Nitric Oxide Production In LPS-stimulated RAW Research Articles

Evaluation of In Vitro Antihypertensive and Anti-Inflammatory Properties of Dairy By-Products

Sweet whey (SW) and yogurt acid whey (YAW) are dairy by-products of the cheese-making process and Greek-style yogurt production, respectively. Both of them are considered pollutants with huge volumes of SW and YAW produced due to the growing demand for dairy products worldwide. Moreover, whey-derived peptides, resulting from fermentation as well as from further hydrolysis during digestion, have been associated with various biological activities. In the present study, the angiotensin-converting enzyme (ACE)-inhibitory activity of 48 SW samples and 33 YAW samples from bovine, ovine, caprine, and ovine/caprine milk obtained were evaluated. Additionally, the SW and YAW digestates and two of their fractions (smaller than 10 kDa, SW-D-P10 and YAW-D-P10, and smaller than 3 kDa, SW-D-P3 and YAW-D-P3), which were obtained after in vitro digestion and subsequent ultrafiltration, were also subjected to evaluation. Our data indicated that the D-P10 and D-P3 fractions exhibited higher ACE-inhibitory activity compared to the corresponding values before digestion. The ACE-inhibitory capacity after in vitro digestion was higher for the ovine SW samples compared to their bovine and caprine counterparts. The effect of the D-P3 fraction on the inhibition of nitric oxide (NO) production and the expression of a selected panel of immune-response-related genes in LPS-stimulated RAW 264.7 macrophages was also evaluated. Fractions from both dairy by-products inhibited NO production in LPS-stimulated RAW 264.7 cells. Especially, ovine SW-D-P3 showed a strong NO inhibitory activity and suppressed inducible nitric oxide synthase (Nos2) mRNA levels. However, YAW-D-P3 could not trigger neither the gene expression of inflammatory macrophage mediators Nos2 and cyclooxygenase-2 (Ptgs2) nor tumor necrosis factor-α (Tnf) and interleukin 6 (Il6) in LPS-stimulated murine macrophages regardless of animal origin. These findings suggest that in vitro digestion could enhance the production of ACE-inhibitory peptides in both dairy by-products, while SW from ovine origin displays higher potential as an anti-inflammatory agent, effectively preventing excessive NO production.

Anti-Inflammatory Effect of Ebractenoid F, a Major Active Compound of Euphorbia ebracteolata Hayata, through Inhibition of Nuclear Factor-κB Activation.

Euphorbia ebracteolata Hayata (Euphorbiaceae family) is a perennial plant that is widely distributed in Korea, Japan, and China. Its roots contain bioactive diterpenes that have anti-inflammatory properties. However, the anti-inflammatory mechanisms are not yet fully understood. This study aimed to identify the most active anti-inflammatory compound from the roots of E. ebracteolata Hayata, using bioassay-guided fractionation and a combinative method of high-speed countercurrent chromatography (HSCCC) and preparative high-performance liquid chromatography (HPLC). Then, we investigated its anti-inflammatory mechanism in lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages. Ebractenoid F was identified as the most potent bioactive compound of E. ebracteolata Hayata. Ebractenoid F significantly decreased nitric oxide (NO) production and nuclear factor-κB (NF-κB) activation induced by LPS in RAW 264.7 macrophages. Moreover, ebractenoid F decreased the degradation of inhibitory κB-α, the nuclear translocation of the p65 and p50 subunits of NF-κB, and the expression of NF-κB downstream genes. Furthermore, ebractenoid F inhibited the phosphorylation of Akt and mitogen-activated protein kinases (MAPKs), such as extracellular signal-regulated kinase (ERK) and c-Jun NH2 terminal kinase (JNK), in LPS-stimulated RAW 264.7 cells. In conclusion, ebractenoid F exerts the most potent anti-inflammatory effect by suppressing NF-κB-mediated NO production in LPS-stimulated RAW 264.7 cells. Ebractenoid F may be a useful therapeutic compound for the prevention or treatment of inflammation-associated diseases.

In vitro antiproliferative, anti-inflammatory effects and molecular docking studies of natural compounds isolated from Sarcocephalus pobeguinii (Hua ex Pobég).

