Evaluation of antioxidant and anti-inflammatory activity and identification of bioactive compound from the marine diatom, Odontella aurita extract

Increased production of reactive oxygen species (ROS) leads to oxidative stress, with its damaging effect extending to the mitochondria and plasma membrane. Further, prolonged inflammation can result in chronic disease development. The marine microdiatom Odontella aurita is recognized for its potential in food and pharmaceutical development. Moreover, it contains antioxidant and anti-inflammatory properties. However, studies regarding the efficacy of their varying extract forms and their underlying mechanisms remain scarce. Therefore, this study aims to explore the antioxidant and anti-inflammatory effects of Odontella aurita extracts obtained using various extraction methods (hot water, 70% ethanol, and chloroform:methanol (CM)). Among the three Odontella aurita extracts, the CM extract demonstrated superior efficacy in protecting RAW 264.7 cells from H 2 O 2 -induced cytotoxicity. It significantly lowered the levels of ROS and enhanced the expression of superoxide dismutase and glutathione peroxidase. Furthermore, the CM extract outperformed other extracts in inhibiting LPS-induced nitric oxide production, reducing mRNA levels in nitric oxide synthase, cyclooxygenase, and the proinflammatory cytokines interleukin IL-1β, IL-6, TNFα. Additionally, CM extract effectively suppressed the activation of NF-κB/IκBα and JAK2-STAT3 in LPS-induced RAW 264.7 cells. HPLC– UV analysis revealed a remarkable 33-fold higher fucoxanthin content in CM compared to the ethanol extract. GC–MS analysis identified elevated levels of cholest-5-en-3-ol, phytol, eicosapentaenoic acid methyl ester, methyl palmitate, palmitoleic acid methyl ester, and neophytadiene in the CM extract. These findings suggest that Odontella aurita CM extract is a promising antioxidant candidate for preventing or treating inflammatory diseases, consequently emphasizing its potential for further development.


Introduction
"Reactive Oxygen Species" (ROS) is a comprehensive term for molecular oxygen (O 2 ) derivatives that naturally occur as part of aerobic life processes.Increased formation of diverse ROS leads to molecular damage commonly referred to as oxidative stress.Among these ROS, hydrogen peroxide (H 2 O 2 ) has emerged as a primary and immediate inducer of oxidative stress within cells.The damaging effects extend to mitochondria and plasma membrane [1].By treating RAW264.7 cells with H 2 O 2 , researchers can simulate conditions of oxidative stress, allowing them to study the protective effects of compounds with antioxidant properties.This model helps in understanding how compounds may scavenge ROS and mitigate oxidative damage, thereby assessing their antioxidant potential.Superoxide dismutase (SOD) and glutathione peroxidase (GPx) are crucial in cellular defense against oxidative stress [2].SOD serves as a primary antioxidant enzyme, facilitating superoxide anion free radical (O 2

