Oral treatments with a flavonoid-enriched fraction from Cecropia hololeuca and with rutin reduce articular pain and inflammation in murine zymosan-induced arthritis.

ETHNOPHARMACOLOGICAL RELEVANCE: Cecropia Loefl. species (Urticaceae) are widely spread across the rainforest in tropical and subtropical regions of Central and South America. Inhabitants of different regions of Brazil employ leaves, fruits and sprouts of Cecropia hololeuca Miq. mainly as anti-inflammatory, anti-asthmatic, expectorant, fever suppressant, and against cough. AIM OF THE STUDY: To evaluate the antinociceptive and anti-inflammatory activities of an aqueous leaf extract of C. hololeuca in a murine model of zymosan-induced arthritis (ZIA) and characterize compounds contributing to these effects. MATERIALS AND METHODS: The crude aqueous extract of C. hololeuca (CAE) was obtained by infusion, screened for antinociceptive and anti-inflammatory activities, and fractionated (solvent partition; RP-2 and Sephadex G-25 column chromatography), yielding fractions that were chemically and pharmacologically investigated. TLC, HPLC-DAD, HPLC-DAD-ESI-MS/MS and NMR analyses were peformed. The antinociceptive activity was assessed by means of acetic acid-induced writhing, hot-plate and rota-rod tests. ZIA was used to evaluate the anti-arthritic activity of oral treatment with CAE, butanolic (BF) and aqueous fraction (AF), as well as the fractions obtained from BF (F2, F2-A and F2-B). Rutin, a flavonoid found in C. hololeuca, was also tested. Mechanical hypernociception, joint edema, local neutrophil recruitment and articular TNF-α quantification were performed to measure the severity of arthritis and identify the anti-inflammatory potential of C. hololeuca. RESULTS: CAE (0.03-1 g/kg, p.o.) showed a dose-related inhibitory effect on acetic acid-induced writhing test, but did not change the pain latency in the hotplate test, nor the first fall time on the rota-rod test. In addition, CAE (1 g/kg, p.o.) inhibited by 65% the mechanical hypernociception, 46% the joint edema, 54% the neutrophil recruitment and 53% the articular TNF-α concentration levels in ZIA. BF (0.4 g/kg, p.o.), AF (0.6 g/kg), F2 (0.1 g/kg) and F2-A (0.045 g/kg), but not F2-B (0.055 g/kg), inhibited the mechanical hypernociception, joint edema and neutrophil recruitment in ZIA. Rutin (0.001-0.03 g/kg, p.o.) produced dose-related inhibitory effects in the mechanical hypernociception, joint edema and neutrophil recruitment, and at 0.03 g/kg also inhibited articular TNF-α synthesis after intra-articular zymosan injection. Isoorientin, isovitexin, rutin and isoquercitrin were identified in the most active fraction (F2-A), along with luteolin and apigenin derivatives, tentatively identified as isoorientin-2″-O-glucoside and isovitexin-2″-O-glucoside. CONCLUSION: This study corroborates the popular use by oral route of aqueous preparations of C. hololeuca against joint inflammatory disorders, such as rheumatoid arthritis. Our results demonstrated for the first time that oral administration of rutin shows antinociceptive and anti-inflammatory effects in ZIA, indicating that this flavonoid is one of the immunomodulatory compounds involved in the anti-arthritic activity of C. hololeuca.

The Hypnotic, Anxiolytic, and Antinociceptive Profile of a Novel µ-Opioid Agonist.

5'-4-Alkyl/aryl-1H-1,2,3-triazole derivatives PILAB 1-12 were synthesized and a pharmacological screening of these derivatives was performed to identify a possible effect on the Central Nervous System (CNS) and to explore the associated mechanisms of action. The mice received a peritoneal injection (100 µmol/kg) of each of the 12 PILAB derivatives 10 min prior to the injection of pentobarbital and the mean hypnosis times were recorded. The mean hypnosis time increased for the mice treated with PILAB 8, which was prevented when mice were administered CTOP, a µ-opioid antagonist. Locomotor and motor activities were not affected by PILAB 8. The anxiolytic effect of PILAB 8 was evaluated next in an elevated-plus maze apparatus. PILAB 8 and midazolam increased a percentage of entries and spent time in the open arms of the apparatus compared with the control group. Conversely, a decrease in the percentages of entries and time spent in the closed arms were observed. Pretreatment with naloxone, a non-specific opioid antagonist, prior to administration of PILAB 8 exhibited a reverted anxiolytic effect. PILAB 8 exhibited antinociceptive activity in the hot plate test, and reduced reactivity to formalin in the neurogenic and the inflammatory phases. These data suggest that PILAB 8 can activate µ-opioid receptors to provoke antinociceptive and anti-inflammatory effects in mice.