Background: Sarcocephalus pobeguinii (Hua ex Pobég) is used in folk medicine to treat oxidative-stress related diseases, thereby warranting the investigation of its anticancer and anti-inflammatory properties. In our previous study, the leaf extract of S. pobeguinii induced significant cytotoxic effect against several cancerous cells with high selectivity indexes towards non-cancerous cells. Aim: The current study aims to isolate natural compounds from S. pobeguinii, and to evaluate their cytotoxicity, selectivity and anti-inflammatory effects as well as searching for potential target proteins of bioactive compounds. Methods: Natural compounds were isolated from leaf, fruit and bark extracts of S. pobeguinii and their chemical structures were elucidated using appropriate spectroscopic methods. The antiproliferative effect of isolated compounds was determined on four human cancerous cells (MCF-7, HepG2, Caco-2 and A549 cells) and non-cancerous Vero cells. Additionally, the anti-inflammatory activity of these compounds was determined by evaluating the nitric oxide (NO) production inhibitory potential and the 15-lipoxygenase (15-LOX) inhibitory activity. Furthermore, molecular docking studies were carried out on six putative target proteins found in common signaling pathways of inflammation and cancer. Results: Hederagenin (2), quinovic acid 3-O-[α-D-quinovopyranoside] (6) and quinovic acid 3-O-[β-D-quinovopyranoside] (9) exhibited significant cytotoxic effect against all cancerous cells, and they induced apoptosis in MCF-7 cells by increasing caspase-3/-7 activity. (6) showed the highest efficacy against all cancerous cells with poor selectivity (except for A549 cells) towards non-cancerous Vero cells; while (2) showed the highest selectivity warranting its potential safety as a chemotherapeutic agent. Moreover, (6) and (9) significantly inhibited NO production in LPS-stimulated RAW 264.7 cells which could mainly be attributed to their high cytotoxic effect. Besides, the mixture nauclealatifoline G and naucleofficine D (1), hederagenin (2) and chletric acid (3) were active against 15-LOX as compared to quercetin. Docking results showed that JAK2 and COX-2, with the highest binding scores, are the potential molecular targets involved in the antiproliferative and anti-inflammatory effects of bioactive compounds. Conclusion: Overall, hederagenin (2), which selectively killed cancer cells with additional anti-inflammatory effect, is the most prominent lead compound which may be further investigated as a drug candidate to tackle cancer progression.

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.

Anti-inflammatory principles from Balanophora laxiflora

Eighteen known compounds, including lignans, phenylpropanoids, triterpenoids, and phytosterols, were isolated from Balanophora laxiflora (Balanophoraceae). The inhibitory activities of these compounds on the production of nitric oxide (NO), a pro-inflammatory mediator, were evaluated. Compounds isolariciresinol (1), ethyl caffeate (13) and ferulic aldehyde (16) showed potent to mild inhibitory activities on LPS-stimulated NO production in RAW 264.7 macrophages with IC 50 values of 0.81, 7.29, and 98.67 μM, respectively. The results showed that compound 1 had a potent effect on TNF-α production with an IC 50 value of 0.87 μM, but practically no effect on IL-6 synthesis. In addition, an inhibitory effect of compound 1 on nuclear factor-κB (NF-κB) activation was shown by the repression of LPS-stimulated β-lactamase reporter activity. Taken together, these results demonstrated that compound 1 modulates the production of inflammatory mediators through the attenuation of NF-κB transcription signaling.

Hydrolase-treated royal jelly attenuates LPS-induced inflammation and IgE-antigen-mediated allergic reaction

