Food Science and Technology Research, 23 (3), 385_394, 2017Copyright © 2017, Japanese Society for Food Science and Technologydoi: 10.3136/fstr.23.385

http://www.jsfst.or.jp

*To whom correspondence should be addressed. E-mail: golmakani@shirazu.ac.ir

Original paper

Eugenol Enrichment of Clove Bud Essential Oil Using Different Microwave-assisted Distillation Methods

Mohammad-Taghi Golmakani*, Mahdieh Zare and Saeedeh raZZaGhi

Food Science and Technology Department, School of Agriculture, Shiraz University, Shiraz, Iran

Received January 4, 2017 ; Accepted February 10, 2017

Clove bud (Syzygium aromaticum) essential oils (EOs) were extracted by hydro-distillation (HD), steam-distillation (SD), microwave-assisted hydro-distillation (MAHD), and microwave-assisted steam-distillation (MASD) methods. A comparison was made between microwave-assisted extraction and conventional extraction methods in terms of extraction kinetic, chemical composition, eugenol enrichment and antioxidant activity. Significant differences were observed between the ultimate EO yields of HD-based methods (12.98 _ 13.94%) and those of SD-based methods (11.54 _ 12.71%). Nonetheless, chemical compositions of the four extracted EOs were similar, but significant differences occurred among the quantity of compounds in the EOs. The ratio of eugenol per eugenyl acetate in HD-based methods (8.36 _ 11.90%) was higher than those of SD-based methods (5.28 _ 5.81%). These differences result from the hydrolysis of eugenyl acetate to eugenol in HD-based methods. Although, there was no significant difference between antioxidant activity of EOs extracted by microwave-assisted extraction methods and conventional-based methods but, accordingly, there were significant differences between HD-based methods and SD-based methods.

Keywords: antioxidant activity, clove bud, essential oil, eugenol enrichment, microwave-assisted extraction

Introduction Spices and herbs are valuable sources in all over the world, and

being regionally prioritized as food additive, flavors, colors and elements for fragrances (Lucchesi et al., 2004). The Myrtaceae family has approximately 3000 species and 130 _ 150 genera. Clove (Syzygium aromaticum) belongs to this family. It staged a global production of 112,956 tons in 2012. Indonesia and Madagascar produce approximately 64.62% and 20.80%, respectively, and became the largest producers of clove in 2012 (FAOSTAT). Clove is primarily used in the flavoring of foods and tobacco (Burdock, 2009). In the food industry, cloves are often used in the form of grinded powder, oleoresin, and essential oil (EO). These are applied in small amounts because of their intense flavors (Tajik et al., 2014).

Most EOs are complex mixtures of terpenic and sesquiterpenic

hydrocarbons. They can give rise to oxygenated terpenoid and sesquiterpenoid derivatives such as alcohols, aldehydes, ketones, esters, and occasionally carboxylic acids, Aromatic (benzenoid) compounds can also be derived and may yield phenols, phenolic ethers, and aromatic esters (Baser and Buchbauer, 2009). For the past decades, the search for natural bioactive compounds, such as EOs, that can serve as antioxidant agents has increased tremendously. The reasons for it are attributed to the increasing awareness of the harmful nature of reactive oxygen species produced during oxidation processes (Lee and Shibamoto, 2001; Mazidi et al., 2012), beside the increasing concern over the consumption of synthetic additives (Gholivand et al., 2010; Mazidi et al., 2012; Sacchetti et al., 2005). The benefit that results from the rich EOs of clove is actually a manifestation of bioactive substances like eugenol and eugenyl acetate, hence the rise in

M.-T. Golmakani et al.386

consumption by food and cosmetic industries (Guan et al., 2007). Clove EO has high phenolic content in comparison with other common EOs (Gülçin et al., 2004; ViudaMartos et al., 2010; Wang and Wang, 2008). Furthermore, the clove exhibits a relatively strong antioxidant activity, which rivals the activities of the synthetic antioxidants such as butylatedhydroxytoluene and pyrogallol (Dorman et al., 2000; Nassar et al., 2007).

