Anti-Inflammatory Effects of Mitrephora sirikitiae Leaf Extract and Isolated Lignans in RAW 264.7 Cells

Mitrephora sirikitiae Weeras., Chalermglin & R.M.K. Saunders has been reported as a rich source of lignans that contribute to biological activities and health benefits. However, cellular anti-inflammatory effects of M. sirikitiae leaves and their lignan compounds have not been fully elucidated. Therefore, this study aimed to investigate the anti-inflammatory activities of methanol extract of M. sirikitiae leaves and their lignan constituents on lipopolysaccharide (LPS)-induced inflammation in RAW 264.7 mouse macrophage cells. Treatment of RAW 264.7 cells with the methanol extract of M. sirikitiae leaves and its isolated lignans, including (−)-phylligenin (2) and 3′,4-O-dimethylcedrusin (6) significantly decreased LPS-induced prostaglandin E₂ (PGE₂) and nitric oxide (NO) productions. These inhibitory effects of the extract and isolated lignans on LPS-induced upregulation of PGE₂ and NO productions were derived from the suppression of cyclooxygenase 2 (COX-2) and inducible nitric oxide synthase (iNOS) production, respectively. In addition, treatment with 2-(3,4-dimethoxyphenyl)-6-(3,5-dimethoxyphenyl)-3,7-dioxabicyclo[3.3.0]octane (3) and mitrephoran (5) was able to suppress LPS-induced tumor necrosis factor alpha (TNF-α) secretion and synthesis in RAW 264.7 cells. These results demonstrated that M. sirikitiae leaves and some isolated lignans exhibited potent anti-inflammatory activity through the inhibition of secretion and synthesis of PGE₂, NO, and TNF-α.     

Quercetin-Rich Ethanolic Extract of Polygonum odoratum var Pakphai Leaves Decreased Gene Expression and Secretion of Pro-Inflammatory Mediators in Lipopolysaccharide-Induced Murine RAW264.7 Macrophages

Polygonum odoratum var. Pakphai has been used in traditional Thai medicine for the treatment of flatulence and constipation and to relieve the inflammation caused by insect bites. Quercetin (Q), which is abundant in plant-based foods, has been found to exert anti-inflammatory properties. This study evaluated the anti-inflammatory activity of P. odoratum ethanolic extract in RAW264.7 macrophage cells. Leaves were extracted with 50% ethanol, phenolics and flavonoids were then analyzed using UHPLC-QTOF-MS and HPLC-DAD. RAW264.7 cells were induced with lipopolysaccharides (LPSs). They were then treated with the extract and prostaglandin E₂ (PGE₂), and interleukin-6 (IL-6) and tumor necrotic factor-alpha (TNF-α) concentrations were determined. Levels of cyclooxygenase-2 (COX-2), inducible nitric oxide synthase (iNOS), IL-6 and TNF-α mRNAs were analyzed using qRT-PCR. Chemical analysis demonstrated that the extract was abundant with Q while also containing catechin, gallic acid, epicatechin gallate and coumarin. The extract increased the viability of RAW264.7 cells and dose-dependently decreased nitric oxide production, PGE₂, IL-6 and TNF-α levels in the medium from the LPS-induced RAW264.7 cell culture. Consistently, COX-2, iNOS, IL-6 and TNF-α mRNA levels were decreased in a concentration-dependent manner (p < 0.05). Thus, the quercetin-rich ethanolic extract derived from P. odoratum var Pakphai leaves can exert anti-inflammatory activity in LPS-induced RAW264.7 cells through a reduction of the pro-inflammatory mediator response.     

Cissus subtetragona Planch. Ameliorates Inflammatory Responses in LPS-induced Macrophages,HCl/EtOH-induced Gastritis,and LPS-induced Lung Injury via Attenuation of Src and TAK1

