Evaluation of Antioxidant, Antibacterial, and Antifungal Properties of Satureja hortensis Essential Oil
Background: Satureja is a genus belonging to the aromatic plants of Lamiaceae family. The genus Satureja L. (Lamiaceae) comprises more than 30 species of aromatic herbs and shrubs, widely distributed over the Mediterranean region. The genus is represented by 14 species in Iran of which, eight are endemic. Many species of the genus Satureja are reported to have aromatic and medicinal properties. The leaves, ﬂowers, and stems of this plant are used as herbal tea and in treatment of various ailments in traditional medicine.
Objectives: The aim of this study was to investigate antimicrobial and antioxidant activity of essential oil (EO) of Satureja hortensis (Lamiaceae) that grows in Sabalan Mountain (Ardebil province, Iran).
Materials and Methods: This EO was tested in vitro against two bacterial species by disk and agar well diffusion methods and against four bacterial species and three Candida strains by broth microdilution method. Total phenol, flavonoid, and free radical scavenging activity of EO were evaluated.
Results: Total phenolic content was determined by using Folin-Ciocalteu reagent (32.65 mg/g), which was presented as gallic acid equivalent in 1 g of sample. IC50 of EO and ascorbic acid in DPPH method were respectively 277.9 and 19.34 µg. Minimum inhibitory concentration of the EO of S. hortensis against Salmonella typhimurium, Listeria monocytogenes, Escherichia coli, and Bacillus cereus was respectively 2.5%, 2.5%, 5%, and 2.5%. The inhibition zone of EO in disk and agar well diffusion method showed that inhibitory zone on B. cereus was higher than that on S. typhimurium in both methods and B. cereus was more sensitive to EO. MIC to minimal fungicidal concentration (MFC) ratio of S. hortensis EO against Candida albicans, Candida parapsilosis, and Candida krusei in broth microdilution method were respectively 0.048%:0.048%, 0.024%:0.024%, and 0.012%:0.012%.
Conclusions: The data of the study clearly indicated that the EO of S. hortensis has a strong antioxidant, antibacterial, and antifungal activity.
Keywords: Essential Oils; Antioxidants; Traditional Medicine
Satureja is a genus belonging to the aromatic plants of Lamiaceae family. The genus Satureja L. (Lamiaceae) comprises more than 30 species of aromatic herbs and shrubs, widely distributed over the Mediterranean region. These are annual aromatic plants that grow in arid, sunny, rocky habitats (1). The genus is represented by 14 species in Iran of which, eight are endemic. These aromatic species are mainly found in the north, northwestern, and western parts of Iran (2). Many species of the genus Satureja are reported to have aromatic and medicinal properties. The aerial parts of these species have distinctive tastes and can be added to stuffing and sausages. The leaves, ﬂowers, and stems are used as herbal tea and, in traditional medicine, to treat various ailments such as cramps, muscle pains, nausea, and infectious diseases (3). In earlier investigations, Satureja species have been studied with respect to EO composition and have shown to be rich in components such as carvacrol, γ-terpinene, thymol, and p-cymene (4, 5).
We aimed to investigate the antioxidant, antibacterial, and antifungal activities of the isolated EO of Satureja hortensis collected from Sabalan Mountain, Ardebil province, Iran.
3. Materials and Methods
3.1. Plant Material and Preparation of Essential Oil
The aerial part of S. hortensis was collected from Sabalan Mountain, Ardebil, Iran. Air drying of the plant was performed in a shady place for 14 days at room temperature. The plants were verified in Botanical Group of Tehran Medicinal Herbs Research Centre, Tehran University of Medical Science. Plant was extracted via distillation by water for three hours using a Clevenger unit. The EO was dried over anhydrous sodium sulfate (Na2SO4) and stored at 4℃ until further evaluations.
3.2. Flavonoid Contents
Flavonoid contents were estimated according to aluminum chloride colorimetric method. One milliliter of diluted EO of S. hortensis was mixed with 1 mL of 2% AlCl3 methanolic solution. After incubation at room temperature for 15 minutes, the absorbance was measured at 430 nm. Total flavonoids were expressed through the calibration curve of Standard. All samples were analyzed in three replications (6).
3.3. Assay for Total Phenolic Content
Total phenolic constituent of EO of S. hortensis was performed employing the literature methods (7, 8) using Folin-Ciocalteu reagent and gallic acid (both Sigma-Aldrich) as standard. Briefly, 1 mL of various concentration of EO was mixed with Folin-Ciocalteu reagent (5 mL, 1:10 diluted with nanopure water) and incubated in room temperature. After ten minutes, 4 mL of 7.5% Na2CO3 solution was added and the mixture was allowed to stand for 30 minutes with intermittent shaking. Absorbance was measured at 765 nm. The same procedure was repeated for gallic acid and a standard curve was obtained.