Conventional-based extraction methods such as hydro-distillation (HD) and steam-distillation (SD) are the most widely used procedures for the extraction of EOs from medicinal plants (Rashidipoura et al., 2014). However, these extraction methods languish at imperfections such as low extraction efficiency, losses of some volatile compounds, long operation time, high energy consumption, and degradation of unsaturated or ester compounds through thermal or hydrolytic effects on the EO (Bousbia et al., 2009; Chemat and Cravotto, 2013). These shortcomings have led to the consideration of new methods that use less energy while offering the highest extraction efficiency through the shortest extraction time.

Microwave dielectric heating is common in various processes in the food industry all over the world (Farhat et al., 2011). Microwave-assisted extraction (MAE) is more and more applied in EO extraction (Chemat and Cravotto, 2013). Therefore, the advantages of using microwave energy have led to the consideration of new MAE-based methods such as microwave-assisted hydro-distillation (MAHD) and microwave-assisted steam-distillation (MASD).

Many studies have investigated the extraction of clove EO with different conventional-based methods, but no study has yet compared the MAHD and MASD methods as regards the chemical composition of the clove’s EO, its eugenol enrichment and its antioxidant activity.

Materials and MethodsClove buds (Syzygium aromaticum) were purchased from a

local market, Shiraz, Iran. Also, the identity of the genus and species was confirmed by Dr. Khosravi, a senior taxonomist in the herbarium of the Biology Department at Shiraz University, Shiraz, Iran. The moisture content of clove was measured in triplicates according to the method described by Mazidi et al. (2012). The measured moisture content of clove was 5.78 ± 0.06 (%, w/w). All values are reported on a moisture-free basis (Habibi et al., 2016). Free radical 2, 2-diphenyl-1-picrylhydrazyl (DPPHº) and BHT (butylated hydroxyl toluene) were purchased from Sigma–Aldrich (St. Louis, MO, USA). Potassium ferricyanide, trichloroacetic acid, ferric chloride, vitamin C (L-ascorbic acid), methanol and hexane were purchased from Merck (Darmstadt, Germany).

Extraction methods According to Fig. 1b, a modified domestic microwave oven (ME3410W, Samsung Malaysia Electronics, Kuala Lumpur, Malaysia) was used for the method of MAHD. It

operated at 2.45 GHz frequency by a maximum output power of 1000 W, variable at 100 W intervals. Forty grams of dried clove and 400 mL of distilled water were placed in the reactor and heated by microwave radiation. The ratio of clove-to-water was 1:10 (w/w). On the other hand, the MASD method was employed using a microwave oven similar to the one described as above. But in this method, the clove was placed in a glass column, of which the lower and higher parts were connected to a water flask and condenser, respectively (Fig. 1d). The HD (Fig. 1a) and SD (Fig. 1c) were similar to MAHD and MASD, respectively. However, an Electromantle heater (EM2000/C, 335 W, Electrothermal Engineering Ltd., Rochford, UK) was used instead of the microwave oven. According to our preliminary experiments (data not shown), the operation was performed for 240 min (335 W) at 30 min intervals in conventional-based methods. Furthermore, the extraction time lasted for 80 min (1000 W) in MAE-based methods at 5 min intervals. The EOs were obtained from the four extraction methods, dried by anhydrous sodium sulphate and stored in amber vials at 4℃.

Antioxidant activity assays The number of methods to measure the antioxidants in medicinal plants has increased considerably in the last decade. In this study, primary antioxidant activity was evaluated using RSA (Radical scavenging activity), FRAP (Ferrous ion reducing antioxidant power), CUPRAC (Cupric ion reducing antioxidant capacity) and FIC (Ferrous ion chelating) methods.

RSA The RSA or DPPH radical (DPPH°) analysis was carried out according to the method used by Mazidi et al. (2012). The effect of an antioxidant on DPPH° is related to its activity/ability in donating hydrogen to DPPH° solution so as to form the DPPH (non- radical) solution. The RSA is indicated by the value of IC50 which is the concentration of sample that can inhibit 50% of DPPH° activity (Habibi et al., 2016). All the experiments were carried out in triplicates and vitamin C was used as a positive control on antioxidant activity.

FRAP The FRAP analyses of the samples were measured by means of colorimetry according to the method of Fawole et al. (2012). Here, vitamin C is used as a standard (Fawole et al., 2012).

CUPRAC The CUPRAC analysis of the samples was measured according to the method of Apak et al. (2006). Again, vitamin C is used as a standard (Apak et al., 2006).

