Identification of Bioactive Natural Product from the Stems and Stem Barks of Cornus walteri: Benzyl Salicylate Shows Potential Anti-Inflammatory Activity in Lipopolysaccharide-Stimulated RAW 264.7 Macrophages

Cornus walteri (Cornaceae), known as Walter’s dogwood, has been used to treat dermatologic inflammation and diarrheal disease in traditional oriental medicine. As part of an ongoing research project to discover natural products with biological activities, the anti-inflammatory potential of compounds from C. walteri in lipopolysaccharide (LPS)-stimulated mouse RAW 264.7 macrophages were explored. Phytochemical analysis of the methanol extract of the stem and stem bark of C. walteri led to the isolation of 15 chemical constituents. These compounds were evaluated for their inhibitory effects on the production of the proinflammatory mediator nitric oxide (NO) in LPS-stimulated macrophages, as measured by NO assays. The molecular mechanisms underlying the anti-inflammatory activity were investigated using western blotting. Our results demonstrated that among 15 chemical constituents, lupeol and benzyl salicylate inhibited NO production in LPS-activated RAW 264.7 macrophages. Benzyl salicylate was more efficient than NG-monomethyl-L-arginine mono-acetate salt (L-NMMA) in terms of its inhibitory effect. In addition, the mechanism of action of benzyl salicylate consisted of the inhibition of phosphorylation of IκB kinase alpha (IKKα), IκB kinase beta (IKKβ), inhibitor of kappa B alpha (IκBα), and nuclear factor kappa B (NF-κB) in LPS-stimulated macrophages. Furthermore, benzyl salicylate inhibited the expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2). Taken together, these results suggest that benzyl salicylate present in the stem and stem bark of C. walteri has potential anti-inflammatory activity, supporting the potential application of this compound in the treatment of inflammatory diseases.

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ANTI-INFLAMMATORY ACTIVITY OF TINOCRISPOSIDE BY INHIBITING NITRIC OXIDE PRODUCTION IN LIPOPOLYSACCHARIDES-STIMULATED RAW 264.7 CELLS

Asian Journal of Pharmaceutical and Clinical Research ◽

10.22159/ajpcr.2018.v11i4.23739 ◽

2018 ◽

Vol 11 (4) ◽

pp. 149 ◽

Cited By ~ 1

Author(s):

Adek Zamrud Adnan ◽

Muhammad Taher ◽

Tika Afriani ◽

Annisa Fauzana ◽

Dewi Imelda Roesma ◽

...

Keyword(s):

Nitric Oxide ◽

Inflammatory Diseases ◽

Positive Control ◽

Inflammatory Activity ◽

Raw 264.7 Cells ◽

No Production ◽

Raw 264.7 ◽

Dependent Manner ◽

Anti Inflammatory

Objective: The aim of this study was to investigate in vitro anti-inflammatory activity of tinocrisposide using lipopolysaccharides (LPS)-stimulated RAW 264.7 macrophage cells. Tinocrisposide is a furano diterpene glycoside that was isolated in our previous study from Tinospora crispa.Methods: Anti-inflammatory effect was quantified spectrometrically using Griess method by measuring nitric oxide (NO) production after the addition of Griess reagent.Results: The sample concentrations of 1, 5, 25, 50, and 100 μM and 100 μM of dexamethasone (positive control) have been tested against the LPS-stimulated RAW 264.7 cells, and the results showed NO level production of 39.23, 34.00, 28.9, 20.25, 16.3, and 13.68 μM, respectively, and the inhibition level of 22.67, 33.00, 43.03, 60.10, 68.00, and 73%, respectively.Conclusions: From the study, it could be concluded that tinocrisposide was able to inhibit the formation of NO in the LPS-stimulated RAW 264.7 cells in concentration activity-dependent manner, with half-maximal inhibition concentration 46.92 μM. It can be developed as anti-inflammatory candidate drug because NO is a reactive nitrogen species which is produced by NO synthase. The production of NO has been established as a mediator in inflammatory diseases.

