Variations in chemical compositions of essential oil from sour orange (Citrus aurantium L.) blossoms by different isolation methods

Sustainable Chemistry and Pharmacy 10 (2018) 118–124

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Variations in chemical compositions of essential oil from sour orange (Citrus aurantium L.) blossoms by different isolation methods

T

⁎

Aida Mohagheghniapoura, Mohammad Jamal Saharkhiza,b, , Mohammad Taghi Golmakanic, Mehrdad Niakousaric a

Department of Horticultural Science, Faculty of Agriculture, Shiraz University, Shiraz, Iran Medicinal Plants Processing Research Center, Shiraz University of Medical Sciences, Shiraz, Iran c Department of Food Science and Technology, Faculty of Agriculture, Shiraz University, Shiraz, Iran b

A R T I C LE I N FO

A B S T R A C T

Keywords: Essential oil content Extraction methods Limonene Linalool Linalool acetate Sour orange flower

The main goal of this research was to study the impact of different extraction methods on the yield and chemical compositions of essential oil (EO) obtained from sour orange blossoms. Seven different methods were used for the purpose of extracting the EOs, namely, commercial hydrodistillation (CoM), hydrodistillation (HD), steam distillation (SD), Ohmic-assisted hydro distillation (OAHD), solvent-less microwave extraction (SLME), solventfree microwave extraction (SFME), and microwave-assisted hydrodistillation (MAHD). The isolated EOs were analyzed by GC and GC-MS in three replications. The highest EO yield in relation to the extraction time was obtained by the HD method (0.34%) and the lowest one (0.04%) was detected by the SD method. The main compounds and their ranges of concentration in the EOs were as follows: linalool acetate (12.2 ± 0.08–28.9 ± 0.2) %, linalool (22.9 ± 0.07–54. ± 0.2) %, Farnesol (0.2 ± 0.04–10.4 ± 0.07) %, Enerolidol (0.4 ± 0.1–21.4 ± 0.04) % and geranyl acetate (0.97 ± 0.05–9.3 ± 0.08)%. Moreover, SLME caused a selective extraction of E-nerolidol (21.42%) and farnesol (10.45%). The SFME resulted in a two-fold extraction of linalool (54.08%), compared to the COM which yielded an amount of 22.9%. Significant changes in the amounts of limonene (1−14) % and β-pinene (0–9.6)% were also observed by the extraction methods. Overall, the results suggest that MAHD and SFME can be termed as green technologies because of their less energy requirements and less carbon dioxide emissions. Their high-quality EO and cost effective performance for EO extraction can be of importance to pharmaceutical industries.

1. Introduction

Various studies on EOs extracted from different parts of sour orange indicated significant quantitative and qualitative differences in chemical composition. For example, hydrocarbon monoterpenes such as limonene and β-myrcene are the main compounds in EO extracted from the peel. Oxygenated monoterpenes such as linalool, Linalool acetate, limonene, geranyl acetate, and α-terpineol are the main compounds in the EO obtained from leaves and blossoms of sour orange (Dugo et al., 2011). Rahimi et al. (2014) investigated the volatile compounds of sour orange EOs, which were extracted via Hydro-distillation (HD) and ultrasonic-assisted headspace solid phase micro extraction (UA-HSSPME). The main compounds of samples obtained via both methods (i.e. HD and SPME) were linalool acetate, limonene, β-myrcene, geranyl acetate, and neryl acetate; however, considerable differences were observed in the amounts of compounds comprising the EOs extracted by

Among the Citrus species, sour orange (Citrus aurantium L.) is a unique species with applications in cosmetics and pharmaceutical industries. Sour orange blossoms, which are fragrant, brittle, and fragile, are placed along shoot axis in single and/or multiple forms. The sour orange blossom is known as ‘Bahar-Naranj’ in Iran. Sour orange trees usually bloom in the middle of spring and, after a while, the blossoms fall down. The blossoms are used to prepare jam and floral water. Sour orange blossom and its floral water have traditional applications as tranquilizing and diuretic agents (Azadi et al., 2012). Citrus aurantium L. different parts essential oil (EO) contains β-pinene, limonene, trans-βocimene, linalool and α-terpineol as major constituents (Sarrou et al., 2013).

Abbreviations: CoM, commercial hydrodistillation; HD, hydrodistillation; SD, steam distillation; OAHD, Ohmic-assisted hydro distillation; SLME, solvent-less microwave extraction; SFME, solvent-free microwave extraction; MAHD, microwave-assisted hydrodistillation; EO, essential oil ⁎ Correspondence to: Medicinal and Aromatic Plants, Department of Horticultural Sciences, College of Agriculture, Shiraz University, Shiraz, Iran. E-mail address: [email protected] (M.J. Saharkhiz). https://doi.org/10.1016/j.scp.2018.10.008 Received 2 April 2018; Received in revised form 22 October 2018; Accepted 23 October 2018 2352-5541/ © 2018 Elsevier B.V. All rights reserved.

