Hyaluronidase decreases neutrophils infiltration to the inflammatory site.

OBJECTIVE: To evaluate the in vivo anti-inflammatory potential of bovine hyaluronidase (HYAL) using two different models of acute inflammation. METHODS: Air pouches were produced in the dorsal subcutaneous of mice and injected with phosphate saline solution or HYAL. The antiinflammatory action of HYAL was evaluated in carrageenan (Cg)-inflamed air pouches. After 4 and 24 h the cellular influx, protein exudation, cytokines and lipid mediators were evaluated. The action of HYAL on the rolling and adhesion of leukocytes was investigated in the LPS-stimulated mesenteric microcirculation by intravital microscopic. RESULTS: Treatment with HYAL reduced the cellular influx and protein exudation in non-inflamed and inflamed air pouches. HYAL treatment of Cg-inflamed air pouch reduced the production of tumor necrosis factor-alpha (TNF-α), interleukin-8 (IL-8), leukotriene B4 (LTB4) and LTC4, whereas prostaglandins E2 (PGE2) and D2 (PGD2) concentrations were unchanged. Histological analyses showed that HYAL administration diminished cell infiltration in the air-pouch lining. In LPS-stimulated mesenteric microcirculation, HYAL usage decreased rolling and adhesion of leukocytes, but did not affect the blood vessels diameters. CONCLUSION: The results demonstrate that HYAL inhibited cellular recruitment, edema formation and pro-inflammatory mediators production, resulting in decreased adherence of leukocytes to blood vessels and tissue infiltration. Our data suggest that HYAL may be considered an effective candidate to ameliorate acute inflammation.

Isolation of Flavonoids from Deguelia duckeana and Their Effect on Cellular Viability, AMPK, eEF2, eIF2 and eIF4E.

Preparations of Deguelia duckeana, known in Brazil as timbó, are used by indigenous people to kill fish. Reinvestigation of its extracts resulted in the isolation and identification of 11 known flavonoids identified as 3,5,4'-trimethoxy-4-prenylstilbene (1), 4-methoxyderricidine (2), lonchocarpine (3), 4-hydroxylonchocarpine (4), 4-methoxylonchocarpine (5), 5-hydroxy-4',7-dimethoxy-6-prenylflavanone (6), 4'-hydroxyisolonchocarpine (7), 4'-methoxyisolonchocarpine (8), 3',4',7-trimethoxyflavone (9), 3',4'-methylenedioxy-7-methoxyflavone (10), and 2,2-dimethyl-chromone-5,4'-hydroxy-5'-methoxyflavone (11). Except for 1, 3, and 4 all of these flavonoids have been described for the first time in D. duckeana and the flavanone 6 for the first time in nature. Compounds 2, 3, 4, 7, 9, and 10 were studied for their potential to induce cell death in neuronal SK-N-SH cells. Only the chalcone 4 and the flavanone 7 significantly induced lactate dehydrogenase (LDH) release, which was accompanied by activation of caspase-3 and impairment of energy homeostasis in the MTT assay and may explain the killing effect on fish. Interestingly, the flavone 10 reduced cell metabolism in the MTT assay without inducing cytotoxicity in the LDH assay. Furthermore, the flavonoids 2, 3, 4, 7, and 10 induced phosphorylation of the AMP-activated protein kinase (AMPK) and the eukaryotic elongation factor 2 (eEF2). The initiation factor eIF4E was dephosphorylated in the presence of these compounds. The initiation factor eIF2alpha was not affected. Further studies are needed to elucidate the importance of the observed effects on protein synthesis and potential therapeutic perspectives.

Hyaluronidase modulates inflammatory response and accelerates the cutaneous wound healing.

Hyaluronidases are enzymes that degrade hyaluronan an important constituent of the extracellular matrix. They have been used as a spreading agent, improving the absorption of drugs and facilitating the subcutaneous infusion of fluids. Here, we investigated the influence of bovine testes hyaluronidase (HYAL) during cutaneous wound healing in in vitro and in vivo assays. We demonstrated in the wound scratch assay that HYAL increased the migration and proliferation of fibroblasts in vitro at low concentration, e.g. 0.1 U HYAL enhanced the cell number by 20%. HYAL presented faster and higher reepithelialization in in vivo full-thickness excisional wounds generated on adult Wistar rats back skin already in the early phase at 2nd day post operatory compared to vehicle-control group. Wound closured area observed in the 16 U and 32 U HYAL treated rats reached 38% and 46% compared to 19% in the controls, respectively. Histological and biochemical analyses supported the clinical observations and showed that HYAL treated wounds exhibited increased granulation tissue, diminished edema formation and regulated the inflammatory response by modulating the release of pro and anti-inflammatory cytokines, growth factor and eicosanoids mediators. Moreover, HYAL increased gene expression of peroxisome proliferator-activated receptors (PPAR) γ and PPAR ß/δ, the collagen content in the early stages of healing processes as well as angiogenesis. Altogether these data revealed that HYAL accelerates wound healing processes and might be beneficial for treating wound disorders.

