Microsoft Word - 30-Bio_41848 606 Bioscience Journal Original Article Biosci. J., Uberlândia, v. 36, n. 2, p. 606-618, Mar./Apr. 2020 http://dx.doi.org/10.14393/BJ-v36n2a2020-41848 STUDY AND EVALUATION OF ANTIMICROBIAL ACTIVITY AND ANTIOXIDANT CAPACITY OF DRY EXTRACT AND FRACTIONS OF LEAVES OF Raphanus sativus var. oleiferus Metzg. ESTUDO QUÍMICO E AVALIAÇÃO DA ATIVIDADE ANTIMICROBIANA E DA CAPACIDADE ANTIOXIDANTE DO EXTRATO SECO E FRAÇÕES DE FOLHAS DE Raphanus sativus var. oleiferus Metzg. Ana Flavia da SILVA1, *; Marisa de Oliveira LOPES1; Claudio Daniel CERDEIRA1; Ingridy Simone RIBEIRO2; Isael Aparecido ROSA4; Jorge Kleber CHAVASCO2; Marcelo Aparecido da SILVA1; Marcos José MARQUES3; Geraldo Alves da SILVA1 1. Department of Food and Drugs, School of Pharmaceutical Sciences, Federal University of Alfenas, UNIFAL, Alfenas, Minas Gerais, Brazil. afsfarma@hotmail.com*; 2. Department of Microbiology and Immunology and Pathology, Institute of Biomedical Sciences, Federal University of Alfenas, UNIFAL, Alfenas, Minas Gerais, Brazil; 4. Department of Chemistry, Federal University of Lavras, UFLA, Lavras, Minas Gerais, Brazil. ABSTRACT: The radish (Raphanus sativus L.) is a vegetable of the Brassicaceae family cultivated worldwide and has several medicinal properties. Its biological activities are related to various secondary metabolites present in the species, especially phenolics. Thus, the objectives of this study were the chemical analysis and evaluation of the antioxidant and antimicrobial activities of the dry extract and fractions of the fodder turnip leaves (R. sativus var. oleiferus Metzg.). Samples were analyzed by mass spectrometry and the antioxidant activity was evaluated using the 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical method and the reducing power method. Antimicrobial activity was determined by the agar diffusion and microdilution methods. The total phenols were concentrated in the butanol fraction (121.27 mg GAE/g) and the flavonoids were concentrated in the ethyl acetate fraction (98.02 mg EQ/g). The ethyl acetate fraction showed the best antioxidants results, with 83.45% of free radical scavenging and 11.34% of ferric ions reduction. The analysis of antimicrobial activity showed that the dry extract had the highest average zone of inhibition against Bacillus subtilis (18.67 mm). Smaller values of the minimum inhibitory concentration for Micrococcus luteus were, and the ethyl acetate fraction showed a lower minimum inhibitory concentration (0.1 mg/ml) for that microorganism. There was a strong correlation between the antioxidant activity and the content of phenols and flavonoids. The results showed the potential antioxidant and antimicrobial activities of this extract with the ethyl acetate fraction being most promising for further studies. KEYWORDS: Fodder turnip. Phenolic compounds. Brassicaceae. Medicinal plants. INTRODUCTION The use of plants for medicinal purposes is an old practice and is associated with popular knowledge from different parts of the world (VEIGA; PINTO; MACIEL, 2005). The study of the chemical composition of plants indicates their potential biological properties and can encompass both the therapeutic and toxicological properties (SOUZA-MOREIRA; SALGADO; PIETRO, 2010). The discovery of new plant products can significantly contribute to global health, as well as providing alternative therapeutic approaches. There is also the possibility of isolating substances with greater efficacy, lower cost, lower toxicity, milder side effects and greater availability of