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\title{Origanum vulgare (Oregano) and its Carvacrol Biocomponent as an Alternative of Antimicrobial Agent}
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             \author[1]{Natalia Kaori Aida, Janaina Priscila Barbosa, Thais Rossini de Oliveira, Vanessa da Silva Cardoso, Simone Nataly Busato de Feiria, Giovana Claudia Boni, Jose Francisco  Hofling}

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\date{\small \em Received: 6 December 2019 Accepted: 31 December 2019 Published: 15 January 2020}

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\begin{abstract}
        


The use of plants as an alternative to medicinal treatments is an old practice. The increased resistance of microorganisms to conventional antimicrobials has made studies with medicinal plants increasingly relevant, and ethnobotanical and ethnopharmacological knowledge is considered essential for the development of new drugs. The essential oil of Origanum vulgare and its isolated compound Carvacrol have antimicrobial effects demonstrated in the literature as antibacterial and antifungal activity. Therefore, the present study evaluated the antibacterial and antifungal activity of O. vulgare and Carvacrol using the broth microdilution method (CLSI, 2008), determining MIC (Minimum Inhibitory Concentration) and MFC and MBC (Minimum Fungicidal Concentration and Concentration Minimum Bactericide). Used as standard comparative the antimicrobials Fluconazoleand Chlorhexidine.

\end{abstract}


\keywords{origanum vulgare; carvacrol; candida spp., streptococcus spp., staphylococcusaureus MRSA.}

