Sargachromenol Purified from Sargassum horneri Inhibits Inflammatory Responses via Activation of Nrf2/HO-1 Signaling in LPS-Stimulated Macrophages

In this study, we isolated sargachromenol (SC) from Sargassum horneri and evaluated its anti-inflammatory effect in lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages. SC did not show cytotoxicity at all concentrations and effectively increased the cell viability by reducing the nitric oxide (NO) and intracellular reactive oxygen species (ROS) production in LPS-stimulated RAW 264.7 macrophages. In addition, SC decreased the mRNA expression levels of inflammatory cytokines (IL-1β, IL-6, and TNF-α) and inflammatory mediators (iNOS and COX-2). Moreover, SC suppressed the activation of nuclear factor kappa-light-chain-enhancer of activated B cells (NFκB) and mitogen-activated protein kinase (MAPK) signaling, whereas activated the nuclear factor erythroid 2-related factor 2/heme oxygenase-1 (Nrf2/HO-1) signaling in LPS-stimulated RAW 264.7 macrophages. Interestingly, the anti-inflammatory effect of SC was abolished by the inhibition of HO-1 in LPS-stimulated RAW 264.7 macrophages. According to the results, this study suggests that the antioxidant capacity of SC leads to its anti-inflammatory effect and it potentially may be utilized in the nutraceutical and pharmaceutical sectors.     

A Sulfated Polysaccharide from Saccharina japonica Suppresses LPS-Induced Inflammation Both in a Macrophage Cell Model via Blocking MAPK/NF-κB Signal Pathways In Vitro and a Zebrafish Model of Embryos and Larvae In Vivo

Inflammation is a complicated host-protective response to stimuli and toxic conditions, and is considered as a double-edged sword. A sulfated Saccharina japonica polysaccharide (LJPS) with a sulfate content of 9.07% showed significant inhibitory effects against lipopolysaccharide (LPS)-induced inflammation in RAW 264.7 macrophage cells and zebrafish. Its chemical and structural properties were investigated via HPLC, GC, FTIR, and NMR spectroscopy. In vitro experiments demonstrated that LJPS significantly inhibited the generation of nitric oxide (NO) and prostaglandin E₂ (PGE₂) via the downregulation of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) expression and suppressed pro-inflammatory cytokines tumor necrosis factor (TNF)-α and interleukin (IL)-1β production via the nuclear factor-kappa B (NF-κB) and mitogen-activated protein kinase (MAPK) signal pathways in LPS-induced RAW 264.7 cells. Moreover, LJPS showed strong protective effects against LPS-induced inflammatory responses in zebrafish, increasing the survival rate, reducing the heart rate and yolk sac edema size, and inhibiting cell death and the production of intracellular reactive oxygen species (ROS) and NO. Its convenience for large-scale production and significant anti-inflammatory activity indicated the potential application of LJPS in functional foods, cosmetics, and pharmaceutical industries.     

New Ophiobolins from the Deep-Sea Derived Fungus Aspergillus sp. WHU0154 and Their Anti-Inflammatory Effects

Deep-sea fungi have become a new arsenal for the discovery of leading compounds. Here five new ophiobolins 1–5, together with six known analogues 6–11, obtained from a deep-sea derived fungus WHU0154. Their structures were determined by analyses of IR, HR-ESI-MS, and NMR spectra, along with experimental and calculated electronic circular dichroism (ECD) analysis. Pharmacological studies showed that compounds 4 and 6 exhibited obvious inhibitory effects on nitric oxide (NO) production induced by lipopolysaccharide (LPS) in murine macrophage RAW264.7 cells. Mechanical study revealed that compound 6 could inhibit the inducible nitric oxide synthase (iNOS) level in LPS-stimulated RAW264.7 cells. In addition, compounds 6, 9, and 10 could significantly inhibit the expression of cyclooxygenase 2 (COX 2) in LPS-induced RAW264.7 cells. Preliminary structure-activity relationship (SAR) analyses revealed that the aldehyde group at C-21 and the α, β-unsaturated ketone functionality at A ring in ophiobolins were vital for their anti-inflammatory effects. Together, the results demonstrated that ophiobolins, especially for compound 6, exhibited strong anti-inflammatory effects and shed light on the discovery of ophiobolins as new anti-inflammatory agents.     

