Macrophage activation by polysaccharides from Atractylodes macrocephala Koidz through the nuclear factor-κB pathway

Abstract

Context: Atractylodes macrocephala Koidz is a traditional herb. Atractylodes macrocephalaon polysaccharides (AMP) have been found to enhance immunity and improve heart function. However, the mechanisms of the immunomodulatory effect have not been investigated.

Objective: We examined whether AMP activated macrophages and explored the mechanisms of activation.

Materials and methods: AMP was prepared and evaluated its immunomodulatory activity (25, 50, 100, and 200?μg/mL) by detecting the phagocytosis and the production of tumor necrosis factor-α (TNF-α), IFN-γ, and nitric oxide (NO) in RAW264.7 macrophages. Furthermore, the role of nuclear factor-κB (NF-κB) pathway was examined in regulating TNF-α and NO production.

Results: The phagocytosis of macrophages was enhanced by AMP in a dose-dependent manner and the maximal phagocytosis of macrophages occurred at concentrations of 100 and 200?μg/mL. NO, TNF-α, and IFN-γ release was also found to be dose dependent by increasing concentrations of AMP and reached the peak at a concentration of 200?μg/mL. In addition, AMP induced inhibitor kappaB (IκB) degradation and the activation of NF-κB by p65 nuclear translocation, and then the activation of NF-κB in nucleus peaked at a concentration of 200?μg/mL. Besides, NF-κB-specific inhibitor pyrrolidine dithiocarbamate (PDTC) decreased AMP-induced NO and TNF-α production.

Discussion and conclusion: These data suggest that AMP may modulate macrophage activities by stimulating NF-κB or activating NF-κB-dependent mechanisms.     

Potential anti-inflammatory,antioxidant and antimicrobial activities of Sambucus australis

Context: Sambucus australis Cham. &; Schltdl. (Adoxaceae) is used in Brazilian folk medicine to treat inflammatory disorders.

Objective: To evaluate the in vitro anti-inflammatory, antioxidant and antimicrobial properties of S. australis.

Materials and methods: The anti-in?ammatory activity of ethanol extracts of the leaf and bark of S. australis (1–100?μg/mL) were studied in lipopolysaccharide/interferon γ stimulated murine macrophages RAW 264.7 cells (24?h incubation) by investigating the release of nitric oxide (NO) and tumour necrosis factor-alpha (TNF-α) and in the TNF-α-induced nuclear factor kappa (NF-κB) assay. Minimum inhibitory concentration (MIC) was determined by the microdilution test (24?h incubation). Antioxidant activity was determined by 2,2-diphenyl-1-picrylhydrazyl (DPPH), ferric reducing antioxidant power (FRAP) and the NO scavenging assays. Chemical composition was assessed by LC-MS/MS.

Results: Antioxidant activities in the DPPH (IC₅₀ 43.5 and 66.2?μg/mL), FRAP (IC₅₀ 312.6 and 568.3?μg/mL) and NO radical scavenging assays (IC₅₀ 285.0 and 972.6?μg/mL) were observed in the leaf and bark ethanol extracts, respectively. Solely the leaf extract showed significant inhibition of NO and TNF-α production in RAW264.7 cells at concentrations of 2 and 100?μg/mL, respectively, and suppression of TNF-α inhibition of NF-κB by 12.8 and 20.4% at concentrations of 50 and 100?μg/mL, respectively. The extract also exhibited antibacterial activity against Salmonella typhimurium (MIC 250?μg/mL) and Klebsiella pneumoniae (MIC 250?μg/mL). LC-MS/MS revealed the presence of chlorogenic acid and rutin as major compounds.

Discussion and conclusion: The results indicate that the ethanol leaf extract of S. australis exhibit prominent anti-in?ammatory effects.     

Role of GABAB receptors and p38MAPK/NF-κB pathway in paclitaxel-induced apoptosis of hippocampal neurons

Context: The effects of the anticancer drug paclitaxel on learning and memory are rarely studied.

Objective: This study investigated changes in GABA_B receptor expression during paclitaxel-induced apoptosis of hippocampal neurons and the role of the p38MAPK/NF-κB pathway in this process.

