The grinding procedure is one of the important factors that has not been sufficiently addressed in the field of SFE. Although the effect of grinding time (GT) has been investigated on the SFE yields of other raw materials, those studies were limited to short GTs. For example, Wilkinson et al. [1Wilkinson N, Hilton R, Hendry D, Venkitasamy C, Jacoby W. Study of process variables in supercritical carbon dioxide extraction of soybeans. Food Sci Technol Int 2014; 20(1): 63-70.
[http://dx.doi.org/10.1177/1082013212469620] [PMID: 23733823] ] investigated the effect of GT on the SFE yield from soybeans at 483 bar and 80 °C. They placed 60 g of soybeans in a mechanical grinder for different GTs from 10 to 60 s. They concluded that increasing the GT enhances the extraction yield due to the reduction in particle sizes. The SFE yield increased from approximately 6% to 22% as the particle diameters decreased from 0.20 mm to 0.07 mm. In another study by Yahya et al. [2Yahya F, Lu T, Santos R, Fryer P, Bakalis S. Supercritical carbon dioxide and solvent extraction of 2-acetyl-1-pyrroline from Pandan leaf: The effect of pre-treatment. J Supercrit Fluids 2010; 55(1): 200-7.
[http://dx.doi.org/10.1016/j.supflu.2010.05.027] ], the SFE yield from pandan leaf increased up to 50% after a 30-s grinding pretreatment step. Hatami et al. [3Hatami T, Johner JCF, Meireles MAA. Investigating the effects of grinding time and grinding load on content of terpenes in extract from fennel obtained by supercritical fluid extraction. Ind Crops Prod 2017; 109: 85-91.
[http://dx.doi.org/10.1016/j.indcrop.2017.08.010] ] investigated the effects of GTs (15 s to 20 min) and mass of raw material in the mill (15 g to 35 g) on both the global SFE yield from fennel and the yields of its major compounds, namely, anethol and fenchone. The extractor was subjected to a pressure of 200 bar, temperature of 40 °C, and supercritical CO₂ flow rate of 1.67×10⁻⁴ kg/s for 10 min. The results showed that a GT of 6 min provided better results than GTs of 15 s or 20 min in terms of contents of anethol and fenchone in the extract. According to this paper [3Hatami T, Johner JCF, Meireles MAA. Investigating the effects of grinding time and grinding load on content of terpenes in extract from fennel obtained by supercritical fluid extraction. Ind Crops Prod 2017; 109: 85-91.
[http://dx.doi.org/10.1016/j.indcrop.2017.08.010] ], although grinding can facilitate SFE by decreasing particles sizes, it also has some disadvantages. One of such drawback is increasing the temperature of the raw material in the mill, which leads to the evaporation of volatile components from the raw material [3Hatami T, Johner JCF, Meireles MAA. Investigating the effects of grinding time and grinding load on content of terpenes in extract from fennel obtained by supercritical fluid extraction. Ind Crops Prod 2017; 109: 85-91.
[http://dx.doi.org/10.1016/j.indcrop.2017.08.010] ]. To avoid this complication and move one-step forward in grinding process, the current study aimed to develop new grinding procedures to cool the raw material and prevent large increases in temperature. Clove bud was selected as the model raw material for this purpose because it is rich in volatile oils, which makes it suitable for evaluating new SFE techniques. Many researchers have studied SFE from clove [4Wei M-C, Xiao J, Yang Y-C. Extraction of α-humulene-enriched oil from clove using ultrasound-assisted supercritical carbon dioxide extraction and studies of its fictitious solubility. Food Chem 2016; 210: 172-81.
[http://dx.doi.org/10.1016/j.foodchem.2016.04.076] [PMID: 27211636] -6Hatami T, Meireles M, Zahedi G. Mathematical modeling and genetic algorithm optimization of clove oil extraction with supercritical carbon dioxide. J Supercrit Fluids 2010; 51(3): 331-8.
[http://dx.doi.org/10.1016/j.supflu.2009.10.001] ], and they reported eugenol as its main component. Eugenol shows anti-inflammatory, anti-bacterial, antioxidant, and neuro-protective properties, and it has many applications in cosmetics, food products, and dentistry [7Tsai T-H, Huang W-C, Lien T-J, et al. Clove extract and eugenol suppress inflammatory responses elicited by Propionibacterium acnes in vitro and in vivo. Food Agric Immunol 2017; 28(5): 916-31.
[http://dx.doi.org/10.1080/09540105.2017.1320357] -11Nagababu E, Rifkind JM, Boindala S, Nakka L. Assessment of antioxidant activity of eugenol in vitro and in vivo.Free Radicals and Antioxidant Protocols 2010; 165-80.
[http://dx.doi.org/10.1007/978-1-60327-029-8_10] ]. GC-MS analysis showed that there are twenty-two other compounds in the clove oils, and these compounds include β-caryophyllene, α-humulene, and eugenyl acetate [12Guan W, Li S, Yan R, Tang S, Quan C. Comparison of essential oils of clove buds extracted with supercritical carbon dioxide and other three traditional extraction methods. Food Chem 2007; 101(4): 1558-64.
[http://dx.doi.org/10.1016/j.foodchem.2006.04.009] ]. In this paper, the impact of grinding procedure was evaluated on the SFE of clove in terms of the extraction of these four components.

