The essential oils were analyzed using a Shimadzu GCMS-QP2010 gas chromatograph mass spectrometer (Shimadzu Corporation Technologies, Inc., Tokyo, Japan) equipped with a split/splitless injector. The sample solution (1 µ l) was injected in the split mode at a ratio of 1:10. Analyses were performed with a Supelco Equity-5 capillary column (30 m x 0.2 mm i.d., 0.2 µ m film thickness, and stationary phase consisting of 5% diphenyl/95% dimethylpolysiloxane). The oven temperature program was as follows: held at 50 ° C for 2 minutes, increased from 50 to 220 ° C at a rate of 2 °C/min, and held at 220 °C for 3 minutes. Helium was used as carrier gas at a flow rate of 1.2 ml/min (constant linear velocity of 39.2 cm/sec). The injector temperature was set at 240 ° C. The interface and ion source temperatures were fixed at 220 and 200 °C, respectively. Quadrupole mass spectrometer was operated in electron impact mode at 70 e V, scanning the range m/z 35-350 in cycles of 0.5 s. The GCMS solution software was used for equipment management and data processing. Compounds were identified by searching the NIST 05 mass spectral library and by comparison of their retention indices relative to C7-C30 n-alkane series with those values found in an online database (El-Sayed, 2018).
Fall armyworm (FAW) (Spodoptera frugiperda) is a polyphagous insect responsible for damage to several crops. Synthetic chemical insecticides and genetically modified plants are the most commonly used methods for FAW control. However, the selection of resistant populations has been reported in several studies, justifying the search for new molecules to be used in the control of S. frugiperda. The aim of the present study was to evaluate the toxicity of lemongrass (Cymbopogonflexuosus) essentialoil (LEO) and its major component (citral) in relation to FAW. Additionally, the anticholinesterase activityof LEO and citral was evaluated using acetylcholinesterase (AChE) from Electrophorus electricus. The LEO was toxic to FAW when added to an artificial diet (LC 50 = 1.35 mg mL
of the problem might become more pronounced due to the proliferation of antibiotic-resistant strains (McAdam et al., 2012). Among the predominant bacteria involved in food-borne diseases, Staphylococcus aureus is a leading cause of gastroenteritis resulting from the consumption of a food in which enterotoxigenic staphylococci have grown and produced toxins. These are considered a potential biological threat because of their stability at high temperatures (100°C for 1 h). While not regarded as highly lethal agents due to the low mortality associated with the illness, staphylococcal enterotoxins can incapacitate individuals for two weeks (Schelin et al., 2011). Moreover it is predictable that S. aureus will continue to develop new virulence characteristics and new patterns of synthetic antibiotic resistance (Fry et al., 2013). A similar drug resistance phenomenon is supposedly occurring with S. epidermidis (Widerström et al., 2012). E. coli is reported to cause bloody diarrhea and can sometimes cause kidney failure and even death. K. pneumoniae is second only to E. coli as a urinary tract pathogen and cases of enteroinvasive Klebsiella pneumoniae and Escherichia coli sepsis from contaminated hamburgers are documented (Sabota et al., 1998; Kim et al., 2005). As in the case of bacteria previously mentioned, Klebsiella infections are encountered far more often now than in the past probably due to the emergence of increasingly resistant strains (Saha et al., 2008). Apart from bacteria, fungal contamination of food is practically inevitable. Candida as many other yeasts and molds is undesirable in many foods manly due to its food spoiling activity, nevertheless, dangerous levels of candida have been found in yoghurt (Montagna et al., 1998). Candida spp. is responsible for the most prevalent opportunistic fungal infections in humans andof particular significance in immunocompromised individuals (Muñoz et al., 2006; Rodloff et al., 2011).