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\let\tabcellsep& 	 	 		 \par
Introduction he use of plants as an alternative to medicinal treatments is an ancient practice, which provide primary health care to 80\% of the world's population. It has also been an important source for new drug discoveries \hyperref[b15]{(Wangchuk \& Tobgay, 2015)}. World Health Organization has estimated that around 80\% of the population of developing countries use traditional herbal medicines as a source of treatment for diseases as a cheap and alternative source, other factors such as the lack of modern health facilities, Cultural priorities and choices also contribute to the use of medicinal plants as a therapeutic alternative \hyperref[b2]{(Aziz et al., 2018)}. Studies on medicinal plants have became increasingly relevant, ethnobotanical and ethnopharmacological knowledge is considered essential for the development of new drugs. Researches studies the effectiveness and use of traditional plants as alternative medicine and adjuvants in a treatment \hyperref[b0]{(Amjad et al., 2017)}. In this context, Origanum vulgare is a plantused since ancient times as a traditional cure for treating infections  {\ref (Karaman et al., 2017)}. Studies show that O. vulgare essential oil is rich in Carvacrol, has anti-inflammatory, antioxidant, antispasmodic, antimicrobial, and antifungal activity \hyperref[b12]{(Król et al., 2019)}. Also, resistant microorganisms have emerged over the years and have been considered a global health threat. Microbial resistance has brought problems in the treatment of infectious diseases, and the development of new antimicrobial agents is required \hyperref[b1]{(Ayaz et al., 2019)}. The occurrence that strains of the genus Candida have been resistant to treatment with commercial antifungals, belonging to the azoles and polyenes family, has been a reason for concern by health professionals. Candida infections mainly affect immunosuppressed patients  {\ref (Barbosa et al., 2019)}. Given this scenario, the plants are promising antimicrobial agent for the development of new drugs, acting in disease prevention and treatment \hyperref[b1]{(Ayaz et al., 2019)}. Therefore, this work aimed to study the antimicrobial activity of Origanum vulgare essential oil and its isolated compound Carvacrol against microorganisms of genus Candida, Streptococci and, Staphylococci. 
\section[{II.}]{II.} 
\section[{Material and Methods}]{Material and Methods} 
\section[{a) Essential oil}]{a) Essential oil}\par
The essential oil was purchased commercially from Quinarí Fragrâncias e Cosméticos LTDA and the isolated compound from Sigma Aldrich. Essential Oil -Lot: 09818DIV Isolated Compound -Lot: S40656V  Origanum vulgare (Oregano) and its Carvacrol Biocomponent as an Alternative of Antimicrobial Agent range of 0.08 to 0.1 equivalent to 5.0 x 10 6 CFU/mL. Then, the inoculum was standardized, diluting to 2.5x 10 3 , according to CLSI, 2008.\par
In asterile microplate was deposited with 100 µl of RPMI, 100 µl of the essential oil or 100 µl of the isolated compound at the initial concentration, followed by serial microdilution and then added with 100 µl of the adjusted inoculum.\par
Groups of test: CG: positive control group; OL: Essential Oil Treatment Group; IC: Treatment group with isolated compound; AC: Treatment group with the commercial antifungal Fluconazole at an initial concentration of 64ug/mL  {\ref (CLSI, 2002)}. Incubated the plates for 48h at 37°C. Defined the MIC as the lowest concentration of compound that did not exhibit visible growth of the microorganism. 
\section[{d) Inoculum adjustment of the Streptococcus spp. and Staphylococcus aureus MRSA}]{d) Inoculum adjustment of the Streptococcus spp. and Staphylococcus aureus MRSA}\par
After growth in BHI liquid culture medium, the inoculum was adjusted by spectrophotometer with a wavelength of 625nm in the range 0.08 to 0.1absorbance, equivalent to 1.0 x 10 8 CFU/mL.\par
In a sterile microplate were deposited 100 µl of BHI, 100 µl of the essential oil or 100 µl of the isolated compound at the initial concentration, followed by serial microdilution, and then added of 100µl of the adjusted inoculum.\par
Groups of test: CG: positive control group; OL: Essential Oil Treatment Group; IC: Treatment group with isolated compound; AC: Treatment group with the commercial antifungal chlorhexidine at the initial concentration of 64ug/mL (CLSI, 2008). Incubation of the plates was made for 24h at 37°C with 10\% CO 2 for Streptococcus strains and aerobically for Staphylococcus aureus MRSA. Defined the MIC as the lowest concentration of compound that did not exhibit visible growth of the microorganism. 
\section[{e) Determination of Minimum Fungicidal Concentration (CFM) and Minimum Bactericidal Concentration (CBM)}]{e) Determination of Minimum Fungicidal Concentration (CFM) and Minimum Bactericidal Concentration (CBM)}\par
In a petri dish containing Sabouraud Dextrose Agar (SDA) for yeast and Brain Heart Infusion Agar (BHI) for bacteria tested the determination of minimum fungicidal concentration (MFC) and minimum bactericidal concentration (MBC). Homogeinized the wells containing target concentrations and transferred the aliquot to the Petri dish with the solid culture medium. After incubation at 37ºC for 24h, established the lowest fungicidal/bactericidal concentration. Determined the MFC/MBC as the lowest concentration of essential oils and isolated compounds that did not allow the growth of any colony of the microorganism on the solid medium after the incubation period. Through visual reading, the inhibition of growth or death provided by the tested substances was confirmed (GULLO et al., 2012). 