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2.2.2. Traditional extraction methods 2.2.2.1. Hydro-distillation (HD). A Clevenger apparatus was used for the extraction of EO with 200 g sour orange blossom and 800 ml water. The amount of EO was recorded after 3 h (Seidi Damyeh et al., 2016).

different methods. In order to identify and utilize the EOs of medicinal plants, they should be extracted from oil sacs or oil glands. Several traditional and novel techniques are used for the purpose of extracting the EOs (Li et al., 2014). However, there are some drawbacks to traditional techniques (e.g. hydrodistillation), which is mainly due to the lengthy procedure of heating in order to achieve the required temperature for evaporation of EOs. Such drawbacks may lead to the loss of components and the decomposition of non-saturated terpenes and esters, along with a high consumption of energy and time. Also, solvent residues may cause poisoning when solvent extraction is applied. Thus, finding the best extraction technique to enhance EO quality in terms of chemical composition can be essential for every special application compatible to international rules (Roohinejad et al., 2017). Recently, different thermal technologies have been used as novel ways to extract EOs, particularly with the aid of microwave and Ohmic heating. These novel techniques have interestingly shown to be a valid alternative to produce high quality EOs. Effective heating, time saving, fast energy transfer, and low operating costs are the main advantages of these technologies over conventional ones. Eblaghi et al. (2016) evaluated the effect of extraction methods (i.e. microwave-assisted hydrodistillation (MAHD) and HD) on the antioxidant activity of EOs extracted from tobacco and wormseed. Their results showed similar EO extraction yields for both methods and significantly shorter extraction durations were reported when using MAHD compared to HD (45 min versus 150 min, respectively). According to this study, MAHD is demonstrated as a rapid extraction method with no adverse effects on the antioxidant and antifungal activities of the EOs. Also, Golmakani and Moayyedi (2016) reported considerably shorter durations of extraction by MAHD compared to HD (15 min versus 120 min, respectively) to obtain lemon peel EO, and consequently microwave assisted extraction was recommended as a rapid, economical and environmentally friendly method. By and large, microwave energy, a volumetric heating source, has several advantages over conventional EO extraction methods. These advantages include more efficient heating, lower heat loss, selective extraction, a smaller instrument size, a lower risk of poisoning via solvent residues, a shorter operation time and less consumed energy, and hence lower production costs (Périno-Issartier et al., 2013). Due to the importance of extraction methods on quantitative and qualitative properties of EOs in pharmaceutical and food industries, a comprehensive study was conducted here for the first time to evaluate the effects of various extraction procedures on EO yield, the ratio of extraction yield to extraction time, and the determination of chemical compositions in the EO of the sour orange blossom which was extracted by different methods.

