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CO2-supercritical extraction, hydrodistillation and steam distillation of essential oil of rosemary (Rosmarinus officinalis)
Journal of Food Engineering 200 (2017) 81e86
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Journal of Food Engineering journal homepage: www.elsevier.com/locate/jfoodeng
CO2-supercritical extraction, hydrodistillation and steam distillation of essential oil of rosemary (Rosmarinus officinalis) ndez a, Jose R. Espinosa-Victoria a, Arturo Trejo b, Lilia A. Conde-Herna Guerrero-Beltra A. n a, * Jose rtir, San Andr Departamento de Ingeniería Química, Alimentos y Ambiental, Universidad de las Am ericas Puebla, Ex Hacienda de Santa Catarina Ma es Cholula, Puebla, 72810, Mexico b leo, Gustavo A. Madero, Mexico City, Mexico Laboratory of Thermodynamics of the Thermophysics Research Area on Solubility, Instituto Mexicano del Petro a
a r t i c l e i n f o
a b s t r a c t
Article history: Received 21 May 2013 Received in revised form 28 June 2016 Accepted 29 December 2016 Available online 30 December 2016
Essential oil of rosemary was obtained by supercritical CO2-extraction (SCE), hydrodistillation (HYDRO), and steam distillation (SD). Quantity of oil, antioxidant activity, and chemical composition (gas chromatography-mass spectrometry, GC-MS) of the essential oils were evaluated. For SCE, oil was obtained at two temperatures (40 and 50 C) and two pressures (10.34 and 17.24 MPa) using a rosemary particle size of 600 ± 50 mm. The yield was between 1.41 and 2.53 g essential oil (EO) 100 g1 of dry rosemary (% w/w). The antioxidant activity values were in the range 29.67e37.55 mg equivalent of Trolox (ET) g1 of EO or 22.66e30.81 mg ascorbic acid (AA) g1 of EO. Yields of essential oil were between 0.35 and 2.35%. The antioxidant activity was found in the range 1.73e2.60 mg ET g1 of EO or 1.50e2.20 mg AA g1 of EO. Camphor, eucalyptol, b-caryophyllene, and borneol acetate were the main chemicals detected by GC-MS in EO. © 2017 Elsevier Ltd. All rights reserved.
Keywords: Rosemary Rosmarinus officinalis Supercritical extraction Hydrodistillation Steam distillation Antioxidant activity
1. Introduction The possibility of replacing the extraction of natural products using organic solvents by supercritical fluid extraction was proposed by Djarmati et al. (1991) and Nguyen et al. (1991). The extraction of antioxidants from plant sources using organic solvents has the disadvantage of causing oxidative transformations during solvent removal (Sebastian et al., 1998). It has been reported that the supercritical fluid extraction may render extracts with higher antioxidant activity than those obtained by using organic solvents. Wenqiang et al. (2007) reported 19.6, 10.1, and 11.5% of essential oil from clove buds using the SCE (50 C, 10 MPa), SD, HYDRO methods, respectively. Goretti et al. (2004), on the other hand, reported 54.0, 34.6, and 73.1% of eugenol in essential oil obtained by the SD, MO (microwave oven distillation), and SCE methods, respectively. The supercritical fluid extraction technology meets some desirable properties for the production of natural products and/or
* Corresponding author. E-mail addresses: [email protected], Guerrero-Beltr (J.A. an). http://dx.doi.org/10.1016/j.jfoodeng.2016.12.022 0260-8774/© 2017 Elsevier Ltd. All rights reserved.
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functional ingredients. According to Rizvi et al. (1994), when a gas is compressed isothermally at pressures beyond its critical pressure, the gas performs as a solvent at its critical temperature; such fluids are called supercritical fluids. A supercritical fluid behaves very well above its triple point. Products of biological origin, such as natural food preservatives or functional ingredients, are sometimes heat labile and easily oxidized; consequently they need to be processed at low temperatures and, if possible, using oxygen-free atmospheres. Carbon dioxide is not oxidant and has a critical temperature of 31.1 C, making it suitable for the extraction of natural sensitive products from parts of plants. The use of supercritical carbon dioxide, for obtaining extracts from plants, is a technique used for recovering of high value-added ingredients (Pourmortazavi and Hajimirsadeghi, 2007; Reverchon and De Marco, 2006; Babovic et al., 2010; Herrero et al., 2010). Today, due to the consumer’s demands and the increasing legal restrictions for delivering healthy foods to consumers, the supercritical CO2-extraction could be the alternative to the solvents extraction of heat labile components. Thus, high quality products, free from solvents could be obtained (Reglero et al., 2005). Despite the above information, the reason for the limited use of the supercritical fluid extraction technology is the high cost of investment
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compared to the solvent extraction technology (Reglero et al., 2005). Nevertheless, in an experimental way, the supercritical technique has been used widely for extracting essential oils of a number of different parts of plants such as seeds of pomegranate (Abbasi et al., 2008), mint leaves (Aghel et al., 2004), lime (AttiSantos et al., 2005), patchouli (Donelian et al., 2009), ocimum leaves (Goretti et al., 2004), just to mention some. Rosemary is an aromatic plant belonging to the Lamiaceae family. Rosemary has been cultivated since ancient times and recognized as one of the plants with the highest content of antioxidants. Substances in R. officinalis, associated with such activity, are carnosol, rosmanol, isorosmanol, rosmadiol, carnosic acid, ~ ez et al., 2003). Tradirosmarinic acid, and methyl carnosate (Iban tionally, rosemary oil is extracted by steam distillation. The extraction with supercritical CO2, as an alternative method for solvents extraction, may retain the essential oil components without any change or degradation; in addition, this solvent may also remove other groups of compounds. It has already been obtained the rosemary essential oil by the supercritical extraction process. Researchers have investigated effects of particle size, flow rate of the solvent, pressure, temperature, and the addition of ethanol to the tested material (Bensebia et al., 2009). Carvalho et al. (2005) have already investigated the effect of the CO2 supercritical fluid extraction of essential oil of rosemary on the kinetics, performance, composition, and antioxidant activity of the oil. However, the extraction conditions may vary according to a number of variables that can change (biological material) or can be changed (type of equipment, working variables). Regarding biological materials, plants may differ in composition depending on the season, growing conditions, variety, pre-treatments such as dying or blanching. In the case of working variables, equipments, depending if they are of a commercial brand or constructed in place, can have different instrumentation for controlling variables such as pressure (pressure gauges), temperature (temperature gauges), flow of gases, among others, that may restrict the use of the equipment. In any case, the working conditions should be optimized for each biomaterial and fluids used for the extraction. Usually, essential oils are complex mixtures of up to 100 components, these can be low molecular weight aliphatic compounds (alkanes, alcohols, aldehydes, ketones, esters, and acids), monoterpenes, sesquiterpenes, and phenylpropanes. There are a number of essential oils which components, such as carvacrol, cinnamaldehyde, citral, p-cymene, eugenol, limonene, menthol, and thymol, have been considered by the FDA as GRAS substances and are registered by the European Commission as food flavorings rez, 2006). (Pe The aim of this study was to characterize the antioxidant activity and chemical composition of the essential oil of rosemary grown in Mexico obtained by the supercritical CO2-extraction, hydrodistillation, and steam distillation methods.