−
) dismutation into molecular O 2 and H 2 O 2 .This process effectively prevents the generation of highly reactive compounds, including peroxynitrite (ONOO −) or hydroxyl radicals (•OH) [3].Conversely, GPxs-a group of oxidoreductases-convert H 2 O 2 into water.Additionally, they catalyze the reduction of peroxide radicals to produce alcohols and O 2 [4].Therefore, enhancing the expression of SOD and GPx, with enhancing the activity of these enzymes, is important in protecting cells from H 2 O 2 -induced oxidative stress.Oxidative stress is a significant contributor to the initiation and progression of inflammation, thereby promoting the development of inflammation-related diseases [5].The interactions between inflammation and ROS significantly contribute to the pathogenesis of various conditions, including endothelial dysfunction, cancer, and other diseases [6].Therefore, employing antioxidant strategies through antioxidants remains important for immune regulation and inflammation suppression [7].
Inflammation is a physiological response of the immune system, serving to protect the body against various inflammatory triggers, such as toxic compounds, infections, and tissue injury [8].When appropriately regulated, acute and controlled inflammatory process serves as a defensive immune reaction.The purpose is to eliminate detrimental external stimuli, promote the healing process, and sustain tissue and organ homeostasis [9].However, prolonged and unchecked inflammation can result in chronic disease development [10].Therefore, identifying treatment approaches that curb excessive inflammatory responses becomes crucial.Macrophages play important roles in immune responses and actively contribute to protective responses during inflammation [11].Macrophage activation, triggered by lipopolysaccharides (LPS) derived from gram-negative bacteria, stimulates Toll-like receptor 4 (TLR4), initiating downstream signaling pathways such as mitogen-activated protein kinases (MAPKs) and nuclear factor (NF)-κB [12].Such activation leads to the release of various inflammatory mediators, including nitric oxide (NO), cyclooxygenase (COX)-2, interleukin (IL)-6, IL-1β, and tumor necrosis factor (TNF)-α [13].Therefore, inhibiting inflammatory mediators produced by macrophages is essential in managing inflammatory diseases.Treating RAW264.7 cells with LPS stimulates an inflammatory response, including the production of pro-inflammatory cytokines and mediators.This model allows researchers to study the anti-inflammatory effects of compounds by assessing their ability to suppress the release of inflammatory molecules and modulate immune responses.Another important inflammatory signaling pathway responsible for mediating immune responses is the Janus kinase (JAK)signal transducer and activator of transcription (STAT) signaling pathway [14].In macrophages, the elevation of inflammatory cytokines induced by LPS activates JAKs, leading to STAT phosphorylation.Subsequently, these phosphorylated STAT translocate to the nucleus, functioning as transcription factors that regulate target gene expression responsible for regulating inflammatory mediators [15].Therefore, strictly regulating or inhibiting JAK-STAT signaling activation is crucial to mitigate inflammation and manage diseases.
Recently, extensive research has been conducted to identify safe antioxidant and antiinflammatory compounds derived from diverse natural sources [16][17][18][19].The findings from these studies have yielded practical applications across diverse fields, specifically in health food development [20].However, further research is required to identify natural products with enhanced antioxidant and antiinflammatory properties.Additionally, improving extraction methods to isolate active ingredients from these natural sources is necessary.Odontella aurita contains several bioactive compounds, including fucoxanthin, eicosapentaenoic acid, chrysolaminarin, pigments, fibers, and phytosterols, which have demonstrated beneficial effects on human health [18,[21][22][23][24][25][26].Thus, it holds the potential for development as a feed, food, and functional material with high value-added properties [26,27].In a previous study, the supplementation of a high-fat diet (HFD) with freeze-dried Odontella aurita was found to mitigate HFD-induced insulin resistance [28].Furthermore, the aqueous extract of Odontella aurita exhibited antioxidant and antiinflammatory effects on human activated neutrophil granulocytes and stimulated human whole blood [29].However, studies on the antioxidant and antiinflammatory activities of various extracts of Odontella aurita and the mechanisms underlying these effects remain scarce.Therefore, this study aims to compare the antioxidant and antiinflammatory effects of Odontella aurita extracts obtained via various methods: hydrothermal (HW), 70% ethanol (EtOH), and chloroform:methanol at a ratio of 2:1 v/v (CM).Additionally, it also aims to reveal unidentified cellular components and fundamental mechanisms responsible for intracellular ROS inhibition and antiinflammatory effects.
Our findings revealed that among the evaluated extracts, the CM extract exhibited the most significant antioxidant and antiinflammatory properties.Moreover, the CM extract significantly reduced ROS levels and enhanced the expression of SOD and GPx family genes in H2O2-induced RAW 264.7 cells.It also inhibited proinflammatory molecules in LPS-induced RAW 264.7 cells, potentially by inhibiting NF-κB/IκBα and JAK-STAT signaling pathways.Therefore, we propose that Odontella aurita CM extract is a potential antioxidant candidate for preventing or treating inflammatory diseases.

