Antioxidant and antimicrobial activities of fennel, ginger, oregano and thyme essential oils

Funding information Istanbul TeknikÜniversitesi,Grant/Award Number: Project no38819 Abstract In this study, the aim was to evaluate the antimicrobial and antioxidant activities of thyme (Thymus vulgaris), oregano (Origanum vulgare), ginger (Zingiber officinale) and fennel (Foeniculum vulgare) essential oils in addition to their chemical compositions. Based on the results of gas chromatography-mass spectrometry (GC—MS) analysis, major components were thymol and p-cymene in thyme, carvacrol, and p-cymene in oregano, α-zingiberene and ar-curcumene in ginger and (E)-anethole in fennel essential oils. Essential oils were investigated for their antimicrobial activities by agar well diffusion and broth microdilution methods against Campylobacter jejuni and Campylobacter coli. The inhibition zone diameters varied from 9.2 ± 0.7 to 28.7 ± 2.1 mm for C. jejuni and 14.7 ± 2.0 to 27.8 ± 2.8 mm for C. coli. While the minimum inhibitory concentrations (MICs) were lower for thyme and oregano EOs (5.65–43.20 μg/ml), the highest MIC valuewas obtained in fennel EOagainstC. jejuni (28530 μg/ml). Total phenolic contents and antioxidant activities of these essential oils were evaluated by using Folin Ciocalteu, 1,1-diphenyl-2-picrylhydrazyl (DPPH), Cupric Reducing Antioxidant Capacity (CUPRAC) and 2, 2-azinobis(3-ethylbenzo-thiazoline)-6-sulphonic acid (ABTS) methods. The total phenolic content of the essential oils ranged between 7.72 (ginger) to 193 (thyme) mg GAE/L. Antioxidant activities of thyme and oregano were found to be the highest according to theABTSmethod,whereas thymewas found to be the highest by the CUPRACmethod and ginger by the DPPHmethod.

of synthetic preservatives because of consumers' negative attitudes (Burt, 2004;Smith-Palmer et al., 1998). A well-known group of medicinal plants, Thymus species, have several biological and pharmacological properties, and their essential oils can be used as nutraceuticals and natural preservatives in foods (Mancini et al., 2015). Fennel (Foeniculum vulgare Mill.; Fam. Umbelliferae) is also an important medicinal and aromatic plant with anti-inflammatory and antimicrobial effects (Mahfouz & Sharaf-Eldin, 2007). One of the oldest and the most widely used herbs, ginger, belongs to the Zingiberaceae family (Singh et al., 2008).
Oregano is another important aromatic plant that is commonly used for flavouring purposes and its essential oil has been used for centuries due to its medicinal properties (Quiroga et al., 2011). The antimicrobial activity of all these medicinal plants' essential oils is mainly associated with their chemical composition and phytochemicals (Borugȃ et al., 2014). In this study, EOs from fennel, ginger, oregano and thyme have been selected considering their wide application due to their characteristic aroma and flavour, as well as their antimicrobial and antioxidant potential and medicinal properties.
Apart from having antimicrobial properties, many of the EOs have also been characterized as natural antioxidants (Ruberto & Baratta, 2000). Since they are natural antioxidants with the virtue of being non-toxic, research on EOs has been gaining more attention due to their potential use as preservatives, supplements, cosmeceuticals or nutraceuticals. This situation is incredibly worthwhile since most prevalent synthetic antioxidants are assumed to have potential adverse health effects. Many herbs and spices such as thyme, clove, cinnamon, rosemary, oregano and plant extracts such as tea contain components with antioxidant activities and are known to effectively hinder oxidation (Amorati et al., 2013;Brewer, 2011). For example, oregano EO, which is thymol and carvacrol-rich, has been shown to have a significant antioxidant effect on the process of lard oxidation (Kulisic et al., 2004). Another study by Tongnuanchan et al. (2013) revealed that root essential oils of ginger, turmeric and plai provided antioxidant activity when incorporated into fish skin gelatin.
There are several methods for determining the antimicrobial activities of EOs against different microorganisms. The most common methods are to measure the zone diameter (Friedman et al., 2002) and determine the minimum inhibition concentration (MIC). The type of microorganisms, test medium and concentration of EOs are important factors in implementing antimicrobial activity test methods (Zaika, 1998). It is also essential to explain the particular mechanisms of antimicrobial action as well as their antimicrobial effects. EOs and their components have activity against a wide range of targets, especially the membrane and cytoplasm, by changing the cell morphology (Nazzaro et al., 2013). The mechanism of their antimicrobial activity was defined by their hydrophobicity resulting in increased cell permeability and leakage of cell constituents (Diao et al., 2014;Lambert et al., 2001).
Moreover, the antimicrobial activity of EOs has correlated with the diffusion ability of EOs (diffusion coefficient, zeta potential and droplet size of EOs) through the cell membrane of microorganisms (Mutlu-Ingok, Firtin et al., 2020b). Different factors affect the antimicrobial activity of EOs, such as microbial cultures, geographical origin, plant part from which EO was derived, extraction method and harvesting time (Mutlu-Ingok, Devecioglu et al., 2020a). Moreover, clinical and standard strains of microorganisms differed in terms of their sensitivities of EOs (Mutlu-Ingok et al., 2019).
Depending on the potential of EOs as antimicrobials and antioxidants, they can be alternatives to synthetic antioxidants/antimicrobials for conventionally produced foods. However, it is also vital to perform bacterial-based studies for their safe use. According to our literature survey, several research studies have investigated the antimicrobial activities of EOs; however, they have not been critically tested against Campylobacter spp. Campylobacter is one of the major foodborne pathogens, and raw poultry and its products are considered to be an important source. Although it was reported that Campylobacter spp.
Depending on the increasing number of infections and resistance of microorganisms against antibiotics, essential oils were thought to be a good alternative to control these microorganisms by using their antimicrobial activities.
Another important aspect of this study is that EOs were investigated with respect to their both antimicrobial and antioxidant activities. This is important in terms of determining potential application areas of EOs by baring their biological activities. Also, considering the necessity of relating these biological activities to the chemical composition adds value to this work. In summary, this study aimed to investigate the antimicrobial and antioxidant activities of thyme, oregano, ginger and fennel EOs against Campylobacter jejuni and Campylobacter coli using different methods.