a kit Keck Sieve Shaker (Cole Parmer, Vernon Hills, IL, USA). Dried samples were packed into plastic bags, sealed under vacuum, protected from light and stored at room temperature until use. 2.3. Supercritical CO2-extraction system The supercritical fluid-extraction equipment was assembled and starting to work at the University of the Americas Puebla. The extraction system is a modification based on a system found in the Laboratory of Thermodynamics of the Thermophysics Research n and Trejo, 2001) and recovery Area on solubility (Eustaquio-Rinco (Avila-Ch avez et al., 2007) of hydrocarbons at the Instituto Mexleo (IMP) in Mexico City. The supercritical fluidicano del Petro extraction equipment consists of three main sections: the inlet or feeding section, the extraction section and the outlet section. 2.3.1. Inlet section This section provides the solvent (CO2) and allows to reach the required pressure conditions for entering into the extraction section. The system of this section is made up of one tank of CO2 (99% purity), one compressor, one pressure gauge, one thermo compressor, one Supercritical Fluid Pump model SFT-10 by Supercritical Fluid Technologies Inc. (Newark, DE, USA), and serpentine flow tubing for driving CO2 to the recollection cell. 2.3.2. Extraction section In this section, the extraction process takes place at the working temperature and pressure conditions. It consists of the following parts: an equilibrium cell for extraction made of 316 stainless steel (6.1 cm in diameter, 18 cm in height, and 0.526 L in volume), a pressure gauge attached to the extraction cell, a heating system made up of light bulbs and fans, and a Cole Parmer Digi-Sense temperature controller R/S (Vernon Hills, IL, USA) attached to a gauge sensor (ASL F200 Precision Thermometers), (Instrumart, Carlsbad, CA, USA). 2.3.3. Outlet section In this section, the extract is separated from the solvent by changing the supercritical conditions at room atmospheric conditions. It consists of the following parts: glass recollection cell for recovering of product, a heating bath and a flowmeter for measuring volumetric flow rate. 2.3.4. Oil extraction 25 g of dried ground rosemary, with a particle size of 600 ± 50 mm, were placed in the equilibrium extraction cell. Extractions were performed at temperatures of 40 and 50 C and pressures of 10.34 and 17.24 MPa. The CO2 volumetric flow rate was 126.24 ± 20.83 mL min1. The recollection cell was immersed in a water bath at 8 ± 0.5 C for condensing all extracted vapors. All tests were performed twice.
2. Materials and methods 2.4. Hydrodistillation and steam distillation 2.1. Materials Fresh rosemary (Rosmarinus officinalis L.) was purchased in the local market of the city of Cholula, Puebla, Mexico. 2.2. Sample preparation Rosemary branches were dried in a foods tray dryer (Excallibur, Pennsylvania, and USA) at a temperature of 35 C for 24 h. Leaves were removed from the branches for obtaining the essential oil. Whole and pulverized, in a mortar, dried rosemary leaves were used for the oil extraction. The pulverized sample was sieved using
The process, for obtaining the essential oil by the SD and HYDRO methods, was conducted in a Clevenger type distillation apparatus. The apparatus is made up of a heat source, a 2 L pear-shaped glass (PSG) container for creating steam by boiling water, a 2 L spherical glass container (SGC) with upper and bottom entrances, a straight glass condenser, and a glass collector for separating and recovering the essential oil; oil appears on top of water in the collector. Steam distillation (SD): sample is placed in the SGC flask and water in the PSG container. Water is then heated and the crated vapor passes though the sample in the SGC container. Consequently, vapor drag the essential oil and is condensed and recovered in the glass
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collector. Hydrodistillation (HYDRO) (the SGC is not required): sample and water in a ratio 1:5 (w/v) are placed into the PSG container. The system is heated and the vapor-oil mixture is condensed and then collected in the glass collector. An experimental design with three factors and two levels: methods (hydrodistillation and steam distillation), material quantity (25 and 50 g) and sample (whole leaves and ground (600 ± 50 mm)). The moisture content of dried sample was 2.991 ± 0.66% (w/w). All extractions were done in duplicate. The yield of essential oil was calculated with the following equation:
Essential:oil:ðgÞ 100 Sample:weight:ðgÞ
Essential:oilð%Þ ¼
(1)
2.5. Antioxidant activity The antioxidant activity was analyzed by the ABTS (2,2 ’-azinobis-(3-ethyl-benzotiazolino-6-sulfonic acid)) (Sigma-Aldrich, St. Louis, USA) method, according to the methodology suggested by Re et al. (1999). The ABTSþ radical was obtained by reacting the ABTS (7 mM) along with potassium persulfate (2.45 mM) (Sigma-Aldrich, St. Louis, USA) during 16 h at room temperature. Once the radical ABTSþ was formed, it was diluted with ethanol to obtain an ABTSþ radical-ethanol solution with an initial absorbance (Ai) of 0.700 ± 0.020, measured at 754 nm. The antioxidant capacity was measured placing 3920 mL of the ABTSþ radical-ethanol solution in a quartz spectrophotometer cell, 80 mL of essential oil (dissolved in ethanol), thoroughly mixed, allowed to react for 7 min, and the final absorbance measured (Af). To calculate the antioxidant activity in samples, standard curves of Trolox (6-hydroxy-2,5,7,8cidocarboxílico) (0e0.2 mg mL1) and ascortetrametilcromo-2-a bic acid (0e0.14 mg mL1) (Omnichem, Puebla, Mexico) were prepared. The antioxidant activity was calculated as mg ET mL1 of essential oil (T) and mg equivalent to AA mL1 of essential oil (AA). The following equations were used for calculating antioxidant activity.
Ið%Þ ¼
Ai Af 100 Ai
(2)
% C½mgT=mL þ 3:256% R2 ¼ 0:996 mgT=mL
Ið%Þ ¼ 387:13
(3) % C½mgAA=mL mgAA=mL þ 0:358% R2 ¼ 0:996
83
of the mass range was m/z 43e350. One mL of sample was injected. The identification of the volatile compounds was performed comparing their mass spectra with mass spectra of the NIST (National Institute of Standards and Technology) database library and information published in literature (Adams, 1989). The GC-MS analysis was performed only to the oil obtained at 10.34 MPa and 40 C. 2.7. Scanning electron microscopy (SEM) The morphology of samples exposed to the entire process of supercritical fluids extraction was examined using a JEOL JSM6610LV scanning electron microscope (Tokyo, Japan). A probe, attached to the microscope, was used to perform the chemical analysis by the dispersive X-ray energy technique. All samples were examined under high vacuum and an acceleration voltage of 20 kV. 3. Results y discussion 3.1. Rosemary oil obtained by SCE Fig. 1 depicts the kinetics of essential oil extraction of rosemary previously performed for stating the working time. It is observed that the longer the extraction time, the higher the yield of rosemary essential oil. The kinetics behavior of the oil extraction was similar to that reported by Bensebia et al. (2009) and Fornari et al. (2012). It is also observed that percentages of the essential oil for 150 and 180 min of extraction were similar (p > 0.05). Therefore, the four experiments were carried out during 180 min twice (Fig. 2). Fig. 2 illustrates the yields of rosemary essential oil obtained at different CO2-supercritical extraction conditions. The final yield of rosemary essential oil was between 1.41 and 2.53 g essential oil/ 100 g dried matter. The sample with lower yield was the one obtained at a pressure of 10.34 MPa and 50 C, while the best performance of the system for rendering the higher rosemary oil content was with the sample treated at 17.24 MPa and 40 C. The best extraction of essential oil was obtained at the higher pressure (17.24 MPa) and the lower temperatures (40 C). At the lower pressure (10.34 MPa) and the higher temperature (50 C) the lower yield of oil was obtained. Bensebia et al. (2009) achieved yields of up to 3% oil in rosemary at 14.87 MPa and 40 C. Carvalho et al. (2005) achieved yields of up to 4% (w/w) at 24.78 MPa and 40 C after 250 min of extraction. Fornari et al. (2012) achieved yields of 3.5% (w/w) at 24.78 MPa, 40 C and 270 min. In all these studies, researchers used higher pressures and extraction times
Ið%Þ ¼ 491:28
(4)
where I is the inhibition. 2.6. GC-MS compounds identification For the characterization of volatile compounds, a gas chromatograph, coupled to a mass selective detector quadrupole 5975 (Agilent Technologies 6850N GC, Santa Clara, CA, USA) was used. A HP5-MS column of 30 m in length and 0.25 mm in diameter, covered with a film of 0.25 mm made of 5% phenyl and 95% dimethylpolysiloxane, was used. The conditions for the GC-MS analysis were as follows: Helium as carrier gas at a flow rate of 15.5 mL min1, a temperature of 250 C in the injector, Split ratio of 10:1, and a temperature program, starting at 60 C, at a rate of 4 C/ min until reach 240 C. Ionization energy was 70 eV. The scanning
Fig. 1. Kinetics of essential oil extraction by supercritical extraction of rosemary.
