Foodborne pathogens are widely diverse in nature and continue to be the major cause of global public health problems in both developed and developing countries. Among these pathogens is Salmonella, responsible for costs with medical care, loss of productivity and expenses with control by the food industry and, mainly, problems for the poultry industry (MAJOWICZ et al., 2010).
The Enteric serotypes usually are the disease causative, such as aviary paratyphoid, with Salmonella enterica subsp. enterica ser. Enteritidis and S. Typhimurium, which stand out in this subgroup, because they are important cause of problems related to food infections in humans (OLIVEIRA et al., 2013), especially in regard to poultry originated products, since eggs and meat are the main transmission path of the disease (KOTTWITZ et al., 2008). In view of the above, natural products such as extracts and essential oils are an excellent resource for the search of new bioactive substances with biological potential, since they have a superior molecular diversity to synthetic products (SIQUEIRA et al., 2014).
The plant Zanthoxylum caribaeum Lam.belongs to the Rutaceae family, with more than 500 species it has worldwide distribution mainly in tropical and subtropical regions. Z. caribaeum is popularly known as espinho-preto (PIRANI& GROPPO, 2015) and has been widely used by the population for its anti-inflammatory properties (VILLALBA et al., 2007). It should be emphasized that there are few studies with this species and none of them are related to the biological action of it. However, according to Patino & Cuca (2011) Zanthoxylum spp. have such a broad ethnobotanical and biological importance, thus a promising source of substances with different biological activities.
Based on this context, the present study aimed at the chemical characterization of the essential oil and evaluation of the antibacterial activity of the oil and plant extracts of Z. caribaeum against different serotypes of Salmonella of poultry origin with higher incidence in the western region of Parana, Brazil.
Material and methods
The experimental analyses were carried out at the Agricultural Biotechnology Laboratory in West of Parana State University. The Leaves of Z. caribaeum were collected from January to April 2015, in the Paulo Gorski ecological park, situated in Cascavel city in the west of Parana state, Brazil (24° 57'51" S 53° 26'2"W). An exsicate of the plant was incorporated in the West of Parana State University Herbarium (UNOP) for botanical identification and registration of the voucher, under the number UNOP 1849.
For the essential oil extraction, 60 g of fresh Z. caribaeum plant material was added in 700 mL of distilled water and submitted to extraction by hydrodistillation for four hours using a Clevenger-type apparatus (SILVA et al., 2012). The essential oil collected was, dried over in hydrated sodium sulphate (~1 g), and stored under refrigeration and shelter from light at an average temperature of 4°C until analyzed.
The constituents of the essential oil were identified through gas chromatography coupled to mass spectrometry (GC-MS) and the identification of the compounds was performed by comparing their retention times with the retention times obtained in the literature (ADAMS, 2007).
To prepare the extracts, the leaves of Z. caribauem were dried and milled in knife mills according to Weber et al. (2014). For the preparation of the aqueous extract, 10 g of crushed plant material were added to 100 mL of distilled water. This mixture was kept on a rotary shaker at 220 rpm for 24 hours. After this period, the solution was filtered using Whatman no. 1 paper filter and centrifuged for 15 minutes at 5000 rpm. The supernatant was collected, thus obtaining the extract at the final concentration of 100 mg.mL-1. The extract was stored at 4 oC. The organic extracts were prepared according to Pandini et al. (2015) with modifications, ethanol (EtOH), methanol (MeOH), hexane (Hex), acetone (AcOH), dichloromethane (CH2Cl2) and ethyl acetate (EtOAc) were used as solvents.
Ten grams of ground vegetable material was added to 100 mL of the desired organic solvent, the solution then was maintained on a rotary shaker at 220 rpm for 24 hours. The solution was filtered using Whatman no. 1 paper filter and centrifuged for 15 minutes at 5000 rpm. After collection of the supernatant, the extracts were submitted to rotaevaporation. The crude extracts were diluted in 10 % dimethylsulfoxide (DMSO) until a final concentration of 400 mg.mL-1 and then stored at 4 oC.
