Journal of Food Quality

Correlation between selected microelements, phenolic compounds and antimicrobial action in medicinal plants extracts

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Journal of Food Quality JFQ-2010-164 Original Article 11-Jun-2010

Ducat, Giseli; UNICENTRO, Qumica Torres, Yohandra; UNICENTRO, Qumica Dalla Santa, Herta; UNICENTRO, ENGENHARIA DE ALIMENTOS Caetano, Isis; UNICENTRO, Qumica Kleinubing, Sirlene; UNICENTRO, Frmacia Stock, Daniele; UNICENTRO, Qumica Quinia, Sueli; UNICENTRO, Quimica Tussoline, Loyse; UNICENTRO Justo, Tais; UNICENTRO phenolic compounds, vegetables, metals, antimicrobian action, klebsiella, staphylococcus aureus

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Correlation between of Selected Microelements, Phenolic Compounds and Antimicrobial Action in medicinal plants Extracts.
GISELI DUCAT, YOHANDRA REYES TORRES, HERTA STUTZ DALLA SANTA, ISIS KAMINSKI CAETANO, SIRLENE ADRIANA KLEINUBING, DANIELE STOCK, LOYSE TUSSOLINE, TAIS HOBOLD JUSTO, AND SUELI PRCIO QUINIA*
Departamento de Qumica, Universidade Estadual do Centro-Oeste, Rua Camargo Varela de S 03, CEP 85040-080, Guarapuava, Parana, Brazil * E-mail address: spquinaia@unicentro.br; Tel.: +55-042-36298330: +55 042- 36298101

Abstract Phenolic compounds, especially flavonoids, are of increasing interest due to their functional properties in the diet for the promotion of human health and antimicrobial effects. The extracts of three different medicinal plants - Calendula officinalis L., Maytenus ilicifolia Martius ex Reissek and Cymbopogon citrate (DC) Stapf - obtained with 70% and 95% v/v ethanol had their antimicrobial activity tested against five pathogenic microorganisms. A quantitative analysis of total phenols, flavonoids and mineral species was performed on the extracts using UV-Vis spectroscopy and atomic absorption spectroscopy. The most active extract was the Cymbopogon citrate extract against Klebsiella pneumoniae. The mineral content of the medicinal herbs varied according to the type of plant; however, the concentration of heavy elements (Cd, Cr and Pb) was null in the extracts. It was observed a direct correlation between Al and the amount of phenolic compounds in the ethanolic extracts suggesting the possibility of quelation. Keywords: Plants, extracts, metals, phenolic compounds, antimicrobial activity, Klebsiella pneumoniae.

1. INTRODUCTION Recently, the use of herbs with pharmacological properties has increased significantly. Moreover, medicinal herbs play an important role in public health, especially in developing countries. In Brazil, the use of plant extracts in the treatment of certain diseases is very common. This habit can be explained at least in part, by the belief that herbs with therapeutic effects do not have toxic effects on the organism. Several studies have proved that medicinal herbs contain many classes of compounds like polyphenols, alkaloids, tannins, carotenoids, therpenoids, and others (Arabbi et al, 2004; Hamburger et al, 2003; Leite et al, 2001). Among these classes are the flavonoids and the phenolic acids, which are known for exhibiting many pharmacological properties such as blood vessel protection, anticarcinogenic, antiviral, anti-inflammatory, as well as antiallergic. Some of these properties have been related to the antioxidant activity of these compounds (Hamburger et al., 2003; Carbajal et al, 1989; Holetz et al, 2002; Puatanachokchai et al, 2002; Runnie et al, 2004; Sartori et al, 2003). There are also various research studies on the level of minerals present in medicinal plants, since these are essential for human nutrition (Fernandez et al, 2002; Almeida et al, 2002; Rashed, 1995). Plants can absorb metals from the soil, and under certain conditions, elevated levels can accumulate in the leaves and other edible parts of the plant. Between the minerals and oligoelements essential to the human organism, Ca, Na, K, Mg and Mn are present in medicinal plants on the order of g/kg, while Cr, Fe, Co, Ni, Cu, Zn are present on the order of mg/kg. Al, in most of its forms, is not harmful to living organisms. However, under certain conditions, with low pH, it tends to form toxic compounds. These are, then, potentially toxic for all living beings, including humans (Street et al, 2006). Pb and Cd, both in their elemental form and organic or inorganic compounds, pose risks of intoxication for exposed organisms. The possibility of