3.4. Determination Antioxidant Activity (DPPH Assay)
DPPH (1, 1-diphenyl-2-picrylhydrazyl) is a free radical generating compound which is used to determine the radical scavenging activity of extracts and EOs (9). The DPPH radical scavenging assay has been used widely to evaluate the radical scavenging activity of the different type of antioxidant substances (10). The color changes will allow the detection of the scavenging activity at 517 nm. The half-maximal inhibitory concentration (IC50) is often used to express the amount or concentration of extracts needed to scavenge 50% of the free radicals. Aliquots (50 μL) of various concentrations of the EO were added to 5 mL of a 0.004% methanol solution of DPPH. After a 30 minutes incubation period at room temperature, the absorbance was read against a blank at 517 nm. Inhibition free radical DPPH in percent (I%) was calculated in following way:
I% = [(Ablank – Asample)/Ablank] ×100,
where Ablank is the absorbance of the control reaction (containing all reagents except the test compound) and Asample is the absorbance of the test compound. The antioxidant activity of the extract was expressed as IC50 (11, 12). Ascorbic acid was used as the positive control and all tests were performed in triplicate.
3.5. Tested Microorganisms
Antibacterial activity of EO of S. hortensis was investigated against two gram-negative bacteria (S. typhimurium ATCC 13311, E. coli ATCC 43894) and two gram-positive bacteria (B. cereus ATCC 11178, L. monocytogenes 19118). This EO also was tested against three fungal strains including Candida albicans ATCC 10231, Candida krusei 22113, and Candida parapsilosis ATCC 22019.
3.6. Antimicrobial Bioassay
The in vitro antibacterial activity of EO was performed by disc diffusion, agar well diffusion, and broth microdilution methods. Antifungal activity of EO was evaluated by broth microdilution assay. Briefly, 200 μL of EO was diluted in 1 mL of 10% DMSO. Then serial dilution of EO of S. hortensis was performed.
3.7. Agar Well Diffusion Test
The used agar well technique was based on the well-established method of Deans and Ritchie (1987) with slight modifications. The freshly prepared inoculums (108 CFU/mL) was swabbed all over the surface of the nutrient agar plate (Merck, Germany) using sterile cotton swab. Four wells were bored in the medium with the help of sterile cork-borer having 6-mm diameter and were labeled properly. Then 35 μL of serially diluted EO were added to the wells. Plates were incubated at 37℃ for 24 hours. After incubation, plates were observed for zone of inhibition (13).
3.8. Disk Diffusion Test
This method is presented as a consensus standard by the National Committee for Clinical Laboratory Standards (NCCLS) (2001). EOs were diluted in 10% DMSO to the test concentration (20%, 10%, 5%, and 2.5%). Antimicrobial tests were performed by disc diffusion method using 100 μL of suspension containing 2× 108 CFU/mL of bacteria. The discs (6 mm in diameter) were impregnated with 35 μL of the diluted oil and placed on the inoculated agar. Negative controls were prepared using the same solvents to dissolve the EO. The inoculated plates were kept at 37℃ for 24 hours. Antimicrobial activity was evaluated by measuring the zone of inhibition against the test microorganisms.
3.9. Broth Microdilution Assay
A broth microdilution method was used to determine the minimum inhibitory concentration (MIC) according to the NCCLS (2003). The inocula of the bacterial strains were prepared from overnight broth cultures and suspensions were adjusted to 0.5 McFarland standard turbidity. The 10% DMSO was used to dissolve the EO and then diluted to the highest concentration (200 μL/mL). A serial doubling dilution of the oil was prepared in a 96 well microliter plate in inoculated nutrient broth (the final concentration in each well was adjusted to 2 × 106 CFU/mL). The plate was incubated for 24 hours at 37℃.
3.10. Determination of Minimum Inhibitory Concentration and Minimum Fungicidal Concentration
For determination MIC, microdilution broth method was used based on the Clinical Laboratory Standards Institute (CLSI, M27-A2). Stocks and dilutions of EO were prepared in 10% DMSO. Final concentrations in the microdilution plates ranged from 12.5% to 0.012% (v/v). The microdilution plates were prepared by using the RPMI 1640 broth medium (Sigma) with L-glutamine and without sodium bicarbonate and buffered at pH of 7.0 with 0.165 mol/L of morpholinepropansulfonic acid (MOPS) (Sigma). Yeast suspensions were prepared after Shacking and adjusting to a 0.5 McFarland standard transmittance at a wavelength of 530 nm. The final yielded inoculum was of 0.5 ×103 to 2.5 × 103 cells/mL. Two wells served as the growth control and sterility check. MICs were visually determined at 24 hours of incubation at 35℃, and were observed for the presence or absence of growth. The growth in each well was compared with that of the growth control EO-free well (14).