In vivo and in vitro anti-inflammatory activity of neorogioltriol, a new diterpene extracted from the red algae Laurencia glandulifera

Neorogioltriol is a tricyclic brominated diterpenoid isolated from the organic extract of the red algae Laurencia glandulifera. In the present study, the anti-inflammatory effects of neorogioltriol were evaluated both in vivo using carrageenan-induced paw edema and in vitro on lipopolysaccharide (LPS)-treated Raw264.7 macrophages. The in vivo study demonstrated that the administration of 1 mg/kg of neorogioltriol resulted in the significant reduction of carregeenan-induced rat edema. In vitro, our results show that neorogioltriol treatment decreased the luciferase activity in LPS-stimulated Raw264.7 cells, stably transfected with the NF-κB-dependent luciferase reporter. This effect on NF-κB activation is not mediated through MAPK pathways. The inhibition of NF-κB activity correlates with decreased levels of LPS-induced tumor necrosis factor-alpha (TNFα) present in neorogioltriol treated supernatant cell culture. Further analyses indicated that this product also significantly inhibited the release of nitric oxide and the expression of cyclooxygenase-2 (COX-2) in LPS-stimulated Raw264.7 cells. These latter effects could only be observed for neorogioltriol concentrations below 62.5 μM. To our knowledge, this is the first report describing a molecule derived from Laurencia glandulifera with anti-inflammatory activity both in vivo and in vitro. The effect demonstrated in vitro may be explained by the inhibition of the LPS-induced NF-κB activation and TNFα production. NO release and COX-2 expression may reinforce this effect.     

The effect of extraction process on polyphenol content, triterpene composition and bioactivity of yellow birch (Betula alleghaniensis Britton) extracts

Crude ethanol extracts from different tissues of Betula alleghaniensis: wood, inner and outer bark, foliage, and twigs were evaluated for their nitric oxide (NO)-radical scavenging activities and inhibitory effects on the production of NO in LPS/INFγ-stimulated RAW 264.7 macrophages. As a renewed interest in plant-derived drugs has led to an increased need for efficient extraction methods, ultrasonic-assisted extraction (UAE) was investigated and compared with conventional maceration. The lower energy consumption by the UAE process compared to maceration determined in our study qualifies also this extraction as an environmentally friendly process. Our results indicate that the foliage extract has the most potent radical scavenging capacity while wood and twigs extracts exert the highest inhibitory effects on the production of NO in LPS/INFγ-stimulated RAW 264.7 macrophages. Extracts obtained by UAE present lower cytotoxic activity on RAW cells. Our results also demonstrate that ultrasounds help to selectively extract the bioactive molecules from foliage, twigs and wood, which inhibit the production of NO by macrophages. The highest total phenol content has been determined for the inner bark extracts and the flavonoids are the major phenolics present in foliage extracts. Poor correlations determined between the total phenols and radical scavenging capacity of the extracts indicates to the synergistic or antagonist effects of molecules present, various polyphenols and triterpenes being identified in the extracts studied in this research.     

Immunostimulative Activity of Low Molecular Weight Chitosans in RAW264.7 Macrophages

Chitosan and its derivatives such as low molecular weight chitosans (LMWCs) have been reported to exert many biological activities, such as antioxidant and antitumor effects. However, complex and molecular weight dependent effects of chitosan remain controversial and the mechanisms that mediate these complex effects are still poorly defined. This study was carried out to investigate the immunostimulative effect of different molecular weight chitosan in RAW264.7 macrophages. Our data suggested that two LMWCs (molecular weight of 3 kDa and 50 kDa) both possessed immunostimulative activity, which was dependent on dose and, at the higher doses, also on the molecular weight. LMWCs could significantly enhance the the pinocytic activity, and induce the production of tumor necrosis factor α (TNF-α), interleukin 6 (IL-6), interferon-γ (IFN-γ), nitric oxide (NO) and inducible nitric oxide synthase (iNOS) in a molecular weight and concentration-dependent manner. LMWCs were further showed to promote the expression of the genes including iNOS, TNF-α. Taken together, our findings suggested that LMWCs elicited significantly immunomodulatory response through up-regulating mRNA expression of proinflammatory cytokines and activated RAW264.7 macrophage in a molecular weight- and concentration-dependent manner.     

