Nitric Oxide Production In RAW Research Articles

Revealing the anti-inflammatory activity of Euphorbia hirta extract: transcriptomic and nitric oxide production analysis in LPS-Induced RAW 264.7 cells

ABSTRACT Excessive production of nitric oxide (NO) dan pro-inflammatory cytokines can lead to tissue damage and chronic inflammation. Euphorbia hirta presents an interesting opportunity for plant-derived anti-inflammatory agents as it contains a range of compounds which have demonstrated anti-inflammatory effects. This study investigates the anti-inflammatory properties of E. hirta extract on gene expression and NO production in LPS-treated RAW 264.7 cells. The results demonstrate that E. hirta extracts effectively suppress NO production in RAW 264.7 cells. Furthermore, the extract inhibits iNOS protein expression and reduces the expression of pro-inflammatory cytokines TNF-α and IL−12. RNA sequencing revealed significant alterations in gene expression, including 167 upregulated and 56 downregulated genes-related inflammation pathways. The study concludes that E. hirta extract possesses anti-inflammatory activity by modulating gene expression and inhibiting NO production in LPS-treated RAW 264.7 cells. The extract influences genes associated with inflammation signalling pathways, including cytokine signalling, NF-κB, iNOS and JAK-STAT pathways.

New alkaloids from Stemona tuberosa and structural revision of tuberostemonols P and R

Three Stemona alkaloids named stemotuberines A–C (1–3) with unique C17N frameworks, presumably formed by elimination of the C-11–C-15 lactone ring of the stichoneurine skeleton, were isolated from the roots of Stemona tuberosa. Their structures were elucidated by spectroscopic analysis, X-ray diffraction, and computational methods. Compounds 2 and 3 showed inhibition (IC50 values of 37.1 and 23.2 μM, respectively) against LPS-induced nitric oxide production in RAW 264.7 cells. In addition, concern was expressed about the reported plant origin (S. sessilifolia) of the recently described alkaloids tuberostemonols O–R (4–7), which should be S. tuberosa. NMR calculations indicated structural misassignment of these compounds except for 6. Isolation of tuberostemonol P (5) from our material of S. tuberosa allowed for a close examination of the spectroscopic data leading to the revised structure 5a. Tuberostemonol R (7) was found to have identical 1H and 13C NMR data to the well-known alkaloid croomine, and therefore its structure including relative stereochemistry must be revised as 7a.

Enhancing the nitric oxide inhibitory activity using a combination of plant essential oils and mixture design approach

The synergistic effects of essential oils (EOs) from three aromatic plant species, Foeniculum vulgare subsp. piperitum (C.Presl) Bég. (FV), Origanum heracleoticum L. (OH) and Lavandula austroapennina N.G.Passal., Tundis & Upson. (LA), were evaluated for their inhibitory properties on nitric oxide production in RAW 264.7 macrophages stimulated with lipopolysaccharide (LPS). We utilized a Design of Experiments (DoE) methodology to enhance a formulation by combining three Essential Oils (EOs), while simultaneously taking into account two response variables, maximization of NO inhibition with minimum cytotoxicity. The optimal blend of components was predicted, and the statistical outcome's efficacy was experimentally verified. The combination corresponding to 87.7 % FV, 12.3 % LA and 0.0 % OH showed high inhibitory effect (76.3 %) with negligible cytotoxicity (4.5 %). This research provides new information on the interactions among fennel, oregano and lavender essential oils and shows how they can synergistically inhibit in vitro LPS-induced NO production.

The Inhibition Activity of Natural Methoxyflavonoid from Inula britannica on Soluble Epoxide Hydrolase and NO Production in RAW264.7 Cells.

Soluble epoxide hydrolase (sEH) is an enzyme targeted for the treatment of inflammation and cardiovascular diseases. Activated inflammatory cells produce nitric oxide (NO), which induces oxidative stress and exacerbates inflammation. We identify an inhibitor able to suppress sEH and thus NO production. Five flavonoids 1-5 isolated from Inula britannica flowers were evaluated for their abilities to inhibit sEH with IC50 values of 12.1 ± 0.1 to 62.8 ± 1.8 µM and for their effects on enzyme kinetics. A simulation study using computational chemistry was conducted as well. Furthermore, five inhibitors (1-5) were confirmed to suppress NO levels at 10 µM. The results showed that flavonoids 1-5 exhibited inhibitory activity in all tests, with compound 3 exhibiting the most significant efficacy. Thus, in the development of anti-inflammatory inhibitors, compound 3 is a promising natural candidate.

