Chemical composition and antimicrobial activity of two extract of propolis against isolates of Staphylococcus spp. and multiresistant bacterials

Composição química e atividade antimicrobiana de dois extratos de própolis contra isolados de Staphylococcus spp. e bactérias multirresistentes

Jarbas F. Amarante Márcia F. Ribeiro Mateus M. Costa Fredson G. Menezes Tania M.S. Silva Talita A.B. Amarante Adriana Gradela Liliane M.D. Moura About the authors

ABSTRACT:

There is a growing need to discover and develop alternative therapies for the treatment of mastitis caused by Staphylococcus spp. and multidrug-resistant bacterial infections. This study examined the chemical composition and antimicrobial potential of two propolis extracts (EPA and EPB) against seventy-seven isolates of Staphylococcus spp. obtained from subclinical bovine mastitis; three clinical strains of MRSA and two from clinical strains of S. aureus ATCC, identified as S. aureus ATCC 6538 and S. aureus ATCC 25923. The total phenolic content was determined by the Folin-Ciocalteau method, the total flavonoid content by the Dowd method and the phenolic profile was quantified by HPLC-DAD. The MBC values of the extracts were evaluated by broth microdilution method. The amount of total phenolic and flavonoid compounds was higher in EPA than EPB. Both extracts revealed the presence of caffeic, coumaric, cinnamic, ferulic and 3,4-dihydroxybenzoic acids, with higher concentrations of coumaric and cinnamic acids. Staphylococcus spp. isolates were susceptible to EPA (90.9%), EPB (83.1%) and oxacillin (80.5%). The oxacillin susceptible isolates were also susceptible to EPA (70.1%) and EPB (80.6%), whereas those oxacillin-resistant strains were also susceptible to EPA (40.0%) and to EPB (26.7%). MBC ranged from 34.3 to 68.7μm/mL for EPA and from 68.7 to 137.5μg/mL for EPB. Both extracts inhibited significantly (100%) the clinical strains of MRSA, S. aureus ATCC 6538 and S. aureus ATCC 25923 at the concentration of 68.7μg/mL. It is concluded that both extracts of propolis, whose main constituents are coumaric and cinnamic acids, have high antimicrobial activity against the microorganisms studied, and EPA also against oxacillin-resistant strains. These findings reinforce its potential use for the treatment of bovine mastitis.

INDEX TERMS:
Chemical composition; antimicrobial activity; extract of propolis; Staphylococcus spp.; multiresistant bacterials; phenolic compounds; microdilution; oxacillin-resistant Staphylococcus aureus; propolis; bacterioses

RESUMO:

É cada vez mais oportuna a necessidade de descobrir e desenvolver terapias alternativas para tratamento da mastite causada por Staphylococcus spp. e de infecções bacterianas multirresistentes. Este estudo examinou a composição química e o potencial antimicrobiano de dois extratos etanólicos de própolis (EPA e EPB) contra setenta e sete isolados de Staphylococcus spp. obtidos a partir de mastite bovina subclínica; três estirpes clínicas de MRSA e duas de linhagens clínicas de S. aureus ATCC, identificadas como, S. aureus ATCC 6538 e S. aureus ATCC 25923, ambas metacilina resistentes. O teor total de fenólicos foi determinado pelo método de Folin-Ciocalteau, o teor de flavonoides totais pelo método Dowd e o perfil fenólico foi quantificado por HPLC-DAD. CBM dos extratos foi avaliada pelo método de microdiluição em caldo. A quantidade total de compostos fenólicos e flavonoides foi maior no EPA do que no EPB. Ambos os extratos revelaram a presença dos ácidos cafeico, cumárico, cinâmico, ferúlico e 3,4-di-hidroxibenzóico, com maiores concentrações de ácidos cumárico e cinâmico. Os isolados de Staphylococcus spp. foram sensíveis a EPA (90,9%), EPB (83,1%) e oxacilina (80,5%). Os isolados suscetíveis à oxacilina também foram suscetíveis ao EPA (70,1%) e ao EPB (80,6%), enquanto os do resistente à oxacilina foram suscetíveis ao EPA (40,0%) e ao EPB (26,7%). MBC variou de 34,3 a 68,7μm/mL para EPA e de 68,7 a 137,5μg/mL para EPB. Ambos os extratos inibiram significativamente (100%) as linhagens clínicas de MRSA, S. aureus ATCC 6538 e S. aureus ATCC 25923 na concentração de 68,7μg/mL. Conclui-se que os extratos etanólicos da própolis, cujos principais constituintes são os ácidos cumário e cinâmico, possuem atividade antimicrobiana contra os micro-organismos estudados, e o EPA também contra as cepas resistentes à oxacilina. Estes achados reforçam seu potencial uso para o tratamento da mastite bovina.

TERMOS DE INDEXAÇÃO:
Composição química; atividade antimicrobiana; extrato de própolis; Staphylococcus spp.; bactérias multirresistentes; compostos fenólicos; microdiluição; Staphylococcus aureus oxacilina-resistente; própolis; bacterioses

Introduction

In Brazil, milk production is a very important segment, totaling 25.4 billion liters annually. Mastitis is the main disease that affects dairy production, with a prevalence of 48.64% in the subclinical form (Acosta et al. 2016Acosta A.C., Silva L.B.G., Medeiros E.S., Pinheiro-Júnior J.W. & Mota R.A. 2016. Mastites em ruminantes no Brasil. Pesq. Vet. Bras. 36(7):565-573. <http://dx.doi.org/10.1590/S0100-736X2016000700001>
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), causing changes in the physical-chemical composition and cellularity of the milk, resulting in high economic damages with the reduction in milk production and significant effects on public health (Langoni 2000Langoni H. 2000. Tendências de modernização do setor lácteo: monitoramento da qualidade do leite pela contagem de células somáticas. Revta Ed. Contin. CRMV-SP 3(3):57-64., Ribeiro 2008Ribeiro M.G. 2008. Princípios terapêuticos na mastite em animais de produção e de companhia, p.759-771. In: Andrade S.F. (Ed.), Manual de Terapêutica Veterinária. 3ª ed. Roca, São Paulo.).

Approximately 140 different etiological agents may cause bovine mastitis, mainly in the subclinical form, of which the most prevalent contagious microorganisms have been Staphylococcus aureus, Streptococcus spp., Streptococcus agalactiae, Staphylococcus spp. and Corynebacterium bovis, in pure culture or in association (Costa 2001Costa E.O. 2001. Uso de antimicrobianos na mastite, p.443-445. In: Spinosa H.S., Gorniak S.L. & Bernardi M. (Eds), Farmacologia Aplicada à Medicina Veterinária. Guanabara Koogan, Rio de Janeiro., Santos & Fonseca 2007Santos M.V. & Fonseca L.F.L. 2007. Estratégias para o Controle de Mastite e Melhoria da Qualidade do Leite. Manole, São Paulo. 314p., Ribeiro et al. 2009Ribeiro M.G., Geraldo J.S., Langoni H., Lara G.H.B., Siqueira A.K., Salerno T. & Fernandes M.C. 2009. Microrganismos patogênicos, celularidade e resíduos de antimicrobianos no leite bovino produzido no sistema orgânico. Pesq. Vet. Bras. 29(1):52-58. <http://dx.doi.org/10.1590/S0100-736X2009000100008>
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, Martins et al. 2010Martins R.P., Silva J.A.G., Nakazato L., Dutra V. & Almeida Filho E.S. 2010. Prevalência e etiologia infecciosa da mastite bovina na microrregião de Cuiabá, MT. Ciênc. Anim. Bras. 11(1):181-187. <http://dx.doi.org/10.5216/cab.v11i1.5085>
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, Peixoto et al. 2012Peixoto E.C.T.M., Jardim J.G., Heinzen E.L., Domingues P.F., Padovani C.R. & Orsi R.O. 2012. Própolis no controle da mastite bovina. Arch. Vet. Sci. 17(4):43-52., Saeki et al. 2012Saeki E.K., Mello-Peixoto E.C.T., Matsumoto L.S., Marcusso P.F. & Monteiro R.M. 2012. Mastite bovina por Staphylococcus aureus: sensibilidade às drogas antimicrobianas e ao extrato alcoólico de própolis. Acta Vet. Bras. 5:284-290. <http://dx.doi.org/10.21708/avb.2011.5.3.2172>
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). S. aureus is recognized worldwide as a cause of several purulent diseases in humans and animals (Bean & Griffin 1990Bean N.H. & Griffin P.M. 1990. Foodborne disease outbreaks in the United States, 1973-1987: pathogens, vehicles, and trends. J. Food Protect. 53(9):804-817. <http://dx.doi.org/10.4315/0362-028X-53.9.804> <PMid:31018312>
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) and also an important cause of food poisoning in humans (Omoe et al. 2002Omoe K., Ishikawa M., Shimoda Y., Hu D., Ueda S. & Shinagawa K. 2002. Detection of seg, she, and sei genes in Staphylococcus aureus isolates and determination of the enterotoxin productivities of Staphylococcus aureus isolates harborin seg, seh, or sei genes. J. Clin. Microbiol. 40(3):857-862. <http://dx.doi.org/10.1128/JCM.40.3.857-862.2002> <PMid:11880405>
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). Additionally this, it shows persistence in the mammary tissue, due to the characteristics of its virulence (Dos Santos et al. 2003Dos Santos C.R., Arcenio F., Carvalho E.S., Lúcio E.M.R.A., Araújo G.L., Teixeira L.A., Sharapin N. & Rocha L. 2003. Otimização do processo de extração de própolis através da verificação da atividade antimicrobiana. Revta Bras. Farmacogn. 13:71-4.) and the appearance of resistant strains by the inadequate use of antibiotics in the treatment of diseases (Barberio et al. 2002Barberio A., Gietl H. & Dalvit P. 2002. “In vitro” sensibilidade aos antimicrobianos de Staphylococcus aureus e coliformes isolados de mastite bovina na região de Veneto, Itália, no período de 1996-1999. Napgama 5(1):3-10., Hogeveen et al. 2011Hogeveen H., Huijps K. & Lam T.J. 2011. Economic aspects of mastitis: new developments. N. Z. Vet. J. 59(1):16-23. <http://dx.doi.org/10.1080/00480169.2011.547165> <PMid:21328153>
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). Methicillin-resistant Staphylococcus aureus (MRSA) strains are one of the most prevalent in cases of mastitis and have as main characteristic to be multiresistant to the antimicrobials of the beta-lactam group (Freitas et al. 2005Freitas M.F.L., Pinheiro Junior J.W., Stamford T.L.M., Rabelo S.S.A., Silva D.R., Silveira Filho V.M., Santos F.G.B. & Mota R.A. 2005. Perfil se sensibilidade antimicorbiana in vitro de Staphylococcus coagelusa positivos isolados de leite de vaca com mastite no agreste do Estado e Pernambuco. Arq. Inst. Biológico, São Paulo, 72(2):171-177.).

One of the main tools for the control and treatment of mastitis is antimicrobial therapy (Erskine et al. 2003Erskine R.J., Wagner S. & De Graves F.J. 2003. Mastitis therapy and pharmacology. Vet. Clin. N. Am., Food Anim. Pract. 19(1):109-138.), but its efficiency is compromised in cases of antimicrobial resistance (Russi et al. 2008Russi N.B., Bantar C. & Calvinho L.F. 2008. Antimicroial susceptibility os Staphylooccus aureus causing bovine mastitis in Argentine dairy herds. Revta Argent. Microbiol. 40(2):116-119. <PMid:18705495>, Shi et al. 2010Shi D., Hao Y., Zhang A., Wulan B. & Fan X. 2010. Antimicrobial resistance of Staphylococcus aureus isolanted from bovine mastitis in China. Transbound Emerg. Dis. 57(4):221-224. <PMid:20557495>, Spohr et al. 2011Spohr M., Rau J., Friedrich A., Klittich G., Fetsch A., Guerra B., Hammerl J.A. & Tenhagen B.A. 2011. Methicilin-resistent Staphylooccus aureus (MRSA) in three dairy herds in southwest Germany. Zoonoses Publ. Health 58(4):252-261. <http://dx.doi.org/10.1111/j.1863-2378.2010.01344.x> <PMid:20630047>
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). In addition, the use of antimicrobials causes severe economic losses due to the discarding of animals (Silva et al. 2004Silva L.A.F., Silva E.B., Romani A.F. & Garcia A.M. 2004. Causas de descarte de fêmeas bovinas leiteiras adultas. Revta Bras. Saúde. Prod. Anim. 5:9-17.) and milk (Nero et al. 2007Nero L.A., Mattos M.R., Beloti V., Barros M.A.F. & Franco B.D.G.M. 2007. Resíduos de antibióticos em leite cru de quatro regiões leiteiras no Brasil. Revta Ciênc. Tecnol. Aliment. 27(2):391-393. <http://dx.doi.org/10.1590/S0101-20612007000200031>
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), reduction of milk production and drug costs (Vianni & Lázaro 2003Vianni M.C.E. & Lázaro N.S. 2003. Perfil de suscetibilidade a antimicrobianos em amostras de cocos Gram-positivos, catalase negativos, isolados de mastite subclínica bubalina. Pesq. Vet. Bras. 23(2):47-51. <http://dx.doi.org/10.1590/S0100-736X2003000200001>
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).

