Universidade Federal de Santa Maria
Ci. e nat., Santa Maria, v. 42, Esp. Ed.: UNICEUMA, e9, 2020
DOI: 10.5902/217946041095
ISSN 2179-460X
Received: 03/12/2019 Accepted: 08/12/2019 Published: 09/10/2020
Environment
Antimicrobial and anti-biofilm activities of Alpinia zerumbet (Pers.) B.L. Burtt & R.M. Sm. essential oil against Corynebacterium ulcerans
Pâmela Ruth Santos VianaI
Denes Sousa LeiteII
Victor Elias Mouchrek FilhoIII
Dionney Andrade de SousaIV
Ana Luíza de Mattos-GuaraldiV
Márcia Barros AlvesVI
Wellyson da Cunha Araújo FirmoVII
Priscila Soares SabbadiniVIII
I Universidade CEUMA, São Luís, Brasil; e-mail: ruthvianna789@gmail.com;
II Universidade CEUMA, São Luís, Brasil; e-mail: denessousa27@gmail.com;
III Universidade Federal do Maranhão, São Luís, Brasil; e-mail: victor.mouchreck@ufma.br;
IV Universidade Federal do Maranhão, São Luís, Brasil; e-mail: dionney.andrade@ufma.br;
V Universidade do Estado do Rio de Janeiro, Rio de Janeiro, Brasil; e-mail: aguaraldi@gmail.com;
VI Universidade CEUMA, São Luís, Brasil; e-mail: marcia_barros16@hotmail.com;
VII Universidade CEUMA, São Luís, Brasil; e-mail: well_firmo@hotmail.com;
VIII Universidade CEUMA, São Luís, Brasil; e-mail: priscila.sabbadini@ceuma.br
ABSTRACT
Multiresistant strains of Corynebacterium ulcerans highlight the necessity of researches in new drugs and the pharmacological use of essential oils (EO) could be a therapeutic alternative. Thus, this study aimed to evaluate the antibacterial activity and the effect of subinhibitory concentration of AZEO on bacterial morphology and biofilm formation on polystyrene surface by C. ulcerans isolated in dogs. The AZEO was obtained by steam distillation from the leaves of the plant. The phosphomolybdenum and hemolysis inhibition tests were made to analyse, respectively, the antioxidant and toxicity activity. It was determined the minimum inhibitory and bactericide concentrations (MIC and MBC) of AZEO on C. ulcerans strains and from these results tests were made with subinhibitory concentrations of AZEO. The inactivation of phosphomolybdenum complex by AZEO was lower than 1% and the hemolytic capacity was low. The antimicrobial study results indicated that AZEO inhibited growth of both tested microbial strains. Bacterial filamentation was observed in AZEO presence, as well as bacterial autoaggregation. There was significant difference in biofilm inhibition. We suggest that A. zerumbet may represent an alternative therapy to control C. ulcerans-induced bacterial infections.
Keywords: Antioxidant; Filamentation; Autoaggregation
1 Introduction
The popular use of medicinal plants is fundamental to understand its therapeutic potential. Phytotherapics have its medicinal uses defended for centuries by positive results found in many diseases, though phytotherapics are few studied and have no its chemical components known. This fact awakens the interest of researches in studies that are related to plants pharmacology and phytochemical, improve the knowledges about natural medicine (FIRMO et al., 2011).
The pharmacological studies aim to certify the collateral effects of the products obtained from plants, relating them to tested doses in vitro and in vivo assays, as well as verifying the efficacy and the toxicity of herbs of popular and commercial use (LAPA et al., 1999). However, it is necessary the researches of biological activities of medicinal plants are increased in order to provide details that could serve as basis to obtain new scientific knowledges (SILVA; QUADROS; MARIA NETO, 2015).
Alpinia zerumbet (Pers.) B.L. Burtt & R.M. Sm., a plant transported from Asia to Brazil at the time of its discovery, belong to Zingiberaceae family and it is found in scientific literature as synonym of Alpinia speciosa K. Shum, Costus zerumbet Pers., Languas speciosa Small and Zerumbet speciosum J. C. Wendel (LORENGI; SOUZA, 2001). In southeast and northeast of Brazil, where it is commonly known as Colônia, the leaves of A. zerumbet are used in infusion as diuretic, antihypertensive and antiulcerogenic (MATOS, 1998). The antimicrobial activity proven to A. zerumbet essential oils (AZEO) varies according to its composition (WATTIEZ; STERNON, 1942).
In 1986, it was first described the Corynebacterium gender, a group of bacteria that presented similarity with gram-positive diphtheroid bacillus (LEHMANN; NEUMANN, 1986). The specie that stand out in this gender is Corynebacterium diphtheriae, etiologic agent of diphtheria (BURKOVSKI, 2014). However, the reports of respiratory diphtheria-like illness caused by toxigenic Corynebacterium ulcerans, mainly in Europe, that exceed the cases caused by C. diphtheriae, are increasing (HACKER et al., 2016; OTHIENO et al., 2019).