Comparison of immunomodulatory activity of polysaccharides and soluble dietary fibers and adsorption capacities of insoluble dietary fibers extracted from Lentinus edodes stipes

Stipes are the major waste from the processing of Lentinus edodes. To make full use of L. edodes stipes (LES), different fractions of LES polysaccharides (LESPs) were first obtained by water extraction and gradient ethanol precipitation. Afterwards, the LES residues were treated with an optimal combination of Aspergillus niger and Saccharomyces cerevisiae for the preparation of soluble/insoluble dietary fibers (LESS/LESI) using the response surface methodology and the Box-Behnken design. Subsequently, the in vitro immunomodulatory activity of LESPs and LESS, as well as the adsorption capacities of LESI were evaluated. The results showed that LESPs were neutral polysaccharides, mainly containing glucose. The optimal parameters for modifying the residues of LES were the followings: 4% (w/w) A. niger, 8% (w/w) S. cerevisiae, 31 ℃, 3 d, and a solid–liquid ratio of 1:12.5 in a yield of 14.73%/82.45% of LESS/LESI, respectively. The in vitro immunomodulatory activity assays revealed that LESPs and LESS had potent immunostimulatory activity to increase phagocytosis, acid phosphatase activity and nitric oxide production of RAW264.7 murine cell macrophages. The evaluation of adsorption capacities revealed that LESI owned stronger water holding capacity, oil holding capacity and water swelling capacity. This research could provide an effective way to fully utilize discarded L. edodes stipes with high added-value.Graphical An effective strategy was built to fully utilize discarded L. edodes stipes by preparing polysaccharides (LESPs-20, LESPs-50 and LESPs-80), soluble and insoluble dietary fibers (LESS/LESI). Their products showed immunomodulatory activity and adsorption capacities.

Isolation and characterization of dihydrohomoisoflavonoids from Portulaca oleracea L.

Eight pairs of dihydrohomoisoflavonoids (1−8), including four pairs of enantiomeric aglycones [(R,S)-portulacanones B (1) and C (2) and (R,S)-oleracones C (3) and Q (4)] and four pairs of epimeric glycosides [portulacasides A−D and epiportulacasides A−D (5−8)], were obtained from Portulaca oleracea L. Among them, (R,S)-oleracone Q (4) and four pairs of epimeric glycosides (5−8) were reported for the first time. The 50% EtOH fraction from the 70% EtOH extract prevented HepG2 human liver cancer cell damage induced by N-acetyl-p-aminophenol (APAP), and the cell survival rate was 62.3%. Portulacaside B (6a), which was isolated from the 50% EtOH fraction, exhibited hepatoprotective and anti-inflammatory effects. The compound increased the survival rate of APAP-damaged HepG2 human liver cancer cells from 40.0% to 51.2% and reduced nitric oxide production in RAW 264.7 macrophages, resulting in an inhibitory rate of 46.8%.

Antioxidant, anti-inflammatory, and antibacterial activities against acne-causing bacteria of Miliusa velutina (A.DC.) Hook.f. & Thomson extracts