The use of phytotherapics has been shown to be a very useful tool for the prevention and treatment of mastitis, as well as avoiding the elimination of residues in milk (Schiavon et al. 2011Schiavon D.B.A., Schuch L.F.D., Oyarzabal M.E.B., Prestes L.S., Zani J.L. & Hartwig C.A. 2011. Aplicación de plantas medicinales para la antisepsia de pezones de vacas posordeño. Revta Cubana Plant. Med. 16(3):253-259.). In this sense, propolis, a resinous product produced by honey bees, is being used in the treatment of human and animal diseases (Coelho et al. 2010Coelho M.S., Silva J.H., Oliveira E.R.A., Amâncio A.L.L., Silva N.V. & Lima R.M.B. 2010. A própolis e sua utilização em animais de produção. Arch. Zootec. 59(R):95-112.) and has been shown to be a viable and quite promising alternative in the treatment of infections caused by S. aureus (Pinto et al. 2001Pinto M.S., Faria J.E., Message D., Cassini S.T.A., Pereira C.S. & Gioso M.M. 2001. Efeito de extratos de própolis verde sobre bactérias patogênicas isoladas do leite de vacas com mastite. Braz. J. Vet. Res. Anim. Sci. 38(6):278-283. <http://dx.doi.org/10.1590/S1413-95962001000600006>
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, Endler et al. 2003Endler A.L., Oliveira S.C., Amorim C.A., Carvalho M.P. & Pileggi M. 2003. Teste de eficácia da própolis no combate a bactérias patogênicas das vias respiratórias. Ciênc. Biol. Saúde 9(2):17-20., Auricchio et al. 2006Auricchio M.T., Bugno A., Almodóvar A.A.B. & Pereira T.C. 2006. Avaliação da atividade antimicrobiana de prEPArações de própolis comercializadas na cidade de São Paulo. Revta Inst. Adolfo Lutz 65(3):209-212., Zeighampour et al. 2014Zeighampour F., Mohammadi-Sichani M., Shams E. & Naghavi N.S. 2014. Antibacterial activity of propolis ethanol extract against antibiotic resistance bacteria isolated from burn wound infections. Zahedan J. Res. Med. Sci. 16(3):25-30., Shahbaz et al. 2015Shahbaz M., Zahoor T., Randhawa M.A. & Nawaz H. 2015. In vitro antibacterial activity of hydroalcoholic extract of propolis against pathogenic bacteria. Pakistan J. Life Soc. Sci. 13(3):132-136., Chen et al. 2018Chen Y.-W., Ye S.-R., Ting C. & Yu Y.-H. 2018. Antibacterial activity of propolins from Taiwanese green propolis. J. Food Drug Anal. 26(2):761-768. <http://dx.doi.org/10.1016/j.jfda.2017.10.002> <PMid:29567247>
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) due to its proven antibacterial activity (Barbosa et al. 2009Barbosa M.H., Zuffi F.B., Maruxo H.B. & Jorge L.L.R. 2009. Therapeutic properties of propolis for treatment of skin lesions. Acta Paul. Enferm. 22(3):318-322. <http://dx.doi.org/10.1590/S0103-21002009000300013>
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, Araujo & Marcucci 2011Araújo K.C.S. & Marcucci M.C. 2011. Efeito sinergístico da própolis tipificada contra Enterococcus faecalis. Revta Pesq. Inov. Farm. 3:9-14.). However the results using propolis are still conflicting (Pinto et al. 2001Pinto M.S., Faria J.E., Message D., Cassini S.T.A., Pereira C.S. & Gioso M.M. 2001. Efeito de extratos de própolis verde sobre bactérias patogênicas isoladas do leite de vacas com mastite. Braz. J. Vet. Res. Anim. Sci. 38(6):278-283. <http://dx.doi.org/10.1590/S1413-95962001000600006>
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, Loguercio et al. 2006Loguercio A.P., Groff M.C.A., Pedrozzo F.A., Witt M.N., Silva S.M. & Vargas C.A. 2006. Atividade in vitro do extrato de própolis contra agentes bacterianos da mastite bovina. Pesq. Agropec. Bras. 41(2):347-349. <http://dx.doi.org/10.1590/S0100-204X2006000200021>
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, Peixoto et al. 2012Peixoto E.C.T.M., Jardim J.G., Heinzen E.L., Domingues P.F., Padovani C.R. & Orsi R.O. 2012. Própolis no controle da mastite bovina. Arch. Vet. Sci. 17(4):43-52.), due to its distinct and complex chemical constitution, necessitating the development of further research (Lustosa et al. 2008Lustosa S.R., Galindo A.B., Nunes L.C.C., Randau K.P. & Rolim Neto P.J. 2008. Própolis: atualizações sobre a química e a farmacologia. Revta Bras. Farmacogn. 18(3):447-454. <http://dx.doi.org/10.1590/S0102-695X2008000300020>
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, Rufatto et al. 2017Rufatto L.C., Santos D.A., Marinho F., Henriques J.A.P., Ely M.R. & Moura S. 2017. Red propolis: chemical composition and pharmacological activity. Asian Pac. J. Trop. Biomed. 7(7):591-598. <http://dx.doi.org/10.1016/j.apjtb.2017.06.009>
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). In addition, there are few conclusive results regarding their action against MRSA, that play an important role in nosocomial infections (Taubes 2008Taubes G. 2008. The bacteria fight back. Science 321(5887):356-361. <http://dx.doi.org/10.1126/science.321.5887.356> <PMid:18635788>
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) and a serious problem on a global scale (Astani et al. 2013Astani A., Zimmermann S., Hassan E., Reichling J., Sensch K.H. & Schnitzler P. 2013. Antimicrobial activity of propolis special extract GH 2002 against multidrug-resistant clinical isolates. Pharmazie 68(8):695-701. <PMid:24020127>). Brazil is the third largest producer of propolis, contributing 10-15% of the worldwide production (Pereira et al. 2002Pereira A.S., Seixas F.R.M.S. & Aquino Neto F.R. 2002. Propolis: 100 years of research and its future. Quím. Nova 25(2):321-326. <http://dx.doi.org/10.1590/S0100-40422002000200021>
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), for this reason studies about the antibacterial activity of Brazilian propolis would contribute to further increase this participation.

The present study investigated the chemical composition and the antimicrobial activity of two extracts of propolis against isolates of Staphylococcus spp. obtained from subclinical bovine mastitis and also clinical strains of MRSA and Sthaplococcus aureus ATCC 6538 and ATCC 25923.

Materials and Methods

Propolis extracts. The bacterial isolates were tested against two commercial extracts of propolis identified as extract of propolis A (EPA) and extract of propolis B (EPB). The EPA was an 11% green propolis extract (Apis Flora®) originating from the Vassourinha do campo (Baccharis dracunculifolia) and obtained from hives of Jundiai, state of São Paulo. It consists of the following ingredients: green propolis, neutral alcohol (ethanol) 95.1% food grade and purified water. The EPB was also in 11% neutral propolis and alcohol solution (Santa Bárbara®). It was obtained from hives in the State of Bahia, containing in its composition 55% plant resins; 30% beeswax; 8 to 10% of essential oils; and 5% pollen. Both extracts had its chemical composition analysed at the Laboratory of Natural Products of the “Universidade Federal Rural de Pernambuco” (UFRPE), in Recife, Pernambuco.

Determination of total phenolic content. Quantification of phenolic compounds in the EPA and EPB was performed by the Folin-Ciocalteau method using gallic acid as the standard (Singleton & Rossi Junior 1965Singleton V.L. & Rossi Junior J.A. 1965. Colorimetry of total phenolics with phosphomolybdic-phosphotungstic acid reagents. Am. J. Enol. Viticulture 16:144-158.). The standard curve of gallic acid had five points of concentration (4, 8, 16, 24, 36μg/mL), with a wavelength of 760nm, with Y=0.0064x+0.4174, where y is the absorbance and x is the concentration; R2=0.9577. Quantification of phenolic compounds in extracts of propolis was performed in triplicate, with the quantity of phenols expressed in mg of gallic acid equivalent per gram of propolis extract, given the dry extract content (Roesler et al. 2007Roesler R., Malta L.G., Carrasco L.C., Holanda R.B., Sousa C.A.S. & Pastore G.M. 2007. Atividade antioxidante de frutos do cerrado. Ciênc. Tecnol. Aliment. 27(1):53-60. <http://dx.doi.org/10.1590/S0101-20612007000100010>
https://doi.org/10.1590/S0101-2061200700...
).

Determination of total flavonoids. The total flavonoid content in the EPA and EPB was determined by the adapted Dowd method (Meda et al. 2005Meda A., Lamien C., Romito M., Millogo J. & Nacoulma O. 2005. Determination of the total phenolic, flavonoid and proline contents in Burkina Fasan honey, as well as their radical scavenging activity. Food Chem. 91(3):571-577. <http://dx.doi.org/10.1016/j.foodchem.2004.10.006>
https://doi.org/10.1016/j.foodchem.2004....
), with absorbance readings at 300nm, constructing a standard curve of quercetin in five concentrations (1, 5, 10, 20, 40μg/ml), with Y=0.0198x+0.3552, where “y” is the absorbance and “x” is the concentration; R2=0.9807. The total flavonoid content was expressed as mg of quercetin equivalent per gram of propolis extract, given their dry extract content, as described by Lee et al. (2003)Lee Y.K., Yang P., Mishchenko M.I., Baum B.A., Hu Y.X., Huang H.L., Wiscombe W.J. & Baran A.J. 2003. Use of circular cylinders as surrogates for hexagonal pristine ice crystals in scattering calculations of infrared wavelengths. Appled Optics 42(15):2653-2664. <http://dx.doi.org/10.1364/AO.42.002653> <PMid:12777000>
https://doi.org/10.1364/AO.42.002653...
.

Determination of dry residue content. An aliquot of 5 mL of EPA and EPB, free of wax, was transferred to a porcelain capsule in dry form (heated in a laboratory oven at 105°C, for 2h, cooled in a desiccator and weighed), and the whole was taken to the oven preheated to 105°C, where it remained for 2h. After cooling in desiccator, the set was weighed. The process of heating, cooling and weighing the assembly was repeated at intervals of 1h until a constant mass was reached (when the difference between two consecutive weighing did not exceed 5mg). This analysis was realized in triplicate and the dry residue content (soluble solids in methanol) calculated by the ratio of the mass of the residue deposited in the crucible to the initial mass of the extracts of propolis crude corresponding to the aliquot of 5mL in percent (Instituto Adolfo Lutz 1976Instituto Adolfo Lutz 1976. Métodos Químicos e Físicos para Análise de Alimentos. Vol.1. 2ª ed. Normas analíticas do Instituto Adolfo Lutz, Instituto Adolfo Lutz, São Paulo. 1000p., Brasil 2001Brasil. Ministério da Agricultura 2001. Regulamento técnico para fixação de identidade e qualidade de própolis. Instrução Normativa nº 3, Anexo VI, de 19 de janeiro. Ministério da Agricultura, Brasília, DF., European Pharmacopoeia 2002European Pharmacopoeia 2002. European Pharmacopoeia. 4th ed. Council of Europe, Strasbourg.).