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Anti-Inflammatory Activity of 4-((1R,2R)-3-Hydroxy-1-(4-hydroxyphenyl)-1-methoxypropan-2-yl)-2-methoxyphenol Isolated from Juglans mandshurica Maxim. in LPS-Stimulated RAW 264.7 Macrophages and Zebrafish Larvae Model

Pharmaceuticals ◽

10.3390/ph14080771 ◽

2021 ◽

Vol 14 (8) ◽

pp. 771

Author(s):

Su-Hyeon Cho ◽

SeonJu Park ◽

Hoibin Jeong ◽

Song-Rae Kim ◽

Myeong Seon Jeong ◽

...

Keyword(s):

Nitric Oxide ◽

Inflammatory Responses ◽

Inflammatory Activity ◽

Raw 264.7 Cells ◽

No Production ◽

Raw 264.7 ◽

Dependent Manner ◽

Juglans Mandshurica ◽

Zebrafish Larvae ◽

Anti Inflammatory

Juglans mandshurica Maxim., a traditional folk medicinal plant, is widely distributed in Korea and China. In our previous study, we isolated a new phenylpropanoid compound, 4-((1R,2R)-3-hydroxy-1-(4-hydroxyphenyl)-1-methoxypropan-2-yl)-2-methoxyphenol (HHMP), from J. mandshurica. In the present study, we evaluated the anti-inflammatory activity of HHMP on lipopolysaccharide (LPS)-stimulated RAW 264.7 cells and zebrafish larvae. HHMP significantly inhibited LPS-induced nitric oxide (NO) and prostaglandin E2 production in a dose-dependent manner. Moreover, HHMP treatment considerably suppressed LPS-induced expression of inducible nitric oxide synthase and cyclooxygenase-2. We also demonstrated the mechanisms of HHMP inhibition of inflammatory responses in LPS-stimulated RAW 264.7 cells via Western blot analysis and immunofluorescence staining. Furthermore, HHMP significantly inhibited NO production in LPS-stimulated zebrafish larvae. Consequently, we established that HHMP significantly inhibited the LPS-induced activation of NF-κB and MAPK and the nuclear translocation of p65 in RAW 264.7 cells. Taken together, our findings demonstrate the effect of HHMP on LPS-induced inflammatory responses in vitro and in vivo, suggesting its potential to be used as a natural anti-inflammatory agent.

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Chemical Profiles and Anti-inflammatory Activity of the Essential Oils from Seseli gummiferum and Seseli corymbosum subsp. corymbosum

Natural Product Communications ◽

10.1177/1934578x1601101025 ◽

2016 ◽

Vol 11 (10) ◽

pp. 1934578X1601101

Author(s):

Alev Tosun ◽

Jaemoo Chun ◽

Igor Jerković ◽

Zvonimir Marijanović ◽

Maurizio A. Fenu ◽

...

Keyword(s):

Nitric Oxide ◽

Essential Oils ◽

Inflammatory Activity ◽

Raw 264.7 Cells ◽

No Production ◽

Raw 264.7 ◽

Gene Assay ◽

Germacrene D ◽

Ic50 Values ◽

Anti Inflammatory

The anti-inflammatory activity of the essential oils from Seseli corymbosum subsp. corymbosum Pall. ex Sm. (SC) and Seseli gummiferum Boiss. & Heldr. subsp. corymbosum (SG) was investigated for the first time on lipopolysaccharide (LPS)-stimulated RAW 264.7 cells. The main constituents (determined by GC-FID and GC-MS analyses) were germacrene D (54.1%) and sabinene (22.4%) in SG oil and β-phellandrene (29.2%), α-phellandrene (8.2%) and germacrene D (2.5%) in SC oil. SC and SG oils inhibited nitric oxide (NO) production with IC50 values of 56.1 and 108.2 μg/mL, respectively. The oils also inhibited prostaglandin E2 (PGE2) with IC50 values of 49.4 μg/mL (SC oil) and 95.5 μg/mL (SG oil). The inhibitory effect of SC and SG oils was accompanied by dose-dependent decreases of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) protein expressions in LPS-induced RAW 264.7 cells. The research of the reporter gene assay on nuclear factor κB (NF-κB) showed that SC and SG oils inhibited NF-κB transcriptional activity. The obtained results suggest that SC and SG oils exert the anti-inflammatory effects in LPS-stimulated RAW 264.7 cells by suppressing NF-κB activation.