The history of human’s infections caused by C. ulcerans is related to raw milk consumption or the contact with domestic animals (HOGG et al., 2009). This pathogen is a commensal of domestic animals and wild animals, which could serve as reservoir to zoonotic transmission of the bacteria (DIAS et al., 2011; CONTZEN et al., 2011; SIMPSON-LOUREDO et al., 2014). It had already been described by ribotyping that the same strain of this specie was presented in both a diphtheria patient and her dog (LARTIGUE et al., 2005).
Mattos-Guaraldi et al (2008) described the first C. ulcerans strain producing a diphtheria-like toxin isolated from an elderly woman with a fatal pulmonary infection and a history of leg skin ulcers in the Rio de Janeiro, Brazil. In addition to diphtheria toxin, phospholipase D is considered a virulence factor of C. ulcerans. C. ulcerans showed affinity to fibrinogen, fibronectin and type I collagen (SIMPSON-LOUREDO et al., 2019) and moderate susceptibility to some antibiotics (MATTOS-GUARALDI et al., 2008; SIMPSON-LOUREDO et al., 2014).
Little information is available concerning the effects of plants on properties of Corynebacterium spp. Hence, the present study aimed to evaluate the antioxidant, antibacterial and antibiofilm activities of A. zerumbet on C. ulcerans isolated from dogs.
2 Materials and methods
2.1 Plant material and hydrodistillation procedure
The leaves of A. zerumbet were collected in the morning in São Luís, Brazil (2°33'01.7"S 44°13'32.9"W) in April 2017. The plant material was submitted to drying in ambient temperature and the essential oil (EO) was hydrodistillated using a Clevenger-type apparatus (ADAMS, 2007). Sterile dimethyl sulfoxide [DMSO; 1% in phosphate buffered saline (PBS)] was used as an oil solubilizer. Yield of the essential oil (%) was calculated on the basis of the moisture-free material.
2.2 Phosphomolybdenum complex assay
Spectrophotometric evaluation of antioxidant activity through the formation of a phosphomolybdenum complex was carried out according to Emerenciano et al. (2013). Sample solutions (0,4mL) were combined in an tube with 4mL of reagent solution (0.6 M sulfuric acid, 28 mM sodium phosphate and 4 mM ammonium molybdate). The tubes were capped and incubated at 95°C for 90 min. After the samples had cooled to room temperature, the absorbance of aqueous solutions of each was measured at 680nm against a blank. Ascorbic acid and AZEO were tested at 200μg/mL concentration. The antioxidant activity was expressed as the absorbance of the AZEO in relation to ascorbic acid, considering the value of its absorbance was 100% antioxidant action. This assay was performed in triplicate and repeated at least twice.
2.3. Hemolytic activity
The test was made with red blood cells obtained from a human voluntary and healthy (with no recent history of antibiotic therapy or with anti-inflammatory drugs and/or infectious diseases or inflammatory three weeks before the collect of the sample). The study was reviewed and approved by the Human Research Ethics Committee of the Universidade CEUMA, Brazil (CEP-UNICEUMA) under protocol number 1.732.522 and was performed in accordance with the Declaration of Helsinki 1975, as revised in 2008.
Hemolytic assays were performed according to the method previously described by Yang, Sun and Fang (2005) and Oliveira et al (2011), modified. Briefly, 0,5mL aliquots of human erythrocytes 1% in saline solution (0.9%) were added to 0.5mL of EO in concentrations ranging from 1000 to 50µg/mL. After preparation, the solutions were incubated for 60 minutes at 37 ºC and centrifuged at 3000rpm for 5 minutes. From the supernatant fluid, 100μL were transferred to a flat bottom microtiter plate and absorbance was measured using a spectrophotometer (550nm). To eliminate the EO inference in absorbance, control solutions (white) were prepared without addition of the red blood cells solution. Total and no hemolysis were achieved with Triton X-100 (1mg/mL) and saline plus red blood cell suspensions, respectively. The level of percentage hemolysis was calculated according to the following formula:
% Hemolisys= [(At–An) x 100]/(Ac–An)
Here: At is the absorbance of test sample.
An is absorbance of the negative control (saline).
Ac is the absorbance of the positive control (Triton X-100).
2.4 Antibacterial activity
2.4.1 Origin of bacterial strains and culture conditions
The microorganisms (Table 1) were kindly give by Laboratory of Diphtheria and Corynebacteria of Clinical Relevance/Universidade do Estado do Rio de Janeiro, Brazil, and are stocked in GC-glicerol 20% in Laboratory of Bacterial Respiratory and Systemic Diseases/Universidade CEUMA, Brazil.
To assays, the microorganisms were cultivated for 24-48hours/37°C in Trypticase Soy Broth (TSB, Difco), Trypticase Soy Agar (TSA, Difco) and/or sheep blood agar and tellurite chocolate agar medium (SABBADINI et al., 2010).
