Bacillus maritimus. A Physicochemical analysis of S16 exopolysaccharide (BMEPS) was estimated, which pointed to the presence of uronic acid and sulfate . HPLC analysis indicated that mannuronic acid, glucuronic acid, glucose, and mannose are presented in a molar ratio of 0.8:1.0:2.8:2.3, respectively. Furthermore, FT-IR revealed an abundance of β-configurations. The GPC estimated the average molecular weight (Mw) as 4.31 × 104 g/mol. BMEPS inhibited AChE , BChE , and tyrosinase . It also demonstrated a selective anti-inflammatory action against COX-2 rather than COX-1. Moreover, BMEPS exhibited antioxidant capabilities as free radical and oxygen reactive species (ROS) scavenger, metal chelator, reductant agent, and lipid peroxidation suppressor. These activities are due to the distinct chemical composition. The findings of this study indicate that BMEPS could be considered as promising anti-disease Alzheimer's (AD) material in an in-vitro model, which qualifies it for advanced in-vivo studies in the discovery of alternative Alzheimer’s treatment.Four bacterial isolates were obtained from marine sediments collected from Sahl Hashish, Hurghada Red Sea, Egypt. This study was designed to search for promising anti-Alzheimer natural polysaccharide; therefore, four isolates were screened for exopolysaccharides (EPSs) production and acetylcholinesterase inhibition. The isolate S16 provided the highest EPS yield (7.51 g/L) and acetylcholinesterase inhibition. It was identified morphologically and genetically using 16S rRNA gene sequence analysis as  Polysaccharides are common biological macromolecules that take part in a wide range of physiological functions in humans. It performs a wide range of biological functions, including controlling immunological function, blood pressure, blood sugar, and circulation of blood1. Industrial polysaccharides are frequently derived from plants, animals, algae, and microbes. Microorganisms secrete soluble or insoluble polymers called EPSs2. Furthermore, microorganisms are considered highly reproducible structures among all polysaccharide providers and are closely regulated, whereas exopolysaccharides (EPS) structures produced from plant and animal sources are influenced by climatic, environmental, and feed circumstances. Mainly, marine environments constitute a sizable and distinctive setting where various bacterial populations are required for essential functions for the survival of the planet's ecology. On the other hand, EPSs are frequently utilized as viscosifying, stabilizing, gelling, or emulsifying agents in the food industry because of their distinctive physical and rheological qualities2. Microbial polysaccharides are incorporated into new targets, such as bioflocculants, bio absorbents, heavy metal removal, and drug delivery agents3. Additionally, antitumor, antiviral, immunostimulatory, and anti-inflammatory actions are among the biological effects of EPSs4. Among microorganisms, Bacillus sap. strains produce many types of EPS such as levan, β--glucan5, acidic EPS from marine B. amyloliquefaciens 3MS 20176, and acidic EPS from Bacillus sp. NRC57. Some Bacillus EPSs demonstrated exceptional emulsifying, flocculating, heavy metal removal, or medicinal properties5.The most prevalent organic substance in the world is polysaccharide8. Currently, about ≃ 50 million people are diagnosed with AD, and by 2050, this figure is anticipated to exceed 130 million9. The abnormalities associated with several important physiological operations are to blame for the multidimensional toxicity, which also includes cholinergic toxicity, amyloid burden, metal ion toxicity, tau toxicity, biomolecular damage, oxidative stress, immune outrage, neurovascular toxicity, calcium ion dyshomeostasis, lymphatic dysfunction, mitochondrial dysfunctions, α-synuclein mediated toxicity, synaptic malfunctions, membrane toxicity, apoptosis malfunctions, deterioration in telomerase activity, aberrant post-translational modifications, microbial imbalance and infection, hyperglycemia, endoplasmic reticulum stress, hypercholesterolemia, autophagy malfunctions, genetic risk, and insulin resistance as well as diabetes10. In the CNS , under normal conditions, metal ions  play the role of cofactors for enzymes and deliver mitochondrial, and neuronal functions11. On the contrary, ZnII, CuII, and FeII coordinate with Aβ and speed up the amyloid accumulation and formation of metal-dependent plaques. Aβ-Cu and Aβ-Fe complexes induce the production of excess reactive intermediate species (RIS). RIS are crucial components to induce oxidative stress and neuro-inflammation8. Therefore, discovering metal chelators materials is a promising therapeutic approach. Overproduction of RIS  encourages critical oxidative stress that injures lipids and proteins, leading to neuronal death. AD brain tissues considerably suffer from excessive RIS levels8. The redox-active metal ions (CuII and FeIII) capture Aβ peptide, stabilize oligomeric species, and work as a depot to produce excessive RIS12. Therefore, oxidative stress is the base of AD advancement and a potential target in AD treatments10. The neuronal plasma membrane contains a high amount of polyunsaturated fatty acids that are susceptible to peroxidation by RIS, which induces neurotoxic components like 4-hydroxynonenal13. In AD brain tissue, oxidative stress-mediated cholesterol microdomains inhibit the antioxidant vitamin E in the lipid membrane. Based on the previous explanation, oxidative stress is the key step in cellular dysfunctions that are related to AD. In parallel, many polysaccharides obtained from organisms have been proven to have antioxidant capacities, including i) metal ion capture  capture; ii) inhibition of ROS and RNS production; iii) protection of lipids from peroxidation; and iv) neutralization of free radicals. For example, EPS from Adansonia digitata14, EPS from Novel Bacillus sp. M315, EPS from paenibacillus lactes NRC116, EPS from B. amyloliquefaciens 3MS 20176, and EPS from Bacillus sp. NRC57. On the other hand, cholinergic neurons play a crucial role in a variety of cognitive processes, including memory, attention, response, and the processing of sensory information. The impairment of cholinergic neurons is associated with cholinergic toxicity. Thus, improvement of cholinergic neurotransmission remains the main approach in the symptomatic treatment of cognitive and behavioral disorders in the early stages of AD10. When acetylcholinesterase (AChE) is present in the synaptic cleft, acetylcholine hydrolyzes into choline and acetic acid. Choline acetyltransferase (ChAT) and AChE enzymes control ACh synthesis and breakdown10. The defect in ChAT activity or hyperactivity of AChE in AD patients prompts a considered reduction in ACh content at the synaptic cleft in the cortex, hippocampus, and amygdala. Thus, repairing cholinergic neuronal malformations is a target for improving cognitive disorders in AD patients. Accordingly, AChE inhibitors prevent brain ACh hydrolysis, which increases the concentrations of brain ACh and improves the deficiency of brain cholinergic neurotransmission. Many polysaccharides from organisms have an AChE inhibitory effect, such as EPS from Achromobacter piechaudii NRC217, EPSs from Isochrysis galbana and Nannochloropsis oculate18, and polysaccharides from mushrooms19.AD is a chronic neurodegeneration-related disorder20. Tyrosinase inhibitors are medications that can suppress tyrosinase activity. Several polysaccharides of natural origin showed anti-tyrosinase activity, such as the fruit's pericarp polysaccharides, longan21, Usnea longissima polysaccharides22.On the other hand, tyrosinase catalysis is the conversion of the amino acid L-tyrosine into the compound L-3,4-dihydroxyphenyl amine (L-DOPA). The crucial neurotransmitters norepinephrine and epinephrine are precursors to the key neurotransmitters L-DOPA and dopamine, respectively. Tyrosinase is activated by 14-3-3 proteins through phosphorylation-dependent binding. Tyrosinase and neurodegenerative disorders like Alzheimer's, Parkinson's, and Huntington's diseases are indirectly related due to the 14-3-3 proteins. Also, tyrosinase can injure neurons by producing dopamine quinones, which can oxidize dopamine's catechol ring to produce the extremely reactive compound dopamine quinone. Oxidation of dopamine inhibits dopamine locomotion, glutamate locomotion, and mitochondrial respirationThe current study was designed to search for a new bioactive exo-polysaccharide (EPS) from marine sources accompanied by its isolation, identification, and characterization, followed by an in-vitro route examination of this polysaccharide in treating Alzheimer’s risk factors: oxidative stress, inflammation progression, neurotransmitter degradation, and accumulation of amyloid-β.Four isolates of marine bacteria, S4, S7, S13, and S16, were evaluated, and they were found to have a relatively high yield of EPSs 6.99, 8.66, 7.22, and 7.51 g/L, respectively  was subjected to morphological, physiological, and biochemical characteristics. The colony and morphological characteristics of isolated S16, three days of 37 °C incubation followed the streaking of the pure cultures onto plates. Moreover, the strain formed glistening, slimy, mucoid colonies on agar plates and was capable of producing lengthy strands when expanded using an inoculation loop, exhibiting the typical properties of EPS-producing phenotypes. S16 was identified based on morphological and cultural features, as well as physiological and biochemical studies. Based on the S16 rRNA sequences of strain S16, a phylogenetic tree was created. Bacterial isolate (S16) was primarily identified as gram-positive, endospore-forming, smooth texture, rod shape  of the BMEPS to be 4.31 × 104 g/mol and the average molecular number (Mn) 3.87 × 104 g/mol. The disparity, which is defined as the ratio of Mw per Mn was equal to 1.1.The high level of EPS was 7.51 g/L when fermented at 37 °C for 3 days, as determined by the phenol–H3+ form into the reduced Fe2+ion in the ferricyanide compound. Thereby, the yellow color of Fe3+ transforms into numerous green hues, and blue Fe2+ is formed based on the reducing power of antioxidant samples. BMEPS showed a weak Fe3+ reductive effect, compared to reference materials  compared to LAA (62.99% ± 1.01 to 98.19% ± 1.82) or BHT (52.41% ± 1.59 to 99.09% ± 0.90) at the same concentrations. It could be concluded that IC50 of BMEPS (827.46 μg/ml) was greater than LAA or BHT IC50 values; 26.36 and 68.88 μg/ml, respectively.BMEPS showed moderate Feals Fig. B. It pre2−/ H2O2), it will be oxidized and form hydroperoxides. In the thiocyanate system, ferrous ion is oxidized by linoleate radicals to create ferric ion, which is then measured spectrophotometrically. The antioxidant components restrained the oxidation of ferrous ions leading to prevent the linoleic radical formation in the system24.Lipid peroxidation is a set of chain reactions involving free radicals that can cause a variety of health harm. The unsaturated fatty acids are attacked by ROS, and initiated radical peroxidation chain reactions. When Linoleic acid is incubated with an initiator  in comparison with LAA (64.73% ± 2.20) or BHT (60.31% ± 1.69) at the same concentration. Elevating BMEPS concentration to 1000 µg/ml elevated the inhibition of linoleic acid oxidation to 60.22% ± 1.78, compared to LAA (100% ± 0.0) or BHT (100% ± 0.0) at the same concentrations , compared to LAA (75.17% ± 1.30) or BHT 80.46% ± 2.52) at the same concentrations  compared to LAA (66.32% ± 2.68) or BHT (62.18% ± 1.81). In accordance, increasing concentration to 1000 µg/ml scavenged more amount of ABTS+ cation radical (99.52% ± 0.51) compared to LAA (100.00% ± 0.00) or BHT (100.00% ± 0.00). Insignificant differences were recorded among BMEPS, LAA, and BHT at 750 and 1000 µg/ml. IC50 of BMEPs was 333.22 μg/ml whereas LAA or BHT