Bacterial strains
C. jejuni (ATCC 33560) and C. coli (NCTC 12525) isolates were used in this study. The bacteria were kept in a medium containing 20% glycerol at -80 • C. Bacterial strains were obtained from Refik Saydam National Type Culture Collection (RSKK), Sıhhiye, Ankara.

Gas chromatography and gas chromatography-mass spectrometry
Gas chromatography (GC) analysis was performed using an Agilent 7890B GC system with a flame ionization detector (FID). The chromatographic separations were achieved by the Agilent HP-Innowax column (60 m × 0.25 mm Ø, with 0.25 μm film thickness). The flow rate of helium as the carrier gas was set as 0.7 ml/min. GC oven temperature was kept as 60 • C for 10 min and increased to 220 • C at a rate of 4 • C/min and then kept constant at 220 • C for 10 min. Finally, the temperature was programmed to increase to 240 • C at a rate of 1 • C/min. The split ratio was 40:1, and the temperatures of injector and flame ionization detectors were set at 250 • C. The relative percentage of essential oil compounds were calculated by using FID chromatograms.
GC-mass spectrometry (GC/MS) analysis was performed using an Agilent 7890B GC coupled with a 5977B MSD (Agilent, Palo Alto, CA; SEM A. S., Istanbul, Turkey). Analytical conditions and the column were the same as both GC/MS and GC/FID. The mass range was recorded from m/z 35 to 450. The temperature of the injector was set to 250 • C.
Mass spectra were taken at 70 eV. Relative retention indices (RRI) were calculated by using alkanes. The identification of EO constituents was achieved using Wiley 9-Nist 11 mass spectral database and standard Alkan series (C 7 -C 40 ). Moreover, results were supported by comparing the retention indices with the literature.