To evaluate the antibacterial activity, 11 serotypes of Salmonella enterica subsp. enteric of greater occurrence in the western region of the State of Parana, Brazil: S. Enteritidis, S. Infantis, S. Typhimurium, S. Mbandaka, S. Orion, S. Shwarzengrund, S. Cubana, S. Montevideo, S. Senftenberg, S. Grumpensis e S. Tennessee provided by the veterinary laboratory MercoLab, Cascavel, Parana, Brazil.
The minimum inhibitory concentration (MIC) of the essential oil and plant extracts was determined according to the broth microdilution method proposed by Scur et al. (2014). For the tests, each bacterial suspension was standardized with 1 × 105 UFC.mL-1 concentration. Both the essential oil and the plant extracts were solubilized in methanol (100%) and Mueller-Hinton broth (MHB). For the oil, concentrations were tested ranging from 7000 to 1.68 μg.mL- 1 and for plant extracts, concentrations of 200 to 0.09 mg.mL-1. Microbial wells were reserved for control of broth sterility (MHB only), bacterial growth (bacterial suspension and MHB), reference antimicrobial action (bacterial suspension, MHB and gentamicin 200 mg.mL-1) and solvent (bacterial suspension, MHB and methanol). From the wells that showed no visible bacterial growth prior to the addition of TTC 10% (triphenyltetrazolium chloride) (SCUR et al., 2014), an aliquot of 2 μL was drawn out and inoculated on the surface of MH agar medium. The plaques were incubated for 24 hours at 36 ± 0.1°C. After this period, MBC was defined as the lowest concentration of essential oil capable of causing the death of the inoculum (PANDINI et al., 2015).
The MIC and MBC of the essential oil and the extracts were classified according to the criteria proposed by Sartoratto et al. (2004). For the essential oil, the activity was classified as high (between 50-500 μg.mL-1), moderate (between 600 a 1.500μg.mL-1), low (above 1.500 μg.mL-1). For the extracts, the classification was high (≤12.5 mg.mL-1), moderate (12. 5 a 25 mg.mL- 1), low (50 a 100 mg.mL-1) and very low (>100 mg.mL-1) Pandini et al. (2015).
Results and discussion
The essential oil output obtained through hydrodistillation was 1,24 % for Z. caribaeum. Of the chemical constituents detected in the leaves essential oil, 60,92 % was sesquiterpenes e 2,96 % monoterpenes, representing a total of 63,88 %, the majority compounds being Germacrene D (20,77 %), α-Panasinsene (14,40 %) and β-Selinene (11,68 %) (Table 1).
Table 1. Chemical composition of the essential oil of the leaves of Z. caribaeum obtained by hydrodistillation and analysed by GC-MS
Among the eleven serotypes of Salmonella test, five were susceptible to the leaves essential oil of Z. caribaeum in the tested concentrations (Table 2). The essential oil showed inhibition to the serotypes Mbandaka Shwarzengrund Cubana, Senftenberg e Enteritidis, while the best action was reported about the serotype Senftenberg with a MIC 437.5 μg.mL-1 and MBC 1750 μg.mL-1, so the activity was considered elevated, according to the criteria of Sartoratto et al. (2004).
Table 2. Minimum Inhibitory Concentration (MIC) e Minimum Bactericidal Concentrarion (MBC) of the leaves essential oil of Z. caribaeum against serotypes of Salmonella entérica
Nanasombat & Wimuttigosol (2011) also demonstrated antimicrobial potential within the genus Zanthoxylum, being the essential oil of the species Z. limonella showing a MIC of 20 mg.mL- 1 against the serotype S. Rissen. According to Gutierrez et al. (2008) this diversity of combinations of chemical constituents, presented by the essential oils, can manage to control bacteria that show consistently high resistance to antimicrobial agents, such as the case of the Salmonella genus.