Several Cissus species have been used and reported to possess medicinal benefits. However, the anti-inflammatory mechanisms of Cissus subtetragona have not been described. In this study, we examined the potential anti-inflammatory effects of C. subtetragona ethanol extract (Cs-EE) in vitro and in vivo, and investigated its molecular mechanism as well as its flavonoid content. Lipopolysaccharide (LPS)-induced macrophage-like RAW264.7 cells and primary macrophages as well as LPS-induced acute lung injury (ALI) and HCl/EtOH-induced acute gastritis mouse models were utilized. Luciferase assays, immunoblotting analyses, overexpression strategies, and cellular thermal shift assay (CETSA) were performed to identify the molecular mechanisms and targets of Cs-EE. Cs-EE concentration-dependently reduced the secretion of NO and PGE₂, inhibited the expression of inflammation-related cytokines in LPS-induced RAW264.7 cells, and decreased NF-κB- and AP-1-luciferase activity. Subsequently, we determined that Cs-EE decreased the phosphorylation events of NF-κB and AP-1 pathways. Cs-EE treatment also significantly ameliorated the inflammatory symptoms of HCl/EtOH-induced acute gastritis and LPS-induced ALI mouse models. Overexpression of HA-Src and HA-TAK1 along with CETSA experiments validated that inhibited inflammatory responses are the outcome of attenuation of Src and TAK1 activation. Taken together, these findings suggest that Cs-EE could be utilized as an anti-inflammatory remedy especially targeting against gastritis and acute lung injury by attenuating the activities of Src and TAK1.     

Differential regulation of inducible nitric oxide synthase and cyclooxygenase-2 expression by superoxide dismutase in lipopolysaccharide stimulated RAW 264.7 cells

Inducible nitric oxide synthase (iNOS) and cyclooxygenase 2 (COX-2) have been known to be involved in various pathophysiological processes such as inflammation. This study was performed to determine the regulatory function of superoxide dismutase (SOD) on the LPS-induced expression of iNOS, and COX-2 in RAW 264.7 cells. When a cell-permeable SOD, Tat-SOD, was added to the culture medium of RAW 264.7 cells, it rapidly entered the cells in a dose-dependent manner. Treatment of RAW 264.7 cells with Tat-SOD led to decrease in LPS-induced ROS generation. Pretreatment with Tat-SOD significantly inhibited LPS-induced expression of iNOS and NO production but had no effect on the expression of COX-2 and PGE₂ production in RAW 264.7 cells. Tat-SOD inhibited LPS-induced NF-κB DNA binding activity, IκBα degradation and activation of MAP kinases. These data suggest that SOD differentially regulate expression of iNOS and COX-2 in LPS-stimulated RAW 264.7 cells.     

Anti-Inflammatory and Anti-Diabetic Effect of Black Soybean Anthocyanins: Data from a Dual Cooperative Cellular System

Obesity is characterized by elevated infiltration of macrophages into adipose tissue, leading to the development of insulin resistance. The black soybean seed coat is a rich source of anthocyanins with antioxidative and anti-inflammatory activities. This study investigated the effects of black soybean anthocyanin extract (BSAn) on obesity-induced oxidative stress, the inflammatory response, and insulin resistance in a coculture system of hypertrophied 3T3-L1 adipocytes and RAW264 macrophages. Coculture of adipocytes with macrophages increased the production of reactive oxygen species and inflammatory mediators and cytokines (NO, MCP-1, PGE₂, TNFα, and IL-6) and the release of free fatty acids but reduced anti-inflammatory adiponectin secretion. BSAn treatment (12.5, 25, 50, and 100 μg/mL) alleviated the coculture-induced changes (p < 0.001) and inhibited coculture-induced activation of JNK and ERK signaling (p < 0.01). BSAn also blocked the migration of RAW264.7 macrophages toward 3T3-L1 adipocytes. In addition, treatment with BSAn increased PPARγ expression and glucose uptake in response to insulin in hypertrophied 3T3-L1 adipocyte and RAW264.7 macrophage coculture (p < 0.01). These results demonstrate that BSAn attenuates inflammatory responses and improves adipocyte metabolic function in the coculture of hypertrophied 3T3-L1 adipocytes and RAW264.7 macrophages, suggesting the effectiveness of BSAn for obesity-induced insulin resistance.     

New sesquiterpenes from Inula japonica Thunb. with their inhibitory activities against LPS-induced NO production in RAW264.7 macrophages

Twenty-two new sesquiterpenes were isolated from the aerial parts of Inula japonica Thunb., together with fifteen known ones. Their structures were determined by detailed spectroscopic analysis, X-ray diffraction studies, and modified Mosher method. All 37 compounds were evaluated for the inhibition of LPS-induced nitric oxide (NO) production in RAW264.7 macrophages, and most of isolates significantly inhibited the NO production with IC₅₀ values in the range of 3.5-20 μM. Besides, results obtained in our studies provided a structure-activity relationship that would be used to design anti-inflammatory agents in the future.     