Background: Royal jelly (RJ) is one of the most effectual and beneficial remedies for human beings and currently utilized in many sectors, ranging from the pharmaceutical and food industries to cosmetic and manufacturing sectors due to RJ possessing many bio-therapeutical activities including anti-tumor, antimicrobial and antioxidant activities, vasodilative and hypotensive activities, as well as growth-stimulating, infection-preventing, anti-hypercholesterolemic and anti-inflammatory activities. However, some reports showing direct consumption of RJ can lead to severe allergic reaction and has been linked with acute asthma, dermatitis, and life-threatening anaphylaxis. Thus, this research purposes to explore the potential anti-inflammatory and anti-allergic activities of hydrolyzed RJ as a function of enzyme and the extent of hydrolysis.Methods: RJ was enzymatically hydrolyzed with three commercial enzymes (AlcalaseÒ, FlavourzymeÒ and ProtamexÒ). Anti-inflammatory activity of the hydrolysates was measured by their inhibitory effect on nitric oxide (NO) production of lipopolysaccharide (LPS)-stimulated RAW264.7 macrophage cells. Anti-allergy was determined from the ability of the hydrolysates to inhibit b-hexsosaminidase (b-HEX) release from RBL-2H3 mast cells. Cytotoxicity was also investigated in both RAW264.7 macrophage cells and RBL-2H3 mast cells.Results: The electrophoretic profiles indicated that AlcalaseÒ and FlavourzymeÒ hydrolysates did not show the presence of proteins causing allergic reaction after 60 mins of hydrolysis while these allergens disappeared from ProtamexÒ hydrolysate at the hydrolysis time of 240 min. It was observed that hydrolyzed RJ showed no toxicity on RAW264.7 and RBL-2H3 cells. With the progression of hydrolysis, IC50 values of NO production inhibition significantly decreased while degree of hydrolysis (DH) was increased in all hydrolyzed samples (p < 0.05). Results of b-HEX release inhibition were found in the same fashion. FlavourzymeÒ hydrolysate at the 240 min time point effectively mitigated the oxidative stress and protected DNA in a dose dependent manner.Conclusions: RJ hydrolysates from FlavourzymeÒ resulted in peptides with anti-inflammatory activity as determined by the inhibition of NO production in LPS-stimulated RAW264.7 macrophage cells and anti-allergic property as measured by the suppression of degranulation of sensitized RBL-2H3 cells. Anti-inflammatory effect may be due to their anti-oxidative capability. Inhibition of b-HEX release may be due to their membrane-stabilizing effects or/and blockade of IgE antibody binding to its receptors.Keywords: anti-inflammation, enzymatic hydrolysate, royal jelly, anti-allergy

The Inhibitory Activity of Picria fel-terrae Lour Herbs Extract on Nitric Oxide Production toward RAW 264.7 Cells Induced by Lipopolysaccharide.

AIM:The objective of this study was to evaluate the inhibitory activity of Picria fel-terrae Lour on Nitric Oxide production toward RAW 264.7 cells.METHODS:The extraction was obtained by maceration method using n-hexane, ethyl acetate and ethanol solvents and then nitric oxide (NO) production was obtained using Griess reagent.RESULTS:Extract of Picria fel-terrae Lour herbs can reduce the NO production toward RAW 264.7 cells with induced by lipopolysaccharide has obtained nitric concentrations 12.5 and 25 μg/mL from n-hexane extract (72.50 ± 4.51 and 10.42 ± 1.82), ethyl acetate extract: (88.33 ± 6.51 and 30.83 ± 6.86), ethanol extract: (75.00 ± 1.91 and 22.08 ± 2.53).CONCLUSION:n-hexane extract of Picria fel-terrae Lour Herbs has a high potential to reduce the NO production in LPS-stimulated RAW 264.7 cells compared to ethyl acetate and ethanol extracts of Picria fel-terrae Lour Herbs.

Anti-inflammatory and anti-allergic activities of Skipjack tuna (Katsuwonus pelamis) dark muscle hydrolysates evaluated in cell culture model

