The objective of this study was to evaluate the chemical profile of extracts (aqueous and ethanol) and the essential oil of Myrocarpus frondosus Allem?o, the sensitivity of strains of Escherichia coli , Staphylococcus aureus , Salmonella typhimurium and Listéria monocytogenes , front the extracts and essential oil, by means of the microdilution method in broth, the antioxidant activity by the ABTS method, and the content of phenolic compounds present in the extracts and oil. For the preparation of extracts from plant leaves used with ethanol and water, then separated, the chemical identification of compounds was performed by high-performance liquid chromatography (CLAE-DAD) and gas chromatography coupled to mass spectrum (CG/MS). With the chemical analysis of the extracts obtained the presence of the major compound rutin, and oil major compound was found germacrene B. In the microdilution method in broth, oil and extracts showed inhibition against all the bacteria tested in the concentrations 1 mg/ml to 0.25 mg/ml, except for Staphylococcus aureus at a concentration of 0.25 mg/ml of the essential oil and trans-caryophyllene. The results of Minimum Bactericidal Concentration showed that the essential oil had bactericidal activity at a concentration of 1mg/ml for all bacteria tested and trans-caryophyllene at the same concentration only for Listeria monocytogenes . In relation to the essential oil , antioxidant activity showed higher radical reduction capacity of 40.92% and the content of phenolic compounds ethanol extract showed more 12.72%. The in vitro results support the conclusion that the essential oil is very promising both in antimicrobial action as antioxidant activity and the leaf extracts on antioxidant activity.
Medicinal plants have the largest pharmaceutical source that exists and the use of them is as old as human life. Before the nineteenth century, the plants were the main resource used in traditional medicine [
So today many scientists from different areas are using substances extracted from medicinal plants due to its high efficacy against pathogens and its use was already proven use in traditional medicine [
However, the widespread use of drugs, especially non-prescription, has led to a loss of effectiveness of action against pathogenic microorganisms [
According to Duarte [
According to Calixto [
Traditional medicine has using parts of Myrocarpus frondosus Allemão plant, to treat wounds and bruises, as an expectorant, and other lesions of the respiratory system, anti-inflammatory among other uses [
In this context, this study aims to evaluate the antimicrobial activity, antioxidant of aqueous and ethanolic extracts of leaves, and essential oil from plant seeds Myrocarpus frondosus Allemão (Cabreúva) and the extremely important for pharmaceutical and food industry and identify main chemical constituents present in aqueous and ethanolic extracts of the leaves and the essential oil of the seeds.
The leaves and seeds were collected on the upper slopes of the Northeast in the state of the Rio Grande do Sul. In the anatomy laboratory of wood, the material was dried at 35˚C in an air circulation greenhouse and stored in climate?con- trolled chamber. For identify the species, plant samples were sent to the Herbarium of Forest Engineering at the Federal University of Santa Maria?UFSM, and identified and cataloged under number HDCF n˚ 6215.
For obtaining the aqueous raw extract were used 25 g of crushed dry leaves in knife mills and 100 mL of distilled water. The plant sample and the solvent were placed in a container with 500 mL and kept under stirring of 600 RPM, heating plate with an oil bath at a temperature of 60˚C for 24 hours. The process was repeated until obtaining a clear extract. In the end of extraction, the extract was frozen at −80˚C for subsequent lyophilization, thereby obtaining the dry extract. For obtaining the ethanolic extract followed by the same extraction procedure as above, the solvent used was ethanol. Already the ethanolic extract it evaporated in rotary evaporator to obtain the dry extract. The dried samples were placed in small vials hermetically sealed for later use in chromatographic analysis and in vitro tests.
The essential oil was extracted by hydrodistillation method. Was weight 100 g of the previously crushed seed in a blender along with 1500 mL distilled water and ad ded to a flask attached to Clevenger apparatus. The extraction was performed as described in the Brazilian Pharmacopoeia [
For the identification of metabolites present in the essential oil the same were subjected to chromatographic analysis in equipment GC/MS, brand Shimadzu QP2010, equipped with a splitter split/splitless. With an Rtx-5MS Restek (30 m × 0.25 mm × 0.25 microns) capillary column under the following chromatographic conditions: Helium gas carrier obtained by electron impact fragments to a power of 70 eV rate of 1.2 ml/min, 1:50 split flow and the volume of injected sample of 1 ul. Programmed oven temperature: initial temperature was 40˚C with a heating ramp of 10˚C/min to 280˚C and remained stable at this temperature for 10 minutes. Subsequently the temperature was increased at a rate of 10˚C/minute to 300˚C for a total time of 41 minutes with a injector temperature 250˚C, the interface temperature 300˚C, the compounds were analyzed; NIST08 using GC/MS library.