Materials and methods: Hippocampal neurons isolated from neonatal Sprague–Dawley rats were divided into six groups: Control (C), SB (10?µL of 10-µmol/L SB203580), SN (53?µg/mL SN50), N (1?µmol/L paclitaxel), SB?+?N (10?µmol/L SB203580?+?1?µmol/L paclitaxel) and SN?+?N (53?µg/mL SN50?+?1?µmol/L paclitaxel). Cells in different groups were treated with corresponding agents for 24?h at 37?°C. The apoptosis rate and protein levels of GABA_B1 receptors and NF-κB p65 were evaluated. Rat models of neuropathic pain was induced by paclitaxel and were divided into four groups such as N, B?+?N, SN?+?N and SN?+?B?+?N groups. Rats in the N group received intrathecal injections of normal saline solution. Rats in the B?+?N group received intrathecal injections of 10?μL baclofen (0.05?μg/μL). Rats in the SN?+?N and SN?+?B?+?N groups received intrathecal injections of SN50 and SN50 plus baclofen, respectively. Spatial learning and memory were evaluated in rat models based on the escape latency and the number of crossings over the platform and protein levels of GABA_B1 receptors, NF-κB, IL-1β and TNFα were measured by immunohistochemistry assay and western blot.

Results: The neuronal apoptosis rate was significantly increased in N (49.16?±?3.12)%, SB?+?N (31.18?±?3.02)% and SN?+?N (28.47?±?3.75)% groups, accompanied by increased levels of GABA_B1 receptors and NF-κB p65 (p?p?B1:9.0?±?1.6, NF-κB p65:29.6?±?2.4, IL-1β: 30.4?±?3.4, TNFα: 31.0?±?3.4), B?+?N, SN?+?N and SN?+?B?+?N groups evidently increased levels of GABA_B1 receptor (B?+?N:SN?+?N:SN?+?B?+?N?=?19.4?±?2.1:20.8?±?1.9:28.0?±?1.9) but significantly decreased levels of NF-κB p65 (B?+?N:SN?+?N:SN?+?B?+?N?=?21.2?±?1.5:18.6?±?2.1:12.6?±?1.5), IL-1β (B?+?N:SN?+?N:SN?+?B?+?N?=?22.0?±?1.0:19.6?±?1.8:14.6?±?1.5) and TNF α (B?+?N:SN?+?N:SN?+?B?+?N?=?23.0?±?1.6:22.2?±?0.8:16.6?±?1.7). Similar findings were found in western blot analysis.

Discussions and conclusions: Paclitaxel may reduce cognitive function in rats through the p38MAPK/NF-κB pathway and GABA_B1 receptors.     

Anti-inflammatory effect of pomegranate flower in lipopolysaccharide (LPS)-stimulated RAW264.7 macrophages

Context: Punica granatum L (Punicaceae) flower is an important diabetes treatment in oriental herbal medicine.

Objective: This study investigates the inflammation effects of pomegranate flower (PFE) ethanol extract in LPS-induced RAW264.7 cells.

Materials and methods: PFE (10, 25, 50, 100?μg/mL) was applied to 1?μg/mL LPS-induced RAW 264.7 macrophages in vitro. Levels of nitric oxide (NO), prostaglandin E₂ (PGE₂) and pro-inflammatory cytokines interleukin (IL)-1β (IL-1β), interleukin (IL)-6 (IL-6) and tumor necrosis factor (TNF-α) in the supernatant fraction were determined using enzyme-linked immunosorbent assay (ELISA). Expression of cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS), phosphorylation of mitogen-activated protein kinase (MAPK) subgroups extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK) and P38, as well as nuclear factor-κB (NF-κB) activation in extracts were detected via Western blot.

Results: 10–100?μg/mL PFE decreased the production of NO (IC₅₀ value?=?31.8?μg/mL), PGE₂ (IC₅₀ value?=?54.5?μg/mL), IL-6 (IC₅₀ value?=?48.7?μg/mL), IL-1β (IC₅₀ value?=?71.3?μg/mL) and TNF-α (IC₅₀ value?=?62.5?μg/mL) in LPS-stimulated RAW 264.7 cells significantly. A mechanism-based study showed that phosphorylation of ERK1/2, p38, JNK and translocation of the NF-B p65 subunit into nuclei were inhibited by the PFE treatment.

Discussion and conclusion: These results show that PFE produced potential anti-inflammatory effect through modulating the synthesis of several mediators and cytokines involved in the inflammatory process.     

Chemical composition,antioxidant, antitumor,anticancer and cytotoxic effects of Psidium guajava leaf extracts

Context Psidium guajava L. (Myrtaceae) leaves are used in traditional medicines for the treatment of cancer, inflammation and other ailments.