Agar well diffusion assay
Inhibition zone diameters were determined using a previously described method of Deans and Ritchie (1987), with slight modifica-

Broth microdilution assay
The broth microdilution method was used to determine minimum inhibitory concentrations (MICs) (Wiegand et al., 2008

Total phenolic content
The total phenolic content of the EOs was analyzed using the Folin-Ciocalteu assay (Viuda-Martos et al., 2010). Briefly, 300 μl of an ethanolic solution (containing 1% DMSO) of essential oils (EOs; thyme 0.1 mg/ml; oregano 0.5 mg/ml; ginger 5 mg/ml; fennel 10 mg/ml) was added to the test tubes followed by the addition of 2.5 ml 0.2 N Folin-Ciocalteu's reagent and 2 ml sodium carbonate (7.5% w/v). After vortexing, incubation of the tubes was performed at 50 • C for 5 min.
Absorbance was measured at 760 nm and the calculated results were expressed as Gallic Acid Equivalents (GAE) in milligrams per liter of EO.
All samples were analyzed in triplicate.

Determination of total antioxidant activity
Determination of total antioxidant activities of the EOs were done by three methods: DPPH (1,1-diphenyl-2-picrylhydrazyl), CUPRAC (cupric reducing antioxidant capacity) and ABTS (2,20-azino-bis-3-ethylbenzthiazoline-6-sulphonic acid). All analyses were run in triplicate and ethanol was used as a blank. DPPH method was performed according to Kulisic et al. (2005a). 50 μl of ethanolic solution (containing 10% DMSO) of essential oils with varying concentrations (thyme: 0.1-5 g/L; oregano: 0.1-5 g/L; ginger: 0.01-0.1 g/L; fennel: 10-50 mg/ml) was introduced in a tube. After adding 1 ml of 0.004% ethanolic solution of DPPH, the absorbance of the control sample was measured immediately and the rest of the samples were incubated for 1 h in the dark. The IC 50 value, expressed as the sample amount required for decreasing the absorbance of DPPH by 50%, was calculated by plotting % of inhibition against the sample concentration. The percentage inhibition of the DPPH radical was calculated by using the following equation: where A C(0) is the absorbance of the control at t = 0 min and A EO(t) is the absorbance of the essential oil at the end of the incubation time.
Cupper reducing antioxidant capacity (CUPRAC) was determined according to Apak et al. (2004). 100 μl of ethanolic solution (containing 10% DMSO) of essential oils was placed in a tube and mixed with 1 ml of 10 mM CuCl 2 , 7.5 mM neocuproine and 1 M NH 4 Ac (pH 7). Immediately, 1 ml of distilled water was added to the mixture to make the final volume of 4.1 ml. After 30 min of incubation at room temperature, absorbance was read at 450 nm against a reagent blank.

Statistical analysis
All experiments were conducted in triplicate and the obtained data were reported as mean ± standard deviation. Statistical analysis was carried out using SPSS software (version 20.0, SPSS, Chicago, IL, USA). Mean values were compared using one-way analysis of variance (ANOVA) followed by Tukey post hoc test, and p < 0.05 was considered as significant.

Chemical composition of essential oils
The chemical composition of the EOs, which were identified based on GC and GC-MS, is shown in  (Rota et al., 2008), which can also be supported with the findings of the current work. According to the literature, while cymene does not show antibacterial activity individually, because of the lack of hydroxyl group, its synergistic antibacterial activity has been reported with carvacrol and thymol (Ultee et al., 2002). Oregano EO was found to consist of a mixture of several compounds dominated by two, namely carvacrol (69.9%) and p-cymene (17.0%). The composition of oregano EO has been characterized with carvacrol and thymol as the main compounds (Figiel et al., 2010), but the proportions vary widely depending on the plant's geographical origin, different parts of the plants, mode of extraction and harvesting season (Burt, 2004). Recent results showed that antimicrobial activity of oregano EO is characterized by the phenol constituents, thymol and carvacrol and their two precursor monoterpene hydrocarbons, γ-terpinene and p-cymene.
The high content of (E)-anethole (81.4%) in fennel EO observed in this study is closed to the values stated by Viuda-Martos et al. (2011) and Diao et al. (2014). On the other hand, α-fenchone (4.7%) was the second primary compound detected. Limonene, α-pinene, methyl chavicol, and α-phellandrene were also detected as minor components in the corresponding EO.
It has been mentioned that the specific aroma of ginger is predominantly related to the content of zingiberene (Kamaliroostaa et al., 2013). Similar to other studies, α-zingiberene (31.6%), belonging to the sesquiterpene hydrocarbons, was the main compound of ginger EO in our study (Kamaliroostaa et al., 2013;Sasidharan & Menon, 2010).
Zingiberene (31.6%) was followed by ar-curcumene (19.30%) and βbisabolene (9.2%). While minor variations were detected, the tested essential oils' composition was generally in agreement with the values reported by different authors.

