Introductıon

Aromatic plants constitute a large part of the natural flora and are considered as an important raw material for many branches of industry such as pharmaceutical, flavour and fragrance, perfume and cosmetic (Swamy et al. 2016). Essential oils are volatile, strongly scented and oily mixtures obtained by water vapour distillation from plants or herbal drugs, and are found in the liquid form at room temperature and sometimes they freeze (Özcan et al. 2013). These mixtures may include a wide range of free radical scavenger molecules, such as phenolic compounds, nitrogen compounds, vitamins, terpenoids which are responsible for high antioxidant activity (Elkıran et al. 2018). The use of essential oils for the treatment of various inflammatory diseases such as rheumatism, fever and diabetes suggests that oxidative stress plays a role in human diseases and antioxidant intake may improve human health (Burits et al. 2001; Ekren et al. 2013).

Rutaceae family, which is of economic importance, includes 153 genera and approximately 1800 species in the world (Luo et al. 2010; Aktaş 2017). Citrus L., which is a nutritionally important genus, is a member of the Aurantioideae subfamily in the Rutaceae family and includes many fruit plants such as sour orange, sweet orange, citrons, pummelos, kumquats, lemon, mandarins and grapefruit. This genus is one of the most important horticultural crops with over 80 million tons of agricultural production worldwide (Bourgou et al. 2012; Kumar et al. 2013; Nagano et al. 2018). Citrus plants are indigenous to tropical Asia, but are found in all tropical and subtropical countries. It is an easily obtainable plant widely used in the treatment of various diseases (Suryawanshi 2011). Citrus fruits and fruit juices are sources of bioactive compounds important for human health such as ascorbic acid, flavonoids, phenolic compounds and pectins (Wali et al. 2013). Citrus aurantium L., which belongs to the genus Citrus, known as sour orange or bitter orange is traditionally grown in the Mediterranean region (Sarrou et al. 2013). The essential oils obtained from C. aurantium are used as an antioxidant, antimicrobial, anti-ulcerogenic, neuroprotective, anti-anxiety, anti-inflammatory, anti-cancer, anti-viral and anti-larvicidal agent (Almeida et al. 2015; Radan et al. 2018).

The aim of this study was to determine the composition of essential oils obtained from fruit peel of Citrus aurantium fruits growing in Aydın/Turkey and to evaluate their antimicrobial effects against some highly pathogenic bacteria and fungi.

Materials and Methods

Plant Materials

Fruits of Citrus aurantium plants were obtained as study materials in November and in December 2018 (Fig. 1).

Fig. 1
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Location of Citrus aurantium populations Aydın/Turkey (C1: Central, C2: Germencik, C3: Koçarlı, C4: Nazilli) (https://www.google.com/maps)

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Isolation of Essential Oils and GC-MS Analysis

Essential oils were obtained with a clevenger apparatus and their compositions were determined by GC-MS. The analyses were performed withan Agilent 7890A gas chromatograph coupled with the 5975C inert Mass Selective Detector. Helium was used as a carrier gas. Separation was carried out with a capillary column coated with 5% phenyl-methyl polysiloxane (30.0 m × 250 μm × 0.25 μm). The temperature of injection port with split mode (split ratio 1:10) was set at 250 °C. The GC column temperature was programmed as follows: initial temperature was set at 50 °C and held for 2 min, then raised to 80 °C (held for 2 min), 100 °C (held for 1 min), 150 °C (held for 1 min), 240 °C (held for 1 min) and finally to 270 °C (held for 7 min), with heating rate of 5 °C/min (Rambla et al. 2015; Apaydın 2018). The slower heating rate is supposed to provide better separation of compounds with similar volatility. Mass spectrometer conditions were: ionization voltage of 70 eV and, molecular weight spectrum range was 100–550 Da. The recorded mass spectra were compared with the reference spectra using the computer libraries (Wiley and NIST).

Microorganisms

Four species of gram (+) and gram (−) bacteria, as well as four species of fungi were used in the assays. Enterobacter aerogenes CCM 2531 and Proteus vulgaris FMC 1 were obtained from Fırat University; Gordonia rubripertincta (laboratory isolate), Staphyloccocus cohnii (laboratory isolate) and Yersinia pseudotuberculosis ATCC 911 were acquired from Yeditepe University; Bacillus cereus 702 ROMA and Enterococcus faecalis ATCC 29212 were acquired from Rize University; Escherichia coli ATCC 35218 was obtained from Giresun University; Candida albicans FMC 17 and Candida tropicalis ATCC 13803 were obtained from Fırat University; Candida parapsilosis ATCC 22019 was obtained from Giresun University, and Saccharomyces cerevisiae ATCC 9763 was obtained from Giresun Province Control Laboratory.

