Chemical Composition and in vitro Anti-inflammatory Activity of Wheat Germ Oil Depending on the Extraction Procedure.

This study aimed to examine the chemical composition of wheat germ oil extracted by three different methods, and to evaluate its inhibitory effect on the cyclooxygenase and proteinase activities. The results showed that the contents of policosanols, tocopherols and phytosterols were affected by the extraction procedure. However, the fatty acid composition of the different oil extracts was nearly the same. Among the tested oils samples, cold pressed oil exhibited the strongest inhibitory activity against proteinase (93.4%, IC50 =195.7 µg/mL) and cyclooxygenase 1 (80.5%, IC50 =58.6 µg/mL). Furthermore, the cold pressed oil had the highest content of octacosanol, ß-sitosterol and α-linolenic acid, suggesting that those bioactive compounds could be essential for the potent ani-cyclooxygenase activity. The present data revealed that wheat germ oil contained cyclooxygenase and trypsin inhibitors, which are the promising therapeutic target for the treatment of various inflammatory diseases. Thus, wheat germ oil might be used to develop functional foods and pharmaceutic products for the human health.

Spectral characterization, antioxidant, antimicrobial, cytotoxic, and cyclooxygenase inhibitory activities of Aloysia citriodora essential oils collected from two Palestinian regions.

BACKGROUND: Aloysia citriodora Palau (AC) is commonly known as Lemon Verbena and has been utilized as a medicinal tea in folkloric medicine for the treatment of abdominal spasm, anxiety, and fever. The present investigation aimed to identify the chemical ingredients of AC essential oil (EO) collected from two different locations in Palestine and to assess their antioxidant, antimicrobial, cytotoxic, and cyclooxygenase (COX) inhibitory effects. METHODS: Gas chromatography/mass spectroscopy (GC/MS) technique was used to identify the chemical components of the hydro-distilled EO from both regions, while DPPH, MTS, and COX assays were utilized to estimate the antioxidant, cytotoxic, and COX inhibitory activities of the EOs, respectively. Moreover, a broth microdilution assay was used to assess antimicrobial potentials against seven microbial strains. RESULTS: The GC/MS technique revealed the presence of 17 compounds from the AC collected from the Umm al-Fahm region and 13 compounds from the sample from the Baqa al-Gharbiyye region, while α-citral was the major component of both EOs, representing 47.62 and 43.46%, respectively. The Baqa al-Gharbiyye AC EO exerted more potent antioxidant activity than the Umm al-Fahm EO, with IC50 values of 11.74 ± 0.18 and 35.48 ± 0.14 µg/mL, respectively, while the positive control Trolox had antioxidant IC50 values of 2.45 ± 0.01 µg/mL. Interestingly, both EOs inhibited more potential activity against Methicillin-Resistant Staphylococcus aureus (MRSA) and Proteus vulgaris than Ciprofloxacin and Ampicillin antibiotics and also showed more potent antifungal activity against Candida albicans than Fluconazole. Moreover, the Baqa al-Gharbiyye AC EO had a more potent cytotoxic effect than the Umm al-Fahm EO, with IC50 values of 84.5 ± 0.24 and 33.31 ± 0.01 µg/mL, respectively, compared with Doxorubicin, which had an IC50 dose of 22.01 ± 1.4 µg/mL. The EOs from Baqa al-Gharbiyye showed potent activity against both COX-1 and COX-2 enzymes, with IC50 of 52.93 ± 0.13 and 89.31 ± 0.21 µg/mL, respectively, while the EOs from the Umm al-Fahm region showed weaker activity against these enzymes, with IC50 of 349.99 ± 0.33 and 1326.37 ± 1.13 µg/mL, respectively. CONCLUSION: Both characterized EOs have a huge variety of chemical components. The Baqa al-Gharbiyye AC EO has more potent antioxidant and cytotoxic activities than the Umm al-Fahm EO, but both have potential antimicrobial activity against MRSA, P. vulgaris, and C. albicans. These results suggest the use of AC EOs as promising sources of active ingredients in the food, cosmetic, and pharmaceutical industries.

[Determination of seven non-selective cyclooxygenase inhibitors in milk powder by ultra-performance liquid chromatography-tandem mass spectrometry with dispersive solid phase extraction].

