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Introduction
Multi-resistant bacteria (ESKAPE) represent an inherent problem for the world population. In the United States, the estimated number of annual infections is higher than 2 million, whereas in developing countries, communicable diseases are the main cause of mortality, and emerging and re-emerging infectious diseases represent a major issue [1]. Antibiotic resistance jeopardises the achievements of modern medicine by impeding the treatment and prevention of infections. Some ESKAPE pathogens (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, Escherichia coli) can tolerate transient exposure to high doses of antibiotics without changes in their minimum inhibitory concentration (MIC). This tolerance is associated with the irreversible destruction of the active site of the antibiotic, modification of the bacterial target site, reduction of antibiotic accumulation by mutation or loss of membrane channels and persistence through cells embedded in biofilms [2,3].
The World Health Organization (WHO) and Pan American Health Organization (PAHO) have drawn special attention to multidrug-resistant bacteria, generating a critical priority list that includes dangerous multidrug-resistant bacteria that may be of nosocomial origin or acquired in the community. They are classified by their degree of lethality, treatment and hospitalization time; the ease with which they are transmitted between animals, from animals to people and between people. The list is divided into critical, high, and medium priority levels, which include S. aureus, E. coli, E. faecalis and P. aeruginosa [4].
Additionally, yeasts of the genus Candida are opportunistic human pathogens [5] that affect mucous membranes. More than 90 % of clinical infections are caused by species of the genus Candida, such as C. glabrata, C. albicans, C. krusei and C. tropicalis, highlighting their virulence factors such as membrane and cell wall barriers, dimorphism, biofilm formation, signal transduction pathways, proteins related to stress tolerance, hydrolytic enzymes and toxin production [6]. Therefore, the study of these yeasts, whose incidence has increased in the last three decades, is imperative, due to the increase in the Acquired Immune Deficiency Syndrome (AIDS) epidemic, an increasingly aging population, a greater number of immunocompromised patients and the more widespread use of medical devices permanent [4]. Resistance to antifungals has increased in many Candida species, contributing to treatment failure and amplifying intra-hospital issues [7].
Free radicals are chemical species present in the body that can cause oxidative stress, damaging cells and body functions, which can result in various diseases such as cancer, arthritis and respiratory diseases, among others. Antioxidants have the ability to scavenge free radicals, playing an important role in defending the body against different chronic diseases [8]. It is therefore essential to develop new compounds with antimicrobial and antioxidant activity. In this context, plants are a source of secondary metabolites, many of which have these two effects, and one of these constituents is EOs, which are complex mixtures containing between 20 and 60 components, mainly monoterpenes, sesquiterpenes, aliphatic and aromatic compounds [9].
The composition of essential oils (EOs) varies with temperature, climate, plant maturity and season, among others, and this variability could influence the properties of the EOs [10]. They play an important role in protecting plants from pathogens and predators [11] and are applied in the production of food, flavours, cosmetics and pharmaceuticals [12]. The bioactive compounds of EOs present various biological activities such as anti-inflammatory, analgesic, anti-cancer [13], antimicrobial and antioxidant activities [14,15]. Different EOs from plants of the family Asteraceae have antioxidant and antimicrobial activities [16], such as those from Achillea millefolium subsp. millefolium Afan [17] and Pulicaria inuloides [18]. Some EOs of plants of the Fabaceae family also possess these activities, such as those from Myrocarpus frondosus [19].
Recent studies found that some extracts of aerial parts of Trixis angustifolia, Dalea bicolor, Eupatoriun glabratum and some species of Tagetes have antimicrobial activity against different bacteria [20-22]. However, there are no reports about antimicrobial effects of the EOs of these plants.
In this study, we determined the composition of four EOs from plants of the family Asteraceae, namely essential oil of Trixis angustifolia (EOTA), essential oil of Tagetes parryi (EOTP) and essential oil of Eupatorium glabratum (EOEG), and of one EO from a plant Dalea bicolor of the family Fabaceae, namely EODB. For the first time, the antioxidant capacities of these EOs were evaluated, as well as their antimicrobial activities toward two Gram (+) bacteria and two Gram (-) bacteria and their antifungal activities toward four Candida species.
Materials and methods
General
The aerial parts of T. angustifolia, D. bicolor, T. parryi and E. glabratum, were collected in San Luis Potosí State, México. The plants were identified by the taxonomist José García Pérez, and a voucher specimen of each plant was deposited in the Herbarium Isidro Palacios of the Universidad Autónoma de San Luis Potosí (Table 1).