Table 1 – Origin of partially studied Corynebacterium ulcerans strains isolated from dogs in Pernambuco, Brazil
|
Strains |
Origin |
Isolation site |
tox gene* |
|
2649 |
Asymptomatic dog |
Nasopharynx |
-
|
|
2652 |
Asymptomatic dog |
Nasopharynx |
-
|
|
*, diphtheria toxin; -, negative
|
|||
2.4.2 Minimal Inhibitory and Bactericidal Concentrations
The antimicrobial activity of AZEO was determined by the microdilution. Briefly, each strain was grown on TSA plates at 37◦C for 48h, suspended in saline solution (∼1.5 × 108 CFU/mL) and diluted in TSB culture medium (ratio 1:10). For the determination of minimum inhibitory concentrations (MICs), aliquots of bacterial suspension were incubated in TSB containing EO at different concentrations (60–2.000µg/mL). Samples were then incubated for 48h at 37◦C. After the incubation period, 30μL of resazurin (0.03%) working solution was added to each well. The microtiter plates were then incubated again for 30 minutes at 37 °C. The results were reported visually. Color changes from blue to pink indicated reduction of resazurin which meant bacterial growth. The MIC value was determined as the lowest concentration of the test solution which prevented full color changes of resazurin. Serial dilutions of penicillin (0.015–256µg/mL) were used as positive controls. The effect of the addition of DMSO 1% as an oil solubilizer on MIC was investigated and it was found that this concentration did not interfere with bacterial growth.
For determining the minimum bactericidal concentrations (MBCs), just after the MIC experiments, the cultures were seeded on TSA medium and incubated for 48h at 37◦C. The MBC corresponded to the lowest concentration of the compound to which no viable bacteria was observed (SANTURIO et al., 2007). Experiments were performed twice in triplicate.
2.4.3 Bacterial morphology
The experimental model of bacterial morphology elaborated based on a system used by Gomes et al. (2013) with modifications. Microorganisms [Optical Density (OD) 0.4; 570nm] were grown in TSB medium with or without subinhibitory concentrations (subMIC) of the AZEO (MIC/2 and MIC/4). After 48 hours of incubation at 37ºC, all strains were observed after Gram staining by light microscopy.
2.4.4 Anti-biofilm activity
Biofilm formation was quantified according to the method previously described by Stepanovic et al. (2004). Briefly, aliquots of bacterial suspensions in TSB with OD570 0.2 were applied per well into 96 well cell culture plate with or without subMIC of the AZEO (MIC/2 and MIC/4). DMSO 1%-treated bacteria and TSB without bacteria were used as positive and negative controls, respectively. After 48 hours at 37ºC, the content of each well was aspirated and washed twice with PBS (pH 7.2, 0.01M). The remaining attached bacterial cells were fixed with 99% methanol and were stained with 2% crystal violet. The bound dye was then solubilized with 160µL 33% glacial acetic acid and absorbance was taken at 570 nm using an enzyme immunosorbent assay reader (THERMOLAB). Mean absorbance values of each sample were calculated and compared with the mean values of controls. Experiments were performed twice in triplicate.
2.5 Statistical Analyses
GraphPad Prism 6 program was used for data analyses. The statistical comparison between groups were analyzed using Turkey test. A p<0.05 was considered significant.
3 Results
3.1 A. zerumbet essential oil yield
The essential oil of A. zerumbet showed yield of 0.31%.
3.2 Evaluation of the antioxidant capacity
A low antioxidant activity of 0.51% was obtained for the AZEO as compared to ascorbic acid positive control after the phosphomolybdenum assay (Figure 1).
Figure 1 - Antioxidant activity of the essential oil of Alpinia zerumbet by the method of the phosphomolybdenum complex
3.3 Hemolytic activity
The hemolytic activity of the AZEO was investigated in vitro in human erythrocytes. The hemolytic percentage 12.61%, 13.24%, 13.55%, 14.88% and 15.14% were obtained for a dose of 50µg/mL, 100µg/mL, 200µg/mL, 500µg/mL and 1000µg/mL, respectively (Figure 2).
Figure 2 - Effect of essential oil of Alpinia zerumbet on hemolytic activity
3.4. A. zerumbet inhibits the growth of C. ulcerans
The antibacterial activity of AZEO was highest against C. ulcerans strain 2652 (MIC 250µg/mL) (Table 2). The MBC could not be determined with the concentrations tested.