Anti-Leishmania amazonensis activity of Serjania lethalis A. St.-Hil.

Extracts of Serjania lethalis A. St.-Hil leaves and stems were tested in order to identify potential agents against Leishmania amazonensis. The hexane fraction (HF) and dichloromethane subfractions (DDF and MDF) showed leishmanicidal effect. The anti-promastigote IC50 values were 10.29 (HF), 11.41 (DDF) and 28.33µg/mL (MDF); whereas those against amastigote were 7.2 (HF), 8.1 (DDF) and 6.5µg/mL (MDF). Among the fractions and subfractions assayed, only HF altered the cell cycle of the parasite, increasing 3-fold the number of cells in the sub-G0/G1 phase. HF also changed the parasite mitochondrial membrane potential (ΔΨm) and the percentage of annexin-V-propidium iodide positive promastigotes. Our evaluations of the IC50 values showed that HF, DDF and MDF decreased NO production in infected macrophages stimulated with IFN-γ and LPS. Moreover, HF increased the production of TNF-α in Leishmania infected macrophages. This paper reports for the first time the leishmanicidal activity of extracts and fractions of Serjania lethalis leaves and also characterizes its leishmanicidal and immunomodulatory properties.

Electrospray ionization tandem mass spectrometry analysis of isopimarane diterpenes from Velloziaceae.

RATIONALE: The study of natural products by electrospray ionization tandem mass spectrometry (ESI-MS/MS) is an important strategy for the characterization of the major fragmentation reactions which can then help to determine the composition of complex mixtures. Application of ESI-MS/MS to a series of isopimarane diterpenes from Velloziaceae allowed the rationalization of their fragmentation mechanisms. METHODS: Velloziaceae diterpenes were isolated by silica gel column chromatography and investigated by ESI-MS/MS analysis. The fragmentation studies were performed on a quadrupole-time-of-flight instrument using N2 as the collision gas. To help rationalize the fragmentation pathways observed, the geometry and sites of reactivity of the diterpenes were obtained by theoretical calculations using the B3LYP/6-31 + G(d,p) model. Fragmentation mechanisms were proposed on the basis of the calculated protonation sites and product ions energies using density functional theory (DFT) methods. RESULTS: The presence of hydroxyl and carbonyl groups on the terpene core influences the protonation site observed. One compound showed a radical cation as the base peak. MS/MS spectra exhibit water elimination as the major fragmentation pathway (via two ways), either when protonation takes place on the oxygen atom, or through elimination after activation from hydrogen migration. After the elimination of water, the formation of an endocyclic double bond induces a sequential retro-Diels-Alder (RDA) reaction as the major fragmentation step. CONCLUSIONS: A thorough rational analysis of the fragmentation mechanisms of protonated Velloziaceae diterpenes was used to propose the dissociation mechanisms in ESI-MS/MS. The presence of esters in the side chain also influenced the intensity or occurrence of the observed protonated or cationized molecules in ESI-MS. These results will aid the identification of analogues in sample extracts in future metabolomics studies.

Antitumoral activity of new polyamine-naphthoquinone conjugates.

Polyamine-naphthoquinone conjugates 5a-c were synthesized by nucleophilic displacement of 2-methoxy-lawsone 3a, 2-methoxylapachol 3b and 2-methoxy-nor-lapachol 3c with the polyamine N1-Boc-N5-Bn-spermidine 4. Unprotected derivatives 6a-c were synthesized to evaluate the effect of the protective Boc group on the activity of compounds 5a-c. The colorimetric MTT assay was used to evaluate their cytotoxic activity. All compounds were active against human lines of promyelocytic leukemia (HL-60), lung cancer (GLC4), Burkitt lymphoma (Daudi) and a mouse breast tumor (Ehrlich carcinoma), but only unprotected 6a-c showed activity against the human line of melanoma (MV-3). IC50 values were obtained from dose response curves by linear regression. DNA fragmentation was measured by quantification of the subG1 peak of the cell cycle. Apoptosis of HL-60 treated with 5c was dose-dependent. The amount of DNA fragmentation observed by exposure of HL-60 to 25 microM of compounds 5a-c and 6a-c is compatible with the decrease in viability induced by the drugs at this concentration. Production of ROS was measured by H2-CFDA. Kinetics of HL-60 DNA fragmentation and ROS formation by 5c indicated that production of ROS precedes cell death. In conclusion, spermidine-1,4-naphthoquinone conjugates exhibited an increase in activity compared with the natural products and induced apoptosis of tumor cell lines by a mechanism that is mediated, at least in part, by ROS production.