Table 1 Data about plant species, and yield of EOs.
| Plant Species | Date and place | Coordinates | Plant part | Yield (w/w) | Voucher number |
|---|---|---|---|---|---|
| Trixis angustifolia | February 2008, 1 km from the junction to Guadalcázar, SLP | 22°38'23.7"N 100°30'49.0"W | Aerial parts | 0.64 | SLPM44557 |
| Dalea bicolor | February 2014, at the Cañada del Lobo dam, San Luis Potosí, SLP | 22°05'44.0"N 100°57'56.9"W | Aerial parts | 0.45 | SLPM57550 |
| *Tagetes parryi | November 2013, Agua Blanca, Municipality of Villa de Zaragoza, SLP | 22°03'35.7"N 100°37'11.5"W | Aerial parts | 0.54 | SLPM31975 |
| Eupatorium glabratum | February 2008, in the Realejo, community of Guadalcázar, SLP | 22°39'57.4"N 100°25'04.4"W | Aerial parts | 0.19 | SLPM44553 |
*Previosly reported by González-Velasco [23].
Essential oil extraction
The EOs were obtained by hydrodistillation from the aerial parts of the fresh plants. They were extracted with diethyl ether, and this solvent was eliminated under reduced pressure at 20 °C. The EOs were then stored at 5 °C.
Composition of the EOs
The composition of EOs was determined by GC-MS using a chromatograph (Agilet Technology, model 6890N) connected to a selective mass detector model 5973 Network (MSD, Agilent Technologies, Wilmington, DE, USA). An HP-5MS capillary column (30 m length, 0.25 mm internal diameter, and 0.25 µm film width) (J&W, Folsom, CA, USA) was used for the separation. The EOs samples (10 µL) were diluted with acetone (1 mL) and the injector temperature was 240 °C, operated in the splitless mode, and the carrier gas was helium at 1mL/min. The oven temperature was programmed at 50 °C/3 min, with a heating rate of 3 °C/min up to 240 °C/2 min. The MSD was operated at 70 eV, the ion source was set a 150 °C, and the transfer line was at 240 °C and the mass range was analyzed 15-600 m/z. The software MSD ChemStation (Agilent B.04.02) was used for data recording and the compounds were identified based on their mass spectra by comparison with the spectra reported in the Wiley 09 and NIST11 libraries. In addition, the Kovak index was calculated for each peak, with reference to the n-alkane standards (C6-C26) running under the same conditions.
Microorganisms
We used four yeast and four bacterial species. The yeasts, Candida albicans ATCC 10231, C. glabrata ATCC 32554, C. krusei ATCC 90878 and C. tropicalis ATCC 750, were inoculated in sterile Sabouraud dextrose broth and incubated at 37 °C/24-48 h. The bacteria, Staphylococcus aureus ATCC 6538, Enterococcus faecalis ATCC 29212, Escherichia coli ATCC 8739 and Pseudomonas aeruginosa ATCC 9027, were inoculated in sterile tryptocasein soy broth and incubated at 37 °C/24 h.
Inoculum preparation
First, 100 µL of bacterial and yeast suspensions were individually inoculated in 8 mL of sterile tryptocasein soy broth and sterile Sabouraud dextrose broth and incubated at 37 °C for 24-48 h. The microorganisms were then adjusted to a density of 105 colony-forming units (CFU)/ mL (corresponding to 0.5 McFarland standards). Finally, the suspensions were diluted to 1:1,000 with saline solution [24].
Determination of the minimum inhibitory concentration (MIC)
The antimicrobial activity of the EOs was evaluated by the microdilution technique in 96-well plates to determine the MIC. First, 50 µL of sterile tryptocasein soy broth (for bacteria) [24] and sterile Sabouraud dextrose broth (for yeasts) [25] were pipetted into 96-well plates. Then, 50 µL of EOTA, EODB, EOTP and EOEG were added, and a serial dilution of each extract was subsequently carried out to obtain concentrations of 500, 250, 125, 62.5, 31.25, 15.6, 7.8, 3.9, 1.95 and 0.97 µg/mL. Finally, 50 µL of the 1:1,000 dilution of bacterial or yeast inoculate was added and incubated at 37 °C/24 h. As positive inhibition controls, we used fluconazole and itraconazole (250 to 0.12 µg/mL) for yeasts and ciprofloxacin (100 to 0.95 µg/mL) for bacteria. The MIC was determined at an absorbance of 625 nm. The activity of the EOs was compared with those of the respective controls; all tests were carried out six times.
Antioxidant activity 2,2-Diphenyl-1-picrylhydrazyl DPPH assay
The DPPH test was performed according to the method of Williams [26], with modifications. The reaction mixture contained 100 µL of 0.208 mM DPPH and 100 µL of the EOs dissolved in methanol [400-12.5 µg/mL]. The negative control consisted of 100 µL of 0.208 mM DPPH with 100 µL methanol. We used TROLOX (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid; 0-40 µg/mL) as a positive control. Absorbance was determined at a wavelength of 517 nm after 20 min in the dark. The reductive capacity of the EOs was determined using the following equation:
where Acontrol is the absorbance of the negative control, and AEO is the absorbance of the EO. The concentrations of the samples responsible for a 50 % decrease in the initial activity of the DPPH free radical (IC50) were calculated by linear regression.
