Influence of pre-treatments on yield, chemical

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J. of Supercritical Fluids 95 (2014) 468–473

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Influence of pre-treatments on yield, chemical composition and antioxidant activity of Satureja montana extracts obtained by supercritical carbon dioxide Senka Vidovic´ a , Zoran Zekovic´ a , Biljana Maroˇsanovic´ b , Maja Pandurevic´ Todorovic´ b , Jelena Vladic´ a,∗ a b

Faculty of Technology, University of Novi Sad, Bulevar Cara Lazara 1, 21000 Novi Sad, Serbia SP Laboratorija a.d., Industrijska 3, 21220 Beˇcej, Serbia

a r t i c l e

i n f o

Article history: Received 22 July 2014 Received in revised form 13 October 2014 Accepted 13 October 2014 Available online 22 October 2014 Keywords: Satureja montana Supercritical extraction Pretreatments Carvacrol Antioxidants

a b s t r a c t Main objective of this work was to investigate the influence of pre-treatments of Satureja montana herbal material on supercritical extraction process, in terms: influence on extraction yield, composition and antioxidant activity of extracts. Four different pretreatments were investigated: water pre-treatment, ethanol pre-treatment, ultrasound and high pressure pre-treatment. Extraction yields were in the range from 1.68 to 2.35 g/100 g. Pre-treatments with water and ethanol increase the yield of extraction for 25% and 17%, respectively. According to GC/MS results the main compound of obtained extracts was carvacrol. Analyses confirmed that carvacrol content in extracts can be significantly increased by application of pretreatments. Ultrasound pre-treatment and high pressure pre-treatment yielded highest concentration of carvacrol in extracts (around 66%). Using these two pre-treatments of herbal material, concentration of carvacrol, in extracts obtained by supercritical carbon dioxide extraction, could be increased for around 25%. Antioxidant activity of all extract was very high and in the range of well-known antioxidants. © 2014 Elsevier B.V. All rights reserved.

1. Introduction Herbs and spices have always been used as additives because of their ability to improve organoleptic properties of food. Today, they are valuated, not only for improvement of organoleptic properties, but also for their nutritive and medicinal benefits. One of such, valuated herbal species, is Satureja montana L. (winter savory), well-known aromatic plant, frequently used as spice and as a traditional medicinal herb. Beside traditional use in folk medicine, many scientific studies confirmed S. montana significant pharmacological activity (Table 1). S. montana contains various biologically active constituents including essential oil, triterpenes [22] and flavonoids [23]. Main components are aromatic monoterpens. The most dominant compound, carvacrol, is responsible for essential oil’s characteristic taste. Herbal plant and spices today are used not only in the native plant form, but also in the form of different extracts (produced by different extraction techniques) in which increase

∗ Corresponding author. Tel.: +381 21 485 3731; fax: +381 21 450 413. ´ E-mail address: [email protected] (J. Vladic). http://dx.doi.org/10.1016/j.supflu.2014.10.019 0896-8446/© 2014 Elsevier B.V. All rights reserved.

concentration of certain constituent of interest (with desirable biological activity or of some other desirable properties) could be achieved. Today, special attention is focused on essential oils and extracts contains essential oils, for which a significant pharmacological and antioxidative activity is confirmed and which can help to maintain nutritive and sensor quality of food [9]. According to Grosso et al. [24] many problems which occurs using standard method of extractions (thermal degradation, hydrolysis of individual components or the use of organic solvents) were exceeded by applying supercritical extraction. Several works have been published in order to report the use of SFE (supercritical fluid extraction) for isolation of different groups of plant metabolites, among them supercritical extraction of S. montana was also investigated [11,17,24]. The most frequent extraction fluid in supercritical extraction process is carbon dioxide. It is non-toxic and non-explosive extraction fluid, easy removed from the obtained products-extracts, thus allowing the preparation of solvent free extracts. Carbon dioxide possesses low critical temperature and pressure (Tc = 31.1 ◦ C, Pc = 73.8 bar), therefore making it possible to avoid thermo-degradation of thermo-sensitive constituents. Regarding mild temperature extraction conditions and possibility of selective extraction (by tuning pressure and

