Result and discussion
Optimization by central composite design
An experimental procedure based on the CCD was adopted for three independent variables (extraction time, irradiation power and moisture content), which resulted in 20 experimental runs. Table 1 shows the responses obtained in the CCD experiments and the overall design. The results indicated considerable variations in the extraction efficiency of essential oil and TQ amount. These variations reflected the importance of optimization to attain higher productivity of essential oil and TQ.
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Table 2 comprises the equation of dependent variables and the analysis of variance (ANOVA) of the effects. Second-order polynomial models used to express essential oil extraction yield (YE) and TQ content. The R-squared statistic indicated that the model explains 97.04% and 92.26% of the variability in YE% and TQ%, respectively. The adjusted R-squared statistic was 0.93 for YE% and 0.83% for TQ%. The p-value less than 0.05 indicate the model terms are significant. The “lack of fit p-values” more than 0.05 implies the lack of fit is not significant relative to the pure error, which indicated that the models were accurate and satisfactory. The significance of each coefficient was determined by F-value and p-value listed in Table 2. The data indicate that irradiation power (P) and moisture content (M) have significant effects on both YE% and TQ%, but extraction time (ET) influences only YE%.
The response surface methodology (RSM) is interested as an excellent tool for obtaining the maximum amount of complex information and the best way to predict the effect of the independent variables on the dependent one. Besides, RSM plays an important role in designing, formulating, developing and analyzing new scientific research, as well as improving existing studies and products (Bas and Boyaci 2007). Three-dimensional response surface of multiple non-linear regression models were applied to explain the interactions between extraction time, irradiation power and moisture content on the YE% and TQ% (Fig. 2A-D). Fig. 2A and C depict the interaction between extraction time and irradiation power on the extraction yield of essential oil and its thymoquinone content. Increasing the extraction time from 15 to 30 min with irradiation power from 180 to 450 W enhanced the YE% and TQ%, while both of them started decreasing when the extraction time and irradiation power were increased up to 45 min and 720 W. This might be the reason for the volatilization and decomposition of essential oil and its constituents when the irradiation power and extraction time increased (Qiet al. 2014). The effect of extraction time on TQ% was less than YE%. The results showed that a longer irradiation time and power were not suitable for essential oil extraction. Fig. 2B shows the three-dimensional plot of the response surface for the essential oil extraction yield as related to moisture content and time. The increase of extraction time from 15 to 30 min with an increase of moisture content from 15% to 50% significantly accelerated essential oil extraction, and extraction yield of essential oil decreased when moisture content was higher than 60%. At low moisture content, the evaporation rate was low, resulting in an incomplete extraction. On the contrary, a high proportion of water might lead to hydrolysis of some volatile components (Liet al. 2012).
As illustrated in Fig. 2D, an increase in TQ% was observed by increasing the moisture content from 15 to 100% with extraction time from 15 to 30 min, while the TQ% was not more changed apparently after 45 min. Those results suggested that increasing moisture content from 30% to 50% together with an increase of extraction time from 15 to 30 min, the extraction yield reaches a peak value with a good recovery of TQ. The results showed that the amount of oil extracted increased at first when the power was increased, but started to decrease when the power passed 450 W. This reveals that an increase in power enhanced the mass transfer ratio until a certain value, thus increasing the extraction yield. However, the extraction yield of essential oil decreased slightly at higher irradiation power. This might be due to a quick change of temperature, leading to partially thermal decomposition of volatiles (Qiet al. 2014).
Based on the results, the high content of TQ (20%) in the highest overall yield of essential oil (0.33%) was obtained through MAE extraction conditions of extraction time 30 min, irradiation power 450 W, and moisture content 50%. Verification experiments were conducted six times under these optimal conditions. The resulting mean extraction yield and thymoquinone percent were 0.32% and 19.47% with a relative standard deviation (RSD) of 3.16% and 3.78% respectively.
