Cryptocaryoic acids A - C: New phenyl alkyl acids isolated from the leaves of Australian rainforest plant Cryptocarya mackinnoniana.

Phytochemical investigation of the leaves of the Australian rainforest tree Cryptocarya mackinnoniana led to the discovery of three new oxygenated phenyl alkyl acids, cryptocaryoic acids A - C and two known compounds, cryptocaryone and 2',6'-dihydroxy-4'-methoxychalcone. The structures of all the compounds were determined by detailed spectroscopic analysis. Mosher's analysis was used for absolute stereochemistry determination at C-11, while the remaining stereochemistry determination of the one remaining stereocenter C-13 was based on NOESY correlations. All compounds isolated were also evaluated for their anti-inflammatory properties by assessing their inhibitory effects on LPS and interferon-γ induced nitric oxide (NO) production and TNF- α release in RAW 264.7 macrophages. The new cryptocaryoic acids exhibited weak to moderate anti-inflammatory activity (NO inhibition) ranging from (18.4-56 µM).

Mulgravanols A and B, rare oxidized xanthenes and a new phloroglucinol isolated from the Australian rainforest plant Waterhousea mulgraveana (Myrtaceae).

Phytochemical investigation of the Australian rainforest plant leaves Waterhousia mulgraveana, yielded two rare oxidized xanthenes, mulgravanols A (1) and B (2) along with a new phloroglucinol, mulgravanol C (3). Mulgravanol A (1) is the first reported example of a complex xanthene flanked by a methine bridged phloroglucinol unit. All the compounds displayed moderate inhibitory effects on nitric oxide production and TNF-α release in RAW 264.7 macrophages (IC50) 42-55 µM. The structures of the new compounds were assigned based on a detailed spectroscopic interpretation.

Hydrogen peroxide mediates pro-inflammatory cell-to-cell signaling: a new therapeutic target for inflammation?

Nitric oxide is now universally recognized as an extracellular signaling molecule. Nitric oxide, produced in one cell, diffuses across the extracellular space and acts with targets in an adjoining cell. In this study, we present proof that hydrogen peroxide - like nitric oxide - acts as a true first (intercellular) messenger for a multitude of pro-inflammatory ligands. RAW 264.7 macrophages were activated with three different ligands, lipopolysaccharide, interferon-gamma or advanced glycation end products in the presence of increasing concentrations of (hydrogen peroxide scavenging) catalase. As inflammatory readouts, nitric oxide and tumor necrosis factor were determined. We hypothesize that hydrogen peroxide travels between cells propagating the signal, then a certain percentage of the readout should be inhibited by catalase in a concentration-dependent manner. The experiment showed concentration-dependent inhibition of nitric oxide and tumor necrosis factor-α production in response to all three ligands/ligand combinations (interferon-gamma, lipopolysaccharide, and chicken egg albumin-derived advanced glycation end product) in the presence of increasing concentration of catalase. For example, catalase inhibited 100% of nitric oxide and 40% of tumor necrosis factor-α production at its highest concentration. Our results suggest that hydrogen peroxide travels through cell membranes into the extracellular space and enters and activates adjacent cells. Like nitric oxide, we suggest that it is a ubiquitous first messenger, able to transmit cell-to-cell pro-inflammatory signals such as nitric oxide and tumor necrosis factor-α. In a therapeutic setting, our data suggest that compounds acting as hydrogen peroxide scavengers might not even need to enter the cell to act as anti-inflammatory drugs.

Chronic Microglial Activation in the GFAP-IL6 Mouse Contributes to Age-Dependent Cerebellar Volume Loss and Impairment in Motor Function.

Chronic microglial activation is a prominent feature of many chronic neurodegenerative diseases, including Parkinson's and Alzheimer's disease. To investigate the effects of chronic microglial activation on cerebellar structure and motor function throughout the lifespan, the transgenic GFAP-IL6 mouse model was used. The aim of the study was to examine inflammatory markers and neuronal degeneration while simultaneously characterizing the motor performance of GFAP-IL6 mice at 3, 6, 14, and 24 months of age in comparison to WT (C57BL/6) mice. In respect to markers of neuroinflammation in the cerebellum, increased numbers of Iba1+ microglia were observed as early as at 3 months of age. In addition, TNF-α levels proved to be significantly higher in the GFAP-IL6 compared to WT mice at all time points. A difference in cerebellar volume between the GFAP-IL6 and WT mice was observed later in life, starting at 6 months and increasing to a loss of about 50% in aged (24 months old) GFAP-IL6 mice. Synaptic deficits were also assessed by using pre- (synaptophysin) and post-synaptic (PSD95) markers. While synaptophysin levels remained unchanged, PSD95 levels decreased in the aging GFAP-IL6 mice compared to their WT littermates from 14 months onward. To assess the effect of microglia activation and neurodegeneration on behavior, a variety of motor function tests, semi-quantitative cerebellar ataxia score, accelerod, beam walking, and open field tests were performed. An age-dependent difference between the genotypes was observed in many of the motor function tests. For example, reduced performance on the accelerod and higher ataxia scores were observed at 6 months of age, followed by the beam walking test showing differences at 14 months of age. In summary, this study constitutes a comprehensive, age-dependent examination of inflammatory, synaptic and neurodegenerative changes in the brains of GFAP-IL6 mice leading to a deterioration in motor performance. The results also indicate that early chronic microglia activation in the GFAP-IL6 mouse leads to observable cerebellar volume loss and motor deficits later in life.