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Table 1 Pharmacological activity of different Satureja extracts. Pharmacological activity

Satureja extract

Reference

Diuretic activity Antidiarrheal Antispasmodic activity Antiviral activity Antibacterial and fungicidal activity Antioxidant activity

Infusion, ethanolic extract and essential oil Essential oil Essential oil Ethanolic extract, aqueous extract Essential oil, methanolic extracts, supercritical extract, soxhlet extract Ethanolic extract, methanolic extracts, supercritical extract, soxhlet extract, essential oil, powder extract obtained by spray drying of ethanolic extract Powder extract obtained by spray drying of ethanolic extract Essential oil Supercritical extract, soxhlet extract, essential oil Essential oil, ethanolic extract

[1] [2,3] [2] [4,5] [6,7,8,9,10,11] [12,13,14,9,10,15,16,17]

Ethanolic extract

[20,21]

ACE inhibitory activity Antiproliferative activity Anticholinesterase Antinociceptive and antiinflammatory effects Treatment of premature ejaculation

temperature), supercritical carbon dioxide extraction is recognized as “method of choice” for extraction of essential oils and extracts containing constituents of essential oils. The pre-treatment of herbal material to be extracted can change the “direction of extraction process”, leading to the higher extraction yields, to the production of extracts with increase content of certain constituents, and production of extracts of desirable properties, sensory or biological. Several pre-treatments have been investigated in the case of supercritical extraction with carbon dioxide (SECD): mechanical pre-treatment, rapid gas decompression, different drying pre-treatments, ultrasound pre-treatment etc. Most of them are used for disruption of plant material structures and cells. Ivanovic´ et al. [25] investigated influence of two different pre-treatments (mechanical pre-treatments, using different types of mills, and rapid gas decompression) on extraction of Usnea barbata by supercritical carbon dioxide. They found out that recovery of usnic acid from U. barbata using supercritical carbon dioxide was dependent on the pre-treatment and extraction conditions [25]. Crampon et al. [26] also focused their research on the influence of pre-treatments (two drying methods and one mechanical pre-treatment-grinding) upon extraction kinetics and yields in the case of Nannochloropsis oculata supercritical extraction. They found out that drying under air flow leads to faster extraction kinetics. The extraction curve obtained in this research illustrated that the smaller particle size, the higher the extraction kinetics [26]. A positive effect on extraction kinetics was observed after CO2 decompression for materials of the Lamiaceae species, as well as valerian and ginger roots [27,28]. The rapid gas decompression, in the case of St. John’s wort by SECD, led to increase of extraction yield [29]. During the exposure to compressed CO2 prior to extraction – beside static extraction of compounds to the surrounding dense gas atmosphere – the gas sorbs into plant material. In the case of St. John’s wort extractables are stored within various secretory structures. The rapid gas decompression leads to disruption of cell therefore certain amount of extractables being stored in the discrete secretory structures becomes accessible for the extraction [29]. Several studies confirmed the positive influence of the ultrasound pre-treatment on the efficacy of supercritical extraction. Riera et al. [30] showed that the presence of ultrasound can increase the yield of almond oil by 20% compared to traditional SECD. Luo et al. [31] also showed that SC-CO2 reverse microemulsion extraction of ginsenosides from ginseng was significantly accelerated by applying ultrasound. Gao et al. [32] reported that the using of ultrasound can significantly increase the yield of lutein esters. The present study was undertaken to investigate the influence of different pre-treatments on S. montana on further supercritical extraction process, composition and antioxidant activity of obtained extracts. SECD has been applied regarding its efficiency for extraction of aromatic and thermolabile volatile compounds,

[15] [18] [11] [19]

Table 2 Methods of pretreatment applied prior S. montana SECD, and obtained extracts. Extract