HPLC-DAD analysis. The High Performance Liquid Chromatography (HPLC) system consisted of two SCL-10Avp solvent pumps, equipped with a SPDM2O diode array detector (HPLC-DAD; Shimadzu, Corp., Kyoto, Japan). Samples were injected into a Rheodyne 7125i type injector with a 20mL capacity loop. Chromatographic separation was done with a C-18 column (25cm x 4.6mm x 5mm, Shimpack CLC-ODS), pre-column C-18 SULPELCO 4.0mm. Water: formic acid (99: 1, solvent A) and methanol (solvent B) were used as the mobile phase and water was used for the acid derivatives: formic acid (95:5, solvent A) and methanol (solvent B). The chromatographic condition was: 0-15min 20% B, 15-20min 30% B, 20-30min 40% B, 30-40min 40% B, with flow rate of 1.0mL/min. For monitoring, the wavelength of 290nm and temperature of 40°C (Dalmora et al. 1997Dalmora S., de Oliveira J.E., Affonso R., Gimbo E., Ribela M.T. & Bartolini P. 1997. Analysis of recombinant human growth hormone directly in osmotic shock fluids. J. Chromatogr. A 782(2):199-210. <http://dx.doi.org/10.1016/S0021-9673(97)00493-7> <PMid:9368400>
https://doi.org/10.1016/S0021-9673(97)00...
) were used. The identification of phenolics was based on retention times, UV-spectra and chromatographic comparison (co-injection) with authentic markers.

The caffeic, p-coumaric, ferulic, cinnamic, and 3,4-dihydroxybenzoic acids identified in propolis were quantified using the external standard method based on EPAk area. Analyses were made by drawing a calibration curve. To make the calibration curve of each phenolic compound, appropriate volumes from each stock solution were diluted with methanol to obtain working solutions in the concentration range of 0.5-40mg/mL that were correlated with the measured area. The area of these EPAk was drawn and the corresponding concentration of phenolics was calculated based on the calibration curve. For each sample, the quantitative analyses were performed in triplicate at 290nm.

Reagents and standards. Ferulic, 3-hydroxy-4-methoxycinnamic, caffeic, p-coumaric, cinnamic, sinapic, 4- methoxycinnamic, 3,4- dihydroxybenzoic, 4-hydroxybenzoic and syringic acids were obtained from Sigma-Aldrich (Hamburg, Germany), gallic and vanillic acids were obtained from Fluka Chemie AG (Buchs, Switzerland). All reagents used were analytical grade, as well as formic acid (Vetec, Brazil) and methanol (TEDIA).

Tested samples. The antimicrobial activity of EPA and EPB was analysed at the Animal Microbiology and Immunology Laboratory of the “Universidade Federal do Vale do São Francisco” (Univasf), in Petrolina, Pernambuco State, using seventy-seven isolates of Staphylococcus spp. obtained from cases of subclinical bovine mastitis in dairy farms in the Northeast region of Brazil. They were also tested three multiresistant isolates, one being Methicillin-resistant Staphylococcus aureus (S. aureus) (MRSA) and two from clinical strains of S. aureus ATCC, identified as ATCC 6538 and ATCC 25923, both methicillin-resistant.

Microdilution and minimum bactericidal concentration (MBC). The antimicrobial activity of the EPA and EPB was determined as the minimum bactericidal concentration (MBC) against Staphylococcus spp. isolates (n=77) and clinical strains of MRSA (n=3), S. aureus ATCC 6538 (n=1) and S. aureus ATCC 25923 (n=1). The samples plated on the TSA culture medium (Tryptone Soy Agar), were inoculated in tubes containing 3mL of Mueller Hinton (MH) broth medium, in order to perform microdilution according to Clinical And Laboratory Standards Institute (2006)Clinical and Laboratory Stardards Institute . National Committee for Clinical Laboratory Standards. Methods for broth dilution susceptibility testing of bacteria isolated from aquatic animals; approved guideline. Wayne, PA: 2006. (CLSI/NCCLS Document M49-A). After 24h, the medium was turbid at 0.5 on the Mac Farland scale (1x108 CFU/ml) and 0.1mL of this suspension was inoculated into tubes containing 9.9mL of MH broth (NCCLS 2002NCCLS 2002. Performance Standards for Antimicrobial Susceptibility Testing. National Committee For Clinical Laboratory Standards, Wayne, PA, p.133.). Subsequently, microdilution was performed by placing 200μL of pure, sterile MH broth in each well of the microplate and then 200μL of each extract in the first well, followed by a 1:2 dilution and discarding the last 200μL, with the concentration varying from 0.26μg/ml to 550μg/ml. Then each well was inoculated with 10μL of the suspension containing the microorganisms. These wells and the positive control wells and negative control were incubated for 24h at 37°C. The contents of each of these wells were inoculated into petri dishes containing MH agar medium and incubated for 24h at 37°C. The lowest concentration of extract in which there was no growth of the microorganisms in the plaques was considered the MBC (NCCLS 2002NCCLS 2002. Performance Standards for Antimicrobial Susceptibility Testing. National Committee For Clinical Laboratory Standards, Wayne, PA, p.133.). All samples were tested in triplicate.

Oxacillin susceptibility test. The sensitivity test was performed in all samples, in triplicate, by the modified Kirby-Bauer disk diffusion method (Clinical And Laboratory Standards Institute 2006Clinical and Laboratory Stardards Institute . National Committee for Clinical Laboratory Standards. Methods for broth dilution susceptibility testing of bacteria isolated from aquatic animals; approved guideline. Wayne, PA: 2006. (CLSI/NCCLS Document M49-A)), with microbial turbidity on the 0.5 scale of Mac Farland in MH broth. Samples were transferred with sterile swab to MH agar plates, in which the oxacillin-containing discs (1μg) were applied. The plates are incubated in an oven for 24h at 37°C. The breakpoint was determined according to (Clinical And Laboratory Standards Institute 2006Clinical and Laboratory Stardards Institute . National Committee for Clinical Laboratory Standards. Methods for broth dilution susceptibility testing of bacteria isolated from aquatic animals; approved guideline. Wayne, PA: 2006. (CLSI/NCCLS Document M49-A)).

Statistical analysis. The hypotheses are related to the antibacterial activity potential in vitro of two commercial extracts of propolis against bovine mastitis caused by Staphylococcus spp., in addition to relating them to resistance to oxacillin. The analyzes were performed observing the significance levels of the samples at 1% and 5%. The results were analyzed using analysis of variance (ANOVA) and the probability p=0.05 was considered the critical value for all tests. The Tukey post-hoc test was used to separate statistically significant means. The SAS software Proc Gun model software was used for statistical analysis.

Results

Analysis of the propolis extracts

The content of total phenolic and of total flavonoid varied between the samples. The EPA had a total phenolic content of 126.22mg (12.62%) and of total flavonoids of 51.06mg (5.10%) and the EPB of 73.12mg (7.31%) and 17.45mg (1.74%), respectively. The dry residue (soluble solids in methanol) content was 11.52% to the EPA and 10.37% to the EPB.

The components of each extract were identified by comparison with retention times of known chemical standards commonly found in propolis. The HPLC-DAD chromatograms of EPA and EPB indicated a similar profile of phenolic compounds in both extracts, with the presence, mainly, of cinnamic, ferulic, caffeic, coumaric, and 3,4-dihydroxybenzoic acids in all samples, which were detected according to their retention time and the UV spectral characteristics in comparison to those of standards.

The HLPC-DAD analysis revealed that the EPA presented a concentration of 226.55μg of coumaric acid, 222.55μg of cinnamic acid, 106.87μg of caffeic acid, 7.04μg of ferulic acid and 2.2μg of 3,4-hydroxybenzoic in 5mg of dry extract of propolis A, and for the EPB the concentrations were, respectively, 130.03μg, 130.03μg, 54.86μg, 3.99μg and 1.05μg in 5mg of dry extract of propolis B.

Antimicrobial activity of the propolis extracts

MBC against Staphylococcus spp. isolates ranged from 8.6μg/mL to 275μg/mL for the EPA, with a higher (P<0.01) number of sensitive isolates at the concentration of 68.7μg/mL (22/70, 31.4%), followed by the concentration of 34.3μg/mL (19/70, 27.1%). MBC ranged from 2.1μg/mL to 275μg/mL for the EPB, with a higher (P<0.01) number of sensitive isolates at the concentration of 137.5μg/mL (19/70, 56.2%), followed by concentration of 68.7μg/mL (19/64, 29.7%). These results indicated that EPA was more effective (P<0.01) in equal (22/70, 31.4%) or lower (33/70, 47.1%) concentration than 68.7μg/mL, while the EPB was more effective (P<0.01) in a higher concentration (38/64, 59.4%), presenting similar results at equal concentration (19/64, 29.7%) and lower result at concentrations below 68.7μg/mL (7/64, 10.9%) (Table 1). MBC against clinical strains of MRSA, ATCC 6538 and ATCC 25923 was also 68.7μg/ml for both EEP-A and EEP-B.

Table 1.
Minimum bactericidal concentration (MBC) of the extracts of propolis against isolates of Staphylococcus spp.

The analysis of the antimicrobial activity of the extract of propolis showed that 90.9% (70/77) of the 77 isolates of Staphylococcus spp. were susceptible to EPA, 83.1% (64/77) to EPB, 80.5% (62/77) susceptible to oxacillin and 19.5% (15/77) resistant to oxacillin. Among Staphylococcus spp. isolates susceptible to oxacillin 70.1% (44/62) were also susceptible to EPA and 80.6% (50/62) to EPB, whereas among those resistant to oxacillin 40.0% (6/15) was susceptible to EPA and 26.7% (4/15) to EPB (Fig.1).

Fig.1.
Antimicrobial activity of extract of propolis A (EPA) and B (EPB) on isolates of Staphylococcus spp. susceptible to several antibiotics and susceptible or resistant to oxacillin.

Clinical strains of MRSA and S. aureus ATCC 6538 and S. aureus ATCC 25923 exhibited 100% susceptibility to both extracts.

Discussion

This study brings important contributions to the scientific community due to the number of clinical strains of Staphylococcus spp. evaluated, as well as by the potential use of the propolis in the treatment of clinical cases of bovine mastitis and other infections caused by MRSA.