Cytotoxic clerodane diterpenes from Zuelania guidonia.

The leaves of Zuelania guidonia yielded eight new clerodane diterpenes, namely, zuelaguidins A-H (1-8), and the known clerodane diterpene esculentin A (9). Some of these structures contained a 3,6-dihydro-1,2-dioxin moiety. The new compounds were isolated and identified using 1D- and 2D-NMR experiments. All compounds were evaluated for cytotoxicity against the CCRF-CEM (human acute lymphocytic leukemia), CEM-ADR5000 (human acute lymphocytic leukemia resistant to doxorubicin), and MIA-PaCa-2 (human pancreatic carcinoma) cell lines as well as for their selectivity against peripheral blood mononuclear cells from healthy human subjects. Zuelaguidins B, C, and E were the most potent compounds against the CCRF-CEM cell line, with IC50 values ranging from 1.6 to 2.5 µM.

Structural and conformational analysis of proanthocyanidins from Parapiptadenia rigida and their wound-healing properties.

Structure elucidation and conformation analysis of four proanthocyanidins isolated from the bark of Parapiptadenia rigida were performed by two-dimensional NMR spectroscopy, HRESIMS, CD, and molecular mechanics (MM+) force field calculations. The known prodelphinidin, epigallocatechin-(4ß→8)-epigallocatechin-3-O-gallate (1) was accompanied by the new epigallocatechin-(4ß→8)-4'-O-methylgallocatechin (2), epicatechin-(4ß→8)-4'-O-methylgallocatechin (3), and (4α→8)-bis-4'-O-methylgallocatechin (4). Compound 4 was previously published but the earlier structure must presumably be revised to 4'-O-methylgallocatechin-(4α→8)-4'-O-methylepigallocatechin. Conformational studies showed the compact rotamer with B and E rings in quasi-equatorial orientations as the preferred conformation for compounds 1-3, whereas 4 consists of two stable rotamers, each with a quasi-equatorial orientation of ring B and E, respectively. The isolated compounds were studied for their wound-healing effects in a scratch assay and showed promising results.

Catechin derivatives from Parapiptadenia rigida with in vitro wound-healing properties.

Analysis of the ethanolic extract of the bark from Parapiptadenia rigida resulted in the isolation of the new catechin derivatives 4',3''-di-O-methylapocynin-D (10), 4',3''-di-O-methylapocynin-B (11), epigallocatechin-3-O-ferulate (8), and 4'-O-methylepigallocatechin-3-O-ferulate (9) and the catechins 4'-O-methylepigallocatechin-3-O-gallate (6) and 4'-O-methylepicatechin-3-O-gallate (7). These compounds, isolated for the first time from a natural source, are accompanied by the five known catechins 4'-O-methylgallocatechin (1), 4'-O-methylepigallocatechin (2), 3'-O-methylepicatechin (3), epigallocatechin-3-O-gallate (4), and epicatechin-3-O-gallate (5). Compounds 5 and 7 displayed promising wound-healing effects in a scratch assay. Some of the catechin derivatives showed inhibitory effects on NF-κB DNA binding and p38α MAPK activity.

Further sesquiterpene lactones from Viguiera robusta and the potential anti-inflammatory activity of a heliangolide: inhibition of human neutrophil elastase release.

In addition to known heliangolides, a new eudesmanolide was isolated from the leaf rinse extract of Viguiera robusta (Asteraceae). Structural elucidation was based on spectral analysis. It is the first report on eudesmanolides in members of the subgenus Calanticaria of Viguiera. In this work, the main isolated compound, the furanoheliangolide budlein A, besides its previously reported in vitro and in vivo anti-inflammatory activities, inhibited human neutrophil elastase release. The inhibition was at the concentration of (16.83 +/- 1.96) microM for formylated bacterial tripeptide (fMLP) stimulation and (11.84 +/- 1.62) microM for platelet aggregation factor (PAF) stimulation, being slightly less active than the reference drug parthenolide. The results are important to demonstrate the potential anti-inflammatory activities of sesquiterpene lactones and corroborate the previous studies using other targets.

Sesquiterpene lactones from Dimerostemma species (Asteraceae) and in vitro potential anti-inflammatory activities.

Two Brazilian species of Dimerostemma (Asteraceae) were chemically investigated. Two known sesquiterpene lactones (STLs), a germacrolide and an eudesmanolide, were isolated from D. episcopale while D. brasilianum afforded the new germacranolide 1beta,5beta/10alpha-trihy-droxy-8alpha-angeloyloxy-germacra-3,11(13)-dien-6alpha,12-olide in addition to a known one. Structure identification based on NMR and MS analyses. 1beta,10alpha,4alpha,5beta-Diepoxy-8alpha-angeloyloxy-costunolide isolated from D. brasilianum was studied for its anti-inflammatory activity. This STL completely inhibited DNA binding of the transcription factor NF-kappaB at a concentration of 5 microm and 10 microM in Jurkat T and Raw 264.7 cells, respectively. Elastase release from human neutrophils was reduced to 50% at a concentrations of 23 microM after stimulation with PAF and of 27 microM after stimulation with fMLP without showing cytotoxic effects. Additionally, elastase was also directly inhibited.