Method of pre-treatment and extraction

E0 E1 E2 E3 E4

Extraction of herbal material without pre-treatment Extraction of herbal material with water pre-treatment Extraction of herbal material with high pressure pre-treatment Extraction of herbal material with ultrasound pre-treatment Extraction of herbal material with ethanol pre-treatment

which are the main constituents of S. montana essential oil. Influence of four different pre-treatments was observed and analyzed: water pre-treatment, ethanol pre-treatment, ultrasound and pretreatment with high pressure. 2. Material and methods 2.1. Chemicals Commercial carbon dioxide (Messer, Novi Sad, Serbia) was used for laboratory supercritical fluid extraction. All other chemicals were of analytical reagent grade. 2.2. Plant material S. montana was cultivated at Institute of Field and Vegetable Crops, Baˇcki Petrovac, Serbia, in year 2012. The collected plant material was air dried and stored in a paper bags, at a room temperature. The dried S. montana was grounded in a domestic blender prior extraction, and the particle size of grounded material were determined using sieve sets (Erweka, Germany). 2.3. Pretreatment of S. montana material Before SECD, S. montana herbal material was the subject of different pre-treatments. All experiments were performed in three replicates. In pre-treatment with water 60 g of investigated herbal material was moisturized by addition of 36 g of water in extraction vessel. This mixture was vigorously stirred for 1 h. In the same extraction vessel obtained wet sample was extracted by high pressure carbon dioxide. This way extract E1 was obtained (Table 2). In a pre-treatment with high pressure 60 g of investigated herbal material was placed in extractor vessel and exposed to carbon dioxide at pressure of 100 bar and temperature of 40 ◦ C for 1 h. After pre-treatment with high pressure such herbal material was extracted by high pressure carbon dioxide. This way extract E2 was obtained (Table 2). In pre-treatment with ultrasound 60 g of investigated herbal material was placed in glass flask, without addition of any solvent,

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and exposed to ultrasound treatment during 1 h. Ultrasonic power was 125 W and frequency 50 Hz. After ultrasound pre-treatment such herbal material was placed in high extraction plant extractor vessel and extracted by high pressure carbon dioxide. This way extract E3 was obtained (Table 2). In pre-treatment with ethanol 60 g of investigated herbal material was moisturized by addition of 10 g of 96% ethanol. This mixture was vigorously stirred for 15 min in extraction vessel. In the same extraction vessel obtained wet sample was extracted by high pressure carbon dioxide. This way extract E4 was obtained (Table 2). The moisture content before and after herbal pre-treatment was measured. According to official Pharmacopeia moisture content in investigated materials was determined using standard procedure by drying of sample at 105 ◦ C until constant mass (Ph. Jug. V). All experiments were performed in three replicates.

Table 3 Extraction yields obtained in SECD of S. montana, with and without application of pre-treatments.

2.4. Supercritical carbon dioxide extraction (SECD)

where Asample is the absorbance of sample solution and Ablank is the absorbance of control. Antioxidant activity was also expressed as IC50 which represents the concentration of test (SE) solution required for obtaining the 50% of radical scavenging capacity.