More than 300 constituents have been identified in different propolis samples (Marcucci 1995Marcucci M. 1995. Propolis: chemical composition, biological properties and therapeutic activity. Apidologie 26(2):83-99. <http://dx.doi.org/10.1051/apido:19950202>
https://doi.org/10.1051/apido:19950202...
, Bankova et al. 2000Bankova V., Castro S.L. & Marcucci M.C. 2000. Propolis: Recent advances in chemistry and plant origin. Apidologie 31(1):3-15. <http://dx.doi.org/10.1051/apido:2000102>
https://doi.org/10.1051/apido:2000102...
, De Castro 2001De Castro S.L. 2001. Propolis: biological and pharmacological activities. Therapeutic uses of this bee-product. ARBS 3:49-83., Park et al. 2002Park Y.K., Alencar S.M. & Aguiar C.L. 2002. Botanical origin and chemical composition of Brazilian propolis. J. Agric. Food Chem. 50(9):2502-2506. <http://dx.doi.org/10.1021/jf011432b> <PMid:11958612>
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, Pietta et al. 2002Pietta P.G., Gardana C. & Pietta A.M. 2002. Analytical methods for quality control of propolis. Fitoterapia 73(Suppl.1):S7-S20. <http://dx.doi.org/10.1016/S0367-326X(02)00186-7> <PMid:12495705>
https://doi.org/10.1016/S0367-326X(02)00...
, Alencar et al. 2007Alencar S.M., Oldoni T.L., Castro M.L., Cabral I.S., Costa-Neto C.M., Cury J.A., Rosalen P.L. & Ikegaki M. 2007. Chemical composition and biological activity of a new type of Brazilian propolis: red propolis. J. Ethnopharmacol. 113(2):278-283. <http://dx.doi.org/10.1016/j.jep.2007.06.005> <PMid:17656055>
https://doi.org/10.1016/j.jep.2007.06.00...
), which proportions depend upon of the place and time of collection (Park et al. 2002Park Y.K., Alencar S.M. & Aguiar C.L. 2002. Botanical origin and chemical composition of Brazilian propolis. J. Agric. Food Chem. 50(9):2502-2506. <http://dx.doi.org/10.1021/jf011432b> <PMid:11958612>
https://doi.org/10.1021/jf011432b...
). Although the chemical composition and biological properties of propolis are variable, it generally has in its composition resin and vegetable balsam (50%), wax (30%), essential and aromatic oils (10%), pollen, as well as several other substances (5%) (Burdock 1998Burdock G.A. 1998. Review of the biological properties and toxicity of bee propolis (Propolis). Food Chem. Toxicol. 36(4):347-363. <http://dx.doi.org/10.1016/S0278-6915(97)00145-2> <PMid:9651052>
https://doi.org/10.1016/S0278-6915(97)00...
, Park et al. 2002Park Y.K., Alencar S.M. & Aguiar C.L. 2002. Botanical origin and chemical composition of Brazilian propolis. J. Agric. Food Chem. 50(9):2502-2506. <http://dx.doi.org/10.1021/jf011432b> <PMid:11958612>
https://doi.org/10.1021/jf011432b...
, Pietta et al. 2002Pietta P.G., Gardana C. & Pietta A.M. 2002. Analytical methods for quality control of propolis. Fitoterapia 73(Suppl.1):S7-S20. <http://dx.doi.org/10.1016/S0367-326X(02)00186-7> <PMid:12495705>
https://doi.org/10.1016/S0367-326X(02)00...
). The most important active compounds are flavonoids, terpenoids and phenylpropanoids (García-Lafuente et al. 2009García-Lafuente A., Guillamón E., Villares A., Rostagno M.A. & Martínez J.A. 2009. Flavonoids as anti-inflammatory agentes: implications in cancer and cardiovascular disease. Inflamm. Res. 58(9):537-552. <http://dx.doi.org/10.1007/s00011-009-0037-3> <PMid:19381780>
https://doi.org/10.1007/s00011-009-0037-...
), aromatic acids and phenolic compounds (Garza-González et al. 2010Garza-González E., Morfin-Otero R., Llaca-Diaz J.M. & Rodriguez-Noriega E. 2010. Staphylococcal cassette chromosome mec (SCC mec) in methicillin-resistant coagulase-negative staphylococci. A review and the experience in a tertiarycare setting. Epidemiol. Infect. 138(5):645-654. <http://dx.doi.org/10.1017/S0950268809991361> <PMid:19961645>
https://doi.org/10.1017/S095026880999136...
). The total phenolics and flavonoids contents may vary due to different factors, such as flora ecology (Park et al. 2002Park Y.K., Alencar S.M. & Aguiar C.L. 2002. Botanical origin and chemical composition of Brazilian propolis. J. Agric. Food Chem. 50(9):2502-2506. <http://dx.doi.org/10.1021/jf011432b> <PMid:11958612>
https://doi.org/10.1021/jf011432b...
); resin collection period (Dos Santos et al. 2003Santos F.G.B., Mota R.A., Silveira V.M., Souza H.M., Oliveira M.B.M., Johner J.M.Q., Leal N.C., Almeida A.M.P. & Balbino T.C.L. 2003. Tipagem molecular de Staphylococcus aureus isolados do leite de vacas com mastite subclínica e equipamentos de ordenha procedentes do estado de Pernambuco. Napgama 6(1):19-23.); genetics of the queen bee (Park et al. 1998Park Y.I.A., Ikegaki M., Abreu J.A.S. & Alcici N.M.F. 1998. Estudo da prEPAração dos extratos de própolis e suas aplicações. Ciênc. Tecnol. Aliment. 18(3):313-318. <http://dx.doi.org/10.1590/S0101-20611998000300011>
https://doi.org/10.1590/S0101-2061199800...
); local flora and collection region (Bankova 2005Bankova V. 2005. Chemical diversity of propolis and the problem of standardization. J. Ethnopharmacol. 100(1):114-117. <http://dx.doi.org/10.1016/j.jep.2005.05.004> <PMid:15993016>
https://doi.org/10.1016/j.jep.2005.05.00...
), among others. In spite of this, the values found in this paper were in accordance with the minimum limits set by the “Ministério de Agricultura, Pecuária e Abastecimento” (MAPA) of 5% for total phenolic and 0.5% for total flavonoids (Brasil 2001Brasil. Ministério da Agricultura 2001. Regulamento técnico para fixação de identidade e qualidade de própolis. Instrução Normativa nº 3, Anexo VI, de 19 de janeiro. Ministério da Agricultura, Brasília, DF.).

The obtained results confirmed that, the EPA and EPB contain considerable amounts of phenolic and flavonoids compounds, which were higher in EPA than EPB. In both extracts the total phenol content was higher than the total flavonoids content, a common finding for propolis from Brazil (Alencar et al. 2007Alencar S.M., Oldoni T.L., Castro M.L., Cabral I.S., Costa-Neto C.M., Cury J.A., Rosalen P.L. & Ikegaki M. 2007. Chemical composition and biological activity of a new type of Brazilian propolis: red propolis. J. Ethnopharmacol. 113(2):278-283. <http://dx.doi.org/10.1016/j.jep.2007.06.005> <PMid:17656055>
https://doi.org/10.1016/j.jep.2007.06.00...
, Moreira et al. 2008Moreira L., Dias L.G., Pereira J.A. & Estevinho L. 2008. Antioxidant properties, total phenols and pollen analysis of propolis samples from Portugal. Food Chem. Toxicol. 46(11):3482-3485. <http://dx.doi.org/10.1016/j.fct.2008.08.025> <PMid:18804144>
https://doi.org/10.1016/j.fct.2008.08.02...
, Kalogeropoulos et al. 2009Kalogeropoulos N., Konteles S.J., Troullidou E., Mourtzinos I. & Karathanos V.T. 2009. Chemical composition, antioxidant activity and antimicrobial properties of propolis extracts from Greece and Cyprus. Food Chem. 116(2):452-461. <http://dx.doi.org/10.1016/j.foodchem.2009.02.060>
https://doi.org/10.1016/j.foodchem.2009....
, Nunes et al. 2012Nunes C.F., Finger P.F., Fischer G., Castro C.C., Hübner S.O., Paulino N., Marcucci M.C., Vieira O., Martes P.E. & Vargas G.D. 2012. Padronização de uma Amostra de Extrato Etanólico de Própolis Verde. Revta Fitos 7(1):67-72., Coelho 2013Coelho J.P.M. 2013. Identificação e quantificação de compostos fenólicos em própolis da região sul do Brasil. Avaliação da atividade antioxidante por técnicas espectroscópicas e eletroquímicas. Master’s Thesis, Universidade de Salamanca, Bragança, Portugal. 57p.), Greece, Cyprus (Kalogeropoulos et al. 2009Kalogeropoulos N., Konteles S.J., Troullidou E., Mourtzinos I. & Karathanos V.T. 2009. Chemical composition, antioxidant activity and antimicrobial properties of propolis extracts from Greece and Cyprus. Food Chem. 116(2):452-461. <http://dx.doi.org/10.1016/j.foodchem.2009.02.060>
https://doi.org/10.1016/j.foodchem.2009....
), Czech Republic, Ireland and Germany (Al-Ani et al. 2018Al-Ani I., Zimmermann S., Reichling J. & Wink M. 2018. Antimicrobial activities of European propolis collected from various geographic origins alone and in combination with antibiotics. Medicines, Basel 5(1):2-17. <http://dx.doi.org/10.3390/medicines5010002> <PMid:29301368>
https://doi.org/10.3390/medicines5010002...
). The total phenol and flavonoids contents observed for EPA were similar to those of Kumazawa et al. (2004)Kumazawa S., Hamasaka T. & Nakayama T. 2004. Antioxidant activity of propolis of various geographic origins. Food Chem. 84(3):329-339. <http://dx.doi.org/10.1016/S0308-8146(03)00216-4>
https://doi.org/10.1016/S0308-8146(03)00...
, El Sohaimy & Masry (2014)El Sohaimy S.A. & Masry S.H.D. 2014. Phenolic content, antioxidant and antimicrobial activities of Egyptian and Chinese propolis. Am. Eurasian J. Agric. Environ. Sci. 14(10):1116-1124. and Silva et al. (2015)Silva W.E.L., Ferrari Junior W.D., Rosa P.R., Peixoto R.M., Tenório J.A.B., Silva T.M.S. & Costa M.M. 2015. In vitro activit of propolis: Synergism in combination with antibiotic against Staphylococcus spp. African J. Microbiol. Res. 9(1):1-5. <http://dx.doi.org/10.5897/AJMR2014.7173>
https://doi.org/10.5897/AJMR2014.7173...
, lower than Alencar et al. (2007)Alencar S.M., Oldoni T.L., Castro M.L., Cabral I.S., Costa-Neto C.M., Cury J.A., Rosalen P.L. & Ikegaki M. 2007. Chemical composition and biological activity of a new type of Brazilian propolis: red propolis. J. Ethnopharmacol. 113(2):278-283. <http://dx.doi.org/10.1016/j.jep.2007.06.005> <PMid:17656055>
https://doi.org/10.1016/j.jep.2007.06.00...
, Cabral et al. (2012)Cabral I.S.R., Oldoni T.L.C., Alencar S.M., Rosalen P.L. & Ikegaki M. 2012. The correlation between the phenolic composition and biological activities of two varieties of Brazilian propolis (G6 and G12). Braz. J. Pharm. Sci. 48(3):557-564. <http://dx.doi.org/10.1590/S1984-82502012000300023>
https://doi.org/10.1590/S1984-8250201200...
, Peixoto et al. (2012)Peixoto E.C.T.M., Jardim J.G., Heinzen E.L., Domingues P.F., Padovani C.R. & Orsi R.O. 2012. Própolis no controle da mastite bovina. Arch. Vet. Sci. 17(4):43-52. and El Sohaimy & Masry (2014)El Sohaimy S.A. & Masry S.H.D. 2014. Phenolic content, antioxidant and antimicrobial activities of Egyptian and Chinese propolis. Am. Eurasian J. Agric. Environ. Sci. 14(10):1116-1124., and higher than Tiveron et al. (2016)Tiveron A.P., Rosalen P.L., Franchin M., Lacerda R.C.C., Bueno-Silva B., Benso B., Denny C., Ikegaki M. & Alencar S.M. 2016. Chemical characterization and antioxidant, antimicrobial, and anti-inflammatory activities of south Brazilian organic propolis. PLoS One 11(11):e0165588. <http://dx.doi.org/10.1371/journal.pone.0165588> <PMid:27802316>
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. Cunha et al. (2004)Cunha I.B.S., Sawaya A.C.H.F., Caetano F.M., Shimizu M.T., Marcucci M.C., Drezza F.T., Povia G.S. & Carvalho P.O. 2004. Factors that influence the yield and composition of brazilian propolis extracts. J. Braz. Chem. Soc. 15(6):964-970. <http://dx.doi.org/10.1590/S0103-50532004000600026>
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observed a variation of 6.41 to 15.24% in the total phenol content, while Gonsales et al. (2006)Gonsales G.Z., Orsi R.O., Fernandes Junior A., Rodrigues P. & Funari S.R.C. 2006. Antibacterial activity of propolis collected in different regions of Brazil. J. Venom. Anim. Toxins Trop. Dis. 12(2):276-284. <http://dx.doi.org/10.1590/S1678-91992006000200009>
https://doi.org/10.1590/S1678-9199200600...
verified variations between 0.05 and 0.63% in the contents of flavonoids and Sousa et al. (2007)Sousa J.P.B., Furtado N.A.J.C., Jorge R., Soares A.E.E. & Bastos J.K. 2007. Perfis físico-químico e cromatográfico de amostras de própolis produzidas nas microrregiões de Franca (SP) e Passos (MG), Brasil. Braz. J. Pharmacogn. 17(1):85-93. <http://dx.doi.org/10.1590/S0102-695X2007000100017>
https://doi.org/10.1590/S0102-695X200700...
verified a total flavonoid content of 0.06 to 0.38% for samples from São Paulo (Franca region) and from 0.12 to 2.11% for those from Minas Gerais (Passo region). This high variability in the total phenol and flavonoid content occurs due to the different sources of vegetable exudate, as well as by the location of the apiary (Park et al. 2002Park Y.K., Alencar S.M. & Aguiar C.L. 2002. Botanical origin and chemical composition of Brazilian propolis. J. Agric. Food Chem. 50(9):2502-2506. <http://dx.doi.org/10.1021/jf011432b> <PMid:11958612>
https://doi.org/10.1021/jf011432b...
, Gonsales et al. 2006Gonsales G.Z., Orsi R.O., Fernandes Junior A., Rodrigues P. & Funari S.R.C. 2006. Antibacterial activity of propolis collected in different regions of Brazil. J. Venom. Anim. Toxins Trop. Dis. 12(2):276-284. <http://dx.doi.org/10.1590/S1678-91992006000200009>
https://doi.org/10.1590/S1678-9199200600...
, Sousa et al. 2007Sousa J.P.B., Furtado N.A.J.C., Jorge R., Soares A.E.E. & Bastos J.K. 2007. Perfis físico-químico e cromatográfico de amostras de própolis produzidas nas microrregiões de Franca (SP) e Passos (MG), Brasil. Braz. J. Pharmacogn. 17(1):85-93. <http://dx.doi.org/10.1590/S0102-695X2007000100017>
https://doi.org/10.1590/S0102-695X200700...
). The soluble solids content in the both extract of propolis tested were higher than those observed by Sousa et al. (2007)Sousa J.P.B., Furtado N.A.J.C., Jorge R., Soares A.E.E. & Bastos J.K. 2007. Perfis físico-químico e cromatográfico de amostras de própolis produzidas nas microrregiões de Franca (SP) e Passos (MG), Brasil. Braz. J. Pharmacogn. 17(1):85-93. <http://dx.doi.org/10.1590/S0102-695X2007000100017>
https://doi.org/10.1590/S0102-695X200700...
and met the current legislation standards (Brasil 2001Brasil. Ministério da Agricultura 2001. Regulamento técnico para fixação de identidade e qualidade de própolis. Instrução Normativa nº 3, Anexo VI, de 19 de janeiro. Ministério da Agricultura, Brasília, DF.).