Culture and dry Odontella aurita sample
Odontella aurita diatoms were sourced from the National Marine Biodiversity Institute of Korea and cultured at JDK Biotech Ltd. (Korea).The diatoms were cultivated in 20 L plastic transparent water bottles containing cultivation medium, with an inoculation of 0.1 g of Odontella aurita per 1 L of medium.The cultivation medium was prepared by mixing 20 L of Jeju lava seawater, which has been autoclaved at 121 °C for 15 min, with 10 mL of F/2 Broth (AlgaBoost (2000x) F/2; AusAqua Pty Ltd., AU), 1.5 g of Sodium Nitrate (1476; DUKSAN PURE CHEMI-CALS CO., LTD., Korean), and 2.25 mL of Sodium Silicate Solution (Si-Star Liquid; Myoungjeonbio Co., Ltd., Korean).In a culture room with an average PAR condition of 147, Jeju lava seawater was introduced into the water tank to maintain an average temperature of 20 °C.A 20-L incubator was placed in the water tank, and the air was injected using an air pump (HP-40, TECHNO TAKATSUKI CO., LTD., Japan) for 16 h daily.The incubation period lasted for 7 days.The incubated Odontella aurita underwent vacuum filtration using 5-µM qualitative filter paper.The raw material, dehydrated to < 85% moisture, was collected.Subsequently, the raw material was exposed to hot air at 60 °C in a Forced Convection Drying Oven (HB-502L; HANBAEK SCIENTIFIC CO., Korea) for 24 h, resulting in a hot air-dried sample containing < 5% moisture.

Extraction
Odontella aurita samples were subjected to extraction using three different methods following established protocols [30].Briefly, for the hot water (HW) extraction, powdered samples were mixed with water, autoclaved at 125 °C (15 min), for ethanol extractions, samples were combined with 400 mL of 70% ethanol, sonicated (45 min at 25 °C), and regarding the CM extractions, samples were mixed with 400 mL of CM, sonicated (45 min at 25 °C).After being maintained at 26 °C for 24 h, the extracts were filtered through a 150 mm filter paper.The resultant extracts were mixed with 400 mL of water and transferred to a separating funnel.After thorough mixing, the aqueous layers were separated.Finally, the extracts were collected, followed by evaporation using a rotary evaporator, and then freeze-dried at -65 °C for 72 h.

Determination of total polyphenol content
The measurement of total polyphenol content involved combining 125 μL of the samples with 1.375 mL of distilled water, followed by the addition of 500 μL of Folin-Ciocalteu's reagent.After a 3-min incubation in darkness, 1 mL of 10% Na 2 CO 3 was introduced, and the mixture underwent a 30-min reaction in darkness.The absorbance was measured at an optical density (OD) of 700 nm using a microplate reader.The data are expressed as milligrams of gallic acid per gram of equivalent extract.

Determination of DPPH• (2,2-diphenyl-1-picrylhydrazyl) radical scavenging activity assay
The DPPH radical scavenging activity was determined following established protocols [31].Each Odontella aurita extract and 200 μM DPPH solution prepared by dissolving in ethanol were mixed, 40 μL and 160 μL, respectively.The resulting mixture was then incubated in the dark at 37 °C for 30 min.Following this, the absorbance was measured at OD 517 nm.DPPH• scavenging ability was calculated using the formula: Activity (%) = (A -B)/A × 100 (where A represents absorbance of control, and B denotes absorbance of sample extract or standard), with catechin serving as the positive control.The halfmaximal effective concentration (EC 50 ) value for each sample was calculated using GraphPad Prism 9.3.1 software (GraphPad, Inc., La Jolla, CA, USA).

Determination of ABTS• (2,2ʹ-azino-bis 3-ethylbenzothiazoline-6-sulfonic acid) radical scavenging activity assay
The ABTS• radical scavenging assay was conducted following established protocols [31].After mixing 900 μL ABTS solution (7 mM ABTS with 2.45 mM potassium persulfate) and 100 μL each Odontella aurita extract for 3 min at 25 °C, absorbance was measured at D 734 nm.The ABTS• scavenging ability was calculated using the formula: Activity (%) = (A -B)/A × 100 (where A is the absorbance of control B represents the absorbance of sample extract or standard), with α-tocopherol serving as the positive control.The EC 50 value for each sample was calculated in the same way as the EC 50 value for DPPH scavenging capacity mentioned above.