The extraction process was carried out on laboratory scale high pressure extraction plant (HPEP, NOVA, Swiss, Effertikon, Switzerland). The main plant parts and properties, by manufacturer specification, were: gas cylinder with CO2 , the diaphragm type compressor (with pressure range up to 1000 bar), extractor with heating jacket for heating medium (with internal volume 200 ml, maximum operating pressure of 700 bar and temperature 100 ◦ C), separator with heating jacket for heating medium (with internal volume 200 ml, maximum operating pressure of 250 bar), pressure control valve, temperature regulation system and regulation valves [33]. The extraction of S. montana herbal material, after appropriate pre-treatment, was conducted using supercritical carbon dioxide at pressure of 100 bar and temperature of 40 ◦ C. The extraction time was the same in all cases (4.5 h). The separator conditions were 15 bar and 23 ◦ C. After extraction, obtained extracts were placed in the glass bottles, sealed and stored at 4 ◦ C to prevent any possible degradation until further analysis. All experiments were performed in three replicates. 2.5. Chemical analysis—Chromatographic procedure GC/MS analysis was run on Agilent GC6890N system coupled to mass spectrometer model Agilent MS 5795. An HP-5MS column (30 m length, 0.25 mm inner diameter and 0.25 ␮m film thicknesses) was used. Injected volume of sample solution in methanol was 5 ␮l with split ratio 30:1. The compounds were identified using the NIST 05 and Wiley 7n mass data base and by comparing their retention times to those in mass spectral libraries. The GC/MS operating conditions were as follows: injector temperature 250 ◦ C, temperature program was: from 60 ◦ C to 150 ◦ C (4 ◦ C/min), carrier gas He with flow rate 2 ml/min. Quantifications of dominant aromatic compound carvacrol was performed by FID detector and calibration curve for compound. The percentage composition was calculated from the peak area. The GC/FID operating conditions were: injector temperature 250 ◦ C, temperature program from 60 ◦ C to 150 ◦ C (4 ◦ C/min), detector temperature 300 ◦ C. 2.6. Antioxidant assay—DPPH test The free radical scavenging activity of supercritical extracts (SE) of S. montana was determined using simple and fast spectrophotometric method as described by Espin et al. [34]. Briefly, prepared extracts were mixed with methanol (96%) and 90 ␮M 2,2-diphenyl-1-picrylhydrazyl (DPPH) to give different final concentration. After 60 min at room temperature, the absorbance of samples was

SE

Extraction yield (%, w/w)

E0 E1 E2 E3 E4

1.88 2.35 1.68 1.73 2.20

± ± ± ± ±

0.010 0.001 0.002 0.002 0.010

measured at 517 nm. Radical scavenging capacity (%RSC) was calculated by following equation:



% RSC = 100 −



Asample × 100 Ablank

(1)

3. Results and discussion Mean particle size of S. montana, used in investigation of different pre-treatments influence on extraction yield, extract composition and antioxidant activity of extract obtained by SECD, was determined using sieve sets. Particle size of this ground herbal material was 0.28 mm. Using standard procedure, according to official Pharmacopeia, moisture content of herbal material before and after pre-treatment was determined. Before pre-treatment moisture content in investigated herbal material was 11.34%. The moisture content of herbal material after water pre-treatment was much higher, 42.71%. The moisture content of herbal material after ethanol pre-treatment was 16.09%, while after ultrasound and high pressure pre-treatments was the same as the moisture content in herbal material before pre-treatment (11.34%). After adequate pre-treatment, herbal material was extracted using supercritical carbon dioxide, and this way different SE were obtained. Extraction yields obtained in SECD of S. montana, with and without application of pre-treatments, ranged from 1.68 to 2.35% (w/w) (Table 3). A great number of studies confirmed that yield and selectivity of supercritical fluid extraction depends on moisture of the material being investigated, which can be explained by the chemical structure of active components of the herbal drug [35–42]. The influence of moisture on mass transition and solubility of the oil have been shown as negligible if the moisture of the drug is maintained between 3 and 12% [36]. If the moisture content of the extracted drug is increased for as much as 25%, a higher yield of the extraction can be achieved [40]. Extraction yield, obtained in SECD of S. montana without application of pre-treatment, was higher than yield obtained in case of SECD with high pressure-pretreatment, and higher than the yield obtained in the case S. montana SECD with ultrasound pretreatment. Lower yields indicating negative impact of these two pre-treatments on extraction yield in further SECD process. These results may be explained by degradation of certain compounds in S. montana herbal material, provoked by high pressure and/or ultrasound exposure. The yields higher than the extraction yield obtained in SECD without pre-treatment have been achieved using water and ethanol pre-treatments. The highest extraction yield, achieved in the case of SECD of herbal material pre-treated with water, can be explained by highest moisture content. This result is in accordance to the results and statements of Eggers, Goodrum and Kilgo, and Balachandran et al. [35,36,40]. There are two possible explanations related to the

S. Vidovi´c et al. / J. of Supercritical Fluids 95 (2014) 468–473 Table 4 GC/MS analysis of S. montana SE (relative percent, %).