presented 18.61 and 33.44 μg/ml, respectively.BMEPS showed a potent ABTSner Fig. B. The lo2), anionic oxygen gas (O2), is a main cause of oxidative stress. O2·−is generated in phenazine methosulphate (PMS)-nicotinamide adenine dinucleotide (NADH) systems by oxidation of NADH and assayed by the reduction of nitro blue tetrazolium (NBT). In this method, O2 reduces the yellow dye NBT2+ to produce the blue formazan which is measured spectrophotometrically at 560 nm. Generally, antioxidants can inhibit blue NBT formation. The decrease in absorbance indicates the consumption of superoxide anion in the reaction mixture meaning the presence of antioxidant material.Superoxide  and BHT (73.22 ± 2.78 to 98.11% ± 1.89) at the same concentrations. The greatest concentration of BMEPS, LAA, and BHT showed O2 scavenging ability was nearly equal. BMEPS concentration to inhibit 50% of generated O2 radicals was 19.96 µg/ml, in comparison with LAA and BHT; 22.35 and 20.551 µg/ml, respectively.BMEPS captured Oals Fig. C. In rea.) released from SNP has a powerful NO+ which can change the structure and functionality of numerous cellular components. When NO interacts with superoxide to generate the peroxy nitrite anion (. ONOO-), its toxicity rises which is a potentially powerful oxidant that can break down to produce OH and NO226.Nitric oxide  or BHT (65.99% ± 2.00 and 95.88% ± 1.89). IC2O2 released from leuco-dichlorofluorescein (1-DCF) oxidation in the presence of phenol. Polysaccharide anti-inflammatory effectiveness was examined in comparison to celecoxib as an anti-inflammatory drug with more selectivity towards COX-2 than COX-1. The repressive effect of BMEPs on COX-1 was less than its effect on COX-2  compared to inhibition with celecoxib; 2.36% ± 0.77 and 17.24% ± 1.51 at the same concentrations. The IC50 of BMEPS (6367.38 μg/ mL) was higher than that of celecoxib (3820.64 μg/ mL) which means this polysaccharide is safer than celecoxib on physiological system.The BMEPS anti-inflammatory activity was measured by calculating the inhibition percentage of HX-2 Fig. A. COX-1 COX-2 activity by about 20.74% ± 1.00, 30.34% ± 1.52, 49.67% ± 1.63, 62.88% ± 1.85, and 78.09% ± 2.00 at 100, 250, 500, 750, and 1000 μg/ mL, correspondingly, compared to celecoxib .BMEPS had a moderate 50 values of BMEPS at all incubation periods  were less than kojic acid  at the same durations which means the tested polysaccharide is more promising as an anti-tyrosinase inhibitor than kojic acid.Tyrosinase is an oxidoreductase that contains copper. It speeds up the process of ortho-hydroxylation of monophenols and aerobic oxidation of catechol. The activity of the enzyme was measured by spectrophotometrically measuring the oxidation of 3, 4-dihydroxyphenylalanine (L. DOPA) to the red dopachrome. The anti-tyrosinase activity of BMEPs was determined during three incubation periods , and compared to kojic acid as a standard inhibitor. BMEPS showed potent anti-tyrosinase character compared to kojic acid, in concentration and time-dependent action Fig. . Anti-ty27. Four isolates of marine bacteria S4, S7, S13, and S16 were assessed for Eps production and the isolate S16 is found to have a relatively highest EPSs yield and acetylcholinesterase inhibitor efficacy. The promising bacterial isolate (S16) belongs to the gamma subdivision of the Proteobacteria phylum and is closely related to Bacillus maritimus MSM1.Marine bacteria of different genera are a promising source for the production of several bioactive substances with a variety of biotechnological uses. Many bacterial compounds known as EPSs have a wide range of applications and outstanding physiological activities such as crystallization, emulsification, and antioxidant agentsMw) of the BMEPS as 4.31 × 104 g/mol and the average molecular number (Mn) as 3.87 × 104 g/mol. The disparity, which is defined as the ratio of Mw per Mn was equal to 1.1. Pentoses, hexoses, or uronic acids are typically structured in repeating units and make up the majority of the EPSs produced by marine bacteria. Due to the fact that location in the gut is determined by chemical composition, the EPSs' composition of glucose and mannose creates an unmixed water layer in the gut, which reduces the absorption of carbohydrates and lipids. Soluble fibers are therefore somewhat effective in the management of diabetes since they can reduce the postprandial rise in blood sugar28. Most EPSs produced by marine bacteria have OH- and COO- groups, which give them a temporary negative charge and acidic characteristics. The majority of marine EPSs are generally linear, have a range of lengths, and have a mean molecular mass of 1 × 105 to 3 × 105 g/mol29. EPS-secreting deep sea hydrothermal bacteria have been reported in recent years to have a high molecular weight, up to 1 × 106 g/mol, frequently acidic, and include uronic acid at a concentration of ~ 30%.The chemical composition of BMEPS was evaluated. Monosaccharide investigation was performed using HPLC and compared to standards. BMEPS was made up of mannuronic acid, glucouronic acid, glucose, and mannose in a molar ratio of 0.8:1.0:2.8:2.3, respectively. The GPC analysis estimated the average molecular weight . It is currently unknown what causes the pathological alterations in Alzheimer's disease . Several hypotheses were proposed as a cause for AD but two of them are believed to be the main cause: some believe that an impairment in the cholinergic function is a critical risk factor for AD, while others suggest that alteration in amyloid β-protein production and processing is the main initiating factor. However, no widely accepted theory for the pathogenesis of AD exists at this time31.A specific type of brain disease is Alzheimer's disease. It is brought on by harm to the brain's neurons, which are nerve cells. The neurons in the brain are necessary for all human activities, including walking, talking, and thinking. The first neurons to suffer damage in Alzheimer's are those in regions of the brain involved in memory, language, and thought. As a result, memory, language, and thinking issues are frequently the first symptoms. Despite the fact that these symptoms are novel to the individual affected, the brain alterations that cause them are believed to have begun 20 years or more before to symptoms32. Recent research has revealed that natural substances have a neuroprotective effect, which they have long been utilized as therapeutic agents for treating a variety of degenerative disorders. Natural substances have been shown to offer therapeutic potential for AD in both in vitro and in vivo studies, allowing some of them to move into the clinical trial stages. The first natural substance to enter clinical trials for AD was nicotine. Subsequently, additional substances, including as vitamins C, E, and D, attracted increasing interest due to their protective effects against neuroinflammation and oxidative damage. Recently, bryostatin, a macrolide lactone extract from bryozoan Bugula neritina, has been evaluated and showed the ability to induce α-secretase activity, reduce Aβ production, and enhance the learning and memory in an AD mice model33. Other natural compounds used in folk medicine  have shown promising results in the treatment of Alzheimer's disease by working on several mechanisms34.Around 24 million people have been reported to have Alzheimer's disease worldwide as of right now, and by 2050, experts predict that number will have quadrupled. Even though AD is an issue of public health, only two kinds of medications are currently approved to treat AD: cholinesterase enzyme inhibitors , and N-methyl D-aspartate (NMDA) antagonistsB. maritimus MSM1 was evaluated. In general, BMEPS showed good antioxidant features where it; (i) neutralized free radicals (DPPH. and ABTS+), (ii) scavenged RS (O2− and NO), (iii) chelated Fe+2 ions, and (iv) inhibited lipid peroxidation, in comparison with two reference materials LAA and BHT. In accordance, the antioxidant properties of natural polysaccharides were documented in several studies. Our study was supported by Ibrahim et al.14 findings on EPSs from Adansonia digitate, El-Newary et al.6 acidic EPS from marine B. amyloliquefaciens 3MS 2017, acidic EPS produced from Bacillus sp. NRC57.In the present study, the antioxidant capacities of BMEPS from 4), sulfate content and binding position, molecular weight linkages, and molecular weight35. The antioxidant characteristics of current polysaccharide (PS) could be explained depending on many axes as follows: First axis, the monosaccharides content of PS is associated with antioxidant characteristics, particularly, rhamnose and mannose36. The second axis, glycosyl linkages type plays an essential function in the antioxidant properties, specifically, arabinose (1– 4) and mannose (1–2) of the side chain that is strongly linked to the reductive ability, meanwhile glucose (1–6) and arabinose (1– 4) are closely correlated with DPPH radical scavenging activity37. The third axis, uronic acids in acidic PS make them potent antioxidants agents38 and uronic acids exerted promising free radical scavenging and reductive capacity in the following order; poly-galactouronic acid > glucuronic acid > galactouronic acid38. The fourth axis, molecular weight has a strong effect on antioxidant capacities, where low molecular weight polysaccharide is superior to molecules with high masses. PS with low molecular weight has potent reducing power to neutralize the free radicals. Xing et al.39 demonstrated that chitosan that has a low molecular weight (9 kDa) exhibited O2− scavenger efficacy better than chitosan with a high molecular weight (760 kDa). Additionally, Rice polysaccharides, with low molecular weights, have promising reducing power, metal chelation, and free radicals scavenging abilities. The fifth axis is attached function groups, sulfated low molecular weight polysaccharides such as Ulva pertusa polysaccharides have better antioxidant abilities than the sulfated polysaccharide with high molecular weight PS40. Finally, sulfate groups increased the ability of sulfated polysaccharides as free radicals’ scavengers, metal chelators, and inhibition of lipid peroxidation formation38. High sulfate-content polysaccharides were more effective than the low ones41. Scavenging free radicals of materials is via electron transfer or hydrogen donates from the antioxidant materials to this radical to be a stable form24. The PS can donate hydrogen atoms because hydrogen bonds have low dissociation energies. Polysaccharide hydrogen bonds are weak because of the position of the sulfur in the sulfated polysaccharides. Also, sulfate groups of sulfated PS trapped free radicals electrostatically42. The sixth axis is pH, the antioxidant abilities of acidic low molecular weight polysaccharides are better than acidic high molecular weight polysaccharides43. According to the structure–activity relationship mentioned before, it could be suggested that the antioxidant properties of BMEPS could be associated with its low molecular weight (4.31 × 104 g/mol), uronic acids (24.7%), sulfate (18.3%), monosaccharaides composition; mannuronic acid, glucouronic acid, glucose and mannose in a molar ratio of 0.8:1.0:2.8:2.3, respectively.BMEPS antioxidant properties could be discussed depending on its chemical composition. The natural polysaccharides' antioxidant activity is affected by several factors including structural characteristics, the number of active groups  Nitric Oxide (NO) and prostaglandin synthesis, (ii) NF kappa B expression, (iii) reactive oxygen species (ROS), (iv) migration of leukocytes, and finally (v) the pro-inflammatory cytokines i.e. TNF, IL6, and IL1AChE or BChE. Several polysaccharides have AChE inhibitory action. For example, proteoglucan (PGM) from mushroom45, EPSs from the L. delbrueckii subsp. bulgaricus B3 and L. plantarum GD246, EPSs produced from brown macroalgae