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Effect of ethyl acetate fraction of urucum extract on cell viability of peripheral blood mononuclear cells and antimicrobial activity in human pathogenic bacteria.

Adrielle Zagminan1, José L. Viana1, Rodrigo B. Dutra1, Aruanã J. Pinheiro1, Letícia T. Carvalho2, Marco Aurélio B. Paschoal2, Angela Falcai1, Lídio G. Neto1, Letícia M. Gonçalves2 and Eduardo M. Sousa1*
  • 1 Universidade CEUMA, Post-Graduate Program in Parasite Biology, Brazil
  • 2 Universidade CEUMA, Post-Graduate Program in Dentistry, Brazil

In the last century, the Streptococcus pyogenes infections caused many deaths, especially in cases of puerperal fever (sepsis after childbirth). Streptococcus pyogenes is one of the most frequent pathogens of humans. It is estimated that 5-15% of normal subjects have bacteria in the respiratory tract without disease signs. As microorganisms, can infect S. pyogenes when the immune system is compromised or when the organisms are able to penetrate the constitutive defenses of innate immunity. When bacteria are introduced or transmitted to the vulnerable tissues, a variety of types of suppurative infections may occur. Acute tonsillitis is extremely frequent in the pediatric population, and although palatine tonsils show bacterial colonization by polymicrobial flora, including anaerobic and aerobic (e.g. S. mutans and S. pyogenes, Staphylococcus aureus, Moraxella catarrhalis, Haemophilus influenzae, Prevotella sp, Bacteroides fragilis and Fusobacterium sp. the major concern of professionals in the area of child health still is Streptococcus pyogenes, beta hemolytic microorganism classified in group A. Approximately 30-40% of acute tonsillitis are streptococci etiology, and the importance of this indeed due to the possibility of non-suppurative late complications caused by this pathogen, as for example, acute glomerulonephritis, and rheumatic fever, which is 90% of surgery indications for heart valve replacement in children in Brazil. Multidrug resistance is now the norm among these pathogens. Staphylococcus aureus is the pathogen of greatest concern perhaps due to its intrinsic virulence, the ability to cause a wide range of life-threatening infections, and its ability to adapt to different environmental conditions. The mortality caused by S. aureus bacteremia remains approximately 20-40%, despite the availability of effective antimicrobials. S. aureus is now the leading global cause of nosocomial infections and, as more patients are treated outside the hospital, is a growing concern in the community. S. aureus isolates from intensive care units and blood culture isolates around the world are becoming increasingly resistant to a greater number of antimicrobial agents. This inevitably leave less effective bactericidal antibiotics to treat these infections often life threatening. As quickly as new antibiotics are introduced, staphylococci developed efficient mechanisms of resistance The potential of medicinal plants are far from exhausted there is a need for social and economic development based on renewable resources. New knowledge and new needs to find the new diversity of plants with therapeutic activity of new molecules with applications in pharmaceutical technology and in developing more effective antimicrobial molecules. The development of alternative drugs to cope with various infectious diseases from natural products can provide an important source of chemical diversity. In Brazil, it is estimated that a quarter (25%) of the pharmaceutical industry revenues are comprised of plant-derived medicines. Although our country has the highest plant diversity in the world, with about 60.000 plant species cataloged above, only 8% were studied for research of bioactive compounds and 1.100 species were assessed in its medicinal properties. Bixa orellana L., urucum, or urucu, a native tropical tree of Central and South American rain forests is used to treat various diseases in popular medicine. The indigenous peoples have used Amazon for body painting urucum for centuries and it is believed that the original Aztec chocolate drink contained urucum seed. Furthermore, annatto seeds and leaves of the tree were used to prepare Aztec remedies for a variety of diseases such as tonsillitis, asthma, pleurisy, rectal disturbances, headache, jaundice, insolation and burns. In Brazil, urucum seeds are used for treating elevated blood lipid levels, as colorants for foods among other applications. Published studies have indicated an antioxidant effect, antibacterial, antifungal, analgesic and anti-inflammatory action. Due to widespread use of urucum for a large medicinal application, it is necessary to investigate its antimicrobial efficacy over infectious agents. This study was performed to determine the effects of ethyl acetate fraction of urucum extract (BoEtOAc) on peripheral blood mononuclear cells (PBMC) and evaluate to its antimicrobial activity against some bacteria human pathogens. To extract preparation fresh leaves of Urucum (i.e., Bixa orellana L.) were collected at Ático Seabra Herbarium from Federal University of Maranhão (UFMA), air-dried and macerated with water-alcohol solution (70% ethanol, v/v). The Bixa orellana hydroalcoolic extract (BoHA) were concentrated on a rotary evaporation. After that, the dried extract was suspended in a methanol/water solution (9:1) and the ethyl acetate (EtOAc) fraction was obtained after the partitioning of the methanol/water solution with different polarity solvents (hexane, chloroform and then ethyl acetate). To investigate the effect in PBMC, blood sample was collected from healthy human volunteers (non-smoker donors who had not taken any drug for at least 15 days prior to sampling, aged 18–35 years old) and isolated by the standard method of density-gradient centrifugation over Histopaque®-1119 (Sigma-Aldrich®). PBMCs were washed and ressuspended in supplemented DMEM culture medium plus fetal bovine serum 10% (Sigma-Aldrich®),100 μg/ml streptomycin, and 100 U/ml penicillin. Cell viability was determined to be viable by the trypan blue exclusion test; 93%. PBMCs were plated in 96-well plates (2 x105 cells/well) and added 100 μL of BoEtOAc at ranged from 3,33 to 33,3 mg/ml or DMEM as control. These sets were incubated at 37°C in a 5% CO2 for 24 h. At the end of the culture, an aliquot of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT, 2 mg/mL in PBS) was added to each well and incubated for another 4 hour at 37°C protected from light. Then, all the solution consists of complete medium and MTT (Sigma-Aldrich®) was discarded. The formazan crystals were dissolved in DMSO (100uL) and the absorbance was determined at 450 ƞm using a multi-plate (Thermo Plate®) reader. The antimicrobial activity was evaluated by agar diffusion method to BoEtOAc extract in Muller Hinton plates which were scattered each studied bacteria for 24h. The assays according to Clinical Laboratory Standards Institute (CLSI) were used to test the antimicrobial activity of the BoEtOAc extract the concentrations of the EtOAc fraction tested ranged from 3,33 to 33,3 mg/ml against Staphylococcus aureus (ATCC25923), and Streptococcus pyogenes (ATCC19615). The tests were performed in triplicate and the results expressed as the arithmetic average of the diameter of the inhibition zones formed around the disks