Inhibitory effects of Piper umbellatum and Piper peltatum extracts towards myotoxic phospholipases A2 from Bothrops snake venoms: isolation of 4-nerolidylcatechol as active principle.

Phospholipases A(2) (PLA(2)) are important constituents of snake venoms, being responsible for several of their toxic actions. Extracts from plants used in folk medicine were screened for inhibition of the enzymatic activity of myotoxin I, a PLA(2) from Bothrops asper. Piper umbellatum and Piper peltatum extracts tested positive, and their fractionation resulted in the isolation of 4-nerolidylcatechol. Its inhibitory effects towards toxic activities of two Bothrops myotoxins, representing catalytically active (Asp49) and catalytically inactive (Lys49) types of group II PLA(2)s, respectively, were characterized. The enzyme activity of B. asper myotoxin I was completely inhibited by 4-nerolidylcatechol at an inhibitor:toxin ratio of 10:1 (wt/wt) with an IC50 of approximately 1mM. In addition, 4-nerolidylcatechol inhibited representatives of groups I and III of PLA(2)s. Its preincubation with Bothrops myotoxins significantly reduced their myotoxic and edema-inducing activities in animal experiments. However, when 4-nerolidylcatechol was administered in situ, immediately after toxin injection, its inhibitory ability was substantially lower or negligible. This might be explained by the rapid action of these toxins in vivo, together with the slow inactivation of PLA(2) activity observed in vitro. Electrophoretic and chromatographic analyses of myotoxins ruled out major changes in protein charge, hydrophobicity, or gross molecular mass being involved in the inhibition mechanism. Mass spectrometry determinations are consistent with the covalent modification of myotoxin by one molecule of 4-nerolidylcatechol. Finally, a novel compound was isolated from both Piper species, sharing the nerolidyl skeleton, but nevertheless not being inhibitory towards the PLA(2)s studied.

Sesquiterpene lactones from Montanoa hibiscifolia that inhibit the transcription factor NF-kappa B.

The reinvestigation of the aerial parts of Montanoa hibiscifolia afforded four new eudesmanolides (1-4), three of them with a rare endoperoxide structural element and the fourth with a rare carbonyl function. It also afforded three unusual montabibisciolides (5-7), two (5 and 7) of which are new natural compounds. Additionally, seven germacrolides (8-10 and 12-15) and one melampolide (11) could be isolated, including two new germacrolides (8 and 9). Their structures were elucidated using 1D and 2D NMR measurement as well as ESI, CIMS, and HRMS analyses. Low-energy conformations were obtained by molecular mechanics calculations. The (13)C NMR data of five compounds are reported for the first time. Six sesquiterpene lactones (4, 6, 10, 11, 12, and 14) were investigated for their inhibitory activity on DNA binding of the transcription factor NF-kappa B using Jurkat T cells as well as RAW 264.7 cells. Besides the alpha-methylene-gamma-butyrolactone moiety the epoxy group in the acyl residue might take part in the NF-kappa B inhibitory activity of sesquiterpene lactones.

Guaianolides from Viguiera gardneri inhibit the transcription factor NF-kappaB.

Five guaianolides and a germacrolide were isolated from the leaf rinse extract of Viguiera gardneri (Asteraceae), together with known compounds. All compounds were detected in glandular trichomes collected from the leaves and were analyzed by HPLC. Structure elucidation was based on the analysis of spectroscopic data. Low energy conformations were obtained by quantum mechanical calculations. Three closely related guaianolides which were isolated as the main compounds were studied for their anti-inflammatory activity using the transcription factor NF-kappaB as molecular target. NF-kappaB DNA binding was inhibited at sesquiterpene lactones concentrations of 10 or 50 microM.

Anti-inflammatory activity of two different extracts of Uncaria tomentosa (Rubiaceae).

We assessed in vivo the anti-inflammatory activity of two Cat's claw bark extracts, by comparing a spray-dried hydroalcoholic extract against an aqueous freeze-dried extract, to determine which extract was more effective. We used the carrageenan-induced paw edema model in mice. In addition, to assess the molecular mechanism of action, we determined the inhibition of NF-kappa B through the Electrophoretic Mobility Shift Assay (EMSA) and the effects on cycloxygenase-1 and -2. Results showed that the anti-inflammatory activity was significantly higher using the hydroalcoholic compared with the aqueous extract (P<0.05). The extracts also showed little inhibitory activity on cyclooxygenase-1 and -2. It cannot be excluded that the slight inhibitory activity on DNA binding of NF-kappa B is due to cytotoxic effects.