Objective The current study explores scientific validation for this traditional medication.

Materials and methods We used ferric-reducing antioxidant power (FRAP) and 2,2-diphenyl-1-picryl hydrazil (DPPH) assays to estimate antioxidant activity of P. guajava leaf extracts (methanol, hexane and chloroform). Antitumour and in vivo cytotoxic activities were determined using potato disc assay (PDA) and brine shrimp lethality assay, respectively. Three human carcinoma cell lines (KBM5, SCC4 and U266) were incubated with different doses (10–100?μg/mL) of extracts and the anticancer activity was estimated by MTT assay. NF-κB suppressing activity was determined using electrophoretic mobility shift assay (EMSA). Chemical composition of the three extracts was identified by GC-MS. Total phenolic and flavonoid contents were measured by colorimetric assays.

Results and discussions The order of antioxidant activity of three extracts was methanol?>?chloroform?>?hexane. The IC₅₀ values ranged from 22.73 to 51.65?μg/mL for KBM5; 22.82 to 70.25?μg/mL for SCC4 and 20.97 to 89.55?μg/mL for U266 cells. The hexane extract exhibited potent antitumour (IC_50? value?=_?65.02?μg/mL) and cytotoxic (LC_50? value?=_?32.18?μg/mL) activities. This extract also completely inhibited the TNF-α induced NF-κB activation in KBM5 cells. GC-MS results showed that pyrogallol, palmitic acid and vitamin E were the major components of methanol, chloroform and hexane extracts. We observed significant (p?<?0.05) difference in total phenolic and flavonoid contents of different solvent extracts.

Conclusion The present study demonstrates that P. guajava leaf extracts play a substantial role against cancer and down-modulate inflammatory nuclear factor kB.     

The anti-inflammatory effect of Sonchus oleraceus aqueous extract on lipopolysaccharide stimulated RAW 264.7 cells and mice

Context: Sonchus oleraceus L. (Asteraceae) (SO) is a dietary and traditional medicinal plant in China. However, its underlying mechanism of action as an anti-inflammatory agent is not known.

Objective: This study evaluates the anti-inflammatory activity of aqueous extract of SO.

Materials and methods: The extract of SO was used to treat RAW 264.7 cells (in the working concentrations of 500, 250, 125, 62.5, 31.3 and 15.6?μg/mL) for 24?h. Pro-inflammatory cytokines and mediators produced in LPS-stimulated RAW 264.7 cells were assessed. Meanwhile, the expression level of TLR-4, COX-2, pSTATs and NF-κB was tested. Moreover, the anti-inflammatory activity of the extract in vivo was assessed using xylene-induced mouse ear oedema model and the anti-inflammatory compounds in the extracts were analyzed by HPLC-MS.

Results: SO extract significantly inhibited the production of pro-inflammatory cytokines and mediators at gene and protein levels with the concentration of 31.3?μg/mL, and suppressed the expression of TLR-4, COX-2, NF-κB and pSTAT in RAW 264.7 cells. The anti-inflammatory activity of SO in vivo has significant anti-inflammatory effects with the concentration of 250 and 125?mg/kg, and less side effect on the weights of the mice at the concentration of 250?mg/kg. Moreover, HPLC-MS analysis revealed that the anti-inflammatory compounds in the extract were identified as villosol, ferulaic acid, β-sitosterol, ursolic acid and rutin.

Discussion and conclusion: This study indicated that SO extract has anti-inflammatory effects in vitro and in vivo, which will be further developed as novel pharmacological strategies in order to defeat inflammatory diseases.     

6-Gingerol inhibits Vibrio cholerae-induced proinflammatory cytokines in intestinal epithelial cells via modulation of NF-κB

Context The effect of 6-gingerol (6G), the bioactive component of Zingiber officinale Roscoe (Zingiberaceae), in the reduction of Vibrio cholerae (Vibrionaceae)-induced inflammation has not yet been reported.

Materials and methods Cell viability assay was performed to determine the working concentration of 6G. Elisa and RT-PCR were performed with Int 407 cells treated with 50?μM 6G and 100 multiplicity of infection (MOI) V. cholerae for 0, 2, 3, 3.5, 6 and 8?h to determine the concentration of IL-8, IL-6, IL-1α and IL-1β in both protein and RNA levels. Furthermore, the effect of 50?μM 6G on upstream MAP-kinases and NF-κB signalling pathways was evaluated at 0, 10, 15, 30, 60 and 90?min.