the raw material (MENEZES et al., 2009). The Brassicaceae family has about 350 genera and 4000 species, originating in the northern hemisphere and distributed in temperate zones around the New World (ZECCA, 2008). Several horticultural varieties are found, especially Brassica oleracea var. acephala (kale), B. oleracea var. capitata (cabbage), B. oleracea var. borytis (cauliflower), B. oleracea var. italica (broccoli), B. nigra and Sinapis sp. (mustard), Raphanus sativus L. var. radicle (radish) and Eruca sativa (arugula) (SOUZA; LORENZI, 2005). The common radish (R. sativus L.) is a vegetable root crop with numerous varieties, among them: radicle, niger, mougri and oleifera (GUTIERREZ; PEREZ, 2004; ARUNA; YERRAGUNI; RAJU, 2012). Different parts of the plant (leaves, seeds and roots) are used in folk medicine for gastrointestinal disorders and biliary, liver, urinary, respiratory and cardiovascular ailments (DEVARAJ; KRISHNA; VISWANATHA, 2011; SHIN et al., 2015). In addition, the plant Received: 06/05/18 Accepted: 20/11/19 607 Study and evaluation… SILVA, A. F. et al. Biosci. J., Uberlândia, v. 36, n. 2, p. 606-618, Mar./Apr. 2020 http://dx.doi.org/10.14393/BJ-v36n2a2020-41848 contains bioactives with anti-inflammatory (PARK; SONG, 2017; MANIVANNAN et al., 2019), antimicrobiana, antioxidante, antimutagênica (GUTIERREZ and PEREZ, 2004; ALQASOUMI; AI-HOWIRINY; RAFATULLAH, 2008; SHIN et al., 2015; LUO et al., 2018; MANIVANNAN et al., 2019), antiviral (GUTIERREZ; PEREZ, 2004; SHIN et al, 2015; PARK et al., 2017) e antidiabética (BANIHANI, 2017; MANIVANNAN et al., 2019) properties. The major secondary metabolites of the species are polyphenols, flavonoids, alkaloids, tannins, volatile oils and glycosinolates (ARUNA; YERRAGUNI; RAJU, 2012). However the flavonoids are undoubtedly are the largest group of bioactive chemicals (JAHANGIR et al., 2009). R. sativus var. oleiferus Metzg., popularly known as fodder turnip, is a variety of radish little studied from the chemical and biological point of view. Therefore, this study aimed to perform chemical analysis and to evaluate the antioxidant and antimicrobial potential of fodder turnip, in order to correlate the main chemical constituents detected with the biological activities. MATERIAL AND METHODS Plant material The leaves of fodder turnip were collected in June 2013, on a plot situated at Avenida Afonso Pena, the city of Alfenas (latitude 21°25'22.87"S, longitude 45°57'29.42"W), Minas Gerais, Brazil. The climate of the site at the time was cold and dry, with an average temperature of 14 °C. The identity of the plant was confirmed by Prof. Dr. Geraldo Alves da Silva. A voucher specimen of the plant material was deposited in the Herbarium of the Federal University of Alfenas and registered under number 2279. Preparation of the dry extract and fractions The leaves were dried in an oven with air circulation at 45 °C for 72 hours and then pulverized. Three hundred grams of plant material was submitted to the percolation method (PRISTA; ALVES; MORGADO, 1992) using a mixture of ethanol: water (7:3, v/v) as liquid extactor. Subsequently, the extract obtained was concentrated on a rotaevaporator and finally dried by lyophilization, with a final yield of 29.198%. Ten grams of the dry extract was solubilized in water and subjected to a process of liquid-liquid extraction with increasingly polar solvents. After extraction and removal of the organic solvent the hexane (FrHex), ethyl acetate (FrAcOEt), butanol (FrBuOH) and aqueous (FrAq) fractions were obtained. The yields from each