Antimicrobial activities of essential oils
The antimicrobial activities of thyme, oregano, ginger and fennel EOs against Campylobacter spp. examined in the current study and their potential was evaluated by the diameter of inhibition zones and minimum inhibition concentrations. Minimum bactericidal concentrations were also detected. The antibacterial activities of essential oils measured by diffusion and dilution methods are shown in Tables 2 and 3.
Some studies refer to the high antimicrobial effect of EOs and plant extracts against Campylobacter spp. (Aslim & Yucel, 1998;Friedman et al., 2002). However, there are also controversial results reported, which indicates the resistance of Campylobacter spp. against EOs and plant extracts (Klančnik et al., 2010, Smith-Palmer et al., 1998. It has been suggested that for plant EOs, inhibition zone diameter not only depends on the uniform diffusion of the antimicrobial agent into the test medium but also the release of vapor from the oil on bacteria (Friedman et al., 2002).
In  (Wannes, Mhamdi, & Marzouk, 2009) 3 ). c Retention indices relative to standard Alkan series (C 7 -C 40 ), d Peak areas were obtained by GC-FID, e Compounds lower than 1% or not identified. The technical variation of samples did not exceed 10%. 2.1 mm). These differences might be due to the composition, concentration and/or the type of EOs (Aslim & Yucel, 1998). The extraction method, as well as the differences in the strain, were also effective on the antimicrobial activity of EOs. For the orange-based fractions, the diameter of the inhibition zones has been reported to be between 11 and 44 mm against 14 different strains of C. jejuni as published by Nannapaneni et al. (2009). In another study for C. jejuni (ATCC 33560), MIC values ranged from 0.08 to 0.31 μg/ml depending on the rosemary extract formulations (Piskernik et al., 2011).

Diameter of inhibition zone (mm)
The antimicrobial activities of thyme and oregano EOs were compared favorably with streptomycin (Tables 2 and 3 Determination of minimum inhibition and bactericidal concentrations are more sensitive than the agar well diffusion method (Smith-Palmer et al., 1998). The microdilution method is also economical because of less waste of time and resources and provides accurate results. According to Klančnik et al. (2010), MIC values obtained by the microdilution method were the same or lower compared to other dilution methods. It means that the microdilution method results are more susceptible than other antimicrobial activity testing methods.
Minimum inhibition and bactericidal concentrations of thyme, oregano, ginger and fennel EOs obtained by broth microdilution method were also examined to express the antimicrobial activity. As seen in Tables 2 and 3, lower inhibition and bacteriostatic concentrations were required for oregano EO. Thus, C. jejuni and C. coli were the most sensitive to oregano among all tested EOs. Aslim and Yucel (1998) (2002) reported that fennel seed oil was the most ineffective oil compared to cardamon, cumin seed, dill weed, ginger, oregano and thyme EOs. In our study, fennel and ginger EOs showed the lowest antimicrobial effect against C. jejuni and C. coli by both agar diffusion and microdilution assay methods. In contrast with the current study, Friedman et al. (2002) reported ginger EO as effective against C. jejuni.
Numerous studies examined the minimum inhibition and bacteriostatic concentrations of EOs on some pathogen microorganisms, but studies related to Campylobacter spp. are still limited. Moreover, most of the studies are about the antimicrobial effects of EOs but their action mechanism is still unclear (Lambert et al., 2001).