Nitric oxide production assay
RAW 264.7 cells (10 5 cells/well) were cultured following established protocols [30].RAW 264.7 cells were starved for 24 h in DMEM medium (1% FBS (v/v) and 1% antibiotics) and then pretreated with Odontella aurita extract (concentrations of 8, 16, and 32 μg/mL) for 2 h before exposure to 200 ng/mL LPS for 22 h.Then, 100 μL of sodium nitrite standard solution or culture medium was mixed with 100 μL of Griess reagent, incubated at 25 °C for 10 min, and the OD 550 nm value of each sample was measured using a microplate reader.

Reactive oxygen species assay
The intracellular levels of ROS were assessed following established protocols [32].After a 6-h pre-treatment with the samples, RAW 264.7 cells were exposed to 500 μM H 2 O 2 for 1 h.Subsequently, 10 μM H 2 DCFDA was introduced to each dish for a 20-min incubation period, and the collected cells were analyzed using a flow cytometer from BD Biosciences Co., Franklin, NJ, USA.

qRT-PCR analysis
Total RNA was extracted using the TRIzol reagent, and it served as a template (total RNA 1 ng) for complementary DNA synthesis using the ReverTra Ace kit (Toyobo Co., Ltd., Osaka, Japan).The qRT-PCR products were analyzed using Bio-Rad CFX Manager 2.0 software (Bio-Rad Laboratories, Hercules, CA, USA).Table S1 lists the primer sequences used in this study.

Western blot assay
Western blotting was conducted following a previously described protocol [32].After incubation or treatment, cells were lysed using RIPA lysis buffer (Sigma Chemical Co., St. Louis, MO, USA).After quantitative protein analysis, cell lysates were separated using SDS-PAGE, and the separated proteins were then transferred to PVDF membranes.Following this, the membranes underwent incubation with various primary antibodies, with most used at 1:1000 dilutions, except for β-Actin (utilized at 1:10,000).Secondary antibodies (anti-rabbit and antimouse immunoglobulin G [IgG]; Vector Laboratories, Burlingame, CA, USA) were used at a 1:5000 dilution.Protein bands were detected using a BS E (Biosesang, Seongnam, South Korea).

Cell viability assays
The cell viability assay was conducted as previously described [32].RAW 264.7 cells were treated with various concentrations of Odontella aurita extract, cultured for 24 h, and then exposed to MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide) solution (100 μL).After incubation in MTT solution for 2 h, 150 μL of DMSO was added per well, and the microplates was shaken for 30 min in the dark.Absorbance at 570 nm was measured using a microplate reader, and all experiments were performed in triplicate.

High-performance liquid chromatography (HPLC) analysis
Fucoxanthin quantification in Odontella aurita extracts was conducted using high-performance liquid chromatography (DGU-20A 3R degassing UNIT , SHIMADZU Inc., Kyoto, Kyoto, Japan).Each extract (1 mg) was diluted in 1 mL MeOH, filtered through a 0.2 mm filter, and subjected to HPLC analysis.The mobile phase gradient was set up using 100% water (A) and 100% methanol (B), with the following steps: starting at 100% solvent A, it was held for 5 min before gradually decreasing to 50% in  3 min.Subsequently, the gradient decreased from 50 to 25% solvent A in 4 min before decreasing from 25 to 10% in 6 min.This was further reduced from 10 to 5% in 8 min.Finally, solvent A was reduced to 0% and maintained for 15 min before being increased back to 100% and held for 10 min.The injection volume was 20 μl/sample, with a flow rate of 1 ml/min, and the absorbance was recorded at 445 nm.Fucoxanthin content was determined using a standard curve at five different concentrations and expressed in milligrams per gram of dry-weight powder extract.All experiments were performed in triplicate.