An ultra-performance liquid chromatography-tandem mass spectrometry with dispersive solid phase extraction (dSPE-UPLC-MS/MS) method was developed to determine seven non-selective cyclooxygenase inhibitors in milk powder. The samples were extracted with 0.01 mol/L pH 2.5 ascorbic acid-acetonitrile-ethyl acetate solution (2:5:5, v/v/v), and then purified with a mixture of anhydrous sodium sulfate, octadecyl carbon silica gel (C18-N) sorbent and ethylenediamine-N-propyl carbon silica gel (NH2-PSA) sorbent. The analysis was performed using a UPLC-MS/MS system with Waters CORTECS UPLC C18 (100 mm×2.1 mm, 1.6 µm) column. The mobile phase consisted of 0.1% (v/v) formic acid aqueous solution and acetonitrile containing 0.1% (v/v) formic acid by gradient elution, and the multiple reaction monitoring (MRM) mode was used. Five linear calibration curves were obtained with correlation coefficients (r2) ≥ 0.9965. The recoveries were determined at three spiked levels and ranged from 76.4% to 89.8%. The limits of quantification (LOQs) were from 2 to 5 µg/kg. This method is suitable for the determination of seven non-selective cyclooxygenase inhibitors in milk powder.

Bioactive properties of commercialised pomegranate (Punica granatum) juice: antioxidant, antiproliferative and enzyme inhibiting activities.

Pomegranate juice and related products have long been used either in traditional medicine or as nutritional supplements claiming beneficial effects. Although there are several studies on this food plant, only a few studies have been performed with pomegranate juice or marketed products. The aim of this work is to evaluate the antioxidant effects of pomegranate juice on cellular models using hydrogen peroxide as an oxidizing agent or DPPH and superoxide radicals in cell free systems. The antiproliferative effects of the juice were measured on HeLa and PC-3 cells by the MTT assay and pharmacologically relevant enzymes (cyclooxygenases, xanthine oxidase, acetylcholinesterase and monoamine oxidase A) were selected for enzymatic inhibition assays. Pomegranate juice showed significant protective effects against hydrogen peroxide induced toxicity in the Artemia salina and HepG2 models; these effects may be attributed to radical scavenging properties of pomegranate as the juice was able to reduce DPPH and superoxide radicals. Moderate antiproliferative activities in HeLa and PC-3 cancer cells were observed. However, pomegranate juice was also able to inhibit COX-2 and MAO-A enzymes. This study reveals some mechanisms by which pomegranate juice may have interesting and beneficial effects in human health.

Functionalized magnetic nanoparticles coupled with mass spectrometry for screening and identification of cyclooxygenase-1 inhibitors from natural products.

Development of simple and effective methods for high-throughput, high-fidelity screening and identification of cyclooxygenase-1 (COX-1) inhibitors from natural products are important for drug discovery to treat inflammation and carcinogenesis. Here, we developed a new screening assay based on cyclooxygenase-1 (COX-1) functionalized magnetic nanoparticles (i.e. Fe3O4@SiO2-COX-1) for solid phase ligand fishing, and then mass spectrometry (MS) was applied for structural identification. Incubation conditions were optimized. High specificity for isolating COX-1 inhibitors was achieved by testing positive control, indomethacin, with active and inactive COX-1. Moreover, high stability of immobilized COX-1 (remained 95.3% after ten consecutive cycles) allows the analysis reproducible. When applied to turmeric extract, four curcuminoids (i.e. curcumin, demethoxycurcumin, bisdemethoxycurcumin, and 1-(4-hydroxy-3,5-dimethoxyphenyl)-7-(4-hydroxy-3-methoxyphenyl)-(1E,6E)-1,6-heptadiene-3,5-dione), difficult to be distinguished from original MS spectrum of turmeric extract, were isolated as main COX-1 inhibitors. Their structures were characterized based on their accurate molecular weight and diagnostic fragment ions. The results indicated that the proposed method was a simple, robust and reproducible approach for the discovery of COX-1 inhibitors from complex matrixes.

Ardisia crispa roots inhibit cyclooxygenase and suppress angiogenesis.