Synthesis and Anti-Inflammatory Activity of 1-Methylhydantoin Cinnamoyl Imides

In this study, 1-methylhydantoin cinnamic imides were synthesized from 1-methylhydantoin and trans-cinnamic acid, and their anti-inflammatory activity was investigated. The anti-inflammatory activity in vitro was evaluated by measuring the contents of NO, TNF-α and IL-1β in the supernatant of RAW264.7 cells stimulated by LPS. The cytotoxicity of 1-methylhydantoin cinnamoyl imides on RAW264.7 cells was detected using the CCK-8 method. The results showed that compounds 2 and 4 can significantly inhibit the release of NO and reduce the secretion of TNF-α and IL-1β. Compound 3 inhibited the production of TNF-α. The inhibition rate of COX was evaluated in vitro. The in vivo anti-inflammatory activities of the five compounds were evaluated by establishing an animal model of xylene ear swelling. The results showed that 1-methylhydantoin cinnamic imides could alleviate xylene-induced ear edema in mice in a dose-dependent manner. Among them, the effect of compound 5 was the most significant. Under the action of high dosage, its ear swelling inhibition rate was as high as 52.08%.     

Anti-Inflammatory Activities of the Ethanol Extract of Prasiola japonica,an Edible Freshwater Green Algae,and Its Various Solvent Fractions in LPS-Induced Macrophages and Carrageenan-Induced Paw Edema via the AP-1 Pathway

Prasiola japonica possesses several biological activities. However, reports on the anti-inflammatory activities and molecular mechanisms of its different solvent fractions remain limited. In this study, we investigated the potential anti-inflammatory activities of P. japonica ethanol extract (Pj-EE) and four solvent fractions of Pj-EE made with hexane (Pj-EE-HF), chloroform (Pj-EE-CF), butanol (Pj-EE-BF), or water (Pj-EE-WF) in both in vitro (LPS-induced macrophage-like RAW264.7 cells) and in vivo (carrageenan-induced acute paw edema mouse models) experiments. The most active solvent fraction was selected for further analysis. Various in vitro and in vivo assessments, including nitric oxide (NO), cytokines, luciferase assays, real-time polymerase chain reactions, and immunoblotting analyses were performed to evaluate the underlying mechanisms. In addition, the phytochemical constituents were characterized by Liquid chromatography-tandem mass spectrometry. In in vitro studies, the highest inhibition of NO production was observed in Pj-EE-CF. Further examination revealed that Pj-EE-CF decreased the expression of inflammation-related cytokines in LPS-induced RAW264.7 cells and suppressed subsequent AP-1-luciferase activity by inhibition of phosphorylation events in the AP-1 signaling pathway. Pj-EE-CF treatment also demonstrated the strongest reduction in thickness and volume of carrageenan-induced paw edema, while Pj-EE-BF showed the lowest activity. Furthermore, Pj-EE-CF also reduced gene expression and cytokines production in tissue lysates of carrageenan-induced paw edema. These findings support and validate the evidence that Pj-EE, and especially Pj-EE-CF, could be a good natural source for an anti-inflammatory agent that targets the AP1 pathway.     

Diterpenoids Isolated from Podocarpus macrophyllus Inhibited the Inflammatory Mediators in LPS-Induced HT-29 and RAW 264.7 Cells