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intoxication by Pb in the human population usually occurs through occupational exposure (Midio and Martins, 2000). Non-occupational exposure is restricted to diet and represents the principal source of daily absorption of inorganic compounds for the population; however, there are few reported cases of intoxication through this type of exposure, since organic Pb compounds are not present in foods (Midio and Martins, 2000). Recently, there has been emphasis on the importance of determining the level of toxic metals in aqueous infusions and formulations made from medicinal plants (Veiga Jr. et al, 2005), considering not only the benefits but also the safety of the use of teas and other formulations made from medicinal plants. This study emphasizes three medicinal plants cultivated and used by the population of Guarapuava, Paran, Brazil: Calendula officinalis L., Maytenus ilicifolia Martius ex Reissek and Cymbopogon citrates (DC) Stapf. The prime objective was to quantify metal ions present in ethanolic extracts, considering both essential and non-essential ions to the human organism. The analysis was performed using flame atomic absorption spectrometry. The level of total phenolic compounds and flavonoids was determined by Uv-vis spectroscopy. Antimicrobial activity of the phenolic extracts was also evaluated against foodborne pathogens or food spoilage organisms or clinically isolates microorganisms. Finally, principal component analysis (PCA) was used to evaluate a possible correlation among metallic ions, amounts of organic compounds, and the antimicrobial activity of the alcohol extracts, as well as similarity among the samples. 2. MATERIALS AND METHODS Instrumental Determinations of Cd, Pb, Cr, Fe, Ca, Mg, Mn, Cu, Zn, Al, Na and K were performed using a flame atomic absorption spectrometer - FAAS (Varian modelo AA 220), equipped with Varian brand hollow cathode lamps. All of the absorbance measured was carried out in area integration mode. Samples were prepared in triplicate and their signals subtracted from their blanks. Determinations of the total phenols and flavonoids were performed using a Varian Cary 50 Bio UVVis spectrophotometer. For the phenolic acids, a wavelength of 760 nm was used, and for the flavonoids, a wavelength of 425 nm (Andrade et al, 2007; Kujala et al, 2000). Samples and reagents The samples of Calendula officinalis L (CA), Maytenus ilicifolia Martius (ES) and Cymbopogon citrates (LG), dried and packaged, were provided by CERCCOPA (Central Regional de Comercializao do Centro Oeste do Paran). The reagents and solvents used in the spectrophotometric analyses were of analytic grade. All standards used for the analyses of metal ions by FAAS were obtained from J.T. Baker Instra-Analysed (1000 g mL-1). All solutions were prepared with ultrapure water (HUMAN UP 900). In the determination of the total phenols, were used: a stock solution of gallic acid 1000 g mL-1 in methanol, a saturated solution of sodium carbonate (20 g) and sodium tartarate (1.2 g) in 100 mL of ultrapure water and the colorimetric reagent Folin- Ciocalteu. For the determination of flavonoids were used: a stock solution of quercetin 1000 g mL-1 in methanol and a solution of dihydrated aluminum chloride 5% m/v in methanol. For the antimicrobial testes, the following pathogenic microorganisms were used: Grampositive bacteria Staphylococcus aureus (American Type Culture Collection) ATCC 25923, Listeria monocytogenes ATCC 19111; Gram-negative bacteria Pseudomonas aeruginosa ATCC 27853 and clinically isolated Klebsiella pneumonia; and the clinically isolated yeast Candida albicans, which were cultivated in tripticase soy agar (TSA). Ethanolic extracts To obtain the alcohol extracts from the samples of medicinal plants, ethanol solutions were prepared in the proportions of 70 % and 95 % v/v. 25 g of each plant (Maytenus ilicifolia Martius and Cymbopogon citrates leaves; Calendula officinalis L flowers) were transferred to 1000 mL 2
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beckers and 400 mL of ethanol was added to each container. The containers were subjected to magnetic agitation for 24 h, after which the contents were filtered, and the solvents were evaporated in a rotary evaporator with a water bath of 60 C under reduced pressure, to obtain the dry extracts. The extracts obtained were named according to the concentration of the ethanol solution, EE70 (ethanolic extract 70 %) and EE95 (ethanolic extract 95 %). Determination of the concentration of metal ions in the ethanol extracts In order to determine the concentration of metal ions in the ethanol extracts, 0.01 g of each plant extract was weighed, dissolved in ethanol (95 %) in 10 mL volumetric flask (in triplicate). The quantification was carried out through FAAS using as blank the metals standard solutions in ethanol. Total phenol quantification by Folin Ciocalteu Method To obtain the analytical curve aliquots of gallic acid in methanol (5 to 70 g mL-1) were used, along with 500 L of Folin- Ciocalteau reagent and 500 L of a saturated solution of sodium carbonate and sodium tartarate in a 5 mL volumetric flask. The solutions were allowed to rest for 2 hours, and then the measure of absorbance in the wavelength of = 760 nm was performed. For the phenol content in the extracts, 10 mg of each plant extract was dissolved in 5 mL of methanol and transferred to 10mL volumetric flasks. An aliquot of 150 L was taken from each of these solutions, and the same procedure was used to obtain the calibration curves. For each extract, the experiment was performed in triplicate. Quantification of flavonoids by complexation with aluminum chloride Methanolic quercetin solutions of known concentrations (5-60 g mL-1) prepared from a stock solution were used to obtain the analytical curve. In the preparation of these solutions 250 L of an aluminum chloride solution 5 % m/v was placed in 5 mL volumetric flasks. After 30 minutes the measure of absorbance at = 425 nm was carried out. The flavonoid content in the plant extracts were performed with solutions of 10mg of each extract in 10 mL of methanol. Aliquots of 300 L were taken from each of these samples, repeating the same procedure described for the solutions used to obtain the analytical curve. For each extract, the analysis was made in triplicate. Determination of antimicrobial activity Agar Diffusion Susceptibility Test The EE70 and EE95 extracts were tested in the concentration of 10% m/v against the microorganisms cultures. The extracts were filtered through a sterilized membrane (0.22 m). All the materials utilized for the antimicrobial tests were sterilized in an autoclave for 15 minutes at 120 C. The microorganism inoculation procedure and the addition of extracts were carried out in a laminar flux chamber. The microorganisms cultivated in tripticase soy broth (TSB (Difco)) and remained for 24 hours at 35 C in a bacteriologic greenhouse. These microbial suspensions were standardized in a sterile saline solution (0.9 %) for a concentration of 106 CFU(colony forming units) mL-1, compared with the scale 0.5 of McFarland, according to NCCLS/CLSI, 2005 standards. The microbial suspensions were sown with a sterile swab on a Petri dish containing 18 mL of tripticase soy agar (TSA (Difco)). After this, 8mm orifices were made in the microbial suspensions using Duran tubes. With a micropipette 100 L of the extracts were added to each orifice. Ethanol 70% and 95% were used as a negative control, according to the tested extract. The Petri dishes were incubated at 37 C for 24 hours for the bacteria and 25 C for 48 hours for the yeast. The susceptibility to the plant extracts was expressed in terms of diameter of the microbial growth inhibition halo, measured with a caliper.