Antibacterial and Antifungal Activities

Antibacterial and antifungal activities of the essential oils were estimated by determination of MIC. MIC can be defined as the lowest concentration of the essential oil that prevents the growth of the test microorganisms. For MIC, a broth microdilution assay was used. Two-fold serial dilutions (in DMSO), from 0.01953 to 20 µL/mL of the C. aurantium essential oils, were prepared in a 96-well microplate. Into each well 95 µL of Müller Hinton Broth and 5 µL of the inoculum were added. An aliquot of 100 µL of essential oil was placed into the first well in a plate. Then, 100 µL from the serial dilutions were transferred into seven consecutive wells. The last well containing 195 µL of nutrient broth without compound and inoculum on each row was used as a negative control. Plates were incubated at 30 °C for 48 h for fungi and at 37 °C for 24 h for bacteria (Güllüce et al. 2004).

Results and Discussions

Chemical Composition of the Essential Oils

The components of essential oil from peel of C. aurantium fruits are gathered in Table 1 and the GC-MS chromatograms are presented in Fig. 2, 3, 4 and 5.

Table 1 Composition of the essential oil from C. aurantium fruit peel
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Fig. 2
figure 2

Gas chromatogram of the essential oil of C. aurantium (Central region)

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Fig. 3
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Gas chromatogram of the essential oil of C. aurantium (Germencik region)

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Fig. 4
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Gas chromatogram of the essential oil of C. aurantium (Koçarlı region)

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Fig. 5
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Gas chromatogram of the essential oil of C. aurantium (Nazilli region)

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According to the results of the GC-MS analysis, the essential oil obtained from fruits of C. aurantium grown in the Central region contained the highest amount of dl-limonene (72.51%) and hexasiloxane (13.28%), whereas in the oil samples extracted from fruits picked in the Germencik region limonene (77.27%) and hexasiloxane (6.19%) were found to be in the highest concentration. In the case of the Koçarlı population of Citrus aurantium, essential oil contained mainly limonene (79.77%). Similarly, limonene was the main component of essential oil extracted from fruits grown in the Nazilli region and it constitutes 95.70%, whereas β‑myrcene with concentration of 0.76% was the in the second place (Table 1).

In the earlier studies, chemical composition of essential oil extracted from peel, flowers and leaves of C. aurantium was studied. Bozkurt et al. (2017) found that d‑limonene (89.29%) and linalool (3.43%) were the main components of essential oils from C. aurantium fruit peels purchased from the local markets in Adana/Turkey. Similar conclusions have been drawn by Radan et al. (2018) who determined the chemical composition of the essential oils from C. aurantium fruit peels (region of Split, Dalmatia) and found that limonene constituted from 51.3 to 91.1% and linalool—from 1.5 to 17.2%. Azhdarzadeh and Hojjati (2016) investigated the chemical composition of the essential oils obtained from leaves, and peels of mature and immature C. aurantium fruits from southwest Iran. It was found that concentrations of the following components were the highest: linalool (32.6%) and α‑terpineol (17.24%) in leaves, limonene (81.6%) and linalool (2.19%)—in mature peels, and limonene (59.88%) and alloaromadendrene (5.88%)—in immature peels. Rowshan and Najafian (2015) from Fars Research Center for Agriculture and Natural Resources in Fars province (South of Iran) investigated composition of essential oil obtained from peels of Citrus aurantium fruits harvested in three different periods. They found that limonene (78.14%) and linalool (8.19%) were dominant components in essential oil obtained from peel of immature fruits of green colour, limonene (94.72%) and myrcene (1.87%)—from peel of semi-mature fruits of green colour, and limonene (93.47%) and myrcene (1.78%)—from peel of mature fruits of orange colour. According to Gölükçü et al. (2015) limonene (94.00–94.65%) was the most important component of the oil extracted from Citrus peel. Sevindik et al. (2018) studied the chemical composition of essential oil of C. aurantium flowers collected from Aydın province. As a result of the study, they identified nerolidol (28.07%) and linalool (14.47%) as the most important components. Sarrou et al. (2013) investigated the chemical composition of the essential oils obtained from peel, flowers and leaves of C. aurantium grown in Greece. In the oil from flowers they detected mainly linalool (29.14%) and β‑pinene (19.08%), whereas essential oil from peel contained mainly limonene (94.67%) and myrcene (2.00%). There were differences in composition of essential oils extracted from young and old leaves. Linalool (58.21%) and linalyl acetate (12.42%) dominated in oil from young leaves, and linalool (36.03%) and linalyl acetate (23.00%) were main components of oil from old leaves. Trabelsi et al. (2016) examined the chemical composition of the essential oils obtained from flowers, leaves and fruit peel of C. aurantium grown in Nabeul region of Northern Tunisia. They found that linalyl acetate (53.76%) and linalool (22.11%) had the highest concentration in oil from flowers, linalool (39.74%) and linalyl acetate (25.44%) dominated in oil in leaves, whereas limonene (87.02%) and α‑terpineol (6.74%) were main components in oil from fruit peel. As a result, in our study, it was found that limonene had the highest concentration among all chemical components of the essential oils obtained from C. aurantium fruit peel. Limonene is one of the most common essential oil components of aromatic plants, which is widely used as a flavour and fragrance additive in products such as perfumes, beverages, detergents and soaps. Another application is a component in household cleaning products (Erasto and Viljoen 2008).