Table 2- Minimum inhibitory and bactericidal concentrations of Alpinia zerumbet essential oil on Corynebacterium ulcerans
|
Strains |
MIC1 |
MBC2 |
|
|
AZEO3 |
Penicillin 0.03 |
AZEO |
|
|
2649 |
500 |
1 |
ND4 |
|
2652 |
250 |
1 |
ND |
|
1MIC, Minimum Inhibitory Concentration is expressed in µg/mL. 2MBC, Minimum Bactericidal Concentration. 3AZEO, Alpinia zerumbet essential oil. 4ND, Not determined with the concentrations evaluated. Penicillin was positive control. |
|||
3.5 A. zerumbet subMIC effect on bacterial morphology
C. ulcerans strains 2649 and 2652 showed an increase in bacterial autoaggregation after growth in the presence of subinhibitory concentrations (MIC/2 and MIC/4) of AZEO, however C. ulcerans strain 2652 showed a greater increase in aggregation (Figure 3).
Morphological changes suggesting bacterial filamentation were also observed in C. ulcerans strain 2652 (Figure 3). Microorganisms cultured in the presence of 1% DMSO did not present morphological alterations or increase in the bacterial grouping.
Figure
3 – Photomicrographs of Gram-stained cells of C. ulcerans strain 2652
grown in the absence (control) (A) or in the presence of MIC/4 of Alpinia
zerumbet essential oil. Arrow and arrow heads indicate bacterial filamentation
and grouping, respectively. Magnification x1000
3.6 A. zerumbet subMIC decreases biofilm formation
We attempted to analyze the effects of subMIC of AZEO (MIC/2 and MIC/4) on the ability of C. ulcerans strains 2649 and 2652 to form biofilm. As depicted on Figure 4, AZEO decreased biofilm formation by these bacteria at both tested concentrations. However, the correlation test showed significant difference (p<0.05) only for the C. ulcerans 2649 strain. It was observed that 1% DMSO did not influence in the biofilm formation in analyzed strains.
Figure 4 – Effect of Alpinia zerumbet essential oil (MIC/2 and MIC/4) on biofilm formation in Corynebacterium ulcerans strains. *p<0.05, compared with DMSO 1%-treated bacteria (Turkey test). Experiments were performed twice in triplicate. Each bar represents mean ± SD. The absorbance values were obtained in nm.
4 Discussion
It is necessary to understand the plant potential and their chemical components as agents to be used in the prevention and/or treatment of diseases, especially infectious. Thus, the development of studies with vegetal species is fundamental, particularly in Brazil, where the biodiversity is significant (RAMESHA et al., 2011).
Oil used in this study showed low yield (0.31%). According to Santos et al (2012), the lowest values of AZEO were observed in the morning, followed by an increase in the afternoon, reaching the maximum concentration at 14:33 h (0.48%). Essential oils generally reach their highest contents in the morning (MARCHESE; FIGUEIRA, 2005), however, with the advancement of the researches, different responses have been verified, depending on each species. Moreover, distillation time has been shown to have an influence on essential oil yield and composition of crops such as peppermint, lemongrass, palmarosa (CANNON et al., 2013) and lavender (ZHELJAZKOV et al., 2013).
Several studies have been presented about the antioxidant capacity of Brazilian plants (DE MORAIS et al., 2006; FIRMO et al., 2011; VIANA et al., 2017). Despite the low antioxidant activity found in AZEO in this study, evaluated by phosphomolybdenum complex methodology, Tu and Tawata (2015) mentioned a strong AZEO antioxidant potential on 2.2-diphenil-1-picrilhydrazyl free radicals. Methanolic extracts of A. zerumbet presented antioxidant activity equivalent to ascorbic acid (WONG; LIM; OMAR, 2009). Also, in Wong, Lim and Omar (2009) study, polyphenols were found in methanolic extracts of A. zerumbet. The polyphenols are antioxidants and act as donor of hydrogen and reducer of reactive oxygen species (BALASUNDRAM; SUNDRAM; SAMMAN, 2006).
According to Sucupira et al (2012), due to the diversity of methods with varied fundamentals and interferences, the comparison of the results of antioxidant activity becomes difficult. Moreover, the environmental conditions (light, humidity, temperature and predators) may represent an important differential factor in the production of secondary metabolites in the plant, which could directly reflect on the antioxidant capacity (GOBBO-NETO; LOPES, 2007). Kuraya et al. (2017) revealed that the antioxidant activity and yield of AZEO differed significantly between individual plants and collecting seasons.
The emergence of antimicrobial resistant microorganisms has increased the search for new therapeutic agents, encouraging the researches with medicinal plants, since many of them may have antimicrobial properties. However, it is fundamental medicinal plants and its extracts, or its essential oil be considered safe, without sensitizing manifestations that could endanger the user health. Lysis of erythrocytes cells is easily obtained by measuring the release of hemoglobin, constituting a good tool for toxicity studies. Determination of hemolysis activity is quick, reproducible, and inexpensive (OLIVEIRA et al, 2011; MAPFUNDE; SITHOLE; MUKANGANYAMA, 2016).
Here, the hemolytic effect of AZEO is seen to increase with the incresing concentrations of the oil. Thus the AZEO have shown dose dependent hemolytic activity. Ralph et al (2009) through testing for hemolytic activity rated the degree of in vitro toxicity according to the observed mortality rate: 0 to 9% = non-toxic, 10 to 49 % = slightly toxic, 50 to 89 % = toxic; 90 to 100 % = highly toxic. In this study, AZEO was reported to possess weakly hemolytic activity towards human erythrocytes.