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Minimum inhibition concentration The extracts that showed positive results in the diffusion test in agar were tested to check the minimum inhibition concentration (MIC) through the serial dilution test with a system of multiple tubes (Rios et al, 1998). The test was performed by adding 1 mL of Miller Hinton (Difco) culture medium in serial tubes. After this, 1 mL of the plant extract previous diluted in distillated water (1:2) was added in tube 1, homogenized and dilution in tubes 2 and 3 were done in the proportion of 1:10. Nothing was added to tube 4 which was used as a negative control. In this way, the final concentrations of the extracts were 25 mg mL-1; 2.5 mg/mL and 0.25 g mL-1 for tubes 1, 2 e 3, respectively. Following this, all the tubes received 1 mL of microbial suspension, appropiate diluted, and were incubated at 37 C for the bacteria and at 25 C for the yeast, both for 24 h. MIC was considered to be the lowest concentration that inhibited the microbial growth, in other words, without clouding the medium in the tube. Minimal bactericide concentration An aliquot was taken from the MIC test tubes in which there was no clouding of the medium, duly diluted and placed in a culture medium of TSA. The results were analyzed after a rest of 24 hours at 37C for bacteria and 25C for the yeast. Petri dishes without any colonies of microorganisms were considered to be MBC. Analyses of correlations between the parameters evaluated The statistical tools used were principal components analysis (PCA). PCA permits the evaluation of the data set, reducing its dimension, conserving most of the useful statistical information present in the original data (Correia and Ferreira, 2007). HCA is carried out through the determination of the distances among the sample data, which can be calculated considering the closest K-neighbor or the mean of the samples. The data can be the original (scaled or centered on the mean) or the scores generated by the PCA. The statistical operations were performed using the MATLAB program, version 6.0. 3. RESULTS AND DISCUSSION