Fig. (1) shows the clove samples, the milled clove before and after extraction, the material distribution inside the mill, and the extract oil. Clove is almost cylindrical in shape, but the milled clove is approximately spherical (A). The distinct differences between the milled material before and after SFE (B and C in Fig. 1) indicate the high performance of the SFE in obtaining the extractable oil. This figure (part D) also shows that the raw material mostly accumulates close to the cutting edges of the mill. Part E of this figure shows that the color of essential oil is yellow (color is one of the indicators of the quality of the essential oil of clove.

Fig. (1) A schematic of (A) the raw material before grinding, (B) raw material after grinding, (C) raw material after SFE, (D) clove distribution inside the crushing chamber of the mill, (E) and extract oil obtained from clove using SFE.

Fig. (2) shows the SFE yields of eugenol, β-caryophyllene, α-humulene, and eugenyl acetate. The similarities in the trends of the yields of these four components could be explained by the fact that their enthalpies of vaporization under standard conditions are very close to each other, namely, 66.30 kJ/mol for eugenol [13 Chemeo [homepage on the Internet]. Chemical Properties of Eugenol [updated 2016; cited 2018 Feb 12]. Available from: https://www.chemeo.com/cid/45-324-2/Eugenol], 65.50 kJ/mol for β-caryophyllene [14 Chemeo [homepage on the Internet]. Chemical Properties of Caryophyllene [updated 2016; cited 2018 Feb 12]. Available from: https://www.chemeo.com/cid/70-147-1/Caryophyllene], 49.4 kJ/mol for α-humulene [15 ChemSpider [homepage on the Internet]. a-Humulene [updated 2015; cited 2018 Feb 12]. Available from: http://www.chemspider. com/Chemical-Structure. 4444853.html#suppInfoTab], and 56.80 kJ/mol for eugenyl acetate [16 Chemeo [homepage on the Internet]. Chemical Properties of Acetyl eugenol [updated 2016; cited 2018 Feb 12]. Available from: https://www.chemeo.com/cid/85-505-7/Acetyl%20eugenol]. As shown in this figure, eugenol had by far the highest yield (9.61 to 11.20 g / 100 g clove), followed by eugenyl acetate (2.09 to 2.55 g / 100 g clove), and β-caryophyllene (1.34 to 1.58 g / 100 g clove). Grinding procedure “E” gave the highest extraction yield of the aforementioned components, while grinding procedures “A” and “C” gave the lowest yields.

Fig. (2) Effect of grinding procedure on the extraction yield (g / 100 g clove) of eugenol, β-caryophyllene, α-humulene, and eugenyl acetate from clove by SFE at 150 bar, 40 °C, and a supercritical CO2 flow rate of 1.03 × 10−4 kg/s during a dynamic period of 15 min.

Among the continuous grinding procedures (“A”, “B”, and “D”), grinding procedure “B” provided the best performance (Fig. 2). The superiority of grinding procedure “B” over “A” and “D” can be attributed to the average diameters and the increasing temperature of the clove in the mill achieved for the various grinding procedures, and these data are shown in (Table 2). It is clear from the table that the temperature was substantially influenced by the grinding procedure, and it increased from 5.5 °C for “A” to 10.1 °C for “D”. According to Table 2, increasing the GT had two opposing effects on the SFE yield. On one hand, the particle diameter decreased, which had a positive effect on the extraction yield due to enhancing the specific surface area of the particles in the SFE bed. On the other hand, the temperature of material in the mill was increased by increasing GT, and this accelerated the evaporation of the volatile compounds, which tended to reduce the extraction yield of the aforementioned components. These results are in line with those previously published by Hatami et al. [3Hatami T, Johner JCF, Meireles MAA. Investigating the effects of grinding time and grinding load on content of terpenes in extract from fennel obtained by supercritical fluid extraction. Ind Crops Prod 2017; 109: 85-91.
[http://dx.doi.org/10.1016/j.indcrop.2017.08.010] ] on the effect of GT on the SFE yields of anethol and fenchone from fennel. Comparison of the performances of “B” and “C” on one hand and “D” and “E” on the other hand is interesting as each of these pairs have the same particle diameter; 0.39 mm for “B” and “C” and 0.37 mm for “D” and “E”. Although grinding procedure “B”, a continuous procedure, provided a higher yield than grinding procedure “C”, a non-continuous procedure, comparing “D” and “E” showed the opposite trend; “E” gave a far higher extraction yield of the aforementioned components than “D”. This can be justified based on the impact of another important factor, the total GT, which includes the duration of both crushing and stopping. The second column of (Table 2) shows the values of parameter for the whole grinding procedure. It is evident that increasing both the total GT and the temperature increase the evaporation of the volatile components in the raw material. Based on the obtained results, it could be reasonably concluded that for “B” and “C”, total grinding time is the key factor; thus, more evaporation occurs in “C” despite the lower temperature reached. However, for “D” and “E”, the temperature increase is the key factor, which results in “E” providing a higher yield than “D”. These results show that the total grinding time and the temperature increase might have different effects in different grinding procedures.