Results The effective dose (ED₅₀) value of 6G was found to be 50?μM as determined by cell viability assay. Pre-treatment with 50?μM 6G reduced V. cholerae infection-triggered levels of IL-8, IL-6, IL-1α and IL-1β by 3.2-fold in the protein level and two-fold in the RNA level at 3.5?h. The levels of MAP-kinases signalling molecules like p38 and ERK1/2 were also reduced by two- and three-fold, respectively, after 30?min of treatment. Additionally, there was an increase in phosphorylated IκBα and down-regulation of p65 resulting in down-regulation of NF-κB pathway.

Conclusion Our results showed that 6G could modulate the anti-inflammatory responses triggered by V. cholerae-induced infection in intestinal epithelial cells by modulating NF-κB pathway.     

Methanol extract of the aerial parts of barley (Hordeum vulgare) suppresses lipopolysaccharide-induced inflammatory responses in vitro and in vivo

Abstract

Context: Recently, there has been renewed interest in barley (Hordeum vulgare L. Poaceae) as a functional food and for its medicinal properties.

Objective: This study examines the anti-inflammatory potential of the active fractions of barley and the mechanisms involved.

Materials and methods: The macrophages were exposed to 100?μg/mL of each of the barley extracts in the presence of 1?μg/mL lipopolysaccharide (LPS) and after 24 or 48?h of incubation, cells or culture supernatants were analyzed by various assays. The anti-inflammatory potential of barley fractions was also investigated using the LPS-injected septic mouse model. The active constituents in the fractions were identified using gas chromatography-mass spectrometry (GC-MS).

Results: The active fractions, named F₄, F₇, F₉ and F₁₂, inhibited almost completely the LPS-induced production of nitric oxide (NO) and inducible NO synthase. Pre-treatment with these fractions at 100?μg/mL diminished the tumor necrosis factor-α (TNF-α) levels to 19.8, 3.5, 1.2 and 1.7?ng/mL, respectively, compared to LPS treatment alone (41.5?ng/mL). These fractions at 100?μg/mL also suppressed apparently the secretion of interleukin (IL)-6 and IL-1β and the DNA-binding activity of nuclear factor-κB in LPS-stimulated cells. Mice injected intraperitoneally with LPS (30?mg/kg BW) showed 20% survival at 48?h after injection, whereas oral administration of the fractions improved the survival rates to 80%. GC-MS analysis revealed the presence of the derivatives of benzoic and cinnamic acids and fatty acids in the fractions.

Discussion and conclusion: The aerial parts of barley are useful as functional food to prevent acute inflammatory responses.     

Bioactivities of six sterols isolated from marine invertebrates

Context: Epidioxy sterols and sterols with special side chains, such as hydroperoxyl sterols, usually obtained from marine natural products, are attractive for bioactivities.

Objective: To isolate and screen bioactive and special sterols from China Sea invertebrates.

Materials and methods: Two hydroperoxyl sterols (1 and 2) from the sponge Xestospongia testudinaria Lamarck (Petrosiidae), three epidioxy sterols (3–5) from the sea urchin Glyptocidaris crenularis A. Agassiz (Glyptocidaridae), sponge Mycale sp. (Mycalidae) and gorgonian Dichotella gemmacea Milne Edwards and Haime (Ellisellidae) and an unusual sterol with 25-acetoxy-19-oate (6) also from D. gemmacea were obtained and identified. Using high-throughput screening, their bioactivities were tested toward Forkhead box O 3a (Foxo3a), 3-hydroxy-3-methylglutaryl CoA reductase gene fluorescent protein (HMGCR-GFP), nuclear factor kappa B (NF-κB) luciferase, peroxisome proliferator-activated receptor-γ co-activator 1α (PGC-1α), protein-tyrosine phosphatase 1B (PTP1B), mitochondrial membrane permeabilization (MMP) and adenosine monophosphate-activated protein kinase.

Results: Their structures were determined by comparing their nuclear magnetic resonance data with those reported in the literature. Three epidioxy sterols (3–5) showed inhibitory activities toward Foxo3a, HMGCR-GFP and NF-κB-luciferase with the IC₅₀ values 4.9–6.8?μg/mL. The hydroperoxyl sterol 29-hydroperoxystigmasta-5,24(28)-dien-3-ol (2) had diverse inhibitory activities against Foxo3a, HMGCR-GFP, NF-κB-luciferase, PGC-1α, PTP1B and MMP, with IC₅₀ values of 3.8–19.1?μg/mL.