Species of Podocarpus are used traditionally in their native areas for the treatment of fevers, asthma, coughs, cholera, chest pain, arthritis, rheumatism, and sexually transmitted diseases. To identify natural products having efficacy against inflammatory bowel disease (IBD), we identified a new, 16-hydroxy-4β-carboxy-O-β-D-glucopyranosyl-19-nor-totarol (4) together with three known diterpenoids from P. macrophyllus. Furthermore, all the extracts, fractions, and isolates 1–4 were investigated for their anti-inflammatory effects by assessing the expression on nitric oxide (NO) production and proinflammatory cytokines in lipopolysaccharide (LPS)-stimulated RAW 264.7 and HT-29 cells. Among them, nagilactone B (2) exhibited a potent anti-inflammatory effect against NO production on RAW 264.7 cells; therefore, nagilactone B was further assessed for anti-inflammatory activity. Western blot analysis revealed that nagilactone B significantly decreased the expression of LPS-stimulated protein, inducible nitric oxide synthase (iNOS), cyclooxygenase (COX)-2, and phosphorylated extracellular regulated kinase (pERK)1/2. In addition, nagilactone B downregulated tumor necrosis factor (TNF)-α, interleukin (IL)-6, and IL-8 levels in LPS-induced macrophages and colonic epithelial cells. To our best knowledge, this is the first report on the inhibitory effect of nagilactone B (pure state) and rakanmakilactone G against NO production in LPS-stimulated RAW 264.7 cells. Thus, diterpenoids isolated from P. macrophyllus could be employed as potential therapeutic phytochemicals for IBD.     

In vitro anti-inflammatory activities of naucleoffieine H as a natural alkaloid from Nauclea officinalis Pierrc ex Pitard,through inhibition of the iNOS pathway in LPS-activated RAW 264.7 macrophages

Abstract

Naucleoffieine H, a natural indole alkaloid, was isolated and identified from Nauclea officinalis Pierrc ex Pitard which is a traditional Chinese medicine used for the treatment of various diseases, such as cold, fever, bronchitis, pneumonia, acute tonsillitis, etc. In the present study, the effect of naucleoffieine H on the anti-inflammatory activities was investigated in LPS-induced RAW 264.7 macrophages. The results showed that naucleoffieine H significantly inhibited the release of nitric oxide (the level of nitrite as a stable biomarker of NO production) and tumor necrosis factor-α (TNF-α). Interesting, naucleoffieine H down-regulated the overexpression of inflammatory protein induced nitric oxide synthase (iNOS), but no effect on the expression cyclooxygenase-2 (COX-2) protein. In addition, this bioactive alkaloid suppressed enzymatic activity of iNOS activated by LPS. The above results indicated that naucleoffieine H suppress NO and TNF-α overproduction via block the iNOS pathway in LPS-induced RAW 264.7 macrophages.     

A new coumarin isolated from Sarcandra glabra as potential anti-inflammatory agent

One new coumarin, 3,5-dihydroxy-7-O-α-L-rhamno pyranosyl-2H-chromen-2-one (1), was isolated from the whole plant of Sarcandra glabra. The structure was elucidated by spectroscopic methods. Our results indicated that 1 significantly inhibit nitric oxide (NO) production in LPS-induced RAW264.7 macrophages. RT-PCR analysis indicated it inhibited iNOS mRNA expression. In addition, Western blot analysis showed that 1 attenuated LPS-induced synthesis of iNOS protein in the macrophages. These results suggest that 1 could be potential anti-inflammatory agent by down-regulating iNOS expression.     

Camel Grass Phenolic Compounds: Targeting Inflammation and Neurologically Related Conditions

Background: The use of plants for therapeutic purposes has been supported by growing scientific evidence. Methods: This work consisted of (i) characterizing the phenolic compounds present in both aqueous and hydroethanol (1:1, v/v) extracts of camel grass, by hyphenated liquid chromatographic techniques, (ii) evaluating their anti-inflammatory, antioxidant, and neuromodulation potential, through in vitro cell and cell-free models, and (iii) establishing a relationship between the chemical profiles of the extracts and their biological activities. Results: Several caffeic acid and flavonoid derivatives were determined in both extracts. The extracts displayed scavenging capacity against the physiologically relevant nitric oxide (^•NO) and superoxide anion (O₂^•−) radicals, significantly reduced NO production in lipopolysaccharide (LPS)-stimulated macrophages (RAW 264.7), and inhibited the activity of hyaluronidase (HAase), acetylcholinesterase (AChE) and butyrylcholinesterase (BChE). Some of these bioactivities were found to be related with the chemical profile of the extracts, namely with 3-caffeoylquinic, 4-caffeoylquinic, chlorogenic, and p-coumaric acids, as well as with luteolin and apigenin derivatives. Conclusions: This study reports, for the first time, the potential medicinal properties of aqueous and hydroethanol extracts of camel grass in the RAW 264.7 cell model of inflammation, and in neurologically related conditions.     