Determination of metal ions The concentration of metal ions essential and toxic to the organism was evaluated in the ethanol extracts, in order to verify which elements are extracted along with the phenolic compounds and flavonoids. Table 1 shows the measured levels of metal ions present in the 70 % and 95 % v/v ethanolic extracts. It was determined that the 70 % v/v solvent extracted the elements more efficiently due to the increase in polarity of the solvent as a result of the greater quantity of water present. Ions of Cd, Pb, Fe, Cr and Mn were not detected in any of the extracts. Figures 1, 2 and 3 are a graphic representation of the levels of metals in each ethanolic extract. It can be seen in Table 1 that, of all the ions extracted, Al is the only element that is not essential to the human organism. The elevated presence of Al in the extracts can be explained by the possible complexation that occurs between the element and the organic compounds in the plants. Normally, the level of Al found in plants is very high, on the order of mg Al kg-1 (Street et al. 2006; Malik et al, 2008). This high level can be justified by the absorption of Al by plant in acidic soils. In general, soil pH values below 5.5 can cause serious Al toxicity problems for plants. A wide variety of plant species create mechanisms that allow them to grow in acidic soils, in which the toxic concentrations of Al ions in the form of monomers, positively charged, could limit their growth. Organic acids, which liberate ions like polyphenolates, play a fundamental role in these mechanisms of Al tolerance, through the formation of chelates, though making it less toxic to plants (Da costa et al, 2009).

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Figure 2 Figure 3 Table 1 Determination of total phenolic compounds and flavonoids The 95 % ethanol solvent extracted greater quantities of phenolic compounds and flavonoids from the Calendula officinalis L and Maytenus ilicifolia Martius than the 70 % ethanol (Table 1). However, for the Cymbopogon citrates, the 70 % ethanol was more efficient in the extraction of phenolic compounds, and the two solvents were similar in the extraction of flavonoids. Various factors can influence the quantity of phenolic compounds in the plants, such as time of harvest and position or age of the leaves, since larger quantities are present in younger leaves (Gorbo-Neto and Lopes, 2007). The values of total phenols in the ethanolic extracts, when converted into the respective masses of the leaves of Maytenus ilicifolia Martius, Cymbopogon citrates and Calendula officinalis L flower used, were found to be on the order of 30.65 a 57.95 mg g-1 for the 70 % v/v extract and between 11.14 and 41.44 mg g-1 for the 95 % v/v extract. For the flavonoids, the levels were on the order of 4.5 to 8.41 mg g-1 (70 % v/v) and 2.5 to 5.3 mg g-1 (95 % v/v). The levels obtained in this study were on the same order of scale as those obtained by Yariwakei et al. 2005, who determined the level of total phenols in leaves of Maytenus ilicifolia Martius obtained from specimens cultivated in Ribeiro Preto, So Paulo, Brazil. The levels varied from 21.96 to 45.92 mg g-1 (expressed in mg tannic acid per g of dry leaves) and from 1.3 a 3.8 mg (flavonoids per g of dry leaves). Antimicrobial activity of the ethanolic extracts None of the ethanolic extracts of the three plants studied showed any activity against the microorganisms Pseudomonas aeruginosa ATCC 27853, Listeria monocytogenes ATCC 19111 and Candida albicans (clinically isolated). For the microorganisms Staphylococcus aureus ATCC 25923, and Klebsiella sp., the results are depicted in Table 1. The ES-70 and CA-95 extracts showed weak antibacterial activity against Staphylococcus aureus, while the other extracts showed no antimicrobial activity. All tested extracts showed activity against Klebsiella sp., with the LG-70 extract showing the greatest activity, with an inhibition zone of 9.00 0.42 mm. This extract also showed the greatest concentration of flavonoids (41.542.69), which suggests that the antimicrobial activity might be correlated with the presence of this group of molecules (Middleton and Kandaswami, 1993; Atty and Das, 1988; Hackett, 1986). Antimicrobial activity of plants extracts is also evaluated through determination of the quantity of the substance which is necessary to inhibit the growth of the test microorganism; this value is known as minimum inhibition concentration MIC. The extracts that showed antimicrobial activity were used to determine the MIC (minimum inhibitory concentration) and the MBC (minimum bactericide concentration). The results are shown in Table 2. ES95%, inhibited the development of S. aureus in concentrations of 25, 2.5, and 0.25 mg/mL, so the MIC for this extract is 0.25 mg/mL. However, after placement in a culture medium, absence of microbial development was observed only in the 25 mg/mL concentration, which indicates that this concentration is the MBC of this extract against S. aureus. According to Holetz et al, 2002, a plant or its extract is considered to have antimicrobial potential when it has a MIC smaller than 1.0 mg mL-1; using these criteria, the ES95 extract can be considered to have important antimicrobial activity against S. aureus. The CA70 extract showed inhibitory activity in concentrations of 25 and 2.5 mg mL-1 (MIC); however, the MBC occurred at 25 mg mL-1 . This result suggests the presence of inhibitory molecules in this extract, but with insignificant antimicrobial potential versus S. aureus. The antimicrobial activity of the extracts was more efficient against Klebsiella sp., since the results indicate that the MIC is 0.25 mg mL-1 for the ES95, ES70, CA95, LG95 and LG70 extracts. Moreover, the MBC was also determined to be 0.25 mg mL-1 for the ES95, LG95 e LG70 extracts. Klebsiella sp. showed a much greater susceptibility than S. aureus to the extracts tested.