2.2.2.2. Steam distillation (SD). Extraction was performed by hydrostream distillation apparatus (using 200 g sour orange blossom) and the amount of EO was measured after 3 h (Xavier et al., 2011). 2.2.3. Novel extraction methods 2.2.3.1. Ohmic-assisted hydrodistillation (OAHD). The Ohmic extractor that was used in this study was designed and assembled in the Transport Properties Lab at the Department of Food Science and Technology, Shiraz University. The system was made up of a Teflon cylindrical chamber (7 cm internal diameter and 0.25 m length) and was equipped with two Titanium coated 316 stainless steel electrodes. The system was fully automated, and the voltage (0–350 V) and current (0–16 A) could be measured and recorded (Seidi Damyeh et al., 2016). Extraction was performed using 100 g sour orange blossom, 400 ml water, and 1.5 g NaCl (to create sufficient conductivity) for 50 min. 2.2.3.2. Microwave-assisted extraction. Three different methods were applied to extract EOs with the aid of microwave, including merely 200 g of sour orange blossom in the extraction flask without the solvent (SFME), 200 g of sour orange blossom and 100 ml water (SLME), and 200 g of sour orange blossom with 200 ml water (MAHD). Finally, the amount of EO was measured after 50 min (Farhat et al., 2011). The results were reported in grams of EOs per 100 g of fresh blossoms. Each experiment was performed in triplicate. The EOs were dried by anhydrous sulphate and stored at 4 °C so as to maintain them for further analysis. 2.3. Gas chromatography (GC) and gas chromatography/mass spectrometry (GC/MS) identification techniques Initially, the GC/FID oil analysis was conducted in order to arrive at a desirable analytical condition. The work was performed on a gas chromatograph Agilent technologies model 7890 A apparatus attached to HP-5 column (25 m × 0.32 mm, 0.52 µm film Thickness) and connected to a flame ionization detector (FID). Nitrogen gas was utilized as carrier gas with a flow rate of 1 ml/min and split ratio was 1:30. The injector temperature was 250 °C, and detector temperature was 280 °C, while the column temperature was linearly programmed from 60° to 250°C (at a rate of 5 °C/min) and was held for 10 min at 250 °C. Then, 1 µl of anhydrous and diluted EO samples were consecutively injected (1/10, v/v, essential oil/hexane). The above method was then applied for GC/MS analysis. This process was done using a gas chromatograph (7890 A, Agilent Technologies, Santa Clara, CA) coupled with a mass spectrometry (5975 C, Agilent Technologies, Santa Clara, CA) operating at 70 eV ionization energy, 0.5 s/scan and a mass range of 35–400 amu (u), equipped with a DB-5MS capillary column (Phenyl Methyl Siloxane, 30 m × 0.25 mm ID., Agilent technologies). One microliter of the obtained EO sample was injected into the GC/MS in split mode (split ratio: 1/100). Helium was utilized as carrier gas with the same flow rate as for GC/FID (1 ml/min). The mass spectrometer was acquired in EI mode (70 eV) in a mass range of 30–600 m/z. The injector and detector temperatures were at 280 °C. The oven temperature was planned to start at 60 °C and gradually heated up to 210 °C at a rate of 3 °C/min. Thereafter, the rate of temperature elevation became 20 °C/min until 240 °C was reached, whereupon the temperature was held constant for 8.5 min. The MSD ChemStation Software (G1701EA, E.02.01.1177, Agilent Technologies, Santa Clara, CA) was employed to analyze mass spectra and chromatograms. The compounds were identified by comparing their mass spectral fragmentation patterns with those kept in the data

2. Materials and methods 2.1. Material Sour orange blossoms were collected from Fasa, Shiraz, Iran (Latitude: 28° 31 ´- 29° 24 ´ N, Longitude: 53° 19 ´- 54° 15 ´ E). The plant species was identified and authenticated by A. Khosravi, a plant taxonomist at Shiraz University, Iran. The voucher specimen (No: 625) was deposited in the herbarium. The EO samples were extracted from the fresh blossoms. 2.2. EO extraction Five different major extraction procedures were used in this study as follows: 2.2.1. Commercial HD (CoM) This method was considered as a control in this study, in which 150 kg of fresh sour orange blossoms were extracted with 400 L of water in an industrial distillation device for 3 h (Moazeni et al., 2014). 119

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followed by SFME and MAHD. Similar results were obtained by Salehi Sourmaghi et al. (2015), in which a higher EO yield of coriander was obtained by HD compared to MAHD. However, no such significant difference in EO yield was observed by Eblaghi et al. (2016) after extracting EOs from Tanacetum polycephalum and Artemisia chamaemelifolia by HD and MAHD. Likewise, no significant difference was observed by Golmakani and Moayedi (2016) via HD, SLME, and MAHD when extracting EOs from Lemon peel (Citrus limon var. Eureka). Conversely, our results appeared contradictory to those reported by Hashemi- Moghaddam et al. (2012) on Biebersteinia multifida and by Aberoomand Azara et al. (2010) on Carum copticum where a higher extraction yield was obtained via MAHD rather than HD, and via SFME rather than HD, respectively. It is noteworthy to mention that CoM, which is industrially used for EO extraction, resulted in an EO yield (0.1%) that was lower than the performance of HD and three different microwave-assisted extraction techniques. These results are in agreement with those reported by Fathi and Sefidkon (2012), in which higher Eucalyptus sargentii EO was obtained via HD (3.39%) compared to water-steam distillation (2.89%) and CoM (1.35%) methods.

bank (Wiley/NBS) and with mass spectral data taken from the relevant literature (Jiang et al., 2011; Lima et al., 2013; Hashemi et al., 2011; Gavahian et al., 2012). Also, quantitative analyses of EO constituents were under the same chromatographic conditions using a GC coupled with a flame ionization detector (FID). The relative data for percentages were obtained from the electronic integration of chromatogram peak areas. The amount and type of each compound were determined using the retention index and the kovats index (Adams, 2007). 2.4. Statistical analysis All extractions and evaluation of chemical composition of the obtained EOs were conducted in triplicate and the results were expressed as the mean values ± standard deviation (SD). A general linear model (GLM) procedure in SAS software (Statistical Analysis Software, Version 9.1, SAS Institute Inc., Cary, NC) was used for the comparison of mean values. 3. Results and discussion 3.1. Essential oil content