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Fig. 4. Antioxidant activity of rosemary essential oil obtained by supercritical extraction at selected temperatures and pressures.
Fig. 2. Rosemary essential oil yield obtained by CO2-supercritical extraction.
than the used in this study. It is important to mention that the season of the year, variety, among other variables, may affect the amount of essential oils in plants. Fig. 5. Antioxidant activity of rosemary essential oil obtained by steam distillation and hydrodistillation at selected pressures and temperatures.
3.2. Rosemary oil obtained by HYDRO and SD Fig. 3 shows the yields of rosemary essential oils obtained by the HYDRO and SD methods. The lower yield was 0.35% (w/w) when extracting 25 g of ground rosemary using the HYDRO method. The higher yield (2.35%) was obtained by the SD method using 50 g of whole sample. It is important to say that the extraction time for each experiment was 90 min. Better yields were obtained with the SD method using 50 g of whole sample. Yields obtained with the HYDRO method in ground rosemary were low. The highest yield was obtained with the CO2supercritical extraction process. Rasooli et al. (2008) obtained a yield of rosemary oil of 1% when the extraction was performed with the HYDRO method. Boutekedjiret et al. (2003) reported 1.2 and 0.44% of rosemary oil obtained by steam distillation and hydrodistillation, respectively. Flamini et al. (2002), Angioni et al. (2004), and Jamshidi et al. (2009) reported maximum yields of 1.44, 2.13, and 2.6% of oil, respectively, using the HYDRO method. 3.3. Oils antioxidant activities Figs. 4 and 5 ilustrates the antioxidant activity characterisitcs of
rosemary oil obtained by CO2-supercritical extraction (Fig. 4) and steam destilation and hydrodestillation, respectively (Fig. 5). The rosemary oil that showed the greatest antioxidant activity (37.55 ± 0.86 mg Trolox/g of essential oil) was the one obtained at 17.24 MPa and 40 C; the lower antioxidant activity (29.67 ± 0.52 mg Trolox/g essential oil) was observed at the extraction conditions of 10.34 MPa and 50 C (Fig. 5). Rosemary oils obtained at 17.24 MPa showed the higher antioxidant activities. With regard to oils extracted by HYDRO and SD (Fig. 5), the SD oil from 25 g of ground rosemary showed a higher antioxidant activity while the oil obtained from 50 g of entire rosemary by HYDRO presented the lowest antioxidant activity. High antioxidant activities were observed in oils obtained from ground samples. It can be clearly observed an increased antioxidant activity in oils obtained by supercritical extraction (Fig. 4); almost 14 times more than quantities obtained by SD or HYDRO. Liu et al. (2012) reported antioxidant activity values between 0.5 and 6.5 mg Trolox/g of oil in pomegranate seed oil (Punica granatum L.) obtained by supercritical extraction. Samec et al. (2010) reported an antioxidant capacity of 0.50e3.00 mg Trolox mL1 of an infusion obtained from flowers and leaves of Teucrium arduini L. In this study, much higher antioxidant activity values were found for the extracts obtained by supercritical extraction compared with those reported by researchers mentioned above. 3.4. Chemical compounds identification by GC-MS
Fig. 3. Yield of rosemary essential oil obtained by the HYDRO and SD methods. W: whole, G: Ground. SD: Steam Distillation, HYDRO: Hydrodistillation.
Table 1 shows the compounds identified by GC-MS of rosemary oils obtained by supercritical extraction, SD, and HYDRO. As can be seen, the major components of the essential oil of Rosmarinus officinalis L., obtained by supercritical extraction, were camphor, b~ ez et al. (1999) identified caryophyllene, and eucalyptol. Iban camphor fractions, ranging from 4.81 to 40.85%, and eucalyptol fractions between 1.79 and 12.29% in rosemary oil obtained also by supercritical extraction. These authors also found b-pinene, linalool, borneol, a-terpineol, verbenone, borneol acetate, a-caryophyllene ~ ez et al. (humulene), and g-muurolene (g-cardinene). However, Iban (2000) reported carnosic acid and rosmanol as the main components. In the rosemary oil obtained by SD, 19 compounds were
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Table 1 Compounds identified in the essential oil of rosemary by GC-MS. Compound
a-Pinene Camphene b-pinene Octenol a-Phellandrene a-Terpinene Eucalyptol b-trans-Ocimene g-Terpinene Terpinolene Linalool Camphor Borneol a-Terpineol Verbenone Safrole Borneol acetate b-Caryophyllene a-Caryophyllene ¼ Humelene g-Muurolene ¼ g-cadinene Epoxycaryophylene Palmitic acid Copaene a b c d e
SCEa
SDb
HYDROc
RTd (min)
PAe (%)
RTd (min)
PAe (%)
RTd (min)
PAe (%)
7.44 8.22 9.91 e 11.82 e 13.34 e 14.76 e 17.16 18.74 19.66 20.61 21.05 e 23.30 27.19 28.07 29.80 31.37 39.82 e
2.77 2.20 1.68 e 1.25 e 10.60 e 1.36 e 3.32 28.42 3.41 3.07 7.98 e 8.14 12.18 5.14 1.54 1.22 0.90 e
7.22 7.93 9.47 10.26 11.44 12.03 12.92 13.40 14.34 15.61 e 18.21 18.99 19.82 20.37 22.90 22.81 26.64 27.44 e 30.71 e e
11.96 9.29 6.01 1.15 11.78 4.71 15.36 1.10 4.27 1.99 e 14.76 3.82 1.98 0.80 1.20 2.97 4.34 0.59 29.93 0.65 e e
7.60 8.39 10.05 e 11.35 12.69 13.61 e 14.89 16.19 e 19.03 19.94 20.85 e e 23.51 27.35 28.22 1.66 31.54 e 25.93
5.03 3.74 0.70 e 5.35 1.28 12.59 e 1.78 1.00 e 21.51 3.95 2.77 e e 10.53 10.22 5.56 e 5.31 e 0.83
Supercritical extraction. Steam distillation. Hydrodistillation. Retention time. Pick area.
identified and 17 compounds by the HYDRO method. Angioni et al. (2004) reported a-pinene (23%), camphene (7.6%), borneol (16%), camphor (4.5%), verbenone (9.4%), and borneol acetate (10.4%) in rosemary oil obtained by SD. Gachkar et al. (2007) reported apinene (14.9%), linalool (14.9%), and eucalyptol (1,8-cineole) (7.43%)
as major components of rosemary oil content by SD. Boutekedjiret et al. (2003) found 1,8-cineole (52.4%), camphor (12.6) and bpinene (5.7%) as major components in rosemary oil obtained by HYDRO and 1,8 cineol (31.9%), camphor (19.7%) and a-Terpineol (12.8%) extracted by SD as the primary compounds. Differences in
a
b
c
d
e
Fig. 6. Micrographs of whole rosemary supercritical extracted. A) Untreated, b) 10.34 MPa, 40 C, c) 10.34 MPa, 50 C, d) 17.24 MPa, 40 C, e) 17.24 MPa, 50 C.