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Table 2 Chemometric study Principal components analysis (PCA) was applied to the schedule experimental data of the levels of metal ions, phenols, flavonoids, and antimicrobial activity obtained from the plant extracts to verify possible similarities and differences among the samples and their correlation between the variables (Correia and Ferreira, 2007). The first, second, and third principal components captured 38.0 %, 26.5 % and 19.4 % of the variance of the experimental data, totaling 83.9 % of the variance captured. The first and second principal components (Fig. 4) separated the EE95 obtained from the LG, CA and ES from the others in function of similar Na levels. The EE70 of the LG differentiated from the others because of the highest levels of Al, flavonoids and activity against Klebsiella sp. The EE70 of CA separated due to its highest levels of Cu, Ca, Zn and intermediate quantity of K. Finally, EE70 of ES stood out for having highest levels of Mg and activity against S. aureus. In the first principal component, there was a direct correlation between the variables level of flavonoids, activity against Klebsiella, and level of Al in the ethanolic extracts, indicating that Al could be complexed with the flavonoids. The remaining metal ions and the activity against S. aureus were inversely related to the levels of flavonoids. The loadings on the second principal component show levels of Al, Ca, Cu, Zn, flavonoids and the activity against Klebsiella to be directly correlated, once again making possible the hypothesis of the formation of phenol/flavonoid chelates with Al, including other metals like Ca, Cu, Zn. These compounds can contribute to the diminution of the toxicity of Al through the formation of complexes. Da costa et al., 2009, observed that Al is predominantly in the inert form in infusions of erva mate (Ilex paraguariensis (50 to 70 %), suggesting the complexation of the element with polyphenolic compounds present in the erva mate. With the use of chemical speciation methods, it is possible to investigate the fractions of the species of higher-toxicity Al present in plant extracts to know the fractions that can simply be in the hydrated form showing elevated lability, or inert through the formation of stable chelates.