3.2. Essential oil rate

The impact of extraction methods on EO content is illustrated in Fig. 1. The results demonstrated significant differences in EO content obtained by seven different methods (P ≤ 0.05). According to Fig. 1, the HD resulted in the highest EO (0.36%), and the lowest EO content was obtained by SD and OAHD methods (0.040% and 0.047%, respectively). This may be due to a mass formation because of the sticking feature of thin petals on the sour orange blossoms, when they come into contact with steam, which prevents the proper effect of steam on severing the EO containing glands in petals, thereby barring the complete extraction of EO (Jiang et al., 2011). However, no significant differences were observed by Gavahian et al. (2012) in the EO yield of Thymus vulgaris when comparing the two methods of HD and OAHD. The MAHD led to a significantly higher EO yield compared to OAHD (0.14% and 0.047%, respectively), and this may result from a more efficient heating procedure performed by MAHD which is capable of extracting EOs optimally. By contrast, no significant difference was observed by Seidi Damyeh et al. (2016) regarding the EO yield of Satureja macrosiphonia obtained by HD, OAHD, and MAHD. Three different microwave-assisted extraction methods, namely, MAHD, SLME, and SFME, gave rise to an acceptable extraction yield, which were higher than those obtained by CoM, OAHD, and SD methods, but lower than that of the HD method. This could be due to the accelerated rupture of EO glands in the presence of microwaves (Mohammadi et al., 2013). Amongst these three microwave-assisted methods, the highest EO yield (0.21% w/w) was extracted by SLME,

The rate of EO accumulation (%/h) by different extraction methods are presented in Fig. 2. The values of EO rate vary significantly (P ≤ 0.01) with regard to the extraction method. The SLME method demonstrated the highest EO rate (0.26%/h), which was followed by SFME (0.2%/h) and MAHD (0.15%/h). A prominent reason for this observation is the more efficient heating performed by the microwave compared to other methods. The rate of heating by microwave is more rapid than other methods which is due to the fact that microwaves readily penetrate the samples and materials. The absorption of microwaves by dielectric materials is followed by the transfer of microwave energy into the material, with a consequential rise in temperature. Thus, microwave-assisted extraction techniques are able to warm up the mixture faster than the other applied methods and they consequently reduce the extraction time, which results in a higher EO extraction rate. Our results are in line with the findings of Golmakani and Moayyedi (2016) on lemon peel EO with 0.08 and 0.07 g/min extracted via SLME and MAHD, respectively. Moreover, Seidi Damyeh et al. (2016) observed a higher rate of EO accumulation by MAHD (0.068 ml/ min) and OAHD (0.028 ml/min) compared to HD (0.016 ml/min). In this study, a higher rate of EO extraction was obtained by HD (0.11%/h) compared to OAHD (0.056%/h), CoM (0.03%/h), and SD (0.013%/h). It is evident that the extraction rate of EO by HD was higher than the other three examined microwave-assisted techniques. These results are in stark contrast to previous findings reported by Seidi Damyeh et al. (2016), on Satureja macrosiphonia, and by Gavahian et al.

Fig. 1. Effect of different extraction methods on sour orange blossom essential oil yield. CoM*: Commercial hydrodistillation, HD: Hydro-distillation, SD: Steam distillation, OAHD: Ohmic-assisted hydro-distillation, MAHD: Microwave-assisted hydro-distillation, SLME: Solvent microwave extraction, SFME: Solvent-free microwave extraction. **Columns with different letters are significantly different (P ≤ 0.05).

Fig. 2. Effect of different extraction methods on yield of sour orange blossom essential oil in relation to time. CoM*: Commercial hydrodistillation, HD: Hydro-distillation, SD: Steam distillation, OAHD: Ohmic-assisted hydro-distillation, MAHD: Microwave-assisted hydro-distillation, SLME: Solvent microwave extraction, SFME: Solvent-free microwave extraction. **Columns with different letters are significantly different (P ≤ 0.05). 120

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Table 1 Chemical composition of sour orange blossom essential oils obtained through seven extraction methods; HD: Hydro-distillation; OAHD: Ohmic-assisted hydrodistillation; MAHD: Microwave-assisted hydrodistillation; SFME: solvent-free microwave extraction; SLME: solvent-less microwave extraction; CoM* ; commercial hydrodistillation; SD: steam distillation (compositions are expressed as percent of chromatographic area). Number

Compounds

RIc

RIr

Rt (min)

Relative peak area (%) SFME

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25

Sabinene β- Pinene β - Myrcene Limonene cis- Ocimene trans- β- Ocimene cis-Linalool oxide alpha-Terpineol Linalool Banzil nitril Terpinen− 4-ol α-terpinol Nerol Linalool acetate Indole Methyl anthranilate Neryl acetate Geranyl acetate E-Nerolidol Farnesol (2Z,6E) Monoterpene Hydrocarbons oxygenated Monoterpenes Oxygenated Sesquiterpenes Other compounds Total oxygenated compounds