Context: Acne is often associated with bacterial infection, inflammation, and oxidative stress. In Thailand, Miliusa velutina has traditionally been used to relieve pain and to heal wounds. Given its biological properties, M. velutina extracts may represent an alternative approach to treat acne. Aims: To characterise M. velutina extracts regarding their antioxidant and anti-inflammatory properties and antibacterial activity against acne-causing bacteria. Methods: We prepared crude methanolic and aqueous extracts of M. velutina leaves and stem bark by maceration. We evaluated the antioxidant activity of the extracts by using four in vitro methods and determined the total phenolic and flavonoid contents. We tested the anti-inflammatory activity of the extracts by examining their ability to inhibit nitric oxide production in RAW 264.7 macrophages. We tested the antibacterial activity against acne-causing bacteria by using the agar diffusion method and by determining the minimum inhibitory concentration (MIC) and the minimum bactericidal concentration (MBC). Results: The methanolic bark extract showed potent antioxidant properties, denoted by its ability to scavenge the ABTS and DPPH radicals and its iron-reducing power and total antioxidant activity. The methanolic leaf extract presented the best ability to inhibit nitric oxide production, with a half maximal inhibitory concentration (IC50) of 7.20 ± 1.92 µg/mL. Additionally, M. velutina extracts exhibited antibacterial activity against Cutibacterium acnes, Staphylococcus epidermidis, Staphylococcus aureus, methicillin-resistant S. aureus and Pseudomonas aeruginosa, with the MIC and MBC ranging from 0.49 to 62.50 mg/mL. Conclusions: These findings indicate that M. velutina extract can be used as a novel therapeutic agent for acne treatment owing to its antioxidant, anti-inflammatory and antibacterial properties.

HPPO-Derived Meroterpenoids from the Marine-Derived Fungus Penicillium sp. SCSIO 41691.

Seven new 4-hydroxy-6-phenyl-2H-pyran-2-one (HPPO) derived meroterpenoids, 1-methyl-12a,12b-epoxyarisugacin M (1), 1-methyl-4a,12b-epoxyarisugacin M (2), 2,3-dihydroxy-3,4a-epoxy-12a-dehydroxyisoterreulactone A (3), 2-hydroxy-12a-dehydroxyisoterreulactone A (4), 3'-demethoxyterritrems B' (5), 4a-hydroxyarisugacin P (6), and 1-epi-arisugacin H (7), together with two known analogues (8 and 9), were isolated from the marine-derived fungal strain Penicillium sp. SCSIO 41691. Their structures were elucidated by spectroscopic methods, and the absolute configurations of compounds 1 and 3 were determined by single-crystal X-ray diffraction. Among them, 1 and 2 had a unique methyl migration in the basic meroterpenoid skeleton with a 12a,12b-epoxy or 4a,12b-epoxy group, and 3 was a highly oxygenated HPPO-derived meroterpenoid featuring a rare 6/5/6/6/6/6 hexacyclic system with a 3,4a-epoxy group. Biologically, 5 exhibited inhibitory activity against lipopolysaccharide-induced nitric oxide production in RAW 264.7 cells with an IC50 value of 21 μM, more potent than the positive control indomethacin.

Anti-inflammatory monoterpenes from morinda (Morinda officinalis How.)

Morinda (Morinda officinalis How.) is widely consumed as a functional food owing to its potential to promote health. This study investigated the anti-inflammatory phytochemicals of morinda and isolated 30 monoterpenes, including 6 undescribed iridoids (1, 6, 9–11 and 25), 2 undescribed acyclic monoterpenoids (28, 29), a known acyclic monoterpenoid and 21 known iridoids. Their chemical and stereo-structures were elucidated based on HR–ESI–MS, NMR, 13C-NMR calculations, ECD data and ECD calculations. Notably, compounds 11, 12 and 20 exerted pronounced inhibitory effects on lipopolysaccharide-induced nitric oxide production in RAW 264.7 macrophages, with IC50 values of 28.51 ± 1.70, 25.45 ± 4.17 and 29.17 ± 3.71 μM respectively (indomethacin, IC50 of 33.68 ± 2.19 μM). The same compounds exert anti-inflammatory effects by blocking nuclear translocation of nuclear factor κ-B, and down-regulating the expression of inflammatory cytokines such as cyclooxygenase-2, inducible nitric oxide synthase, interleukin-1β and interleukin-6 at mRNA and protein levels in a dose-dependent manner. These results suggest that moderate consumption of morinda helps prevent and reduce the occurrence of inflammatory-related diseases.

Metabolites Profile of Extracts and Fractions of Erythroxylum mexicanum Kunth by UHPLC-QTOF-MS/MS and its Antibacterial, Cytotoxic and Nitric Oxide Inhibitory Activities.