Gas chromatography-mass spectrometry (GC-MS) analysis
The GC-MS analysis was carried out following a previously established protocol [32], utilizing a GCMS-QP-2010 Plus spectrometer equipped with a DB-5MS GC column (0.25 mm internal diameter, 0.25 µm film thickness, 30 m length) from Shimadzu Co., Nakagyoku, Kyoto, Japan.Each sample was injected at a volume of 1 µL in splitless mode.Helium served as the carrier gas at a flow rate of 1 mL/min.The temperature gradient exposure ranged from 80 °C-300 °C, with the following sequence: an initial 80 °C (5 min); then from 80 °C-280 °C (10 min; 5 °C/min); 280 °C-300 °C (10 min; 10 °C/min), resulting in total run time of 67 min.Mass spectra were detected using the W9N08 Wiley library 9.0 at 85% similarity.

Statistical analysis
The data presented are mean values with standard deviation obtained from triplicate experiments.Statistical analysis was conducted using one-way analysis of variance (ANOVA) with Tukey's multiple comparison tests in GraphPad Prism 9.3.1 software.Statistical significance was set at p < 0.05.

Extraction yield and total polyphenol content (TPC) of Odontella aurita extracts
Extraction yield (mass of extract/mass of dry matter × 100%) served as an indicator to analyze the effects of extraction conditions on the samples.Table 1 presents the results, showing varying extraction yields with the use of different solvents.The highest extract yield was observed with HW (23.4%), followed by that of EtOH (20.4%), and CM exhibited the lowest yield at 2.4%.Phenolics and flavonoids are known for their beneficial biological properties [33].Owing to their benefits, phenolic compounds have been extensively discussed as potential pharmaceutical agents [34].In the present study, the TPCs of different Odontella urita extracts were evaluated (Table 1).The findings reveal that the CM solvent extracts exhibited the highest TPC (5.086 ± 0.46 mg GAE/g dry weight), followed by EtOH (0.894 ± 0.20 mg GAE/g dry weight), with the lowest observed in the HW solvent extracts (0.534 ± 0.13 mg GAE/g dry weight) (Table 1).These findings highlight the effect of different solvents on the extraction of Odontella aurita in yielding varying levels of TPC.

Antioxidant activity
To assess the antioxidant activity, DPPH and ABTS radical scavenging assays were conducted on HW, EtOH, and CM extracts of Odontella aurita.The ABTS• radical scavenging activities of the various solvent extracts increased with rising extract concentrations from 2-8 mg/mL (Fig. 1A).Similarly, all the extracts indicated dosedependent DPPH radical scavenging activity (Fig. 1B).The CM extract displayed significantly higher radicalscavenging activity in both antioxidant assays compared to extracts obtained with other solvents.The CM extract exhibited the highest antioxidant activity, with EC 50 values of 1.821 ± 0.25 and 5.463 ± 0.47 mg/mL for the ABTS• and DPPH• assays, respectively.Conversely, no significant differences in the antioxidant properties were found between HW and EtOH.These findings suggest that the

Protective efficacy against H 2 O 2 -induced cytotoxicity in RAW 264.7 cells
Before investigating the antioxidant effects of Odontella aurita extract, we conducted a cytotoxicity assessment for each extract in a concentration-dependent manner using the MTT assay.In the cell viability assay, the HW extract exhibited no significant cytotoxicity up to 64 μg/ mL, while EtOH and CM demonstrated cytotoxic effects at 64 μg/mL (Additional file 1: Fig. S1A).As all extracts demonstrated no cytotoxicity up to 32 μg/mL, further confirmation of the antioxidant effects of each Odontella aurita extract was conducted at concentrations of 8, 16, and 32 μg/mL.