SLME

MAHD

OAHD

SD

HD

CoM* b

ND ND ND 1.09 f ± 0.07 ND 1.08 f ± 0.01 3.53a ± 0.07 ND 28.33 f ± 0.11 0.71b ± 0.01 ND 5.33a ± 0.09 ND 17.40 f ± 0.13 ND 0.85a ± 0.01

ND 0.45e ± 0.04 ND 3.47e ± 0.10 ND 0.77 g ± 0.02 ND ND 45.11c ± 0.12 ND ND 3.41c ± 0.04 ND 18.81e ± 0.11 ND ND

ND 2.44d ± 0.16 1.46d ± 0.07 4.66d ± 0.06 0.21b ± 0.01 2.51d ± 0.06 0.52d ± 0.02 0.18c ± 0.01 47.62b ± 0.22 0.13e ± 0.00 0.06d ± 0.00 1.57e ± 0.03 2.51d ± 0.06 12.20 g ± 0.08 0.70c ± 0.04 0.77b ± 0.02

ND 2.71c ± 0.06 1.43d ± 0.04 6.66c ± 0.09 0.23b ± 0.02 4.21b ± 0.05 0.38e ± 0.01 0.12d ± 0.00 29.36e ± 0.08 0.13e ± 0.01 0.09 cd ± 0.00 2.10d ± 0.05 4.21a ± 0.05 27.17b ± 0.24 0.91b ± 0.03 0.26e ± 0.01

1.02 ± 5.84b ± 2.01b ± 9.15b ± 0.16c ± 3.24c ± 0.88b ± 0.24b ± 34.22d ± 0.32d ± 0.12c ± 4.23b ± 3.24b ± 20.95d ± 2.27a ± 0.62c ±

0.01 0.13 0.06 0.12 0.02 0.04 0.04 0.01 0.20 0.01 0.00 0.09 0.04 0.07 0.05 0.01

1.69a ± 0.09 9.63a ± 0.12 2.79a ± 0.03 14.07a ± 0.11 0.41a ± 0.03 7.52a ± 0.09 0.11 f ± 0.01 0.47a ± 0.01 22.90 g ± 0.07 0.84a ± 0.03 0.23b ± 0.02 0.33 g ± 0.05 ND 25.43c ± 0.11 0.84b ± 0.03 0.32e ± 0.02

990 1029 1036 1048 1037 1088 1103 1141 1180 1194 1229 1263 1296 1344

990 1029 1037 1050 1072 1089 1096 1138 1177 1188 1229 1257 1291 1337

5.40 6.70 6.81 7.18 7.65 8.53 9.72 11.46 12.68 13.55 14.76 15.55 15.90 18.40

ND 0.49e ± 0.05 1.72c ± 0.09 3.64e ± 0.1 0.49a ± 0.02 1.93e ± 0.07 0.64c ± 0.01 ND 54.08a ± 0.15 0.44c ± 0.04 1.53a ± 0.05 0.96 f ± 0.04 2.71c ± 0.02 28.86a ± 0.26 0.42c ± 0.01 0.42d ± 0.02