The present study shows the untargeted metabolite profiling and in vitro antibacterial, cytotoxic, and nitric oxide (NO) inhibitory activities of the methanolic leaves extract (MLE) and methanolic stem extract (MSE) of Erythroxylum mexicanum, as well as the fractions from MSE. Using ultra-high performance liquid chromatography/quadrupole time-of-flight tandem mass spectrometry (UHPLC-QTOF-MS/MS), a total of 70 metabolites were identified; mainly alkaloids in the MLE, while the MSE showed a high abundance of diterpenoids. The MSE fractions exhibited differential activity against Gram-positive bacteria. Notably, the hexane fraction (HSF) against Streptococcus pyogenes ATCC 19615 (MIC=62.5 μg/mL) exhibited a bactericidal effect. The MSE fractions exhibited cytotoxicity against all cancer cell lines tested, with selectivity towards them compared to a noncancerous cell line. Particularly, the HSF and chloroform fraction (CSF) showed the highest cytotoxicity against prostate cancer (PC-3) cells, with IC50 values of 19.9 and 18.1 μg/mL and selectivity indexes of 3.8 and 4.2, respectively. Both the HSF and ethyl acetate (EASF) fractions of the MSE inhibited NO production in RAW 264.7 macrophages, with NO production percentages of 50.0 % and 51.7 %, respectively, at a concentration of 30 μg/mL. These results indicated that E. mexicanum can be a source of antibacterial, cytotoxic, and anti-inflammatory metabolites.

Nitrogenous compounds from Aesculus wilsonii seeds

Eight nitrogenous compounds including five undescribed ones, aeswilnitrousol A (1), aeswilnitrousosides BD (2–4), and 6-(2-hydroxy-3-methylbutylamino)-8-oxoadenine (5) were isolated from the seeds of Aesculus wilsonii. Their structures and absolute configurations were established based on spectroscopic determination, calculated electronic circular dichroism (ECD) analysis, as well as chemical reaction methods. Among the three known compounds, 7 and 8 were obtained from the Aesculus genus for the first time, and 6 was gained from this plant initially. The 13C NMR data of 7 and 8 were reported for the first time. Moreover, the inhibitory effect of all the isolates against LPS-induced nitric oxide production in RAW264.7 macrophages was evaluated. As a result, compounds 2 and 8 exhibited anti-inflammatory activity in a concentration-dependent manner at 10, 25, and 50 μM.

Enhanced antioxidant, tyrosinase inhibition, and anti-inflammatory activities of Praeparatum mungo and three of its derivatives

The main objective of this study is to explore the functions of Praeparatum mungo (PM) and three of its derivatives, Praeparatum mungo/turmeric (PM/T), Praeparatum mungo/bromelain (PM/B), and Praeparatum mungo/inorganic elements (PM/IE). The results indicated that additives included in the fermentation process of PM enhanced PM’s antioxidant properties. PM/B exhibited the highest total phenolic content (19.18 ± 0.46 mg gallic acid equivalent/g), DPPH free radical scavenging activity, and ferric reducing power. PM/IE exhibited the highest ABTS free radical scavenging activity and chelating ferrous ion activity. PM/T exhibited the best inhibitory tyrosinase activity. The 625 μg/mL PM extract can extensively reduce nitric oxide production of RAW264.7 macrophages stimulated by 1 μg/mL LPS and exhibited no cytotoxicity for anti-inflammatory applications. Additives in PM natural fermentation process can enhance antioxidant, tyrosinase inhibition, and anti-inflammatory properties of PM for future applications.

Pharmacological Evaluation of Anti-Diabetic Activity of Myricetin in Different Cell Lines

The aim of this research work was to perform pharmacological evaluation of Myricetin by using different cell lines. To check the short-term cytotoxic effect of Myricetin in HepG2 and L6 cells, a colorimetric based MTT assay was performed also the glucose utilization in both HepG2 and L6 cells was estimated in order to measure the in vitro anti-diabetic activity of Myricetin. For Lipid accumulation T3-L1 preadipocytes cells are used. For measurement of nitric oxide (NO) the RAW-264.7 cells are used. The alpha-amylase assay and alpha-glucosidase inhibition assay was performed to check if our drug is having effect in term of inhibiting α-amylase and α-glucosidase respectively. Western blot analysis was performed to check the expressions of iNOS protein in LPS treated RAW 264.7 cells before and after the exposure of Myricetin. The cytotoxicity data suggested that Myricetin displayed low level of cytotoxicity in HepG2 cells and in L6 myoblast cell lines. For glucose utilization experiment, it discovered that glucose uptake level was notably increase in HepG2 cells and in L6 myoblast cell lines with the increasing concentration of Myricetin. Myricetin increase lipid accumulation in 3T3-L1 preadipocytes and also inhibits nitric oxide production in RAW 264.7 cell lines. Research was found that Myricetin had no significant effect in order to alter the expression of alpha-amylase and alpha-glucosidase. Our research concludes that Myricetin can be used as a potent anti-diabetic agent.