FIC The FIC analysis of the samples was ascertained by the method of Decker and Welch (1990). The FIC was calculated according to according to the following equation

Chelating effect (%) = (Ab _ As / Ab ) ×100 ······Eq. 1

Where, Ab is the absorbance of the blank without EO or EDTA and As is the absorbance in the presence of EO or EDTA (Decker and Welch, 1990).

Physical properties Specific gravity, visual appearance, and

Eugenol Enrichment of Clove Essential Oil Using Microwave 387

refractive index of EO were measured according to the methods suggested by Food Chemical Codex (FCC)3. Moreover, the color of the EOs was determined (L*: lightness, a*: redness-greenness, and b*: blueness-yellowness) according to the method described by Golmakani and Moayyedi (2015). In the L*a*b* system, color differences can be expressed as a single numerical value (∆Eab), which is calculated according to the following equation:

ΔEab = √＿＿＿＿＿＿＿＿＿＿＿＿＿＿(ΔL*)2 + (Δa*)2 + (Δb*)2 ······Eq. 2

Gas Chromatography/Mass Spectrometry Identification of EO constituents was made by a gas chromatography (7890A, Agilent Technologies, Santa Clara, CA, USA) coupled with a mass spectrometer (5975C, Agilent Technologies, Santa Clara, CA, USA) operating at 70 eV ionization energy, 0.5 s/scan and a mass range of 35 _ 400 a tomic mass un i t s (amu) . The gas chromatography was equipped with a HP-5MS capillary column (5% phenyl polysilphenylene- siloxane; 30 m length; 0.25 mm internal diameter; 0.25 μm film thickness). The injector and detector temperatures were maintained at 280℃. The temperature timeline of the oven was programmed accordingly; the initial temperature was set to be 60℃ and was then allowed to increase to 210℃ (at a rate of 3℃/min). Finally, the temperature increased to

240℃ (at a rate of 20℃/min) whereupon 240℃ was held constant for 8.5 min. A volume of 1 μL of the sample was injected into the GC/MS with the injector being in the split mode (split ratio of 1/100). Helium was used as the carrier gas with a flow rate of 0.9 mL/min. Then, the MSD ChemStation Software (G1701EA, E.02.01.1177, Agilent Technologies, Santa Clara, CA, USA) was applied to handle mass spectra and chromatograms. Most constituents were tentatively identified by comparison of their retention indices (RIs), established in accordance with reference to an homologous series of C5

_ C28 n-alkanes that had been injected following the injection of the EOs under the same chromatographic conditions. Identifications were confirmed when possible by making comparisons between their mass spectral fragmentation patterns and those stored in the database bank (Wiley libraries/NBS) and also with the mass spectra of relevant data in the literature (Nassar et al., 2007). Furthermore, quantitative analysis of EO constituents was carried out under the same chromatographic conditions using a gas chromatography coupled with a flame ionization detector.

Scanning electron microscopy Scanning electron microscopy (SEM) images of cloves were obtained for the untreated samples and also for those that followed treatment with conventional-based

Fig. 1. Schematic diagram of (a) hydro-distillation, (b) microwave-assisted hydro-distillation, (c) steam-distillation, and (d) microwave-assisted steam-distillation used in this study.

M.-T. Golmakani et al.388

methods (after 240 min) and MAE-based methods (after 80 min). The treated cloves were freeze-dried using a freeze drier (Armfield, UK) and were fixed on the specimen holder by aluminum tape. The treated cloves were then sputtered by gold in a sputter coater (Polaron SC7640, UK). All the specimens were examined by SEM (Cambridge, UK) under high vacuum conditions at an accelerating voltage of 20.0 kV and a working distance of 3 _ 39 mm (i.e. the distance between the surface of the sample and the microscope lens).

Statistical analysis All experiments were performed in triplicate. In order to determine significant differences among the sample means, one-way analysis of variance (ANOVA) was done and the Tukey’s test at the confidence level of 95% (P < 0.05) was conducted using SAS software (Statistical Analysis Systems Institute Inc., version 9.1, Cary, NC, USA).