1365 1384 1656 1723 –

1361 1381 1563 1723 –

20.18 21.08 28.67 34.76 –

0.42e ± 0.02 0.42 f ± 0.02 0.42 f ± 0.14 0.42 g ± 0.04 7.11e ± 0.23

3.64a ± 0.01 6.17b ± 0.06 21.42a ± 0.17 10.45a ± 0.07 2.17 g ± 0.08

ND 9.35a ± 0.08 13.66b ± 0.15 4.97d ± 0.06 4.69 f ± 0.08

0.90d ± 0.05 0.97e ± 0.05 11.52c ± 0.13 9.08c ± 0.07 11.71d ± 0.37

1.07c ± 0.03 1.62d ± 0.06 8.02d ± 0.08 9.33b ± 0.07 15.98c ± 0.26

1.25b ± 0.04 2.23c ± 0.04 5.29e ± 0.04 2.72 f ± 0.04 22.27b ± 0.35

0.13 f ± 0.01 2.26c ± 0.03 5.51e ± 0.03 4.52e ± 0.05 36.58a ± 0.30

–

–

–

78.42a ± 0.35

64.40d ± 0.54

76.68b ± 0.35

65.59c ± 0.41

64.60d ± 0.46

66.19c ± 0.41

51.39e ± 0.18

–

–

–

12.91e ± 0.18

31.87a ± 0.24

18.63c ± 0.21

21.06b ± 0.20

18.06d ± 0.15

8.24 g ± 0.00

10.03 f ± 0.02

–

–

–

1.56c ± 0.07

1.56c ± 0.02

0e

1.64c ± 0.06

1.36d ± 0.06

3.30a ± 0.07

2.00b ± 0.04

–

–

–

91.33b ± 0.53

96.27a ± 0.78

95.31a ± 0.56

86.65c ± 0.61

82.66d ± 0.61

74.43e ± 0.41

61.42 f ± 0.16

Note: Compounds are listed in order of their elution time from a HP-5MS capillary column. In each row, means with different letters are significantly different (P ≤ 0.05). SFME, solvent-free microwave extraction; SLME, solvent microwave extraction; MAHD, microwave-assisted hydrodistillation; OAHD, Ohmic-assisted hydrodistillation; SD, steam distillation; HD, hydrodistillation; CoM, commercial hydrodistillation; RI, retention indices; RT, retention time. RIc: The retention index is related to the compounds. RIr: The retention index is related to the resources. ND: Not detected.

(2012) on Thymus vulgaris. The mentioned reports claimed higher rates of EO accumulation by OAHD compared to HD.

3.3. Chemical composition of essential oil in different extraction methods 3.3.1. Main compounds The chemical compositions of EOs extracted from sour orange blossoms varied because of differences in the seven extraction methods (Table 1). Twenty components comprised more than 99% of the total composition. Significant differences (P ≤ 0.01) in the quantities of the main EO components were observed by different extraction methods. The amount of linalool in EOs extracted by different methods are illustrated in Fig. 3. As can be seen, the highest and lowest amounts of linalool were obtained by OAHD (48.69%) and CoM (22.90%), respectively. On the other hand, the amount of linalool significantly increased (P ≤ 0.05) by 53% according to the applied extraction method. Linalool acetate was seen as the second major component of the EO, and its amounts varied in response to different extraction methods (Fig. 3). This compound showed a different trend compared to linalool. More amounts of linalool acetate were extracted mostly by conventional methods compared to the novel methods, although apart from the SFME. However, the highest (28.28%) and the lowest (12.47%) amounts of linalool acetate were extracted by SD and OAHD, respectively (P ≤ 0.05). The extraction method significantly affected the

Fig. 3. Effect of different extraction methods on linalool and linalool acetate content of sour orange blossom essential oil. CoM*: Commercial hydrodistillation, HD: Hydro-distillation, SD: Steam distillation, OAHD: Ohmic-assisted hydro-distillation, MAHD: Microwave-assisted hydro-distillation, SLME: Solvent microwave extraction, SFME: Solvent-free microwave extraction. ** Columns with different letters are significantly different (P ≤ 0.05).

amount of this component and caused an increase of 55%. According to Fig. 4, a decreasing trend is observed for the limonene content in EOs extracted by the seven different methods. Higher amounts of limonene were obtained by conventional methods, where the highest limonene content was obtained by CoM (14.07%), followed by HD (9.4%) and

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of E-nerolidol were obtained by the three conventional methods, among which the CoM generated the lowest amount (5.51%). Differences in the amounts of farnesol (6E, 2Z), as a component, are shown in Fig. 5. The highest amount of this compound was obtained by SLME (10.45%) followed by SD (9.7%) and OAHD (9.2%) methods. The lowest concentration of this components was detected by using SFME (0.42%). 3.3.2. Minor compounds Minor compounds of EO were also significantly (P ≤ 0.01) affected by the applied extraction methods (Table 1). Sabinene, as a minor EO constituent, was only found in the EOs extracted by CoM (1.7%) and HD (1%). The highest content of β-myrcene was extracted by CoM (2.79%). However, this compound was not detected in EOs obtained by SLME and MAHD methods. The highest and the lowest amounts of nerol were obtained by SD (4.21%) and OAHD (2.51%). Furthermore, this compound was not observed in EOs extracted by CoM, SLME, and MAHD. According to Table 1, a lower amount of neryl acetate was extracted by the CoM method (0.13%) compared to the other extraction methods. The highest amount (3.64%) of this compound was obtained via SLME, whereas none of it was found in the EO extracted by MAHD. The highest geranyl acetate was extracted by MAHD (9.3%) and the lowest (0.97%) was obtained by OAHD.

