscle relaxation, neurotransmission ,7, and tThe second gas transmitter, CO, is a product of heme catabolism, and is usually regarded as a toxic species that disrupts cellular respiration. However, recently, CO has been found to be an important signaling molecule and protector of tissues against injuries induced by several types of stress .The purpose of this review is focused on description of the gasotransmitters from various perspectives. In the paper, the basic chemical and biological properties of CO and NO are addressed to provide a proper foundation for further discussion. Selected pharmaceutical data are presented later in the text.Both CO and NO are diatomic gaseous oxides of carbon or nitrogen, respectively. CO is a colorless, odorless, and tasteless flammable gas that is slightly less dense than air . NO is oCO is a stable, naturally occurring compound with carbon in a 2+ oxidation state. The molecule has ten valence electrons, distributed among three bonds and one lone pair on each atom  21]. NO. NO21]. −, NO+ and with N2. Occupied nonbonding orbitals on the carbon and on the oxygen point away from the molecule and can interact with transition metals (M) via covalent  bonds ..33].2 along with cofactors such as calmodulin and tetrahydrobiopterin (BH4) . In. In33]. Chemical Entities of Biological Interest under the CHEBI entry: 17,245 for CO  16,480 fCO and NO as chemical compounds were discovered, similar to oxygen, in 1770 by Joseph Priestley . Researc−6 M) generated by NOS2 reacts with various reactive oxygen species to produce reactive nitrogen species, which exert indirect effects, such oxidation, nitration, and nitrosation. At low concentrations ranging from 10−12 to 10−9 [NO is a small and diffusible free radical that acts as a secondary messenger throughout the human body . NO is n to 10−9 , circula to 10−9 . For exa to 10−9 . Therefo to 10−9 .2 carrying capacity of blood and disrupts the release of O2 from Hb for its use in tissues.The toxicity of CO has been known since ancient Greek and Roman times. The mechanism of CO toxicity is not complicated since it does not create any toxins in the body. However, it does not poison the body with complex chemical compounds; CO works simply and effectively, displacing oxygen from the organs and blocking its transport. . Therefo2 storage in muscle cells by binding to and displacing O2 from myoglobin. Though all human tissues are vulnerable to carbon monoxide-induced hypoxic injury, those with the highest O2 demand are especially vulnerable, including the brain and heart. Due to this the heme proteins are among the most important cellular targets of CO. Another potential target of CO is the cytochrome P450 family of enzymes. P450s are involved in oxidative metabolism of drugs and xenobiotics. Physiological levels of CO are usually too low to inhibit P450s, however exogenous exposure to CO can lead to inhibition of P450s isoenzymes such as CYP2D6, CYP3A and CYP2C affecting drug metabolism and vascular tone [Through similar mechanisms, CO diminishes the Olar tone ,78. Ferrlar tone .CO can also bind to and activate sGC similarly but less effectively than NO and induces vasorelaxation in rat-tail arteries  and it a3 per day [Some CO is also produced in normal animal metabolism in low quantities  and  per day . The phy per day . CO admi per day . Notably per day .2 molecule [2 or even CO. Hb scavenges NO through the high-affinity ferrous binding sites on the heme moiety  identifier (NCT number): NCT03616002 [Carbon monoxide, with cytoprotective and homeostatic properties , recentl03616002 ; NCT030603616002 ; NCT025303616002 ; NCT001203616002 ; NCT046103616002 ; NCT024203616002 . The tre03616002 ,112.1/2 < 1 min), (iii) the reactivity of metals with biological substances , (iv) central metal toxicity [3BCO2), boranocarbamates (H3BCO), hexamethylenetetramine carboxyborane (HMTA-CB or HMTA-BH2COOH), methylene chloride (MC) [3 and one equivalent of hydroxides upon CO release [An alternative to the oral or injection administration of CO is CORM-stable solid complexes of CO . Anothertoxicity ,126,127.ide (MC) ,129. HowCORMs have shown a variety of activities, such as vasodilatory, anti-inflammatory, antiapoptotic, anti-ischemic, anticancer, and cardioprotective activities, and have also been proven to regulate mitochondrial activity, respiration experimental colitis, chemically induced liver injury, and ischemia reperfusion kidney injury ,134,135.Many studies have demonstrated that CO inhalation at low doses (<250 ppm) offers protection against inflammation and ischemic injury in the heart, liver, and kidney ,137 NCT0Direct NO gas administration can be used in clinical settings since it can enhance arterial oxygenation in patients with acute respiratory distress syndrome . NO gas Drugs that generate NO are known as nitrovasodilators, e.g., organic nitrates (RONO) including nitroglycerin   amyl nit2+ complexed with NO and five CN− ions. Acting as a prodrug, SNP reacts with sulfhydryl groups on erythrocytes  to produce NO [A water-soluble sodium salt, SNP, is comprised of Feoduce NO . Upon bioduce NO . Among coduce NO . This maoduce NO . NO has oduce NO . This isoduce NO . When the body cannot generate sufficient amounts of NO to maintain homeostatic functions, the administration of exogenous NO is a practical method of supplementation, e.g., in patients affected by osteoporosis . As discNO-NSAID consists of a traditional nonsteroidal anti-inflammatory drug (NSAID) to which a NO-releasing moiety is covalently attached . NO-NASIThe importance of NO was discovered in the 1970s, and several years later (in 1987), the physiological role of CO was also proven, leading to a much better understanding of both biochemistry and the development of NO/CO-based pharmaceuticals. Their medical applications are developing rapidly, and it is likely that new drugs based on NO/CO will be used in medical therapies in the near future. However, it seems unquestionable that currently, NO-NSAIDs and CORMs are groups of compounds capable of transporting and releasing controlled NO/CO in cell systems, which constitute a reliable tool for studying the pharmacological effects of these gases or identifying their mechanisms of action. The results of the biology and chemistry of NO-NSAIDs and CORM obtained so far indicate that these compounds are good candidates for the introduction of drugs that release these gases.2S) small molecules are accepted as gaseous neurotransmitters. The research continues to expand, including in the mediator group more gaseous compounds, such as acetaldehyde (CH3CHO), methane (CH4), sulfur dioxide (SO2), dinitrogen oxide (N2O), ammonia (NH3) and hydrogen (H2) [There has been significant continuous progress in the area of gasotransmitters since Wang  first ingen (H2) ,100,177.