Results and DiscussionTemperature profiles According to Fig. 2, two phases were

observed in the process of increasing extraction temperature in HD, SD, MAHD, and MASD methods. The first phase represents the heating phase from 20℃ to 100℃ and distillation of the first EO droplets. In the second part, the extraction temperature was constant and equal to boiling temperature of water (100℃) until the end of extraction. The rate at which the temperature was set to rise channeled through by determining the slope of linear parts of the temperature profile. As shown in Table 1, the rate of temperature rise in MAE-based methods were clearly higher than conventional-based methods and the rate of temperature rise in MAHD and MASD methods were almost 5 and 6 fold higher than those of HD and SD methods, respectively. This higher temperature rise is due to the fact that MAE-based methods apply three ways of heat transfer, namely radiation, conduction, and convection, for the samples. In conventional-based methods, however, heat transfers only take place through conduction and convection (Golmakani and Rezaei, 2008). According to Table 1, there were significant differences in the rate of temperature rise between HD-based methods and SD-based methods. These data indicate that the MAHD method had the highest rate of temperature rise.

The starting point for the second part of extraction coincides with the starting time of EO accumulation. To reach this point, it was necessary to heat the samples for 22.0 and 30.0 min by HD and SD methods, respectively, while MAHD and MASD methods needed only 4.0 and 4.5 min, respectively. These results are mainly due to the better heating efficiency in the MAE-based methods compared to the conventional-based methods (Golmakani and Rezaei, 2008).

Extraction yield and extraction kinetics Table 1 shows the effects of different extraction methods on total extraction time, final EO yield and average rate of EO accumulation. Final EO yields extracted by MAE-based methods (13.94% in MAHD and 12.71% in MASD) were significantly higher in comparison to the corresponding values in conventional-based methods (12.98% in HD and 11.54% in SD). These results are in good agreement with the findings of Golmakani and Rezaei (2008) because MAHD led to a considerable increase in ultimate thyme EO yield in comparison with HD. Final EO yields in HD-based methods were 12.98% and 13.94% in HD and MAHD, respectively. These were significantly greater compared to their corresponding values in the SD-based methods (11.54% and 12.71% in HD and MAHD, respectively). Furthermore, MAHD (13.94%) showed the highest final EO yield compared to other extraction methods.

According to Table 1, there are significant differences among the average rate of EO accumulation (g/min) based on the total extraction time. Results show that the average rates of EO accumulation by MAE-based methods were greater than conventional-based methods. Also, data show that MAHD had the highest average rate of EO accumulation (0.17 g/min). Additionally, the findings revealed that the shorter extraction time taken in MAE-based methods is not only caused by their earlier onset of extraction, but also by their higher extraction rates which is mainly due to the more efficient heat transfer conducted by the microwave (Golmakani and Rezaei, 2008). Its shorter extraction time is compared with the longer duration required for conventional based methods.

According to Fig. 2, MAE-based methods reduce the extraction time while providing an equal yield. The extraction yield in MAHD

Table 1 . Effects of extraction method on extraction kinetic of clove essential oil (EO)

Extraction parameter Extraction method

HD* SD MAHD MASD

Rate of temperature increase 3 .60c** 2 . 70d 20 .00a 17 .80b

Starting time of EO accumulation (min) 22 .00b 30 .00a 4 .00c 4 .50c

Total extraction time (min) 240 .00a 240 .00a 80 .00b 80 .00b

Extraction duration (min) 218 .00a 210 .00b 76 .00c 75 .50c

Final EO yield (%, w/w) 12 .98b 11 .54c 13 .94a 12 .71b

Average rate of EO accumulation (g/min) 0 .05c 0 . 05d 0 .17a 0 .16b

*HD, hydro-distillation; SD, steam-distillation; MAHD, microwave-assisted hydro-distillation; MASD, microwave-assisted steam-distillation. **In each row, means with different superscript letters are significantly different (P < 0.05).

Eugenol Enrichment of Clove Essential Oil Using Microwave 389

after 60 min was similar to the final yield in HD after 240 min. These data show that for obtaining the same amounts of EO, the time required for HD was 4 times higher than that in MAHD. These results are in good agreement with the findings of Mazidi et al. (2012) for Black Zira (Bunium persicum Boiss.). They found that the extraction duration required for HD was almost 4 times higher than that in MAHD, but with an equal yield of extraction (Mazidi et al., 2012). Moreover, the extraction yield in MASD after 50 min of operation equaled to the final extraction yield in SD after 240 min. This means that the MASD managed to operate 4.8 times faster than SD.