The hemolytic activity of plants is related to their phytochemical composition (ZOHR; FAWZIA, 2014). Some studies have demonstrated the presence of saponin in the AZEO (COSTA et al., 2015). Saponins induce hemolysis through interaction with erythrocyte cell membrane components, causing membrane deformation and consequent disruption (ARABSKI et al., 2012; CARVALHO; OLIVEIRA, 2012).
In an in vitro model using human leukocytes, the AZEO did not induce genotoxicity at concentrations among 50-300µg/mL (CAVALCANTI et al., 2012). Acetone and methanolic extracts tested in carcinogenic cell lines did not presented no significant toxicity (CÔRREA; COSTA, 2008). The AZEO (400mg/kg) did not show a mutagenic profile in peripheral blood cells and bone marrow in mice (CAVALCANTI et al., 2012).
The EO obtained from A. zerumbet rhizome showed antibacterial activity on several Gram positive and negative species as: Streptococcus mutans, Staphylococcus aureus, Micrococcus luteus, Salmonella tiphy and Pseudomonas aeruginosa (INDRAYAN; TYAGI; AGRAWAL, 2010). The chloroform extracts of A. galanga was identified with the MIC and MBC values against S. aureus at 128 and 256µg/mL, respectively (VORAVUTHIKUNCHAI; PHONGPAICHIT; SUBHADHIRASAKUL, 2005).
We have also demonstrated that AZEO was active against both strains of C. ulcerans. MBC could not be defined with the concentrations evaluated. However, it was required a lower AZEO concentration to inhibit C. ulcerans strain 2652 (MIC value of 250µg/mL) in comparison with strain 2649 (MIC value of 500µg/mL). The MIC value of penicillin (1µg/mL) was much lower in comparison to MICs of AZEO for the test organisms. From the study it was clearly observed that AZEO have anti bacterial effect but is not potent like antibiotic penicillin.
Various in vitro experiments have established the fact that a combination of plants and antibiotics possess a synergistic effect, which results in a significant decrease in levels of MIC for the antibiotics. The combination may be helpful in the preclusion of the emergence of resistant bacteria and reducing the drug toxicity (CRISTO et al., 2016; RAFIQ et al., 2017).
Studies using low concentrations of antimicrobial compounds have shown that, besides these concentrations did not kill the bacteria, they can modify the physical or chemical structure of their cell wall, which may impact, for example, on motility, adhesion and toxin production (SUBRT; MESAK; DAVIES, 2011; GOMES et al, 2013).
Here, the growth of C. ulcerans strain 2652 in the presence of subMIC of AZEO induced morphological alterations which suggest filamentation. Sutton et al. (2011) reported that subMIC of penicillin generated during treatment of C. diphtheriae infections may result in difficulties in diagnosis and/or treatment, as antimicrobial-mediated bacterial filamentation might resemble fungal hyphae, especially in body fluids where the antibiotics may reach sublethal concentrations. This misinterpretation during clinical diagnostic procedures may lead to inappropriate treatment.
We observed an increased in bacterial aggregation, especially for C. ulcerans strain 2652, after growth with AZEO subMIC. Phytochemical screening showed the presence of tannin in A. zerumbet (AL-ENAZI, 2018). Tannins have great ability to interact with proteins and other macromolecules, which give them the capacity to interconnect different structures (CARVALHO; OLIVEIRA, 2012). Moreover, Gomes et al (2013) reported that subinhibitory concentrations of antimicrobial agents enhanced cell-surface hydrophobicity of C. diphtheriae.
AZEO may present terpenes in its constitution (OLIVEIRA et al., 2017). According Dalleau et al. (2008), terpenes are substances that penetrate in lipid layers and that can alter cellular permeability and membrane fluidity, suggesting that these alterations cause superficial and morphological changes, reducing the cellular adherence and impacting in biofilm formation. In the present study, it was proved that AZEO subMIC inhibited the biofilm production on polystyrene surface for both C. ulcerans strains, although a greater bacterial aggregation was observed. Subinhibitory concentrations of crude hydroalcoholic extracts of Pisidium guajava, Tithonia diversifolia e Anacardium occidentale were also capable to significantly inhibit the biofilm production on polystyrene by C. ulcerans (FIRMO et al., 2018). C. diphtheriae strains showed an increase in biofilm production on polystyrene in the presence of subinhibitory concentrations of erythromycin (GOMES et al, 2013).
5 Conclusions
A. zerumbet essential oil demonstrated antibacterial properties as the capacity of promoting morphological alterations and reducing the biofilm production. Moreover, AZEO showed low toxicity proving to be safe for human use. The results strengthen the hypothesis of its potential to pharmacological use, since the plant has now been used for several diseases, as well as demonstrate the possibility of its use in the therapy of infections caused by C. ulcerans. In this regard active ingredient present in essential oil extracted from the leaves of A. zerumbet needs to be separated and purified for further study.