The chemical profile of the oil revealed a high proportion of sesquiterpenes, especially hydrocarbonated sesquiterpenes. The essential oils, due to the lipophilicity of these substances of terpenic structure, allows the breaking of cell membrane lipids, thus increasing the permeability of the same (BAKKALI et al., 2008). This feature can increase the action of antimicrobials in cell interior, so justifying the activity encountered for the different serotypes of Salmonella.
However, the compounds that are found in lesser quantity, like in this case α- Felandrene e D-Limonene, can too contribute to the antimicrobial activity of the oils, it’s possible they are involved in a synergic action with other active compounds (FRUTUOSO et al., 2013).
The plant extracts that showed the best results in relation to de antimicrobial activity against the tested salmonellas were AcOH, AcEt e MeOH, because all of the eleven serotypes analysed showed themselves sensitive to them (Table 3). The aqueous extract showed bacteriostatic activity only for S.Mbandaka, while the Hex and CH2Cl2 extracts didn’t show inhibitory action, only against the serotype S. Infantis. However, EtOH extract presented antimicrobial activity to 81. 81 % (9/11) of the serotypes tested, with the exception of S. Tennessee and S.Enteritidis.
It was verified that, in general, AcEt extract presented the best values of MIC and MBC for the different serotypes of Salmonella which varied from 25 to 50 mg.mL-1, when compared to the other extracts, it’s activity being considered moderate to low. However, the serotypes Grumpensis e Tennessee (25 e 50 mg.mL- 1) presented the most sensitivity to MeOH extract.
In the literature, wasn’t found studies regarding the antimicrobial activity of the plant extracts of Z. caribaeum against the serotypes of Salmonella. However, Zhang et al. (2014) reported the antimicrobial potential within the genus against Gram-negative bacteria. According to the authors, ethanolic extract, ethyl acetate, acetone and methanolic of Zanthoxylum bungeanum showed MIC of 4.61 mg.mL- 1, 2.32 mg.mL-1, 5.7 mg.mL-1 and 3.1 mg.mL-1, respectively, against the strain E. coli, corroborating with our study.
Table 3. Minimum Inhibitory Concentration (MIC) e Minimum Bactericidal Concentration (MBC) of the different leaves extract of Z. caribaeum against the serotypes of Salmonella enteric
Among the extracts tested, the AcEt presented wide spectrum of antimicrobial action, showing activity for different serotypes of Salmonella. The AcEt is a solvent of medium polarity, a trait that enables the extraction of the following chemical classes: flavonoids, tannins, xanthenes, triterpenic acids, saponins and phenolic compounds in general (CECHINELFILHO & YUNES, 1998). The antimicrobial activities of some plant extracts are due to the high grade of phenolic compounds (AL-HABIB et al., 2010; KUMAR et al., 2011).
Still, it can be noticed that the usage of solvents with different polarities in the achievement of the extracts, promoted a differentiated antimicrobial activity, being that such activity might be related to the action of several phytochemical constituents of the plant. Therefore, raw extracts of vegetal species can. many times, present a greater antimicrobial action against pathogens, due to the synergism between the bioactive compounds that are extracted by the solvent or according to the method of extraction employed (LEE & LEE, 2010; DELGADO-ADAMEZ et al., 2012).
Besides that, the different susceptibility presented by the serotypes of Salmonella enterica is commonly reported in the literature, can be explained by the defense mechanisms present in this Gram-negative bacteria. This elevated genetic variability that genus Salmonella exhibit is resulting of a dynamic interaction between the pathogens, the environment and the different hosts (LIU et al., 2011; SCUR et al., 2016).
Lastly, it is concluded that the essential oil and the plant extracts of the leaves of Z. caribaeum present bioactive constituents with antimicrobial activity for different serotypes of Salmonella enterica, suggesting its future potentialities for the poultry sector, in search of safety and quality of the food consumed, as well as, new perspectives for studies with natural products in the industry.
This article was originally published in Revista Brasileira de Saúde e Produção Animal. vol.18 no.3 Salvador July/Sept. 2017. http://dx.doi.org/10.1590/s1519-99402017000300005. This is an Open Access article distributed under the terms of the Creative Commons Attribution License.