Extratos glicólicos de Pfaffia paniculata K E Juglans regia L: avaliação do potencial antimicrobiano, anti-inflamatório, citotóxico e genotóxico / Glycolic extracts of Pfaffia paniculata K and Juglans regia L: evaluation of antimicrobial, anti-inflammatory, cytotoxic and genotoxic potential

A resistência antimicrobiana atingiu proporções alarmantes em todo o mundo, sendo que na Europa mortes causadas por micro-organismos multirresistentes superam os índices de mortalidade da AIDS, tuberculose e a gripe. Assim a fitoterapia desponta no combate a esta problemática, com as diversas atividades biológicas de plantas e seus derivados. Portanto os objetivos do presente estudo foram avaliar a ação antimicrobiana, anti-inflamatória, citotoxicidade, genotoxicidade e constituição fitoquímica dos extratos glicólicos de P. paniculata e J. regia. A ação sobre bactérias anaeróbias (Porphyromonas gingivalis, P. endodontalis, Parvimonas micra e Fusobacterium nucleatum) foi realizada por meio dos testes de microdiluição em caldo (Protocolo M11-A8 - CLSI) e sobre biofilmes monotípicos. Já a ação sobre aeróbios foi realizada sobre 3 cepas de Klebsiella pneumoniae multirresistente, com testes sobre culturas planctônicas (Protocolo M7-A9) e biofilmes; Foi realizada a verificação da atividade antimicrobiana sobre biofilmes heterotípicos de Candida albicans associada a Staphylococcus aureus, Enterococcus faecalis, Streptococcus mutans ou Pseudomonas aeruginosa. A citotoxicidade e a genotoxicdade dos extratos foram avaliadas sobre macrófagos de camundongos (RAW 264.7) e queratinócitos humanos (HaCat) pelos testes de MTT e micronúcleos, respectivamente. O potencial antiinflamatório foi verificado dosando os níveis de TNF-⍺, IL-10 e IL-1ß pelo teste de ELISA. Os dados obtiveram distribuição normal sendo a análise estatística realizada por ANOVA e Tukey (p<0,05%). Os extratos de P. paniculata e J. regia promoveram CMM de 50 mg/mL para as anaeróbias. Os biofilmes de P. gingivalis e P. micra foram eliminados com 100 e 200 mg/mL dos extratos (5 min) e com as concentrações de 50 e 100 mg/mL por 24 h; F. nucleatum e P. endodontalis obtiveram reduções variando de 80 a 90%. Os biofilmes heterotípicos de C. albicans e S. mutans obtiveram reduções de até 80% após contato por 5 min. com J. regia e 71% para P. paniculata. Os biofilmes multirresistentes de K. pneumoniae obtiveram reduções na atividade metabólica de até 67,9%. P. paniculata promoveu viabilidade celular variando de 61,1% a 133,8% sobre queratinócitos humanos após 24 h de contato com as concentrações de 12,5 a 0,39 mg/mL, enquanto J. regia obteve 43,9 a 128,4% de viabilidade. Os macrófagos de camundongo obtiveram viabilidade de 18,1 a 101,9% com P. paniculata e 35,4 a 60,6% para J. regia. P. paniculata promoveu a redução nos níveis da citocina pró-inflamatória IL-1ß e aumento nos níveis da citocina antiinflamatória IL-10. Já J. regia promoveu a redução da citocina pró-inflamatória TNF-⍺. Ambos os extratos não promoveram genotoxicidade frente as linhagens celulares. A análise fitoquímica evidenciou a presença de benzofenonas e ácido cafeoilquínico nos extratos de P. paniculata e J. regia, respectivamente. Em conclusão, os extratos de P. paniculata e J. regia demonstraram ação antimicrobiana sobre bactérias aeróbias e anaeróbias e multirresistentes com destaque a eliminação dos biofilmes de P. gingivalis, P. endodontalis, P. micra e K. pneumoniae (multirresistentes). Os extratos demonstraram a ausência de toxicidade e genotoxicidade conforme tempo de aplicação e concentração utilizada, além de possuírem potencial anti-inflamatório(AU)