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Bioactive Phytochemicals from Mulberry: Potential Anti-Inflammatory Effects in Lipopolysaccharide-Stimulated RAW 264.7 Macrophages

International Journal of Molecular Sciences ◽

10.3390/ijms22158120 ◽

2021 ◽

Vol 22 (15) ◽

pp. 8120

Author(s):

Dahae Lee ◽

Seoung Rak Lee ◽

Ki Sung Kang ◽

Ki Hyun Kim

Keyword(s):

Nitric Oxide ◽

Ethanol Extract ◽

Phytochemical Analysis ◽

Raw 264.7 ◽

Dependent Manner ◽

Concentration Dependent Manner ◽

Raw 264.7 Macrophages ◽

Iκb Kinase ◽

Cox 2 ◽

Anti Inflammatory

The fruits of the mulberry tree (Morus alba L.), known as white mulberry, have been consumed in various forms, including tea, beverages, and desserts, worldwide. As part of an ongoing study to discover bioactive compounds from M. alba fruits, the anti-inflammatory effect of compounds from M. alba were evaluated in lipopolysaccharide (LPS)-stimulated mouse RAW 264.7 macrophages. Phytochemical analysis of the ethanol extract of the M. alba fruits led to the isolation of 22 compounds. Among the isolated compounds, to the best of our knowledge, compounds 1, 3, 5, 7, 11, 12, and 14–22 were identified from M. alba fruits for the first time in this study. Inhibitory effects of 22 compounds on the production of the nitric oxide (NO) known as a proinflammatory mediator in LPS-stimulated RAW 264.7 macrophages were evaluated using NO assays. Western blot analysis was performed to evaluate the anti-inflammatory effects of cyclo(L-Pro-L-Val) (5). We evaluated whether the anti-inflammatory effects of cyclo(L-Pro-L-Val) (5) following LPS stimulation in RAW 264.7 macrophages occurred because of phosphorylation of IκB kinase alpha (IKKα), IκB kinase beta (IKKβ), inhibitor of kappa B alpha (IκBα), nuclear factor kappa B (NF-κB) and activations of inducible nitric oxide synthase (iNOS), and cyclooxygenase-2 (COX-2). Cyclo(L-Pro-L-Val) (5) significantly suppressed phosphorylations of IKKα, IKKβ, IκBα, and NF-κB and activations of iNOS and COX-2 in a concentration-dependent manner. Taken together, these results indicate that cyclo(L-Pro-L-Val) (5) can be considered a potential therapeutic agent for the treatment of inflammation-associated disorders.

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3-Amino-alkylated indoles: unexplored green products acting as anti-inflammatory agents

Future Medicinal Chemistry ◽

10.4155/fmc-2019-0234 ◽

2020 ◽

Vol 12 (1) ◽

pp. 5-17 ◽

Cited By ~ 4

Author(s):

Sarah Mazzotta ◽

Luca Frattaruolo ◽

Matteo Brindisi ◽

Cristina Ulivieri ◽

Francesca Vanni ◽

...

Keyword(s):

Nitric Oxide ◽

Inflammatory Activity ◽

Raw 264.7 ◽

Nitric Oxide Production ◽

Inhibitory Effects ◽

Green Products ◽

Raw 264.7 Macrophages ◽

Oxide Production ◽

Green Approach ◽

Anti Inflammatory

Aim: Over the years, indole has proved to be a versatile scaffold for the design of molecules acting as anti-inflammatory agents. Materials & Methods: A small library of 3-amino-alkylated indoles has been obtained by an optimized Mannich green approach. The anti-inflammatory activity of the new 3-amino-alkylated indoles, GLYC 0–10, was evaluated in RAW 264.7 macrophages. Results: The anti-inflammatory activity of the new 3-amino-alkylated indoles, GLYC 0–10, was evaluatedn and, among them, GLYC 4, 5 and 9 displayed the greatest inhibitory effects on nitric oxide production, with IC50 values of 5.41, 4.22 and 6.3 μM, respectively. Conclusion: Our outcomes, overall, highlight the importance of the indole substitution in the anti-inflammatory activity of these compounds, exerted by acting on the interlinked NF-κB/ERK1/2 pathways.