Investigation Into the Effects of Tenilsetam on Markers of Neuroinflammation in GFAP-IL6 Mice.

PURPOSE: To test the short- and long-term effects of Tenilsetam on chronic neuroinflammation in the GFAP-IL6 mouse. METHODS: From 3 months of age, GFAP-IL6 mice were divided into 2 groups and fed with Tenilsetam enriched food pellets or control food pellets, respectively, for either 5 or 15 months. Total numbers of Iba-1+ microglia, TSPO+ cells were determined using an unbiased stereological method. Levels of methylglyoxal and TNF-α in the cerebellar homogenate were tested using HPLC and ELISA, respectively. RESULTS: Tenilsetam decreased the total number of Iba-1+ microglia in both the cerebellum and the hippocampus of GFAP-IL6 mice at 8 months and in the cerebellum at 18 months. In the cerebellum, it decreased the density of microglia in GFAP-IL6 mice to a similar level after 5 and 15 months' feeding. Tenilsetam prevented the volume loss of the cerebellum at 8 months. It also significantly decreased TNF-α in the cerebellum of GFAP-IL6 mice to a similar level of WT mice after 15 months of feeding. CONCLUSION: Tenilsetam has anti-inflammatory effects evidenced by the decreased number of microglia in both the cerebellum and hippocampus, and decreased TNF-α levels in the GFAP-IL6 Tenilsetam fed animals.

Activation of Macrophages and Microglia by Interferon-γ and Lipopolysaccharide Increases Methylglyoxal Production: A New Mechanism in the Development of Vascular Complications and Cognitive Decline in Type 2 Diabetes Mellitus?

Methylglyoxal (MGO), a dicarbonyl compound derived from glucose, is elevated in diabetes mellitus and contributes to vascular complications by crosslinking collagen and increasing arterial stiffness. It is known that MGO contributes to inflammation as it forms advanced glycation end products (AGEs), which activate macrophages via the receptor RAGE. The aim of study was to investigate whether inflammatory activation can increase MGO levels, thereby completing a vicious cycle. In order to validate this, macrophage (RAW264.7, J774A.1) and microglial (N11) cells were stimulated with IFN-γ and LPS (5 + 5 and 10 + 10 IFN-γ U/ml or µg/ml LPS), and extracellular MGO concentration was determined after derivatization with 5,6-Diamino-2,4-dihydroxypyrimidine sulfate by HPLC. MGO levels in activated macrophage cells (RAW264.7) peaked at 48 h, increasing 2.86-fold (3.14±0.4 µM) at 5 U/ml IFN-γ+5 µg/ml LPS, and 4.74-fold (5.46±0.30 µM) at 10 U/ml IFN-γ+10 µg/ml LPS compared to the non-activated controls (1.15±0.02 µM). The other two cell lines, J774A.1 macrophages and N11 microglia, showed a similar response. We suggest that inflammation increases MGO production, possibly exacerbating arterial stiffness, cardiovascular complications, and diabetes-related cognitive decline.

Anti-Inflammatory Chemical Profiling of the Australian Rainforest Tree Alphitonia petriei (Rhamnaceae).

Chronic inflammation is an important pathological condition in many human diseases, and due to the side effects of the currently used non-steroidal anti-inflammatory drugs, discovery of novel anti-inflammatory drugs is of general interest. Anti-inflammatory activity guided compound isolation from the plant Alphitonia petriei led to the isolation of the known plant sterols emmolic acid (1), alphitolic acid (2), trans- and cis-coumaroyl esters of alphitolic acid (3 and 4) and betulinic acid (5). A detailed spectroscopic analysis led to the structure elucidation of the alphitolic acid derivatives (1-5), and the semi-synthetic emmolic acid acetate (6). When tested in LPS (Lipopolysaccharides) + IFN-γ (Interferon gamma) activated RAW 264.7 macrophages, all compounds except (1) exhibited potent anti-inflammatory activity (IC50 values as low as 1.7 µM) in terms of downregulation of NO and TNF-α production, but also demonstrated some considerable cytotoxicity.

Anti-inflammatory activity of cinnamon (C. zeylanicum and C. cassia) extracts - identification of E-cinnamaldehyde and o-methoxy cinnamaldehyde as the most potent bioactive compounds.