Discussion and conclusion: The bioactivities of 3–5 showed that 5α,8α-epidioxy is the active group. Otherwise, the most plausible biosynthesis pathway for 1 and 2 in sponge involves the abstraction of an allylic proton by an activated oxygen, such as O₂, along with migration of carbon–carbon double bond. Therefore, the bioactive and unstable steroid should be biosynthesized in sponge under a special ecological environment to act as a defensive strategy against invaders.     

Anti-cryptococcal activity of ethanol crude extract and hexane fraction from Ocimum basilicum var. Maria bonita: mechanisms of action and synergism with amphotericin B and Ocimum basilicum essential oil

Context: Ocimum basilicum L. (Lamiaceae) has been used in folk medicine to treat headaches, kidney disorders, and intestinal worms.

Objective: This study evaluates the anti-cryptococcal activity of ethanol crude extract and hexane fraction obtained from O. basilicum var. Maria Bonita leaves.

Materials and methods: The MIC values for Cryptococcus sp. were obtained according to Clinical and Laboratory Standards Institute in a range of 0.3–2500?μg/mL. The checkerboard assay evaluated the association of the substances tested (in a range of 0.099–2500?μg/mL) with amphotericin B and O. basilicum essential oil for 48?h. The ethanol extract, hexane fraction and associations in a range of 0.3–2500?μg/mL were tested for pigmentation inhibition after 7?days of treatment. The inhibition of ergosterol synthesis and reduction of capsule size were evaluated after the treatment with ethanol extract (312?μg/mL), hexane fraction (78?μg/mL) and the combinations of essential oil?+?ethanol extract (78?μg/mL?+?19.5?μg/mL, respectively) and essential oil?+?hexane fraction (39.36?μg/mL?+?10?μg/mL, respectively) for 24 and 48?h, respectively.

Results: The hexane fraction presented better results than the ethanol extract, with a low MIC (156?μg/mL against C. neoformans T₄₄₄ and 312?μg/mL against C. neoformans H99 serotype A and C. gattii WM779 serotype C). The combination of the ethanol extract and hexane fraction with amphotericin B and essential oil enhanced their antifungal activity, reducing the concentration of each substance needed to kill 100% of the inoculum. The substances tested were able to reduce the pigmentation, capsule size and ergosterol synthesis, which suggest they have important mechanisms of action.

Conclusions: These results provide further support for the use of ethanol extracts of O. basilicum as a potential source of antifungal agents.     

Anti-inflammatory effect of torilidis fructus ethanol extract through inhibition of Src

Context: Torilidis fructus, fruits of Torilis japonica Decadolle (Umbelliferae), is a medicinal herb traditionally used as a pesticide, an astrictive, or a medicine for various inflammatory diseases.

Objectives: Due to the lack of pharmacological studies on this herbal medicine, we explored the inhibitory activity of torilidis fructus on the macrophage-mediated inflammatory response using its ethanol extract (Tf-EE).

Material and methods: The Griess assay and prostaglandin (PGE₂) ELISA assay were conducted with Tf-EE (0-75?µg/mL) and LPS (1?µg/mL) treated RAW264.7 cells in cultured media. Tf-EE pretreated RAW264.7 cells were incubated with LPS for 6?h and semi-quantitative PCR was performed. Reporter gene assays, overexpression of target enzymes and immunoblotting were performed on macrophages to determine the molecular targets of Tf-EE.

Results: Tf-EE markedly suppressed the inflammatory response of macrophages, such as lipopolysaccharide (LPS)-induced nitric oxide (NO) and PGE₂ production with IC₅₀ values of 35.66 and 62.47?µg/mL, respectively. It was also found that Tf-EE reduced the expression of inducible NO synthase (iNOS) and cyclooxygenase (COX)-2 by 80%. Nuclear translocation and activation of nuclear factor (NF)-κB (p65 and p50) were declined by 60% and 30% respectively, and their regulatory events including the phosphorylation of AKT, IκBα, Src, and the formation of complexes between Src and p-p85 were also recognized to be diminished.