The HLPC-DAD analysis revealed mainly the presence of simple phenolic acids as caffeic, coumaric and cinnamic acids and methylated phenolic acids such as ferulic acid, and also 3,4-dihydroxybenzoic acid. These results agree with Al Naggar et al. (2016)Al Naggar Y., Sun J., Robertson A., Giesy J.P. & Wiseman S. 2016. Chemical characterization and antioxidant properties of Canadian propolis. J. Apicultural Res. 55(4):305-314. <http://dx.doi.org/10.1080/00218839.2016.1233700>
https://doi.org/10.1080/00218839.2016.12...
that also found coumaric acid, ferulic acid and caffeic acid in Canadian propolis. Previous studies also have had describe presence of caffeic acid (Marcucci et al. 2001Marcucci M.C., Ferreres F., Garcıa-Viguera C., Bankova V.S., Castro S.L., Dantas A.P., Valente P.H.M. & Paulino N. 2001. Phenolic compounds from Brazilian propolis with pharmacological activities. J. Ethnopharmacol. 74(2):105-112. <http://dx.doi.org/10.1016/S0378-8741(00)00326-3> <PMid:11167028>
https://doi.org/10.1016/S0378-8741(00)00...
, Bankova et al. 2002Bankova V., Popova M., Bogdanov S. & Sabatini A.G. 2002. Chemical composition of European propolis: expected and unexpected results. Z. Naturforsch. J. Biosci. C 57(5/6):530-533. <http://dx.doi.org/10.1515/znc-2002-5-622> <PMid:12132697>
https://doi.org/10.1515/znc-2002-5-622...
, Kartal et al. 2002Kartal M., Kaya S. & Kurucu S. 2002. GC-MS analysis of propolis samples from two different regions of Turkey. Z. Naturforsch. 57(9/10):905-909. <http://dx.doi.org/10.1515/znc-2002-9-1025> <PMid:12440732>
https://doi.org/10.1515/znc-2002-9-1025...
, 2003Kartal M., Yıldız S., Kaya S., Kurucu S. & Topçu G. 2003. Antimicrobial activity of pro- polis samples from two different regions of Anatolia. J. Ethnopharmacol. 86(1):69-73. <http://dx.doi.org/10.1016/S0378-8741(03)00042-4> <PMid:12686444>
https://doi.org/10.1016/S0378-8741(03)00...
, Salomão et al. 2004Salomão K., Dantas A.P., Borba C.M., Campos L.C., Machado D.G., Aquino Neto F.R. & De Castro S.L. 2004. Chemical composition and microbicidal activity of extracts from Brazilian and Bulgarian propolis. Lett. Appl. Microbiol. 38(2):87-92. <http://dx.doi.org/10.1111/j.1472-765X.2003.01458.x> <PMid:14746537>
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, Ahn et al. 2007Ahn M.-R., Kumazawa S., Usui Y., Nakamura J., Matsuka M., Zhu F. & Nakayama T. 2007. Antioxidant activity and constituents of propolis collected in various areas of China. Food Chemistry 101(4):1383-1392. <http://dx.doi.org/10.1016/j.foodchem.2006.03.045>
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); coumaric acid (Mohammadzadeh et al. 2007Mohammadzadeh S., Shariatpanahi M., Hamedi M., Ahmadkhaniha R., Samadi N. & Ostad S.N. 2007. Chemical composition, oral toxicity and antimicrobial activity of Iranian propolis. Food. Chem. 103(4):1097-1103. <http://dx.doi.org/10.1016/j.foodchem.2006.10.006>
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, Afrouzan et al. 2018Afrouzan H., Tahghighi A., Zakeri S. & Es-Haghi A. 2018. Chemical composition and antimicrobial activities of Iranian Propolis. Iranian Biomed. J. 22(1):50-65. <PMid:28558440>, Al-Ani et al. 2018Al-Ani I., Zimmermann S., Reichling J. & Wink M. 2018. Antimicrobial activities of European propolis collected from various geographic origins alone and in combination with antibiotics. Medicines, Basel 5(1):2-17. <http://dx.doi.org/10.3390/medicines5010002> <PMid:29301368>
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); cinnamic acid (Salomão et al. 2004Salomão K., Dantas A.P., Borba C.M., Campos L.C., Machado D.G., Aquino Neto F.R. & De Castro S.L. 2004. Chemical composition and microbicidal activity of extracts from Brazilian and Bulgarian propolis. Lett. Appl. Microbiol. 38(2):87-92. <http://dx.doi.org/10.1111/j.1472-765X.2003.01458.x> <PMid:14746537>
https://doi.org/10.1111/j.1472-765X.2003...
, Yang et al. 2011Yang H., Dong Y., Du H., Shi H., Peng Y. & Li X. 2011. Antioxidant compounds from propolis collected in Anhui, China. Molecules 16(4):3444-3455. <http://dx.doi.org/10.3390/molecules16043444> <PMid:21512452>
https://doi.org/10.3390/molecules1604344...
, Coelho 2013Coelho J.P.M. 2013. Identificação e quantificação de compostos fenólicos em própolis da região sul do Brasil. Avaliação da atividade antioxidante por técnicas espectroscópicas e eletroquímicas. Master’s Thesis, Universidade de Salamanca, Bragança, Portugal. 57p., El Sohaimy & Masry 2014El Sohaimy S.A. & Masry S.H.D. 2014. Phenolic content, antioxidant and antimicrobial activities of Egyptian and Chinese propolis. Am. Eurasian J. Agric. Environ. Sci. 14(10):1116-1124., Tiveron et al. 2016Tiveron A.P., Rosalen P.L., Franchin M., Lacerda R.C.C., Bueno-Silva B., Benso B., Denny C., Ikegaki M. & Alencar S.M. 2016. Chemical characterization and antioxidant, antimicrobial, and anti-inflammatory activities of south Brazilian organic propolis. PLoS One 11(11):e0165588. <http://dx.doi.org/10.1371/journal.pone.0165588> <PMid:27802316>
https://doi.org/10.1371/journal.pone.016...
, Al-Ani et al. 2018Al-Ani I., Zimmermann S., Reichling J. & Wink M. 2018. Antimicrobial activities of European propolis collected from various geographic origins alone and in combination with antibiotics. Medicines, Basel 5(1):2-17. <http://dx.doi.org/10.3390/medicines5010002> <PMid:29301368>
https://doi.org/10.3390/medicines5010002...
); and ferulic acid (Bankova et al. 2002Bankova V., Popova M., Bogdanov S. & Sabatini A.G. 2002. Chemical composition of European propolis: expected and unexpected results. Z. Naturforsch. J. Biosci. C 57(5/6):530-533. <http://dx.doi.org/10.1515/znc-2002-5-622> <PMid:12132697>
https://doi.org/10.1515/znc-2002-5-622...
, Kartal et al. 2002Kartal M., Kaya S. & Kurucu S. 2002. GC-MS analysis of propolis samples from two different regions of Turkey. Z. Naturforsch. 57(9/10):905-909. <http://dx.doi.org/10.1515/znc-2002-9-1025> <PMid:12440732>
https://doi.org/10.1515/znc-2002-9-1025...
, Salomão et al. 2004Salomão K., Dantas A.P., Borba C.M., Campos L.C., Machado D.G., Aquino Neto F.R. & De Castro S.L. 2004. Chemical composition and microbicidal activity of extracts from Brazilian and Bulgarian propolis. Lett. Appl. Microbiol. 38(2):87-92. <http://dx.doi.org/10.1111/j.1472-765X.2003.01458.x> <PMid:14746537>
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, Ahn et al. 2007Ahn M.-R., Kumazawa S., Usui Y., Nakamura J., Matsuka M., Zhu F. & Nakayama T. 2007. Antioxidant activity and constituents of propolis collected in various areas of China. Food Chemistry 101(4):1383-1392. <http://dx.doi.org/10.1016/j.foodchem.2006.03.045>
https://doi.org/10.1016/j.foodchem.2006....
, Alencar et al. 2007Alencar S.M., Oldoni T.L., Castro M.L., Cabral I.S., Costa-Neto C.M., Cury J.A., Rosalen P.L. & Ikegaki M. 2007. Chemical composition and biological activity of a new type of Brazilian propolis: red propolis. J. Ethnopharmacol. 113(2):278-283. <http://dx.doi.org/10.1016/j.jep.2007.06.005> <PMid:17656055>
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, Mohammadzadeh et al. 2007Mohammadzadeh S., Shariatpanahi M., Hamedi M., Ahmadkhaniha R., Samadi N. & Ostad S.N. 2007. Chemical composition, oral toxicity and antimicrobial activity of Iranian propolis. Food. Chem. 103(4):1097-1103. <http://dx.doi.org/10.1016/j.foodchem.2006.10.006>
https://doi.org/10.1016/j.foodchem.2006....
, Barbarić et al. 2011Barbarić M., Mišković K., Bojić M., Lončar M.B., Smolčić-Bubalo A., Debeljak Z. & Medić-Šarić M. 2011. Chemical composition of the ethanolic propolis extracts and its effect on HeLa cells. J. Ethnopharmacol. 135(3):772-778. <http://dx.doi.org/10.1016/j.jep.2011.04.015> <PMid:21515353>
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, Coelho 2013Coelho J.P.M. 2013. Identificação e quantificação de compostos fenólicos em própolis da região sul do Brasil. Avaliação da atividade antioxidante por técnicas espectroscópicas e eletroquímicas. Master’s Thesis, Universidade de Salamanca, Bragança, Portugal. 57p., El Sohaimy & Masry 2014El Sohaimy S.A. & Masry S.H.D. 2014. Phenolic content, antioxidant and antimicrobial activities of Egyptian and Chinese propolis. Am. Eurasian J. Agric. Environ. Sci. 14(10):1116-1124., Niculae et al. 2015Niculae M., Stan L., Pall E., Paștiu A.I., Balaci J.M., Muste S. & Spînu M. 2015. In vitro synergistic antimicrobial activity of romanian propolis and antibiotics against Escherichia coli isolated from bovine mastitis. Notulae Botanicae Horti Agrobotanaci 43(2):327-334. <http://dx.doi.org/10.15835/nbha43210074>
https://doi.org/10.15835/nbha43210074...
, Oldoni et al. 2015Oldoni T.L.C., Oliveira S.C., Andolfatto S., Karling M., Calegari M.A., Sado R.Y., Maia F.M.C., Alencar S.M. & Lima V.A. 2015. Chemical characterization and optimization of the extraction process of bioactive compounds from propolis produced by selected bees Apis mellifera. J. Braz. Chem. Soc. 26:2054-2062., Bakdash et al. 2018Bakdash A., Almohammadi O.S., Taha N.A., Abu-Rumman A. & Kumar S. 2018. Chemical Composition of Propolis from the Baha Region in Saudi Arabia. Czech J. Food Sci. 36:1-10.).