Ecklonia radiata47. In agreement, Park et al.48 demonstrated that sulfated polysaccharide produced from Ecklonia cava exhibited competitive and non-competitive suppressor effects on acetylcholinesterase in PC12 cells induced by H2O2. In addition, sulfated polysaccharides produced from marine algae such as E. maxima, G. pristoides, U. lactuca, U. rigida, and G. gracilis, significantly reduced the acetyl cholinesterase activity in treated cells with Zn (50 μM) alone50.Additionally, BMEPS appeared anti-cholinesterase activity against either AChE is significantly affected by metal toxicity as Ze and Cu. In an experiment on fish Leporinus obtusidens (piava), exposure to zinc and copper significantly activated AChE51. Also, exposure of zebrafish to ZnCl2 higher than 1 ppm produced Alzheimer’s disease (AD)-like syndrome including suppression of ACh, decrease locomotor behavior, and impairment in short-term memory due to activation on AChE. Moreover, ZnCl2 induces amyloid-β and phosphorylated Tau protein levels in brains52. Therefore, capturing excess of these metals from media could inhibit AChE, and increases ACh content in the synaptic area. In this study, BMEPS exhibited metal chelating ability, which could be the reason for it being anticholinergic in in-vivo applications. On the other hand, sulfated compounds can react to the anionic site of AChE and reduce its activity48 which means that the used sulphated polysaccharide in this study may in part show its inhibitory action on AChE in this way.On the other hand, 53, (iii) Copper chelation that can regulate tyrosinase activity due to the copper structure of tyrosinase as kojic acid54 and (iv) H2O2 scavenging materials, where H2O2 activated tyrosinase and served as the second substrate55. According to the previously presented explanation, we could conclude that the anti-tyrosinase activity of BMEPS is related to being a metal chelator, H2O2 scavenger, and its content of sulfated groups.The other mechanism that was examined in this study is the tyrosinase pathway. There are many tyrosinase inhibitors, both from synthetic and natural sources. The mechanisms of tyrosinase inhibition include one or more of the following; (i) dopaquinone reduction like ascorbic acid, (ii) o-dopaquinone scavenging as in thio-containing compoundsBacillus maritimus MSM1 was characterized by Physico-chemical analysis using UV, FTIR, HPLC, and GPC analyses. These analyses proved that it is a sulphated low molecular weight polysaccharide with uronic substitution  and the monosaccharides are presented in molar ratio 0.8:1.0:2.8:2.3 for mannuronic acid, glucouronic acid, glucose, and mannose, respectively, with the abundance of β-configurations. The GPC estimated the average molecular weight (Mw) as 4.31 × 104 g/mol. BMEPS exhibited promising antioxidant characters and selective inhibitory action against COX-2. The proved efficacies support the incorporation of this polysaccharide as a promising alternative drug in treating inflammatory and oxidative stress-related diseases. Additionally, BMEPS shows anti-cholinesterase and anti-tyrosinase activities which are relevant to neurodegenerative diseases. The promising biological features of BMEPS may be attributed to being rich in SO3 and COO − groups, β-glycosidic linkage, in addition, its low molecular weight. It is evident from the study that BMEPS can be a candidate in further advanced medical studies as a new natural raw material for Alzheimer’s treatment drugs.In conclusion, the marine Exo-polysaccharide produced from 2O2, ABTS , Ascorbic acid (vitamin C), butylated hydroxytoluene (BHT), nicotinamide adenine dinucleotide (NADH), nitroblue tetrazolium salt (NBT), phenazine methosulphate (PMS), sodium nitroprusside (SNP), sulfanilamide, ortho-H3PO4, naphthylethylene diamine dihydrochloride, diammonium salt, 3-(2-pyridyl)-5,6-bis (4-phenyl-sulfonic acid)-1,2,4-triazine (ferrozine), Ferrous chloride, trichloroacetic acid (TCA), potassium ferricyanide, Leuco-2,7-dichlorofluorescien diacetate, hematin, arachidonic acid, Cyclooxygenases enzymes , acetylcholine esterase enzyme, acetylthiocholine iodide, DTNB, butyryl thio-choline Iodide, butyrylcholinesterase enzyme, kojic acid, tyrosinase enzyme, and L-Dopa were purchased from Sigma-Aldrich, USA. Ammonium thiocyanate was purchased from E. Merck. All chemicals and solvents are analytical grades.1,1-diphenyl-2-picryl-hydrazyl (DPPH), peroxidase, HThree samples were collected from marine sediment at different locations in Sahl Hashish, Hurghada Red Sea, Egypt.56. Finally, 50 μL of the supernatant of each dilution was inoculated on marine nutrient agar, and the plates were incubated at 37 °C for 24 h. The colonies that appeared per plate of each sample were subjected to purification. The production of EPS was shown by the formation of a mucous colony, which was followed by the alcohol-based precipitation of EPS. Fresh cultures of marine isolates were applied to solid agar medium (g/L): glucose 20, CaCO3 0.1, NH4NO3 0.8, K2HPO4 0.6, KH2PO4 0.05, MnSO4.4H2O 0.1, yeast extract 1.0 and agar 15, and they were incubated separately at 37ºC. Mucous colony formation was examined by observing the presence of slimy mucous colonies on the plate for 3 days. Furthermore, loopful colonies were immersed in alcohol to observe the precipitation of EPSs. When a loopful of culture is mixed with pre-chilled absolute ethanol, colonies precipitate out from the solution, confirming the EPSs producer.The collected samples (10 g) were placed in 100 mL of sterile seawater and homogenized by shaking at 200 rpm for 20 min, and a serial dilution was performed57. The medium was distributed in an Erlenmeyer flask of 250 mL, containing 50 mL of working volume. The flasks were autoclaved and inoculated using an actively growing culture and incubated at 37 °C for 3 days. The fermented broth was collected and centrifuged at 5000 rpm at 4 °C for 30 min at Sigma-Laborzentrifugen, 2K15, to remove bacteria cells. TCA (5%) was added and left overnight at 4 °C, then was centrifuged at 5000 rpm to remove protein58. The pH of the clear solution was adjusted to 7.0 with 0.1 M NaOH, and the crude EPS was precipitated by the addition of 4 volumes of pre-chilled absolute ethanol to the supernatant liquid; this was stored overnight at 4 °C. The sample was then centrifuged, and after decanting off the ethanol solution, the recovered pellet was re-dissolved in deionized water. The dissolved crude EPS was subjected to another precipitation step with pre-chilled absolute ethanol, followed by subsequent centrifugation and decantation of ethanol as described above. The pellet obtained was then re-dissolved in a minimum of deionized water and dialyzed three times (1000 mL × 3) against flowing tap water using dialysis tubing (Cut off MWCO 3500 Da) for 24 h. The dialyzed solution was subjected to fractional precipitated by 1, 2, 3, and 4 volumes of pre-chilled absolute ethanol. The major yield fraction obtained by 1 volume of absolute ethanol was dried under vacuum at 40 °C59. The UV absorption spectrum was recorded using a UV–Vis Spectrophotometer  between 200 and 800 nm, to examine the existence of proteins and nucleic acids3. The main fraction of EPS from 1 volume ethanol (BMEPS) was assayed in-vitro against Acetylcholinesterase inhibition.Isolates were screened for the production of EPSs in a modified MY medium with 75% seawater. The medium is composed of (g/L): peptone 4.0, yeast extract 2.0, and sucrose 20.0, pH 761 The strain was confirmed with the 16S ribosomal RNA gene sequence and compared with other bacterial sequences by using NCBI BLAST. The taxonomic affiliation of the sequences was retrieved from the classifier program of the ribosomal database project63.The promising isolate (S16), which produced high amounts of EPS and acetylcholine esterase inhibitor, was identified based on the biochemical, morphological, and physiological characteristics of the potential producer as determined by adopting standard methodshttp://www.ncbi.nlm.nih.gov/blast) against the 16S ribosomal RNA sequence database, with the mega X software  to generate the phylogenetic tree from the national center for biotechnology.The genomic DNA of the isolates was extracted using the Bacterial Genomic DNA Extraction Kit. The amplification process took 2:35 total time. On a 0.8% agarose gel dyed with a DNA-safe stain, the PCR products were seen. Finally, the PCR products were sequenced, and the obtained sequence data were analyzed using the basic local alignment search tool (BLAST) software (http://www.ncbi.nlm.nih.gov/genbank) and was used for phylogenetic analysis.The 16S rRNA gene fragment was compared with the NCBI nucleotide database using Blastn. The following bacterial 16S rDNA from taxonomically characterized homologues were collected from the Genbank database on NCBI 6.BMEPS (50 mg) was subjected to hydrolysis with 6 N HCl for 4 h at 100 °C in a sealed tube. In a water bath at 40 °C, the HCl was removed, and (1 mL × 3) co-distilled with water−1 on a Vector 22 Spectrophotometer Bucker14.UV–Vis spectroscopy was conducted on the 2401PC UV–Vis spectrophotometer Shimadzu, Japan, in the wavelength range of 190–700 nm. FTIR was measured in the range of 400–4000 cmMw) of the BMEPS was measured using high-performance gel permeation chromatography (HP-GPC) using Agilent HPLC1100 series system according to You et al.55.The weight average molecular weight  and Ascorbic Acid (LAA) were prepared as 100, 250, 500, 750, and 1000 µg/ml in methanol. The activity expressed as IC50 was calculated by log-probit analysis.The Fe2+ chelating activity of BMEPS was estimated according to the method of Dinis et al.67 and was evaluated by comparing it with two standard compounds (BHT and Ascorbic Acid at the same conditions). The percentage of inhibition of ferrozine-Fe2+complex formation was given by the formula:A0 was the absorbance of the control, and A1 was the absorbance in the presence of the sample of polysaccharide and standards.The Fe.23, the free radical scavenging activity of BMEPS was determined using 1,1-diphenyl-2-picryl-hydrazil (DPPH•). According to the following equation DPPH radical scavenging activity was calculated:A0 was the absorbance of the control and A1 was the absorbance in the presence of MBES.According to the method of Yamaguchi et al68, and modification by Arnao et al.69, The ABTS radical cation scavenging activity of BMEPS was estimated. The ABTS radical cation scavenging activity was calculated as follows:Based on the method described by Miller and Rice-Evans70. The inhibition of lipid peroxidation in percentage was calculated by the following equation:1, A2, A3, andA4 was the absorbance of the sample at 1st day, 2nd, 3rd, and 4th days.The ability of BMEPS to inhibit lipid peroxidation was determined according to the method of Gülçin et al.2−) scavenging activity of BMEPS was based on the method described by Liu et al.71. O2− scavenging was calculated using the following formula:A0 was the absorbance of the control, and A1 was the absorbance of polysaccharide or standard samples.The measurement of the superoxide anion , the NO73 and Celecoxib was used as the standard compound.The cyclooxygenase inhibition activity of BMEPS was performed according to Larsen et al.Acetylcholinesterase (AchE) and Butyrylcholinesterase (BChE) inhibitory assessment.74. The percent inhibition was calculated using the formula:The enzymatic activity of BMEPS was analyzed using the method of Ingkaninan75. The anti-tyrosinase ability of BMEPS was compared to kojic acid . The percentage of tyrosinase inhibition was calculated as follows:Tyrosinase inhibition assay of BMEPS was performed according to the methods of Liu et al.In-vitro antioxidant data were analyzed by t-test in one-way ANOVA (n = 3 replicates) using MIB-SPSS, 25.0 software. The P value was less than 0.05.The data were presented as median ± SE. Supplementary Figure S1.