Fig. 4. Effect of different extraction methods on limonene, β-Pinene and and trans-β-ocimene content of sour orange blossom essential oil. CoM*: Commercial hydrodistillation, HD: Hydro-distillation, SD: Steam distillation, OAHD: Ohmic-assisted hydro-distillation, MAHD: Microwave-assisted hydrodistillation, SLME: Solvent microwave extraction, SFME: Solvent-free microwave extraction. **Columns with different letters are significantly different (P ≤ 0.05).

then SD (6.9%). Regarding novel extraction techniques, OAHD resulted in a higher amount of limonene (4.7%) in the extracted EO. However, microwave-assisted extraction methods were not generally efficient in extracting this particular compound. For instance, using the SLME resulted in the lowest amount of limonene (1.09%). The highest β-pinene content was obtained by CoM (9.63%), followed by HD (6%) and then SD (2.8%). OAHD gave rise to a higher amount of β-pinene compared to the three microwave-assisted extraction methods, but nonetheless obtained lower amounts compared to the conventional methods. The lowest amount of β-Pinene was extracted through SFME (0.49%), followed by MAHD (0.45%). Also, no β-Pinene was found in the EO extracted by SLME (Fig. 4). Significant changes in trans-β-ocimene content was also observed (Fig. 4). Conventional extraction methods led to a higher amount of the compound and the highest concentration of transβ-ocimene was obtained via the CoM method (7.52%), followed by SD (4.3%) and HD (3.3%). Lower amounts of this particular compound were extracted by the novel techniques compared to the conventional methods. However, OAHD resulted in a higher amount of trans-β-ocimene than the amounts obtained by the other three microwave-assisted extraction techniques, with MAHD being capable of the lowest amount (0.77%). A different trend was observed regarding the amount of E-nerolidol in EOs extracted by the seven different methods (Fig. 5). To be more explicit, the novel extraction techniques resulted in a higher amount of E-nerolidol. The highest amount (21.42%) of this compound was obtained by SLME, which was followed by MAHD (13.6%), OAHD (11.7%), and SFME (10.6%), respectively. Conversely, lower amounts

3.3.3. Changes in different classes of chemical compounds in sour orange blossom EO 3.3.3.1. Monoterpene hydrocarbons. As can be seen in Table 1, the quantities of monoterpene hydrocarbons varied based on the extraction method. The highest and the lowest amount of these compounds were extracted by CoM (2.17%) and SLME (36.5%), respectively. Generally, compared to the high efficiency of CoM in extracting monoterpene hydrocarbons, this compound occurred in significantly lower amounts when using the other six methods for the extraction of EO (P ≤ 0.05). 3.3.3.2. Oxygenated monoterpenes. Interestingly, higher amounts of oxygenated monoterpenes were extracted by other extraction methods compared to CoM (P ≤ 0.05). The highest and the lowest amounts of oxygenated monoterpenes were extracted by SFME (78.42%) and CoM (51.39%), respectively. This may be attributed to a rapid heating of polar compounds by microwave, and consequently a shorter extraction time. Less amounts of water were used in the SFME method compared to other methods in this study. This reduces the level of degradation occurring to the main oxygenated compounds. It should be taken into account that as a result of heating and hydrolytic reactions, oxygenated compounds may undergo certain changes and create other compounds that are less valuable. Accordingly, the application of microwave, here as SLME, led to a higher yield of main oxygenated compounds but a lower yield of minor compounds (Akhbari et al., 2018; Filly et al., 2014). 3.3.3.3. Oxygenated sesquiterpens. Several extraction methods were more capable of yielding oxygenated sesquiterpenes compared to the CoM. Meanwhile, HD yielded the lowest amount of these compounds (8.24%). The highest amount of oxygenated sesquiterpens was obtained by SLME (31.87%) (P ≤ 0.05). In general, oxygenated compounds are considered to be more valuable because of their characteristic aroma, antimicrobial, and antioxidant ability. Interestingly, the chemical composition of the EOs mostly consisted of oxygenated compounds. This amounted to over 90% of the EOs extracted by microwave-assisted extraction methods and over 80% of the EO contents extracted by OAHD and SD. The lowest amount of these valuable compounds was extracted by CoM (61.42%). Thus, applying novel extraction techniques could result in a better quality of EO with regard to the chemical composition. Salehi Sourmaghi et al. (2015) also demonstrated an increase in oxygenated

Fig. 5. Effect of different extraction methods on E-nerolidol and farnesol content in sour orange blossom essential oil. CoM*: Commercial hydrodistillation, HD: Hydro-distillation, SD: Steam distillation, OAHD: Ohmic-assisted hydrodistillation, MAHD: Microwave-assisted hydro-distillation, SLME: Solvent microwave extraction, SFME: Solvent-free microwave extraction. **Columns with different letters are significantly different (P ≤ 0.05). 122

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ultimately ensue, making them competitive methods for the commercial extraction of EOs.