Coumaric and cinnamic acids were the main components in EPA and EPB, contrasting with the results by previous reports that found ferulic acid (Alencar et al. 2007Alencar S.M., Oldoni T.L., Castro M.L., Cabral I.S., Costa-Neto C.M., Cury J.A., Rosalen P.L. & Ikegaki M. 2007. Chemical composition and biological activity of a new type of Brazilian propolis: red propolis. J. Ethnopharmacol. 113(2):278-283. <http://dx.doi.org/10.1016/j.jep.2007.06.005> <PMid:17656055>
https://doi.org/10.1016/j.jep.2007.06.00...
, Barbarić et al. 2011Barbarić M., Mišković K., Bojić M., Lončar M.B., Smolčić-Bubalo A., Debeljak Z. & Medić-Šarić M. 2011. Chemical composition of the ethanolic propolis extracts and its effect on HeLa cells. J. Ethnopharmacol. 135(3):772-778. <http://dx.doi.org/10.1016/j.jep.2011.04.015> <PMid:21515353>
https://doi.org/10.1016/j.jep.2011.04.01...
, El Sohaimy & Masry 2014El Sohaimy S.A. & Masry S.H.D. 2014. Phenolic content, antioxidant and antimicrobial activities of Egyptian and Chinese propolis. Am. Eurasian J. Agric. Environ. Sci. 14(10):1116-1124., Niculae et al. 2015Niculae M., Stan L., Pall E., Paștiu A.I., Balaci J.M., Muste S. & Spînu M. 2015. In vitro synergistic antimicrobial activity of romanian propolis and antibiotics against Escherichia coli isolated from bovine mastitis. Notulae Botanicae Horti Agrobotanaci 43(2):327-334. <http://dx.doi.org/10.15835/nbha43210074>
https://doi.org/10.15835/nbha43210074...
) caffeic acid (Kartal et al. 2002Kartal M., Kaya S. & Kurucu S. 2002. GC-MS analysis of propolis samples from two different regions of Turkey. Z. Naturforsch. 57(9/10):905-909. <http://dx.doi.org/10.1515/znc-2002-9-1025> <PMid:12440732>
https://doi.org/10.1515/znc-2002-9-1025...
, Melliou & Chinou 2004Melliou E. & Chinou I. 2004. Chemical analysis and antimicrobial activity of Greek propolis. Planta Med. 70(6):515-519. <http://dx.doi.org/10.1055/s-2004-827150> <PMid:15229802>
https://doi.org/10.1055/s-2004-827150...
, El Sohaimy & Masry 2014El Sohaimy S.A. & Masry S.H.D. 2014. Phenolic content, antioxidant and antimicrobial activities of Egyptian and Chinese propolis. Am. Eurasian J. Agric. Environ. Sci. 14(10):1116-1124.); p-coumaric acid (Jorge et al. 2008Jorge R., Furtado N.A.J.C., Sousa J.P.B., Silva Filho A.A., Gregorio J.L.E., Martins C.H.G., Soares A.E.E., Bastos J.K., Cunha W.R. & Silva M.L.A. 2008. Brazilian propolis: seasonal variation of the prenylated p-coumaric acids and antimicrobial activity. Pharm. Biol. 46(12):889-893. <http://dx.doi.org/10.1080/13880200802370373>
https://doi.org/10.1080/1388020080237037...
, Salomão et al. 2008Salomão K., Pereira P.R.S., Campos L.C., Borba C.M., Cabello P.H., Marcucci M.C. & Castro S.L. 2008. Brazilian propolis: correlation between chemical composition and antimicrobial activity. Evid. Based Complement. Alternat. Med. 5(3):317-324. <http://dx.doi.org/10.1093/ecam/nem058> <PMid:18830454>
https://doi.org/10.1093/ecam/nem058...
, Afrouzan et al. 2018Afrouzan H., Tahghighi A., Zakeri S. & Es-Haghi A. 2018. Chemical composition and antimicrobial activities of Iranian Propolis. Iranian Biomed. J. 22(1):50-65. <PMid:28558440>) and cinnamic acid (Katircioglu & Mercan 2006Katircioglu H. & Mercan N. 2006. Antimicrobial activity and chemical compositions of of Turkish propolis from different regions. African J. Biotechnol. 5(11):1151-1153., Mohammadzadeh et al. 2007Mohammadzadeh S., Shariatpanahi M., Hamedi M., Ahmadkhaniha R., Samadi N. & Ostad S.N. 2007. Chemical composition, oral toxicity and antimicrobial activity of Iranian propolis. Food. Chem. 103(4):1097-1103. <http://dx.doi.org/10.1016/j.foodchem.2006.10.006>
https://doi.org/10.1016/j.foodchem.2006....
, Silva Filho et al. 2009Silva Filho A.A., Resende D.O., Fukui M.J., Santos F.F., Pauletti P.M., Cunha W.R., Silva M.L., Gregório L.E., Bastos J.K. & Nanayakkara N.P. 2009. In vitro antileishmanial, antiplasmodial and cytotoxic activities of phenolics and triterpenoids from Baccharis dracunculifolia D. C. (Asteraceae). Fitoterapia 80(8):478-482. <http://dx.doi.org/10.1016/j.fitote.2009.06.007> <PMid:19540316>
https://doi.org/10.1016/j.fitote.2009.06...
) as the main component. Therefore, this was the first study to relate the joint action of cinnamic and coumaric acids to the antimicrobial activity of propolis, confirming that the propolis composition varies according to botanical and geographical origin (Bankova et al. 2002Bankova V., Popova M., Bogdanov S. & Sabatini A.G. 2002. Chemical composition of European propolis: expected and unexpected results. Z. Naturforsch. J. Biosci. C 57(5/6):530-533. <http://dx.doi.org/10.1515/znc-2002-5-622> <PMid:12132697>
https://doi.org/10.1515/znc-2002-5-622...
, Salomão et al. 2004Salomão K., Dantas A.P., Borba C.M., Campos L.C., Machado D.G., Aquino Neto F.R. & De Castro S.L. 2004. Chemical composition and microbicidal activity of extracts from Brazilian and Bulgarian propolis. Lett. Appl. Microbiol. 38(2):87-92. <http://dx.doi.org/10.1111/j.1472-765X.2003.01458.x> <PMid:14746537>
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, Popova et al. 2005Popova M., Silici S., Kaftanoglu O. & Bankova V. 2005. Antibacterial activity of Turkish propolis and its qualitative and quantitative chemical composition. Phytomedicine 12(3):221-228. <http://dx.doi.org/10.1016/j.phymed.2003.09.007> <PMid:15830845>
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, Sahinler & Kaftanoglu 2005Sahinler N. & Kaftanoglu O. 2005. Natural product propolis: chemical composition. Nat. Prod. Res. 19(2):183-188. <http://dx.doi.org/10.1080/14786410410001704877> <PMid:15715264>
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, Barbarić et al. 2011Barbarić M., Mišković K., Bojić M., Lončar M.B., Smolčić-Bubalo A., Debeljak Z. & Medić-Šarić M. 2011. Chemical composition of the ethanolic propolis extracts and its effect on HeLa cells. J. Ethnopharmacol. 135(3):772-778. <http://dx.doi.org/10.1016/j.jep.2011.04.015> <PMid:21515353>
https://doi.org/10.1016/j.jep.2011.04.01...
, Huang et al. 2014Huang S., Zhang C.P., Wang K., Li G.Q. & Hu F.L. 2014. Recent advances in the chemical composition of propolis. Molecules 19(12):19610-19632. <http://dx.doi.org/10.3390/molecules191219610> <PMid:25432012>
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, Shahbaz et al. 2015Shahbaz M., Zahoor T., Randhawa M.A. & Nawaz H. 2015. In vitro antibacterial activity of hydroalcoholic extract of propolis against pathogenic bacteria. Pakistan J. Life Soc. Sci. 13(3):132-136., Afrouzan et al. 2018Afrouzan H., Tahghighi A., Zakeri S. & Es-Haghi A. 2018. Chemical composition and antimicrobial activities of Iranian Propolis. Iranian Biomed. J. 22(1):50-65. <PMid:28558440>, Bakdash et al. 2018Bakdash A., Almohammadi O.S., Taha N.A., Abu-Rumman A. & Kumar S. 2018. Chemical Composition of Propolis from the Baha Region in Saudi Arabia. Czech J. Food Sci. 36:1-10.).

The results showed that both EPA and EPB showed antimicrobial activity against isolates of Staphylococcus spp., in agreement with previous studies indicated that the antibacterial activity of propolis is more pronounced against Gram-positive bacteria (Langoni et al. 1996Langoni H., Domingues P.F., Funari S.R.C., Chande C.G. & Neves I.R. 1996. Efeito antimicrobiano in vitro da propolis. Arq. Bras. Vet. Zoot. 48(2):227-229., Sforcin et al. 2000Sforcin J.M., Fernandes Junior A., Lopes C.A., Bankova V. & Funari S.R. 2000. Seasonal effect on Brazilian propolis antibacterial activity. J. Ethnopharmacol. 73(1/2):243-249. <http://dx.doi.org/10.1016/S0378-8741(00)00320-2> <PMid:11025162>
https://doi.org/10.1016/S0378-8741(00)00...
, Pinto et al. 2001Pinto M.S., Faria J.E., Message D., Cassini S.T.A., Pereira C.S. & Gioso M.M. 2001. Efeito de extratos de própolis verde sobre bactérias patogênicas isoladas do leite de vacas com mastite. Braz. J. Vet. Res. Anim. Sci. 38(6):278-283. <http://dx.doi.org/10.1590/S1413-95962001000600006>
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, Fernandes Júnior et al. 2003Fernandes Júnior A., Balestrin E.C.C. & Cunha M.L.R.S. 2003. Anti-Staphylococcus aureus activity of bee propolis extracts prepared with different ethanol concentrations. Revta Ciênc. Farm. 24:147-152., Auricchio et al. 2006Auricchio M.T., Bugno A., Almodóvar A.A.B. & Pereira T.C. 2006. Avaliação da atividade antimicrobiana de prEPArações de própolis comercializadas na cidade de São Paulo. Revta Inst. Adolfo Lutz 65(3):209-212., Trusheva et al. 2010Trusheva B., Todorov I., Ninova M., Najdenski H., Daneshmand A. & Bankova V. 2010. Antibacterial mono- and sesquiterpene esters of benzoic acids from Iranian propolis. Chem. Central J. 4(8):1-5. <http://dx.doi.org/10.1186/1752-153X-4-8> <PMid:20350297>
https://doi.org/10.1186/1752-153X-4-8...
, Aguiar et al. 2014Aguiar C.G., Lima L.G. & Athayde L.A. 2014. Efeito antimicrobiano da própolis verde frente a cEPAs de Staphylococcus aureus resistentes à meticilina (MRSA). Revta Bras. Pesq. Ciênc. Saúde 1(1):12-16.). The antibacterial activity of the EPA, with 90.9% susceptibility, was similar to the Loguercio et al. (2006)Loguercio A.P., Groff M.C.A., Pedrozzo F.A., Witt M.N., Silva S.M. & Vargas C.A. 2006. Atividade in vitro do extrato de própolis contra agentes bacterianos da mastite bovina. Pesq. Agropec. Bras. 41(2):347-349. <http://dx.doi.org/10.1590/S0100-204X2006000200021>
https://doi.org/10.1590/S0100-204X200600...
and Coelho et al. (2010)Coelho M.S., Silva J.H., Oliveira E.R.A., Amâncio A.L.L., Silva N.V. & Lima R.M.B. 2010. A própolis e sua utilização em animais de produção. Arch. Zootec. 59(R):95-112. which showed susceptibility of 94.4% in coagulase-positive Staphylococcus. When comparing the two extracts, it was verified that isolates of Staphylococcus spp. susceptible to common antibiotics, as well as those resistant to oxacillin, showed higher susceptibility to EPA, while isolates susceptible to oxacillin showed greater susceptibility to EPB. It is believed that the higher levels of phenolic compounds in EPA have been responsible for the greater susceptibility of isolates resistant to oxacillin, since phenolic compounds to bind to bacterial cell walls and prevent cell division and gr owth, as also observed in previous reports (Stapleton et al. 2004Stapleton P.D., Shah S., Anderson J.C., Hara Y., Hamilton-Miller J.M.T. & Taylor P.W. 2004. Modulation of beta-lactam resistance in Staphylococcus aureus by catechins and gallates. Int. J. Antimicrob. Agents 23(5):462-467. <http://dx.doi.org/10.1016/j.ijantimicag.2003.09.027> <PMid:15120724>
https://doi.org/10.1016/j.ijantimicag.20...
, El Sohaimy 2014El Sohaimy S.A. 2014. Chemical composition, antioxidant and antimicrobial potential of artichoke. Open Nutraceuticals J. 7:15-20., Al-Ani et al. 2018Al-Ani I., Zimmermann S., Reichling J. & Wink M. 2018. Antimicrobial activities of European propolis collected from various geographic origins alone and in combination with antibiotics. Medicines, Basel 5(1):2-17. <http://dx.doi.org/10.3390/medicines5010002> <PMid:29301368>
https://doi.org/10.3390/medicines5010002...
).