Furthermore, CuCoNC+Oxone has a smaller activation energy for RDMB elimination and maintains its superior effectiveness for removing RDMB in various water conditions. The computational chemistry insights have revealed the RDMB degradation mechanism. This study reveals that CuCoNC is a useful activator for Oxone to eliminate RDMB.Even though transition metals can activate Oxone to degrade toxic contaminants, bimetallic materials possess higher catalytic activities because of synergistic effects, making them more attractive for Oxone activation. Herein, nanoscale CuCo-bearing N-doped carbon (CuCoNC) can be designed to afford a hollow structure as well as CuCo species by adopting cobaltic metal organic frameworks as a template. In contrast to Co-bearing N-doped carbon (CoNC), which lacks the Cu dopant, CuCo alloy nanoparticles (NPs) are contained by the Cu dopant within the carbonaceous matrix, giving CuCoNC more prominent electrochemical properties and larger porous structures and highly nitrogen moieties. CuCoNC, as a result, has a significantly higher capability compared to CoNC and Co Conventionally, WOTs are associated with production of radicals that exhibit strong redox powers to decompose contaminants. As the Fenton’s reaction is commonly employed for llutants , SO4•−-rotential ,3,4, mak4•−, a few reagents would be accessible, such as Oxone®, which, however, requires activation for quickly releasing SO4•− from Oxone. Amid several activating strategies [xFe3−xO4 nanocatalysts were also reported to activate Oxone for the removal of RDMB [To produce SOrategies ,6,7,8, hrategies ,4. For irategies . CoxFe3− of RDMB . Likewis of RDMB ,12.3O4) have been fabricated for activating Oxone [Even though Co has been validated as the most efficient metal and a few cobaltic materials  might encounter challenges, including the aggregation of NPs, inconvenient recovery of NPs, and vulnerable instability ,21. ThusOn the other hand, as carbon is the most Earth-abundant, highly accessible, and stable, carbon materials would be favorable media for supporting/embedding CuCo NPs ,23. CarbEven though embedding NPs onto carbon media is feasible by additional fabrication, such an additional fabrication strategy unfortunately faces many issues, including uneven distribution, agglomeration, and insufficient loading of active species ,27. AdopWhile Co-ZIF has a cubic geometry and sub-micron sizes, the derived Co/carbon hybrids are quite challenging to access by Oxone ,29. ThusTo further examine the catalytic activities of CuCoNC, a Co-bearing N-doped carbon (CoNC), which is an analogous to CuCoNC without the Cu dopant, was also prepared.The fabrication of CuCoNC was implemented as depicted in k:For calculating the elimination rate constant, the following equation was adopted for the estimatiation of Morphologies and Compositional analysesSince CuCoNC was derived from Co-ZIF, the appearance of Co-ZIF was firstly characterized as displayed in 2+, and then pyrolysis, the resulting material (+), permeating into Co-ZIF, eventually affording a hollow structure. Moreover, the transmission electron microscopic image also indicated that the NPs decorated ranged from 4 to 11 nm, demonstrating that these NPs were extremely small and the sizes were relatively similar.Via modification by tannic acid as well as Cumaterial e maintaimaterial f furtherMoreover, In particular, a few peaks were observed at 25.6°, 37.0°, 43.4°, 44.3°, 50.5°, and 74.1°. While the broad peak at 25.6° could be attributed to carbon, the noticeable peaks at 43.4, 50.5, and 74.1° would be ascribed to the formation of CuCo  as a resThese results suggest that the resultant NPs adorned on the hollow cubic product were comprised of Cu/Co species, formulating the nanoscale Cu/Co-bearing N-doped carbon (CuCoNC).2+ dopant in 2+, which contained peaks at 25.6°, 44.3°, 51.6°, and 75.9°. While the peak at 25.6° could be ascribed to carbon, the rest of the peaks can be well-indexed to Co0. Thus, the ZIF without Cu2+ dopant became Co-bearing N-doped carbon (CoNC) after the etching and carbonization treatments.A similarity to CuCoNC was also fabricated in the absence of CuTextural properties2 sorption of CoNC, which was actually similar to that of CuCoNC but with a much smaller N2 sorption volume. the surface area of CuCoNC was 70 m2/g, while CoNC merely showed a surface area of 26 m2/g. It would be interesting to examine the magnetism of CuCoNC. In 3O4  for CuCoNC+Oxone would be 0.219 min−1, which was greater than the rate constants for Co3O4 NP+Oxone  and CoNC+Oxone (0.089 min−1), indicating that CuCoNC was certainly superior to the commercial Co3O4 NP and CoNC.In addition, the rate constants for RDMB degradation are then summarized in 3O4 NP were significantly lower . K values of Oxone decomposition are also summarized in k using CuCoNC was 0.092 min−1, the k by CoNC was 0.037 min−1, and that by Co3O4 NP was 0.020 min−1, confirming that the Oxone decomposition by CuCoNC was significantly faster and more efficient than CoNC and Co3O4 NP, thereby causing faster RDMB degradation.As CuCoNC led to the much faster degradation of RDMB, it was interesting to study Oxone decomposition by CuCoNC. In 0, whereas the peaks at 780.3 and 796.2 eV were Co2+ [0 and Cu+ in CuCo alloy [2+ species, as the oxidation of CuCo can occur in contact with O2 as seen in the XRD pattern. Furthermore, Oxone activation correlates to surficial chemistry between Oxone and catalysts . Since Cere Co2+ . MoreoveCo alloy ; the ban0, Cu+, and Cu2+, that act as additional mediating groups for Oxone activation [Similarly, the N1s in CoNC  also hadtivation ,41.In addition, CuCoNC also showed a different result for nitrogen components, with pyridinic N, pyrrolic N, and quaternary N of 37.0%, 45.3%, and 17.7%, respectively , while C−1, corresponding to F2g, gE, and A1g [F2g peak of CoNC was located at 190 cm−1  result of CuCoNC is shown in −1 to 0.329 min−1. When it further rose to 50 °C, the rate of RDMB degradation increased to 0.520 min−1, indicating the favorable effect of elevated temperatures on RDMB elimination. Furthermore, these rate constants at different temperatures were correlated using the Arrhenius equation [As CuCoNC+Oxone eliminated RDMB effectively, temperature and pH would also impact RDMB degradation. equation :(2)lnk=laE was 27.7 kJ/mol, much lower than the reported aE values of RDMB elimination in the literature  was used to be a probe agent for •OH, while MeOH would be used to identify presence of both SO4•− and •OH. After TBA was added to the solution, RDMB degradation proceeded noticeably slower  was adopted to examine superoxide (O2•−). k = 0.034 min−1, demonstrating that O2•− might be present in RDMB elimination. Moreover, NaN3 was used to study singlet oxygen (1O2), and NaN3 also inhibited RDMB elimination with k = 0.024 min−1, revealing that 1O2 is also produced from CuCoNC+Oxone.For further unraveling the degradation mechanism, a few probe agents were then employed for elucidating ROS during RDMB degradation. At first, y slower , and the50 min−1 , signify4•− and •OH as a function of the reaction time. Nevertheless, the concentration of SO4•− seemed much higher than that of •OH, which agrees with the above-mentioned discussions.The occurrence of these radicals at different reaction times were then determined ,52. In p1O2 was associated with furfuryl alcohol (FFA) consumption [1O2.Further, the occurrence of sumption . Figure 4 and DMPO-OH, as displayed in 4•− and •OH. Moreover, the same test was then implemented in MeOH to examine whether O2•− was present  was employed here to measure radicals species in RDMB degradation. Firstly, when 5,5-Dimethyl-1-pyrroline N-oxide (DMPO) was adopted, no distinct feature was observed in the case of Oxone alone . When DM present , and a n1O2  was used, no notable signal was seen for Oxone alone. Once TEMP, Oxone, and CuCoNC were all present, a strong signal was observed and ascribed to TEMP-1O2 , confirm4•−, •OH, and 1O2 would be electrophilic species, the HOMO at the xanthene group of RDMB would be the most susceptible to electrophilic degradation by these species. As CuCoNC can promote Oxone activation to eliminate RDMB, it was essential to study the RDMB elimination process. To this end, the First-Principle-based calculation was employed here ,55. In Ff–, f0, and f+ indicate the electrophilic, radical, and nucleophilic attacks, separately. As 1O2 is an electrophilic molecule, fractions of RDMB with larger f-values would be more susceptible to electrophilic degradation. Thus, 6C exhibits the highest f-values, indicating that 6C might be the initial site for receiving electrophilic degradation (as seen in f0 values are also more susceptible to radical degradation; similarly, 6C might be more susceptible to radical degradation during RDMB elimination (as seen in The Fukui values of RDMB are also generated and summarized in 2 and H2O.To further examine the degradation process of RDMB using CuCoNC+Oxone, the mass spectrometry of RDMB elimination  was thenIn this study, CuCoNC was fabricated to reveal a special hollow-configured morphology and Cu/Co using the template of Co-ZIF followed by the straightforward etching and Cu dopant to afford a practical activator for Oxone. This Cu doping enabled the encapsulation of CuCo into NC, enabling CuCoNC to possess enhanced porous structures, more active sites, and strengthened electrochemistry. Thus, CuCoNC showed much higher catalytic activities than CoNC for Oxone activation to eliminate RDMB. In addition, CuCoNC+Oxone exhibited a lower activation energy for RDMB elimination and retained its effectiveness in eliminating RDMB. These results render CuCoNC a promising activator for Oxone to degrade RDMB.