Anti-Inflammatory Activity of Tanzawaic Acid Derivatives from a Marine-Derived Fungus Penicillium steckii 108YD142

Chemical investigation of a marine-derived fungus, Penicillium steckii 108YD142, resulted in the discovery of a new tanzawaic acid derivative, tanzawaic acid Q (1), together with four known analogues, tanzawaic acids A (2), C (3), D (4), and K (5). The structures of tanzawaic acid derivatives 1–5 were determined by the detailed analysis of 1D, 2D NMR and LC-MS data, along with chemical methods and literature data analysis. These compounds significantly inhibited nitric oxide (NO) production and the new tanzawaic acid Q (1) inhibited the lipopolysaccharide (LPS)-induced inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) proteins and mRNA expressions in RAW 264.7 macrophages. Additionally, compound 1 reduced the mRNA levels of inflammatory cytokines. Taken together, the results of this study demonstrated that the new tanzawaic acid derivative inhibits LPS-induced inflammation. This is the first report on the anti-inflammatory activity of tanzawaic acid Q (1).     

Particulate Matter-Induced Inflammation/Oxidative Stress in Macrophages: Fucosterol from Padina boryana as a Potent Protector,Activated via NF-κB/MAPK Pathways and Nrf2/HO-1 Involvement

Fucosterol is a phytosterol that is abundant in marine brown algae and is a renowned secondary metabolite. However, its ability to protect macrophages against particulate matter (PM) has not been clarified with regard to inflammation; thus, this study aimed to illustrate the above. Padina boryana, a brown algae that is widespread in Indo–Pacific waters, was applied in the isolation of fucosterol. Isolation was conducted using silica open columns, while identification was assisted with gas chromatography-mass spectroscopy (GC-MS) and NMR. Elevated levels of PM led the research objectives toward the implementation of it as a stimulant. Both inflammation and oxidative stress were caused due the fact of its effect. RAW 264.7 macrophages were used as a model system to evaluate the process. It was apparent that the increased NO production levels, due to the PM, were mediated through the inflammatory mediators, such as inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2) and pro-inflammatory cytokines (i.e., interleukin-6 (IL-6), interleukin-1 (IL-1β) and tumor necrosis factor-α (TNF-α), including prostaglandin E2 (PGE₂)). Further, investigations provided solid evidence regarding the involvement of NF-κB and mitogen-activated protein kinases (MAPKs) in the process. Oxidative stress/inflammation which are inseparable components of the cellular homeostasis were intersected through the Nrf2/HO-1 pathway. Conclusively, fucosterol is a potent protector against PM-induced inflammation in macrophages and hence be utilized as natural product secondary metabolite in a sustainable manner.     

Screening of In-Vitro Anti-Inflammatory and Antioxidant Activity of Sargassum ilicifolium Crude Lipid Extracts from Different Coastal Areas in Indonesia

Sargassum brown seaweed is reported to exhibit several biological activities which promote human health, such as anticancer, antimicrobial, antidiabetic, anti-inflammatory, and antioxidant activity. This study aimed to investigate the anti-inflammatory and antioxidant activity of crude lipid extracts of Sargassum ilicifolium obtained from four different coastal areas in Indonesia, namely Awur Bay–Jepara (AB), Pari Island–Seribu Islands (PI), Sayang Heulang Beach–Garut (SHB), and Ujung Genteng Beach–Sukabumi (UGB). Results showed that treatment of RAW 264.7 macrophage cells with UGB and AB crude lipid extracts (12.5–50 µg/mL) significantly suppressed the nitric oxide production after lipopolysaccharide stimulation, both in pre-incubated and co-incubated cell culture model. The anti-inflammatory effect was most marked in the pre-incubated cell culture model. Both two crude lipid extracts showed 2,2-diphenyl-1-picrylhydrazyl radical scavenging activity and high ferric reducing antioxidant power, which were amounted to 36.93–37.87 µmol Trolox equivalent/g lipid extract and 681.58–969.81 µmol FeSO₄/g lipid extract, respectively. From this study, we can conclude that crude lipid extract of tropical S. ilicifolium can be further developed as a source of anti-inflammatory and antioxidant agent.     

Morphological and Proteomic Analyses Reveal that Unsaturated Guluronate Oligosaccharide Modulates Multiple Functional Pathways in Murine Macrophage RAW264.7 Cells