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chemical composition of rosemary oil from different studies can be attributed to factors such as plant variety, country of origin, time of harvest, climatic factors, among other factors. The main components of the essential oil of Rosmarinus officinalis L. obtained in this work were camphor and eucalyptol; compounds widely used in the food and pharmaceutical industries. It has been shown that compounds containing menthol and camphor may reduce the respiration rate in infants with acute bronchitis, may reduce cough in adults, and may improve mucociliary clearance in adults. The FDA (Food and Drug Administration) approved camphor to relieve coughs, but the permitted limit concentration in the preparations is 11% (Paul et al., 2010). 3.5. Scanning electron microscopy Fig. 6 illustrates the micrographs of rosemary exposed to supercritical extraction. There is a smooth surface of the sample without treatment. Structures of rosemary treated with SCE show an irregular surface. Also, samples used for the supercritical extraction showed some holes on the surface of rosemary. Most of the samples appear collapsed and ruptured. 4. Conclusions Higher yields of essential oil and antioxidant activity were observed in the rosemary oil obtained by the CO2-supercritical extraction, followed by steam distillation, and finally by hydrodistillation. The use of whole rosemary leaves improved the oil extraction compared to the oil rendered from powdered leaves. Also, higher yields of oil were observed using 50 g of sample. The antioxidant activity of oil obtained by supercritical extraction was approximately 14 times higher than that obtained in oils extracted by SD or HYDRO. In oils obtained by the three methods, camphor and eucalyptol were the mayor compounds. In the rosemary essential oil obtained by SCE and HYDRO, 17 compounds were identified, and 19 compounds in the oil obtained by SD; however, the oil obtained by supercritical extraction was richer in camphor. Acknowledgements ndez thanks the National Author Lilia Alejandra Conde-Herna Council for Science and Technology (CONACYT) and the Universidad de las Americas Puebla (UDLAP) for the financial support to complete her doctoral studies. References Abbasi, H., Rezaei, K., Rashidi, L., 2008. Extraction of essential oils from the seeds of pomegranate Using organic solvents and supercritical CO2. J. Am. Oil Chem. Soc. 85, 83e89. Adams, R.P., 1989. Identification of Essential Oil Components by Ion Trap Mass Spectroscopy. Academic Press, Inc, San Diego, California, USA. Aghel, N., Yamini, Y., Hadjiakhoondi, A., Pourmortazavi, S.M., 2004. Supercritical carbon dioxide extraction of Mentha pulegium L. essential oil. Talanta 62, 407e411. Angioni, A., Barra, A., Cereti, E., Barile, D., Coïsson, J.D., Arlorio, M., Dessi, S., Coroneo, V., Cabras, P., 2004. Chemical composition, plant genetic differences, antimicrobial and antifungal activity investigation of the essential oil of Rosmarinus officinalis L. J. Agric. Food Chem. 52, 3530e3535. Atti-Santos, A.C., Rossato, M., Atti, L., Cassel, E., Moyna, P., 2005. Extraction of essential oils from lime (Citrus latifolia Tanaka) by hydrodistillation and supercritical carbon dioxide. Braz. Archiv. Biol. Technol. 48 (1), 155e160. vez, M.A., Eustaquio-Rinco n, R., Reza, J., Trejo, A., 2007. Extraction of hyAvila-Cha drocarbons from crude oil tank bottom sludges using supercritical ethane. Sep. Sci. Technol. 42, 2327e2345. Babovic, N., Djilas, S., Jadranin, M., Vajs, V., Ivanovic, J., Petrovic, S., Zizivic, I., 2010. Supercritical carbon dioxide extraction of antioxidant fractions from selected Lamiaceae herbs and their antioxidant capacity. Innov. Food Sci. Emerg. Technol. 11, 98e117. Bensebia, O., Barth, D., Bensebia, B., Dahmani, A., 2009. Supercritical CO2 extraction of rosemary: effect of extraction parameters and modeling. J. Supercrit. Fluids
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Solubility of n-Octadecane in supercritical Eustaquio-Rinco carbon dioxide at 310, 313, 333, and 353 K, in the range 10-20 Mpa. Fluid Phase Equilib. 185, 231e239. Flamini, G., Cioni, P.L., Morelli, I., Macchia, M., Ceccarini, L., 2002. Main agronomicproductive characteristics of two ecotypes of Rosmarinus officinalis L. and chemical composition of their essential oils. J. Agric. Food Chem. 50, 3512e3517. Fornari, T., Ruiz-Rodriguez, A., Vicente, G., Vazquez, E., Garcia-Risco, M.R., Reglero, G., 2012. Kinetic study of the supercritical CO2 extraction of different plants from Lamiaceae family. J. Supercrit. Fluids 64, 1e8. Gachkar, L., Yadegari, D., Rezaei, M.B., Taghizadeh, M., Astaneh, S.A., Rasooli, I., 2007. Chemical and biological characteristics of Cuminum cyminum and Rosmarinus officinalis essential oils. Food Chem. 102, 898e904. Goretti, M., de Abreu, F.J., Oliveira, P.R., Oliveira, F., Tavares, M., 2004. Composition of essential oils from three Ocimum species obtained by steam and microwave distillation and supercritical CO2 extraction. ARKIVOC 6, 66e71. ~ ez, E., 2010. Supercritical fluid Herrero, M., Mendiola, J.A., Cifuentes, A., Ib an extraction: recent advances and applications. J. Chromatogr. A 1217, 2512e2520. ~ ez, E., Oca, A., Murga, G., Lo pez-Sebastia n, S., Tabera, J., Reglero, G., 1999. SuIban percritical fluid extraction and fractionation of different preprocessed rosemary plants. J. Agric. Food Chem. 47, 1400e1404. ~ ez, E., Cifuentes, A., Crego, A.L., Sen ~ ora ns, F.J., Cavero, S., Reglero, G., 2000. Iban Combined use of supercritical fluid extraction, micellar electrokinetic chromatography, and reverse phase high performance liquid chromatography for the analysis of antioxidants from rosemary (Rosmarinus officinalis L.). J. Agric. Food Chem. 48, 4060e4065. ~ ez, E., Kuba tov ~ ora ns, F.J., Cavero, S., Reglero, G., Hawthorne, S.B., 2003. Iban a, A., Sen Subcritical water extraction of antioxidant compounds from rosemary plants. J. Agric. Food Chem. 51, 375e382. Jamshidi, R., Afzali, Z., Afzali, D., 2009. Chemical composition of hydrodistillation essential oil of rosemary in different origins in Iran and comparison with other countries. Am. Eurasian J. Agric. Environ. Sci. 5, 78e81. Liu, G., Xu, X., Gong, Y., He, L., Gao, Y., 2012. Effects of supercritical CO2 extraction parameters on chemical composition and free radical-scavenging activity of pomegranate (Punica granatum L.) seed oil. Food Bioprod. Process. 