Acknowledgment
The authors thank Fundação de Amparo à Pesquisa do Estado do Maranhão – FAPEMA for grant.
References
ADAMS, RP. Identification of essential oils components by gas chromatography/mass spectroscopy. 4 st ed. Carol Stream: Allured Publishing Corporation; 2007.
AL-ENAZI NM. Phytochemical screening and biological activities of some species of Alpinia and Convolvulus plants. Int J Pharmacol. 2018;14(3):301-309.
ARABSKI M, WĘGIEREK-CIUK A, CZERWONKA G, LANKOFF A, KACA W. Effects of saponins against clinical E. coli strains and eukaryotic cell line. BioMed Res. Int. 2012;2012.
BALASUNDRAM N, SUNDRAM K, SAMMAN S. Phenolic compounds in plants and agri-industrial by-products: Antioxidant activity, occurrence, and potential uses. Food. Chem. 2006;99(1):191-203.
BURKOVSKI A. Diphtheria and its etiological agents. In: Corynebacterium diphtheriae and related toxigenic species. Springer. 2014;1-14.
CANNON JB, CANTRELL CL, ASTATKIE T, ZHELJAZKOV VD. Modification of yield and composition of essential oils by distillation time. Ind. Crop. Prod. 2013;41:214-220.
CARVALHO MS, OLIVEIRA DA. Estudo da atividade citotóxica de Myracrodrun urundeuva FR. Allemao. Revista Eletrônica de Biologia (REB). 2012;5(3):1-7.
CAVALCANTI BC, FERREIRA JR, CABRAL IO, MAGALHÃES HI, OLIVEIRA CC, RODRIGUES FA et al. Genetic toxicology evaluation of essential oil of Alpinia zerumbet and its chemoprotective effects against H2O2-induced DNA damage in cultured human leukocytes. Food. Chem. Toxicol. 2012;50(11):4051-4061.
CONTZEN M, STING R, BLAZEY B, RAU J. Corynebacterium ulcerans from diseased wild boars. Zoonoses Public Hlth. 2011;58(7):479-488.
CORRÊA AJC, COSTA MCCD. Estudo da atividade antimicrobiana e citotóxica de extratos brutos de Alpinia speciosa K. Shum. Jornada de iniciação científica; 25-29 de agosto de 2008; Recife;BR. Universidade Católica de Pernambuco;2008.
COSTA GMM, SUGA RG, OLIVEIRA PL, MAGALHÃES PP, FARIAS LM, DUARTE LP et al. Bioactivity of extracts from Alpinia zerumbet against sinusitis-causing bacterial pathogens. Fitos. 2015;9(3):161-252.
CRISTO JS, MATIAS EFF, FIGUEREDO FG, SANTOS JFS, PEREIRA NLF, JUNIOR JGAS et al. HPLC profile and antibiotic-modifying activity of Azadirachta indica A. Juss (Meliaceae). Ind. Crops. Prod. 2016;94903–908.
DALLEAU S, CATEAU E, BERGÈS T, BERJEAUD JM, IMBERT C. In vitro activity of terpenes against Candida biofilms. Int. J. Antimicrob. Agents. 2008:31(6):572-576.
DE MORAIS SM, CATUNDA FEA, DA SILVA ARA, NETO JM, RONDINA D, CARDOSO JHL. Atividade antioxidante de óleos essenciais de espécies de Croton do nordeste do Brasil. Quím. Nova. 2006;29(5):907.
DIAS AASO, SANTOS LS, SABBADINI PS, SANTOS CS, SILVA JUNIOR FC, NAPOLEÃO F et al. Difteria pelo Corynebacterium ulcerans: uma zoonose emergente no Brasil e no mundo. Rev. Saude Publ. 2011;45:1176-1191.
EMERENCIANO DP, CRUZ AMF, PEREIRA JDS, MOURA MFV, MACIEL MAM. Determinação da propriedade antioxidante e teores de minerais presentes nas folhas de Azadirachta indica A. Juss. Fitos. 2013;8:73-160.
FIRMO WCA et al. Bioprospecção de plantas medicinais do cerrado maranhense com propriedades antioxidante, antibacteriana contra Corynebacterium spp e toxicidade: aporte para o desenvolvimento de novos medicamentos[thesis]. São Luís: Universidade Federal do Maranhão/UFMA; 2018.
FIRMO WCA, MENEZES VJM, PASSOS CEC, DIAS CN, ALVES LPL, DIAS ICL et al. Contexto histórico, uso popular e concepção científica sobre plantas medicinais. Cadernos de pesquisa. 2011;18:90-95.
GOBBO-NETO L, LOPES NP. Plantas medicinais: fatores de influência no conteúdo de metabólitos secundários. Quím. Nova. 2007;30(2):374-381.