9 2004; Rhoades et al., 2013). Several examples of this effective role of natural compounds against biofilms have already been reported. Husain et al. (2013) showed that the biofilm forming capability of A. hydrophila WAF-79 was considerably reduced by clove oil. The use of sanitizing detergents containing thyme (Thymus vulgaris) and lemongrass (Cymbopogon citratus) essential oils diminished the biofilm formed by A. hydrophila on stainless steel coupons (Millezi et al., 2013). Citrus vapour, a vaporized blend of citrus essential oils (orange:bergamot, 1:1 (v/v)), reduced surface contamination by VRE and methicilin-resistant Staphylococcus aureus (MRSA) and has a potential application in clinical settings (Laird et al., 2012). Other study showed that Myrcia ovata essentialoil was effective against E. faecalis biofilm after 5, 10 and 30 min of exposure (Cândido et al., 2010). Veras et al. (2014) reported in vitro enterococcal biofilm reduction with Lippia sidoides essentialoil, as a basis for the possible utilization as adjuvant in the treatment of root canals colonized by E. faecalis.
2011). In this regard, essential oils (alternative antimicrobial agents) have been showing potential to be used in fish production (Sutili et al. 2018). They are efficient, inexpensive, biodegradable and rapidly metabolized, with a low risk of accumulation in tissues (Teixeira et al. 2017, Da Cunha et al. 2018). Essential oils may affect the host and act directly on bacterial cells, causing changes in the morphology and lipid profile of bacterial cell membranes, increasing membrane permeability and leading to disruption with cytoplasmic leakage (Nazzaro et al. 2013). Essential oils can be used as nutritional additives in fish diets, showing promising results for health and welfare, such as increased resistance against bacterial infection (Zheng et al. 2009, De Souza et al. 2019b), improved activityof intestinal enzymes (De Souza et al. 2019a), intestinal microbiota (Mahmoud et al. 2004, Navarrete et al. 2010) and immunity (Sutili et al. 2016, Ghafari Farsani et al. 2018). In addition, analysis of biochemical, hematological and enzymatic variables can reveal physiological dysfunctions in fish (Hrubec & Smith 2001), helping in the diagnosis and prognosis of fish diseases.
Plant essential oils and their components have been known to exhibit biological activities, especially antimicrobial , antifungal [2,3], Antibacterial [4-6], Antimycotic  and antioxidant activities . Cymbopogoncitratusis aromatic spice, that belong to the family poaceae and is cultivated almost in all tropical and subtropical countries . Some studies proved that its essentialoil have antimalarial ,anti-Leishmaniase ,Antifungal , Antinociceptive, insecticidal [14,15] andantimicrobial [16 ] activities. Its major component is citral which can be used in hemi-synthesis reaction to give else components more active .
Since the induction of chemical defensives in agriculture, human beings have suffered from our own actions, caused by the indiscriminate and abusive use of those substances; therefore, researches have been motivated to look for alternative ways, aiming to use plant inputs to control pathogenic agents in agriculture. As the essential oils from the species Eucalyptus globulus (eucalyptus), Callis- temon viminalis (weeping bottlebrush), Cymbopogon winterianus (citronella grass) and Tetradenia riparia (misty plume bush) have proved effectiveness and an immense applicability, this research studied the use of those essential oils, aiming the effectiveness against plant pathogens. The plants were collected from Universidade do Vale do Sapucaí, Pouso Alegre (MG). The fungi’s samples belong to the mycology collection from the institution and the tests were based on the mycelial development comparison of the control fungi on the dishes with essential oils. Notice that the inhibition caused by the oils over fungi’s mycelial developing and the analysis of the data have been made through Turkey’s statistic. From the data analyzed, it was possible to realize that the citronella’s grass essentialoil was efficient to control the mycelial development of fungi analyzed, followed by, in order of efficiency, the eucalyptus oil, the weeping bottlebrush oiland the misty plume bush oil.