90, 573e578. Nguyen, U., Evans, D.A., Frakman, G., 1991. Supercritical fluid extraction. In: Rizvi, S.S.H. (Ed.), Supercritical Fluid Processing of Food and Biomaterials. Blackie Academic and Professional, London, UK. Paul, I.M., Beiler, J.S., King, T.S., Clapp, E.R., Vallati, J., 2010. Vapor rub, petrolatum, and no treatment for children with nocturnal cough and cold symptoms. Pediatrics 126, 1092e1099. rez, A.T.F., 2006. Efectividad de los vapores de aceites de tomillo y ore gano como Pe ricas Puebla, agentes antibacterianos. M.Sc. thesis. Universidad de las Ame Puebla, Mexico. Pourmortazavi, S.M., Hajimirsadeghi, S.S., 2007. Supercritical fluid extraction in plant essential and volatile oil analysis. J. Chromatogr. A 1163, 2e24. Rasooli, I., Fakoor, M.H., Yadegarinia, D., Gachkar, L., Allameh, A., Rezaei, M.B., 2008. Antimycotoxigenic characteristics of Rosmarinus officinalis and Trachyspermum copticum L. essential oils. Int. J. Food Microbiol. 122, 135e139. Re, R., Pellegrini, N., Proteggente, A., Annala, A., Yang, M., Rice-Evans, C., 1999. Antioxidant activity applying an improved ABTS radical cation decolorization assay. Free Radic. Biol. Med. 26, 1231e1237. ~ ora ns, J.F., Iban ~ ez, E., 2005. Supercritical fluid extraction: an alterReglero, G., Sen native to isolating natural food preservatives. In: Barbosa-C anovas, G.V., Tapia, M.S., Cano, M.P. (Eds.), Novel Food Processing Technologies. CRC Press, Boca Raton, FL, USA, p. 539. Reverchon, E., De Marco, I., 2006. Supercritical fluid extraction and fractionation of natural matter. J. Supercrit. Fluids 38, 146e166. Rizvi, S.S.H., Yu, Z.R., Bhaskar, A.R., Chidambara-Raj, C.B., 1994. Fundamentals of processing with supercritical fluids. In: Rizvi, S.S.H. (Ed.), Supercritical Fluid Processing of Food and Biomaterials. Blackie Academic and Professional, London, UK. Samec, D., Gruz, J., Strnad, M., Kremer, D., Kosalec, I., Jurisic Grubesic, R., Karlovic, K., Lucic, A., Piljac-Zegarac, J., 2010. Antioxidant and antimicrobial properties of Teucrium arduini L. (Lamiaceae) flower and leaf infusions (Teucrium arduini L.) antioxidant capacity. Food Chem. Toxicol. 48, 113e119. n ~ ez, E., Bueno, J.M., Ballester, L., Tabera, J., Reglero, G., Sebastian, S.L., Ramos, E., Iba 1998. Dearomatization of antioxidant rosemary extracts by treatment with supercritical carbon dioxide. J. Agric. Food Chem. 47, 13e19. Wenqiang, G., Shufen, L., Ruixiang, Y., Shaokun, T., Can, Q., 2007. Comparison of essential oil of clove buds extracted with supercritical carbon dioxide and other three traditional extraction methods. Food Chem. 101, 1558e1564.
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Journal of Food Engineering journal homepage: www.elsevier.com/locate/jfoodeng
CO2-supercritical extraction, hydrodistillation and steam distillation of essential oil of rosemary (Rosmarinus officinalis) ndez a, Jose R. Espinosa-Victoria a, Arturo Trejo b, Lilia A. Conde-Herna Guerrero-Beltra A. n a, * Jose rtir, San Andr Departamento de Ingeniería Química, Alimentos y Ambiental, Universidad de las Am ericas Puebla, Ex Hacienda de Santa Catarina Ma es Cholula, Puebla, 72810, Mexico b leo, Gustavo A. Madero, Mexico City, Mexico Laboratory of Thermodynamics of the Thermophysics Research Area on Solubility, Instituto Mexicano del Petro a
a r t i c l e i n f o
a b s t r a c t
Article history: Received 21 May 2013 Received in revised form 28 June 2016 Accepted 29 December 2016 Available online 30 December 2016
Essential oil of rosemary was obtained by supercritical CO2-extraction (SCE), hydrodistillation (HYDRO), and steam distillation (SD). Quantity of oil, antioxidant activity, and chemical composition (gas chromatography-mass spectrometry, GC-MS) of the essential oils were evaluated. For SCE, oil was obtained at two temperatures (40 and 50 C) and two pressures (10.34 and 17.24 MPa) using a rosemary particle size of 600 ± 50 mm. The yield was between 1.41 and 2.53 g essential oil (EO) 100 g1 of dry rosemary (% w/w). The antioxidant activity values were in the range 29.67e37.55 mg equivalent of Trolox (ET) g1 of EO or 22.66e30.81 mg ascorbic acid (AA) g1 of EO. Yields of essential oil were between 0.35 and 2.35%. The antioxidant activity was found in the range 1.73e2.60 mg ET g1 of EO or 1.50e2.20 mg AA g1 of EO. Camphor, eucalyptol, b-caryophyllene, and borneol acetate were the main chemicals detected by GC-MS in EO. © 2017 Elsevier Ltd. All rights reserved.
Keywords: Rosemary Rosmarinus officinalis Supercritical extraction Hydrodistillation Steam distillation Antioxidant activity
1. Introduction The possibility of replacing the extraction of natural products using organic solvents by supercritical fluid extraction was proposed by Djarmati et al. (1991) and Nguyen et al. (1991). The extraction of antioxidants from plant sources using organic solvents has the disadvantage of causing oxidative transformations during solvent removal (Sebastian et al., 1998). It has been reported that the supercritical fluid extraction may render extracts with higher antioxidant activity than those obtained by using organic solvents. Wenqiang et al. (2007) reported 19.6, 10.1, and 11.5% of essential oil from clove buds using the SCE (50 C, 10 MPa), SD, HYDRO methods, respectively. Goretti et al. (2004), on the other hand, reported 54.0, 34.6, and 73.1% of eugenol in essential oil obtained by the SD, MO (microwave oven distillation), and SCE methods, respectively. The supercritical fluid extraction technology meets some desirable properties for the production of natural products and/or
* Corresponding author. E-mail addresses: [email protected], Guerrero-Beltr (J.A. an). http://dx.doi.org/10.1016/j.jfoodeng.2016.12.022 0260-8774/© 2017 Elsevier Ltd. All rights reserved.