Chlamydomonas reinhardtii and Chlorella vulgaris) when exposed to different concentrations of nitrogen, phosphorus, and sulfur. Results show a significant microalgal growth (3.69 g L−1) and carbohydrates (163 mg g−1) increase in C. reinhardtii under low nitrogen concentration. Also, higher lipids content was produced under low sulfur concentration (246 mg g−1). It was observed that sulfur variation could affect in a negative way proteins production in C. reinhardtii culture. In the case of C. vulgaris, a higher biomass production was obtained in the standard culture medium (1.37 g L−1), and under a low-phosphorus condition, C. vulgaris produced a higher lipids concentration (248 mg g−1). It was observed that a low concentration of nitrogen had a better effect on the accumulation of fatty acid methyl esters (FAMEs) (C16-C18) in both microalgae. These results lead us to visualize the effects that the variation in macronutrients can have on the growth of microalgae and their possible utility for the production of microalgae-based subproducts.The production of biomolecules by microalgae has a wide range of applications in the development of various materials and products, such as biodiesel, food supplements, and cosmetics. Microalgae biomass can be produced using waste and in a smaller space than other types of crops , which shows microalgae’s great potential as a source of biomass. Among the produced biomolecules of greatest interest are carbohydrates, proteins, lipids, and fatty acids. In this study, the production of these biomolecules was determined in two strains of microalgae ( In this process, microalgae use CO2 from the atmosphere or other sources like flue gas streams for biomass production; it has been determined that producing 1 g of microalgae biomass represents the capture of 1.8 g of CO2 ; it reached 0.86 g L−1 of biomass growth at the 10th and 1.37 g L−1 at the 15th culture day  and at high phosphorus concentration [P+] (3.12 g L−1 at 10th day)  a,b. In tnstrated .Isochrysis galbana  presented enhanced cell growth at the nitrogen concentration of 144 mg L−1 in comparison to the nitrogen concentration of 288 mg L−1 . This result is in agreement with those reported by Lovio Fragoso et al. (2019)  has a more negative effect on microalgae growth than the effect obtained with high concentration [P+]. The same effect was reported by Li et al. (2022)  and high [S±] sulfur concentrations at the 15th day, respectively  , the exponential phase is reached in C. reinhardtii culture. The exponential phase of this microalgae can be reached usually at the 8th or 10th day of culture, even in light stress, salinity stress, or nutrient stress conditions. This behavior could be related to nutrients depletion and less light penetration because of its high cell density  concentration, a higher microalgae biomass was produced in all the experiments at the 10th day of culture (3.69 g L−1). In the case of C. vulgaris culture, it was determined by the ANOVA–Tukey pairwise analysis that at medium [Control] concentration of macroelements, a higher microalgae biomass was produced in all the experiments at the 15th day of culture (1.37 g L−1). These results demonstrate that the growth behavior of microalgae with different nitrogen, phosphorus, and sulfur concentrations depends on the microalgae strain; however, some similar trends were found. In the case of Chlorella vulgaris, it can be concluded that the variations made negatively affected cell growth, because the variation was excessive for the culture to adapt in such a short time. However, since the culture did not reach a stationary phase, it was not discarded that in later days the behavior would have changed.From ANOVA–Tukey pairwise analysis of C. reinhardtii produced a higher concentration of carbohydrates with medium macroelements concentration [Control] in most microalgae cultures  [Chlamydomonas malina (Chlorophyta) RCC2488 reached approximately 200 mg g−1 at the 10th culture day.At medium macroelements concentration [Control], . (2020) , where C−] and sulfur [S−] concentration at the 5th culture day in C. reinhardtii culture, 163 mg g−1 (50% greater than medium concentration) and 149 mg g−1 (37% greater than medium concentration) were obtained, respectively. The effect of nitrogen and sulfur deprivation in enhancing the carbohydrate content in microalgae biomass has been reported by different authors and using different microalgae strains. For example, in the case of C. reinhardtii CC-124, an increase of up to 4.3-fold was obtained , where 339 mg g−1 (22% greater than medium [Control] concentration), 244 mg g−1 (8% greater than control), and 174 mg g−1 of carbohydrates (15% greater than control) were obtained at the 5th, 10th, and 15th culture days, respectively. These results can be compared to those obtained by Cheng et al. (2017)  in C. vulgaris culture, but only on days 5 and 15 of the microalgae culture, when 355 mg g−1 (28% greater than control) and 265 mg g−1 of carbohydrates (>50% greater than control) were obtained, respectively. It was shown in previous studies that low phosphorus concentrations could enhance the production of carbohydrate. For example, in Chlorella spp. culture, >50% of carbohydrates were obtained under phosphorus limitation  in C. vulgaris culture, the same behavior was observed that was obtained with low nitrogen and phosphorus concentration, since 306 mg g−1 (10% greater than control) and 282 mg g−1 of carbohydrates (25% greater than control) were reached at the 5th and 10th culture days, respectively. These results are similar to those found by Wang et al. (2022)  and low nitrogen [N−] concentration conditions produced a higher carbohydrates content in all the experiments at the 15th day of culture (175 mg g−1) and at the 5th day of culture (163 mg g−1), respectively. In the case of the ANOVA–Tukey pairwise analysis of carbohydrates in C. vulgaris culture, it was determined that at low phosphorus [P−] concentration, a higher carbohydrates content was produced in all the experiments at the 5th day of culture (355 mg g−1). As it was possible to observe in the case of C. reinhardtii, a higher concentration was obtained in the control, a result which is given at the 15th day of culture, which leads us to consider that this microalga tends to accumulate a greater amount of carbohydrates over time. However, in the case of low nitrogen concentration [N−], a greater amount of carbohydrates can be obtained on the 5th day of culture. Therefore, if its production is sought, its harvest can be considered at this time.According to the ANOVA–Tukey pairwise analysis results of carbohydrates in In this way, it can be shown that the deprivation of these macroelements could enhance the production of carbohydrates in both microalgae; however, it is important to remark that each strain has a different behavior under these conditions. The increase in carbohydrates in microalgae biomass due to nutrients limitation conditions could be related to the fact that cells storing energy in the form of carbohydrates use less energy than they do by storing it in lipids (50% less ATP and 45% less NADPH in TAG synthesis process) . NitrogeUnder phosphorus limitation conditions, the exchange of triose phosphate  could be reduced by the lack of cytoplasmic orthophosphate, generating an accumulation of precursors  of carbohydrates production such as starch biosynthesis ,53. The C. reinhardtii produced a higher concentration of proteins with medium macroelements concentration [Control] in most microalgae cultures, except in the cases of low nitrogen [N−] and high phosphorus [P+] concentration at the 5th culture day and low phosphorus [P−] concentration at the 15th culture day   in . (2023) , where Cl., 2021 , who cul−] and high phosphorus [P+] concentration at the 5th culture day, C. reinhardtii obtained 130 mg g−1 (5% greater than medium concentration) and 145 mg g−1 (17% greater than medium concentration), respectively. These results are similar to those found by Kamalanathan et al. (2016) [C. reinhardtii obtained a higher amount of protein in conditions without nitrogen in the first five days of culture. The increase in proteins at low nitrogen [N−] concentration could be related to the use of intracellular nitrogen stored in macromolecules such as chlorophyll. Similar to these results, C. reinhardtii culture under nitrogen limitation for the first cycles of cell division used the nitrogen found in the cells, and therefore, a slight increase (≤13%) in the protein content was observed in the culture  concentrations increased protein accumulation, which is in agreement with the results found in our work. Particularly, higher accumulation was reported of proteins involved in ribosome structure and synthesis, as well as in DNA and RNA metabolism [+] leads to a reduction in the production of carbohydrates in the cell [Other authors have reported that tabolism . Moreovethe cell . SimilarC. vulgaris obtained a greater amount of proteins at low nitrogen concentration [N−], where 228 mg g−1 (18% greater than medium [Control] concentration) was obtained at the 10th culture day. Under nitrogen limitation, microalgae can up-regulate proteins related to lipids (1-Acylglycerol-3-phosphate O-acyltransferase and Glycerol-3-phosphate O-acyltransferase) and carbohydrates  synthesis [+] of C. vulgaris culture, an increase in proteins content was observed, since it reached 335 mg g−1 (16% greater than medium concentration) at the 5th culture day. These results agree with those found in C. reinhardtii experiments.In the experiments on nitrogen variations, ynthesis . Also, a−] of C. vulgaris culture, a greater amount of proteins was obtained, but only on days 10 and 15 of the microalgae culture, with 242 mg g−1 (25% greater than control) and 210 mg g−1 of proteins (14% greater than control), respectively. In Chlorella sp. culture, it was found that phosphorus limitation could enhance protein production more than high phosphorus concentration  condition, since 70 mg g−1 (43% less than medium concentration), 45 mg g−1 (51% less than medium concentration), and 32 mg g−1 of proteins (>60% less than medium concentration) were obtained at the 5th, 10th, and 15th culture days, respectively. In addition, high sulfur concentration [S+] resulted in 341 mg g−1 (>50% greater than control) at the 10th culture day of C. vulgaris culture. Contrarily, C. vulgaris at low sulfur [S−] concentration produced a protein content decrease, which can be related to the affectation in biosynthesis of the sulfur amino acids such as cysteine and methionine  concentration, a higher protein content was produced in all the experiments at the 5th day of culture (145 mg g−1). On the other hand, from the ANOVA–Tukey pairwise analysis of protein in C. vulgaris culture, it was determined that at high sulfur [S+] and high phosphorus [P+] concentration was produced a higher protein content in all the experiments at the 10th day of culture (341 mg g−1) and at the 5th day of culture (335 mg g−1), respectively.From the ANOVA–Tukey pairwise analysis of protein content in C. reinhardtii at medium macroelements concentration [Control] produced 84 mg g−1, 154 mg g−1, and 127 mg g−1 of lipids at the 5th, 10th, and 15th culture days . On the other hand, C. vulgaris obtained an improved production of lipids with low macroelements concentrations in some microalgae cultures  produced more lipids under nitrogen limitation conditions; however, a complete nitrogen deprivation reduced lipid production  concentration at the 5th culture day in C. reinhardtii, a higher production of lipids (134 mg g−1) was obtained (37% greater than medium concentration). Similar to C. reinhardtii, in the experiments on phosphorus variations, C. vulgaris obtained a greater amount of lipids under low phosphorus concentration [P−], reaching values of 212 mg g−1 (>50% greater than medium [Control] concentration), 183 mg g−1 (34% greater than medium concentration), and 248 mg g−1 (45% greater than medium concentration) at the 5th, 10th, and 15th culture days, respectively. This behavior is similar to that found in other microalgae such as Scenedesmus sp., which at lower phosphorus concentration produced an increase of approximately 50% in lipid content. The phosphorus limitation enhances the expression of genes related to fatty acid biosynthesis such as 3-ketoacyl-CoA synthase and 2-enoyl-CoA reductase [−], since a similar carbohydrate content to that obtained by the control was observed on certain sampling days  was obtained in C. reinhardtii culture. C. reinhardtii