fraction compared to the initial amount of dry extract were 10.26%, 5.88%, 30.40% and 22.12%, respectively. Analysis by mass spectrometry with electrospray ionization (ESI-MS) Mass spectra were obtained using an LTQ XL Linear Thermo Scientific 2D mass spectrometer, equipped with direct insertion of the sample via continuous flow injection (FIA). The extract and the fractions were analyzed in electrospray ionization mode (ESI) with fragmentation in multiple stages held in an ion trap type interface (IT). Negative mode was selected for the generation and analysis of the first-order mass spectra (MS) as well as for other experiments in multiple stages (MSn) using the following conditions: capillary voltage of -41 V voltage spray -5 kV, capillary temperature 280 °C, carrier gas (N2) flow 60 (arbitrary units). The track acquisition was m/z 50–2000 with two or more sweep events performed simultaneously in the LTQ XL mass spectrometer. Determination of total phenols The total phenolic content was determined by the colorimetric method of Folin-Ciocalteau (SINGLETON; ORTHOFER; LAMUELA- RAVENTOS, 1999). The samples were dissolved in ethanol (500 µ g/ml) and 0.5 mL of each solution was added to 2.5 mL Folin-Ciocalteu reagent 10% (v/v). They were then added to 2.0 mL sodium carbonate (Na2CO3) 4% (w/v). The absorbance was measured in a spectrophotometer at 750 nm. The standard used to construct the calibration curve was gallic acid in concentrations from 5 to 100 μg/mL. The results were expressed in mg of GAE (gallic acid equivalents) per gram of sample. Determination of total flavonoid The flavonoid content was determined by a colorimetric method using an aluminum chloride reagent (KALIA et al., 2008). The samples were dissolved in ethanol (1000 µ g/mL) and 0.5 mL of each solution was added to 0.1 mL aluminum chloride (AlCl3.6H2O) 10% (w/v) and to 0.1 mL of potassium acetate (CH3COOK) 1M (w/v). The absorbance was measured in a spectrophotometer at 425 nm. The standard used to construct the calibration curve was quercetin in a concentration range of 5 to 100 μg/mL. The results were expressed in mg of EQ (quercetin equivalents) per gram of sample. 608 Study and evaluation… SILVA, A. F. et al. Biosci. J., Uberlândia, v. 36, n. 2, p. 606-618, Mar./Apr. 2020 http://dx.doi.org/10.14393/BJ-v36n2a2020-41848 Evaluation of antiradical activity The antiradical activity was determined based on the 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging method (YEN; CHANG; DUH, 2005). The samples and standards (ascorbic acid and BHT) were dissolved in ethanol (25–400 µ g/mL) and 2.0 mL of each solution was added to 0.5 mL DPPH (2, 2-diphenyl-1-picrylhydrazyl) solution (0.5 mM). The absorbance was measured at 517 nm and the results were expressed as percentages of DPPH radical scavenged. Assessment of reducing power The reducing power of the samples was measured according to previously described methods (YILDRIM et al., 2001a; YILDRIM et al., 2001b). The samples and standards (quercetin, ascorbic acid and BHT) were dissolved in ethanol (25–400 µ g/mL). To 2.0 mL of each solution was added 2.5 mL phosphate buffer 0.2 M (pH 6.6), 2.5 mL potassium ferricyanide (K3[Fe(CN6)]) (1% w/v) and 2.5 mL trichloroacetic acid (10% w/v). To 2.5 mL of this mixture was added 0.5 mL ferric chloride (FeCl3) 0.1% w/v. The absorbance was measured at 700 nm and the results were expressed as the percentages of ferric ions reduction. Evaluation of antimicrobial activity The dry extract and the fractions were dissolved in dimethyl sulfoxide (DMSO) (50 mg/mL) and