471

Table 5 Quantitative analysis of carvacrol—dominant compound present in of S. montana SE.

Compound

E0

E1

E2

E3

E4

Compound

E0

E1

E2

E3

E4

␤-Myrcene ␣-Terpinene p-Cymene Eucalyptol ␣-Terpinene Trans-Sabinene hydrate Cis-Sabinene hydrate Linalool Borneol Terpinene 4-ol ␣-Terpineol Carvon Carvacrol Trans-Caryophyllene ␣-Amorphen ␤-Bisabolene ␥-Cadinene ␦-Cadinene Caryophyllene oxyde Heptacosane Nonacosane ␣-Terpinolene Percentage of total identified compounds (%)

0.138 0.400 7.126 0.703 0.481 0.798 0.264 0.514 2.422 0.695 0.078 0.816 67.581 2.796 0.554 0.893 0.586 1.016 1.575 0.594 2.739 – 92.733

– – – – – 0.262 0.171 0.339 1.345 0.399 – 2.761 78.950 1.924 0.329 0.528 0.533 0.694 1.639 0.435 0.618 – 90.927

– 0.094 1.951 0.408 0.082 0.410 0.199 0.385 1.273 0.545 0.103 0.321 82.121 1.997 0.343 0.559 0.403 0.684 1.273 0.306 0.455 – 93.912

– – 1.887 0.342 0.065 0.375 0.185 0.274 1.008 0.416 0.080 0.365 86.292 1.958 0.335 0.539 0.317 0.648 0.945 0.246 0.283 0.080 96.640

0.123 0.061 1.817 0.153 0.043 0.447 – 0.617 1.419 0.374 0.058 1.672 82.279 1.818 0.280 0.579 0.310 0.593 1.214 0.476 2.927 – 97.314

Carvacrol (g/100 g)

52.97

57.03

66.10

66.46

64.02

increase of extraction yield with increase in moisture content: the dissolution of water into the supercritical phase enhances solubility of solute and the water-swelling of plant tissue influences the internal or solid mass transfer resistance and movement of solute to the particle surface [35,40]. In the case of SECD of herbal material pre-treated with ethanol higher extraction yield can be explained synergistically, by influence of higher moisture content of herbal material (moisture content of ethanol pre-treated herbal material was 16.09%, higher than moisture content in material without pretreatment) and by enhanced extraction of certain constituents, as in the case of Tanaka et al. study [43]. Twenty five percent higher extraction yield, in the case of water pre-treatment, and around 17% higher in the case of ethanol pretreatment, indicating positive impact of these two pre-treatments on extraction yields in SCDE of S. montana. The highest extraction yield was achieved in the case of SCDE with water pre-treatment. Therefore, in the SCDE of S. montana water content plays an important role as the increase of water content leads to the increase of extracted constituents from this herbal material. Obtained extracts were characterized by qualitative and quantitative GC/MS. The volatile components determined in SE are presented in Table 4. Applied GC/MS analysis resulted in identification of 22 different constituents. Qualitative GC/MS analysis revealed that water pre-treatment (higher moisture content in herbal material to be extracted) had negative influence on isolation of certain constituents of S. montana, like p-cymene and eucalyptol. According to the results of GC/MS analysis the most dominant component in all extracts was phenol compound-carvacrol, with relative content ranging from 67.581 to 86.292% (Table 4). Grosso et al. [24] determined that content of carvacrol in S. montana extracts, obtained from herbal material without pre-treatment, using supercritical carbon dioxide, ranged from 41.7 to 64.5%. Silva et al. [11] state the content of carvacrol in SFE to be 52.7%. According to the results of GC/MS analysis the lowest concentration of carvacrol was measured in extract E0. Highest concentration of carvacrol in extract was achieved in the case of SCDE with ultrasound pre-treatment. Other three pre-treatments (water, ethanol and high pressure) yielded important increase of carvacrol concentration in SE, in comparison to extract obtained without pre-treatment (E0). These findings confirming that application of