Comparison of MAE with HD
Compared with MAE method, conventional hydrodistillation (HD) was used as a reference method for essential oil extraction from N. sativa seeds (Table 1). The results revealed that the extraction yields of MAE for 30 min (0.316 0.01%, w/w) were higher than HD for 3 h (0.23 0.035%, w/w). Also, as seen in Table 1, the content of TQ obtained from HD dropped drastically (3.71%) compared with MAE. In contrast to HD, MAE could decrease the rate of oxidation and hydrolysis of bioactive compounds by reducing the extraction time (Qiet al. 2014). In the HD process, the samples were heated by the thermal conductivity from the outside to the inside of samples. However, in the MAE process, heat transfer arises from the samples center to the outer colder environment. Moreover, the internal heating of the in situ water produces areas of compression in the plant, resulting in the serious rupture of glands and oleiferous receptacles (Lucchesiet al. 2007). This might cause the considerable difference in the extraction yield of essential oil between two extraction methods. Hence, MAE is an efficient, environmentally friendly and energy-saving extraction method. Consequently, microwave-assisted extraction is a promising alternative to extract essential oils from natural products.
Structural changes after extraction
SEM was employed to evaluate the structural changes of N. sativa seeds when subjected to different oil extraction procedures. Fig. 3A shows a micrograph of the untreated seeds (before extraction), and Fig. 3B and C are the SEM images of samples that have treated by HD (3h) and MAE (30 min), respectively. As illustrated in Fig. 3A, the presence of numerous essential oil cells with a full balloon shape is observed. Most of them became atrophic, rupture and appeared wrinkled after extraction by HD (Fig. 3B). In HD process, the heat transfer is mainly performed by conduction and convection only, while in the process of MAE, it is implemented in three ways: radiation, conduction and convection (Maet al. 2012). As a result, in MAE process, heat is produced from within the glands as well as from the outside. When the glands were subjected to more severe thermal stresses and localized high pressures, as in the case of microwave heating, the pressure build-up within the glands could have exceeded their capacity for expansion, and caused their rupture more rapidly and completely than in conventional extraction (Lucchesiet al. 2007; Qiet al. 2014). After MAE, most of cells appeared completely disrupted explaining that all the cell walls are finally damaged and collapsed, and have resulted into undefined boundaries (Fig. 3C).
Gas chromatography-mass spectrometry
The components of essential oil from N. sativa seeds obtained by MAE and HD were analyzed by GC-MS. The detected constituents, their retention indices and relative percentages are given in Table 3. Thirty components were identified in the essential oil obtained by MAE and HD. The number of identified compounds was lower than that reported by Benkaci-Ali et al. (2007) and Liu et al. (2013). The different origin of the seeds and/or the lower sample injection volume may be the reason for this. The essential oils contained mainly of monoterpenes hydrocarbons (59.93% for MAE and 76.36% for HD) together with noticeable contents of oxygenated monoterpenes (24.61 for MAE and 8.2% for HD) and smaller amounts of sesquiterpenes hydrocarbons (5.57 for MAE and 5.69% for HD). In both methods, the monoterpene hydrocarbons were noticeably dominated by p-cymene (41.99% for MAE and 52.82% for HD) while Î-thujene, Î-pinenes, Î²-pinene, sabinene and terpinene were present at lower amounts. Among the oxygenated monoterpenes, TQ was the major bioactive constituent especially in oil isolated by MAE (20.41%), together with lower contents of linalool, terpinen-4-ol and carvacrol. In the HD method, the oxygenated monoterpene content of the essential oil was substantially lower than that obtained with MAE, because of the decomposition or hydrolysis of thymoquinone (see Table 3). These results are similar to those of Benkaci-Ali et al. (2007) who reported that the content of oxygenated monoterpenes of the oil obtained by MAE was higher than that by HD for N. sativa seeds. Li et al. (2012) suggested that during the procedure of MAE, microwave irradiation highly accelerates the extraction process without causing considerable changes in the essential oil composition, although the percentages of some components depend on the technique applied. Moreover, Benkaci-Ali et al. (2006) reported that MAE can reduce the time of extraction of N. sativa seeds essential oil to less than 10 min. However, the composition of the major products presented a fluctuation according the extraction time.