The ethanolic and aqueous extracts were analyzed by HPLC-DAD (Shimadzu). The reverse phase chromatography was conducted with Phenomenex C-18 column (4.6 mm × 250 mm), using as mobile phase A (2% acetic acid) and B (methanol) at a flow rate 0.8 ml/min and injection volume of 40 µL according to the elution method and Piglet gradient Evaristo and Leitão [
The antimicrobial activity of the extracts, oil essential, rutin and caryophyllene oxide were individually tested with four patterns strains: Escherichia coli ATCC25922, Staphylococcus aureus ATCC25923, Listeria monocytogenes ATCC 7644 and Salmonellatyhimurium ATCC 13311.
The standards used were the Trans-Caryophyllene, ≥98.5% 001.321.406 lot and Rutin Hydrated, ≥94.0%, BCBH0339V lot, purchased from Sigma-Aldrich.
In a test tube, containing saline 0.85% to prepare suspensions of the test strains that were standardized according to Mac Farland 0.5 range, corresponding roughly to the concentration of 1.5 × 108 Colony Forming Units (CFU/mL). For standardize the optical density of the inoculum was read in a spectrophotometer at a wavelength λ = 625 nm in an absorbance of 0.08 - 0.10 according to NCCLS (2003) with modifications [
The broth susceptibility test was performed using the method NCCLS (2003) with modifications, for the determination of MIC. All tests were conducted in trypticase soy broth (TSB). To perform the antimicrobial activity, two standards were used, rutin, major compound of the extracts and the trans-caryophyllene only major compound of essential oil available for purchase, extracts (aqueous e ethanolic) e oil essential. Samples and standards were diluted in DMSO with the initial concentration of 1mg/ml. Prepared a bacterial suspension with a concentration of 5 × 105 CFU/mL. In a total volume of 100 μl of the suspension of standardized microorganisms they were inoculated into each well, and then added to 100 μl of the sample solution. The plates were incubated at 37˚C for a period of 24 h. The MIC was calculated as the highest dilution, showing complete inhibition of standard strain used.
The inhibition of control were used antibiotics against Gram+ and Gram−, positive and negative control and in microdilution method was used dimethylsulfoxide (DMSO).
The results of the MIC, the wells showing complete absence of growth in each well were identified and 10 μl of each well were transferred to a Mueller Hinton Agar plates and incubated at 37˚C for 24 h. The complete absence of growth, was considered as the minimum bactericidal concentration.
For the determination of total phenols used the method described by Lachman [
The radical ABTS is obtained after the reaction ABTS (0.0192 g) of potassium persulfate (5 ml) incubated at room temperature (±25˚C) and in the dark for 24 h. Once the ABTS radicals formed diluted with ethanol to obtain an absorbance ranging from 149 (0.70 ± 0.2) to 734 nm (wavelength of maximum absorption) in a UV spectrophotometer. The first absorption is measured is ethanol which is white then an ABTS radical being measured time zero (T0) and A734. Then the last 6 minutes is measured by diluting 20 μl of DMSO in 2 mL of ABTS radical time of six minutes. For samples prepared from the extracts 20 mg of sample is diluted in 10 ml of DMSO then add 20 ml of diluting the sample in 2 ml of ABTS radical was generated by absorbance A734 was determined continuously 6 as minutes after dilution.
For the test was used 2.5 ml of phenol reagent of Folin-Ciocalteu and 5 ml of Na2CO3 solution with a concentration of 20% is used, it was added to 0.5 ml aqueous solution at concentration 20 mg in 10mL of DMSO. The mixture remained 1 hour at 50˚C before measuring the absorbance at 750 nm. It was measured with a spectrophotometer of UV. The absorption of the developed blue color is measured at 750 nm and the total phenols content was determined from the calibration curve of standard solutions of gallic acid (1 - 20 mg/l) and expressed as mg gallic acid equivalents (GAE).
Each test was performed in triplicate and the mean values were calculated. The data were reported as mean ± standard deviation (SD).