pre-treatments by water addition, ethanol addition, high pressure exposure, and ultrasound exposure, could be used for the increase of concentration of dominant compound-carvacrol in S. montana SE. For precise analysis and quantification of carvacrol in S. montana extracts GC/FID analysis was provided (Table 5). According the results of GC/FID analysis content of carvacrol in obtained extracts ranged from 52.97 g/100 g to 66.46 g/100 g. Ultrasound pre-treatment of S. montana, as well as high pressure pre-treatment, yielded highest concentration of carvacrol in SE (around 66%). Using these two pre-treatments of investigated herbal material concentration of carvacrol, in extract obtained by SCDE could be increased for around 25% in comparison to carvacrol concentration in SE obtained without previous pre-treatment (E0). There are scientific findings showing accelerating increase in concentration of certain compound in SE obtained from material pre-treated with ultrasound pre-treatment [30,31]. Balachandran et al. [40] reported that under the influence of ultrasound, the yield of pungent compounds from ginger can increase dramatically, with an improvement up to 30% compared with traditional SCDE, extraction without pre-treatment. Increase of carvacrol concentration in SE obtained from herbal material pre-treated with exposure to high pressure, can be explained by higher solvent permeation in the inner of cells and therefore higher and faster extraction of certain constituents. Slightly lower increase of carvacrol concentration in extract, for around 20%, could be achieved by application of ethanol pre-treatment, and this could be explained by modified solubility power of carbon dioxide and increased selectivity to certain constituents. The content of the second most dominant component p-cymene, in extract obtained using SCDE of herbal material, without any pretreatment, was 7.126%. In extracts obtained with application of pre-treatments concentration of p-cymene was much lower, indicating that in the case of this compound application of pretreatments influence negatively on its content in SE. Other, less dominant components, detected in all S. montana extracts obtained by SCDE are: borneol, trans-caryophyllene, ␦-cadinene, caryophyllene oxyde and nonacosane (Table 4). In extract E3, according to GC/MS data from Table 4, content of p-cymene is considerably lower (around 4 times) then content of same compound in extract E0. Also, contents of borneol and caryophyllene oxyde, are lower around 2 times, while content of nanocosane is lower around 10 times in extract E3 in comparison to the content of same compounds in extract E0. The lower contents of these, and some other compounds in E3 extract, could be explainable by possible degradation of these compounds provoked by ultrasound in herbal pre-treatment. This could also explain the lower yields obtained in extraction after ultrasound pre-treatment in comparison to extraction yields obtained without pre-treatment. The enhance extraction of carvacrol and production of extract with increased concentration of carvacrol could be of great importance regarding carvacrol use in food and pharmaceutical industry. Accordingly, Dorman and Deans [44] concluded that of seven individual oil components, tested against 25 bacterial strains, carvacrol was the one with the widest spectrum. It was shown to have a bactericidal effect toward Salmonella in pieces of fish stored at 4 ◦ C [45]. Carvacrol can be applied as an antimicrobial and as a flavoring compound to products associated with outbreaks of B. cereus (e.g., rice, pasta, and soup). Ultee et al. showed that carvacrol has biological effects at concentrations relevant for baked goods

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Table 6 Antioxidant activity of S. montana SE. Extracts

IC50 (␮g/ml)

E0 E1 E2 E3 E4

11.9 25.2 23.1 17.7 24.5

± ± ± ± ±

0.002 0.001 0.003 0.0001 0.0003

Table 7 Antioxidant activity of well-known antioxidants. Component

IC50 (␮g/ml)