Acknowledgements

Financial support: FAPEMA (BM-01883/14; BIC-03438/14; BM-01281/15) and CEUMA University.

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Keywords: Urucum extract, Antimicrobial activitiy, peripheral blood mononuclear cells (PBMC), Viability cell, pathogenic bacteria

Conference: IMMUNOCOLOMBIA2015 - 11th Congress of the Latin American Association of Immunology - 10o. Congreso de la Asociación Colombiana de Alergia, Asma e Inmunología, Medellin, Colombia, 13 Oct - 16 Oct, 2015.

Presentation Type: Poster Presentation

Topic: Infectious and parasitic diseases

Citation: Zagminan A, Viana JL, Dutra RB, Pinheiro AJ, Carvalho LT, Paschoal MB, Falcai A, Neto LG, Gonçalves LM and Sousa EM (2015). Effect of ethyl acetate fraction of urucum extract on cell viability of peripheral blood mononuclear cells and antimicrobial activity in human pathogenic bacteria.. Front. Immunol. Conference Abstract: IMMUNOCOLOMBIA2015 - 11th Congress of the Latin American Association of Immunology - 10o. Congreso de la Asociación Colombiana de Alergia, Asma e Inmunología. doi: 10.3389/conf.fimmu.2015.05.00373

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Received: 29 May 2015; Published Online: 16 Sep 2015.

* Correspondence: Dr. Eduardo M Sousa, Universidade CEUMA, Post-Graduate Program in Parasite Biology, São Luís, Maranhão, 65.075-120, Brazil, edmsousa@hotmail.com