Table 2
The total grinding time (crushing + stopping), temperature increase, and average clove diameter for the various grinding procedures.

To show the impact of the milling conditions on the extraction yield with respect to the influence of other typical SFE parameters such as temperature, pressure, and CO₂ flow rate, it is interesting to compare the results obtained in this study with those reported in the literature. As the major component of clove oil is eugenol, the eugenol recoveries of the various reports can be compared. (Table 3) compared the impact of the developed grinding procedure with the impact of temperature, pressure, extraction time, and CO₂ flow rate (F) reported by Chatterjee and Bhattacharjee [17Chatterjee D, Bhattacharjee P. Supercritical carbon dioxide extraction of eugenol from clove buds. Food Bioprocess Technol 2013; 6(10): 2587-99.
[http://dx.doi.org/10.1007/s11947-012-0979-2] ], Wenqiang et al. [12Guan W, Li S, Yan R, Tang S, Quan C. Comparison of essential oils of clove buds extracted with supercritical carbon dioxide and other three traditional extraction methods. Food Chem 2007; 101(4): 1558-64.
[http://dx.doi.org/10.1016/j.foodchem.2006.04.009] ], and Yang et al. [18Yang YC, Wei MC, Hong SJ. Ultrasound-assisted extraction and quantitation of oils from Syzygium aromaticum flower bud (clove) with supercritical carbon dioxide. J Chromatogr A 2014; 1323: 18-27.
[http://dx.doi.org/10.1016/j.chroma.2013.10.098] [PMID: 24290173] ].

Table 3
The significance of temperature, pressure, extraction time, CO₂ flow rate, and grinding procedure on the SFE yield of eugenol from clove.

Chatterjee and Bhattacharjee [17Chatterjee D, Bhattacharjee P. Supercritical carbon dioxide extraction of eugenol from clove buds. Food Bioprocess Technol 2013; 6(10): 2587-99.
[http://dx.doi.org/10.1007/s11947-012-0979-2] ] studied the extraction of eugenol from clove buds using SFE, and they investigated the effect of temperature (40 to 130 °C), pressure (250 to 500 bar), and extraction time (90 to 150 min) using response surface methodology [17Chatterjee D, Bhattacharjee P. Supercritical carbon dioxide extraction of eugenol from clove buds. Food Bioprocess Technol 2013; 6(10): 2587-99.
[http://dx.doi.org/10.1007/s11947-012-0979-2] ]. They charged the extractor with 20 g of clove powder with a diameter of 0.5 mm under a constant CO₂ flow rate of 2 l/min. Despite obtaining the maximum eugenol yield (13.292 g eugenol / 100 g clove) at 90 °C, they recommended a temperature of 60 °C, a pressure of 250 bar, and an extraction time of 90 min as the optimum operational condition for maximizing the extraction yield of eugenol (12.986 g eugenol / 100 g clove). The higher yield achieved at an extraction time of 90 min compared to that at 150 min in their work may be due to the depletion of the oil in the clove particles for t > 90 min, which means that the flow of CO₂ not only does not bring any oil to the collection vial but also causes evaporation of the volatile oils from collection vial out the outlet tube. The impacts of temperature, pressure, and extraction time on the yield of eugenol based on the results obtained by Chatterjee and Bhattacharjee [17Chatterjee D, Bhattacharjee P. Supercritical carbon dioxide extraction of eugenol from clove buds. Food Bioprocess Technol 2013; 6(10): 2587-99.
[http://dx.doi.org/10.1007/s11947-012-0979-2] ] are shown in the first three rows of Table 3. As shown in this table, increasing the temperature from 40 to 60 °C increases the eugenol yield by 112%, while increasing the pressure from 250 to 500 bar or increasing the extraction time from 90 to 150 min changed the eugenol yield by 16% and 15%, respectively.