GOMES DLR, PEIXOTO RS, BARBOSA EAB, NAPOLEÃO F, SABBADINI PS, SANTOS KRN et al. SubMICs of penicillin and erythromycin enhance biofilm formation and hydrophobicity of Corynebacterium diphtheriae strains. J. Med. Microbiol. 2013;62:750-760.
HACKER EA, MATTOS-GUARALDI CA, BURKOVSKI AA, TAUCH A. Corynebacterium ulcerans, an emerging human pathogen. Future Microbiol. 2016;11(9):1191-1208.
HOGG RA, WESSELS J, HART J, EFSTRATIOU A, DE ZOYSA A, MANN G et al. Possible zoonotic transmission of toxigenic Corynebacterium ulcerans from companion animals in a human case of fatal diphtheria. Vet. Rec. 2009;165:691-692.
INDRAYAN AK, TYAGI PK, AGRAWAL NK. Chemical composition and antimicrobial activity of the essential oil of Alpinia speciosa K. Schum. rhizome from India. J Essent Oil Res. 2010;22(2):179-182.
KURAYA E, YAMASHIRO R, TOUYAMA A, NAKADA S, WATANABE K, IGUCHI A et al. Aroma Profile and Antioxidant Activity of Essential Oil from Alpinia zerumbet. Nat Prod Commun. 2017;12(8):1321-1325.
LAPA AJ, SOUCCAR C, LIMA-LANDMAN MTR, GODINHO RO, LIMA TCM. Farmacologia e toxicologia de produtos naturais. In: Simões CM, Schenkel EP, Gosmann G, Mello JCP, Mentz L A, Petrovick PR, editors. Farmacognosia: da planta ao medicamento. Porto Alegre: UFRGS; 1999;181-95.
LARTIGUE MF, MONNET X, FLECHE A, GRIMONT PA, BENET JJ, DURRBACH A et al. Corynebacterium ulcerans in an immunocompromised patient with diphtheria and her dog. J. Clin. Microbiol. 2005;43(2):999-1001.
LEHMANN KB, NEUMANN RO. Atlas und Grundriss der Bakteriologie und Lehrbuch der speziellen bakteriologischen Diagnostik. 1896
LORENGI H, SOUZA HM. Plantas ornamentais no Brasil: arbustivas, herbáceas e trepadeiras. 3 st ed. Nova Odessa: Plantarum;2001.
MAPFUNDE S, SITHOLE S, MUKANGANYAMA S. In vitro toxicity determination of antifungal constituents from Combretum zeyheri. BMC Complement Altern Med. 2016;16(1):162.
MARCHESE JA, FIGUEIRA GM. O uso de tecnologia de pré e pós-colheita e boas práticas agrícolas na produção de plantas medicinais e aromáticas. Rev. Bras. Plantas Med. 2005;7:86-96.
MATOS FJA. Farmácias vivas. 2 st ed. Fortaleza: UFC; 1998.
MATTOS-GUARALDI AL, SAMPAIO JLM, SANTOS CS, PIMENTA FP, PEREIRA GA, PACHECO LGC et al. First detection of Corynebacterium ulcerans producing a diphtheria-like toxin in a case of human with pulmonary infection in the Rio de Janeiro metropolitan area, Brazil. Mem. Inst. Oswaldo Cruz. 2008;103:396-400.
OLIVEIRA DR, COSTA ALM, LEITÃO GG, CASTRO JPS, LEITÃO SG. Estudo etnofarmacognóstico da Saracumirá (Ampelozizyphus amazonicus Ducke), uma planta medicinal usada por comunidades quilombolas do Município de Oriximiná-PA, Brasil. Acta Amaz. 2011;41(3):383- 392.
OLIVEIRA SM, ALMEIDA FR, BOTELHO PDA, PESSOA CV, COSTA FND. Desenvolvimento farmacotécnico de um elixir de colônia (Alpinia zerumbet) [Internet]; 2017 Dezembro 06; Ceará, Brasil. 2017 [cited 2019 aug 02]. Available from: http://201.20.115.105/home/bitstream/123456789/1154/1/1991-3982-1-PB.pdf
OTHIENO R, MARK K, ETHERSON M, FOSTER G, MURRAY S, KALIMA P et al. A case of cutaneous toxigenic Corynebacterium ulcerans likely acquired from a domestic dog. Access Microbiology. 2019.
RAFIQ Z, NARASIMHAN S, HARIDOSS M, VENNILA R, VAIDYANATHAN R. Punica granatum rind extract: Antibiotic potentiator and e ux pump inhibitor of multidrug-resistant Klebsiella pneumoniae clinical isolates. Asian. J. Pharm. Clin. Res. 2017;10:1–5.