Abiesanordines A-N: fourteen new norditerpenes from Abies georgei

Fourteen new norditerpenoids (abiesanordines A-N, 1-14), including a novel podocarpene bearing a rare enolic structure (abiesanordine A, 1), were isolated from Abies georgei together with eight known ones. Their structures were determined mainly by detailed analysis of 1D and 2D NMR spectroscopic data including HSQC, DQF COSY, HMBC, and NOESY. All the isolates were tested for inhibitory activities against LPS-induced NO production in RAW264.7 macrophages, abiesanordine I (9) showed the strongest activity with the IC₅₀ value of 17.0 μg/mL. Furthermore, it exhibited no cytotoxicity against RAW264.7 macrophages under the concentration of 50 μg/mL.     

New Benzil and Isoflavone Derivatives with Cytotoxic and NO Production Inhibitory Activities from Placolobium vietnamense

The phytochemical investigation of Placolobium vietnamense stems led to the isolation of a new isoflavone derivative (1) and three new benzil derivatives (2–4), together with four known pyranoisoflavones (5–8). The structures of all isolated compounds were determined on the basis of extensive spectroscopic analyses, including NMR and HRMS spectral data, as well as comparison of their spectroscopic data with those reported in the literature. The cytotoxicity of all isolated compounds was assessed against the human liver hepatocellular carcinoma (Hep G2) cell line, and compound 1 displayed the most significant cytotoxicity with an IC₅₀ value of 8.0 μM. Furthermore, all isolated compounds were also tested for their inhibitory activity against NO production in RAW 264.7 macrophages. Of these, compound 1 exhibited the strongest inhibitory efficacy against the LPS-induced NO production with the IC₅₀ value of 13.7 μM.     

Anti-inflammatory and anti-oxidant activities of Mallotus oppositifolius (Geisel) methanol leaf extracts

The methanol leaf extract of Mallotus oppositifolius was evaluated for anti-inflammatory activity in rats and mice using acute and chronic anti-inflammatory models with acetylsalicylate acid (aspirin) as the reference drug. The antioxidant activity was done in vitro using ferric reducing antioxidant power (FRAP) and 1,1-diphenyl-2-hydrazyl (DPPH) spectrophotometric assays. The extract dose dependently and significantly reduced paw edema volume in rats induced by carrageenan (p < 0.01), decreased croton oil-induced ear inflammation (p < 0.05), inhibited cotton pellet-induced granuloma in mice and reduced the rat paw thickness in formalin-induced arthritis.     

In Vitro Anti-Inflammatory Activity of Essential Oil and β-Bisabolol Derived from Cotton Gin Trash

Natural α-bisabolol has been widely used in cosmetics and is sourced mainly from the stems of Candeia trees that have become endangered due to over exploitation. The in vitro anti-inflammatory activity of cotton gin trash (CGT) essential oil and the major terpenoid (β-bisabolol) purified from the oil were investigated against lipopolysaccharide (LPS)-stimulated RAW264.7 macrophages as well as the 3t3 and HS27 fibroblast cell lines. Nitric oxide (NO), prostaglandin E₂ (PGE₂), tumor necrosis factor-alpha (TNF-α), interleukin 6 (IL-6), and interleukin 8 (IL-8) were measured using Greiss reagent, enzyme-linked immunosorbent assay (ELISA), and cytokine bead array (CBA)-flow cytometry. Non-toxic concentrations of CGT oil and β-bisabolol (1.6–50.0 µg/mL) significantly inhibited the production of the inflammatory mediators in a dose-dependent manner. Maximal inhibition by β-bisabolol was 55.5% for NO, 62.3% for PGE₂, and 45.3% for TNF-α production in RAW cells. β-Bisabolol induced a level of inhibition similar to an equal concentration of α-bisabolol (50.0 µg/mL), a known anti-inflammatory agent. These results suggest β-bisabolol exerts similar in vitro effects to known topical anti-inflammatory agents and could therefore be exploited for cosmetic and therapeutic uses. This is the first study to report the in vitro anti-inflammatory activity of β-bisabolol in CGT essential oil.     