Conclusions: The signalling events managed by Src and p85 complex seemed to be critically involved in Tf-EE-mediated anti-inflammatory response. This might suggest that Tf-EE exhibited anti-inflammatory effects through Src-targeted inhibition of NF-κB.     

Attenuation of berberine on lipopolysaccharide-induced inflammatory and apoptosis responses in β-cells via TLR4-independent JNK/NF-κB pathway

Context: Toll-like receptor 4 (TLR4)-independent inflammatory and apoptosis responses contribute to β-cell failure in diabetes mellitus (DM). Berberine (BBR), a bioactive isoquinoline derivative alkaloid, ameliorates the inflammatory response in DM.

Objective: This study explored the protective mechanisms of BBR on TLR4-independent inflammation response in β cells.

Materials and methods: Lipopolysaccharide (LPS; 100?ng/ml) was used to induce the inflammatory response in NIT-1 and rat insulinoma (INS-1) cells for 24?h. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and colony formation assays were used for the determination of cell viability. The levels of monocyte chemoattractant protein (MCP-1), interleukin 6 (IL-6), tumor necrosis factor α (TNF-α) and insulin in cultured supernatant were detected by enzyme-linked immunosorbent assay kits. Western blot analysis was performed for the expression of p-c-Jun N-terminal kinase (JNK) and p65 NF-κB in NIT-1 cells, and p65 NF-κB in INS-1 cells.

Results: BBR (1.25, 2.5 and 5?μM) or TLR4 inhibitor (TAK-242, 1?μM) increased remarkably NIT-1 cell viability by 72.6?±?5.0, 85.9?±?9.3, 94.7?±?7.1 and 92.6?±?8.4%. The EC₅₀ of BBR was 1.14?μM. Colony formation assay showed that BBR increased the number of colonies of NIT-1 and INS-1 cells. BBR, TAK-242 or SP-600125 (1?μM) could significantly reduce the levels of MCP-1, IL-6 and TNF-α, insulin and JNK and NF-κB phosphorylation in NIT-1 cells, as well as the p65 NF-κB in INS-1 cells.

Discussion and conclusion: BBR could ameliorate LPS-induced β-cell injury through the TLR4-independent JNK/NF-κB pathway. Thus, this pathway may be a potential target for the prevention and treatment of DM.     

Protection by genistein on cortical neurons against oxidative stress injury via inhibition of NF-kappaB,JNK and ERK signaling pathway

Abstract

Context: Genistein, one of the isoflavones derived from soybean seeds, has been reported to exert multiple bioactivities. However, the mechanism of its action on the central nervous system is not fully understood.

Objective: To investigate the cytoprotection of genistein and its molecular mechanism against H₂O₂-induced cell death in primary rat cortical neurons.

Materials and methods: Genistein (0.01, 0.1, and 1?μM) were added into the primary rat neurons 24?h before and co-cultured with 500?μM H₂O₂ for 1?h. Neuronal injury was assessed by MTT, lactate dehydrogenase (LDH) assay, and Hoechst33258 staining. Intracellular reactive oxygen species (ROS) generation induced by H₂O₂ was determined. Neuronal apoptosis was evaluated by Bcl-2/Bax ratio as well as by caspase-9 and caspase-3 activities. The protein levels and phosphorylation of NF-κB/p65, IκB, JNK, and ERK were detected by western blots.

Results: Genistein pretreatment attenuated H₂O₂-mediated neuronal viability loss, nuclear condensation, and ROS generation in a concentration-dependent manner. Genistein exerted anti-apoptotic effects by reversing the apoptotic factors Bcl-2 and Bax ratio, along with the suppression of caspase-9 and caspase-3 activities. In addition, genistein down-regulated the expression of NF-κB/p65, and suppressed the phosphorylation of p65 and IκB. Genistein also inhibited H₂O₂-induced activation of the MAPK-signaling pathway including JNK and ERK.

Discussion and conclusion: The results indicated that genistein effectively protects cortical neurons against oxidative stress at least partly via inactivation of NF-κB as well as MAPK-signaling pathways, and suggested the possibility of this antioxidant for the prevention and treatment of stroke.     

Anti-neuroinflammatory effects of cudraflavanone A isolated from the chloroform fraction of Cudrania tricuspidata root bark

Context: Cudrania tricuspidata Bureau (Moraceae) is an important source of traditional Korean and Chinese medicines used to treat neuritis and inflammation.