BACKGROUND: In our previous studies conducted on Ardisia crispa roots, it was shown that Ardisia crispa root inhibited inflammation-induced angiogenesis in vivo. The present study was conducted to identify whether the anti-angiogenic properties of Ardisia crispa roots was partly due to either cyclooxygenase (COX) or/and lipoxygenase (LOX) activity inhibition in separate in vitro studies. METHODS: Benzoquinonoid fraction (BQ) was isolated from hexane extract by column chromatography, and later analyzed by using gas chromatography-mass spectrometry (GC-MS). Anti-angiogenic effect was studied on mouse sponge implantation assay. Ardisia crispa ethanolic rich fraction (ACRH), quinone-rich fraction (QRF) and BQ were screened for COX assay to evaluate their selectivity towards two isoforms (COX-1 and COX-2), The experiment on soy lipoxygenase (LOX) inhibitory assay was also performed to determine the inhibitory effect of ACRH, QRF and BQ on soy LOX. RESULTS: BQ was confirmed to consist of 2-methoxy-6-undecyl-1,4-benzoquinone, when compared with previous data. Antiangiogenesis study exhibited a reduction of mean vascular density (MVD) in both ACRH and QRF, compared to control. In vitro study showed that both ACRH and QRF inhibited both COX-1 and COX-2, despite COX-2 inhibition being slightly higher than COX-1 in BQ. On the other hand, both ACRH and QRF were shown to have poor LOX inhibitory activity, but not BQ. CONCLUSIONS: In conclusion, ACRH and QRF might possibly exhibit its anti-angiogenic effect by inhibiting cyclooxygenase. However, both of them were shown to possess poor LOX inhibitory activity. On the other hand, BQ displayed selectivity to COX-2 inhibitory property as well as LOX inhibitory effect.

Antioxidant and anti-inflammatory assays confirm bioactive compounds in Ajwa date fruit.

Ajwa, a variety of date palm Phoenix dactylifera L., produces the most expensive date fruits. Percentages of seed, moisture, fructose, glucose, soluble protein, and fiber in Ajwa dates were 13.24, 6.21, 39.06, 26.35, 1.33, and 11.01, respectively. The ethyl acetate, methanolic, and water extracts of Ajwa dates, active at 250 µg/mL in the MTT assay, inhibited lipid peroxidation (LPO) by 88, 70, and 91% at 250 µg/mL and cyclooxygenase enzymes COX-1 by 30, 31, and 32% and COX-2 by 59, 48, and 45% at 100 µg/mL, respectively. Bioactivity-guided purifications afforded compounds 1-7, in addition to phthalates and fatty acids. Compounds 1-3 showed activity at 100 µg/mL in the MTT assay; inhibited COX-1 enzyme by 59, 48, amd 50% and COX-2 enzyme by 60, 40, amd 39% at 50 µg/mL; and inhibited LPO by 95, 58, amd 66% at 100 µg/mL, respectively. The soluble protein fraction was also very active in both antioxidant and anti-inflammatory assays.

Phenolics content and antioxidant and anti-inflammatory activities of legume fractions.

Two faba bean (Vicia faba L.) subspecies major and minor and lentil seeds grown in Algeria were separated into cotyledons and hulls. These fractions, together with their corresponding whole seeds, were extracted with two solvents, aqueous (70%) acetone and (80%) ethanol, and evaluated for antioxidant activity in relation to their phenolic contents. Acetone selectively extracted tannins from faba beans. The hulls always exhibited high antioxidant activity, measured using the reducing power (RP), antiradical activity (DPPH) or oxygen radical absorbance capacity (ORAC) assays. Aqueous ethanol (80%) extract of lentil hulls exhibited high antioxidant and anti-inflammatory activities preferentially inhibiting 15-LOX (IC(50), 55 µg/ml), with moderate COX-1 (IC(50), 66 µg/ml) and COX-2 (IC(50), 119 µg/ml) inhibitory effects on the COX pathway, whereas faba bean hull extracts exerted relatively mild LOX inhibitory activity.

Inhibitory effects of bioactive leads isolated from Pseudomonas aeruginosa PS3 and Pseudomonas fluorescens PS7 on MAP kinases and down regulation of pro inflammatory cytokines (TNF-α, IL-1ß) and mediators (NO, iNOS and COX).