Effect of Odontella aurita extract on antioxidant enzyme superoxide dismutase and glutathione peroxidase
SOD and GPx play pivotal roles in cellular defense against oxidative stress, serving as essential antioxidants that protect against damage caused by free radicals [2].Given the inhibitory effect of Odontella aurita extracts on ROS levels in H 2 O 2 -stimulated RAW 264.7 cells, we conducted qRT-PCR analysis to investigate the expression of antioxidation genes (SOD and GPx family).As shown in Fig. 3, gene expression levels of antioxidation genes decreased in these cells following H 2 O 2 exposure.Pretreatment with the CM extract significantly enhanced the expression levels of SOD-1, SOD-2, GPx1, GPx2, GPx3, GPx4, GPx5, and GPx7 in H 2 O 2 -treated RAW 264 cells, whereas pre-treatment with EtOH and HW resulted in insignificant changes in gene expression levels, except SOD-2 at EtOH 16 and 32 µg/mL treated (Fig. 3A).Furthermore, pre-treatment with the CM extract had a pronounced effect on SOD activity in H 2 O 2 -treated RAW 264 cells, whereas pre-treatment with EtOH and HW did not change significantly (Fig. 3B).Additionally, pretreatment with the CM extract had a pronounced effect on GPx activity in H 2 O 2 -treated RAW 264 cells higher than other extracts (Fig. 3B).These findings indicate that the CM extract exhibits a protective effect against H 2 O 2 -induced cytotoxicity and demonstrates a superior intracellular ROS inhibition effect than the other extracts

Inhibition of NO production in LPS-stimulated RAW 264.7 cells
Maintaining a delicate balance between ROS and NO levels is important for cellular signaling, homeostasis, and the regulation of physiological functions [38,39].However, an excessive presence of NO or ROS has the potential to disrupt cellular homeostasis, induce irreversible DNA damage, and lead to the progression of diverse diseases, including cancer.Therefore, a promising therapeutic strategy involves modulating inflammatory responses by balancing the ROS and NO levels.LPS, a prevalent recognized activatior of macrophage, binds to specific receptors such as TLR4, triggering the secretion of proinflammatory cytokines [40,41].The Griess assay is a commonly employed method for evaluating the antiinflammatory effect of secondary metabolites or natural extracts.This is achieved by quantifying the levels of NO production in RAW 264.7 cells stimulated with LPS [40,42,43].In this study, the inhibitory effect of Odontella aurita extract on LPS-induced NO in RAW 264.7 cells was investigated.As shown in Fig. 4A and B, LPS exposure increased NO production in these cells, whereas pretreatment with a nontoxic concentration of CM extract demonstrated a suppressive effect on the level of NO production in LPS-stimulated RAW 264.7 cells.In contrast, HW and EtOH did not exhibit inhibitory effect on NO production.

Gene expression inhibition of proinflammation cytokines IL-1β, IL-6, TNFα, nitric oxide synthase (iNOS), and cyclooxygenase-2 (COX-2).
The excessive production of NO in the body is catalyzed by iNOS, a soluble enzyme that exhibits cytotoxicity in its dimeric form [44]. Various triggers, including bacterial pathogens, infections, and immunostimulatory cytokines, activate iNOS, leading to the generation of high NO concentrations through the activation of inducible nuclear factors such as NF-kB [44].Furthermore, in response to external stimuli, macrophages undergo phagocytosis of these pathogens and release pro-inflammatory cytokines like IL-1, IL-6, and TNFα.This process initiates and attracts other inflammatory cells to the inflammation site [45,46].TNFα, interleukin-I, tissue plasminogen activator, and LPS can trigger COX-2 enzymes, catalyzing the synthesis of prostaglandins from arachidonic acid.These prostaglandins serve as mediators and regulators of inflammation in the body [47].Therefore, inhibiting the expression of iNOS, COX-2, IL-1β, IL-6, and TNFα is important to mitigate excessive inflammatory responses.To further explore the anti-inflammatory properties of the CM extract, we examined changes in the expression of key pro-inflammatory cytokines.The mRNA expressions of iNOS, COX-2, IL-1β, IL-6, and TNFα induced by LPS (Fig. 5) were significantly reduced by CM extract.These findings collectively indicate that the CM extract exhibited the most potent anti-inflammatory activity among all Odontella aurita extracts.