CC-124 shows an increase in lipids during sulfur deprivation of approximately 35% [−1 (>50% greater than control) was obtained under low concentration [S−] conditions at the 10th culture day in C. vulgaris. This behavior could be related to the overexpression of genes encoding acyltransferases related to TAG production such as phospholipid diacylglycerol acyltransferase (PDAT1) and diacylglycerol acyltransferase (DGTT1)  concentration, a higher lipids content was produced in all the experiments at the 15th day of culture (246 mg g−1). On the other hand, from the ANOVA–Tukey pairwise analysis of lipid production in C. vulgaris culture, it was determined that at low phosphorus [P−] concentration, a higher lipids content was produced in all the experiments at the 15th day of culture (248 mg g−1).From the ANOVA–Tukey pairwise analysis of lipids production in C. reinhardtii in medium concentration of macronutrients [Control] and after 15 days of cultivation produced 20 ± 3 mg g−1  of palmitic acid (C16:0), 12 ± 1.5 mg g−1 (17%TFA) of oleic acid (C18:1n9c), 13 ± 2.5 mg g−1 (16%TFA) of linoleic acid (C18:2n6c), and 15 ± 2.5 mg g−1 (18%TFA) of α-linoleic acid (C18:3n3)  a–c. Thes−] in C. reinhardtii culture, 34 ± 8 mg g−1 [28%TFA] of palmitic acid, 36 ± 10 mg g−1 [27%TFA] of oleic acid, 23 ± 5 mg g−1 [19%TFA] of linoleic acid, and 19 ± 3 mg g−1 [16%TFA] of α-linoleic acid were produced at the 15th culture day, the highest result in the whole experiment with this microalga strain. In the literature, similar results can be found. For example, Phaeodactylum tricornutum produced a higher percentage of α-linoleic acid and palmitic acid under a low-nitrogen condition  conditions produced 35 ± 12 mg g−1 of palmitic acid (5-fold greater than control), 6 ± 1.2 mg g−1 of oleic acid (2-fold greater than control), 34 ± 12 mg g−1 of linoleic acid (8-fold greater than control), 21 ± 8 mg g−1 of α-linoleic acid (6-fold greater than control), and 3.8 ± 0.75 mg g−1 of arachidonic acid (2-fold greater than control) at the 15th culture day  culture, which, under low nitrogen concentration, produced higher C16 and C20 fatty acids  conditions, C. reinhardtii produced 5% more arachidonic acid (C20:4n6) than control conditions. This result is in agreement with other studies performed with C. reinhardtii culture, in which phosphorus stress generated longer-chain fatty acids  compared to control conditions [−] conditions in C. vulgaris culture , α-linoleic acid (>50% greater), and arachidonic acid (>50% greater) were obtained compared to control.In low phosphorus  resulted in 23%TFA of oleic acid (10% more than control) in C. reinhardtii culture. Also, 2-fold greater control of arachidonic acid was obtained at low sulfur concentration. It has been demonstrated that high sulfur concentration causes negative effects on FAMEs production, except in the case of stearic acid, since it obtained >20% production compared to control. The increase in FAMEs concentration under low sulfur concentration could be related to the overexpression of genes related to acetyl-CoA carboxylase, 3-ketoacyl-ACP synthase, and 3-ketoacyl-ACP reductase production, which are involved in fatty acid synthesis   and α-linoleic acid (>50% greater) were determined compared to the control.ion [S−] e. These observed . In addiThe reagents used for culture medium preparation and to perform the carbohydrates, lipids, and FAMEs analysis were purchased from Sigma-Aldrich . Lowry Protein Assay Kit was purchased from Thermo Fisher Scientific . The spectrophotometric measurements were taken with an absorbance microplate reader from BGM Labtech .Chlamydomonas reinhardtii and Chlorella vulgaris were purchased from UTEX . BG11 culture medium was used to perform the experiments with both microalgae strains. The BG11 culture medium composition was as follows: NaNO3 1.5 g L−1, K2HPO4 40 mg L−1, CaCl2·2H2O 36 mg L−1, MgSO4·7H2O 75 mg L−1, Citric Acid·H2O 6 mg L−1, C6H8FeNO7 6 mg L−1, Na2EDTA·2H2O 1 mg L−1, Na2CO3 20 mg L−1, H3BO3 2.86 mg L−1, MnCl2·4H2O 1.81 mg L−1, ZnSO4·7H2O 0.22 mg L−1, Na2MoO4·2H2O 0.39 mg L−1, CuSO4·5H2O 0.079 mg L−1, and Co(NO3)2·6H2O 0.04 mg L−1. The stock cultures were kept at 21 ± 1 °C with continuous light at 100 μmol photons m−2 s−1 and with 1.5 L m−2 min−1 of aeration (OPTIMA 4.5-Watt pump with an air filter of 0.20 µm).The strains Chlamydomonas reinhardtii and Chlorella vulgaris inocula, with an absorbance of 1 at the wavelength of 750 nm, were cultivated separately in lab glass bottles containing 900 mL of BG11. Before inoculation, the biomass was washed twice with bidistilled water. The cultures were maintained under the same conditions mentioned in the previous section. A factorial design was used for this research and applied to each experiment set  of each strain was defined, as shown in Equations (1) and (2). Samples of 1 mL were obtained every day to assess the biomass growth curve by spectrophotometry. The dry weight was determined by filtering 10 mL of serial algal culture dilutions using pre-weighed Whatman GF/C glass microfiber filters. The filter holding the algal biomass was dried at 100 °C for 2 h. Then, filters with the algal biomass were cooled at room temperature inside a vacuum desiccator and weighed gravimetrically.Prior to the experiments, an equation of relationship between absorbance (750 nm) and dry weight  by using a glucose standard curve.Carbohydrate content was quantified using the sulfuric acid–UV assay described by López-legarda et al. . BrieflyChlamydomonas reinhardtii and Chlorella vulgaris. Firstly, 1 mL of Lowry’s reagent was added to 200 µL of washed biomass solution. Secondly, the tube was vortexed. After 10 min, 100 µL of Folin reagent was added, and the mixture was vortexed again. After 30 min of reaction in the dark, the absorbance of the samples was measured at 750 nm in an absorbance microplate reader from BGM Labtech. Finally, the protein concentration was obtained using a BSA calibration curve.The Modified Lowry Protein Assay Kit was used to determine the total protein produced by Chlamydomonas reinhardtii and Chlorella vulgaris was quantified using the sulfo-phospho-vanillin assay described by Mishra et al. (2014) [The lipid content produced by . (2014) . Brieflyv/v/v), respectively, into 150 mg of dried biomass. The resulting mixture was mixed for 30 s and then centrifuged at 4800 rpm for 10 min at 4 °C. After centrifugation, the lower layer was carefully recovered and filtered using a PTFE syringe filter . Then, it was transferred into a pre-weighed glass tube. The remaining chloroform was dried in a concentrator at 45 °C for 2 h at 15 mm Hg. The extracted lipids were gravimetrically weighted to estimate the total lipid content according to the Bligh and Dyer method with some modifications [Chloroform, methanol, and distilled water were added in ratios of 1:2:0.4 (ications .Fatty acids were determined through derivatization to fatty acid methyl esters (FAMEs) in closed vial by using (2 mL) of methanol at 7% with sulfuric acid using triundecanoin (C11:0) as internal standard. Mixture was heated at 80 °C for 1.5 h. Immediately after reaching room temperature, sample was extracted with 3 mL of hexane while manually shaking for 1 min. The upper phase formed was recovered, and the inferior phase was extracted again with 3 mL of hexane. Recovered phases were placed in a volumetric flask and brought to 10 mL with hexane.m/z = 30–550. For FAMEs identification, the retention time of a typical chromatogram of the 37-component standard reference for FAME was used, and the quantification was realized used the standard internal method, using triundecanoin as the internal standard .Fatty acid profile was analyzed by a gas chromatograph with a mass spectrometer  by using an HP-88 capillary column . The carrier gas was helium at a constant flow of 1 mL/min. The initial oven temperature was 140 °C (held for 5 min), and it was increased by 4 °C/min until it reached the final temperature 240 °C (held for 15 min). Split ratio was 1:10, injector temperature was 265 °C, mass spectrometer analysis used EI ion source, electron energy was 70 eV, the temperature of source and interface were 210 °C, and range of All data were analyzed by Minitab 21 . They were analyzed by an ANOVA with a Tukey pairwise comparison for each type of microalga (α = 0.05). The heatmaps for FAMEs expression were created with R-studio using ggplot2 package.Chlamydomonas reinhardtii and Chlorella vulgaris). It was shown that some microalgae (C. reinhardtii) could produce more biomass under low nitrogen concentration (3.69 g L−1) than standard conditions. It was shown that low concentrations of nitrogen and phosphorus could enhance carbohydrates and lipids in microalgae culture. In addition, surprisingly, the impact that sulfur variation can have on the production of biomolecules in microalgae was found, especially in the case of lipids and proteins. Interestingly, it was determined that low concentration of nitrogen had a better effect on accumulation of some FAMEs  in both microalgae. These results could be useful for increasing productivity of some biomolecules in large-scale microalgae cultures, especially in those intended for the production of FAMEs (C16-C:18).The results found in this study allow us to visualize the impact of three macronutrients’  variation on the production of biomolecules of interest in the same microalgae strain (


Histopathological examination of the heart, and biochemical examinations for heart function (creatine phosphokinase (CPK), lactate dehydrogenase (LDH), and aspartate aminotransferase (AST)), inflammation  and interleukin 10 (IL-10)), oxidative stress , glutathione (GSH), superoxide dismutase (SOD), catalase, nitric oxide (NO), and uric acid), kidney function (urea and creatinine), and minerals  were carried out. Cardiomyopathy induced by DOXO treatment  was ascertained via pathological alterations seen in heart tissue and verified biochemically via increases (P < 0.001) in CPK, LDH, AST, TNF-α, IL-10, MDA, NO, Na, and K levels along with decreases (P < 0.001) in GSH, SOD, catalase, and uric acid. Glycine co-treatment, using doses of 100, 150, and 200 mg/kg, in a dose-dependent manner, displayed ameliorated heart architecture, significantly (P < 0.001) improved biochemical heart function tests, reduced oxidative stress and inflammation, and controlled mineral levels. The positive actions of glycine in DOXO-induced cardiotoxicity amelioration via modulating oxidative stress, inflammation, and immunity are confirmed. Glycine antioxidative properties may be behind its positive outcomes. Finally, we present glycine as a worthy possible option against DOXO-induced heart damage after more validation.Doxorubicin (DOXO) is a well-known cancer chemotherapeutic. However, its toxic effect on the heart limits its clinical application. This study aimed to assess the effectiveness of glycine administration to counteract the DOXO-induction of cardiomyopathy in mice. Fifty male albino mice were divided into five groups ( Heart diseases are the top cause of death worldwide, followed by cancer. In 2019, heart diseases were the cause of 17.9 million deaths ; above three-quarters of these deaths were connected to low- and middle-income nations WHO .Heart toxicity is a well-noted negative outcome of anthracyclines, including doxorubicin. This anthracyclines-induced cardiomyopathy limits their clinical use  drug  is one of the anthracyclines antibiotics. Naturally, it is produced by the fungus Amino acid glycine, composed of a carbon atom attached to an amino group, a carboxylic acid group, and two hydrogens, is the simplest among amino acids. Our body can synthesize glycine, so it is considered non-essential  acquired from Theodor Bilharz Research Institute, Giza, Egypt, were employed. They were housed following the standards in the “Guide for the Care and Use of Laboratory Animals” prepared by the National Academy of Science, published by the National Institute of Health, and approved by the Animal House of Biochemistry, Chemistry Department, Faculty of Science, Damietta University, Egypt. Mice were conserved under controlled conditions  and fed with a standard mice pellet diet and water ad libitum.Control group: mice received normal saline daily for ten days.DOXO group [Cardiomyopathy (cardiac toxicity)]: mice received DOXO   in three equal injections over ten days  for a cumulative dose of 15 mg/kg   for ten days.Gp150 group: concomitant with DOXO, mice received glycine  for ten days.Gp200 group: concomitant with DOXO, mice received glycine  for ten