antimicrobial activity was assessed according to the agar diffusion method according to the methodology proposed in the documents M7-A6 (CLSI, 2003) for bacteria, M24-A2 (CLSI, 2011) for mycobacteria and M44-A2 (CLSI, 2009) for fungi. The microorganisms used consisted of Gram- negative bacteria: Escherichia coli (ATCC 25922), Pseudomonas aeruginosa (ATCC 27853), Proteus mirabilis (ATCC 25922), Salmonella tiphy (ATCC 14028), Enterobacter cloacae LMI-Unifal; Gram- positive bacteria: Enterococcus faecalis (ATCC 51299), Micrococcus luteus (ATCC 9341), Bacillus subtilis (ATCC 6633), Staphylococcus aureus (ATCC 6538), Bacillus cereus (ATCC 11778); Fungi: Candida albicans (ATCC 10231) and Saccharomyces cerevisiae (ATCC 2601) and mycobacteria: Mycobacterium tuberculosis (ATCC 25177) (H37Ra) and Mycobacterium bovis (BCG sample). The chlorhexidine 0.12% was used as positive control and the distilled water as negative control. The minimum inhibitory concentration (MIC) was determined in samples that inhibited microbial growth in the agar diffusion test, according to the document M27A3 (CLSI, 2008). The tests were performed in sterile 96-well microplates. The dry extract and the fractions were dissolved in DMSO to obtain initial concentration of 25 mg/mL. From this stock solution, serial dilutions were performed to achieve a concentration of 0.025 mg/mL. Evaluation of cytotoxicity Peritoneal murinos macrophages were maintained in RPMI 1640 to 37° C and 5% CO2, arranged in 24 wells in the ratio 8x105 per well, to which were added the extract and fractions to be evaluated in various concentrations (0.1 to 160 μg/mL) and incubated for 72 hours. After the incubation period were added 50 µ l of MTT (3-(4,5- dimethylthiazol-2-yl )-2,5diphenyltetrazolium bromide) to each well, with new incubation for 4 hours. The cells were lisadas with DMSO and evaluated in UV/VIS spectrophotometer Shimadzu, double-beam, 2550 model to 570 nm compared to the control without adding drugs (PEREIRA et al., 2010). Statistical analysis The experiments were performed in triplicate and the mean and standard deviation of the results were determined. Values were submitted to analysis of variance (ANOVA) followed by Scott and Knott (1974) test (P<0.05). Correlation analyses were assessed using Spearman correlation coefficients (r). RESULTS AND DISCUSSION In chemical analyses by mass spectrometry, it was found that compounds of molecular masses 564, 578, 594 and 610 g/mol (Figure 1) are common to the extract and all fractions, but with different intensities in each. These compounds probably correspond to flavonoids that were deprotonated and recorded as their respective ions ([M-H]-) (GATES; LOPES, 2012). Fragmentations in multiple stages (MSn) were obtained for these ions, which made it possible to propose their molecular structures based on the literature and likely fragmentation mechanisms. Analyzing the data in MS2 (Table 1), it was observed that the fragmentation of the ion with m/z 609 ([M-H]-) resulted in a base peak of m/z 447 and another peak of m/z 463, suggesting the loss of mass 162 (hexose) and 146 (6-deoxy-hexose) sugars, respectively (WOLFENDER et al. 1992; GATES; LOPES, 2012). 