Antioxidant activity ABTS assay
The radical scavenging capacity of the EOs was determined with ABTS (2,2’-azinobis-3-ethylbenzothiazoline-6-sulfonic acid) as described elsewhere [27]. An ABTS+radical solution was prepared by mixing 7 mM ABTS solution and 2.45 mM potassium persulphate (K2S2O8) in a 1:1 (v/v) ratio. The solution was incubated at room temperature in the dark for 12 h and subsequently diluted with water to obtain an emerald-green solution with an absorbance close to 1,000. The negative control consisted of 20 μL methanol and 180 μL ABTS+; TROLOX was used as a positive control (0-40 µg/mL). The assay was performed in a 96-well plate, where 20 μL of EO dissolved in methanol in a range of 500-100 μg/mL was mixed with 180 μL ABTS+ solution, incubated for 20 min at room temperature in the dark and read at a wavelength of 734 nm. The RSA % was determined according to the following equation:
where Ac is the control absorbance, and As is the sample absorbance. The concentrations of the samples responsible for a 50 % decrease in the initial activity of the ABTS free radical (IC50) were calculated by linear regression.
Statistical analysis
The data obtained between MIC and four EOs against four species of Candida and MIC of four EOs against Gram (+) and Gram (-), species were analyzed, by ANOVA test. The data obtained calculating the DPPH and ABTS indexes were analysed by Tukey’s test. The data was analyzed using statistical program inerSTAT20-a v. 1.3. A p-value of less than 0.05 was considered statistically significant.
Results
Chemical composition of the EOs
The chemical composition of the EOs was determined by GC-MS [28]. We found the three EOs (EOTA, EOTP, EOEG) oxygenated compounds predominate 89.58 %, 69.14 %, 40.59 %, respectively. In the case of EODB the oxygenated compounds represent only 24.8 %. The table 2 is shown for the first time the composition of EOTA. Overall, 34 compounds were identified, accounting for 86.47 % of the oil; the main compounds were piperitone (38.67 %), 1,8-cineole (14.14 %) and α-terpineol (6.38 %).
Table 2 The chemical composition of EOTA.
| Compound | Rt (min) | Relative Abundance (% ± SD) | RIR | RIE |
|---|---|---|---|---|
| Isovaleric acid | 6.11 | 2.37 ± 0.37 | 816 | 808 |
| 2-Methylbutyric acid | 6.76 | 2.45 ± 0.57 | 839 | 838 |
| α-Phellandrene | 10.38 | 0.53 ± 0.01 | 1007 | 1003 |
| p-Cymene | 11.30 | 0.47 ± 0.01 | 1011 | 1022 |
| 1,8-Cineole | 11.62 | 14.14 ± 0.42 | 1023 | 1029 |
| β-cis-Ocimene | 12.45 | 0.19 ± 0.00 | 1024 | 1047 |
| Linalool | 14.83 | 1.07 ± 0.03 | 1082 | 1097 |
| (E)-p-Menth-2-en-1-ol | 15.73 | 0.44 ± 0.06 | 1123 | 1117 |
| cis-p-Menth-2-en-1-ol | 16.60 | 0.39 ± 0.02 | 1118 | 1136 |
| 4-Terpineol | 18.34 | 0.18 ± 0.04 | 1175 | 1173 |
| 3,9-Epoxy-1-p-menthene | 18.70 | 0.12 ± 0.03 | 1178 | 1181 |
| α-Terpineol | 19.03 | 6.38 ± 0.07 | 1172 | 1188 |
| trans-2-Hydroxy-1,8-cineole | 20.68 | 0.11 ± 0.05 | 1228 | 1224 |
| Piperitone | 22.16 | 38.67 ± 0.48 | 1243 | 1257 |
| β-Bourbonene | 27.55 | 0.12 ± 0.00 | 1386 | 1378 |
| β-Elemene | 27.92 | 0.78 ± 0.02 | 1387 | 1387 |
| α-Gurjunene | 28.60 | 0.34 ± 0.00 | 1412 | 1402 |
| Caryophyllene | 29.00 | 1.34 ± 0.00 | 1421 | 1412 |
| α-Bergamotene | 29.77 | 0.23 ± 0.02 | 1427 | 1431 |
| Humulene | 30.40 | 0.25 ± 0.02 | 1454 | 1447 |
| Aromandendrene | 30.70 | 0.41 ± 0.01 | 1455 | 1454 |
| α-Muurolene | 32.39 | 0.13 ± 0.03 | 1494 | 1496 |
| δ-Cadinene | 33.33 | 4.11 ± 0.08 | 1514 | 1520 |
| Elemol | 34.36 | 0.86 ± 0.03 | 1535 | 1545 |
| Palustrol | 34.98 | 0.80 ± 0.03 | 1562 | 1561 |
| Spathulenol | 35.41 | 0.22 ± 0.02 | 1569 | 1571 |
| Guaiol | 36.23 | 1.95 ± 0.03 | 1588 | 1592 |
| Ledol | 36.37 | 0.43 ± 0.00 | 1597 | 1595 |
| 2-(4a,8-Diethyl-2,3,4,4a,5,6,7,8-octahydro-2-naphthalenyl)-2-propanol | 36.56 | 1.14 ± 0.02 | 1598 | 1600 |
| Agarospirol | 37.24 | 0.55 ± 0.08 | 1631 | 1619 |
| δ-Cadinol | 38.06 | 0.28 ± 0.12 | 1646 | 1641 |
| β-Eudesmol | 38.15 | 0.18 ± 0.01 | 1644 | 1644 |
| α-Cadinol | 38.36 | 0.38 ± 0.03 | 1641 | 1650 |
| Bisabolol | 39.49 | 2.06 ± 0.05 | 1683 | 1681 |
| Total identified | 86.47 | |||
| Total unidentified | 13.53 |
Retention time (Rt), retention indexes in the literature (RIR), and retention indexes calculated (RIE), Standard Deviation (SD) duplicated analysis.