days.The effect of glycine treatment on cardiac toxicity induced by DOXO was studied by random dividing the fifty animals into five groups of ten animals each Fig.  as folloRatio = [HW (g)/BW (g)] × 100. Next, a part of the heart tissue was fixed in 10% buffered formalin for histological studies. Tissue homogenate  was prepared in ice-cold PBS (pH 7.2) and centrifuged at 4000 rpm for 15 min at 4 °C. The resultant clear supernatant was used for biomarkers estimations in tissue.The food intake of groups was observed daily. Body weight was registered every five days over the experimental time to calculate the body weight difference (BWD). Three days later, after the last treatment, mice were fasted for eight h and sacrificed by cervical dislocation. Just before scarification, blood specimens  were collected via cardiac puncture and left to clot. Clotted specimens were centrifuged for 15 min at 3000 rpm to separate sera. Finally, sera were stored at – 20 °C until used for estimations. The heart organ was quickly separated, washed with normal saline, blotted dry on filter papers, and weighed. Heart weight (HW)/body weight (BW) ratio was calculated: HW/BW 2O2 degradation was the basis of measuring the activity of tissue catalase according to catalase kit , and tissue nitric oxide (NO) was estimated via applying the NO kit  procedures depending on the formation of a bright reddish purple azo-dye which can be measured at 540 nm.Colorimetrically, the concentration of tissue malondialdehyde (MDA) as an indicator for lipid peroxides was estimated using the MDA kit purchased from BIODIAGNOSTIC, Giza, Egypt, based on the thiobarbituric acid method, GSH level was estimated by the GSH kit  depending on the breakdown of 5,5‾-dithiobis (2-nitrobenzoic acid) resulting in yellow color that measured at 405 nm, the activity of tissue superoxide dismutase (SOD) was assessed relying on the SOD’s ability to inhibit the phenazine methosulfate-mediated reduction of nitro blue tetrazolium dye as stated in the SOD kit , HLevels of TNF-α and interleukin-10 (IL-10) were measured in tissue based on the sandwich enzyme-linked immunosorbent assay (sandwich-ELISA) principle following the instructions of their commercial immunoassay kits .2O2. H2O2 reacted with 3,5-dichloro-2-hydroxy-benzene sulfonic acid and 4-amino antipyrine in the presence of peroxidase, forming a red dye that was read at 578 nm. By urease, urea was decomposed into ammonia and carbamic acid. Carbamic acid decomposed into NH3 and CO2. In an alkaline sodium hypochlorite solution, the released ammonium reacted with salicylate and nitroferricyanide, forming a green dye. The absorbance of the green color was read at 578 nm. CPK level was determined by following the rate of NADPH formation at 340 nm.Serum levels of uric acid, creatinine, urea, and creatine phosphokinase (CPK) were determined using kits obtained from BIOMED DIAGNOSTICS, Germany, according to the manufacturer’s procedure. In brief, creatinine and picric acid reacted in an alkaline medium, forming a yellow–orange color that was read at 492 nm using a colorimeter. By uricase, uric acid was oxidized to allantoin and H+ at 340 nm was monitored to determine lactate dehydrogenase (LDH) activity using the LDH kit . Aspartate aminotransferase (AST) activity was measured using the kit from DIAMOND DIAGNOSTICS, Germany, depending on the measurement of the amount of oxalate formed through a definite time.In addition, the rate of conversion of NADH/NAD2 to give a blue color that was measured at 640 nm. While potassium ions were let to react with sodium tetraphenyl boron to develop a colloidal solution, which was read at 420 nm. Regarding sodium level determination, sodium ions with excess uranyl acetate and magnesium acetate formed sodium magnesium uranyl acetate. The residual uranyl acetate was let to react with potassium ferrocyanide forming a colored complex, which was read at 545 nm.Calcium, phosphorus, sodium, and potassium kits (BIODIAGNOSTIC (diagnostic and research reagents), Giza, Egypt) instructions were followed for determination of their serum levels. Briefly, in an alkaline medium, calcium ions were let to react with methyl thymol blue forming a blue color, which was measured at 585 nm. In a reaction between phosphate and molybdic acid, the phosphomolybdate compound was formed that was reduced using SnClFixed heart tissues were dehydrated by passing in ascending series of alcohol, cleared with xylene, and immersed in paraffin wax. Slices of the tissues of 5–6 μm thickness were prepared, stained with hematoxylin and eosin (H&E) dye, and examined under light microscopy. The pathologist assessing sections was uninformed of the mice’s treatment.P values of < 0.05, < 0.01, and < 0.001 were assumed significant, very significant, and extremely significant, respectively, for all analyses.Statistically, results were expressed as mean ± S.D., and the statistical analysis was performed using Instat software, version 3.10 . For comparison between groups, the one-way analysis of variance (ANOVA) test was applied. P < 0.001) with a BWD of − 3.94 ± 0.9. Treated groups  displayed an approximate 30% increase in weight compared with DOXO alone with BWD of + 4.27 ± 0.72, + 5.37 ± 1.14, and + 5.1 ± 2.33, respectively. By comparing each glycine dose group with the other two glycine doses groups, all differences in mice weights on day 0, 5, or 10 were non-significant, except weight differences on day zero or day five between mice of Gp200 and Gp150 were significant (P < 0.001) compared to Gp100.As shown in Table P < 0.001) the HW by about 54% and HW/BW ratio by about 28% (0.051 g and 0.29 for the DOXO group versus 0.11 g and 0.4 for the control group). The HW raised significantly to approximately twofold in the glycine-treated groups as compared to the DOXO group . Also, HW/BW ratio depending on glycine dose was significantly elevated by about 31%, 34.5%, and 45% in Gp100, Gp150, and Gp200, respectively . Comparing the HW and HW/BW ratio in each glycine dose group with the other two glycine doses groups, all differences were non-significant (Table Administration of doxorubicin in the DOXO group markedly lowered (P < 0.001) in the DOXO group compared to the control group. The treated groups  displayed, on the contrary, dose-dependent significant reductions in these activities. Respectively, Gp100 showed reductions by 67%, 76%, and 36%, Gp150 by 70%, 77%, and 39%, and Gp200 by 73%, 78%, and 46% when compared to the DOXO group (P < 0.001). There were non-significant differences found in the three enzymes when Gp100, Gp150, and Gp200 groups compared to each other except for AST in Gp200 when compared to that of Gp100  and for LDH in Gp200 when compared to Gp100  or to Gp150 .In Fig. To explore the protection mechanisms of glycine on the heart, we determined the inflammation, oxidative stress, and antioxidant indices.P < 0.001) in levels of TNF-α to about twofold (11.91 ± 0.59 ng/mL), IL-10 to about 11-fold (27.69 ± 0.57 ng/mL), MDA to about twofold (0.12 ± 0.013 nM/g ptn), and NO to about twofold (5.31 ± 0.42 mmol/L) in the DOXO group, compared to controls . By treatment with glycine, in a dose-dependent manner, all these levels were decreased to the control/or to lower than the control range except for IL-10 levels were reduced but still higher than the normal control range in all the treated groups. TNF-α, IL-10, MDA, and NO levels were decreased by 45%, 82%, 42%, and 59%, respectively in Gp100, by 51%, 86%, 50%, and 71%, respectively in Gp150, and by 55%, 88%, 51%, and 74%, respectively in Gp200. Regarding comparing groups Gp100, Gp150, and Gp200 to each other, significantly IL-10 level was decreased by 24% in Gp150 and by 34% in Gp200 compared to Gp100 (P < 0.001), by 13% in Gp200 compared to Gp150 (P < 0.01), TNF-α level was declined by 11% in Gp150, and by 18% in Gp200 compared to Gp100 (P < 0.001), MDA level was decreased by 16% in Gp200 compared to Gp100 (P < 0.05), and NO level was reduced by 30% in Gp150, and by 38% in Gp200 compared to Gp100 , 0.014 ± 0.0006 (≈fourfold), and 0.43 ± 0.102 (≈twofold) in Gp100, to 1.52 ± 0.12 (≈fourfold), 0.016 ± 0.001 (≈fourfold), and 0.39 ± 0.08 (≈twofold) in Gp150, and 1.61 ± 0.18 (≈fourfold), 0.0175 ± 0.003 (≈fourfold), and 0.40 ± 0.081 (≈twofold) in Gp200. There were no significant differences noticed comparing NO/IL-10 changes in Gp100, Gp150, and Gp200 groups with each other. But for TNF-α/IL-10, only Gp150 and Gp200 showed significant increments by 18% and 25% compared to Gp100 (P < 0.001), while for MDA/IL-10, only Gp200 displayed a significant increase by 25% compared to Gp100 or by 9% compared to Gp150  from 2.05 ± 0.05, 0.025 ± 0.003, and 0.91 ± 0.16, respectively in control to 0.43 ± 0.026, 0.004 ± 0.0005, and 0.19 ± 0.02, P < 0.001) in the DOXO group compared to the control group . Glycine treatment caused elevations in antioxidants in a concentration-dependent manner. By glycine treatment, the SOD level was significantly increased to a level higher than that in both the DOXO and control groups. These increments were by 74%, 76%, and 84% in Gp100, Gp150, and Gp200, respectively, compared to the DOXO group, and by 12%, 13%, and 19%, respectively, compared to the control group. GSH level was also increased to about threefold compared to the DOXO group (P < 0.001), but to a level still lower than the control . Catalase and uric acid levels as well showed a dose-dependent increase within the glycine-treated groups, but their ranges were still lower than those in the control and DOXO groups. Changes in the levels of GSH, uric acid, SOD, or catalase between the Gp100, Gp150, and Gp200 groups were statistically non-significant when each group was compared to the other two  in the DOXO group, which were significantly reduced by glycine treatment, depending on glycine concentration. Na, K, urea, and creatinine, respectively, showed reduced levels by about 45%, 50%, 54%, 70% in Gp100; 46%, 51%, 65%, and 70% in Gp150; and 47%, 61%, 65%, and 71% in Gp200. In Fig. P < 0.001); all comparisons between each glycine dose group, Gp100, Gp150, or Gp200, and the other two groups in the levels of Ca, P, Na, K, and creatinine were statistically non-significant.Figure Histopathologically, heart tissue in the control group showed normal histology Fig. a. Heart In the current study, DOXO injection seemed connected with substantial declined body weight gain, collapsed heart, and low HW/BW ratio as outcomes of the cardiac damage linked with DOXO-induced toxicity ascertained by the congestion of the heart, necrosis, severe inflammation, and inflammatory cell infiltration seen in the histopathology of heart sections. These marked drops might be due to increased catabolism because of declined food intake attributed to the noticed loss of appetite and fatigue. This is in agreement with Abdelatty et al.  and IkewCPK and LDH leak mainly from damaged cardiac tissue. AST chiefly leaks from damaged hepatic tissues but also leeks from other damaged tissues, including cardiac tissues . In this regard, one can assume that during uncontrollable cardiotoxicity stimulated by DOXO, most of the macrophages have switched to the M2 phenotype, the more anti-inflammatory phenotype, which assists in tissue remodeling (Akata and van Eeden In heart failure, Na and K are the widely disturbed electrolytes. Na is required for the maintenance of typical blood volume and pressure. Association is established between hypernatremia and developing stroke and cardiovascular diseases (Patel and Joseph A limitation of our study is that we could not perform Echocardiography to evaluate cardiac function.In conclusion, DOXO-induced heart toxicity displayed a marked lowering in HW/BW ratio, significant elevations in the activities of CPK, AST, and LDH enzymes, significant elevations in Na, K, TNF-α, IL-10, MDA, and NO levels, and significant drops in levels of GSH and SOD. Glycine treatment reversed all these levels toward normal in a dose-dependent manner. Furthermore, histopathology findings were supportive of the biochemical findings. In addition, our findings indicate the dominance of M1 over M2 in control and glycine-treated groups but the dominance of M2 in the DOXO group. Upon all findings, the present study demonstrated that glycine protected mice’s myocardium against DOXO-induced cardiotoxicity by modulating oxidative stress, inflammation, and immunity. It seems that the anti-oxidative criteria of glycine are behind its exerted positive actions. Finally, we introduce glycine as a potentially good option against DOXO-induced myocardial damage.