Antimicrobial resistance has reached alarming proportions worldwide, with deaths in Europe caused by multi-resistant microorganisms exceeding the mortality rates from AIDS, tuberculosis and influenza. Thus phytotherapy emerges in the fight against this problem, with the various biological activities of plants and their derivatives. Therefore, the objectives of the present study were to evaluate the antimicrobial, antiinflammatory, cytotoxicity, genotoxicity and phytochemical constitution of the glycolic extracts of P. paniculata and J. regia. The action on anaerobic bacteria (Porphyromonas gingivalis, P. endodontalis, Parvimonas micra and Fusobacterium nucleatum) was carried out by means of broth microdilution tests (Protocol M11-A8 - CLSI) and on monotypic biofilms. The action on aerobes was performed on 3 strains of multi-resistant Klebsiella pneumoniae, with tests on planktonic cultures (Protocol M7-A9) and biofilms; The verification of antimicrobial activity on heterotypic biofilms of Candida albicans associated with Staphylococcus aureus, Enterococcus faecalis, Streptococcus mutans or Pseudomonas aeruginosa was also performed. The cytotoxicity and genotoxicity of the extracts were evaluated on mouse macrophages (RAW 264.7) and human keratinocytes (HaCat) by MTT and micronucleus tests, respectively. The anti-inflammatory potential was assessed by the ELISA test, TNF-⍺, IL-10 and IL-1ß levels were measured. The data obtained a normal distribution and the statistical analysis was performed by ANOVA and Tukey (p <0.05%). The extracts of P. paniculata and J. regia promoted CMM of 50 mg / mL for anaerobes. The biofilms of P. gingivalis and P. micra were eradicated with 100 and 200 mg / mL of the extracts (5 min) and with the concentrations of 50 and 100 mg / mL (24 hours); F. nucleatum and P. endodontalis obtained reductions ranging from 80 to 90%. The heterotypic biofilms of C. albicans and S. mutans obtained reductions of up to 80% after contact for 5 minutes with J. regia and 71% for P. paniculata. The multidrug-resistant strains of K. pneumoniae obtained reductions in metabolic activity of up to 67.9%. The P. paniculata extract promoted cell viability ranging from 61.1% to 133.8% on human keratinocytes after 24 h of contact with concentrations of 12.5 to 0.39 mg / mL, while J. regia obtained 43, 9 to 128.4% viability. Mouse macrophages obtained viability from 18.1 to 101.9% with P. paniculata and 35.4 to 60.6% for J. regia. P. paniculata promoted a reduction in the levels of the pro-inflammatory cytokine IL-1ß and an increase in the levels of the anti-inflammatory cytokine IL-10. J. regia promoted the reduction of the pro-inflammatory cytokine TNF-⍺. Both extracts did not promote genotoxicity against cell lines. Phytochemical analysis showed the presence of benzophenones and caffeoylquinic acid in the extracts of P. paniculata and J. regia, respectively. In conclusion, the extracts of P. paniculata and J. regia demonstrated antimicrobial action on aerobic and anaerobic and multiresistant bacteria, with emphasis on the elimination of the biofilms of P. gingivalis, P. endodontalis and P. micra, as well as the reductions of the biofilms of K. pneumoniae multidrug-resistant. The extracts demonstrated the absence of toxicity and genotoxicity according to the time of application and concentration used, in addition to having anti-inflammatory potential(AU)

Food-dependent, exercise-induced anaphylaxis in a patient allergic to peach.

Determining the single factor that triggered anaphylactic shock can be challenging. We present an interesting case of a 25-year-old female patient with recurrent anaphylactic reactions developing after eating various foods, particularly in presence of co-factors of allergic reactions. Symptoms occurred after consumption of various kinds of foods - peach, pancakes with cottage cheese and fruit, a meal from a Chinese restaurant - all eaten on other occasions without symptoms. During diagnosis, skin prick tests were negative for all tested allergen extracts (both inhalatory and food) from Allergopharma. Prick by prick tests were positive for the peach - wheal diameter - 6 mm, nectarine - 4 mm (histamine 4 mm, negative control 0 mm). Increased levels of asIgE were found for allergens of peach (0.55 kU/L).Open challenge test with one mid-size peach combined with the physical exercise challenge test was positive. ImmunoCAP ISAC test indicated increased levels of IgE specific for the lipid transfer protein (LTP) for walnut (nJug r 3), peach (Pru p 3), wheat (rTri a 14) and plane tree (rPla a 3). The patient was diagnosed with food-dependent, exercise-induced anaphylaxis associated with an allergy to lipid transport proteins (LTPs).