[email protected]
functional ingredients. According to Rizvi et al. (1994), when a gas is compressed isothermally at pressures beyond its critical pressure, the gas performs as a solvent at its critical temperature; such fluids are called supercritical fluids. A supercritical fluid behaves very well above its triple point. Products of biological origin, such as natural food preservatives or functional ingredients, are sometimes heat labile and easily oxidized; consequently they need to be processed at low temperatures and, if possible, using oxygen-free atmospheres. Carbon dioxide is not oxidant and has a critical temperature of 31.1 C, making it suitable for the extraction of natural sensitive products from parts of plants. The use of supercritical carbon dioxide, for obtaining extracts from plants, is a technique used for recovering of high value-added ingredients (Pourmortazavi and Hajimirsadeghi, 2007; Reverchon and De Marco, 2006; Babovic et al., 2010; Herrero et al., 2010). Today, due to the consumer’s demands and the increasing legal restrictions for delivering healthy foods to consumers, the supercritical CO2-extraction could be the alternative to the solvents extraction of heat labile components. Thus, high quality products, free from solvents could be obtained (Reglero et al., 2005). Despite the above information, the reason for the limited use of the supercritical fluid extraction technology is the high cost of investment
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compared to the solvent extraction technology (Reglero et al., 2005). Nevertheless, in an experimental way, the supercritical technique has been used widely for extracting essential oils of a number of different parts of plants such as seeds of pomegranate (Abbasi et al., 2008), mint leaves (Aghel et al., 2004), lime (AttiSantos et al., 2005), patchouli (Donelian et al., 2009), ocimum leaves (Goretti et al., 2004), just to mention some. Rosemary is an aromatic plant belonging to the Lamiaceae family. Rosemary has been cultivated since ancient times and recognized as one of the plants with the highest content of antioxidants. Substances in R. officinalis, associated with such activity, are carnosol, rosmanol, isorosmanol, rosmadiol, carnosic acid, ~ ez et al., 2003). Tradirosmarinic acid, and methyl carnosate (Iban tionally, rosemary oil is extracted by steam distillation. The extraction with supercritical CO2, as an alternative method for solvents extraction, may retain the essential oil components without any change or degradation; in addition, this solvent may also remove other groups of compounds. It has already been obtained the rosemary essential oil by the supercritical extraction process. Researchers have investigated effects of particle size, flow rate of the solvent, pressure, temperature, and the addition of ethanol to the tested material (Bensebia et al., 2009). Carvalho et al. (2005) have already investigated the effect of the CO2 supercritical fluid extraction of essential oil of rosemary on the kinetics, performance, composition, and antioxidant activity of the oil. However, the extraction conditions may vary according to a number of variables that can change (biological material) or can be changed (type of equipment, working variables). Regarding biological materials, plants may differ in composition depending on the season, growing conditions, variety, pre-treatments such as dying or blanching. In the case of working variables, equipments, depending if they are of a commercial brand or constructed in place, can have different instrumentation for controlling variables such as pressure (pressure gauges), temperature (temperature gauges), flow of gases, among others, that may restrict the use of the equipment. In any case, the working conditions should be optimized for each biomaterial and fluids used for the extraction. Usually, essential oils are complex mixtures of up to 100 components, these can be low molecular weight aliphatic compounds (alkanes, alcohols, aldehydes, ketones, esters, and acids), monoterpenes, sesquiterpenes, and phenylpropanes. There are a number of essential oils which components, such as carvacrol, cinnamaldehyde, citral, p-cymene, eugenol, limonene, menthol, and thymol, have been considered by the FDA as GRAS substances and are registered by the European Commission as food flavorings rez, 2006). (Pe The aim of this study was to characterize the antioxidant activity and chemical composition of the essential oil of rosemary grown in Mexico obtained by the supercritical CO2-extraction, hydrodistillation, and steam distillation methods.
a kit Keck Sieve Shaker (Cole Parmer, Vernon Hills, IL, USA). Dried samples were packed into plastic bags, sealed under vacuum, protected from light and stored at room temperature until use. 2.3. Supercritical CO2-extraction system The supercritical fluid-extraction equipment was assembled and starting to work at the University of the Americas Puebla. The extraction system is a modification based on a system found in the Laboratory of Thermodynamics of the Thermophysics Research n and Trejo, 2001) and recovery Area on solubility (Eustaquio-Rinco (Avila-Ch avez et al., 2007) of hydrocarbons at the Instituto Mexleo (IMP) in Mexico City. The supercritical fluidicano del Petro extraction equipment consists of three main sections: the inlet or feeding section, the extraction section and the outlet section. 2.3.1. Inlet section This section provides the solvent (CO2) and allows to reach the required pressure conditions for entering into the extraction section. The system of this section is made up of one tank of CO2 (99% purity), one compressor, one pressure gauge, one thermo compressor, one Supercritical Fluid Pump model SFT-10 by Supercritical Fluid Technologies Inc. (Newark, DE, USA), and serpentine flow tubing for driving CO2 to the recollection cell. 2.3.2. Extraction section In this section, the extraction process takes place at the working temperature and pressure conditions. It consists of the following parts: an equilibrium cell for extraction made of 316 stainless steel (6.1 cm in diameter, 18 cm in height, and 0.526 L in volume), a pressure gauge attached to the extraction cell, a heating system made up of light bulbs and fans, and a Cole Parmer Digi-Sense temperature controller R/S (Vernon Hills, IL, USA) attached to a gauge sensor (ASL F200 Precision Thermometers), (Instrumart, Carlsbad, CA, USA). 2.3.3. Outlet section In this section, the extract is separated from the solvent by changing the supercritical conditions at room atmospheric conditions. It consists of the following parts: glass recollection cell for recovering of product, a heating bath and a flowmeter for measuring volumetric flow rate. 2.3.4. Oil extraction 25 g of dried ground rosemary, with a particle size of 600 ± 50 mm, were placed in the equilibrium extraction cell. Extractions were performed at temperatures of 40 and 50 C and pressures of 10.34 and 17.24 MPa. The CO2 volumetric flow rate was 126.24 ± 20.83 mL min1. The recollection cell was immersed in a water bath at 8 ± 0.5 C for condensing all extracted vapors. All tests were performed twice.
2. Materials and methods 2.4. Hydrodistillation and steam distillation 2.1. Materials Fresh rosemary (Rosmarinus officinalis L.) was purchased in the local market of the city of Cholula, Puebla, Mexico. 2.2. Sample preparation Rosemary branches were dried in a foods tray dryer (Excallibur, Pennsylvania, and USA) at a temperature of 35 C for 24 h. Leaves were removed from the branches for obtaining the essential oil. Whole and pulverized, in a mortar, dried rosemary leaves were used for the oil extraction. The pulverized sample was sieved using
The process, for obtaining the essential oil by the SD and HYDRO methods, was conducted in a Clevenger type distillation apparatus. The apparatus is made up of a heat source, a 2 L pear-shaped glass (PSG) container for creating steam by boiling water, a 2 L spherical glass container (SGC) with upper and bottom entrances, a straight glass condenser, and a glass collector for separating and recovering the essential oil; oil appears on top of water in the collector. Steam distillation (SD): sample is placed in the SGC flask and water in the PSG container. Water is then heated and the crated vapor passes though the sample in the SGC container. Consequently, vapor drag the essential oil and is condensed and recovered in the glass
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collector. Hydrodistillation (HYDRO) (the SGC is not required): sample and water in a ratio 1:5 (w/v) are placed into the PSG container. The system is heated and the vapor-oil mixture is condensed and then collected in the glass collector. An experimental design with three factors and two levels: methods (hydrodistillation and steam distillation), material quantity (25 and 50 g) and sample (whole leaves and ground (600 ± 50 mm)). The moisture content of dried sample was 2.991 ± 0.66% (w/w). All extractions were done in duplicate. The yield of essential oil was calculated with the following equation:
Essential:oil:ðgÞ 100 Sample:weight:ðgÞ
Essential:oilð%Þ ¼
(1)
2.5. Antioxidant activity The antioxidant activity was analyzed by the ABTS (2,2 ’-azinobis-(3-ethyl-benzotiazolino-6-sulfonic acid)) (Sigma-Aldrich, St. Louis, USA) method, according to the methodology suggested by Re et al. (1999). The ABTSþ radical was obtained by reacting the ABTS (7 mM) along with potassium persulfate (2.45 mM) (Sigma-Aldrich, St. Louis, USA) during 16 h at room temperature. Once the radical ABTSþ was formed, it was diluted with ethanol to obtain an ABTSþ radical-ethanol solution with an initial absorbance (Ai) of 0.700 ± 0.020, measured at 754 nm. The antioxidant capacity was measured placing 3920 mL of the ABTSþ radical-ethanol solution in a quartz spectrophotometer cell, 80 mL of essential oil (dissolved in ethanol), thoroughly mixed, allowed to react for 7 min, and the final absorbance measured (Af). To calculate the antioxidant activity in samples, standard curves of Trolox (6-hydroxy-2,5,7,8cidocarboxílico) (0e0.2 mg mL1) and ascortetrametilcromo-2-a bic acid (0e0.14 mg mL1) (Omnichem, Puebla, Mexico) were prepared. The antioxidant activity was calculated as mg ET mL1 of essential oil (T) and mg equivalent to AA mL1 of essential oil (AA). The following equations were used for calculating antioxidant activity.