Salmonella Typhimurium secondary infection induced macrophages apoptosis NO-dependent during Mycobacterium neoaurum infection

Mycobacterium neoaurum (M. neoaurum) and Salmonella Typhimurium (S. Typhimurium) are important zoonotic pathogens, and both are intracellular bacteria, which can induce cellular immunity. Coinfection is prevalent worldwide, even more prevalent than single infection. This study aimed to investigate the effect of M. neoaurum/S. Typhimurium coinfection on the percentage property of C57BL/6 mice regulatory T cells (Tregs) and the immune activity of RAW264.7 cells. The secretion of TNF-α, IFN-γ, IL-1β, IL-12 and iNOS in RAW264.7 cells was determined by ELISA. The expression of CD40+ , CD80+ and CD86+ costimulatory molecules on the surface of macrophages was analyzed by flow cytometry. A Nitric oxide (NO) assay was used to detect the production of NO in RAW264.7 cells. The apoptosis of RAW264.7 cells was detected by flow cytometry. The results showed that macrophage expressed a large number of cytokines and surface costimulatory molecules to enhance immune activity. S. Typhimurium secondary infection significantly increased the expression of iNOS and generation of NO, and M. neoaurum/S. Typhimurium-induced apoptosis was NO-dependent. Our data provide a theoretical basis for secondary infections by other pathogens after Nontuberculous Mycobacterium (NTM) infection, and lay a foundation for further research.

Triterpenoids from the fruits of Aphanamixis polystachya and their inhibitory activities on nitric oxide production

Nineteen triterpenoids, including five previously unknown (four triucallane-type derivatives and one highly oxidized A, B-seco limonoids), together with fourteen known triterpenoids, were isolated from the fruits of Aphanamixis polystachya. Their structures were elucidated by extensive spectroscopic analysis. All isolates were evaluated their anti-inflammatory activities. The result showed that all compounds inhibit LPS-induced nitric oxide production in RAW264.7 macrophages with their IC50 value ranging from 95 to 1332 uM, and compound 6 exhibited obvious anti-inflammatory activity comparable to that of the positive control, with IC50 values of 94.96 uM.

Acylated flavonoid glycosides from Barringtonia pendula and their inhibition on lipopolysaccharide-induced nitric oxide production in RAW264.7 cells

Six new acylated flavonoid glycosides namely barringosides J − O (1–6) along with tephrokaempferoside and barringoside D were isolated from the branches and leaves of Barringtonia pendula. The structural elucidation was confirmed by extensive analysis of their spectroscopic data including HRQTOFMS, 1D and 2D NMR experiments. Moderate inhibitory effects on LPS-induced NO production in RAW264.7 cells were observed for barringosides M (4) and N (5) with IC50 values of 48.40 ± 3.01 and 56.61 ± 3.87 μM, whereas weak inhibition was found for compounds 1–3, 6, and 7 with IC50 values ranging from 64.91 ± 3.68 to 79.80 ± 3.90 μM.

Anti-inflammatory and anti-oxidant activity-guided investigation of phytoconstituents from Lactuca indica L.