monoterpenes which play an important role in coriander EO properties when using MAHD compared to HD. Similar results were observed in another study conducted by Filly et al. (2014) in which oxygenated compounds were extracted more than hydrocarbon monoterpenes by MAHD, compared to HD. According to Table 1, the main compounds of the EO obtained from sour orange blossom are linalool acetate (12.20–28.86) %, linalool (22.90–54.08) %, limonene (1.09–14.07) %, farnesol (0.42–10.45) %, E-nerolidol (0.42–21.42) %, β-pinene (0–9.63) %, trans-β-ocimene (0.77–4.21) % and geranyl acetate (0.97–9.35) %. The results of a previous study by Sarrou et al. (2013) on the EO of Citrus aurantium, collected from northern Greece, appear to be in line with our results, in which the main compounds were linalool (29.14%), β-pinene (19.08%), trans-β-ocimene (6.06%) and trans-farnesol (5.14%). However, our results are different compared to some relevant research. Monsef-Esfahani et al. (2004) carried out a research on EO extracted from the Iranian sour orange blossom obtained by HD, CoM, and traditional methods. The blossoms were obtained from the Darab region in Fars province, Iran. The main EO compounds thereof were geraniol, α-terpineol, linalool, and benzene acetaldehyde extracted by the HD method. Furthermore, linalool, methyl anthranilate, and cis-linalool oxide were obtained efficiently by the traditional method. The 1–8-cineol, linalool and α-terpineol were better extracted by CoM. Conversely, the present study showed no geraniol and cineol in its EOs. Boussaada and Chemli (2006) also observed linalool, linalyl acetate, limonene, α-terpineol, βpinene, geraniol and sabinene as the main compounds of Citrus aurantium EO (var. Amara, from Tunisia) obtained by HD. The main compounds of the EO extracted from Citrus aurantium var. Aurantium (collected from Morocco) were linalool, α-terpineol, 6-methyl-5-hepten2-L, geraniol, phenyl ethyl alcohol, benzyl nitrile, methyl anthranilate, and indole (Jeannot et al., 2005). Accordingly, there are differences between the chemical compositions of EOs in the present study when compared to those of previous works. These differences may be attributed to factors such as variety, growth stage, climate, drying and extraction methods (Jiang et al., 2011). From the results of the current work, it could be concluded that a proper extraction method can assist in performing a selective extraction from the plant material, thereby obtaining particular compounds of interest in the EO. Our results showed that linalool and linalool acetate could be extracted selectively by OAHD and SD, respectively. Moreover, limonene, β-pinene, β-myrcene, and trans-β-ocimene are able to be extracted more by the CoM method, whereas E-nerolidol, farnesol, nerol, and geranyl acetate can be extracted selectively by MAHD.

Acknowledgments We would like to thank Shiraz University Research and Technology Council, Shiraz, Iran and Shiraz University of Medical Sciences, Shiraz, Iran for providing the grant (Project Number: 94-01-70–9344) of this project. Mohsen Hamedpour-Darabi is also acknowledged for editing the English language of the paper. References Aberoomand Azara, P., Motaghianpour, Z., Sharifian, A., Larijani, K., 2010. Studies on the Effect of extraction method on chemical composition and antimicrobial activity of Carum copticum essential oil. JFTN 7 (2), 10–18. Adams, R.P., 2007. Identification of Essential Oil Components by Gas Chromatography/ Mass Spectrometry, 4th ed. Allured Publishing, Carol Stream, IL. Akhbari, M., Masoum, S., Aghababaei, F., Hamedi, S., 2018. Optimization of microwave assisted extraction of essential oils from Iranian Rosmarinus officinalis L. using RSM. JFST 1–11. Azadi, B., Nichavar, B., Amin, G., 2012. 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4. Conclusion The extraction yields differed significantly per method. The highest EO yield was obtained via the conventional method (HD), followed by SLME which needed a considerably shorter extraction time. Moreover, microwave-assisted extraction techniques led to a higher rate of EO accumulation when compared to the use of other methods. SLME yielded the highest EO extraction rate. GC/MS analysis revealed significant differences among the percentages of compounds in EOs. Accordingly, the identified main compounds were linalool and linalool acetate, which are oxygenated monoterpenes. It was demonstrated that CoM yielded the lowest amount of linalool compared to the other six extraction methods. However, limonene and β-pinene were extracted more through CoM, compared to the other extraction methods. All in all, SFME and MAHD showed considerable advantages over other methods used in this study. These methods demonstrated shorter extraction durations as well as better qualities of EOs with higher amounts of oxygenated compounds (i.e. active and important compounds of EO). Less energy is consumed because of shorter extraction durations taken by microwave-assisted extraction methods. As a result, these particularities can be considered as cost effective and environmentally friendly because less carbon dioxide emissions will 123

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