Pure lead molecules, showing anti-inflammatory effect were isolated from the marine Pseudomonas aeruginosa PS3 (GenBank Accession No. EF488968) and Pseudomonas fluorescens PS7 (GenBank Accession No. EF488969) using solvent extraction procedures, subsequent column fractionation, followed by bio activity based screening. The structures of the lead molecules (3S, 8aS)-3-isobutylhexahydropyrrolo[1,2-a]pyrazine-1,4-dione (Compound 1) and (8aS)-3-(4-hydroxybenyl) hexahydropyrrolo[1,2-a]pyrazine-1,4-dione (Compound 2) obtained from P. aeruginosa PS3 and P. fluorescens PS7 respectively were established employing spectral analysis. Compounds 1 and 2 at their IC(50) values of 84 and 53µM concentrations respectively down regulated expression of tumor necrosis factor-α (TNF-α) and interleukin 1-ß (IL-1ß) in peripheral blood mononuclear cells (PBMCs) and inducible nitric oxide synthase (iNOS) gene in RAW 264.7 cells. Immunoblot analysis revealed the inhibitory effect of pure compounds on phosphorylation of all the three mitogen activated protein kinases (MAPK) such as ERK, JNK and p38 MAPK. The results of the present investigation revealed that the pure compounds are anti-inflammatory in nature.

Anti-inflammatory, antioxidant, anti-tyrosinase and phenolic contents of four Podocarpus species used in traditional medicine in South Africa.

ETHNOPHARMACOLOGICAL RELEVANCE: Species of Podocarpus are used traditionally in their native areas for the treatment of fevers, asthma, coughs, cholera, chest complaints, arthritis, rheumatism, venereal diseases and distemper in dogs. AIMS OF THE STUDY: To investigate the antioxidant, anti-inflammatory and anti-tyrosinase activities of four Podocarpus species, Podocarpus elongatus, Podocarpus falcatus, Podocarpus henkelii and Podocarpus latifolius, used in traditional medicine in South Africa. Phytochemical analysis to determine the phenolic contents was also carried out. MATERIALS AND METHODS: DPPH, FRAP and ß-carotene-linoleic acid assays were used to determine the antioxidant/radical scavenging activities of these species. Anti-inflammatory activity of these species was assayed against two cyclooxygenase enzymes (COX-1 and COX-2). Tyrosinase inhibition activity was analysed using the modified dopachrome method with l-DOPA as the substrate. Phenolics were quantitatively determined using spectrophotometric methods. RESULTS: Stems of Podocarpus latifolius exhibited the lowest EC(50) (0.84 µg/ml) inhibition against DPPH. The percentage antioxidant activity based on the bleaching rate of ß-carotene ranged from 96% to 99%. High ferric reducing power was observed in all the extracts. For COX-1, the lowest EC(50) value was exhibited by stem extracts of Podocarpus elongatus (5.02 µg/ml) and leaf extract of Podocarpus latifolius showed the lowest EC(50) against COX-2 (5.13 µg/ml). All extracts inhibited tyrosinase activity in a dose-dependent manner with stem extract of Podocarpus elongatus being the most potent with an EC(50) value of 0.14 mg/ml. The total phenolic content ranged from 2.38 to 6.94 mg of GAE/g dry sample. CONCLUSION: The significant pharmacological activities observed support the use of these species in traditional medicine and may also be candidates in the search for modern pharmaceuticals in medicine, food and cosmetic industries.

Anthocyanin content, lipid peroxidation and cyclooxygenase enzyme inhibitory activities of sweet and sour cherries.

Cherries contain bioactive anthocyanins that are reported to possess antioxidant, anti-inflammatory, anticancer, antidiabetic and antiobese properties. The present study revealed that red sweet cherries contained cyanidin-3-O-rutinoside as major anthocyanin (>95%). The sweet cherry cultivar "Kordia" (aka "Attika") showed the highest cyanidin-3-O-rutinoside content, 185 mg/100 g fresh weight. The red sweet cherries "Regina" and "Skeena" were similar to "Kordia", yielding cyanidin-3-O-rutinoside at 159 and 134 mg/100 g fresh weight, respectively. The yields of cyanidin-3-O-glucosylrutinoside and cyanidin-3-O-rutinoside were 57 and 19 mg/100 g fresh weight in "Balaton" and 21 and 6.2 mg/100 g fresh weight in "Montmorency", respectively, in addition to minor quantities of cyanidin-3-O-glucoside. The water extracts of "Kordia", "Regina", "Glacier" and "Skeena" sweet cherries gave 89, 80, 80 and 70% of lipid peroxidation (LPO) inhibition, whereas extracts of "Balaton" and "Montmorency" were in the range of 38 to 58% at 250 microg/mL. Methanol and ethyl acetate extracts of the yellow sweet cherry "Rainier" containing beta-carotene, ursolic, coumaric, ferulic and cafeic acids inhibited LPO by 78 and 79%, respectively, at 250 microg/mL. In the cyclooxygenase (COX) enzyme inhibitory assay, the red sweet cherry water extracts inhibited the enzymes by 80 to 95% at 250 microg/mL. However, the methanol and ethyl acetate extracts of "Rainier" and "Gold" were the most active against COX-1 and -2 enzymes. Water extracts of "Balaton" and "Montmorency" inhibited COX-1 and -2 enzymes by 84, and 91 and 77, and 87%, respectively, at 250 microg/mL.