For EOTP, 21 constituents were determined [23], according for 87.49 % of the EO (Table 3); the main compounds were dihydrotagetone (25.77 %) and verbenone (31.13 %).
Table 3 The chemical composition of EOTP.
| Compound | Rt (min) | Relative Abundance (% ± SD) | RIR | RIE |
|---|---|---|---|---|
| 3-Hexenol-1-ol | 5.66 | 0.16 ± 0.00 | 838 | 806.3 |
| β-Phellandrene | 10.03 | 0.37 ± 0.04 | 964 | 957.0 |
| β-Pinene | 10.13 | 0.32 ± 0.02 | 961.7 | 960.3 |
| β-Myrcene | 10.86 | 0.21 ± 0.01 | 979 | 985.6 |
| α-Phellandrene | 11.40 | 0.23 ± 0.00 | 997 | 1000.0 |
| 1,8-Cineole | 12.63 | 1.46 ± 0.02 | 1023 | 1028.2 |
| trans-β-Ocimene | 13.06 | 2.10 ± 0.12 | 1034 | 1037.1 |
| Dihydrotagetone | 13.87 | 25.77 ± 1.57 | 1055 | 1054.1 |
| Chrysanthenone | 17.20 | 0.31 ± 0.10 | 1099 | 1123 |
| Neo-allo-ocimene | 17.46 | 0.17 ± 0.09 | 1131 | 1128.4 |
| Tagetone | 18.70 | 19.76 ± 1.47 | 1124 | 1153 |
| 4-Terpineol | 19.75 | 0.11 ± 0.02 | 1161 | 1188.5 |
| α-Terpineol | 20.42 | 0.55 ± 0.01 | 1172 | 1188.5 |
| 2-Ethylidene-6-methyl-3,5-heptadienal | 21.22 | 0.37 ± 0.05 | 1182 | 1205 |
| Verbenone | 22.95 | 31.13 ± 3.19 | 1228 | 1242.4 |
| Thymol | 23.47 | 0.14 ± 0.06 | 1266 | 1253.6 |
| Isopiperitenone | 24.32 | 2.31 ± 0.34 | 1249 | 1271.9 |
| Eugenol | 29.96 | 1.46 ± 0.05 | 1392 | 1393.3 |
| Caryophyllene | 31.06 | 0.34 ± 0.04 | 1424 | 1418 |
| p-Cresol | 33.12 | 0.11 ± 0.07 | 1503.9 | 1474.1 |
| Elemol | 36.61 | 0.10 ± 0.01 | 1535 | 1551.2 |
| Total identified | 87.49 | |||
| Total unidentified | 12.51 |
Retention time (Rt), retention indexes in the literature (RIR), and retention indexes calculated (RIE).
Standard Deviation (SD) duplicated analysis. This composition was reported for González-Velasco [23].
In EODB, we identified 46 compounds (Table 4), accounting for 65.98 % of the total EO; the main component was β-pinene (27.25 %), followed by tau-cadinol (6.73 %), β-myrcene (6.23 %) and camphene (3.85 %).