609 Study and evaluation… SILVA, A. F. et al. Biosci. J., Uberlândia, v. 36, n. 2, p. 606-618, Mar./Apr. 2020 http://dx.doi.org/10.14393/BJ-v36n2a2020-41848 Figure 1. Spectrum mass in full scan mode, obtained by direct injection of dry extract. The ion of m/z 593 ([M-H]-) showed a base peak of m/z 447, which is likely to be due to the loss of a sugar of mass 146 (6-deoxy-hexose) (WOLFENDER et al., 1992; RANA et al., 2015). The peak with m/z 301 is the aglycone quercetin (SUN et al., 2007; DEVARAJI; KRISHNA; VISWANATHA, 2011; GATES; LOPES, 2012; RANA et al., 2015; VALLVERDÚ-QUERALT et al., 2015), formed after the loss of sugars. In the MS2 spectrum the ion of m/z 577 ([M-H]-) observed with a base peak at m/z 431 and another peak at m/z 285 may result from the loss of one and two sugar molecules of mass 146 (6-deoxy-hexose), respectively (WOLFENDER et al., 1992; ZANUTTO et al., 2013; RANA et al., 2015). The ion with m/z 563 ([M-H]-) showed a base peak of m/z 417 and another peak of m/z 430, which may be the result of loss of a sugar of mass 146 (6-deoxy- hexose) and a mass 132 sugar (pentose), respectively (WOLFENDER et al., 1992). The peak with m/z 285 corresponds to the aglycone kaempferol (SUN et al., 2007; DEVARAJ; KRISHNA; VISWANATHA, 2011; RANA et al., 2015; VALLVERDÚ-QUERALT et al., 2015) formed after the loss of sugar molecules. Generally, radishes are characterized by a high content of phenolic compounds, including the flavonoids quercetin and kaempferol (SHIN et al., 2015; KIM; BASKAR; PARK et al., 2016; RICARDO et al., 2018; IYDA et al., 2019). Other compounds such as, alkaloids, glycosinolates, pigments and proteoglycans may also be present and exhibit antimicrobial, antioxidant, antitumor and antiviral activities (ARUNA; YERRAGUNI; RAJU, 2012; SHIN et al., 2015). Table 1. Proposed structures of some flavonoids found in dry extract and the fractions. Flavonoids R1 R2 R3 R4 R5 [M-H]- Fragments main 1 O-hex OH O-6-dhex OH OH 609 463, 447, 301 2 OH OH O-6-dhex H O-6-dhex 593 447, 301 3 H OH O-6-dhex OH O-6-dhex 577 431, 285 4 OH OH O-6-dhex H pent 563 417, 430, 285 Legend: hex (hexose); pent (pentose); 6-dhex (deoxy-hexose). The phenol contents (Table 2) were higher in the fractions obtained after the concentration of the dry extract, and were most concentrated in the butanol fraction. The flavonoid contents were also higher in the fractions than in the dry extract except for the aqueous fraction, and were most concentrated in the ethyl acetate fraction. Chorol (2019) in his study with methanolic and acetonic extracts from R.sativus L. leaves obtained phenolic compound concentrations of 20 mg GAE/g e 3.6 mg GAE/g, respectively. Lower values than the values obtained in this work. Phenolic compounds and vitamin C are found in higher amounts in antioxidant Brassicaceae family plants (PODSEDEK, 2007). Flavonoids are reducing agents which can act to capture and neutralize oxidizing species, acting synergistically with other antioxidants such as vitamins C and E. The capacity of flavonoids to chelate metals also prevents these acting as catalysts in the formation of free radicals (SILVA; VALE; FELÍCIO, 2015). This suggests the protective role of these metabolites in reducing the risk of cardiovascular disease and other diseases associated with the formation of free radicals (PEREIRA; CARDOSO, 2012). 610 Study and evaluation… SILVA, A. F. et al. Biosci. J., Uberlândia, v. 36, n. 2, p. 606-618, Mar./Apr. 2020 http://dx.doi.org/10.14393/BJ-v36n2a2020-41848 Table 2. Total phenolic and flavonoid content of dry extract and fractions of leaves of R. sativus var. oleiferus Samples Total phenols content Total flavonoid content (mg GAE/g of sample) * (mg QE/g of sample) * EF FrHex FrAcOEt FrBuOH FrAq 47,02 ± 0,6a 96,09 ± 3,1c 99,95 ± 1,4c 121,27 ± 1,1d 52,48 ± 0,3b 20,36 ± 0,6b 70,93 ± 0,4c 98,02 ± 0,2e 89,87 ± 0,6d 3,28 ± 0,4a Legend: EF: dry extract of the leaves; FrHex: hexane