Background: Oxidative stress and inflammation are inextricably linked and play major roles in the onset and development of Non-communicable diseases (NCD) which are the most common cause of death and disability in modern world. Hydrolyzed proteins have also been suggested to be used to manage adverse food allergic reaction. Therefore, this study aimed to investigate anti-inflammatory and anti-allergy activities of dark muscle tuna hydrolysates using biological cell line systems as a function of enzyme, the extent of hydrolysis and molecular weight range.Methods: Dark muscle tuna hydrolysates were prepared with two different enzyme types; Alcalase and Flavourzyme. Anti-inflammation activity was measured by inhibitory effect of nitric oxide (NO) production on lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophage cells. Anti-allergy was determined from ability of hydrolysates to inhibit b-hexsosaminidase (b-HEX) release from RBL-2H3 mast cells. Cytotoxicity was also investigated in both RAW 264.7 macrophage cells and RBL-2H3 mast cells.Results: No cytotoxic effect on RAW 264.7 macrophage cells and RBL-2H3 mast cells was observed. The NO inhibition and b-HEX release were found significant in dose dependent manner (p<0.05). Alcalase hydrolysates demonstrated greater anti-inflammatory and anti-allergic activities than Flavourzyme hydrolysates (p<0.05). IC50 of both effects were lower than the unhydrolyzed control, > 45.44 mg/ml for NO inhibition and > 65.23 mg/ml for b-HEX release inhibition. These effects increased with the extent of hydrolysis and enzyme concentration. The peptide of lowest molecular weight range (< 3 KDa) was highest in anti-inflammatory and anti-allergic actions. Reducing secretion of TNF-a, IL-6 and IL-1b was found greater in Alcalase hydrolysate than Flavourzyme one.Conclusions: Skipjack tuna dark muscle hydrolysates from Alcalase resulted in peptides with anti-inflammation activity, as determined by NO production in LPS-stimulated RAW 264.7 macrophage cells and anti-allergic properties as measured by a suppression of degranulation of sensitized RBL-2H3 cells. Anti-inflammatory effect may be due to their anti-oxidative capacity and relevant inflammatory factors attenuated with hydrolysate by reducing secretion of pro-inflammatory cytokine (TNF-a, IL-6 and IL-1b). Inhibition of b-HEX release by peptides may be due to membrane-stabilizing action or/and blockade of IgE antibody at fragment region.Keywords: Skipjack tuna, anti-inflammation, enzymatic hydrolysate, dark muscle, anti-allergy

Bioactive compounds from the aerial parts of Hypericum sampsonii

Hypericum sampsonii is an important medicinal plant used in Vietnam to treat many diseases such as backache, burns, diarrhea, and swelling. In order to study the chemical constituents in the aerial parts of H. sampsonii, five compounds, including two xanthones (1–2), and three benzophenones (3–5) were isolated from the aerial parts of the H. sampsonii with various chromatographic separations. Their chemical structures were established on the basis of spectroscopic data such as 1D- and 2D-NMR, HR-ESI-MS. Their anti-inflammatory activities were investigated by measuring nitric oxide (NO) production in lipopolysaccharide (LPS)-induced RAW264.7 cells. Moreover, the DPPH radical scavenging was evaluated. As the obtained results, compound 5 showed the strongest inhibitory activity against LPS-stimulated NO production in RAW264.7 cells with IC50 value ranging from 2.00 ± 0.34 µM.

Optimization of an anti-inflammatory screening model on the RAW 264.7 macrophage cell

Inflammation is the response of living tissues to the injury. Prolonged or inappropriate inflammation has been involved with many diseases such as, cancer, diabetes, heart disease… These days, inflammation has been treated by nonsteroidal and steroidal anti-inflammatory drugs, which have a lot of side effects. It opens the need and interest of new drugs discovery. Particularly, scientific and pharmaceutical communities show great interest in finding new anti-inflammatory compounds in traditional medicinal plants. This study aimed to optimize an in vitro anti-inflammatory model. Cell heterogeneity, cell density, LPS concentration and LPS incubation time were chosen to optimize the production of nitric oxide (NO) in RAW 264.7 macrophage cells. Our results show that 104 cells/well, FBS 1 %, starvation for 6 h, LPS 0.5 µg/mL in 24 h were optimized parameters in the model. Then, extracts from Hedyotis capitellata, a traditional medicinal plant used by K’ho minority, Bidoup Nuiba national park, Lam Dong province, Vietnam, was chosen to evaluate the in vitro antiinflammatory model. The anti-inflammatory activity was tested by measuring the production of NO in lipopolysaccharide-activated RAW 264.7 macrophage cells. The experimental data indicate that the extracts of this plant decreased NO production in LPS-stimulated RAW 264.7, particularly, the petroleum ether fraction at the concentration of 23.8 µg/mL inhibited NO production by 128.20 %; whereas dexamethasone 50 µM inhibited NO accumulation to 112 %.