Table 4 The chemical composition of EODB.
| Compound | Rt (min) | Relative Abundance (% ± SD) | RIR | RIE |
|---|---|---|---|---|
| (E)-2-Hexenal | 7.35 | 0.12 ± 0.00 | 822.4 | 810.8 |
| Camphene | 11.25 | 3.85 ± 0.09 | 943 | 926.3 |
| Benzaldehyde | 11.79 | 0.36 ± 0.01 | 927.2 | 942.5 |
| β-Pinene | 12.59 | 27.25 ± 0.53 | 961 | 966.1 |
| β-Myrcene | 13.29 | 6.23 ± 0.04 | 981 | 986.9 |
| α-Phellandrene | 13.89 | 0.06 ± 0.01 | 997 | 1003.1 |
| (3E)-3-Hexenyl acetate | 14.07 | 0.05 ± 0.01 | 983 | 1006.8 |
| 3-methyl-3-vinylciclohexanone | 14.23 | 0.03 ± 0.01 | 1115 | 1009.9 |
| α-Terpinene | 14.49 | 0.04 ± 0.01 | 1008 | 1015.2 |
| p-Cymene | 14.89 | 0.05 ± 0.00 | 1025 | 1023.2 |
| Limonene | 15.10 | 1.88 ± 0.01 | 1018 | 1027.3 |
| 1,8-Cineole | 15.23 | 0.25 ± 0.01 | 1020 | 1030.0 |
| β-Ocimene | 16.11 | 1.15 ± 0.09 | 1024 | 1047.5 |
| γ-Terpinene | 16.62 | 0.06 ± 0.02 | 1047 | 1057.6 |
| trans-Sabinene hydrate | 17.05 | 0.08 ± 0.00 | 1050 | 1066.3 |
| Terpinoleno | 18.13 | 0.11 ± 0.00 | 1080 | 1087.7 |
| Linalool | 18.71 | 1.14 ± 0.06 | 1082 | 1099.3 |
| Pinocarveol | 20.66 | 0.10 ± 0.02 | 1143 | 1137.7 |
| Camphor | 20.97 | 0.14 ± 0.01 | 1146 | 1143.7 |
| Endo-Borneol | 22.04 | 0.15 ± 0.01 | 1148 | 1164.7 |
| 4-Terpinenol | 22.62 | 0.25 ± 0.04 | 1162 | 1176.1 |
| α-Terpineol | 23.29 | 0.70 ± 0.01 | 1172 | 1189.1 |
| Myrtenol | 23.58 | 0.17 ± 0.02 | 1212.8 | 1195.0 |
| cis-3-Hexenyl valerate | 25.34 | 0.05 ± 0.01 | 1243 | 1232.0 |
| Bornyl acetate | 27.97 | 2.34 ± 0.04 | 1270 | 1287.9 |
| Lavandulyl acetate | 28.18 | 0.13 ± 0.00 | 1292 | 1292.2 |
| Myrtenyl acetate | 29.84 | 0.21 ± 0.08 | 1299 | 1327.6 |
| δ-Elemene | 30.43 | 0.62 ± 0.03 | 1334 | 1340.1 |
| Eugenol | 31.29 | 0.12 ± 0.01 | 1363 | 1358.4 |
| Methyl cinnamate | 32.46 | 0.54 ± 0.01 | 1380 | 1383.3 |
| β-Elemene | 32.94 | 0.12 ± 0.10 | 1387 | 1393.5 |
| Caryophyllene | 34.21 | 0.77 ± 0.00 | 1421 | 1422.8 |
| Humulene | 35.73 | 0.33 ± 0.05 | 1454 | 1459.0 |
| γ-Muurolene | 36.71 | 0.19 ± 0.01 | 1471 | 1481.8 |
| δ-cadinene | 38.03 | 0.20 ± 0.02 | 1514 | 1513.2 |
| 6-Epishyobunone | 38.31 | 2.06 ± 0.16 | 1538 | 1519.8 |
| 6-Epi-shyobunol | 38.57 | 0.68 ± 0.00 | 1555 | 1525.9 |
| Elemol | 39.76 | 1.09 ± 0.14 | 1535 | 1554.2 |
| Elemicin | 39.98 | 0.14 ± 0.06 | 1531 | 1559.2 |
| Caryophyllene oxide | 41.26 | 0.78 ±0.09 | 1575 | 1589.5 |
| Viridiflorol | 41.60 | 0.55 ± 0.08 | 1594 | 1597.6 |
| Guaiol | 41.80 | 1.48 ± 0.07 | 1588 | 1602.4 |
| Dehydroxy-isocalamendiol | 42.17 | 2.06 ± 0.24 | 1593 | 1612.2 |
| tau-cadinol | 43.54 | 6.73 ± 0.52 | 1628 | 1647.8 |
| 7R,8R-8-Hydroxy-4-isopropylidene-7-methylbicyclo[5.3.1]undec-1-ene | 46.59 | 0.40 ± 0.01 | 1754 | 1727.1 |
| Isocalamendiol | 47.55 | 0.18 ± 0.04 | 1725 | 1752.2 |
| Total identified | 65.98 | |||
| Total unidentified | 34.02 |
Retention time (Rt), retention indexes in the literature (RIR), and retention indexes calculated (RIE). Standard Deviation (SD) duplicated analysis.