A liquid chromatography/tandem mass spectrometry method for detecting UGT-mediated bioactivation of drugs as their N-acetylcysteine adducts in human liver microsomes.

The detection of the reactive metabolites of drugs has recently been gaining increasing importance. In vitro trapping studies using trapping agents such as glutathione are usually conducted for the detection of reactive metabolites, especially those of cytochrome P450-mediated metabolism. In order to detect the UDP-glucuronosyltransferase (UGT)-mediated bioactivation of drugs, an in vitro trapping method using N-acetylcysteine (NAC) as a trapping agent followed by liquid chromatography/tandem mass spectrometry (LC/MS/MS) was developed in this study. After the test compounds (diclofenac and ketoprofen) had been incubated in human liver microsomes with uridine diphosphoglucuronic acid (UDPGA) and NAC, the NAC adducts formed through their acyl glucuronides were analyzed using LC/MS/MS with electrospray ionization (ESI). The NAC adduct showed a mass shift of 145 units as compared to its parent, and the characteristic ion fragmentations reflected the parent. This is a concise and high-throughput method for evaluating reactive metabolites by UGT-mediated bioactivation.

Celecoxib concentration predicts decrease in prostaglandin E2 concentrations in nipple aspirate fluid from high risk women.

BACKGROUND: Epidemiologic studies suggest that long term low dose celecoxib use significantly lowers breast cancer risk. We previously demonstrated that 400 mg celecoxib taken twice daily for 2 weeks lowered circulating plasma and breast nipple aspirate fluid (NAF) prostaglandin (PG)E2 concentrations in post- but not premenopausal high risk women. We hypothesized that circulating concentrations of celecoxib influenced PGE2 response, and that plasma levels of the drug are influenced by menopausal status. To address these hypotheses, the aims of the study were to determine: 1) if circulating plasma concentrations of celecoxib correlated with the change in plasma or NAF PGE2 concentrations from baseline to end of treatment, and 2) whether menopausal status influenced circulating levels of celecoxib. METHODS: Matched NAF and plasma were collected from 46 high risk women who were administered celecoxib twice daily for two weeks, 20 subjects receiving 200 mg and 26 subjects 400 mg of the agent. NAF and plasma samples were collected before and 2 weeks after taking celecoxib. RESULTS: In women taking 400 mg bid celecoxib, plasma concentrations of the agent correlated inversely with the change in NAF PGE2 levels from pre- to posttreatment. Nonsignificant trends toward higher celecoxib levels were observed in post- compared to premenopausal women. There was a significant decrease in NAF but not plasma PGE2 concentrations in postmenopausal women who took 400 mg celecoxib (p = 0.03). CONCLUSION: In high risk women taking 400 mg celecoxib twice daily, plasma concentrations of celecoxib correlated with downregulation of PGE2 production by breast tissue. Strategies synergistic with celecoxib to downregulate PGE2 are of interest, in order to minimize the celecoxib dose required to have an effect.

Lipid peroxidation and cyclooxygenase enzyme inhibitory activities of acidic aqueous extracts of some dietary supplements.

The botanical supplement market is growing at a fast pace with more and more people resorting to them for maintaining good health. Echinacea, garlic, ginkgo, ginseng, Siberian ginseng, grape seed extract, kava kava, saw palmetto and St John's wort are some of the popular supplements used for a variety of health benefits. These supplements are associated with various product claims, which suggest that they possess cyclooxygenase (COX) enzyme and lipid s inhibitory activities. COX enzymes are found to be at elevated levels in inflamed and cancerous cells. To test some of the product claims, selected supplements were analysed for their ability to inhibit COX-1 and -2 enzymes and lipid peroxidation in vitro. The supplements were extracted with acidified water (pH 2) at 37 degrees C to simulate the gastric environment. The supplements tested demonstrated varying degrees of COX enzyme inhibition (5-85% for COX-1 and 13-28% for COX-2). Interestingly, extracts of garlic (Meijer), ginkgo (Solaray), ginseng (Nature's Way), Siberian ginseng (GNC, Nutrilite, Solaray, Natrol), kava kava (GNC, Sundown, Solaray) and St John's wort (Nutrilite) selectively inhibited COX-2 enzyme. These supplements also inhibited lipid peroxidation in vitro (5-99%). The results indicated that the consumption of these botanical supplements studied possess health benefits.