Alkaloids from Tetrastigma hemsleyanum and Their Anti-Inflammatory Effects on LPS-Induced RAW264.7 Cells

Alkaloids 1–10 were isolated from the aerial parts of Tetrastigma hemsleyanum (APTH) and obtained from species of the genus Tetrastigma for the first time. The chemical structures of the isolated compounds were identified by NMR, UV, and MS analyses. Their anti-inflammatory activities were investigated by measuring nitric oxide (NO) production in lipopolysaccharide (LPS)-induced RAW264.7 macrophages. Among all the isolates, compounds 6, 7 and 10 showed potent inhibitory activity against LPS-stimulated NO production in RAW264.7 cells (IC50: 31.9, 25.2 and 6.3 μM, respectively). Furthermore, APTH and S-(−)-trolline (10) inhibited induction of inflammatory cytokines or mediators such as interleukin-1β (IL-1β) and inducible nitric oxide synthase (iNOS) via suppression of nuclear factor κB (NF-κB) translocation into the nucleus. In addition, 10 suppressed extracellular signal-regulated protein kinase 1/2 (ERK1/2) mitogen-activated protein kinase (MAPK) phosphorylation in a dose-dependent manner. These results conclusively demonstrated that compound 10 displays anti-inflammatory activity via suppression of NF-κB activation and the ERK-MAPK signaling pathway in LPS-stimulated RAW264.7 cells.

Ethanol Extract of Lilium Bulbs Plays an Anti-Inflammatory Role by Targeting the IKK[Formula: see text]/[Formula: see text]-Mediated NF-[Formula: see text]B Pathway in Macrophages.

Lilium bulbs have long been used as Chinese traditional medicines to alleviate the symptoms of various human inflammatory diseases. However, mechanisms of Lilium bulb-mediated anti-inflammatory activity and the bioactive components in Lilium bulbs remain unknown. In the present study, the anti-inflammatory activity of Lilium bulbs and the underlying mechanism of action were investigated in macrophages using Lilium bulb ethanol extracts (Lb-EE). In a dose-dependent manner, Lb-EE inhibited nitric oxide (NO) production in lipopolysaccharide (LPS)-stimulated RAW264.7 cells and bone marrow-derived macrophages (BMDMs) without causing significant cytotoxicity. Lb-EE also down-regulated mRNA expression of inflammatory genes in LPS-stimulated RAW264.7 cells, which included inducuble nitric oxide synthase (iNOS), cyclooxygenase-2 (COX2), and tumor necrosis factor-[Formula: see text] (TNF-[Formula: see text]). Furthermore, Lb-EE markedly restored LPS-induced morphological changes in RAW264.7 cells to a normal morphology. HPLC analysis identified quercetin, luteolin, and kaempferol as bioactive components contained in Lb-EE. Mechanistic studies in LPS-stimulated RAW264.7 cells revealed that Lb-EE suppressed MyD88- and TRIF-induced NF-[Formula: see text]B transcriptional activation and the nuclear translocation of NF-[Formula: see text]B transcription factors. Moreover, Lb-EE inhibited IKK[Formula: see text]/[Formula: see text]-induced activation of the NF-[Formula: see text]B signaling pathway and IKK inhibition significantly reduced NO production in LPS-stimulated RAW264.7 cells. Taken together, these results suggest that Lb-EE plays an anti-inflammatory role by targeting IKK[Formula: see text]/[Formula: see text]-mediated activation of the NF-[Formula: see text]B signaling pathway during macrophage-mediated inflammatory responses.

In-vitro antioxidative, antiinflammatory properties of Aurea helianthus leaf extract a Korean traditional medicinal plant.

The leaf of Aurea helianthus (A. helianthus Jinhuakui) is popularly used in China traditional medicine, however, scientific evidence on its antioxidant properties rarely studied. In this study, biological activities of A. helianthus leave’s 80% ethanol extract (AHL) were investigated. The measured total polyphenol and flavonoid content of AHL was 184.24 ± 5.01 mg GAE/g and 102.53 ± 0.98 mg NAR/g. AHL showed the highest α, α-diphenyl-β-picrylhydrazyl (DPPH) and 2,2′-azino-bis-3-ethylbenzo-thiazoline-6-sulfonic acid (ABTS) radical scavenging activities of 98.30 ± 0.18% at 1000 µg/mL. DPPH and ABTS radical scavenging activities significantly increased in a AHL concentration-dependent manner. AHL treatment significantly suppressed the generation of pro-inflammatory mediators, including nitric oxide (NO), in lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages. AHL demonstrated strong anti-inflammatory activity that reduced NO production in LPS-stimulated RAW 264.7 cells. To test the potential protective effect of AHL, the antioxidant capacity, on the cell growth, viability of a human hepatoma cell (HepG2) and Raw 264.7 cell were investigated. AHL also enhanced cytotoxicity on the proliferation of HepG2 cells and was capable of inhibiting 56% against LPS at 400 µg/mL. The results of this study the potential of AHL as an excellent antioxidant substance for inhibiting inflammatory mediators. Therefore, AHL may be used as a therapeutic approach to various inflammatory diseases.