Cognitive dysfunction is a frequent complication of type 2 diabetes mellitus (T2DM), usually accompanied by metabolic disorders. However, the metabolic changes in diabetic cognitive dysfunction (DCD) patients, especially compared to T2DM groups, are not fully understood. Due to the subtle differences in metabolic alterations between DCD groups and T2DM groups, the comprehensive detection of the untargeted metabolic profiles of hippocampus and urine samples of rats was conducted by LC–MS, considering the different ionization modes and polarities of the examined compounds, and feature-based molecular networking (FBMN) was performed to help identify differential metabolites from a comprehensive perspective in this study. In addition, an association analysis of the differential metabolites in hippocampus and urine was conducted by the O2PLS model. Finally, a total of 71 hippocampal tissue differential metabolites and 179 urine differential metabolites were identified. The pathway enrichment results showed that glutamine and glutamate metabolism, alanine, aspartate, and glutamate metabolism, glycerol phospholipid metabolism, TCA cycle, and arginine biosynthesis in the hippocampus of DCD animals were changed. Seven metabolites (AUC > 0.9) in urine appeared as key differential metabolites that might reflect metabolic changes in the target tissue of DCD rats. This study showed that FBMN facilitated the comprehensive identification of differential metabolites in DCD rats. The differential metabolites may suggest an underlying DCD and be considered as potential biomarkers for DCD. Large samples and clinical experiments are needed for the subsequent elucidation of the possible mechanisms leading to these alterations and the verification of potential biomarkers. In the wake of the widespread changes in lifestyle, diabetes is becoming more and more prevalent . As a coAs is well known, diabetes is a worldwide metabolic disease , and theFeature-based molecular networking (FBMN) has been from molecular networking combined with feature detection methods ,17. ThisThe purpose of the present work was to comprehensively identify differential metabolites and characterize the metabolic alterations in DCD rats versus T2DM rats. To acquire comprehensive metabolic profiles of urine and hippocampal tissue of diabetic rats with cognitive dysfunction, positive and negative ion modes and reverse-phase (RP) and hydrophilic interaction chromatography (HILIC) columns were used in LC-MS analyses, respectively. Furthermore, FBMN was conducted to fully attribute the metabolites. The detailed workflow is shown in LC-MS-grade acetonitrile and formic acid were purchased from Thermo Fisher Scientific . The water used for the LC-MS analysis was obtained from Watsons. The citric acid/sodium citrate buffer (Lot No. A20HR180485) and streptozocin  were purchased from Shanghai yuanye Bio-Technology .Twenty-four male SD rats  2019-0010) were housed. The temperature was controlled at 21–25 °C, the relative humidity was 55–65%, and the cycle of day and night was 12 h. The rats adapted to the environment for 7 days. All experiments were approved by the Ethics Committee for Animal Care and Treatment at Beijing University of Chinese Medicine (BUCM-4-2022033002-1051).−1) dissolved in the citric acid/sodium citrate buffer , and the control (CON) group rats were injected with vehicle. Seventy-two hours after the STZ injection, the rats with a blood glucose value > 16.7 mmol·L−1 were recognized as diabetic rats and were selected for the subsequent experiments. At the 13th week, the rats were divided into a CON group, a T2DM group , and a DCD group  by Morris water maze experiments. The flow diagram of animal grouping is shown in Eight rats were fed a normal diet, and 16 rats were fed a high-fat diet (HFD12491). After 5 weeks of HFD feeding, the rats were intraperitoneally injected with STZ .−1 solution, and the sample was homogenized at 60 Hz for 120 s at 4 °C. After centrifuging at 13,600× g for 15 min at 4 °C, 650 μL of the supernatant was taken and dried in nitrogen. The residue was subsequently redissolved in 70 μL of 50% methanol and centrifuged at 13,600× g for 10 min at 4 °C. The supernatant was stored for the RP analysis. To monitor the stability of the Q-Exactive Orbitrap MS system, quality control (QC) samples, which were pooled form 10 μL of each sample, were detected after every eight samples. As for the HILIC analysis, another part of the hippocampal tissue was treated with precooled 50% methanol to obtain a 20 mL·g−1 solution. Then, the sample was homogenized at 60 Hz for 120 s at 4 °C and centrifuged at 13,600× g for 15 min at 4 °C. The supernatant was taken for subsequent analyses. The QC samples were prepared from 10 μL of each sample and injected after every eight samples.Firstly, the hippocampal tissue was thawed at room temperature. Then, precooled methanol was added to a part of the hippocampal tissue to obtain a 20 mL·gg for 15 min at 4 °C, and the supernatant was collected for LC-MS analysis. The QC samples were obtained by mixing 10 μL of each sample. In addition, another 100 μL of urine was added to 400 μL of a methanol–acetonitrile solution . Then, the mixture was vortexed for 30 s and centrifuged at 13,600× g for 15 min at 4 °C. The supernatant was prepared for the HILIC analysis. The QC samples were prepared by mixing 10 μL of each sample and lined at regular intervals (every 8 samples).For the RP analysis, after thawing at room temperature, 100 μL urine was diluted with 100 μL water and vortexed for 30 s. Then, the mixture was centrifuged at 13,600× The UPLC analysis was conducted by a Vanquish UPLC system . The chromatographic conditions were as follows.v/v) (A) and 0.1% formic acid–acetonitrile (v/v) (B). The solvent program was set as follows: 0–1 min, 1% B; 1–4 min, 1–20% B; 4–6.5 min, 20–50% B; 6.5–8 min, 50–98% B; 8–10 min, 98% B; 10–10.1 min, 98–1% B; 10.1–13 min, 1% B. The flow rate was set at 0.4 mL·min−1, and the injection volume was 5 μL. For HILIC detection, a BEH amide column  was used at 40 °C. The mobile phase A was composed of water with 10 mM ammonium formate and 0.125% formic acid, the mobile phase B was composed of acetonitrile modified by the addition of 10 mM ammonium formate and 0.125% formic acid and followed at a rate of 0.4 mL·min−1 with a 13 min gradient: 0–2 min, 95% B; 2–7.7 min, 95–70% B; 7.7–9.5 min, 70–40% B; 9.5–10.5 min, 40–30% B; 10.5–11 min, 30–95% B; 11–13 min, 95% B. The flow rate was set to 0.3 mL·min−1, and the injection volume was 3 μL.For the RP analysis, a HSS T3 column  was used at 40 °C. The mobile phase consisted of 0.1% formic acid–water (v/v), while the mobile phase B was acetonitrile. The solvent gradient was set as follows: 0–4 min, 1% B; 4–12 min, 1–50% B; 12–16 min, 50–98% B; 16–18 min, 98% B; 18–18.1 min, 98–1% B; 18.1–20 min, 1% B. The oven temperature was kept at 40 °C, the flow rate was set to 0.3 mL·min−1 and the injection volume was 5 μL. For the HILIC analysis, the mobile phase A was composed of water with 10 mM ammonium formate and 0.1% formic acid (v/v), the mobile phase B was composed of acetonitrile/water  with 10 mM ammonium formate and 0.1% formic acid (v/v). A gradient run was set up as follows: 0–2 min, 95% B; 2–4.5 min, 95–85% B; 4–7.5 min, 85–75% B; 7.5–8.5 min, 75–70% B; 8.5–9.5 min, 70–40% B; 9.5–10 min, 40–30% B; 10–10.5 min, 30–95% B; 10.5–12 min, 95% B. The flow rate was kept at 0.3 mL min−1 with 40 °C, and the injection volume was 3 μL.For the RP analysis, the mobile phase A was 0.1% formic acid–water  was performed. The acquisition settings were as follows: spray voltage, 3.5 kV or −2.8 kV; capillary temperature, 275 °C; auxiliary gas heater, 350 °C; sheath gas, 40 ; auxiliary gas, 10 (Arb), and mass range, 70–1050 Da (hippocampus samples) or 60–900 Da (urine samples).R-m/z ion pairs, sample names, and peak intensifiers and then imported into the multivariate statistical software SIMCA-P 14.0  to perform an orthogonal partial least-squared discriminant analysis (OPLS-DA) analysis. Besides, the data processed by QI software were be imported into the Global Natural Products Social Molecular Networking (GNPS) website  for FBMN and visualized via Cytoscape software. In addition to GNPS, the HMDB database , mzcloud , CFM-ID 4.0 , and ClassyFire website  were used to characterize differential metabolites. Metabolic pathway analysis was performed using the MetaboAnalyst website  and KEGG database . The raw data were denoised, identified, aligned, and normalized by Progenesis QI software . The raw data were converted into a data matrix including tStatistical analysis was conducted on SPSS 22.0 . The statistical differences between two groups were compared by the Student’s t-test, and the statistical differences between multiple groups were compared by one-way ANOVA. Particularly, Student’s t-test and ANOVA analysis were conducted for normally distributed values, and the Mann–Whitney test was used for non-parametric values.−1) in SD rats after 5 weeks of high-fat feeding. Then, the diabetic rats were fed a high-fat diet for another 8 weeks. Finally, 13 surviving diabetic rats were subjected to Morris water maze experiments, and 6 rats with cognitive impairment were screened out by escape latency analysis. As shown in p < 0.01). The prolonged escape latency indicated a decline in cognitive ability in the DCD rats [T2DM was induced by intraperitoneal injection of STZ  was firstly identified as N-acetylaspartylglutamic acid on the online database. Next, m/z 388.0611 was assigned to the class of dipeptides by ClassyFire according to their common characteristic fragments with m/z of 58.0298, 96.0091, 128.0353, 146.0459, 155.0462, 285.0725, 303.0832 [The richness and complexity of metabolite information make it difficult to annotate differential metabolites. For the sake of identifying differential metabolites, FBMN through the GNPS platform, HMDB database, and ClassyFire website was carried out. In addition to online matching known metabolites with mass spectral libraries through the GNPS platform, FBMN can also allow the attribution of unknown metabolites by comparison with known metabolites based on similarity cosine scoring of the MS/MS spectra. For example, as shown in 303.0832 ,24. Fina303.0832 . As GNPSTo identify the key metabolic pathways of DCD, the differential metabolites were imported to MetaboAnalyst for pathway enrichment. As shown in Glutamate is the main excitatory neurotransmitter for the activation of the N-methyl-D-aspartic acid receptor (NMDAR) . When ex2 [As is known to all, the TCA cycle plays an important role in the energy metabolism of organisms ,33. The 2 ,35. The 2 ,37, whicArginine metabolism is related to the development of AD ,39,40. Ap ≤ 0.05 as threshold was conducted. The results showed that the metabolites in the hippocampal tissue had a strong correlation with those in urine. For example, glutamate, which acts as a neurotransmitter in the brain [As the target tissue of cognitive dysfunction, metabolic alterations in the hippocampal tissue are of great significance to elucidate the underlying mechanism, while urine is the analyte of first choice for metabolomics research due to its easy access and abundant metabolites. In order to further explore the potential biomarkers of urine related to those in the hippocampal tissue in rats with DCD, a model of O2PLS was constructed. The model shows excellent performance in the integrated analysis of multiple metabolomics data, and its estimated values are close to the actual parameters in both low- and high-dimensional data . The tophe brain , was neghe brain ,49,50, wThe diagnostic value of urine metabolites associated with those of hippocampus was evaluated by the ROC curve shown in C. AUC > With the prevalence of diabetes, DCD has attracted more and more attention. However, the issue of how to control DCD is still pending. Specifically, the molecular mechanisms of DCD have not been understood, and there is little information about the biomarkers of the disease. Since metabonomics research on the target sample of DCD is beneficial to uncover the molecular mechanisms of this dysfunction and the urine sample is the first choice to screen biomarkers, the analysis of differential metabolites in urine of DCD rats that could reflect the metabolic changes in the hippocampus is of great significance. The integration of metabonomics and FBMN will make the identification of differential metabolites more comprehensive and play an important role in the revelation of possible mechanisms and potential biomarkers.Our study indicates that FBMN facilitates the identification of differential metabolites based on the comprehensive detection of untargeted metabolic profiles in DCD rats. Totally, 71 hippocampus differential metabolites and 179 urine differential metabolites in DCD rats were identified. Glutamine and glutamate metabolism, alanine, aspartate, and glutamate metabolism, TCA cycle, and arginine biosynthesis appeared as the critical pathways in the hippocampal tissue of DCD rats. Seven metabolites (AUC > 0.9) in urine appeared as key differential metabolites that might reflect metabolic changes in the target tissue in DCD rats. The identification and analysis of differential metabolites allowed the description of metabolic alterations in DCD rats, providing new insights for the diagnosis and molecular mechanism of this disease. Large samples and clinical experiments are needed for the subsequent elucidation of the possible mechanisms underlying this condition and the verification of potential biomarkers.