Antibacterial and Antifungal Activity of the Essential Oil

MIC values of the essential oils were given in Table 2. Low MIC values indicates high antimicrobial activity (Abubakar 2009). The investigated essential oils had the similar activities against gram (−) bacteria except for that collected from Koçarlı region, which showed higher antibacterial activity against Y. pseudotuberculosis than other oil samples. The highest antibacterial activity was obtained for essential oil from Aydın region against gram (+) bacteria. Also essential oil extracted from fruits of C. aurantium grown in Germencik region showed good activity against gram (+) bacteria. MIC values for gram (−) bacteria ranged from 2.5 to 5 µL/mL, whereas those for gram (+) strains ranged from 0.3125 to 5 µL/mL The present study revealed that gram (+) bacteria were more susceptible to the tested essential oils compared with gram (−) bacteria species.

Table 2 MIC values (unit should be here) of essential oils from C. aurantium (µL/mL) fruit peel
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The different sensitivity of bacteria can be explained by differences in cell composition between gram (+) and gram (−) strains. Generally, gram (−) bacteria are more resistant than gram (+) bacteria to essential oils, as the cell wall of gram (−) bacteria is more complex (Trombetta et al. 2005; Vasconcelos et al. 2018). When antifungal effects of essential oils were compared, the best result was found for essential oil originated from Nazilli region against C. parapsilosis (0.3125 µL/mL). Generally the highest antifungal activity was shown by essential oil from Germencik region. The investigated essential oils have rather similar activity against S. cerevisiae, C. tropicalis and C. albicans. Bendeha et al. (2016) studied the properties of essential oils isolated from fresh peel of C. aurantium grown in Eastern Morocco. They reported that tested essential oils were active against Listeria monocytogenes, Staphylococcus aureus, E. coli, Citrobacter freundii, S. cerevisiae, Fusarium sp., Aspergillus sp., Penicillium sp. and Alternaria sp. Our results are in aggrement with this research, because we also found that E. coli and S. cerevisiae were susceptible to the essential oils from C. aurantium fruit peels. Hsouna et al. (2013) investigated antimicrobial activity of the essential oil extracted from fresh C. aurantium flowers cultivated in North East of Tunisia. It was found that the growth of all tested microorganisms was inhibited at varying degree. MIC values were also found 1.25 µL/mL, 0.625 µL/mL and 1.25 µL/mL for E. coli, B. cereus and E. faecalis, respectively. MIC values in our study were found 5 µL/mL, 0.625 µL/mL- 2.5 µL/mL and 0.625 µL/mL- 2.5 µL/mL against E. coli, B. cereus and E. faecalis, respectively. Differences in results can be explained by different origin of plant samples. Kırbaşlar et al. (2009) investigated antimicrobial activity of essential oils from Citrus fruits collected from Antalya, Turkey. It was found that peel oil from bitter orange (C. aurantium) had activity against E. coli, B. cereus, P. vulgaris and C. albicans, which is in agreement with our results. Bozkurt et al. (2017) reported that essential oil from sour orange (C. aurantium) demonstrated antimicrobial activity of against B. cereus and E. faecalis but did not show any effect on E. coli. In contrast to this study, we found that Citrus peel oil inhibited the growth of E. coli. Our results are also consistent with those obtained by Azhdarzadeh and Hojjati (2016) who found that essential oil of unripe and ripe peel of C. aurantium had antimicrobial activity against B. cereus, E. coli and S. cerevisiae

GC-MS analysis of the essential oils obtained from the fruit peel of C. aurantium of four different populations (Central, Germencik, Koçarlı, Nazilli) revealed that limonen was the major volatile component. According to the results obtained in this study, it is possible to conclude that peel oils show antimicrobial and antifungal activities against gram (−) bacteria (E. aerogenes, Y. pseudotuberculosis, E. coli, P. vulgaris), gram (+) bacteria (B. cereus, G. Rubripertincta, E. faecalis, S. cohnii) and fungi (S. cerevisiae, C. tropicalis, C. albicans, C. parapsilosis). The findings of this study can be a guide for biotechnological, pharmaceutical and medical studies on C. aurantium in the future.