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Figure 4 4. CONCLUSION

The 95 % v/v ethanolic extracts of the Calendula officinalis L and the Maytenus ilicifolia Martius showed greater values of total phenolic compounds than the 70 % v/v extracts. In the case of the Cymbopogon citrates, the inverse occurred. The extracts that showed the greatest concentrations of flavonoids were the 70 % and 95 % v/v extracts of LG and the 95 % v/v extract of ES. The 95 % v/v ES extract can be considered to have important antimicrobial activity against S.aureus, as shown by the study of MIC. The 70 % and 95 % ES, 70 % and 95 % LG, and 95 % v/v CA extracts showed important antimicrobial activity against Klebsiella sp. The presence of ions of Mn, Fe, Cr, Pb, and Cd was not detected in the ethanolic extracts. The other metal ions were better extracted in 70 % v/v ethanol. Through PCA, it was possible to determine some correlations between metal ions and the level of phenolic compounds and flavonoids, and between antimicrobial activity and phenolic compounds. Particularly, it was observed a direct correlation between Al and the amount of phenolic compounds in the ethanolic extracts suggesting the possibility of quelation. It is also possible that Ca and Al compete for the same active site on the plant, and because of this they appear in inverse correlation in the PCAs.

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Table Captions Table 1 Levels of metal ions present (g g-1), total phenolic compounds and flavonoids (mg g-1 of extract) and antimicrobial activity of the ethanolic extract by diffusion in Agar (mean of inhibition zones, mm) in the EE70 and EE95 of the medicinal plants Maytenus ilicifolia Martius (ES), Calendula officinalis L (CA) and Cymbopogon citrates (LG), n=3. Table 2 Minimum inhibition concentration (MIC) and minimum bactericide concentration (MBC) (mg/mL) of the extracts against Staphylococcus aureus and Klebsiella sp., n=3.

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Figure Captions

Figure 1: Extracted metal ions levels in Maytenus ilicifolia Martius extracts. Figure 2: Extracted metal ions levels in Calendula officinalis L extracts. Figure 3: Extracted metal ions levels in Cymbopogon citrates extracts. Figure 4: Scores (A) and loadings (B) of the first and second principal components for the alcohol extract samples. Kleb. Klebsiella sp ; Staf. S. Aureus.

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Table 1 Levels of metal ions present (g g-1), total phenolic compounds and flavonoids (mg g-1 of extract) and antimicrobial activity of the ethanolic extract by diffusion in Agar (mean of inhibition zones, mm) in the EE70 and EE95 of the medicinal plants Maytenus ilicifolia Martius (ES), Calendula officinalis L (CA) and Cymbopogon citrates (LG), n=3. Metals ions ES CA LG EE70 Cu Zn Mn Fe Na Cr Al Mg Ca K Pb Cd Phenolic acids Flavonoids Staphylococc us aureus Klebsiella sp.
ND = not detected

EE95 ND 0.8760.01 ND ND ND

EE70 10.790.01 10.040.01 ND ND ND

EE95 0.1770.00 1.030.00 ND ND ND 9.620.01

EE70 6.150.02 4.390.02 ND ND 71.220.05 ND 24.800.01 98.900.19

EE95 0.600.003 0.8540.00 ND ND 24.940.09 ND 21.870.02 7.150.06

2.200.00 8.140.01 ND ND 61.930.03 ND

31.890.07 153.620.12 82.920.37

306.180.41 122.490.15 363.680.01 169.890.14 420.390.20 90.070.15 1129.150.5 46.090.06 384.000.04 2

63.700.08

9274.460.1 1907.520.2 9578.940.3 924.350.08 2801.060.0 913.850.13 2 3 2 3

ND ND 230.44 0.40

20.60 1.25 39.00 1.10 13.550.55 18. 95 0.45 41.54 2.69 38.20 2.04 1.45 0.35 2.45 0.64 ND 2.70 0.28 ND 0.73 0.11 ND ND 6.10 0.57

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29.610.02 167.270.13 18.870.07

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ND ND 518.28 1.30

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ND ND

ND ND 120.26 0.90

ND ND 285.97 0.84

ND ND 170.00 0.50

92.35 0.50

3.48 1.10

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9.00 0.42

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Table 2 Minimum inhibition concentration (MIC) and minimum bactericide concentration (MBC) (mg/mL) of the extracts against Staphylococcus aureus and Klebsiella sp., n=3. Staphylococcus aureus Klebsiella sp Plant Test EE70 EE95 EE70 EE95 MIC 2.5 ND ND 0.25 CA MBC 25 ND ND 2.5 MIC LG MBC MIC ES MBC
ND = not detected

ND ND ND ND

ND ND 0.25 25

0.25 0.25 0.25 2.5

0.25 0.25 0.25 0.25

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Figure 1

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Figure 2

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Figure 3

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Journal of Food Quality

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Figure 4

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