Secondary metabolites produced by an endophytic fungus Cordyceps ninchukispora from the seeds of Beilschmiedia erythrophloia Hayata

Four new amide derivatives, designated as cordycepiamides A–D (1–4), together with 14 known compounds (5–18), were isolated from the EtOAc-soluble fraction of the 95% EtOH extract of long-grain rice fermented with the endophytic fungus C. ninchukispora BCRC 31900, derived from the seeds of medicinal plant Beilschmiedia erythrophloia Hayata. Their structures were elucidated by means of spectroscopic and mass-spectrometric analyses, particularly 1D and 2D NMR spectroscopy as well as HRESIMS. All known isolates except 11, were isolated for the first time from this species. The antiinflammatory activities of selected isolated 10 compounds (1, 2, 4–6, 9–12, and 14) were evaluated as inhibitory activities against lipopolysaccharide (LPS) induced nitric oxide (NO) production in RAW264.7 cell lines. Compound 3→4 was shown to have modest anti-inflammatory effects through inhibition of NO production in LPS-stimulated RAW264.7 cells.

Anti-inflammatory activity of a sulfated polysaccharide isolated from an enzymatic digest of brown seaweed Sargassum horneri in RAW 264.7 cells.

BACKGROUND/OBJECTIVESSargassum horneri is an edible brown alga that grows in the subtidal zone as an annual species along the coasts of South Korea, China, and Japan. Recently, an extreme amount of S. horneri moved into the coasts of Jeju Island from the east coast of China, which made huge economic and environmental loss to the Jeju Island. Thus, utilization of this biomass becomes a big issue with the local authorities. Therefore, the present study was performed to evaluate the anti-inflammatory potential of crude polysaccharides (CPs) extracted from S. horneri China strain in lipopolysaccharide (LPS)-stimulated RAW 264.7 cells.MATERIALS/METHODSCPs were precipitated from S. horneri digests prepared by enzyme assistant extraction using four food-grade enzymes (AMG, Celluclast, Viscozyme, and Alcalase). The production levels of nitric oxide (NO) and pro-inflammatory cytokines, including tumor necrosis factor (TNF)-α and interleukin (IL)-1β were measured by Griess assay and enzyme-linked immunosorbent assay, respectively. The levels of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2), nuclear factor (NF)-κB, and mitogen-activated protein kinases (MAPKs) were measured by using western blot. The IR spectrums of the CPs were recorded using a fourier transform infrared spectroscopy (FT-IR) spectrometer.RESULTSThe polysaccharides from the Celluclast enzyme digest (CCP) showed the highest inhibition of NO production in LPS-stimulated RAW 264.7 cells (IC50 value: 95.7 µg/mL). Also, CCP dose-dependently down-regulated the protein expression levels of iNOS and COX-2 as well as the production of inflammatory cytokines, including TNF-α and IL-1β, compared to the only LPS-treated cells. In addition, CCP inhibited the activation of NF-κB p50 and p65 and the phosphorylation of MAPKs, including p38 and extracellular signal-regulated kinase, in LPS-stimulated RAW 264.7 cells. Furthermore, FT-IR analysis showed that the FT-IR spectrum of CCP is similar to that of commercial fucoidan.CONCLUSIONSOur results suggest that CCP has anti-inflammatory activities and is a potential candidate for the formulation of a functional food ingredient or/and drug to treat inflammatory diseases.