According to Fig. 2, the final EO yields extracted by HD-based methods were higher than those of corresponding SD-based methods but, in the first 50 min of extraction, EO yield of MASD was higher than that of MAHD. During this period, the combination of microwave distilled water heating and microwave dry distillation of cloves occurred in MASD. Since cloves were not submerged in distilled water (Fig. 1d), dry distillation was enacted so as to counterbalance the internal warming of in situ water. Evaporation of in situ water led to a rapid build-up of pressure within the clove and caused faster ruptures in the glands of plant tissues, through a process called solvent-free microwave destruction. During the last 30 min of MASD, surfaces of the cloves were completely moistened and microwave radiation was mostly absorbed by the surrounding water and not by the in situ water of clove. Moreover, the amount of in situ water decreased sharply in the last 30 min of MASD. Indeed, negligible solvent-free microwave destruction happened during the last 30 min of MASD and therefore its EO yield became lower than MAHD.

Structural changes after extraction The clove images obtained by the scanning electron microscopy (SEM) before and after

extraction are shown in Fig. 3. As illustrated in Fig. 3a and Fig. 3b (i.e., before the extraction), many intact EO glands exist immediately beneath the surface of clove leaves. It is logical that a higher amount of clove EO happens when more numbers of EO glands reside in the clove. Images show the cloves after extraction by HD and SD methods, after 240 min, and by MAHD and MASD methods after 80 min (Fig. 3c-f). All four extraction methods destroyed the EO glands in the end. The total extraction time in MAE-based methods was lower, and the disruption of the EO glands was more severe than conventional-based methods. Regarding MAE-based methods, in addition to glands destruction, clove tissue was destroyed as well due to the sudden temperature rise and internal pressure increase. Similar to our findings, Wang et al. (2010) extracted mango flower EO by MAHD and reported that MAHD caused the glandular walls to rupture faster than the conventional HD method (Wang et al., 2010).

Electric consumption Electric consumption is a function of time and power. It was calculated based on the maximum power consumptions of the Electromantle in the conventional-based methods (335 W) and the microwave oven in MAE-based methods (1000 W). The total electric consumption was almost 1.34 kWh for different extraction methods, but the reduced extraction time is clearly an advantage of the MAE-based methods in comparison with that of the conventional-based methods. Furthermore, results of the CO2 rejection into the atmosphere (1.07 kg) were similar to those of electric consumption. There were significant differences between conventional- and MAE-based methods in terms of relative electric consumption. Relative electric consumption in conventional-based methods was higher than MAE-based methods. Also, relative electric consumption in SD-based methods was higher than HD-based methods. Results of relative CO2 rejection

Fig. 2. Time-temperature and time-yield profile of clove essential oils extracted by hydro-distillation (HD), steam-distillation (SD), microwave-assisted hydro-distillation (MAHD), and microwave-assisted steam-distillation (MASD) methods.

M.-T. Golmakani et al.390

were similar to those of relative electric consumption. Indeed, HD-based methods are better than SD-based methods in terms of relative electric consumption and CO2 rejection.

Evaluation of physical constants The extracted EO has physical properties such as specific gravity, refractive index, visual appearance and colors. These properties vary depending on whether the extraction is by conventional-based methods or MAE-based methods (Table 2). Nonetheless, there were no significant differences among specific gravities, refractive indices, visual appearances and colors of EOs extracted by the different extraction methods. For comparison purposes, the physical properties of EOs can be studied according to the FCC standards (Table 2). Specific gravity, refractive index, visual appearance and colors of all the samples were within the range indicated by FCC standards

(Burdock, 2010). Also, Golmakani and Rezaei observed no significant difference between the physical properties (specific gravity, refractive index and appearance) of Zataria multiflora EOs extracted by HD and MAHD methods (Golmakani and Rezaei, 2008). In addition, Mazidi et al. (2012) reported similar specific gravities and refractive indices for Black Zira (Bunium persicum Boiss.) EOs extracted by MAHD and HD methods. There were no significant differences between L*, a*, and b* of EOs extracted by MAE-based methods in comparison with conventional-based methods. EOs extracted by MAHD and MASD methods –– known as fast and efficient extraction methods –– did not bear any remarkable standouts in color of EOs and this was confirmed by their low ∆Eab. In contrast to our results, Mazidi et al. (2012) reported that colors of EOs extracted by MAHD were somewhat

Fig. 3. Scanning electron micrographs of clove (a, b) before extraction and after extraction (c) by hydro-distillation, (d) steam-distillation, (e) microwave-assisted hydro-distillation, and (f) microwave-assisted steam-distillation.