EPA demonstrated potent antibacterial activity against Staphylococcus spp. isolates in lower concentrations than EPB. These results agree with Kareem et al. (2015)Kareem A.A., Abdzaid N.Y., Salman R.M., Mohamed M.K., Dekel A.J. & Abdul-Muhsen R.S. 2015. Study of antibacterial activity in the local Iraqi propolis. J. Contemporary Med. Sci. 1(2):6-8. who also observed this activity in Iranian propolis against Staphylococcus spp. MBC for EPA (34.3-68.6μg/mL) was similar to Cabral et al. (2009)Cabral I.S.R., Oldoni T.L.C., Prado A., Bezerra R.M.N., Alencar S.M., Ikegaki M. & Rosalen P.L. 2009. Composição fenólica, atividade antibacteriana e antioxidante da própolis vermelha brasileira. Quím. Nova 32(6):1523-1527. <http://dx.doi.org/10.1590/S0100-40422009000600031>
https://doi.org/10.1590/S0100-4042200900...
and Rhajaoui et al. (2001)Rhajaoui M., Ourmazil H., Faid M., Lyagoubi M., Elyachioui M. & Benjouad A. 2001. Antibacterial activity of a Moroccan propolis extracts. Sci. Letters 3(3):201-207.; lower than Hayacibara et al. (2005)Hayacibara M.F., Koo H., Rosalen P.L., Duarte S., Franco E.M., Bowen W.H., Ikegaki M. & Cury J.A. 2005. In vitro and in vivo effects of isolated fractions of Brazilian propolis on caries development. J. Ethnopharmacol. 101(1/3):110-115. <http://dx.doi.org/10.1016/j.jep.2005.04.001> <PMid:15913934>
https://doi.org/10.1016/j.jep.2005.04.00...
, Alencar et al. (2007)Alencar S.M., Oldoni T.L., Castro M.L., Cabral I.S., Costa-Neto C.M., Cury J.A., Rosalen P.L. & Ikegaki M. 2007. Chemical composition and biological activity of a new type of Brazilian propolis: red propolis. J. Ethnopharmacol. 113(2):278-283. <http://dx.doi.org/10.1016/j.jep.2007.06.005> <PMid:17656055>
https://doi.org/10.1016/j.jep.2007.06.00...
and Tiveron et al. (2016)Tiveron A.P., Rosalen P.L., Franchin M., Lacerda R.C.C., Bueno-Silva B., Benso B., Denny C., Ikegaki M. & Alencar S.M. 2016. Chemical characterization and antioxidant, antimicrobial, and anti-inflammatory activities of south Brazilian organic propolis. PLoS One 11(11):e0165588. <http://dx.doi.org/10.1371/journal.pone.0165588> <PMid:27802316>
https://doi.org/10.1371/journal.pone.016...
and higher than Santos et al. (2002Santos F.A., Bastos E.M.A., Uzeda M., Carvalho M.A.R., Farias L.M., Moreira E.S.A. & Braga F.C. 2002. Antibacterial activity of Brazilian propolis and fractions against oral anaerobic bacteria. J. Ethnopharmacol. 80(1):1-7. <http://dx.doi.org/10.1016/S0378-8741(02)00003-X> <PMid:11891080>
https://doi.org/10.1016/S0378-8741(02)00...
) and Zeighampour et al. (2014)Zeighampour F., Mohammadi-Sichani M., Shams E. & Naghavi N.S. 2014. Antibacterial activity of propolis ethanol extract against antibiotic resistance bacteria isolated from burn wound infections. Zahedan J. Res. Med. Sci. 16(3):25-30.. According to Cos et al. (2006)Cos P., Vlietinck A.J., Berghe D.V. & Maes L. 2006. Anti-infective potential of natural products: how to develop a stronger in vitro ‘proof-of concept’. J. Ethnopharmacol. 106(3):290-302. <http://dx.doi.org/10.1016/j.jep.2006.04.003> <PMid:16698208>
https://doi.org/10.1016/j.jep.2006.04.00...
, the ideal anti-infective concentration would be generally below 100μg/ml for extracts, confirming the excellent efficiency of the EPA in relation to EPB and to others studies.

Staphylococcus spp. have a high ability to develop mechanisms of antimicrobial resistance, which makes them a serious problem of global public health (Ratti & Sousa 2009Ratti R.P. & Sousa C.P. 2009. Staphylococcus aureus meticilina resistente (MRSA) e infecções nosocomiais. Revta Ciênc. Farm. Básica Apl. 30(2):137-143., Garza-González et al. 2010Garza-González E., Morfin-Otero R., Llaca-Diaz J.M. & Rodriguez-Noriega E. 2010. Staphylococcal cassette chromosome mec (SCC mec) in methicillin-resistant coagulase-negative staphylococci. A review and the experience in a tertiarycare setting. Epidemiol. Infect. 138(5):645-654. <http://dx.doi.org/10.1017/S0950268809991361> <PMid:19961645>
https://doi.org/10.1017/S095026880999136...
, Reddy et al. 2017Reddy B.V., Kusuma Y.S., Pandav C.S., Goswami A.K. & Krishnan A. 2017. Water and sanitation hygiene practices for under-five children among households of Sugali tribe of Chittoor District, Andhra Pradesh, India. J. Environ. Publ. Health 2017:1-7. <http://dx.doi.org/10.1155/2017/7517414> <PMid:28642797>
https://doi.org/10.1155/2017/7517414...
). This is the case with methicillin-resistant Staphylococcus aureus (MRSA) strains, which are resistant to all beta lactam antimicrobials (Adelman 2005Adelman J. 2005. Propolis: variabilidade composicional, correlação com a flora e bioatividade antimicrobiana/antioxidante. Master’s Thesis in Farmaceutical Science, Universidade Federal do Paraná, Curitiba. 186p.), and often presents the multiple resistance phenomenon (Ratti & Sousa 2009Ratti R.P. & Sousa C.P. 2009. Staphylococcus aureus meticilina resistente (MRSA) e infecções nosocomiais. Revta Ciênc. Farm. Básica Apl. 30(2):137-143.). EPA and EPB inhibited significantly (100%) the clinical strains of MRSA and of S. aureus ATCC 6538 and ATCC 25923. Our results were similar to others reports in relation to the MRSA (Vera et al. 2011Vera N., Solorzano E., Ordoñez R., Maldonado L., Bedascarrasbure E. & Isla M.I. 2011. Chemical composition of Argentinean propolis collected in extreme regions and its relation with antimicrobial and antioxidant activities. Nat. Prod. Commun. 6(6):823-827. <http://dx.doi.org/10.1177/1934578X1100600618> <PMid:21815419>
https://doi.org/10.1177/1934578X11006006...
, Astani et al. 2013Astani A., Zimmermann S., Hassan E., Reichling J., Sensch K.H. & Schnitzler P. 2013. Antimicrobial activity of propolis special extract GH 2002 against multidrug-resistant clinical isolates. Pharmazie 68(8):695-701. <PMid:24020127>, Aguiar et al. 2014Aguiar C.G., Lima L.G. & Athayde L.A. 2014. Efeito antimicrobiano da própolis verde frente a cEPAs de Staphylococcus aureus resistentes à meticilina (MRSA). Revta Bras. Pesq. Ciênc. Saúde 1(1):12-16.) and S. aureus ATCC 6538 (Hazem et al. 2017Hazem A., Popescu C.V., Crișan I., Popa M., Chifiriuc M.C., Pircalabioru S. & Lupuliasa D. 2017. Antibacterial efficiency of five propolis extracts planktonic and adherent microbial strains. Farmacia 65(5):813-818.). In opposition, AL-Ani et al. (2018)Al-Ani I., Zimmermann S., Reichling J. & Wink M. 2018. Antimicrobial activities of European propolis collected from various geographic origins alone and in combination with antibiotics. Medicines, Basel 5(1):2-17. <http://dx.doi.org/10.3390/medicines5010002> <PMid:29301368>
https://doi.org/10.3390/medicines5010002...
observed a moderate anti-MRSA efficacy and Katircioglu & Mercan (2006)Katircioglu H. & Mercan N. 2006. Antimicrobial activity and chemical compositions of of Turkish propolis from different regions. African J. Biotechnol. 5(11):1151-1153. a low efficacy against S. aureus ATCC 25923, reinforcing the importance of the evaluation of multiresistant strains in the tests of antimicrobial activity of natural products (Cos et al. 2006Cos P., Vlietinck A.J., Berghe D.V. & Maes L. 2006. Anti-infective potential of natural products: how to develop a stronger in vitro ‘proof-of concept’. J. Ethnopharmacol. 106(3):290-302. <http://dx.doi.org/10.1016/j.jep.2006.04.003> <PMid:16698208>
https://doi.org/10.1016/j.jep.2006.04.00...
). Low MBC, like observed in this study, might be advantageous in therapeutic level for appropriate treatment of bacterial infections with regard to toxicity and stability of formulations (Astani et al. 2013Astani A., Zimmermann S., Hassan E., Reichling J., Sensch K.H. & Schnitzler P. 2013. Antimicrobial activity of propolis special extract GH 2002 against multidrug-resistant clinical isolates. Pharmazie 68(8):695-701. <PMid:24020127>).

The individual phenolic compounds present in the EPA and EPB were not identified and quantified, because submitting the entire extracts to antimicrobial activity studies seems to be more beneficial than to submit isolated constituents, since a bioactive individual component can change its properties in the presence of other compounds present in the extract (Borchers et al. 2004Borchers A.T., Keen C.L. & Gershwin M.E. 2004. Mushrooms, tumors, and immunity: an update. Exp. Biol. Med. 229(5):393-406. <http://dx.doi.org/10.1177/153537020422900507> <PMid:15096651>
https://doi.org/10.1177/1535370204229005...
), corresponding to a synergistic effect.