tion and the preformed biofilm was evaluated according to SLAVERS et al. (2016), with some modifications. Biofilm formation was quantified by crystal violet. Controls were prepared by replacing the inoculum volume by tryptic soy broth (TSB), andessentialoil by sterile water. Positive con- trol was carried out with the S. epidermidis strain ATCC 35984 and as negative control culture medium only, with- out inoculum. The tests were carried out in quadrupli- cate and the optical density measured in absorbance at 450 nm. It was considered a producer of biofilm samples, whose reading of the average for each strain was increased to the value of the cutting point (ODc), defined by the formula expressed on Equation 1:
Sample of the essentialoil was dissolved in 99.9% methanol (Merck) to final concentrations of 3, 5, 8 and 11 µg/mL. Higher concentrations were not tested because of formation of colloid system. Acetylcholinesterase inhibitory assays were performed by using colorimetric method as described previously (Georgiev et al., 2011a). For the assay 0.86 U AChE (type VI-S, Sigma) were dissolved in 1 mL 50 mM phosphate buffer (pH 8), supplied with 0.15 M NaCl (Sigma) and 0.05% (v/v) Tween 80 (Duchefa, The Netherlands). 20 µL of prepared enzyme solution were added into 2 mL 50 mM phosphate buffer (pH 8) mixed with β0 µL of methanol solution ofessentialoil with corresponding concentrations. The samples were incubated at 4ºC for β0 min in darkness, then the reaction was started by adding 20 µL 6 mM (in 50 mM phosphate buffer pH 7) acetylthiocholine iodide (Sigma) and β0 µL 5 mM (in 50 mM phosphate buffer pH 7) 5,5’-dithiobis(2-nitrobenzoic acid) (DTNB) (Sigma). Samples were vigorously mixed on vortex and incubated at γ7ºC for β0 min in darkness, then cooled down in ice. β0 µL of 1.8 mM (in 50 mM phosphate buffer pH 7) eserine salicylate (Sigma) was added to inactivate the enzyme. A blank sample (developed with pure methanol instead ofessentialoil solutions) was prepared as well. A positive controls (100% inhibition) were developed, following the same procedure, but the enzyme was fully inhibited by adding β0 µL eserine salicylate before substrate (acetylthiocholine iodide) addition. Changes in absorption at 405 nm of samples against their positive controls were measured (Shimadzu UV/Vis mini 1240, Japan) and used to calculate corresponding inhibition rates (IR, %) as described previously (Georgiev et al., 2011b). Because of inability to obtain miscible system with higher concentrations ofessentialoil, the half maximal inhibitory concentration (IC 50 )
The results obtained in this work demonstrate that Gram- positive bacteria tend to be more sensitive to the essential oils than Gram-negative ones. Some authors report that this is common for essential oils of plants of the Lamiaceae family (SHAPIro; MeIer; GUGGeNHeIM, 1994; HAMMer; CArSoN; rILeY, 1999). However, this relation should not be used to define the antimicrobialactivity, and thus each case should be carefully evaluated. When observing the results for S. officinalis 1, one can note that the Micrococcus luteus presented the highest MIC (6.93 mg.mL –1 ) although being a Gram-positive
Thirty sheep of both sexes between the ages of 10 to 16 months with an average weight of 22 kg were kept in paddocks and fed with fresh grass, salt-mineral supplement, and water ad libitum. Individual fecal samples were collected to determine the level of natural infection by gastrointestinal nematode using a modified McMaster technique (UENO & GONÇALVES, 1998). Sheep were divided into three homogeneous groups according to the fecal egg count (FEC). Each group (n = 10) received orally the following treatments for three days: G1 - 500 mg/kg CcEO; G2 - 450 mg/kg CcEOn; and G3 (control) - 3% tween 80 and 1% chitosan. The CcEOn dose was adjusted according to the volume ofessentialoil present in the nanoemulsion. The animals of G2 received the smallest dose because the nanoencapsulation may potentiate the anthelmintic effect of CcEO. For the fecal egg count reduction test (FECRT), samples from each animal were collected before and after 7 and 14 days of treatment to determine the FEC using the McMaster technique (UENO & GONÇALVES, 1998) and to perform fecal cultures according to Roberts & O’Sullivan (1950).