Finally, 45 compounds were determined in EOEG, corresponding to a total of 54.00% (Table 5), the major compounds were α-cadinol (7.78 %), bornyl acetate (6.45 %), and caryophyllene oxide (5.96 %).
Table 5 The chemical composition of EOEG.
| Compound | Rt (min) | Relative Abundance (% ± SD) | RIR | RIE |
|---|---|---|---|---|
| β-Pinene | 9.17 | 0.45 ± 0.01 | 961.7 | 949 |
| Myrcene | 9.90 | 0.23 ± 0.00 | 981 | 983 |
| α-Phellandrene | 10.38 | 0.12 ± 0.01 | 1007 | 1002 |
| p-Cymene | 11.27 | 1.16 ± 0.06 | 1025.4 | 1021 |
| Limonene | 11.44 | 0.19 ± 0.00 | 1018 | 1025 |
| 1,8-Cineole | 11.55 | 0.02 ± 0.01 | 1023 | 1027 |
| trans-β-Ocimene | 12.46 | 0.03 ± 0.00 | 1034 | 1046 |
| Linalool | 14.83 | 0.25 ± 0.02 | 1081 | 1085 |
| Fenchol | 15.31 | 0.21 ± 0.03 | 1100 | 1107 |
| Perillen | 15.59 | 0.12 ± 0.01 | 1109 | 1113 |
| (E)-p-2-Menthen-1-ol | 15.72 | 0.25 ± 0.00 | 1123 | 1116 |
| α-Campholenal | 15.94 | 0.11 ± 0.01 | 1120 | 1121 |
| cis-2-p-Menthen-1-ol | 16.60 | 0.19 ± 0.02 | 1118 | 1135 |
| (Z)-β-Terpineol | 16.87 | 0.27 ± 0.00 | 1125 | 1141 |
| Endo-Borneol | 17.73 | 0.41 ± 0.03 | 1148 | 1159 |
| Terpinen-4-ol | 18.32 | 0.18 ± 0.01 | 1175 | 1172 |
| α-Terpineol | 18.95 | 2.03 ± 0.04 | 1172 | 1186 |
| cis-Sabinol | 19.47 | 0.74 ± 0.08 | 1179 | 1197 |
| (E)-Carveol | 19.81 | 0.37 ± 0.03 | 1206 | 1204 |
| cis-Carveol | 20.33 | 0.49 ± 0.08 | 1207 | 1216 |
| Methylthymol | 21.04 | 0.96 ± 0.02 | 1215 | 1232 |
| Bornyl acetate | 23.32 | 6.45 ± 0.14 | 1285 | 1283 |
| Carvacrol | 24.23 | 0.30 ± 0.01 | 1278 | 1303 |
| Myrtenyl acetate | 24.85 | 1.70 ± 0.21 | 1306 | 1317 |
| α-Cubebene | 26.07 | 0.09 ± 0.02 | 1350 | 1345 |
| α-Copaene | 27.17 | 0.17 ± 0.01 | 1376 | 1369 |
| β-Bourbonene | 27.54 | 0.19 ± 0.01 | 1386 | 1378 |
| Alloaromadendrene | 29.79 | 0.69 ± 0.03 | 1459 | 1431 |
| Aristolene | 30.02 | 0.22 ± 0.04 | 1423 | 1437 |
| α-Curcumene | 31.73 | 1.60 ± 0.03 | 1472 | 1479 |
| Carvacryl propionate | 31.91 | 0.43 ± 0.15 | 1484 | |
| β-Bisabolene | 32.13 | 0.81 ± 0.07 | 1500 | 1489 |
| α-Muurolene | 32.39 | 0.96 ± 0.03 | 1494 | 1496 |
| γ-Cadinene | 32.91 | 1.70 ± 0.03 | 1505 | 1509 |
| δ-Cadinene | 33.34 | 3.93 ± 0.03 | 1514 | 1519 |
| Nerolidol | 34.99 | 0.90 ± 0.04 | 1545 | 1560 |
| Spathulenol | 35.42 | 2.18 ± 0.01 | 1577 | 1571 |
| Caryophyllene oxide | 35.61 | 5.96 ± 0.35 | 1576 | 1576 |
| Ledol | 35.94 | 1.77 ± 0.08 | 1597 | 1584 |
| (4-tert-Butylphenoxy)methyl acetate | 36.44 | 0.21 ± 0.09 | 1563 | 1597 |
| Humulene-1,2-epoxide | 36.59 | 0.47 ± 0.06 | 1601 | 1600 |
| Cubenol | 37.35 | 0.76 ± 0.09 | 1631 | 1621 |
| tau-Muurolol | 37.89 | 4.88 ± 0.24 | 1628 | 1636 |
| α-Muurolol | 38.05 | 1.08 ± 0.21 | 1646 | 1641 |
| α-Cadinol | 38.40 | 7.78 ± 0.33 | 1641 | 1650 |
| Total identified | 54.00 | |||
| Total unidentified | 46.00 |
Retention time (Rt), retention indexes in the literature (RIR), and retention indexes calculated (RIE). Standard Deviation (SD) duplicated analysis.