Inhibition of NF-κB/IκBα and JAK-STAT signal in LPS-induced RAW 264.7 cells
Since the CM extract most effectively inhibited LPSinduced inflammation, our next focus was on identifying specific downstream signals of LPS-induced inflammation that were blocked by the CM extract.
Maintaining the homeostasis of the JAK/STAT signaling pathway is important for an organism's immune system because activation of this pathway is closely linked to the regulation of inflammation and immune responses.[5,48].In the presence of inflammation, an excessive generation and spread of pro-inflammatory mediators leads to the JAK/STAT pathway overactivation.STAT activation promotes the abnormal and heightened expression of pro-inflammatory cytokines, creating a positive feedback loop that ultimately exacerbates inflammatory diseases [49].A previous study suggested that JAK2, STAT1, and STAT3 activation contribute to pro-inflammatory responses.Moreover, it specifically activates STAT3, leading to adverse neuroinflammation in microglia [50].Inhibitors targeting JAK1/2, reducing the activation of both JAK1/2 and their downstream signaling molecules STAT1 and STAT3, prove effective in reducing the expression of inflammatory genes during the α-synuclein-induced inflammatory response [51].Similarly, inhibiting the activation of JAK1, JAK2, STAT1, STAT3, and STAT5 has been shown to reduce the expression of proinflammatory genes(IL-1α, IL-1β, IL-6, and IL-27) in the LPinduced inflammatory response in RAW 264.7 cells [52].In this study, we postulated that the CM extract might inhibit the activation of JAK-STAT signaling, given its association with LPS-induced inflammatory signals.Our findings confirmed a significant suppression of JAK2 and STAT3 activation by the CM extract (Fig. 6A).LPS, an extrinsic pathogen, directly binds to macrophage TLR4, activating macrophages and subsequently facilitating pathogen elimination through phagocytosis [53].The activated TLR4 signaling pathway causes excessive activation of downstream signaling pathways, including NF-κB and MAPK, through MyD88-dependent signaling, which ultimately results in overexpression of pro-inflammatory cytokines [54].Pro-inflammatory mediators produced during this process form a positive feedback loop that affects both the cells themselves and peripheral immune cells [55].This ultimately activates various immune cells and stimulates excessive production of proinflammatory cytokines, contributing to various inflammatory diseases [56].Consequently, inhibiting NF-κB/IκBα and/or MAPK activation becomes important to prevent the secretion of excessive pro-inflammatory mediators and the continuous activation of immune cells, forming a key aspect of the treatment strategy for inflammation-related diseases.In this study, we initially hypothesized that CM extract could interfere with the activation of NF-κB/ IκBα and MAPK induced by LPS.However, contrary to expectations, the CM extract did not inhibit LPS-induced MAPK signaling activation (Additional file 1: Fig. S2).Meanwhile, there was no significant difference in the p-IκBα/IκBα ratio between the LPS treatment only group and the control group.This finding contrasts with previous studies that demonstrated a higher protein expression level of p-IκBα/IκBα in the LPS treatment-only group than in the control group after short-term LPS treatment (4 h) [52].These outcomes were likely attributed to the activation of a feedback loop following prolonged LPS treatment (22 h).Nevertheless, our findings confirmed that the CM extract significantly prevents the activation of NF-κB/IκBα signaling pathway (Fig. 6B).In light of these results, we suggest that CM extract inhibits the activation of NF-κB/IκBα and JAK2-STAT3 pathways induced by LPS, thereby reducing the production of inflammatory cytokines such as IL-1b, IL-6, TNFα, and iNOS, COX-2.