Alginate is a natural polysaccharide extracted from various species of marine brown algae. Alginate-derived guluronate oligosaccharide (GOS) obtained by enzymatic depolymerization has various pharmacological functions. Previous studies have demonstrated that GOS can trigger the production of inducible nitric oxide synthase (iNOS)/nitric oxide (NO), reactive oxygen species (ROS) and tumor necrosis factor (TNF)-α by macrophages and that it is involved in the nuclear factor (NF)-κB and mitogen-activated protein (MAP) kinase signaling pathways. To expand upon the current knowledge regarding the molecular mechanisms associated with the GOS-induced immune response in macrophages, comparative proteomic analysis was employed together with two-dimensional electrophoresis (2-DE), matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/TOF MS) and Western blot verification. Proteins showing significant differences in expression in GOS-treated cells were categorized into multiple functional pathways, including the NF-κB signaling pathway and pathways involved in inflammation, antioxidant activity, glycolysis, cytoskeletal processes and translational elongation. Moreover, GOS-stimulated changes in the morphologies and actin cytoskeleton organization of RAW264.7 cells were also investigated as possible adaptations to GOS. This study is the first to reveal GOS as a promising agent that can modulate the proper balance between the pro- and anti-inflammatory immune responses, and it provides new insights into pharmaceutical applications of polysaccharides.     

Total Synthesis and Anti-Inflammatory Bioactivity of (−)-Majusculoic Acid and Its Derivatives

The first total synthesis of marine natural product, (−)-majusculoic acid (1) and its seven analogs (9–15), was accomplished in three to ten steps with a yield of 3% to 28%. The strategy featured the application of the conformational controlled establishment of the trans-cyclopropane and stereochemical controlled bromo-olefination or olefination by Horner–Wadsworth–Emmons (HWE) reaction. The potential anti-inflammatory activity of the eight compounds (1 and 9–15) was evaluated by determining the nitric oxide (NO) production in the lipopolysaccharide (LPS)-induced mouse macrophages RAW264.7. (−)-Majusculoic acid (1), methyl majusculoate (9), and (1R,2R)-2-((3E,5Z)-6-bromonona-3,5-dien-1-yl)cyclopropane-1-carboxylic acid (12) showed significant effect with inhibition rates of 33.68%, 35.75%, and 43.01%, respectively. Moreover, they did not show cytotoxicity against RAW264.7 cells, indicating that they might be potential anti-inflammatory agents.     

Modulation of Lipopolysaccharide Stimulated Nuclear Factor kappa B Mediated iNOS/NO Production by Bromelain in Rat Primary Microglial Cells

Background:

Microglial cells act as the sentinel of the central nervous system .They are involved in neuroprotection but are highly implicated in neurodegeneration of the aging brain. When over-activated, microglia release pro-inflammatory factors, such as nitric oxide (NO) and cytokines, which are critical in eliciting neuroinflammatory responses associated with neurodegenerative diseases. This study examined whether bromelain, the pineapple-derived extract, may exert an anti-inflammatory effect in primary microglia and may be neuroprotective by regulating microglial activation.

Methods:

Following the isolation of neonatal rat primary microglial cells, the activation profile of microglia was investigated by studying the effects of bromelain (5, 10, 20, and 30 µg/ml) on the levels of NO, inducible nitric oxide synthase (iNOS), and nuclear factor kappa B (NF-κB) in microglia treated with lipopolysaccharide (LPS) (1 µg/ml). Data were analyzed using Student''s t-test. P values less than 0.05 were considered to be statistically significant, compared with the LPS-treated group without bromelain.

Results:

Results showed that pretreatment of rat primary microglia with bromelain, decreased the production of NO induced by LPS (1 µg/ml) treatment in a dose-dependent manner. Bromelain (30 µg/ml) also significantly reduced the expression of iNOS at mRNA level and NF-κB at protein level. Moreover, the study of mitochondrial activity in microglia indicated that bromelain had no cytotoxicity at any of the applied doses, suggesting that the anti-inflammatory effects of bromelain are not due to cell death.

Conclusion:

Bromelain can be of potential use as an agent for alleviation of symptoms in neurodegenerative diseases.     

Sarcocrassocolides M-O, bioactive cembranoids from the Dongsha Atoll soft coral Sarcophyton crassocaule

Three new cembranoids, sarcocrassocolides M-O (1-3), have been isolated from the soft coral Sarcophyton crassocaule. The structures of the metabolites were determined by extensive spectroscopic analysis. Compounds 1-3 were shown to exhibit moderate cytotoxicity toward a limited panel of cancer cell lines and display significant in vitro anti-inflammatory activity in LPS-stimulated RAW264.7 macrophage cells by inhibiting the expression of the iNOS protein.     