Eugenol Enrichment of Clove Essential Oil Using Microwave 391

lighter than those extracted by HD (Mazidi et al., 2012).Gas chromatography/mass spectrometry (GC/MS) The EO

compounds obtained by different extraction methods are presented in Table 3. A total number of 12 compounds were identified by the four extraction methods. The EOs extracted by different extraction methods were qualitatively similar, but there were significant differences in their quantity (relative peak area). According to Table 3, clove EOs contain high amounts of phenolic monoterpene alcohol, eugenol (82.65 _ 88.80%). Many reports indicated that eugenol is the most abundant compound in clove EO (Guan et al.,

2007, Lee and Shibamoto, 2001). Second to eugenol, another main compound was eugenyl acetate (7.46 _ 15.63%), a derivative of eugenol. MAHD method yielded the highest percentage of eugenol (88.80%) and at the same time the lowest percentage of eugenyl acetate (7.46%). These differences are due to the hydrolysis of eugenyl acetate to eugenol (Fig. 5). The EOs extracted by HD-based methods had higher ratios of eugenol per eugenyl acetate in comparison with those of SD-based methods. In contrast to our findings, Guan et al. reported that clove EO extracted by the SD contained the highest percentage of eugenol (58.2%), while clove EO extracted by the HD contained the lowest percentage of eugenol (48.82%) (Guan et al., 2007). Périno-Issartier et al. found that the amount of linalyl acetate in lavandin EO extracted by the SD was about 1.5 fold higher than the HD (Périno-Issartier et al., 2013).Unlike the case in HD, the SD method does not expose lavandin to direct contact with water, which indeed makes the hydrolysis of linalyl acetate less marked in comparison with the HD. The water being directly mixed with lavandin can be used as an initiator of the linalool formation reaction in the HD.

The eugenol yield (g/100 g clove) depends on the final yield and eugenol percentage (Table 3). The concentrations of eugenol obtained by HD-based methods were higher than those by SD-based methods. Also, eugenol yields of HD-based methods were significantly higher than those of SD-based methods.

Antioxidant activities Due to the complexity of antioxidative mechanisms, it is realized that independent analyses are inappropriate to generalize the total antioxidant activity of the sample. Therefore, four complementary evaluations were carried out in the present study, namely, radical scavenging activity (RSA), ferrous ion reducing antioxidant power (FRAP), cupric ion reducing antioxidant capacity (CUPRAC) and ferrous ion chelating (FIC).

RSA RSA is a prevalent method to evaluate antioxidant activity in a relatively short time. According to Table 4, the IC50 values of clove EOs extracted by different extraction methods were in the range of 0.11 _ 0.33 mg/mL. Contrary to our results, Ivanovic et al. reported lower IC50 values for clove EO extracted by the HD (21.50 µg/mL) (Gülçin et al., 2012). IC50 values of the EOs extracted by SD-based methods (0.24 _ 0.33 mg/mL) were slightly higher than HD-based methods (0.11 _ 0.17 mg/mL). It seems that higher antioxidant activity (i.e. lower IC50 values) in HD-based methods is a result of higher eugenol content. Also, Lee and Shibamoto reported that antioxidant activity of clove EO was mainly due to its high eugenol content (Lee and Shibamoto, 2001).

According to Table 4, there were no significant differences in terms of IC50 values between conventional- and MAE-based methods. These results are in good agreement with the findings of Mazidi et al. (2012). They found no significant differences between the IC50 values of Black Zira EOs extracted by the HD and MAHD methods.

FRAP In FRAP analysis, the presence of antioxidant

Tabl

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Ph

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of c

love

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entia

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(EO

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Spec

ific

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1.04

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241.

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211.

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1.53

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421.

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M.-T. Golmakani et al.392

Fig. 4. Electric energy consumptions of hydro-distillation (HD), steam-distillation (SD), microwave-assisted hydro-distillation (MAHD), and microwave-assisted steam-distillation (MASD) for extraction of clove essential oil (EO). For each parameter, means with different letters are significantly different (P < 0.05).

Fig. 5. Hydrolysis of eugenyl acetate to eugenol and acetic acid.