The way propolis exerts its antimicrobial action is complex and occur, among other things, through inhibition of the bacteria growth by inhibiting of its enzymatic activity diminishing their effects on biological systems (Zeighampour et al. 2014Zeighampour F., Mohammadi-Sichani M., Shams E. & Naghavi N.S. 2014. Antibacterial activity of propolis ethanol extract against antibiotic resistance bacteria isolated from burn wound infections. Zahedan J. Res. Med. Sci. 16(3):25-30.); inhibition of cell division; collapse of the bacterial cytoplasm, cell membranes, and cell walls; bacteriolysis; and protein synthesis inhibition (Takaisi-Kikuni & Schilcher 1994 apud Fernandes Junior et al. 2005Fernandes Júnior A., Balestrin E.C., Betoni J.E.C., Orsi R.O., Cunha M.L.R.S. & Montelli A.C. 2005. Propolis: anti-Staphylococcus aureus activity and synergism with antimicrobial drugs. Mem. Inst. Oswaldo Cruz 100(5):563-566. <http://dx.doi.org/10.1590/S0074-02762005000500018> <PMid:16184236>
https://doi.org/10.1590/S0074-0276200500...
). In vitro studies have attributed the antimicrobial activity of propolis to the presence of phenolic compounds, flavonoids, phenolic acids and their esters (Pinto et al. 2001Pinto M.S., Faria J.E., Message D., Cassini S.T.A., Pereira C.S. & Gioso M.M. 2001. Efeito de extratos de própolis verde sobre bactérias patogênicas isoladas do leite de vacas com mastite. Braz. J. Vet. Res. Anim. Sci. 38(6):278-283. <http://dx.doi.org/10.1590/S1413-95962001000600006>
https://doi.org/10.1590/S1413-9596200100...
, Freitas et al. 2005Freitas M.F.L., Pinheiro Junior J.W., Stamford T.L.M., Rabelo S.S.A., Silva D.R., Silveira Filho V.M., Santos F.G.B. & Mota R.A. 2005. Perfil se sensibilidade antimicorbiana in vitro de Staphylococcus coagelusa positivos isolados de leite de vaca com mastite no agreste do Estado e Pernambuco. Arq. Inst. Biológico, São Paulo, 72(2):171-177., Katircioglu & Mercan 2006Katircioglu H. & Mercan N. 2006. Antimicrobial activity and chemical compositions of of Turkish propolis from different regions. African J. Biotechnol. 5(11):1151-1153., Mohammadzadeh et al. 2007Mohammadzadeh S., Shariatpanahi M., Hamedi M., Ahmadkhaniha R., Samadi N. & Ostad S.N. 2007. Chemical composition, oral toxicity and antimicrobial activity of Iranian propolis. Food. Chem. 103(4):1097-1103. <http://dx.doi.org/10.1016/j.foodchem.2006.10.006>
https://doi.org/10.1016/j.foodchem.2006....
, Barbarić et al. 2011Barbarić M., Mišković K., Bojić M., Lončar M.B., Smolčić-Bubalo A., Debeljak Z. & Medić-Šarić M. 2011. Chemical composition of the ethanolic propolis extracts and its effect on HeLa cells. J. Ethnopharmacol. 135(3):772-778. <http://dx.doi.org/10.1016/j.jep.2011.04.015> <PMid:21515353>
https://doi.org/10.1016/j.jep.2011.04.01...
) or to the synergistic action of flavonoids and other active principles (Santos et al. 2002Santos F.A., Bastos E.M.A., Uzeda M., Carvalho M.A.R., Farias L.M., Moreira E.S.A. & Braga F.C. 2002. Antibacterial activity of Brazilian propolis and fractions against oral anaerobic bacteria. J. Ethnopharmacol. 80(1):1-7. <http://dx.doi.org/10.1016/S0378-8741(02)00003-X> <PMid:11891080>
https://doi.org/10.1016/S0378-8741(02)00...
, Da Silva Filho et al. 2004Silva Filho A.A., Bueno P.C.P., Gregorio L.E., Silva M.L.A., Albuquerque S. & Bastos J.K. 2004. In vitro trypanocidal activity evaluation of crude extract and isolated compounds from Baccharis dracunculifolia D. C. (Asteraceae). J. Pharm. Pharmacol. 56(9):1195-1199. <http://dx.doi.org/10.1211/0022357044067> <PMid:15324490>
https://doi.org/10.1211/0022357044067...
, Barros et al. 2007Barros M.P., Sousa J.P., Bastos J.K. & de Andrade S.F. 2007. Effect of Brazilian green propolis on experimental gastric ulcers in rats. J. Ethnopharmacol. 110(3):567-571. <http://dx.doi.org/10.1016/j.jep.2006.10.022> <PMid:17126509>
https://doi.org/10.1016/j.jep.2006.10.02...
, Sousa et al. 2007Sousa J.P.B., Furtado N.A.J.C., Jorge R., Soares A.E.E. & Bastos J.K. 2007. Perfis físico-químico e cromatográfico de amostras de própolis produzidas nas microrregiões de Franca (SP) e Passos (MG), Brasil. Braz. J. Pharmacogn. 17(1):85-93. <http://dx.doi.org/10.1590/S0102-695X2007000100017>
https://doi.org/10.1590/S0102-695X200700...
). Flavonoids (Uzel et al. 2005Uzel A., Sorkun K., Onçağ O., Cogŭlu D., Gençay O. & Salih B. 2005. Chemical compositions and antimicrobial activities of four different Anatolian propolis samples. Microbiol. Res. 160(2):189-195. <http://dx.doi.org/10.1016/j.micres.2005.01.002> <PMid:15881836>
https://doi.org/10.1016/j.micres.2005.01...
, Alencar et al. 2007Alencar S.M., Oldoni T.L., Castro M.L., Cabral I.S., Costa-Neto C.M., Cury J.A., Rosalen P.L. & Ikegaki M. 2007. Chemical composition and biological activity of a new type of Brazilian propolis: red propolis. J. Ethnopharmacol. 113(2):278-283. <http://dx.doi.org/10.1016/j.jep.2007.06.005> <PMid:17656055>
https://doi.org/10.1016/j.jep.2007.06.00...
, Cushnie & Lamb 2011Cushnie T.P.T. & Lamb A.J. 2011. Recent advances in understanding the antibacterial properties of flavonoids. Int. J. Antimicrob. Agents 38(2):99-107. <http://dx.doi.org/10.1016/j.ijantimicag.2011.02.014> <PMid:21514796>
https://doi.org/10.1016/j.ijantimicag.20...
) and phenylethyl ester of caffeic acid apEPAr to act by inhibiting bacterial RNA polymerase (Uzel et al. 2005Uzel A., Sorkun K., Onçağ O., Cogŭlu D., Gençay O. & Salih B. 2005. Chemical compositions and antimicrobial activities of four different Anatolian propolis samples. Microbiol. Res. 160(2):189-195. <http://dx.doi.org/10.1016/j.micres.2005.01.002> <PMid:15881836>
https://doi.org/10.1016/j.micres.2005.01...
), while phenolic compounds, such as caffeic acid, benzoic acid and cinnamic acid, cause functional and structural damages on the membrane or cell wall of microorganisms (Scazzocchio et al. 2005Scazzocchio F., D’auria F.D., Alessandrini D. & Pantanella F. 2005. Multifactorial aspects of antimicrobial activity of propolis. Microbiol. Res. 4(4):327-333. <http://dx.doi.org/10.1016/j.micres.2005.12.003> <PMid:16427259>
https://doi.org/10.1016/j.micres.2005.12...
) or inhibition of bacterial replication (Rastogi et al. 2008Rastogi N., Domadia P., Shetty S. & Dasgupta D. 2008. Screening of natural phenolic compounds for potential to inhibit bacterial cell division protein FtsZ. Indian. J. Exp. Biol. 46(11):783-787. <PMid:19090350>) and galagin and caffeic acid cause enzymatic inhibition in bacteria (Koo et al. 2002Koo H., Rosalen P.L., Cury J.A., Park Y.K. & Bowen W.H. 2002. Effects of compounds found in propolis on Streptococcus mutans growth and on glucosiltransferase activity. Antimicrob. Agents Chemother. 46(5):1302-1309. <http://dx.doi.org/10.1128/AAC.46.5.1302-1309.2002> <PMid:11959560>
https://doi.org/10.1128/AAC.46.5.1302-13...
). The results suggest that the antibacterial activity of EPA and EPB occurred mainly due to the content of phenolic compounds as previously described (Bankova et al. 1995Bankova V., Christov R., Kujumgiev A., Marcucci M.C. & Popov S. 1995. Chemical composition and antibacterial activity of Brazilian propolis. Z. Naturforsch. J. Biosci. 50(3/4):167-172. <PMid:7766255>, Marcucci et al. 2001Marcucci M.C., Ferreres F., Garcıa-Viguera C., Bankova V.S., Castro S.L., Dantas A.P., Valente P.H.M. & Paulino N. 2001. Phenolic compounds from Brazilian propolis with pharmacological activities. J. Ethnopharmacol. 74(2):105-112. <http://dx.doi.org/10.1016/S0378-8741(00)00326-3> <PMid:11167028>
https://doi.org/10.1016/S0378-8741(00)00...
, Popova et al. 2005Popova M., Silici S., Kaftanoglu O. & Bankova V. 2005. Antibacterial activity of Turkish propolis and its qualitative and quantitative chemical composition. Phytomedicine 12(3):221-228. <http://dx.doi.org/10.1016/j.phymed.2003.09.007> <PMid:15830845>
https://doi.org/10.1016/j.phymed.2003.09...
, Estevinho et al. 2008Estevinho L., Pereira A.P., Moreira L., Dias L.G. & Pereira E. 2008. Antioxidant and antimicrobial effects of phenolic compounds extracts of Northeast Portugal honey. Food Chem. Toxicol. 46(12):3774-3779. <http://dx.doi.org/10.1016/j.fct.2008.09.062> <PMid:18940227>
https://doi.org/10.1016/j.fct.2008.09.06...
, Ristivojević et al. 2016Ristivojević P., Dimkić I., Trifković J., Berić T., Vovk I., Milojković-Opsenica D. & Stanković S. 2016. Antimicrobial activity of serbian propolis evaluated by means of MIC, HPTLC, bioautography and chemometrics. Plos One 11(6):1-15. <http://dx.doi.org/10.1371/journal.pone.0157097> <PMid:27272728>
https://doi.org/10.1371/journal.pone.015...
, Al-Ani et al. 2018Al-Ani I., Zimmermann S., Reichling J. & Wink M. 2018. Antimicrobial activities of European propolis collected from various geographic origins alone and in combination with antibiotics. Medicines, Basel 5(1):2-17. <http://dx.doi.org/10.3390/medicines5010002> <PMid:29301368>
https://doi.org/10.3390/medicines5010002...
), in particular by the content of cumaric and cinnamic acids (Popova et al. 2005Popova M., Silici S., Kaftanoglu O. & Bankova V. 2005. Antibacterial activity of Turkish propolis and its qualitative and quantitative chemical composition. Phytomedicine 12(3):221-228. <http://dx.doi.org/10.1016/j.phymed.2003.09.007> <PMid:15830845>
https://doi.org/10.1016/j.phymed.2003.09...
, Sforcin 2007Sforcin J.M. 2007. Propolis and the immune system. Rev. J. Ethnopharmacol. 113(1):1-14. <http://dx.doi.org/10.1016/j.jep.2007.05.012> <PMid:17580109>
https://doi.org/10.1016/j.jep.2007.05.01...
). However, it can not be ruled out that different substances may be involved in the antimicrobial activity, since Cabral et al. (2012)Cabral I.S.R., Oldoni T.L.C., Alencar S.M., Rosalen P.L. & Ikegaki M. 2012. The correlation between the phenolic composition and biological activities of two varieties of Brazilian propolis (G6 and G12). Braz. J. Pharm. Sci. 48(3):557-564. <http://dx.doi.org/10.1590/S1984-82502012000300023>
https://doi.org/10.1590/S1984-8250201200...
observed that G6 propolis, which had the lowest phenolic compound and flavonoid contents, showed the best antibacterial effect.

Conclusion

We could confirm that both extracts of propolis, whose main constituents are cumin and cinnamic acids, have high antimicrobial activity against the microorganisms studied, and EPA had also a high antimicrobial activity against oxacillin-resistant strains. These findings reinforce its potential use for the treatment of bovine mastitis.

Acknowledgments

The authors thank the “Programa de Apoio a Núcleos Emergentes” of “Fundação de Amparo à Ciência e Tecnologia do Estado de Pernambuco” (PRONEM-FACEPE), process no. 0741.1.06/14.

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Publication Dates

  • Publication in this collection
    04 Nov 2019
  • Date of issue
    Sept 2019

History

  • Received
    25 Nov 2018
  • Accepted
    09 May 2019
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