Ið%Þ ¼
Ai Af 100 Ai
(2)
% C½mgT=mL þ 3:256% R2 ¼ 0:996 mgT=mL
Ið%Þ ¼ 387:13
(3) % C½mgAA=mL mgAA=mL þ 0:358% R2 ¼ 0:996
83
of the mass range was m/z 43e350. One mL of sample was injected. The identification of the volatile compounds was performed comparing their mass spectra with mass spectra of the NIST (National Institute of Standards and Technology) database library and information published in literature (Adams, 1989). The GC-MS analysis was performed only to the oil obtained at 10.34 MPa and 40 C. 2.7. Scanning electron microscopy (SEM) The morphology of samples exposed to the entire process of supercritical fluids extraction was examined using a JEOL JSM6610LV scanning electron microscope (Tokyo, Japan). A probe, attached to the microscope, was used to perform the chemical analysis by the dispersive X-ray energy technique. All samples were examined under high vacuum and an acceleration voltage of 20 kV. 3. Results y discussion 3.1. Rosemary oil obtained by SCE Fig. 1 depicts the kinetics of essential oil extraction of rosemary previously performed for stating the working time. It is observed that the longer the extraction time, the higher the yield of rosemary essential oil. The kinetics behavior of the oil extraction was similar to that reported by Bensebia et al. (2009) and Fornari et al. (2012). It is also observed that percentages of the essential oil for 150 and 180 min of extraction were similar (p > 0.05). Therefore, the four experiments were carried out during 180 min twice (Fig. 2). Fig. 2 illustrates the yields of rosemary essential oil obtained at different CO2-supercritical extraction conditions. The final yield of rosemary essential oil was between 1.41 and 2.53 g essential oil/ 100 g dried matter. The sample with lower yield was the one obtained at a pressure of 10.34 MPa and 50 C, while the best performance of the system for rendering the higher rosemary oil content was with the sample treated at 17.24 MPa and 40 C. The best extraction of essential oil was obtained at the higher pressure (17.24 MPa) and the lower temperatures (40 C). At the lower pressure (10.34 MPa) and the higher temperature (50 C) the lower yield of oil was obtained. Bensebia et al. (2009) achieved yields of up to 3% oil in rosemary at 14.87 MPa and 40 C. Carvalho et al. (2005) achieved yields of up to 4% (w/w) at 24.78 MPa and 40 C after 250 min of extraction. Fornari et al. (2012) achieved yields of 3.5% (w/w) at 24.78 MPa, 40 C and 270 min. In all these studies, researchers used higher pressures and extraction times
Ið%Þ ¼ 491:28
(4)
where I is the inhibition. 2.6. GC-MS compounds identification For the characterization of volatile compounds, a gas chromatograph, coupled to a mass selective detector quadrupole 5975 (Agilent Technologies 6850N GC, Santa Clara, CA, USA) was used. A HP5-MS column of 30 m in length and 0.25 mm in diameter, covered with a film of 0.25 mm made of 5% phenyl and 95% dimethylpolysiloxane, was used. The conditions for the GC-MS analysis were as follows: Helium as carrier gas at a flow rate of 15.5 mL min1, a temperature of 250 C in the injector, Split ratio of 10:1, and a temperature program, starting at 60 C, at a rate of 4 C/ min until reach 240 C. Ionization energy was 70 eV. The scanning
Fig. 1. Kinetics of essential oil extraction by supercritical extraction of rosemary.
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Fig. 4. Antioxidant activity of rosemary essential oil obtained by supercritical extraction at selected temperatures and pressures.
Fig. 2. Rosemary essential oil yield obtained by CO2-supercritical extraction.
than the used in this study. It is important to mention that the season of the year, variety, among other variables, may affect the amount of essential oils in plants. Fig. 5. Antioxidant activity of rosemary essential oil obtained by steam distillation and hydrodistillation at selected pressures and temperatures.
3.2. Rosemary oil obtained by HYDRO and SD Fig. 3 shows the yields of rosemary essential oils obtained by the HYDRO and SD methods. The lower yield was 0.35% (w/w) when extracting 25 g of ground rosemary using the HYDRO method. The higher yield (2.35%) was obtained by the SD method using 50 g of whole sample. It is important to say that the extraction time for each experiment was 90 min. Better yields were obtained with the SD method using 50 g of whole sample. Yields obtained with the HYDRO method in ground rosemary were low. The highest yield was obtained with the CO2supercritical extraction process. Rasooli et al. (2008) obtained a yield of rosemary oil of 1% when the extraction was performed with the HYDRO method. Boutekedjiret et al. (2003) reported 1.2 and 0.44% of rosemary oil obtained by steam distillation and hydrodistillation, respectively. Flamini et al. (2002), Angioni et al. (2004), and Jamshidi et al. (2009) reported maximum yields of 1.44, 2.13, and 2.6% of oil, respectively, using the HYDRO method. 3.3. Oils antioxidant activities Figs. 4 and 5 ilustrates the antioxidant activity characterisitcs of
rosemary oil obtained by CO2-supercritical extraction (Fig. 4) and steam destilation and hydrodestillation, respectively (Fig. 5). The rosemary oil that showed the greatest antioxidant activity (37.55 ± 0.86 mg Trolox/g of essential oil) was the one obtained at 17.24 MPa and 40 C; the lower antioxidant activity (29.67 ± 0.52 mg Trolox/g essential oil) was observed at the extraction conditions of 10.34 MPa and 50 C (Fig. 5). Rosemary oils obtained at 17.24 MPa showed the higher antioxidant activities. With regard to oils extracted by HYDRO and SD (Fig. 5), the SD oil from 25 g of ground rosemary showed a higher antioxidant activity while the oil obtained from 50 g of entire rosemary by HYDRO presented the lowest antioxidant activity. High antioxidant activities were observed in oils obtained from ground samples. It can be clearly observed an increased antioxidant activity in oils obtained by supercritical extraction (Fig. 4); almost 14 times more than quantities obtained by SD or HYDRO. Liu et al. (2012) reported antioxidant activity values between 0.5 and 6.5 mg Trolox/g of oil in pomegranate seed oil (Punica granatum L.) obtained by supercritical extraction. Samec et al. (2010) reported an antioxidant capacity of 0.50e3.00 mg Trolox mL1 of an infusion obtained from flowers and leaves of Teucrium arduini L. In this study, much higher antioxidant activity values were found for the extracts obtained by supercritical extraction compared with those reported by researchers mentioned above. 3.4. Chemical compounds identification by GC-MS
Fig. 3. Yield of rosemary essential oil obtained by the HYDRO and SD methods. W: whole, G: Ground. SD: Steam Distillation, HYDRO: Hydrodistillation.