fraction; FrAcOEt: ethyl acetate fraction; FrBuOH: butanol fraction; FrAq: aqueous fraction; GAE: gallic acid equivalents; QE: quercetin equivalents. Evaluation of the antioxidant activity Among the samples analyzed in this work, it was observed that the ethyl acetate fraction presented higher antioxidant capacity (Table 3), possibly due to the higher flavonoid content (Table 2). In addition, the ethyl acetate fraction presented a percentage of free radical sequestration close to the standards of quercetin and ascorbic acid, demonstrating the potential of this fraction. Goyeneche et al. (2015) demonstrated in their study that the leaves of R. sativus L. presented a four times higher concentration of flavonoids in relation to the root, showing an excellent antioxidant capacity. The increase of the activity as a function of the concentration of these compounds was also evidenced in the study of Park et al. (2016) carried out with three cultivars of radish. Eveline and Pasau (2019) determined that the ethyl acetate would be the best type of solvent for the extraction of bioactive compounds in radishes, due to higher flavonoid concentrations and to a higher capability of neutralization of free radicals obtained. Table 3. Antirradicalar activity and reducing power (to 100 µ g/mL) of dry extract and fractions of leaves of R. sativus var. oleiferus. Samples Percentages of DPPH radical scavenging* Percentages of ferric ions reduction* EF FrHex 26,48 ± 0,4a 47,70 ± 0,1c 6,49 ± 0,28a 10,54 ± 0,29d FrAcOEt 83,45 ± 0,5g 11,34 ± 0,29e FrBuOH 57,20 ± 0,3d 10,71 ± 0,29d FrAq 40,16 ± 0,3b 8,33 ± 0,36b Quercetina 81,44 ± 0,2f - Ácido Áscórbico 90,43 ± 0,3h 20,53 ± 0,51f BHT 63,7 ± 0,10e 9,13 ± 0,28c Legend: EF: dry extract of the leaves; FrHex: hexane fraction; FrAcOEt: ethyl acetate fraction; FrBuOH: butanol fraction; FrAq: aqueous fraction; BHT: butylated hydroxytoluene. *Means with different letters in the same column are significantly different (Scott and Knott, P <0.05). In previous studies with plants belonging to the Brassicaceae, the percentages of DPPH radical scavenged were found ranging from 14% to 23% in extracts of rocket (E. sativa) at a concentration of 0.1 mg/mL (ARBOS et al., 2010), 51.05% in broccoli extracts (B. oleracea var. italica), 29.06% in a radish extract (R. sativus L. var. radicle) (MELO; FARIA, 2014) and 57.7 % in essential oils of leaves of Eruca vesicaria (HICHRI et al., 2019), results lower than those obtained in the ethyl acetate fraction of this study. The reducing power method complements the antiradical activity assessment, since it evaluates the capability of the samples in reducing ferric ions into ferrous ions by phenolic hydroxyls (ROGINSKY; LISSI, 2005). The test results confirm the samples can inhibit the oxidative damages, since the ferric ions are catalyzers of the formation of radical species (LLESUY, 2002). Evaluation of the antimicrobial activity The dry extract of the leaves of R. sativus var. oleiferus presented larger inhibition zones of microbial growth against B. subtilis, and this difference was significantly superior to those produced by the fractions (P <0.05) (Table 4). 