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Our results showed that N. sativa seeds purchased from Iran belong to the p-cymene/thymoquinone chemotype, which is in agreement with the results of Hajhashemi et al. (2004). A variety of chemotypes have been described in the literature. Burits and Bucar (2000) reported the chemical composition of the essential oils from N. sativa from Austria. They found thymoquinone (27-57%) and p-cymene (7-15.7%) as the major compounds. Another Iranian N. sativa essential oil was found to be dominated by phenylpropanoid components and displayed a trans-anethole chemotype (Nickavaret al. 2003). N. sativa essential oil from Bangladesh (Liuet al. 2013), Algeria (Benkaci-Aliet al. 2007) and India (Singhet al. 2005) was found to be a p-cymene/thymoquinone chemotype. A chemotype with 33.0% p-cymene and 26.8% thymol and the majority of monoterpenes was reported for N. sativa essential oil from Morocco (Morettiet al. 2004; D’Antuonoet al. 2002) and a chemotype with 60.2% p-cymene and 12.9% Î-terpinene was reported by Wajs et al. (2008) for N. sativa from Poland. This shows that the contents of compositions of essential oils of N. sativa seeds were dependent on the extraction method as well as the species.
Owing to the complicity of essential oils, the antioxidant activities cannot be assessed by only a single method, but at least two test systems have been proposed for the determination of antioxidant activity to establish authenticity (Schlesieret al. 2002). In the present study, the antioxidant activity of N. sativa seeds essential oils extracted by MAE and HD were evaluated in vitro by DPPH scavenging activity and ferric reducing antioxidant power assays, and compared with traditional antioxidants.
- Free radical scavenging activity (DPPH)
DPPH assay is often used as an indicator of free radical scavenging capacity; it is an electron-transfer-based assay, which is an important mechanism of antioxidant action (Bayramogluet al. 2008). As shown in Fig. 4A, DPPH radical-scavenging activity (SC %) increased when the oil concentration increased. A low IC50 value indicates strong antioxidant activity in a sample. The IC50 values of the essential oil, obtained by MAE and HD, were 28.10 and 36.90 µg/ml, respectively. In brief, the DPPH scavenging effect decreased in the order: VC > MAE > HD > VE. The results showed that essential oil obtained by MAE possessed a higher free radical scavenging capacity than HD.
- Ferric reducing antioxidant power (FRAP)
The reduction capacity of an extract or oil may use as an important indicator of its potential antioxidant activity (Maet al. 2012). A higher absorbance indicated a higher ferric reducing power. As shown in Fig. 4B, both the essential oil by different extraction methods and standards showed increased ferric reducing power with the increased concentration. Essential oil extracted by MAE at the highest concentrations analyzed, showed the highest ferric reducing capacity in terms of Fe concentrations (1670 µM Fe /g) with statistical differences with control VC and VE (1400 and 300 µM Fe /g, respectively. The reducing power of essential oil obtained by HD was slightly lower than that of MAE (1580 µM Fe /g). It can be due to the reduction of thymoquinone compound which is a major active chemical component of the essential oil studied. As mentioned above the antioxidant activities of Nigella sativa L. essential oil could be mainly due to the action of thymoquinone existing in the essential oil studied. In brief, the reducing power of essential oils and antioxidants revealed the descending order of: MAE > HD > VC > VE.
Microwave-assisted extraction technique (MAE) was considered for the extraction of essential oil from Nigella sativa L. seeds. Response surface methodology was successfully implemented for optimization of extraction yield of essential oil and its thymoquinone content. The optimum parameters were extraction time 30 min, irradiation power 450 W and moisture content 50%. Thymoquinone content of oil obtained by HD was reduced substantially due to the long time extraction of HD. The antioxidant activity of essential oils extracted by MAE and HD were evaluated by DPPH and reducing power tests, and compared with traditional antioxidants. Based on the results, we conclude that MAE method represents a valuable alternative to traditional HD for the essential oil extraction from Nigella sativa L. seeds owing to the excellent extraction efficiency, higher thymoquinone content and higher antioxidant activity of the essential oil. Further study is recommended to evaluate the antimicrobial activity and other bioactive properties of N. sativa essential oil extracted by MAE and compare this method with other extraction methods. Furthermore, essential oil from Nigella sativa L. seedswould be a novel nature resource for the usage in food and healthy fields.
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