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IN-VITRO ANTI-INFLAMMATORY ACTIVITIES OF EXTRACTS FROM BACTERIA ASSOCIATED WITH MARINE SPONGES: THEONELLA SP.

Jurnal Teknologi ◽

10.11113/jt.v77.6758 ◽

2015 ◽

Vol 77 (25) ◽

Author(s):

Siti Aisha Mohd Radzi ◽

Yosie Andriani ◽

Habsah M. ◽

Tengku Sifzizul Tengku Mohamad ◽

Jasnizat Saidin

Keyword(s):

Nitric Oxide ◽

Secondary Metabolites ◽

Marine Organism ◽

Marine Sponges ◽

Inflammatory Activity ◽

Raw 264.7 Cells ◽

No Production ◽

Raw 264.7 ◽

Crude Extracts ◽

Anti Inflammatory

In recent years, high numbers of research focusing on secondary metabolites produced by terrestrial bacteria towards anti-inflammatory properties but, minority of them used bacteria associated to marine organism. In this study, 25 bacteria colonies were isolated from sponge Theonella sp. which was collected from Bidong Island, Terengganu. Cytotoxicity and anti-inflammatory activity of secondary metabolites produced by these bacteria were studied using macrophage cell lines: RAW 264.7 .Macrophage plays a role in mammalian immune system by providing defense against a foreign harmful substance for example Lipopolysaccharides (LPS) which induces production of pro-inflammatory mediators such as nitric oxide (NO). Inhibition of NO production in LPS stimulated RAW 264.7 cells is highly established assay to screen for anti-inflammatory activity. All isolates were cultured and supernatants were collected for the extraction of secondary metabolites using diaion HP-20 to obtain crude extracts. Evaluations of the cytotoxicity effect of the extracts on RAW 264.7 were done using MTT assay. The highest concentration sample producing lowest toxicity effect was used for anti-inflammatory assay. Macrophages were induced with LPS before treated with different concentrations of crude metabolites in anti-inflammatory assay. The effects of crude secondary metabolites extracted to the induced macrophage were monitored by observing the inhibition level of Nitric Oxide (NO) released by RAW 264.7 into supernatant and estimated from nitrate standard curve. A preliminary assay revealed that isolate label with TM1.8 and TM 1.9 possesses anti-inflammatory activities of 112.06% and 109.7% respectively for every 1ug/L sample. The result suggested that these two crude extracts were more effective in inhibiting NO production compared to positive control N-Monomethyl-L-arginine Monoacetate (L-NMMA) which showed an inhibitory activity at 87.41% for every 1ug/L sample.

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Chemical Characterization of the Hydroethanolic Extract of the Inner Stem Bark of Dilodendron bipinnatum. Comparative Cytotoxic Evaluation and Anti-inflammatory Potential of a Simple Mixture of its Isolates 3-O-β-Glucopyranosyl-β-sitosterol and 3-O-β-Glucopyranosyl-stigmasterol

Natural Product Communications ◽

10.1177/1934578x1901400107 ◽

2019 ◽

Vol 14 (1) ◽

pp. 1934578X1901400 ◽

Cited By ~ 1

Author(s):

Karoline Costa Lima ◽

Domingos Tabajara de Oliveira Martins ◽

Antonio Macho ◽

Ruberlei Godinho de Oliveira ◽

Eduarda Pavan ◽

...

Keyword(s):