A subset of walnut allergic adults is sensitized to walnut 11S globulin Jug r 4.

BACKGROUND: The role of sensitization to commercially available allergens of English walnut (Juglans regia) Jug r 1, 2 and 3 in walnut allergy has been previously investigated in walnut allergic adults and was unable to explain all cases of walnut allergy. OBJECTIVES: Identify recognized walnut allergens, other than the ones previously investigated (Jug r 1-3), in walnut allergic adults and determine the sensitization frequency and diagnostic value. METHODS: Three different in-house walnut extracts were prepared and analysed on SDS-PAGE blots to identify allergenic walnut proteins. Immunoblots and immunoprecipitation, followed by LC-MS analysis, were performed to screen for, and confirm, IgE binding to walnut allergens in selected walnut allergic adults. In a cohort of 55 walnut challenged adults, including 33 allergic and 22 tolerant, sensitization to native and recombinant walnut allergen Jug r 4 was assessed using immunoblotting and immuno-line blot (EUROLINE), respectively. RESULTS: Screening of sera of 8 walnut allergic adults identified Jug r 4 as an allergen in our population. In the total cohort of 55 subjects, 5 were positive for Jug r 4 on immunoblot and 10 on EUROLINE. All but one EUROLINE positive subject had a positive food challenge (sensitivity 27%, specificity 95%, PPV 90%, NPV 47%). All 5 subjects positive on immunoblot were also positive on EUROLINE. LC-MS analysis showed a lack of Jug r 4 in the ImmunoCAP extract. Co-sensitization to other 11S albumins (eg hazelnut Cor a 9) was common in Jug r 4 sensitized subjects, potentially due to cross-reactivity. CONCLUSIONS: Walnut 11S globulin Jug r 4 is a relevant minor allergen, recognized by 27% of walnut allergic adults. It has a high positive predictive value of 90% for walnut allergy. Specific IgE against Jug r 4 occurred mostly with concomitant sensitization to other walnut components, mainly Jug r 1.

Expression of the cyclooxygenase isoforms in the prodromal stage of black walnut-induced laminitis in horses.

OBJECTIVE: To compare the levels of mRNA expression of cycooxygenase (COX)-1 and COX-2 in the digital laminae of normal horses and horses in the developmental stages of laminitis experimentally induced by administration of black walnut extract (BWE). SAMPLE POPULATION: Samples of mRNA extracted from the digital laminae of 5 control horses and 5 horses at the onset of leukopenia after administration of BWE. PROCEDURE: Specimens of laminae were collected from anesthetized horses prior to euthanasia. Expression of COX-1 and COX-2 mRNA in laminae of control and affected horses was evaluated via real-time quantitative polymerase chain reaction techniques. RESULTS: Expression of COX-2 mRNA was significantly increased in the BWE-treated group, compared with that in control horses. In contrast to COX-2 regulation, COX-1 mRNA expression was not significantly different between groups. Interestingly, despite consistent clinical signs such as leukopenia in all BWE-treated horses, distinct differences in COX-2 mRNA expression were detected among those 5 horses (compared with values for control horses, the increase in COX-2 mRNA expression ranged from no increase to a 30-fold increase). CONCLUSIONS AND CLINICAL RELEVANCE: Results indicated that there was a significant upregulation of COX-2 mRNA expression during the developmental stages of laminitis, with no significant change in expression of the COX-1 isoform. These data appear to provide support for aggressive use of nonsteroidal anti-inflammatory drugs in horses at risk for laminitis; further investigation into the clinical value of selective COX-2 inhibitors for treatment of laminitis in horses appears to be warranted.