Total phenolic content and antioxidant activities of essential oils
The total phenolic contents of thyme, oregano, fennel and ginger EOs, as presented in Figure 1, are reported as mg gallic acid equivalent/L essential oil. The total phenolic content (TPC) of thyme EO was found to be significantly higher, which corresponds to 193 mg GAE/L while oregano EO had a value of 163 mg GAE/L. On the other hand, fennel was found to have less TPC (17.7 mg GAE/L) than thyme and oregano, while ginger showed the lowest amount among the four EOs (7.72 mg GAE/L). In a similar study by Viuda-Martos et al. (2010), the TPC of EOs from thyme, oregano, rosemary, clove, and sage was investigated.
Their results also showed that thyme and oregano EOs were the rich- Several studies proved that polyphenols from different origins exhibit antioxidant properties (Cai et al., 2004;Zheng & Wang, 2001), and the content of phenolic compounds might be correlated with their antioxidant activities. In this study, three different assays were used for determining the antioxidant capacities of EOs since a single method may not be sufficient to fully understand the antioxidant activity of a substance as each method has several advantages and disadvantages.
DPPH and ABTS methods are based on the reaction of a potential antioxidant with a colored radical (i.e., DPPH• or ABTS• + ), whereas the CUPRAC method is based on the reaction of an antioxidant compound with Cu 2+ ions having oxidizing properties (Apak et al., 2016). These methods are generally considered as useful in screening the antioxidant content of natural extracts or matrices since they are easy, simple and reasonably costly methods (Alam et al., 2013;Amorati & Valgimigli, 2015). On the other hand, biological methods testing antioxidant activity have several advantages such as better reflecting the real situation in biological systems, but they may be expensive, time-consuming and have a more complex mechanism (Frankel & Finley, 2008).
The total antioxidant capacity (TAC) of the EOs with the DPPH assay was demonstrated in Figure 2, where the results are given as the per cent inhibition. The highest antioxidant activity was observed in ginger EO with an IC 50 value of 1.66 g/L. It was followed by thyme and oregano EOs showing a similar inhibition concentration as 4 and 4.34 g/L, respectively. However, no statistically significant difference was observed between these three EOs (p > 0.05). Fennel EO was found to have the weakest antioxidant activity with an IC 50 value of 270 g/L (p < 0.05). The results obtained for thyme and oregano were similar to the results reported by Bozin et al. (2006), indicating that thyme and oregano showed higher antioxidant activities (with similar IC 50 values of 0.19 and 0.17 μg/ml, respectively) than basil EO or the synthetic antioxidant, BHT. Research on the ginger EO showed that this EO exhibited 100% inhibition of DPPH between the concentrations of 5-20 μl/ml (in this study, the concentration range of ginger oil was 0.011-0.114 μl/ml since higher concentrations caused turbidity in the reaction mixture, thus affected the accuracy) (Amorati et al., 2013). In contrast to our findings, IC 50 values determined by DPPH assay of EOs from three fennel cultivars have shown to vary from 0.35 to 15.33 g/L (Shahat et al., 2011), which have relatively higher antioxidant activities than the fennel EO tested in this study. In a recent study, antioxidant properties of eight EOs from thyme, clove buds, cinnamon, lemon balm, cedar, lemon, mandarin and rosemary have been determined to be within the range of 0.25 to 4.60 mg TEAC/g. Among them, clove and thyme EOs were found to be the most antioxidative oils (4.60 and 4.30 mg TEAC/g, respectively) (Olszowy & Dawidowicz, 2016). Furthermore, Deng et al. (2016) Figure 4 and the results are given as the per cent inhibition. According to the results, there was no significant difference between thyme and oregano essential oils (p > 0.05) and they displayed the same IC 50 value detected as 0.08 g/L. These results are in line with the results obtained from the DPPH assay indicating that the same concentrations of essential oils are required to inhibit the 50% of DPPH or ABTS radicals. However, no reliable data could be obtained for ginger and fennel EOs since they interfered with the reaction solutions and became cloudy. On the other hand, the difference between the magnitude of the values obtained by DPPH and ABTS methods may be attributed to the distinctness of the assays. For example, the background color formed due to the matrix exhibits utmost adverse effects on the precision of color-fading reactions such as DPPH and ABTS (Apak et al., 2016). It has been also reported in the literature that IC50 values obtained with different methods may vary significantly.