611 Study and evaluation… SILVA, A. F. et al. Biosci. J., Uberlândia, v. 36, n. 2, p. 606-618, Mar./Apr. 2020 http://dx.doi.org/10.14393/BJ-v36n2a2020-41848 Table 4. The averages values of inhibition zones of microbial growth (mm) of dry extract and fractions of leaves of R. sativus var. oleiferus evaluated by the agar diffusion method. Microorganisms Averages inhibition zones (mm)* EF FrHex FrAcOEt FrBuOH FrAq Cl Ag Gram-positive B. subtilis 18,67 d 9,00 b 9,34 b 6,00 b 5,30b 28,00 e 0,00 a B. cereus 18,00 c 0,00 a 9,34 b 5,60 b 0,00 a 27,34 e 0,00 a M. luteus 10,67 b 0,00 a 9,67 b 0,00 a 0,00 a 34,67 f 0,00 a E. faecalis 9,34 b 0,00 a 7,34 b 0,00 a 0,00 a 19,67 d 0,00 a S. aureus 13,00 c 8,00 b 9,34 b 0,00 a 0,00 a 27,34 e 0,00 a Gram-negative E. coli 0,00 a 0,00 a 0,00 a 0,00 a 0,00 a 21,34 d 0,00 a P. aeruginosa 0,00 a 0,00 a 0,00 a 0,00 a 0,00 a 21,34 d 0,00 a P. mirabilis 0,00 a 0,00 a 0,00 a 0,00 a 0,00 a 17,00 c 0,00 a S. typhimurium 0,00 a 0,00 a 0,00 a 0,00 a 0,00 a 21,34 d 0,00 a E. cloacae 0,00 a 0,00 a 0,00 a 0,00 a 0,00 a 11,67 b 0,00 a Fungi S. cerevisae 0,00 a 0,00 a 0,00 a 0,00 a 0,00 a 15,34 c 0,00 a C. albicans 0,00 a 0,00 a 0,00 a 0,00 a 0,00 a 15,67 c 0,00 a Mycobacteria M. tuberculosis 0,00 a 0,00 a 0,00 a 0,00 a 0,00 a 20,00 b 0,00 a M. bovis 0,00 a 0,00 a 0,00 a 0,00 a 0,00 a 20,00 b 0,00 a Legend: EF: dry extract of the leaves; FrHex: hexane fraction; FrAcOEt: ethyl acetate fraction; FrBuOH: butanol fraction; FrAq: aqueous fraction; Cl: Chlorhexidine 0.12%; Ag: Distilled water. * Means with different letters in the same column are significantly different (Scott and Knott, P <0.05). The antibacterial activity is related to bioactive compounds present in the dry extract and the fractions of the leaves. However, which chemical compounds, alone or in combination, are active can not be determined in this study. There is a possibility that the compounds act synergistically against B. subtilis, B. cereus, M. luteus, S. aureus and E. faecalis, because the dry extract of the leaves produced larger inhibition zones compared to their fractions. The antimicrobial activity of flavonoids can be attributed to their role causing changes in the physical-chemical properties of cell membranes, such as decreasing their fluidity (TSUCHIYA, 2010), activity of flavonoids can be attributed to the inhibition of nucleic acid synthesis, energy metabolism and cell wall synthesis (TIM; LAMBB, 2011). The dry extract and the fractions of the leaves of fodder turnip proved to be active only against gram-positive bacteria. This absence of antimicrobial activity is likely due to different actions of the active compounds of the extract on the walls or cell membranes of the microorganisms, since these exhibit variations in their chemical constitutions cells. The cell wall of gram-negative microorganisms is more complex, and has an additional outer membrane with a second lipid bilayer which strongly adheres to the peptidoglycan layer, giving greater stiffness and antigenicity (GUIMARÃES; MOMESSO; PUPO, 2010). The high concentration of high molecular weight lipids present in the cell wall of mycobacteria acts as a barrier for polar compounds (ARANTES et al., 2005). The cytoplasmic membranes of fungi include steroids, lipids and also proteins (TORTORA; FUNKE; CASE, 2012). This may explain the resistance of these microorganisms to hydroethanolic extracts. However, several studies have demonstrated the antimicrobial capacity of different extracts of the plant against some of these microorganisms (SHUKLA et al., 2011; AHMAD et al., 2012; UMAMAHESWARI; AJITH; ASOKKUMAR, 2012; JANJUA; SHAHID; ABBAS, 2013; CHIHOUB et al., 2019; DUY et al., 2019; IYDA et al., 2019), suggesting that future experiments with extracts of the fodder turnip of different polarities are promising. The lowest MIC values (Table 5) were obtained against M. luteus, and the ethyl acetate fraction showed the lowest MIC against this microorganism, followed by the aqueous