Chemical constituents from the fruits of Vitex negundo var. cannabifolia and their biological activities in vitro

Chemical constituents from the fruits of Vitex negundo var. cannabifolia and their nitric oxide (NO) inhibitory and cytotoxic activities were investigated. The compounds were isolated and purified by various column chromatography, and their structures were identified by physiochemical properties and spectroscopic data. Thirteen lignans and six phenolic compounds were isolated from the CH2Cl2 extract of the fruits of V. negundo var. cannabifolia, respectively. Their structures were elucidated as 6-hydroxy-4-(4-hydroxy-3-methoxyphenyl)-3-hydroxymethyl-7-methoxy-3,4-dihydro-2-naphthaldehyde (1), vitedoin A (2), vitexdoin F (3), detetrahydroconidendrin (4), vitexdoin E (5), 4-oxosesamin (6), L-sesamin (7), (+)-beechenol (8), ligballinol (9), 2-(4-hydroxyphenyl)-6-(3-methoxy-4-hydroxyphenyl)-3,7-dioxabicyclo[3.3.0]octane (10), (-)-pinoresinol (11), balanophonin (12), thero-guaiacylglycerol-β-coniferyl aldehyde ether (13), trans-p-coumaryl aldehyde (14), coniferyl aldehyde (15), 5,7-dihydroxychromone (16), trans-3,5-dimethoxy-4-hydroxy-cinnamic aldehyde (17), frambinone (18), and alternariol 4-methyl ether (19). Compounds 8-10,14,18,19 were firstly isolated from Verbenaceae family, compound 13 was obtained from Vitex species, and 6,7,12,15-17 from V. negundo var. cannabifolia for the first time, respectively. The isolated compounds were evaluated for their anti-inflammatory and cytotoxic effects in vitro. Eight compounds (3,5,7,10,11,14,15,17) showed inhibition against NO production in LPS-stimulated RAW 267.4 cells (IC₅₀ in the range of 7.8-81.1 μmol•L⁻¹) and four compounds (1-4) showed cytotoxicity on HepG-2 cells (IC₅₀ in the range of 5.2-24.2 μmol•L⁻¹).

Comparative characterization of physicochemical properties and bioactivities of polysaccharides from selected medicinal mushrooms.

Mushroom polysaccharides have been known to possess various pharmacological activities. However, information on their chemical and biological differences between mushrooms remains limited. In this study, we aimed to examine the differences in physicochemical characteristics of polysaccharides prepared from Antrodia cinnamomea (AC-P), Coriolus versicolor (CV-P), Grifola frondosa (GF-P), Ganoderma lucidum (GL-P), and Phellinus linteus (PL-P), followed by evaluating their inhibitory effects on nitric oxide (NO) production in lipopolysaccharide (LPS)-stimulated RAW264.7 macrophages. Results showed that under similar conditions of preparation, the monosaccharide composition of polysaccharides varied between different mushrooms, and glucose was the predominant monosaccharide, followed by galactose and mannose. AC-P and GF-P contained the highest amount of (1,3;1,6)-β-D-glucans. The degree of branching of (1,3;1,6)-β-D-glucans in all polysaccharides ranged from 0.21 to 0.26, with the exception of GF-P (0.38). The molecular weights of different polysaccharides showed diverse distributions; AC-P, CV-P, and GF-P contained two major macromolecular populations (< 30 and >200kDa) and possessed triple-helix conformation, whereas GL-P (10.2kDa) and PL-P (15.5kDa) only had a low molecular weight population without triple-helix structure. These polysaccharides showed different inhibitory potency on NO production in LPS-stimulated RAW264.7 cells.

Attenuation of Inflammatory Mediators (TNF-α and Nitric Oxide) and Up-Regulation of IL-10 by Wild and Domesticated Basidiocarps of Amauroderma rugosum (Blume & T. Nees) Torrend in LPS-Stimulated RAW264.7 Cells.