For example, in a study by Bendaoud et al. (2010), the IC50 value determined by DPPH assay of Schinus molle L. EO was found to be almost 14-fold higher than the IC50 value determined by the ABTS assay. In another study, the IC50 value of pimento EO was app. two-fold higher with DPPH method compared to ABTS method (Padmakumari et al., 2011).
In a study performed by Spagnoletti et al. (2016), the IC 50 value of EO from thyme was determined as 0.58 mg/L, which has markedly lower antioxidant activity than the sample studied in this work. F I G U R E 2 % DPPH radical scavenging activity of thyme, oregano, ginger and fennel essential oils at different concentrations (for thyme and oregano A = 5 g/L, B = 1 g/L, C = 0.5 g/L; for ginger A = 0.1 g/L, B = 0.05 g/L, C = 0.01 g/L; for fennel A = 50 g/L, B = 25 g/L, C = 10 g/L) F I G U R E 3 Cupric ion reducing abilities of thyme, oregano, ginger, and fennel essential oils (*Trolox Equivalent Antioxidant Capacity). Different letters indicate statistically significant differences between EOs (p < 0.05) In contrast with this study, the IC 50 value of thyme oil, measured by the ABTS assay, was reported to be higher than oregano oil (17 and 32 g/L, respectively).
The antioxidant potential of an EO can be more or less predicted by considering its composition. In general, a good antioxidant property is expected if the EO contains high amounts of phenolic compounds (Amorati et al., 2013). However, different fractions of the EOs may act differently in the prevention of oxidation. For example, Kulisic et al. (2005b) examined the effect of different fractions of EOs on lard oxidation inhibition. They revealed that hydrocarbon fraction of several EOs had no effect on the inhibition of oxidation or had a pro-oxidant effect while oxygenated fraction (dominantly containing thymol and carvacrol) of these EOs showed a better effect on the extension of induction time of the lard oxidation.
On the other hand, in some EOs, these two fractions showed a synergistic effect, which means the highest antioxidant activity was obtained when the whole EO is used. In another study, the antioxidant properties of thymol, carvacrol, 6-gingerol, hydroxytyrosol and zingerone were tested in phospholipid liposome systems and a decrease in the peroxidation of phospholipid liposomes was observed when thymol, carvacrol, 6-gingerol and hydroxytyrosol were used. However, zingerone showed only a weak inhibitory effect on the oxidation of phospholipid liposome system (Aeschbach et al., 1994). Considering this information, better antioxidant properties and total phenolic content of thyme F I G U R E 4 Antioxidant activity of thyme, oregano essential oils at different concentration (for thyme and oregano A = 0.1 g/L, B = 0.05 g/L, C = 0.01 g/L) measured by ABTS method and oregano tested in this study may be linked with their thymol and carvacrol content. Ruberto and Baratta (2000) reported that monoterpene hydrocarbons (especially terpinolene, αand γ-terpinene), which are among the tested 99 pure EO components, showed a significant effect on the prevention of oxidation on two lipid model systems.
According to Table 1, the predominant monoterpene hydrocarbons, pcymene, and γ-terpinene are only detected in thyme and oregano EOs.
Thus, their antioxidant activity may also be linked to their p-cymene and γ-terpinene contents.
As a result, although the antioxidant properties of the EOs in this study are mostly in accordance with the literature, their levels and efficacy greatly vary, which may be related to the diversity of the EOs based on plant's geographical origin, different parts of plants, extraction mode and harvesting season (Burt, 2004). In addition to their antioxidant activity, their antimicrobial activities were also crucial for controlling corresponding microorganisms (Mutlu-Ingok & Karbancioglu-Guler, 2017).

CONCLUSIONS
One possible method for preventing or retarding the growth of pathogenic microorganisms and deterioration in agricultural products and foods could be using natural antimicrobials alone or together with other preservation techniques. Although all tested essential oils in the current study exhibited antimicrobial activity against C. jejuni and C. coli, oregano EO exerted the most potent inhibitory effect against Campylobacter spp. On the other hand, thyme and oregano EOs were found to be the most potent antioxidants and rich sources of phenolic compounds among the other EOs. It seems apparent that the antimicrobial activity was primarily associated with the presence of remarkable levels of carvacrol and thymol. The findings of this study showed the potential use of oregano EO as well as other tested EOs for food applications, but high concentrations of EOs may cause organoleptic changes in foods which should be considered in further studies.

CONFLICT OF INTEREST
The authors declare that they have no competing interest.