Composition analysis
Fucoxanthin and its derivatives have been shown to offer numerous health benefits, including anti-obesity [22], anticancer [23], anti-inflammatory [18,24], and antioxidant effects [25].Since fucoxanthin is recognized as one of the predominant carotenoids in Odontella aurita [21], we quantified the fucoxanthin content in each Odontella aurita extract using HPLC-UV.The findings revealed that the CM extract exhibited the highest fucoxanthin content (51.75 ± 3.1 μg/mg dry weight), which was 33.17fold higher than the EtOH extract, while fucoxanthin content was not detected in the HW extract (Fig. 7A and  B).This result implies that the fucoxanthin content of Odontella aurita extracts depended on the solvent used and the extraction process.The highest accumulation of fucoxanthin was observed in the CM extract, aligning with the higher antioxidant and anti-inflammatory effects of the CM extract compared to the other extracts.
These findings indicate a positive correlation between the accumulation of fucoxanthin compounds and the activity of Odontella aurita extract.As anticipated, Pearson's correlation analysis demonstrated a significant positive relationship between fucoxanthin content and antioxidant and anti-inflammatory activities (Fig. 7C).These results demonstrate that fucoxanthin can possible potent candidates for the beneficial effects, anti-inflammatory and anti-oxidant of CM extract.We conducted a compositional analysis of the CM extract using GC-MS to identify its lipophilic constituents (Table 2).The result of the analysis suggested that the predominant compounds in Odontella aurita CM extract were fatty acids, including cholest-5-en-3-ol (3beta) (11.31% peak area), phytol (10.44% peak area), 5,8,11,14,17-eicosapentaenoic acid methyl ester (10.21% peak area), Methyl palmitate (6.35% peak area), Palmitoleic acid methyl ester (5.88% peak area), neophytadiene (2.34% peak area) and methyl gamma linolenoate (1.6% peak area).These compounds have been observed to exhibit antioxidant and anti-inflammatory effects.This implies that cholest-5en-3-ol (3beta), phytol, 5,8,11,14,17-eicosapentaenoic acid methyl ester, methyl palmitate, neophytadiene and methyl gamma linolenoate may contribute to supporting the potential antioxidant and anti-inflammatory activities of CM extracts.However, animal model studies must be performed to validate the in vitro results.Additionally, it will be necessary to evaluate the efficacy and safety profile of the extracts in living organisms, taking into account the individual efficacy, bioavailability, metabolism, and potential side effects of these compounds.Future studies could significantly contribute to a deeper understanding of the therapeutic potential of Odontella aurita extract and promote its development as a promising candidate for managing oxidative stress-related and inflammatory diseases.

Fig. 4
Fig. 4 Significant anti-inflammatory effects of the CM extract in LPS-induced RAW 264.7 cells.Before exposure to 200 ng/mL LPS for 22 h, the cells were pre-treated with Odontella aurita extract at varying concentrations (0, 8, 16, and 32 μg/mL) for 2h.A presents cytotoxic effects; B displays the measurement of Nitric oxide (NO) production levels using the NO assay.*p < 0.05 vs. untreated control group; #p < 0.05 vs. LPS only treated group; ns not significant; HW, hot water extract; EtOH, ethanol extract; CM, chloroform:methanol at a ratio of 2:1, v/v extract

Table 1
Yield extract and total polyphenol content of Odontella aurita extracts To validate the antioxidant effects of the 2 resulted in a dose-dependent and time-dependent reduction in cell viability (Additional file 1: Fig.S1B).Nonetheless, the cytotoxic effects induced by H 2 O 2 in RAW 264.7 cells were alleviated by the CM extract (Fig.2A).Considering that the accumulation of H 2 O 2 -induced oxidative stress may result in cell death, we investigated the potential of Odontella aurita extract to restore cell viability by reducing intracellular ROS levels.As shown in Fig.2B, ROS levels increased in RAW 264.7 cells following H 2 O 2 exposure.However, pre-treatment with Odontella aurita extract substantially reduced ROS production levels in H 2 O 2 -induced RAW 264.7 cells in a concentration-dependent manner.

Table 2
Tentatively identified major lipophilic compounds in Odontella aurita CM extract using GC-MS