Stem Bark ◽

Inflammatory Activity ◽

Raw 264.7 Cells ◽

Spectrometric Analysis ◽

No Production ◽

Raw 264.7 ◽

Anti Inflammatory ◽

Hydroethanolic Extract

Phytochemical investigation of the hydroethanolic extract of the inner stem bark of Dilodendron bipinnatum (HEDb) by column chromatography led to the separation of three major fractions: a) a mixture of phytosterols (ST mixture), including β-sitosterol (1), stigmasterol (2) and campesterol (3); b) a mixture of 3- O-β-glucopyranosyl-β-sitosterol (4) and 3- O-β-glucopyranosyl-stigmasterol (5) (SGP mixture); and c) epicatechin (6), as a single isolate. Their structures were determined by spectrometric analysis using 1H- and 13C-NMR spectroscopy, and GC-MS. The safety profile of the SGP mixture, when evaluated on RAW 264.7 cells, using the alamar blue® assay, exhibited no cytotoxic effects. The anti-inflammatory activity was comparatively analyzed in vivo using the lipopolysaccharide (LPS)-induced peritonitis model in mice, showing a strong reduction of leukocyte migration to the peritoneal cavity using both the SGP mixture and the HEDb. In vitro assessment of nitric oxide (NO) in the macrophagic RAW 264.7 cell line showed an inhibition of NO by the SGP mixture when cells were stimulated with LPS. Taken together, the results show an important contribution of the sterol glucoside mixture on the anti-inflammatory activity of HEDb. Also, one of the mechanisms for such inhibition seems to be a direct inhibition of NO production in stimulated macrophages.

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(3β,16α)-3,16-Dihydroxypregn-5-en-20-one from the Twigs of Euonymus alatus (Thunb.) Sieb. Exerts Anti-Inflammatory Effects in LPS-Stimulated RAW-264.7 Macrophages

Molecules ◽

10.3390/molecules24213848 ◽

2019 ◽

Vol 24 (21) ◽

pp. 3848 ◽

Cited By ~ 2

Author(s):

Seulah Lee ◽

Dahae Lee ◽

Su Cheol Baek ◽

Mun Seok Jo ◽

Ki Sung Kang ◽

...

Keyword(s):

Nitric Oxide ◽

Medicinal Plant ◽

Phytochemical Analysis ◽

No Production ◽

Raw 264.7 ◽

Raw 264.7 Macrophages ◽

Euonymus Alatus ◽

Cox 2 ◽

Anti Inflammatory ◽

Pharmacologically Active

To discover new pharmacologically active natural products, here, we performed the phytochemical analysis of a Korean medicinal plant. Euonymus alatus (Thunb.) Sieb. is a traditional medicinal plant that has been used as a remedy for various diseases in Asian countries. In particular, the cork cambium on the twigs of E. alatus has been used to treat dysmenorrhea, tumors, diabetes, and wound. Phytochemical analysis of the methanolic extract of E. alatus twigs led to the isolation of a sterol, which was identified as (3β,16α)-3,16-dihydroxypregn-5-en-20-one (1) by 1D and 2D nuclear magnetic resonance (NMR) spectroscopy and high-resolution electrospray ionization mass spectrometry. The stereochemistry of 1 was established with nuclear Overhauser effect spectroscopy (NOESY) analysis and comparison of electronic circular dichroism (ECD) data. To the best of our knowledge, the isolation of compound 1 from nature is first reported here, as well as the complete and revised NMR data assignment of 1. In lipopolysaccharide (LPS)-stimulated RAW-264.7 macrophages, compound 1 significantly inhibited nitric oxide (NO) production at an IC50 value of 12.54 ± 0.05 μM as well as the protein expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2). Moreover, the pre-treatment with compound 1 attenuated the LPS-induced phosphorylation of nuclear factor kappa B (NF-κB) p65 through the inhibition of the phosphorylation of IκB kinase alpha (IKKα), IKKβ, and inhibitor of kappa B alpha (IκBα). Compound 1 also inhibited the LPS-induced phosphorylation of p38, c-Jun NH2-terminal kinase (JNK), and extracellular signal-regulated kinase (ERK). Taken together, compound 1 may serve as an anti-inflammatory constituent of E. alatus twigs and its anti-inflammatory property is thought to be associated with the inhibition of NO production via suppression of iNOS and COX-2 expression through inhibition of IKKα/β, I-κBα and NF-κB p65 activation and downregulation of p38, JNK, and ERK mitogen-activated protein kinase signal pathways in RAW 264.7 macrophages. These findings also provide experimental evidence that compound 1 identified from E. alatus twigs could be a candidate for an anti-inflammatory agent.