In the Chromatogram analysis of aqueous and ethanolic extracts of leaves (
The essential oil chemical analysis by GC/MS, fourteen compounds were identified, the B germacrene the major compound with 39.28% of the total oil
compounds, followed by beta-copaene, beta-pinene, and Caryophyllene (
The results of the broth susceptibility test (
Peak | Compound Name | Área% | R. Time |
---|---|---|---|
1 | alpha-Pinene | 1.01 | 4.981 |
2 | Beta-Phellandrene | 0.83 | 5.574 |
3 | Beta-Pinene | 11.03 | 5.641 |
4 | Gamma-Elemene | 0.56 | 11.008 |
5 | Copaene | 1.13 | 11.559 |
6 | (-)beta-Bourbonene | 0.86 | 11.700 |
7 | Beta-Elemene | 1.12 | 11.743 |
8 | Caryophyllene | 7.51 | 12.175 |
9 | Humulene | 1.57 | 12.614 |
10 | Beta-Copaene | 24.51 | 12.963 |
11 | Germacrene B | 39.28 | 13.166 |
12 | Delta-Cadinene | 1.23 | 13.432 |
13 | Gamma-Muurolene | 1.28 | 14.114 |
14 | Spathulenol | 7.03 | 14.160 |
15 | ND | 1.05 | 14.246 |
100.00 |
Strains standards samples | Concentration (mg/mL) | E.c | S.a | L.m | S.t | ||||
---|---|---|---|---|---|---|---|---|---|
MIC | CBM | MIC | CBM | MIC | CBM | MIC | CBM | ||
EAF | 1.0 | − | + | − | − | − | − | − | − |
0.5 | − | + | − | + | − | + | − | + | |
0.25 | − | + | − | + | − | + | − | + | |
EEF | 1.0 | − | + | − | + | − | + | − | + |
0.5 | − | + | − | + | − | + | − | + | |
0.25 | − | + | − | + | − | + | − | + | |
RU | 1.0 | − | + | − | + | − | − | − | + |
0.5 | − | + | − | + | − | + | − | + | |
0.25 | − | + | − | + | − | + | − | + | |
OE | 1.0 | − | − | − | − | − | − | − | − |
0.5 | − | + | − | + | − | + | − | + | |
0.25 | − | + | + | + | − | + | − | + | |
TC | 1.0 | − | + | − | + | − | − | − | + |
0.5 | − | + | − | + | − | + | − | + | |
0.25 | − | + | + | + | − | + | − | + |
Inhibited growth (−); bacterial growth (+); MIC: Minimum Inhibitory Concentration; CBM: Minimum Bactericidal Concentration; E.c: Escherichia coli; S.a: Staphylococcus aureus; L.m: Listeria monocytogenes; S.t: Salmonela thymurium; EAF: aqueous extract of leaves; EEF: ethanolic extract of leaves; RU: Rutin; OE: Óil essential; TC: Trans-cariofillene.
aureus. The compounds found in the essential oil are classified as terpenes, and these are recognized by the antibacterial and antioxidant activity [
The compounds found in the essential oil are classified as terpenes, and these are recognized by the antibacterial and antioxidant activity [
The Minimum Bactericidal Concentration (CMB) is defined as the lowest concentration of oil and/or extract resulting in the death of 99.9% of the inoculum. The results of CMB demonstrated that the essential oil had bactericidal activity at a concentration of 1mg/ml for all bacteria tested and trans-caryophyl- lene at the same concentration only for Listeria monocytogenes.
The 10% DMSO used as a control, there was growth of microorganism, thus confirming that the diluent does not interfere with the tests.
In
The oil tested the antioxidant activity, has a capacity of inhibition of 40.92% (
Galic acid equivalents mg/L | Weight of oil (mg/L) | TOTAL PHENOLIC CONTENT (TPC%) | |
---|---|---|---|
C GAE | TPC % | ||
1FA | 198.979 | 2000 | 9.949 |
2FE | 254.478 | 2000 | 12.724 |
5OE | 120.221 | 2000 | 6.011 |
Where: FA: Aqueous extract of leaves; FE: Ethanolic extract of leaves; OE: Essential oil from the seeds.
may be related to the presence of rutin, one flavonoid, recognized for antioxidant activity and found in different concentrations in plants of the same family of M. frondosus [
The methodology used in this study allowed the identification of rutin in the extracts of leaves and germacrene B in the essential oil as major compounds. The results of this study clearly show that the essential oil had a Myrocarpus frondosus Allemão antioxidant activity in relation to the extracts that can be useful both in the pharmaceutical as in the food industry. In the antimicrobial activity, both as essential oil extracts appear to be promising, against Gram positive bacteria and Gram negative. However, additional studies were needed to isolate and identify active compounds, and to understand the mechanism of action as pharmacological agents in vivo studies.
We thank the laboratories LPPN, Food Microbiology, UPV/EHU, the Federal University of Pelotas and CAPES financial support.
de Santi, I.I., Gatto, D.A., Machado, M.R.G., dos Santos, P.S.B. and Freitag, R.A. (2017) Chemical Composition, Antioxidant and Antimi- crobial Activity of the Oil and Plant Extract Myrocarpus frondosus Allemão. American Journal of Plant Sciences, 8, 1560-1571. https://doi.org/10.4236/ajps.2017.87108