Amauroderma rugosum, commonly known as “Jiǎzī” in China, is a wild mushroom traditionally used by the Chinese to reduce inflammation, to treat diuretic and upset stomach, and to prevent cancer. It is also used by the indigenous communities in Malaysia to prevent epileptic episodes and incessant crying by babies. The aim of this study was to compare the wild and domesticated basidiocarps of A. rugosum for antioxidant and in vitro anti-inflammatory effects in LPS-stimulated RAW264.7 cells. The wild basidiocarps of A. rugosum were collected from the Belum Forest, Perak, Malaysia and the domesticated basidiocarps of A. rugosum were cultivated in the mushroom house located in the University of Malaya, Kuala Lumpur, Malaysia. Both the wild and domesticated basidiocarps were subjected to ethanolic extraction and the extracts were tested for antioxidant and anti-inflammatory activities. In this study, the crude ethanolic extract of wild (WB) and domesticated (DB) basidiocarps of A. rugosum had comparable total phenolic content and DPPH scavenging activity. However, WB (EC50 = 222.90 μg/mL) displayed a better ABTS cation radical scavenging activity than DB (EC50 = 469.60 μg/mL). Both WB and DB were able to scavenge nitric oxide (NO) radical and suppress the NO production in LPS-stimulated RAW264.7 cells and this effect was mediated through the down-regulation of inducible nitric oxide synthase (iNOS) gene. In addition, both WB and DB caused down-regulation of the inflammatory gene TNF-α and the up-regulation of the anti-inflammatory gene IL-10. There was no inhibitory effect of WB and DB on nuclear translocation of NF-κB p65. In conclusion, the wild and domesticated basidiocarps of A. rugosum possessed antioxidant and in vitro anti-inflammatory properties. WB and DB inhibited downstream inflammatory mediators (TNF-α and NO) and induced anti-inflammatory cytokine IL-10 production. No inhibitory effects shown on upstream nuclear translocation of NF-κB p65. WB and DB exhibited antioxidant activity and attenuation of proinflammatory mediators and therefore, A. rugosum may serve as a potential therapeutic agent in the management of inflammation.

Inhibition of nitric oxide production by Solanum melongena and Solanum macrocarpon on RAW 264.7 cells

Nitric oxide (NO) plays an important role in the physiology and pathophysiology of disease. Overproduction of NO is associated with chronic inflammatory diseases and cancers. Several species of Solanaceae have been used traditionally to treat inflammatory-related diseases. To analyse the possible anti-inflammatory properties of these species, the Griess assay was used to evaluate the effects of various Solanum melongena and Solanum macrocarpon extracts on NO production in lipopolysaccharide (LPS)-stimulated RAW 264.7 cells. The cytotoxicity of the extracts on the cell line was tested using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Extracts that significantly inhibited NO production were further evaluated for inducible nitric oxide synthase (iNOS) expression by Western blot. Thin-layer chromatography was used to determine the major compounds in the extracts. All extracts significantly inhibited NO production in LPS-stimulated RAW 264.7 cells in a dose-dependent manner. At 200 µg/ml, ethyl acetate extract of S. macrocarpon showed the highest NO inhibition of 81%, with a median inhibitory concentration (IC50) value of 44.78 ± 0.04 µg/ml. The viability of cells treated with the extracts was greater than 80%. Ethanol and ethyl acetate extracts of S. melongena, together with ethanol, hexane and ethyl acetate extracts of S. macrocarpon, reduced iNOS expression significantly. At 200 µg/ml, ethyl acetate extract of S. macrocarpon inhibited iNOS protein expression by 79%. Phytochemical analysis of the extracts showed that fluorescent, double-bond compounds, phenols, flavonoids and terpenoids were mainly present in the extracts. Taken together, the results show the potential of ethanol and ethyl acetate extracts of S. melongena, and hexane and ethyl acetate extracts of S. macrocarpon, as agents for the prevention and treatment of inflammatory-related diseases.

New ent-kauranes from the fruits of Annona glabra and their inhibitory nitric oxide production in LPS-stimulated RAW264.7 macrophages

Three new ent-kaurane diterpenoids, 7β,16α,17-trihydroxy-ent-kauran-19-oic acid (1), 7β,17-dihydroxy-16α-ent-kauran-19-oic acid 19-O-β-d-glucopyranoside ester (2), 7β,17-dihydroxy-ent-kaur-15-en-19-oic acid 19-O-β-d-glucopyranoside ester (3) along with five known compounds, paniculoside IV (4), 16α,17-dihydroxy-ent-kaurane (5), 16β,17-dihydroxy-ent-kaurane (6), 16β,17-dihydroxy-ent-kauran-19-al (7), and 16β,17-dihydroxy-ent-kauran-19-oic acid (8) were isolated from the fruits of Annona glabra. Their chemical structures were elucidated by physical and chemical methods. All compounds were evaluated for inhibitory activity against nitric oxide (NO) production in LPS-stimulated RAW 264.7 macrophages. As the results, compound 3 showed potent inhibitory LPS-stimulated NO production in RAW 264.7 macrophages with the IC50 value of 0.01±0.01μM; compounds 1 and 7 showed significant inhibitory NO production with the IC50 values of 0.39±0.12μM and 0.32±0.04μM, respectively.