Minimum inhibitory concentration
The antimicrobial activity of the four oils was tested in vitro on four yeasts, two Gram (+) bacteria and two Gram (-) bacteria. As controls were used fluconazole and itraconazole for yeasts and ciprofloxacin for bacteria. The results (table 6) showed that any of the EOs inhibited the growth of C. krusei. However the other three yeasts were sensitive to all EOs, and the highest antimicrobial activity was found against C. albicans, with an inhibition concentration of 62.5 µg/mL. C. tropicalis was inhibited by EOTA, EOTP and EOEG and the oils had activity on C. glabrata at 250 µg/mL. It should be noted that the EOs inhibited the growth of three yeasts examined, with MIC values ranging from 62.5-250 µg/mL, highlighting the inhibitory activity against C. albicans and C. tropicalis (Table 6).
Table 6 Minimum inhibitory concentration of four EOs against four species of Candida.
| Essential oils | MIC of yeast [µg/mL] | |||
|---|---|---|---|---|
| C. krusei | C. glabrata | C. tropicalis | C. albicans | |
| EOTA | 500 | 250 | 125 | 62.5 |
| EODB | 500 | 250 | 250 | 62.5 |
| EOTP | 500 | 250 | 125 | 62.5 |
| EOEG | 500 | 250 | 125 | 62.5 |
| Fluconazole | 0.97 | 1.95 | 0.97 | 0.24 |
| Itraconazole | 0.48 | 0.12 | 0.12 | 0.12 |
p value using ANOVA test p=0.0607
Table 7 shows the antibacterial activities of the EOs, with MIC values ranging from 125-500 μg/mL. The four oils presented mean inhibition of S. aureus and P. aeruginosa at a concentration of 125 μg/mL, except for EOTA, which inhibited P. aeruginosa at 500 μg/mL. However, the activity of EOTA against E. coli was highest at a concentration of 250 μg/mL with respect to EODB, EOTP and EOEG. In contrast, any EOs inhibited the growth of E. faecalis.
Table 7 Minimum inhibitory concentration of four EOs against Gram (+) and Gram (-), species.
| EOs | MIC of bacteria [µg/mL] | |||
|---|---|---|---|---|
| Gram (+) | Gram (-) | |||
| S. aureus | E. faecalis | E. coli | P. aeruginosa | |
| EOTA | 125 | 500 | 250 | 500 |
| EODB | 125 | 500 | 500 | 125 |
| EOTP | 125 | 500 | 500 | 125 |
| EOEG | 125 | 500 | NA | 125 |
| Ciprofloxacin | 0.19 | 0.19 | 0.095 | 0.19 |
NA (not activity). p value using ANOVA test p=0.1104
Antioxidant activity (DPPH and ABTS)
The antioxidant activities of the EOs were calculated in terms of the radical scavenging activity (RSA) %, which reflects the capacity of the EOs to reduce the concentrations of the radicals DPPH and ABTS. The EOs EOTA, EODB and EOTP showed antioxidant activity. With DPPH the IC50 values were 0.814, 1.195 and 1.050 mg/mL, respectively, and with ABTS IC50 values were 0.183, 0.252, 0.137. However, EOEG had a lower antioxidant activity DPPH (IC50 = 3.480 mg/mL) and ABTS (IC50= 0.410 mg/mL) (Table 8).
Table 8 DPPH and ABTs radical scavenging activity of EOs.
| EOs | DPPH | ABTS | ||
|---|---|---|---|---|
| RSA ± SE % | IC50 [mg/mL] | RSA ± SE % | IC50 [mg/mL] | |
| EOTA | 21 ± 1.61a | 0.814 | 14 ± 0.54ab | 0.183 |
| EODB | 20 ± 0.46a | 1.195 | 10 ± 0.44b | 0.252 |
| EOTP | 21 ± 0.32 a | 1.050 | 22 ± 0.14a | 0.137 |
| EOEG | 6 ± 0.091 b | 3.480 | 7 ± 0.05b | 0.410 |
| Trolox | 86 ± 0.70 | 0.005 | 90 ± 2.25 | 0.002 |
DPPH (1,1'-diphenyl-2-picrylhydrazine), ABTS (2,2’-azinobis-3-ethylbenzothiazoline-6-sulfonic acid, RSA (radical scavenging activity). p value between EOs using Tukey´s test DPPH p<0.003 and ABTS p<0.009. Means not joined by the same letter show significant differences.