Cyclooxygenase 2 expression in the spared nerve injury model of neuropathic pain.

Cyclooxygenase-2 (COX-2) after induction peripherally, and within the CNS, plays an important role in producing inflammatory pain. However, its role in neuropathic pain models is controversial. Recently a robust and persistent model of partial nerve injury pain, the spared nerve injury (SNI) model, has been developed. The aim of the present study was to examine the regulation of COX-2 in the rat SNI model and to evaluate the effectiveness of the selective COX-2 inhibitor rofecoxib in preventing neuropathic allodynia and hyperalgesia. RNase protection assays revealed only a very small and transient increase in COX-2 mRNA in the dorsal horn of the spinal cord in the SNI model with a maximum change at 24 h. Immunohistochemical analysis showed a small increase in COX-2 protein in the deep layers of the dorsal horn 10 h following SNI surgery. Rofecoxib (100 microM) did not affect spontaneous excitatory postsynaptic currents or alpha-amino-3-hydroxy-5-methyl-4-isoxazole propanoic acid (AMPA) and N-methyl-d-aspartate (NMDA) responses in lamina II neurons from spinal cords of animals with SNI indicating no detectable action on transmitter release or postsynaptic activity. Furthermore, rofecoxib treatment (1 and 3.2 mg/kg for 5 and 3 days respectively starting on the day of surgery) failed to modify the development of allodynia and hyperalgesia in the SNI model. However, rofecoxib significantly reduced inflammatory hypersensitivity evoked by injection of complete Freund's adjuvant into one hindpaw, indicating that the doses used were pharmacologically active. The pain hypersensitivity produced by the SNI model is not COX-2-dependent.

Cyclooxygenase inhibitory and antioxidant compounds from the mycelia of the edible mushroom Grifola frondosa.

The bioassay-guided isolation and purification of the hexane extract of the cultured mycelia of Grifola frondosa led to the characterization of a fatty acid fraction and three compounds, ergosterol (1), ergostra-4,6,8(14),22-tetraen-3-one (2), and 1-oleoyl-2-linoleoyl-3-palmitoylglycerol (3). The composition of fatty acid fraction was confirmed as palmitic, oleic, and linoleic acids by GC-MS and by comparison with the retention values of authentic samples. The structures of compounds 1-3 were established by spectroscopic methods. The fatty acid fraction and compounds 1-3 showed cyclooxygenase (COX) enzyme inhibitory and antioxidant activities. The inhibition of COX-1 enzyme by the fatty acid fraction and compounds 1-3 at 250 microg/mL were 98, 37, 55, and 67%, respectively. Similarly, COX-2 enzyme activity was reduced by fatty acid fraction and compounds 1-3 at 250 microg/mL by 99, 37, 70, and 4%, respectively. The inhibitions of liposome peroxidation by the fatty acid fraction and compounds 1 and 2 at 100 microg/mL were 79, 48, and 42%, respectively. This is the first report of compounds 2 and 3 from the cultured mycelia of G. frondosa. The COX inhibitory activities of compounds 1-3 are reported here for the first time.

Constituents in evening primrose oil with radical scavenging, cyclooxygenase, and neutrophil elastase inhibitory activities.

Cold-pressed, non-raffinated evening primrose oil was found to contain lipophilic radical scavengers. A highly enriched fraction of these compounds could be obtained from the oil by extraction with aqueous ethanol and subsequent liquid-liquid partitioning with petroleum. LC-DAD-MS analysis revealed that the fraction contained three aromatic compounds with identical UV and ESI-MS spectra. The compounds were isolated by RP-HPLC and their structures established by chemical and spectroscopic means as 3-O-trans-caffeoyl derivatives of betulinic, morolic, and oleanolic acid. The morolic acid derivative was a new compound. The three esters exhibited pronounced radical scavenging activity against the stable 2,2-diphenyl-1-picrylhydrazyl radical and were potent inhibitors of neutrophil elastase and cyclooxygenase-1 and -2 in vitro. Commercial samples of evening primrose oils contained only traces of these lipophilic antioxidants.