Table 3 . Chemical compositions of clove essential oils extracted by different extraction methods

NO. Compounds Retention time Retention index Relative peak area (%)

HD* SD MAHD MASD

1 Benzaldehyde 6 .26 956 0 .07 0 .08 0 .06 0 .052 2-Nonanone 10 .58 1089 0 .04 0 .04 0 .05 0 .043 Benzyl acetate 13 .45 1161 0 .05 0 .04 0 .06 0 .054 Ethyl benzoate 13 .71 1167 0 .01 0 .01 0 .01 0 .015 Methyl salicylate 14 .69 1191 0 .08 0 .08 0 .1 0 .076 Chavicol 17 .22 1251 0 .31 0 .24 0 .22 0 .227 Eugenol 21 .77 1359 87 .26 82 .65 88 .80 83 .398 (E)-Caryophyllene 24 .09 1415 1 .35 0 .91 2 .65 1 .379 α-Humulene 25 .46 1449 0 .19 0 .13 0 .4 0 .2110 Eugenyl acetate 28 .52 1526 10 .43 15 .63 7 .46 14 .3411 Caryophyllene oxide 30 .54 1578 0 .2 0 .17 0 .19 0 .2212 Benzyl benzoate 37 .14 1759 0 .02 0 .03 0 .01 0 .02

Eugenol/eugenyl acetate ratio 8 .36 5 .28 11 .9 5 .81Eugenol yield (g/100 g clove) 11 .32 9 .53 12 .37 10 .59Eugenyl acetate yield (g/100 g clove) 1 .35 1 .80 1 .03 1 .82

* HD, hydro-distillation; SD, steam-distillation; MAHD, microwave-assisted hydro-distillation; MASD, microwave-assisted steam-distillation.

Eugenol Enrichment of Clove Essential Oil Using Microwave 393

compounds in the EOs would result in ferric iron (Fe3+) being reduced to ferrous iron (Fe2+) via electron donation. As shown in Table 4, the Fe reduction equivalent (g vitamin C/100 g EO) in HD-based methods was similar to that in SD-based methods. Moreover, the Fe reduction equivalents (g vitamin C/100 g EO) of conventional-based methods were insignificantly higher than those of MAE-based methods. Phutdhawong et al. (2007) extracted EOs of Cinnamomum iners Reinw. ex Bl. by HD and MAHD methods. They further measured their antioxidant activity by the FRAP method and reported that the antioxidant activity of both EOs were similar, being 387.77 and 387.41 µM in HD and MAHD, respectively (Weerachai Phutdhawong, 2007).

CUPRAC According to Table 4, antioxidant activities of clove EOs extracted by HD-based methods were similar to those of SD-based methods. Also, there were no significant differences between conventional- and MAE-based methods with regard to CUPRAC results. The values for CUPRAC results were 3.62 _ 4.70 g vitamin C/100 g EO which confirm the values for FRAP results, being 4.57 _ 5.30 g vitamin C/100 g EO.

FIC As can be seen in Table 4, the FIC of HD-based methods were higher than SD-based methods. The higher chelating power of EOs obtained by HD-based methods could be due to the presence of higher amounts of eugenol. This results in its higher partial hydrolysis to eugenol and the release of acetic acid during FIC measurement. Eugenyl acetate was hydrolysed during EOs extraction by HD-based methods and acetic acid was released, but then acetic acid remained in the aqueous phase. Furthermore, there were no significant differences between the chelating powers of conventional- and MAE-based methods.

ConclusionA comparison was made between conventional- and MAE-

based methods in terms of extraction kinetics, chemical composition, eugenol enrichment and antioxidant activity of clove EOs. Faster extraction was clearly an advantage of MAE-based methods over conventional-based methods. HD-based methods yielded higher amounts of EO than SD-based methods. EOs of

HD-based methods had higher eugenol contents compared to those of SD-based methods. These differences are due to the hydrolysis of eugenyl acetate to eugenol in HD-based methods. Differences in the amount of eugenol led to higher antioxidant activity of EOs obtained by HD-based methods when compared with SD-based methods. Chemical compositions and antioxidant activities of MAE-based methods were similar to those of conventional-based methods; therefore, microwave radiation did not have any adverse effects on the chemical composition and antioxidant activity of clove EO.

Acknowledgements This research project was financially supported by Shiraz University. We also thank the Iranian editor, Mohsen Hamedpour-Darabi, for natively editing the English of the paper.

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