Vietnamese dandelion(Lactuca indica L.)presents various remedies to cure pustules and several derma-inflammatory diseases in terms of traditional medicine. In this study, the anti-inflammatory and antioxidant effects of several fractions from L. indica were screened by assessing the inhibitory capacity of nitric oxide production in RAW 264.7 cells and the DPPH scavenging activity. Through screening steps, the chloroform extract exhibited extraordinary inhibition on NO synthesis, whereas the ethyl acetate singly showed a remarkable capacity to neutralise the DPPH radical. Bioactivity-guided isolation led to the identification of six compounds from the chloroform and ethyl acetate extracts, including pentadecanoic acid (1), lupeol (2), vanillin (3), 5-hydroxy-3ʹ,4ʹ,7-trimethoxyflavone (4), luteolin-7-O-β-D-glucoside (5), acacetin-7-O-β-D-rutinoside (6). Only compound (5) exhibited notable interruption in NO synthesis (IC50=13.86±1.10 μM) and simultaneously neutralised DPPH radical (IC50=26.93±0.10 μM). This study suggests that compound (5) as well as the chloroform and ethyl acetate extracts could be potential in preventing inflammatory diseases mediated by excessive production of NO. Additionally, (5) and the ethyl acetate extract could potentially be natural anti-oxidants.

Design, synthesis and evaluation of ursodeoxycholic acid-cinnamic acid hybrids as potential anti-inflammatory agents by inhibiting Akt/NF-κB and MAPK signaling pathways

A series of ursodeoxycholic acid (UDCA)-cinnamic acid hybrids were designed and synthesized. The anti-inflammatory activity of these derivatives was screened through evaluating their inhibitory effects of LPS-induced nitric oxide production in RAW264.7 macrophages. The preliminary structure-activity relationship was concluded. Among them, 2m showed the best inhibitory activity against NO (IC50 = 7.70 μM) with no significant toxicity. Further study revealed that 2m significantly decreased the levels of TNF-α, IL-1β, IL-6 and PGE2, down-regulated the expression of iNOS and COX-2. Preliminary mechanism study indicated that the anti-inflammatory activity of 2m was related to the inhibition of the Akt/NF-κB and MAPK signaling pathway. Furthermore, 2m reduced inflammation by a mouse model of LPS-induced inflammatory disease in vivo. In brief, our findings indicated that 2m might serve as a new lead compound for further development of anti-inflammatory agents.

Immunostimulatory Activity of Lactococcus lactis subsp. lactis CAB701 Isolated from Jeju Cabbage.

This study explored the potential of Lactococcus lactis subsp. lactis CAB701 as a probiotic strain, focusing on its immunostimulatory properties. Despite adverse conditions in the gastrointestinal environment, this strain exhibited remarkable survivability, as evidenced by its tolerance to acid, bile, and pancreatin, coupled with its impressive ability to adhere to Caco-2 cells. It also exhibited significant antioxidant activity, similar to the established probiotic Lacticaseibacillus rhamnosus GG (LGG). Our research elucidates the potent immunostimulatory effects of L. lactis subsp. lactis CAB701. This strain significantly enhanced nitric oxide production in RAW 264.7, far exceeding that obtained with LGG. An in-depth examination revealed elevated expression of key inflammatory mediators, including inducible nitric oxide synthase, tumor necrosis factor-alpha, cyclooxygenase-2, interleukin (IL)-1 beta, and IL-6. L. lactis subsp. lactis CAB701 increases the expression of critical signaling proteins in the mitogen-activated protein kinase pathway. This prompted a substantial increase in the expression of phosphorylated c-Jun N-terminal kinases and extracellular signal-regulated kinases, suggesting their role in modulating these immune-related pathways. Overall, these findings demonstrate the significant immunostimulatory capacity of L. lactis subsp. lactis CAB701, positioning it as a potential candidate for probiotic use, especially in applications that enhance immune responses.

Enantiomeric N-acetyldopamine trimers from Cicadae Periostracum and their absolute configurations

Six previously undescribed N-acetyldopamine (NADA) trimmer racemates, percicamides A−F (1−6), were isolated from a 70% ethanol extract of Cicadae Periostracum. Subsequent chiral-phase separation afforded six pairs of enantiomers, (+)- and (−)-percicamides A−F (1a/1b−6a/6b). Their structures including absolute configurations were elucidated by combined extensive spectroscopic data and quantum chemical calculations. Compounds 1−6 represent the first examples of NADA trimmers with a cis-relationship of H-7′/H-8′ or H-7′′/H-8′′. Bioassays verified that all isolated compounds showed weak inhibitory effects on nitric oxide production in RAW 264.7 cells.