Wenqiang et al. [12Guan W, Li S, Yan R, Tang S, Quan C. Comparison of essential oils of clove buds extracted with supercritical carbon dioxide and other three traditional extraction methods. Food Chem 2007; 101(4): 1558-64.
[http://dx.doi.org/10.1016/j.foodchem.2006.04.009] ] extracted eugenol from clove buds using SFE at a constant CO₂ flow rate (2 l/min) and extraction time (120 min) but at various temperatures (30, 40, and 50 °C), pressures (100, 200, and 300 bar), and particle sizes (three degree index, 1#, 2#, and 3#). The best operating conditions for maximizing the eugenol yield were a temperature of 50 °C, pressure of 300 bar, and particle diameter index of 1#. Under these conditions, 13.64 g eugenol / 100 g clove was recovered. The data from their study are summarized in the fourth to sixth rows of Table 3. In particular, increasing the temperature from 30 to 50 °C together with decreasing the particle size of clove from 3# to 1# at a constant pressure of 300 bar enhanced the eugenol yield by 34%. It should be noted that particle diameter and grinding procedure are dependent of each other, and they can be combined in one variable (grinding procedure). However, they are both reported in (Table 3) for clarity.

Yang et al. [18Yang YC, Wei MC, Hong SJ. Ultrasound-assisted extraction and quantitation of oils from Syzygium aromaticum flower bud (clove) with supercritical carbon dioxide. J Chromatogr A 2014; 1323: 18-27.
[http://dx.doi.org/10.1016/j.chroma.2013.10.098] [PMID: 24290173] ] compared traditional SFE to ultrasound-assisted SFE (USFE) for the extraction from clove buds with 0.355 mm mean particle diameter. They evaluated the impacts of static time (10 to 30 min), dynamic time (20 to 140 min), temperature (30 to 50 ^oC), pressure (100 to 200 bar), and CO₂ flow rate (0.4 to 1.8 g/min) on the extraction yield. The highest USFE yield of 22.04 g extract / 100 g clove buds was obtained at static time of 15 min, dynamic time of 100 min, temperature of 40 ^oC, pressure of 150 bar, and CO₂ flow rate of 1.4 g/min, while the corresponding numbers for the highest SFE yield of 19.06 g extract / 100 g clove buds were, respectively, 30 min, 140 min, 40 ^oC, 200 bar, and 1.8 g/min. The current study, however, only focuses on the impacts of flow rate of CO₂ on eugenol yield based on SFE data of their study (the seventh and eighth rows of Table 3). Noticeably, it is assumed in this part of table that the extract composition does not affected by CO₂ flow rate. This table reports that increasing the flow rate from 0.4 to 0.9 g/min and from 0.9 to 1.8 g/min enhanced the eugenol yield by 36% and 37%, respectively.

A general analysis of the whole data in Table 3 reveals that temperature, in particular, and CO₂ flow rate are by far the most important factor in the SFE of eugenol from clove. The impacts of the other factors like pressure and extraction time are comparable to the impact of the grinding procedure in the current study as employing grinding procedure “E” instead of “A” increases the eugenol yield by 17%. Nevertheless, using the grinding procedure to increase the SFE performance offers many advantages over changing the pressure or extraction time including its simplicity and lower costs.

This work highlights the impacts of five grinding procedures (A, B, C, D, and E) on the recoveries of eugenol, β-caryophyllene, α-humulene, and eugenyl acetate in the SFE of clove. GC analysis of the obtained extracts showed that eugenol had by far the highest yield (9.61 to 11.20 g / 100 g clove), followed by eugenyl acetate (2.09 to 2.55 g / 100 g clove), and β-caryophyllene (1.34 to 1.58 g / 100 g clove). Grinding procedure “E” produced the extract with the highest contents of the aforementioned components, while grinding procedures “A” and “C” gave the lowest yields. Among the continuous grinding procedures (“A”, “B”, and “D”), grinding procedure “B” provided the best performance in terms of the SFE yield of the main components due to the crossover effect of the GT, which affects the particle size, in one side, and the temperature of material in the mill, in the other side. The experimental results reveal that particles diameter, temperature increases in the mill, and the total GT, which includes the duration of both crushing and stopping, are all critical factors affecting the SFE yields of the key components of clove oil. It was revealed that employing grinding procedure “E” instead of “A” increases the eugenol yield by 17%, which is very comparable with the impacts of pressure and extraction time in the literature data. The grinding procedure has the potential to play a critical role in SFE for producing higher quality extracts.