Chronic inflammation is a contributing factor in many age-related diseases. In a previous study, we have shown that Sri Lankan cinnamon (C. zeylanicum) was one of the most potent anti-inflammatory foods out of 115 foods tested. However, knowledge about the exact nature of the anti-inflammatory compounds and their distribution in the two major cinnamon species used for human consumption is limited. The aim of this investigation was to determine the anti-inflammatory activity of C. zeylanicum and C. cassia and elucidate their main phytochemical compounds. When extracts were tested in LPS and IFN-γ activated RAW 264.7 macrophages, most of the anti-inflammatory activity, measured by down-regulation of nitric oxide and TNF-α production, was observed in the organic extracts. The most abundant compounds in these extracts were E-cinnamaldehyde and o-methoxycinnamaldehyde. The highest concentration of E-cinnamaldehyde was found in the DCM extract of C. zeylanicum or C. cassia (31 and 34 mg g(-1) of cinnamon, respectively). When these and other constituents were tested for their anti-inflammatory activity in RAW 264.7 and J774A.1 macrophages, the most potent compounds were E-cinnamaldehyde and o-methoxycinnamaldehyde, which exhibited IC50 values for NO with RAW 264.7 cells of 55 ± 9 µM (7.3 ± 1.2 µg mL(-1)) and 35 ± 9 µM (5.7 ± 1.5 µg mL(-1)), respectively; and IC50 values for TNF-α of 63 ± 9 µM (8.3 ± 1.2 µg mL(-1)) and 78 ± 16 µM (12.6 ± 2.6 µg mL(-1)), respectively. If therapeutic concentrations can be achieved in target tissues, cinnamon and its components may be useful in the treatment of age-related inflammatory conditions.

Determination of anti-inflammatory activities of standardised preparations of plant- and mushroom-based foods.

PURPOSE: Chronic inflammatory processes contribute to the pathogenesis of many age-related diseases. In search of anti-inflammatory foods, we have systematically screened a variety of common dietary plants and mushrooms for their anti-inflammatory activity. METHODS: A selection of 115 samples was prepared by a generic food-compatible processing method involving heating. These products were tested for their anti-inflammatory activity in murine N11 microglia and RAW 264.7 macrophages, using nitric oxide (NO) and tumour necrosis factor-α (TNF-α) as pro-inflammatory readouts. RESULTS: Ten food samples including lime zest, English breakfast tea, honey-brown mushroom, button mushroom, oyster mushroom, cinnamon and cloves inhibited NO production in N11 microglia, with IC50 values below 0.5 mg/ml. The most active samples were onion, oregano and red sweet potato, exhibiting IC50 values below 0.1 mg/ml. When these ten food preparations were retested in RAW 264.7 macrophages, they all inhibited NO production similar to the results obtained in N11 microglia. In addition, English breakfast tea leaves, oyster mushroom, onion, cinnamon and button mushroom preparations suppressed TNF-α production, exhibiting IC50 values below 0.5 mg/ml in RAW 264.7 macrophages. CONCLUSION: In summary, anti-inflammatory activity in these food samples survived 'cooking'. Provided that individual bioavailability allows active compounds to reach therapeutic levels in target tissues, these foods may be useful in limiting inflammation in a variety of age-related inflammatory diseases. Furthermore, these foods could be a source for the discovery of novel anti-inflammatory drugs.

Anti-inflammatory effects of five commercially available mushroom species determined in lipopolysaccharide and interferon-γ activated murine macrophages.

Inflammation is a well-known contributing factor to many age-related chronic diseases. One of the possible strategies to suppress inflammation is the employment of functional foods with anti-inflammatory properties. Edible mushrooms are attracting more and more attention as functional foods since they are rich in bioactive compounds, but their anti-inflammatory properties and the effect of food processing steps on this activity has not been systematically investigated. In the present study, White Button and Honey Brown (both Agaricus bisporus), Shiitake (Lentinus edodes), Enoki (Flammulina velutipes) and Oyster mushroom (Pleurotus ostreatus) preparations were tested for their anti-inflammatory activity in lipopolysaccharide (LPS) and interferon-γ (IFN-γ) activated murine RAW 264.7 macrophages. Potent anti-inflammatory activity (IC50<0.1 mg/ml), measured as inhibition of NO production, could be detected in all raw mushroom preparations, but only raw Oyster (IC50=0.035 mg/ml), Shiitake (IC50=0.047 mg/ml) and Enoki mushrooms (IC50=0.099 mg/ml) showed also potent inhibition of TNF-α production. When the anti-inflammatory activity was followed through two food-processing steps, which involved ultrasonication and heating, a significant portion of the anti-inflammatory activity was lost suggesting that the anti-inflammatory compounds might be susceptible to heating or prone to evaporation.