RALPH ACL, FERREIRA SB, FERREIRA VF, LIMA ES, VASCONCELLOS MC. Avaliação da citotoxicidade de naftoquinonas sintéticas em modelo de Artemia franciscana e eritrócitos. 61ª Reunião Anual da SBPC; 2009 July 12-17; Manaus; Brasil;2009.
RAMESHA BT, GERTSCH J, RAVIKANTH G, PRITI V, GANESHAIAH KN, UMA SHAANKER R. Biodiversity and chemodiversity: future perspectives in bioprospecting. Curr. Drug Targets. 2011;12(11):1515-30.
SABBADINI PS, GENOVEZ MRN, SILVA CFD, ADELINO TLN, SANTOS CSD, PEREIRA GA et al. Fibrinogen binds to nontoxigenic and toxigenic Corynebacterium diphtheriae strains. Mem. Inst. Oswaldo Cruz. 2010;105(5):706-711.
SANTOS MS, JEZLER CN, OLIVEIRA ARMF, OLIVEIRA RA, MIELKE MS, COSTA LCB. Harvest time and plant age on the content and chemical composition of the essential oil of Alpinia zerumbet. Hort. Bras. 2012;30:385-390.
Santurio JM, Santurio DF, Pozzatti P, Moraes C, Franchin PR, Alves SH et al. Atividade antimicrobiana dos óleos essenciais de orégano, tomilho e canela frente a sorovares e Salmonella enterica de origem avícola. Cienc. Rural. 2007;37:803-808.
SILVA LE, QUADROS DA, MARIA NETO AJ. Estudo etnobotânico e etnofarmacológico de plantas medicinais utilizadas na região de Matinhos-PR. Ciênc. Nat. 2015;37(2):266-276.
SIMPSON-LOUREDO L, RAMOS JN, PEIXOTO RS, SANTOS LS, ANTUNES CA, LADEIRA EM et al. Corynebacterium ulcerans isolates from humans and dogs: fibrinogen, fibronectin and collagen-binding, antimicrobial and PFGE profiles. Antonie Van Leeuwenhoek. 2014;105(2):343-352.
SIMPSON-LOUREDO L, SILVA CM, HACKER E, SOUZA NF, SANTANA MM, ANTUNES CA et al. Detection and virulence potential of a phospholipase D-negative Corynebacterium ulcerans from a concurrent diphtheria and infectious mononucleosis case. Antonie van Leeuwenhoek. 2019:1-11.
STEPANOVIĆ S, ĆIRKOVIĆ I, RANIN L, SVABIĆ VLAHOVIĆ M. Biofilm formation by Salmonella spp. and Listeria monocytogenes on plastic surface. Lett. Appl. Microbiol. 2004;38(5):428-432.
SUBRT N, MESAK LR, DAVIES J. Modulation of virulence gene expression by cell wall active antibiotics in Staphylococcus aureus. J. Antimicrob. Chemother. 2011;66(5):979-984.
SUCUPIRA NR, SILVA AB, PEREIRA G, COSTA JN. Methods for measuring antioxidant activity of fruits. UNOPAR Cient., Ciênc. Biol. Saúde. 2012;14(4):263-269.
SUTTON B J, PARSONS AC, PALAVECINO EL. Filamentous bacteria masquerading as fungi: a diagnostic pitfall in direct smear interpretation with report of two cases. Am. J. Clin. Pathol.2011; 64(10): 927-929.
TU P, TAWATA S. Anti-oxidant, anti-aging, and anti-melanogenic properties of the essential oils from two varieties of Alpinia zerumbet. Molecules. 2015;20(9):16723-16740.
VIANA PRSV, LEITE DS, MATTOS-GUARALDI AL, FIRMO WCA, SABBADINI PS. Potencial Antioxidante e Antibacteriano contra Corynebacterium ulcerans do óleo essencial de canela. CEUMA Perspectivas. 2017; 30(2):115-123.
VORAVUTHIKUNCHAI SP, PHONGPAICHIT S, SUBHADHIRASAKUL S. Evaluation of antibacterial activities of medicinal plants widely used among AIDS patients in Thailand. Pharm. Biol. 2005;43(8):701-706.
WATTIEZ N, STERNON F. Élements de chimie végétale. 2 st ed. Paris: Masson et Cie;1942.
WONG LF, LIM YY, OMAR M. Antioxidant and antimicrobial activities of some Alpina species. J. Food Biochem. 2009;33(6):835-851.
YANG ZG, SUN HX, FANG WH. Haemolytic activities and adjuvant effect of Astragalus membranaceus saponins (AMS) on the immune responses to ovalbumin in mice. Vaccine. 2005;23(44):5196-5203.
ZHELJAZKOV VD, CANTRELL CL, ASTATKIE T, JELIAZKOVA E. Distillation time effect on lavender essential oil yield and composition. J. Oleo Sci. 2013;62(4): 195-199.
ZOHRA, Mohammedi; FAWZIA, Atik. Hemolytic activity of different herbal extracts used in Algeria. Int J Pharm. Sci. Research. 2014;5:495-500.