Synthesis and Investigation of Flavanone Derivatives as Potential New Anti-Inflammatory Agents

Flavonoids are polyphenols with broad known pharmacological properties. A series of 2,3-dihydroflavanone derivatives were thus synthesized and investigated for their anti-inflammatory activities. The target flavanones were prepared through cyclization of 2′-hydroxychalcone derivatives, the later obtained by Claisen–Schmidt condensation. Since nitric oxide (NO) represents an important inflammatory mediator, the effects of various flavanones on the NO production in the LPS-induced RAW 264.7 macrophage were assessed in vitro using the Griess test. The most active compounds were flavanone (4G), 2′-carboxy-5,7-dimethoxy-flavanone (4F), 4′-bromo-5,7-dimethoxy-flavanone (4D), and 2′-carboxyflavanone (4J), with IC50 values of 0.603, 0.906, 1.030, and 1.830 µg/mL, respectively. In comparison, pinocembrin achieved an IC₅₀ value of 203.60 µg/mL. Thus, the derivatives synthesized in this work had a higher NO inhibition capacity compared to pinocembrin, demonstrating the importance of pharmacomodulation to improve the biological potential of natural molecules. SARs suggested that the use of a carboxyl-group in the meta-position of the B-ring increases biological activity, whereas compounds carrying halogen substituents in the para-position were less active. The addition of methoxy-groups in the meta-position of the A-ring somewhat decreased the activity. This study successfully identified new bioactive flavanones as promising candidates for the development of new anti-inflammatory agents.     

Chemical constituents from Gueldenstaedtia verna and their anti-inflammatory activity

A new triterpenoid glycoside (1) and 15 known compounds (2–16) were isolated from the whole plants of Gueldenstaedtia verna. The new compound (1) was identified as complogenin 22-O-β-d-glucopyranoside by extensive spectroscopic techniques including 1D (¹H and ¹³C) and 2D NMR experiments (HSQC, HMBC and NOESY), HR-DART-MS and chemical methods. Most of the isolates were evaluated for their inhibitory activities on LPS-induced NO production in RAW 264.7 cells. The inhibitory effects of the active compounds, sulphuretin (8) and (22E,24S)-5α,8α-epidioxy-24-methyl-cholesta-6,9(11),22-trien-3β-ol (13), on the production of pro-inflammatory mediators (including IL-6, IL1β and PGE₂) were further estimated in vitro by ELISA in RAW 264.7 macrophages.     

Diterpenoid Compounds Isolated from Chloranthus oldhamii Solms Exert Anti-Inflammatory Effects by Inhibiting the IKK/NF-κB Pathway

Chloranthus oldhamii Solms (CO) is a folk medicine for treating infection and arthritis pain but its pharmacological activity and bioactive compounds remain mostly uncharacterized. In this study, the anti-inflammatory compounds of C. oldhamii were identified using an LPS-stimulated, NF-κB-responsive RAW 264.7 macrophage reporter line. Three diterpenoid compounds, 3α-hydroxy-ent-abieta-8,11,13-triene (CO-9), 3α, 7β-dihydroxy-ent-abieta-8,11,13-triene (CO-10), and decandrin B (CO-15) were found to inhibit NF-κB activity at nontoxic concentrations. Moreover, CO-9 and CO-10 suppressed the expression of IL-6 and TNF-α in LPS-stimulated RAW 264.7 cells. The inhibitory effect of CO-9 on TNF-α and IL-6 expression was further demonstrated using LPS-treated bone marrow-derived macrophages. Furthermore, CO-9, CO-10, and CO-15 suppressed LPS-triggered COX-2 expression and downstream PGE2 production in RAW 264.7 cells. CO-9 and CO-10 also reduced LPS-triggered iNOS expression and nitrogen oxide production in RAW 264.7 cells. The anti-inflammatory mechanism of the most effective compound, CO-9, was further investigated. CO-9 attenuated LPS-induced NF-κB activation by reducing the phosphorylation of IKKα/β (Ser176/180), IκBα (Ser32), and p65 (Ser534). Conversely, CO-9 did not affect the LPS-induced activation of MAPK signaling pathways. In summary, this study revealed new anti-inflammatory diterpenoid compounds from C. oldhamii and demonstrated that the IKK-mediated NK-κB pathway is the major target of these compounds.