and butanol fractions. The ethyl acetate fraction was shown to be more effective, with MICs between 0.1 and 12.5 mg/mL. 612 Study and evaluation… SILVA, A. F. et al. Biosci. J., Uberlândia, v. 36, n. 2, p. 606-618, Mar./Apr. 2020 http://dx.doi.org/10.14393/BJ-v36n2a2020-41848 Table 5. Determinations of Minimum Inhibitory Concentration (MIC) (mg/mL) of dry extract and fractions of leaves of R. sativus var. oleiferus. Bacteria Minimum Inhibitory Concentration (mg/mL) EF FrHex FrAcOEt FrBuOH FrAq B. subtilis 3,12 6,25 0,4 3,12 3,12 B. cereus 12,5 3,12 0,8 1,56 3,12 M. luteus 12,5 nd 0,1 0,4 0,2 E. faecalis 12,5 12,5 12,5 12,5 12,5 S. aureus 6,25 nd 0,1 3,12 3,12 Legend: EF: dry extract of the leaves; FrHex: hexane fraction; FrAcOEt: ethyl acetate fraction; FrBuOH: butanol fraction; FrAq: aqueous fraction; nd: not detected at maximum concentration tested (12.5 mg/mL). Some techniques used for antimicrobial evaluation may also lead to false negative results. The agar diffusion test, for instance, is only efficient for polar substances, allowing the diffusion of these through the culture medium. In addition, factors such as the presence of bacterial enzymes, the composition of the medium, the diffusion of the substance in the medium, the inoculum density, the time and temperature of incubation, the stability of substances and the molecular mass may hinder diffusion in the medium (SILVEIRA et al., 2009), which can prevent the contact of the extracts with microorganisms, leading to negative results. However, the plate microdilution assay allows direct contact of the extract with the microorganisms, avoiding possible limitations of the agar diffusion method. The extracts that present MIC less than 100 µ g/mL can be considered to have good antimicrobial activity, MIC between 100–500 µ g/mL are moderately active, MIC between 500– 1000 µ g/mL are very active and higher MIC than 1000 µg/mL are considered inactive (HOLETS et al., 2002; FABRY et al., 2008). Thus, from table 5, it can be seen that ethyl acetate fractions, followed by aqueous and butanol fractions showed the best antimicrobial activity, as demonstrated by their MIC in the range 100–800 µ g/mL. In a study with aqueous extracts of the fruits of R. sativus L. MIC values of 5 mg/mL against S. aureus were found (EDZIRI et al., 2012), higher than those obtained in the ethyl acetate, butanol and aqueous fractions of the leaves of R. sativus var. oleiferus. Jadoun et al. (2016) obtained MIC values of 0.5 mg/mL for the same microorganism, however using sulfur compounds isolated from seeds of R. sativus L. Chihoub et al. (2019) obtained MIC of 20 mg / mL for E. faecalis using hydroethanolic extracts 80% of R. sativus L leaves. Therefore, the results obtained highlight the fodder turnip as a source of antibacterial compounds. Correlation analyze The results of the correlation analyze between the main constituents detected and the biological activities (Table 6) showed values of positive r, according to the classification of Callegari-Jaques (2003). Table 6. Statistical analysis of the correlation between the parameters evaluated. Correlation r ** Antimicrobian activity MIC x Phenolic 0,40 MIC x Flavonoids 0,33 Antioxidant activity "in vitro" DPPH• method x Phenolic 0,80 DPPH• method x Flavonoids 0,77 Reducing power method x Phenolic 0,70 Reducing power method x Flavonoids 0,64 Legend: r = correlation coefficient; MIC = Minimum Inhibitory Concentration; DPPH• = radical DPPH. **According to the classification of Callegari-Jacques (2003): 0.00 Date accessed: September 23, 2013.