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5,7-Dihydroxyflavone Analogues May Regulate Lipopolysaccharide-Induced Inflammatory Responses by Suppressing IκBα-Linked Akt and ERK5 Phosphorylation in RAW 264.7 Macrophages

Evidence-based Complementary and Alternative Medicine ◽

10.1155/2017/7898973 ◽

2017 ◽

Vol 2017 ◽

pp. 1-12 ◽

Cited By ~ 1

Author(s):

Atsuyoshi Nishina ◽

Kazue Shimizu ◽

Mamoru Koketsu ◽

Masayuki Ninomiya ◽

Daisuke Sato ◽

...

Keyword(s):

Signaling Pathways ◽

Inflammatory Mediators ◽

Inflammatory Responses ◽

Inflammatory Activity ◽

No Production ◽

Raw 264.7 ◽

Raw 264.7 Macrophages ◽

Expression Levels ◽

Anti Inflammatory ◽

Low Cytotoxicity

We studied the anti-inflammatory activity of twelve 5,7-dihydroxyflavone analogues in lipopolysaccharide- (LPS-) stimulated RAW 264.7 macrophages. We found that chrysin (1) and 4′-methoxytricetin (9) showed relatively significant anti-inflammatory activity and low cytotoxicity. Moreover,1and9recovered the expression levels of iNOS and COX2, as well as those of the intracellular inflammatory mediators IL-1βand IL-6, which were upregulated by LPS stimulation. In addition,1and9actively regulated the phosphorylation of IκBα, leading to the activation of NFκB. Phosphorylation of Akt and ERK5 (upstream of NFκB) by LPS stimulation was significantly regulated by1and9, as well as by BIX 02189 and LY 294002, which are phosphorylation inhibitors of ERK5 and Akt, respectively. The results suggest that compounds1and9may suppress the levels of iNOS and COX2 by regulating phosphorylation of Akt, ERK5, and IκBαand thus NFκB-related signaling pathways, resulting in anti-inflammatory effects in the cells. Because1and9showed low cytotoxicity and regulated both PGE2and NO production caused by inflammatory responses, they may hold promise as natural anti-inflammatory agents.

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In Vitro and In Vivo Anti-Inflammatory Effects of Sulfated Polysaccharides Isolated from the Edible Brown Seaweed, Sargassum fulvellum

Marine Drugs ◽

10.3390/md19050277 ◽

2021 ◽

Vol 19 (5) ◽

pp. 277

Author(s):

Lei Wang ◽

Hye-Won Yang ◽

Ginnae Ahn ◽

Xiaoting Fu ◽

Jiachao Xu ◽

...

Keyword(s):

Nitric Oxide ◽

Brown Seaweed ◽

Sulfated Polysaccharides ◽

Raw 264.7 ◽

Raw 264.7 Macrophages ◽

Sargassum Fulvellum ◽

Pharmaceutical Industries ◽

Anti Inflammatory

In the present study, the in vitro and in vivo anti-inflammatory effects of the sulfated polysaccharides isolated from Sargassum fulvellum (SFPS) were evaluated in lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages and zebrafish. The results indicated that SFPS improved the viability of LPS-stimulated RAW 264.7 macrophages from 80.02 to 86.80, 90.09, and 94.62% at the concentration of 25, 50, and 100 µg/mL, respectively. Also, SFPS remarkably and concentration-dependently decreased the production levels of inflammatory molecules including nitric oxide (NO), tumor necrosis factor-alpha, prostaglandin E2, interleukin-1 beta, and interleukin-6 in LPS-treated RAW 264.7 macrophages. In addition, SFPS significantly inhibited the expression levels of cyclooxygenase-2 and inducible nitric oxide synthase in LPS-treated RAW 264.7 macrophages. Furthermore, the in vivo test results indicated that SFPS improved the survival rate of LPS-treated zebrafish from 53.33 to 56.67, 60.00, and 70.00% at the concentration of 25, 50, and 100 µg/mL, respectively. In addition, SFPS effectively reduced cell death, reactive oxygen species, and NO levels in LPS-stimulated zebrafish. Taken together, these results suggested that SFPS possesses strong in vitro and in vivo anti-inflammatory activities, and could be used as an ingredient to develop anti-inflammatory agents in the functional food and pharmaceutical industries.

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