Objective: The anti-neuroinflammatory effects of cudraflavanone A isolated from a chloroform fraction of C. tricuspidata were investigated in LPS-induced BV2 cells.

Materials and methods: Cudraflavanone A was isolated from the root of C. tricuspidata, and its structure was determined by MS and NMR data. Cytotoxicity of the compound was examined by MTT assay, indicating no cytotoxicity at 5–40?μM of cudraflavanone A. NO concentration was measured by the Griess reaction, and the levels of PGE₂, cytokines and COX-2 enzyme activity were measured by each ELISA kit. The mRNA levels of cytokines were analysed by quantitative-PCR. The expression of iNOS, COX-2, HO-1, NF-κB, MAPKs and Nrf2 was detected by Western blot.

Results: Cudraflavanone A had no major effect on cell viability at 40?μM indicating 91.5% viability. It reduced the production of NO (IC₅₀?=?22.2?μM), PGE₂ (IC₅₀?=?20.6?μM), IL-1β (IC₅₀?=?24.7?μM) and TNF-α (IC₅₀?=?33.0?μM) in LPS-stimulated BV2 cells. It also suppressed iNOS protein, IL-1β and TNF-α mRNA expression. These effects were associated with the inactivation of NF-κB, JNK and p38 MAPK pathways. This compound mediated its anti-neuroinflammatory effects by inducing HO-1 protein expression via increased nuclear translocation of Nrf2.

Discussion and conclusions: The present study suggests a potent effect of cudraflavanone A to prevent neuroinflammatory diseases. Further investigation is necessary to elucidate specific molecular mechanism of cudraflavanone A.     

Endophytic fungi associated with Taxus fuana (West Himalayan Yew) of Pakistan: potential bio-resources for cancer chemopreventive agents

Context: Endophytic fungi, being a prolific source of bioactive secondary metabolites, are of great interest for natural product discovery.

Objective: Isolation and partial characterization of endophytic fungi inhabiting the leaves and woody parts of Taxus fuana Nan Li & R.R. Mill. (Taxaceae) and evaluation of biological activity.

Materials and methods: Endophytic fungal isolates were identified by molecular analysis of internal transcribed spacer (ITS) regions of 18S rDNA. Extracts of the endophytic fungi cultured on potato dextrose agar and modified medium were evaluated using cancer chemoprevention bioassays [inhibition of TNF-α-induced NFκB, aromatase and inducible nitric oxide synthase (iNOS); induction of quinone reductase 1 (QR1)] and growth inhibition with MCF-7 cells.

Results: Nine of 15 fungal isolates were identified as belonging to Epicoccum, Mucor, Penicillium, Chaetomium, Paraconiothriym, Plectania or Trichoderma. Five of the 15 extracts inhibited NFκB activity (IC₅₀ values ranging between 0.18 and 17?μg/mL) and five inhibited iNOS (IC₅₀ values ranging between 0.32 and 12.9?μg/mL). In the aromatase assay, only two isolates mediated inhibition (IC₅₀ values 12.2 and 10.5?μg/mL). With QR1 induction, three extracts exhibited significant activity (concentrations to double activity values ranging between 0.20 and 5.5?μg/mL), and five extracts inhibited the growth of MCF-7 cells (IC₅₀ values ranging from 0.56 to 17.5?μg/mL). Six active cultures were derived from woody parts of the plant material.

Conclusion: The endophytic fungi studied are capable of producing pharmacologically active natural compounds. In particular, isolates derived from the wood of Taxus fuana should be prioritized for the isolation and characterization of bioactive constituents.     

Antioxidant,anti-inflammatory and anti-septic potential of phenolic acids and flavonoid fractions isolated from Lolium multiflorum

Context: Interest has recently renewed in using Lolium multiflorum Lam. (Poaceae) (called Italian ryegrass; IRG) silage as an antioxidant and anti-inflammatory diet.

Objective: This study investigated the antioxidant, anti-inflammatory and anti-septic potential of IRG silage and identified the primary components in IRG active fractions.

Materials and methods: Total 16 fractions were separated from the chloroform-soluble extract of IRG aerial part using Sephadex LH-20 column before HPLC analysis. Antioxidant and anti-inflammatory activities of the fractions at doses of 0–100?μg/mL were investigated using various cell-free and cell-mediated assay systems. To explore anti-septic effect of IRG fractions, female ICR and BALB/c mice orally received 40?mg/kg of phenolic acid and flavonoid-rich active fractions F₇ and F₈ every other day for 10 days, respectively, followed by LPS challenge.