Authors

Quarcetin Vitamin C BHT

10.5 16 24

Perez-Rozes et al. [51] Khitam et al. [52] Khitam et al. [52]

(15.75 ppm) and flavoring of foods (e.g., nonalcoholic beverages (0.18 mM/28.54 ppm) [46]. Antioxidant activity of SE of S. montana, characterized as the IC50 value, ranged from 11.9 ␮g/ml to 25.2 ␮g/ml (Table 6). The smallest antioxidant activities were measured in samples obtained by SCDE with water and ethanol pre-treatments of herbal material. Extract obtained by SCDE without any pre-treatment of investigated material from showed highest antioxidant activity characterized with lowest IC50 value. Considering the results of antioxidative activity, it can be concluded that application of pre-treatments in the case of SCDE of S. montana does not have a positive effect on antioxidative activity of obtained SE. Although carvacrol is one of the most active natural antioxidants [47], according to analysis of antioxidative activity, and based on results of quantitative and qualitative analysis of extracts, it can be concluded that carvacrol does not have the main role in antioxidant activity of S. montana SE. This is supported with the observation that extract with lowest concentration of carvacrol (E0) showed the highest antioxidant activity. Therefore, the higher antioxidant activity of extracts with lower concentration of carvacrol, could be explained and could be attributed to synergism of other, less dominant components of S. montana SE. According to scientific references, for some components present in lower concentration in S. montana SE, antioxidative activity was confirmed. Thus, Vardar-Unlu et al. concluded that possible synergistic or antagonistic effects could also be taken into account for constituents of the essential oil, such as borneol, ␣-terpinene, and p-cymene [48]. Also, strong antioxidant activity was previously reported for ␣-terpinene and linalool [49,50]. Regarding literature data on IC50 values of well-known antioxidant compounds (Table 7) it can be concluded that antioxidant activity of S. montana extract (E0), obtained using SCDE, at 100 bar and 40 ◦ C, without pre-treatment of herbal material, is higher than antioxidative activity of vitamin C and BHT (butil-hidroxi-toluol), and similar to the antioxidant activity of quercetin. Bearing this in mind, SE should be considered as very strong natural antioxidant and can be used in such manner for variety of applications. Even though pre-treatments do not have a positive influence on antioxidant activity, extract obtained from herbal material pretreated with ultrasound showed similar antioxidant activity with the Vitamin C and higher antioxidant activity than the activity of BHT. Extracts obtained from herbal material by application of SCDE with others pre-treatments also showed high antioxidant activity (antioxidant activity was similar to the activity of BHT). 4. Conclusion The main objective of this work was to investigate the influence of different pre-treatments on S. montana SCDE (extraction

yield, chemical composition and antioxidative activity of obtained extracts). Four different pre-treatments were applied: water pretreatment, high pressure pre-treatment, ultrasound and ethanol pre-treatment. The higher extraction yields, in comparison to extraction yields obtained without S. montana pre-treatment, have been achieved using water and ethanol pre-treatment. These higher yields indicating positive impact of these two pre-treatments on extraction yields in the case of S. montana SCDE. Pre-treatment had influence on composition of S. montana extracts obtained by SCDE. Using ultrasound and high pressure pre-treatment, concentration of carvacrol has been increased in extracts for around 25%. Slightly lower increase of carvacrol concentration, for around 20%, has been achieved by application of ethanol pre-treatment. Therefore, it can be concluded that carvacrol content in S. montana extracts can be changed and increased by application of these three pretreatments. The application of pre-treatments, on the other hand, has a negative impact on extracts’ antioxidant activity. In spite of that, all obtained extracts showed very high antioxidant activity, similar to that of well-known antioxidants, and therefore could be used in food or pharmaceutical industry as natural antioxidants prepared by application of “green technology”.

Acknowledgement The authors gratefully acknowledge the financial support of this work by the Ministry of Education and Science, Republic of Serbia (Project No. TR31013).

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