Molecular Mechanism of Anti-Inflammatory Activities of a Novel Sulfated Galactofucan from Saccharina japonica

Seaweed of Saccharina japonica is the most abundantly cultured brown seaweed in the world, and has been consumed in the food industry due to its nutrition and the unique properties of its polysaccharides. In this study, fucoidan (LJNF3), purified from S. japonica, was found to be a novel sulfated galactofucan, with the monosaccharide of only fucose and galactose in a ratio of 79.22:20.78, and with an 11.36% content of sulfate groups. NMR spectroscopy showed that LJNF3 consists of (1→3)-α-l-fucopyranosyl-4-SO₃ residues and (1→6)-β-d-galactopyranose units. The molecular mechanism of the anti-inflammatory effect in RAW264.7 demonstrated that LJNF3 reduced the production of nitric oxide (NO), and down-regulated the expression of MAPK (including p38, ENK and JNK) and NF-κB (including p65 and IKKα/IKKβ) signaling pathways. In a zebrafish experiment assay, LJNF3 showed a significantly protective effect, by reducing the cell death rate, inhibiting NO to 59.43%, and decreasing about 40% of reactive oxygen species. This study indicated that LJNF3, which only consisted of fucose and galactose, had the potential to be developed in the biomedical, food and cosmetic industries.     

Anti-Inflammatory and Analgesic Effects of the Marine-Derived Compound Excavatolide B Isolated from the Culture-Type Formosan Gorgonian Briareum excavatum

In recent years, several marine-derived compounds have been clinically evaluated. Diterpenes are secondary metabolites from soft coral that exhibit anti-inflammatory, anti-tumor and cytotoxic activities. In the present study, we isolated a natural diterpene product, excavatolide B, from cultured Formosan gorgonian Briareum excavatum and investigated its anti-inflammatory activities. We found that excavatolide B significantly inhibited the mRNA expression of the proinflammatory mediators, inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2), in lipopolysaccharide (LPS)-challenged murine macrophages (RAW 264.7). We also examined the anti-inflammatory and anti-nociceptive effects of excavatolide B on intraplantar carrageenan-induced inflammatory responses. Excavatolide B was found to significantly attenuate carrageenan-induced nociceptive behaviors, mechanical allodynia, thermal hyperalgesia, weight bearing deficits and paw edema. In addition, excavatolide B inhibited iNOS, as well as the infiltration of immune cells in carrageenan-induced inflammatory paw tissue.     

Characterization of Molecular Species and Anti-Inflammatory Activity of Purified Phospholipids from Antarctic Krill Oil

The phospholipids (PLs) from Antarctic krill oil were purified (>97.2%) using adsorption column chromatography. Forty-nine PL molecular species were characterized by ultrahigh-performance liquid chromatography-quadrupole-time-of-flight mass spectrometry (UHPLC-Q-TOF-MS). Most of molecular species contained eicosapentaenoic acid (EPA, 20:5), docosahexaenoic acid (DHA, 22:6), docosapentaenoic acid (DPA, 22:5), and arachidonic acid (AA, 20:4). Notably, a special species PC (20:5/22:6) (1298.17 nmol/g) and many ether PLs were detected. The Antarctic krill PL liposome (IC₅₀ = 0.108 mg/mL) showed better anti-inflammatory activity than crude Antarctic krill oil (IC₅₀ = 0.446 mg/mL). It could block NF-κB signaling pathway via suppression of IκB-α degradation and p65 activation and dose-dependently reduce the cellular content of inflammatory mediators including nitric oxide (NO), reactive oxygen species (ROS), and inflammatory cytokines in lipopolysaccharide (LPS)-stimulated RAW 264.7 cells. In addition, it can suppress carrageenan-induced mouse paw swelling. Results from the present study could provide a reference for better evaluation of nutritional and medicinal values of Antarctic krill oil.     

Characterization and online detection of surfactin isomers based on HPLC-MS(n) analyses and their inhibitory effects on the overproduction of nitric oxide and the release of TNF-α and IL-6 in LPS-induced macrophages

A rapid method for characterization and online detection of surfactin isomers was developed based on HPLC-MS(n) (n = 1, 2, 3) analyses, and many surfactin isomers were detected and characterized from the bioactive fraction of the mangrove bacterium Bacillus sp. Inhibitory activities of surfactin isomers on the overproduction of nitric oxide and the release of TNF-α and IL-6 in LPS-induced macrophages were systematically investigated. It was revealed that the surfactin isomers showed strong inhibitory properties on the overproduction of nitric oxide and the release of IL-6 on LPS-induced murine macrophage cell RAW264.7 with IC(50) values ranging from 1.0 to 7.0 μM. Structure-activity relationship (SAR) studies revealed that the existence of the free carboxyl group in the structure of surfactin isomers was crucial. These findings will be very helpful for the development of this novel kind of natural product as new anti-inflammatory agents.