Table 1 shows the compounds identified by GC-MS of rosemary oils obtained by supercritical extraction, SD, and HYDRO. As can be seen, the major components of the essential oil of Rosmarinus officinalis L., obtained by supercritical extraction, were camphor, b~ ez et al. (1999) identified caryophyllene, and eucalyptol. Iban camphor fractions, ranging from 4.81 to 40.85%, and eucalyptol fractions between 1.79 and 12.29% in rosemary oil obtained also by supercritical extraction. These authors also found b-pinene, linalool, borneol, a-terpineol, verbenone, borneol acetate, a-caryophyllene ~ ez et al. (humulene), and g-muurolene (g-cardinene). However, Iban (2000) reported carnosic acid and rosmanol as the main components. In the rosemary oil obtained by SD, 19 compounds were
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Table 1 Compounds identified in the essential oil of rosemary by GC-MS. Compound
a-Pinene Camphene b-pinene Octenol a-Phellandrene a-Terpinene Eucalyptol b-trans-Ocimene g-Terpinene Terpinolene Linalool Camphor Borneol a-Terpineol Verbenone Safrole Borneol acetate b-Caryophyllene a-Caryophyllene ¼ Humelene g-Muurolene ¼ g-cadinene Epoxycaryophylene Palmitic acid Copaene a b c d e
SCEa
SDb
HYDROc
RTd (min)
PAe (%)
RTd (min)
PAe (%)
RTd (min)
PAe (%)
7.44 8.22 9.91 e 11.82 e 13.34 e 14.76 e 17.16 18.74 19.66 20.61 21.05 e 23.30 27.19 28.07 29.80 31.37 39.82 e
2.77 2.20 1.68 e 1.25 e 10.60 e 1.36 e 3.32 28.42 3.41 3.07 7.98 e 8.14 12.18 5.14 1.54 1.22 0.90 e
7.22 7.93 9.47 10.26 11.44 12.03 12.92 13.40 14.34 15.61 e 18.21 18.99 19.82 20.37 22.90 22.81 26.64 27.44 e 30.71 e e
11.96 9.29 6.01 1.15 11.78 4.71 15.36 1.10 4.27 1.99 e 14.76 3.82 1.98 0.80 1.20 2.97 4.34 0.59 29.93 0.65 e e
7.60 8.39 10.05 e 11.35 12.69 13.61 e 14.89 16.19 e 19.03 19.94 20.85 e e 23.51 27.35 28.22 1.66 31.54 e 25.93
5.03 3.74 0.70 e 5.35 1.28 12.59 e 1.78 1.00 e 21.51 3.95 2.77 e e 10.53 10.22 5.56 e 5.31 e 0.83
Supercritical extraction. Steam distillation. Hydrodistillation. Retention time. Pick area.
identified and 17 compounds by the HYDRO method. Angioni et al. (2004) reported a-pinene (23%), camphene (7.6%), borneol (16%), camphor (4.5%), verbenone (9.4%), and borneol acetate (10.4%) in rosemary oil obtained by SD. Gachkar et al. (2007) reported apinene (14.9%), linalool (14.9%), and eucalyptol (1,8-cineole) (7.43%)
as major components of rosemary oil content by SD. Boutekedjiret et al. (2003) found 1,8-cineole (52.4%), camphor (12.6) and bpinene (5.7%) as major components in rosemary oil obtained by HYDRO and 1,8 cineol (31.9%), camphor (19.7%) and a-Terpineol (12.8%) extracted by SD as the primary compounds. Differences in
a
b
c
d
e
Fig. 6. Micrographs of whole rosemary supercritical extracted. A) Untreated, b) 10.34 MPa, 40 C, c) 10.34 MPa, 50 C, d) 17.24 MPa, 40 C, e) 17.24 MPa, 50 C.
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chemical composition of rosemary oil from different studies can be attributed to factors such as plant variety, country of origin, time of harvest, climatic factors, among other factors. The main components of the essential oil of Rosmarinus officinalis L. obtained in this work were camphor and eucalyptol; compounds widely used in the food and pharmaceutical industries. It has been shown that compounds containing menthol and camphor may reduce the respiration rate in infants with acute bronchitis, may reduce cough in adults, and may improve mucociliary clearance in adults. The FDA (Food and Drug Administration) approved camphor to relieve coughs, but the permitted limit concentration in the preparations is 11% (Paul et al., 2010). 3.5. Scanning electron microscopy Fig. 6 illustrates the micrographs of rosemary exposed to supercritical extraction. There is a smooth surface of the sample without treatment. Structures of rosemary treated with SCE show an irregular surface. Also, samples used for the supercritical extraction showed some holes on the surface of rosemary. Most of the samples appear collapsed and ruptured. 4. Conclusions Higher yields of essential oil and antioxidant activity were observed in the rosemary oil obtained by the CO2-supercritical extraction, followed by steam distillation, and finally by hydrodistillation. The use of whole rosemary leaves improved the oil extraction compared to the oil rendered from powdered leaves. Also, higher yields of oil were observed using 50 g of sample. The antioxidant activity of oil obtained by supercritical extraction was approximately 14 times higher than that obtained in oils extracted by SD or HYDRO. In oils obtained by the three methods, camphor and eucalyptol were the mayor compounds. In the rosemary essential oil obtained by SCE and HYDRO, 17 compounds were identified, and 19 compounds in the oil obtained by SD; however, the oil obtained by supercritical extraction was richer in camphor. Acknowledgements ndez thanks the National Author Lilia Alejandra Conde-Herna Council for Science and Technology (CONACYT) and the Universidad de las Americas Puebla (UDLAP) for the financial support to complete her doctoral studies. References Abbasi, H., Rezaei, K., Rashidi, L., 2008. Extraction of essential oils from the seeds of pomegranate Using organic solvents and supercritical CO2. J. Am. Oil Chem. Soc. 85, 83e89. Adams, R.P., 1989. Identification of Essential Oil Components by Ion Trap Mass Spectroscopy. Academic Press, Inc, San Diego, California, USA. Aghel, N., Yamini, Y., Hadjiakhoondi, A., Pourmortazavi, S.M., 2004. Supercritical carbon dioxide extraction of Mentha pulegium L. essential oil. Talanta 62, 407e411. Angioni, A., Barra, A., Cereti, E., Barile, D., Coïsson, J.D., Arlorio, M., Dessi, S., Coroneo, V., Cabras, P., 2004. Chemical composition, plant genetic differences, antimicrobial and antifungal activity investigation of the essential oil of Rosmarinus officinalis L. J. Agric. Food Chem. 52, 3530e3535. Atti-Santos, A.C., Rossato, M., Atti, L., Cassel, E., Moyna, P., 2005. Extraction of essential oils from lime (Citrus latifolia Tanaka) by hydrodistillation and supercritical carbon dioxide. Braz. Archiv. Biol. Technol. 48 (1), 155e160. vez, M.A., Eustaquio-Rinco n, R., Reza, J., Trejo, A., 2007. Extraction of hyAvila-Cha drocarbons from crude oil tank bottom sludges using supercritical ethane. Sep. Sci. Technol. 42, 2327e2345. Babovic, N., Djilas, S., Jadranin, M., Vajs, V., Ivanovic, J., Petrovic, S., Zizivic, I., 2010. Supercritical carbon dioxide extraction of antioxidant fractions from selected Lamiaceae herbs and their antioxidant capacity. Innov. Food Sci. Emerg. Technol. 11, 98e117. Bensebia, O., Barth, D., Bensebia, B., Dahmani, A., 2009. Supercritical CO2 extraction of rosemary: effect of extraction parameters and modeling. J. Supercrit. Fluids
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