The oil isolated by hydrodistillation from the aerial part of S. hortensis was found to be yellow liquid and was obtained in yields of 0.9% (v/w).
4.1. Total Phenolic and Flavonoid Contents
The total phenols content, which were measured by Folin-Ciocalteu reagent in terms of gallic acid equivalent (standard curve equation: y = 29.857x + 0.0437; R² = 0.9968), was 32.65 mg/g, presented as gallic acid equivalent in mg/g EO.
4.2. DPPH Assay
Free radical scavenging activity of EO of S. hortensis was measured by DPPH assay. Free radical scavenging increased by increasing EO concentration. The concentration of EO of S. hortensis resulting in 50% inhibition of the free radical (IC50) is shown in Figure 1. The IC50 level of EO of S. hortensis was higher than standard.
The Concentration of Satureja Essential Oil and Ascorbic Acid Resulting in 50% Inhibition of the Free Radical
4.3. Disk Diffusion and Agar Well Diffusion Methods
The antimicrobial activity of EO of S. hortensis against microorganisms was examined in the present study and its potency was assessed by the presence or absence of inhibition zone and MIC values. The results are given in Table 1. The data of the study clearly indicated that the EO has strong antibacterial and antifungal activities.
Inhibition Zone of Different Concentrations of Satureja hortensis Essential Oil on Bacterial Strains in Disk and Agar Well Diffusion Method a
4.4. Broth Microdilution Assay
The data of the study clearly indicated that the EO of Satureja had antibacterial activity against a number of bacteria. MIC of EO of S. hortensis against S. typhimurium, L. monocytogenes, E. coli, and B. cereus was respectively 2.5%, 2.5%, 5%, and 2.5%.
4.5. Antifungal Assay
Table 2 illustrates the inhibitory activity of EO on the growth of different Candida species including C. albicans, C. parapsilosis, and C. krusei. As shown in table, the lower MIC was related to C. krusei with MIC of 0.012 μL/mL.
Antifungal Testing of Satureja hortensis Essential Oil by Broth Microdilution Method a,b
Plant’s EOs are a potentially useful source of antimicrobial compounds. EOs are natural products extracted from vegetal materials that can be used as natural additives in many foods because of their antibacterial, antifungal, antioxidant, and anticarcinogenic properties (15). The data obtained from the disc and agar well diffusion methods indicated that the EO displayed a variable degree of antimicrobial activity on tested strains. The data indicated that gram-negative S. typhimurium was more resistant to the EO and has smaller inhibition zone. Skocibusic et al. reported that inhibition zone of EO of S. hortensis on B. cereus at 10 and 20 μL per disk was 14 and 23 mm, respectively. Similar to our study, The inhibitory eﬀect increased by increasing oil concentration (16). The results of MIC indicate that the oil exhibited varying levels of antimicrobial activity against the investigated food pathogens in a range of 2.5% to 5%. The higher level of MIC was related to the E. coli. Skocibusic et al. reported that S. typhimurium was more resistant to EO than E. coli and B. cereus were (16). In previous studies, the EO of S. hortensis L. showed antifungal activity against phytopathogenic (17) and food spoilage fungi (18). The major component of this oil, carvacrol, is capable of inhibiting aflatoxin production by Aspergillus parasiticus (19) and Aspergillus flavus in a liquid medium and tomato paste (20, 21). The same authors suggested that carvacrol could be useful in controlling aflatoxin contamination of susceptible crops. Adiguzel et al. reported that the oil of S. hortensis exhibited high antifungal activity against Aspergillus niger (MIC/minimal bactericidal concentration [MBC], 0.78 μL/mL), Saccharomyces cerevisiae (MIC/MBC, 0.39/0.20 μL/mL), and C. albicans (MIC/MBC, 0.20 μL/mL). The oil from Turkey did not show any effect against C. albicans (18). According to our results, EO of S. hortensis collected in Sabalan Mountain showed higher activity in comparison to the results from previous studies. The EO of S. hortensis can be considered as a useful natural agent for the treatment of candidiasis. The antioxidant activity of the EO of S. hortensis indicates that the EO has a protective effect against radical oxygen species and therefore, it can be used as a natural preservative ingredient in the food or pharmaceutical industry.
The authors gratefully acknowledge the Microbiology Department of Veterinary Faculty of Tabriz University, Iran, for providing microbial strains.
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|Microorganism||Concentrations of Satureja hortensis Essential Oil|
a Data are presented in millimeter.
a Abbreviations: MIC, minimum inhibitory concentration; and MFC, minimum fungicidal concentration.
b Data are presented as %.