Results: The active fractions showed greater antioxidant and anti-inflammatory potential compared with other fractions. IC₅₀ values of F₇ and F₈ to reduce LPS-stimulated NO and TNF-α production were around 15 and 30?μg/mL, respectively. Comparison of retention times with authentic compounds through HPLC analysis revealed the presence of caffeic acid, ferulic acid, myricetin and kaempferol in the fractions as primary components. These fractions inhibited LPS-stimulated MAPK and NF-κB activation. Supplementation with F₇ or F₈ improved the survival rates of mice to 70 and 60%, respectively, in LPS-injected mice and reduced near completely serum TNF-α and IL-6 levels.

Discussion and conclusion: This study highlights antioxidant, anti-inflammatory and anti-septic activities of IRG active fractions, eventually suggesting their usefulness in preventing oxidative damage and inflammatory disorders.     

Standardized myrtol attenuates lipopolysaccharide induced acute lung injury in mice

Context: Standardized myrtol, an essential oil containing primarily cineole, limonene and α-pinene, has been used for treating nasosinusitis, bronchitis and chronic obstructive pulmonary disease (COPD).

Objective: To investigate the effects of standardized myrtol in a model of acute lung injury (ALI) induced by lipopolysaccharides (LPS).

Materials and methods: Male BALB/c mice were treated with standardized myrtol for 1.5?h prior to exposure of atomized LPS. Six hours after LPS challenge, lung injury was determined by the neutrophil recruitment, cytokine levels and total protein concentration in the bronchoalveolar lavage fluid (BALF) and myeloperoxidase (MPO) activity in the lung tissue. Additionally, pathological changes and NF-κB activation in the lung were examined by haematoxylin and eosin staining and western blot, respectively.

Results: In LPS-challenged mice, standardized myrtol at a dose of 1200?mg/kg significantly inhibited the neutrophile counts (from 820.97?±?142.44 to 280.42?±?65.45, 10³/mL), protein concentration (from 0.331?±?0.02 to 0.183?±?0.01, mg/mL) and inflammatory cytokines level (TNF-α: from 6072.70?±?748.40 to 2317.70?±?500.14, ng/mL; IL-6: from 1184.85?±?143.58 to 509.57?±?133.03, ng/mL) in BALF. Standardized myrtol also attenuated LPS-induced MPO activity (from 0.82?±?0.04 to 0.48?±?0.06, U/g) and pathological changes (lung injury score: from 11.67?±?0.33 to 7.83?±?0.79) in the lung. Further study demonstrated that standardized myrtol prevented LPS-induced NF-κB activation in lung tissues.

Discussion and conclusion: Together, these data suggest that standardized myrtol has the potential to protect against LPS-induced airway inflammation in a model of ALI.     

Inhibitory effects of Pinus massoniana bark extract on hepatitis C virus in vitro

Abstract

Context: Given that Pinus massoniana Lamb (Pinaceae) bark extract (PMBE) is a safe and non-toxic flavonoid found abundantly in nature, it was considered a promising novel candidate agent in the treatment of virus infection.

Object: Experiments were conducted to assay the antiviral character of PMBE against Hepatitis C virus (HCV).

Materials and methods: Assay of PMBE cytotoxicity, HCV replication, infectious HCV production, and its potential influence on the pathways for IFN-ISRE and NS3 protease were conducted.

Results: HCV replication was suppressed when the concentration of PMBE raised greater than 5?μg/mL and its EC₅₀ was 9.58?μg/mL. In the 10?μg/mL group, HCV replication was suppressed for 48?h. When the concentration increased to 40?μg/mL, HCV replication was significantly suppressed (luciferase activity was only 10% at 96?h). PMBE could inhibit HCV virus production efficiently (PMBE group was 5?FFU). Cell viability was affected by 40?μg/mL of PMBE. The F/R ratio ranged from 98% to 101%. The rate of OD₄₅₀ ranged from 96% to 102%. NS3 catalytic activity ranged from 5% (40?μg/mL PMBE) to 45% (5?μg/mL PMBE). Even when used in a low amount (5?μg/mL), NS3 catalytic activity was significantly inhibited (p?<?0.01).

Conclusions: The results suggest that PMBE is effective for use in the stabilization of HCV replication and active liver inflammation.