EO was tested for antibacterial activity using the macrobroth dilution method in broth media Mueller-Hinton (Difco). In these experiments, 0.4 ml of a suspension containing 1 x 10 6 CFU/ml was added to 3.6 ml of susceptibility test broth con- taining serial twofold dilutions of the EO in glass test tubes (13 by 100 mm) fitted with loose plastic nonscrew caps. All tubes were incubated in air at 37°C for 24 hr before being read. The MIC was considered the lowest concentration of the sample that prevented visible growth. Minimum bacteri- cidal concentrations (MBCs) were determined by subculturing, 10 µ l from each negative tube and from the positive growth control. MBCs were de- fined as the lowest concentration yielding nega- tive subcultures or only one colony. All samples were examined in duplicate in three separate ex- periments.
albicans, besides inhibiting the growth of the yeast (Manohar et al., 2001). Other studies to assess the inhibitory effect of these monoterpenes confirmed the inhibition of the growth of 25 microorganisms, including animal and plant pathogens, besides saprophytic fungi, such as Aspergillus flavus and the dermatophytous fungus Trichophyton rubrum (Adan et al., 1998; Dorman and Deans, 2000). The antimicrobial potential of more than 280 plant species has already been investigated against several species of Aspergillus, and approximately 100 of them showed some inhibitory activity over the production of mycotoxins and fungus growth. Thymus and Origanum, which possessed thymol and carvacrol, completely inhibited A. flavus, Penicillium digitatum, P. italicum, Botrytis cinerea and Alternaria citri, the parasites of seeds and fruits (Montes-Belmont and Carvajal, 1998; Arras and Usai, 2001). Thymol reduced the percentage of contamination by A. flavus to 43.7% and 75ppm of carvacrol were enough to reduce the A
Croton zehntneri (Euphorbiaceae) is a native aromatic plant from Northeast region of Brazil. The monoterpenoid estragole (ESL) has been isolated by classical chromatographic methods from the essentialoil (EO) of C. zehnteneri leaves and characterized by GC-FID and GC-MS, its antimicrobialand cytotoxic potentials being assessed. The analysis of the EO enabled the identification of 100% of the integrated constituents, of which yield was about 1.8%. The main components identified were: eucalyptol, estragole (84.7%) and spathulenol. The dosage of 50 μg/disk of ESL presented fairly significant zones of inhibition against Gram-positive bacteria and fungi. The ESL presented toxicity against Artemia salina with LC 50 and
Nowadays EOs became once again popular since the available synthetic drugs are often related with unpleasant side effects and drug resistance may occur (Lang & Buchbauer 2012; Bakkali et al. 2008). Additionally, research has been made in order to apply these substances in the agricultural domain. In fact, diseases caused by plant pathogenic bacteria are an emergent concern of food safety. These bacteria can cause not only considerable losses in productivity and quality of harvests but also harm to the ones who ingest infected products. The management of plant disease is difficult and the complexity increases due to the large number of phytopathogenic bacteria and their easiness to spread along large distances through infected seeds. Despite such problems in disease control, there is a lack ofantimicrobial agents likely to be applied in agriculture. The bactericides currently available in the market have high toxicity and are not biodegradable. The two main groups of bactericides existent are antibiotics and cooper compounds. Antibiotics are forbidden in most countries due to possible production of resistant strains. On the other hand, cooper agents are strictly controlled by the European Union due to their toxicity and environmental impact (Bajpai et al. 2011; Lo Cantore et al. 2009). Also, the green consumerism trends impel the development of new food products, especially the ones derived from plants since consumers prefer natural substance rather than synthetic ones (Lang & Buchbauer 2012; Lo Cantore et al. 2009).