An inhibitor of type-1 cyclo-oxygenase in tissues from human pregnancy.

BACKGROUND: Previous work has shown that first trimester human decidua contains a protein which directly inhibits the activity of type-1 cyclo-oxygenase (COX-1). METHODS: Activity assays for cyclo-oxygenase types I and II were developed. Cell cytosol was prepared from a number of different sources: human placenta and decidua (first and third trimester), two placental cell-lines (BeWo and TCL-1), an endometrial stromal cell-line and K562 erythroleukemia cells. The effects of all cytosols on activity of type I cyclo-oxygenase, and of cytosols from BeWo choriocarcinoma and decidual cells on type II enzyme, were tested. RESULTS: Cytosols from first trimester human placenta, two placental cell-lines, an endometrial stromal cell-line and K562 erythroleukemia cells all inhibited the type I enzyme. The inhibitor protein could not be detected in third trimester human decidual cells after labor, and was present only at very low levels in third trimester decidua prior to the onset of labor. Cytosols from BeWo and decidual cells had no effect on the activity of the type-2 cyclo-oxygenase enzyme. CONCLUSIONS: The inhibitor of type I cyclo-oxygenase was not specific to pregnancy-related tissues, and may be a general regulator of this enzyme. Lower levels of inhibitor were present at term, but the physiological significance of this is unclear. The cytosolic inhibitor appears to be specific to the type I enzyme.

Effect of NSAID administration on tissue levels of immunoreactive prostaglandin E2, leukotriene B4, and (S)-flurbiprofen following extraction of impacted third molars.

Post-operative pain and inflammation are frequently managed with non-steroidal anti-inflammatory drugs (NSAIDs). Despite the prevalence of their use, however, relatively little is known about in vivo tissue concentrations of inflammatory mediators at the site of tissue injury and their modulation by NSAIDs. This study compares the effect of oral administration of the NSAID flurbiprofen, to placebo, on tissue levels of immunoreactive prostaglandin E2 (iPGE2), leukotriene B4 (iLTB4), and (S)-flurbiprofen within the surgical wound using implanted microdialysis probes in the dental impaction pain model. Twenty-four healthy patients in need of extraction of partial to complete bony mandibular third molars were recruited for this randomized, double-blind, placebo-controlled study. Following pre-operative administration of N2O/O2, midazolam i.v., and regional block anesthesia with 3% mepivacaine, each patient underwent surgical removal of their impacted third molars. Immediately following completion of the surgery, two semi-permeable microdialysis probes (3 kDa molecular weight cut-off) were implanted into each mandibular surgical site. Patients were taken to a recovery room and microdialysis samples and patient pain reports (visual analog scale, VAS) were collected at 30 min intervals for 4 h. Patients randomly received either flurbiprofen (200 mg orally) or placebo at the onset of post-operative pain. Dialysate samples were collected, frozen, and later assayed for iPGE2, iLTB4, and (S)-flurbiprofen levels. Results of this study show that flurbiprofen decreased post-operative pain by approximately 70% compared to placebo-treated patients (P < 0.001). During the 4 h post-operative timecourse of this study, flurbiprofen treatment significantly reduced peak tissue levels of iPGE2 (9.2 +/- 2.6 vs. 0.4 +/- 0.15 nM; P < 0.001), without having a significant effect on peak tissue levels of iLTB4 (2.5 +/- 1.4 vs. 1.49 +/- 0.86 nM) compared to placebo treatment. Levels of (S)-flurbiprofen significantly increased within the surgical wound exceeding therapeutic levels by 60 min after administration. Flurbiprofen is able to significantly suppress the local production of iPGE2 and provide significant analgesic efficacy without altering iLTB4 tissue levels in this model of acute post-operative inflammatory pain. These data indicate that NSAIDs selectively alter eicosanoid levels within surgical wound and evoke analgesia at time points coincident with elevated wound levels of the drug. The combined use of microdialysis probes in awake patients who provide simultaneous pain reports may offer insight into peripheral mechanisms of inflammatory mediator release and pain.