Background: Sepsis is a life-threatening condition characterized by immune dysregulation, with significant mortality occurring during the late immunosuppressive phase. While astaxanthin (AST), a marine-derived tetraterpene, has shown anti-inflammatory potential in sepsis, its role in modulating immunosuppression remains unexplored. Objectives: This study aimed to investigate the immunomodulatory effects of AST on immunosuppressed macrophages in vitro, focusing on its ability to restore inflammatory responses and immune function. Methods: Using lipopolysaccharide (LPS)-stimulated RAW264.7 macrophages, we investigated AST’s immunomodulatory effects during immunosuppression. Cells were pretreated with AST followed by LPS stimulation (LPS1st) and restimulation. Cell viability was assessed using the MTT assay. Inflammatory cytokine tumor necrosis factor-alpha (TNF-α) and interleukin-6 (IL-6) production were measured by ELISA. NF-κB activity was determined via ELISA and immunofluorescence (IF). Bactericidal activity was evaluated using an assay with Escherichia coli. Phagocytic capacity was quantified using neutral red uptake. Reactive oxygen species (ROS) and nitric oxide (NO) levels were measured using respective detection kits. Transcriptomic analysis was performed using RNA-Seq, followed by gene set enrichment analysis (GSEA), gene ontology (GO), and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses. Results: The AST showed no cytotoxicity under both normal and LPS-challenged conditions. In immunosuppressed macrophages, AST pretreatment restored inflammatory responses, specifically TNF-α and IL-6 production, NF-κB activity, bactericidal function, and phagocytic capacity, accompanied by increased ROS and NO production. Transcriptomic analysis revealed AST’s regulation of multiple immune-related pathways, with significant enrichment of both innate and adaptive immune response pathways. Conclusions: Our findings demonstrate AST’s novel dual immunomodulatory properties in addressing both hyperinflammation and immunosuppression in sepsis, suggesting its potential as a therapeutic candidate, particularly for late-stage sepsis treatment.

The aim of present research in the first stage was to extract astaxanthin from Haematococcus pluvialis using acid-acetone method and then nanoencapsulation of the pigment using maltodextrin-sodium caseinate coating. In the next step, antioxidant and antibacterial activities of nanocapsules carrying astaxanthin and the free form of the pigment was evaluated. In order to evaluate antibacterial activity of the samples, Listeria monocytogenes, Staphylococcus aureus, Streptococcus iniae, Bacillus subtilis (Gram positive), Yersinia ruckeri, Escherichia coli and Enterobacter aerogenes (Gram negative) were used. The results showed that the antioxidant activity of nanocapsules carrying astaxanthin is significantly higher than the free form of pigment (p<0.05); In addition, this activity was improved by increasing the concentration of samples from 100 to 200 µg/ml (p<0.05). By astaxanthin nanoencapsulation, the diameter of non-growth zone of the studied bacteria increased (p<0.05), but minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of the pigment and its carrier nanocapsules decreased (p<0.05). According to the results of zone of inhibition, Gram positive (except Listeria monocytogenes) and Gram negative bacteria were resistant up to concentrations of 60 and 80 µg/ml of samples, respectively. In the following, the MIC and MBC of the pigment (free and nanoencapsulated forms) for the seven bacteria ranged from 50 to 400 and 100 to 500 µg/ml, respectively. The results of evaluation the antioxidant and antibacterial activities of nanocapsules carrying astaxanthin during storage period (30 days at 4ºC) indicated stability and no significant change of these properties (p>0.05). According to the values of diameter of non-growth zone, MIC and MBC, Listeria monocytogenes was the most sensitive bacteria against astaxanthin and its carrier nanocapsules. Based on the findings, astaxanthin extracted from Haematococcus pluvialis has antioxidant and antibacterial activities, and these properties are improved by the pigment nanoencapsulation using maltodextrin-sodium caseinate coating.

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astaxanthin is a xanthophylls, a fat-soluble, oxygenated pigment a member of the carotenoid family. It has a unique molecular structure that 500 times-gives it powerful antioxidant function. astaxanthin has 100 the antioxidant capacity of Vitamin E and 10 times the antioxidant capacity of beta-carotene and also stronger antioxidant than lutein, lycopene and tocotrienols. Mutagenesis of Phaffia rhodozyma was done with different concentrations of NTG. After many times of selections, mutated colonies were obtained that were resistant to 0.05% 2-Deoxyglucose. These were tested in GPY broth for astaxanthin formation. Four mutant’s colonies obtained with 250mg/ml of NTG. The highly pigmented mutants produced approximately 485 mg of total carotenoid per g yeast estimated as astraxanthin, compared with the parental strain which had 250 mg/g. The results of TLC analysis of carotenoid composition of P. rhodozyma mutant indicated that mutant strain produced five different carotenoids on the basis of polarity and RF values.

astaxanthin and β-Carotene are well-known carotenoids globally, covering more than half of the market demand for carotenoids. Haematococcus pluvialis microalgae are one of the most important sources of natural astaxanthin, consisting of up to 4% of its dry weight. The most critical challenge for this microalgae is the breakdown of the wall and the extraction of the pigment. In this study, chemical methods, including acid, acetone, and ionic solution, and physical processes such as ultrasound waves and magnetic stirrer, were used to break down the cell wall and meas-ure total astaxanthin in H. pluvialis, respectively. Due to the rapid oxidation of the pigment, in the next step, to extract and store astaxanthin from damaged cells, use olive oil. A spectrophotometer examined astax-anthin, monoester, and diester derivatives, and their amount was determined by thin layer chromatography (TLC) and high performance liquid chromatography (HPLC). The results showed that using acid treatment, ultrasound waves, and extraction by acetone is the best method to measure the amount of astaxanthin in the algae. The HPLC results also showed that the amount of astaxanthin monoester (88. 44%) was higher than the free forms (3. 76%) and diester (7. 82%) in the total content of extracted astaxanthin. In addition, the amount of total astaxanthin in the H. pluvialis was about 1. 6% of the dry weight of the algae.