Essential oils have the potential for use in the prevention and treatment of oral diseases. The antimicrobialactivityof Schinus molle L. essentialoil (SMEO) has already been reported. Chitosan, a natural product with antimicrobialactivityand good biocompatibility has potential in biodental applications. In this study, we evaluated the in vitro antimicrobialactivityof SMEO against bacteria associated with periodontal disease in dogs, developed and evaluated the physicochemical properties of a novel chitosan-based buccal delivery system containing SMEO. SMEO showed antimicrobialactivity against Gram positive and Gram negative bacteria associated with canine periodontitis, with MIC values of 750 µg.mL -1 for Staphylococcus spp. and Streptococcus
Biofilms are a big problem in food sectors such as dairy processing, fresh produce, poultry and red meat processing (Chen et al. 2007), and usually are associated with serious hygiene problems causing deterioration and recontamination of the product (Gram et al. 2007). Several reports describe the persistence of some foodborne pathogens affecting the quality and safety of food products. Most outbreaks whose etiologic agents are transmitted by food seems to be associated with biofilms. In recent years, there have been a significant number of reports correlating the persistence of foodborne pathogens on food contact surfaces and the occurrence of biofilms (Simões and Vieira 2009, Yaron and Römling 2015, Bridier et al. 2015). Enterococcus spp. and Staphylococcus spp. are Gram positive pathogens associated with foodborne outbreaks. These bacteria have virulence factors, such as, aggregation substances, extracellular surface proteins, and ability to form biofilms (Chapman 2003, Fisher and Phillips 2009, Marinho et al. 2013). The E. faecalis was responsible for an outbreak with lenezolid resistance (LRE) in a hospital, affecting 13 people between 2004 and 2005 in Tennessee, USA (Kainer et al. 2007). Resistant bacteria may be transferred to humans through the food chain, and colonize the gastrointestinal tracts and/or may be able to transfer resistance genes to the resident microbiota (Fisher and Phillips 2009, Frazzon et al. 2009, Cassenego et al. 2011).
Stem (1000 g), dried at room temperature, was triturated and subjected to cold extraction with ethanol for three days. The obtained solution was distilled under reduced pressure resulting in 105.5 g of the ethanolic extract. Then, the ethanol extract was adsorbed onto silica gel and subjected to the chromatographic column using increasing eluents: hexane, chloroform, ethyl acetate and methanol provide the following fractions: hexane (1.1 g), chloroform (19.6 g), ethyl acetate (17.5 g) and methanol (18.2 g). Chloroform fraction was subjected to silica gel column chromatography using the eluents in increasing order of polarity: hexane (100%), hexane/chloroform (9:1, 8:2, 7:3, 6:4, 1:1, 4:6, 3:7, 2:8, 1:9), chloroform (100%), chloroform/ etOAc (9:1, 8:2, 7:3, 6:4, 1:1, 4:6, 3:7, 2:8, 1:9), etOAc /methanol (9:1, 8:2, 7:3, 6:4, 1:1, 4:6, 3:7, 2:8, 1:9) and methanol (100%), resulting in 499 fractions with 5 ml each. Fractions (F’202- 277), resulted from extraction in chloroform/ethyl acetate 8:2, were grouped by similarity, yielding an off-white material of 20 mg that was purified and analyzed.
Several studies on the synergism among natural products were carried out (3, 7, 8, 35, 36). It is expected that mixtures of propolis and EOs may lead to a better inhibitory action than that achieved by the substance alone (37). Combinations of EOs from several plants were analyzed by Gutierrez et al. (38), in order to find successful interactions among them. These authors also reported that such combinations, especially when oregano was used, were able to potentiate antimicrobialactivity against Bacillus cereus, E. coli and Pseudomonas aeruginosa strains in food systems. Although the oils previously tested were not quite the same as those employed in our research, we aimed to verify the in vitro antimicrobialactivityof propolis (EEP) from Apis mellifera andessential oils from clove, ginger, cinnamon and peppermint on E. coli and S. aureus strains from human specimens. The minimal inhibitory concentration (MIC) was determined through the agar dilution method and the synergism among these natural products was assayed by the time-kill curve method.
content, and which metabolites are being generated. Besides nematocidal effect, plant extracts/compounds are tested for their ability to impair egg hatching and larval development from feces of infected animals treated with those plant extracts. Desired effects can result in reduced re-infection and lighter worm loads leading to decreased pasture contamination levels (Ketzis et al., 2002; Max, 2010). In vivo tests, problems with absorption through the gastrointestinal tract, and compound solubility and stability after oral intake are the main obstacles in devel- oping herbal formulations with good bioavailability and anthelmintic efficacy. According to Stepek et al. (2007), given the sensitivity to pH, it is not surprising that plant enzymes for instance have lower efficacy against stom- ach nematodes in situ than against those residing further down the gastrointestinal tract. It is necessary to evalu- ate additional parameters on ongoing research, such as performance measurements, indicators of immunity, and behavioral observations when considering the potential of such plants (Athanasiadou et al., 2007).