\section[{III.}]{III.} 
\section[{Results}]{Results} 
\section[{a) Evaluation of Minimum Inhibitory Concentration (MIC)}]{a) Evaluation of Minimum Inhibitory Concentration (MIC)}\par
and Minimum Fungicidal / Bactericidal Concentration (CFM / CBM) Tested the Origanum vulgare essential oil and its isolated compound Carvacrol against reference strains of the genus Candida, Streptococcus, and Staphylococcus aureus methicillin-resistant strain to determine its inhibitory effect by broth microdilution technique. Both substances showed inhibitory activity on microbial cells (Table  {\ref 1}).For reference strains of the genus Candida, inhibitory concentrations were between 0.125 mg/mL and 0.5 mg/mL for O. vulgare essential oil and 0.125 mg/mL and 0.0625 mg/mL for Carvacrol isolated compound. The antifungal Fluconazole was also tested against strains to determine the minimum inhibitory concentration by broth microdilution technique. Fluconazole has shown inhibitory activity between 1 and 32 µg/ml (Table  {\ref 1}). For bacterial strains of the genus Streptococcus, the essential oil of O. vulgare behaved similarly, varying the MIC between concentrations of 0.5 mg/mL and 0.250 mg/mL, as well as its isolated compound Carvacrol. The essential oil and Carvacrol inhibited the strain Staphylococcus aureus at concentrations of 0.5 and 0.250 mg/mL respectively. Chlorhexidine antimicrobial was also tested against strains to determine the minimum inhibitory concentration by broth microdilution technique. Chlorhexidine demonstrated inhibitory activity between 3.75 to 7 µg/ml (Table  {\ref 1}).After the determined the MIC values, used an aliquot of the susceptibility test to determine the minimum fungicidal/bactericidal concentration (MFC/MBC) of the strains. For Candida spp. Strains, O. vulgare essential oil, showed fungicidal activity against Candida spp. strains, varying its effect on concentrations between 0.5 mg/mL and 0.250 mg/mL; while Carvacrol showed 0.250 mg/mL fungicidal activity for all Candida strains tested (Table  {\ref 1}). Streptococcus bacterial strains showed a bactericidal concentration ranging from 0.5 mg/mL to 0.250 mg/mL for the essential oil and ranged from 0.5 mg/mL to 0.125 mg/mL for the isolated compound. S. aureus(MRSA) showed a bactericidal concentration of 0.5 mg/mL for both.\par
Table  {\ref 1}: Visual reading results of MIC and CFM/CBM of the strains tested. 
\section[{IV.}]{IV.} 
\section[{Discussion}]{Discussion}\par
Excessive and indiscriminate use of antimicrobials is a major determinant of some emerging infections, selection of resistant pathogens, and the continued development of antimicrobial resistance globally. The increasing emergence of multi-drug resistant organisms and the limited development of new agents available to combat them have caused an imminent crisis with alarming implications \hyperref[b13]{(Sartelli et al., 2016)}.In view of the increasing numbers of cases of conventional drug-resistant microorganisms, researchers are looking for alternatives to biocompounds that have antimicrobial properties against microorganisms. Studies with plants as promising agents in the search for new compounds.\par
In this study, the obtained data showed antimicrobial activity of the tested essential oil, as well as its isolated compound against planktonic cells of Candida spp., oral Streptococcus species, and S. aureus methicillin resistant strain. The essential oil inhibited antimicrobial growth between concentrations of 0.5 to 0.125mg/mL against all strains tested. At the same time, the isolated compound showed antimicrobial activity between concentrations of 0.250 to 0.0625mg/mL against all strains tested. The minimum fungicidal/bactericidal concentration (MFC/MBC) of the essential oil in the strains tested was between 0.5 mg / ml and 0.250 mg / ml. The isolated compound showed MFC/MBC between concentrations of0.5 mg/mL to 0.125 mg/mL (Table  {\ref 1}).\par
These data, initially reveal the antimicrobial action of this essential oil, as well as its isolated compound, corroborating with the literature, pointing out   
\section[{Reference strain}]{Reference strain} 
\section[{O. vulgare Carvacrol}]{O. vulgare Carvacrol}\par
The data obtained in this study added to the data in the recent literature, suggest that the essential oil of O. vulgare and also its isolated compound Carvacrol show antibacterial and antifungal potential.\par
These data open possibilities for many other studies, such as the performance of these oils and biocomponents in mature biofilms and multispecies biofilms, evaluating cell viability and possible morphological changes, added to cytotoxicity tests, action on cancer cells, in an attempt to add more information about this plant and its use as an alternative agent in the treatment of infections and acting as coadjuvants.\par
V. 
\section[{Conclusion}]{Conclusion}\par
? Both the essential oil of O. vulgare, and its isolated compound Carvacrol, can inhibit the growth of the tested microorganisms in low concentrations; ? The isolated compound is more effective when compared to the essential oil, inhibiting the microorganisms in a lower concentration; ? About the microorganisms, essential oil and Carvacrol are more effective against Candida spp. when compared to bacterial strains; ? Both the EO and its main biocomponent Carvacrol show fungicidal/bactericidal activity against the tested strains 		 		\backmatter  			  				\begin{bibitemlist}{1}
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\end{document}