Discussion
Infectious diseases caused by microorganisms and their resistance to antimicrobials have increased the costs of hospital care as well as morbidity and mortality, making them some of the major public health problems [1,29]. Between 2016 and 2020, ESKAPE pathogens were the most isolated in hospitals [1], and candidiasis infections have increased in the last three decades [30]. The results of this study on ATCC microorganisms suggest need future research in clinical isolates.
In the present research, the inhibitory activity of EOTA against Candida strains and Gram (+) and Gram (-) bacteria was observed (Tables 6 and 7). This inhibition could be due to piperitone, whose antifungal effect has been described [31]. 1,8-cineol, a compound present in EOTA, inhibits the growth of different Candida species by blocking hyphal transition, the expression of genes that code for ergosterol biosynthesis (ERG11), and efflux pumps (CDR1 and CDR2) [32]. This suggests that in our study, these compounds are responsible for the inhibition of C. glabrata, C. tropicalis and C. albicans.
1,8-cineole also inhibits the growth of Gram (+) and Gram (-) bacteria by modifying the permeability of the bacterial membrane, an intracellular and morphological alteration of the cell, which could explain the inhibition observed for S. aureus and E. coli (125 and 250 µg/mL, respectively) [33].
Dihydrotagetone, the main bioactive component of EOTP, has antibacterial activity against Gram (+) and Gram (-) bacteria and also decreases the oxidative damage of food [34]. In our study, we observed antibacterial activity of EOTP on S. aureus and P. aeruginosa (125 µg/mL), as well as antifungal activity mainly on C. albicans and C. tropicalis (62.5 and 125 µg/mL, respectively).
Another bioactive compound is β-pinene, which is one of the main bioactive compounds identified in EODB, with antibacterial and antifungal activity. Rivas da Silva [35] documented its ability to inhibit the formation of biofilms in C. albicans and, consequently, the growth of this yeast. This effect is similar to the inhibition of growth observed for C. albicans at 62.5 µg/mL, and EODB was also able to inhibit C. glabrata and C. tropicalis (250 µg/mL). In contrast, the antibacterial activity against S. aureus and P. aeruginosa was 125 µg/mL. This may be related to the lipophilic nature of EO, which allows this oil to easily cross the cell wall, causing microbial death [36].
The main components of EOEG are α-cadinol, caryophyllene oxide and tau-Muurolol. This EO showed antifungal activity to the three yeasts studied, highlighting its activity to C. albicans and C. tropicalis; the antibacterial activity was the same as that presented by EODB to S. aureus and P. aeruginosa. Other authors also reported the antimicrobial activity of caryophyllene oxide to S. aureus [37].
The antimicrobial activities of the EOs tested in this study suggest that they can be used as alternatives in the treatment of nosocomial infections caused by multiresistant bacteria [38]. The EOs have antimicrobial activity, especially against different Candida strains. However, some in vivo studies about the toxicity of these oils will be done in the close future.
Oxidative stress is generated by an excess of free radicals and has been associated with different diseases such as atherosclerosis, cancer, hypertension [39] and infections [40]. The antioxidant capacity of the four oils was determined by scavenging-methods using DPPH and ABTS. These oils diminished stable radicals, but their antioxidant activity was low (table 8). Then, these results suggest that the antimicrobial and antioxidant effect are not related. In this study was determined the antioxidant capacity by two assays, because the DPPH assay determined radical dissolved in organic solvents then this assay is suitable to hydrophobic systems, whereas ABTS assay is useful to lipophilic and hydrophilic antioxidant systems [41]
Conclusions
The rise of multidrug resistant microbes has produced high rates of morbidity and mortality, therefore, one of the main challenges of researches is to find new efficient drugs to treat infectious diseases. Many EOs possess antimicrobial activity, which could be attributed to synergism between their components. In the future might explore the activity of the main compounds and the synergistic mechanism.
The results obtained of this study show that EODB, EOEG, EOTP have a low antioxidant activity, which might relate to their oxygenated components.
This study tested the antifungal activity of these EOs against the yeasts, C. albicans, C. glabrata, C. krusei and C. tropicalis, and against the bacteria, Staphylococcus aureus, Enterococcus faecalis, Escherichia coli and Pseudomonas aeruginosa. The results show that EODB, EOEG, EOTP and EOEG inhibited the growth of bacteria Gram+ and Gram - also, they have antimicrobial activity against C. glabrata, C. tropicalis and C. albicans. The results of this study suggest need future research in clinical isolates.
Acknowledgments: We thank to Yessica Elisa Medina Rivera to participate in the obtaining of essential oil of Dalea bicolor, to Sandra Pecina Martínez and Claudia Alejandra Castillo López to obtaining and characterization of the essential oils of Trixis angustifolia and Eupatorium glabratum.
