Winter savory: Supercritical carbon dioxide extraction and mathematical modeling of extraction process

Accepted Manuscript Title: Winter savory: supercritical carbon dioxide extraction and mathematical modeling of extraction process Author: Jelena Vladi´c Zoran Zekovi´c Stela Joki´c Sandra Svilovi´c Strahinja Kovaˇcevi´c Senka Vidovi´c PII: DOI: Reference:

S0896-8446(16)30163-2 http://dx.doi.org/doi:10.1016/j.supflu.2016.05.027 SUPFLU 3652

To appear in:

J. of Supercritical Fluids

Received date: Revised date: Accepted date:

18-2-2016 29-5-2016 30-5-2016

Please cite this article as: Jelena Vladi´c, Zoran Zekovi´c, Stela Joki´c, Sandra Svilovi´c, Strahinja Kovaˇcevi´c, Senka Vidovi´c, Winter savory: supercritical carbon dioxide extraction and mathematical modeling of extraction process, The Journal of Supercritical Fluids http://dx.doi.org/10.1016/j.supflu.2016.05.027 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Winter savory: supercritical carbon dioxide extraction and mathematical modeling of

2

extraction process

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Running Title: S. MONTANA SUPERCRITICAL EXTRACTS

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Jelena Vladić1, Zoran Zeković1, Stela Jokić2, Sandra Svilović3, Strahinja Kovačević1, Senka

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Vidović1*

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1

Faculty of Technology Novi Sad, University of Novi Sad, Bulevar cara Lazara 1, 21000 Novi

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Sad, Serbia 2

Faculty of Food Technology, Josip Juraj Strossmayer University of Osijek, Franje Kuhača

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20, 31000 Osijek, Croatia 3

Faculty of Chemistry and Technology, University of Split, Teslina 10/V. 21000 Split, Croatia

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Abstract

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Main objective of this work was to investigate the influence of pressure and temperature on

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supercritical carbon dioxide extraction of Satureja montana in terms of extraction yield and

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chemical composition. The most dominant compound in all investigated extracts was

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oxygenated monoterpene-carvacrol. The kinetics of the supercritical carbon dioxide extraction

17

of S. montana as well as the solubility data were investigated by modelling the extraction

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curves using different empirical models and all models used showed similar deviation from

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experimental data. Hierarchical cluster analysis was employed in order to reveal possible

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similarities and dissimilarities among the extracts obtained at different extraction conditions.

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Keywords: Satureja montana, supercritical extraction, carvacrol, mathematical modeling

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Corresponding author: Bulevar cara Lazara 1, 21000 Novi Sad, Serbia; Tel.: +381 63

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8523177, Fax: +381 21 450413, E-mail: [email protected]

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1. Introduction

29 30

One of the most important family of medicinal plants, Lamiaceae, consist of over 3000

31

species [1], most of which are used as additives because of their ability to improve

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organoleptic properties of food, and are also known for their nutritive and medicinal benefits.

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A member of the above mentioned family is a well-known aromatic plant Satureja montana

34

L., commonly known as winter savory, and widely spread in the Balkan region. Various

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biologically active constituents such as essential oil, triterpenes and flavonoids are contained

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in S. montana [2, 3]. The most dominant phenolic compounds of the essential oil of S.

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montana are thymol and carvacrol [4, 5]. Due to pharmacologically significant chemical

38

composition, S. montana and its extracts possess noteworthy biological activities and which

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were proved in numerous scientific articles. Apart from the antioxidant activity [6-13],

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antibacterial and fungicidal activity were affirmed by Panizzi et al. [14], as well as Skočibušić

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and Beţić [15]. In addition, winter savory showed an anti-inflammatory effect on cultured

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human erythroleukemic K562 cells and an important anti-HIV-1 activity [16, 17]. Stanić and

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Samardzija [18] proved that S. montana possesses diuretic effect in vivo.

44 45

Nowadays, herbal plants and spices are not only employed in the native plant form, but also in

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the form of different extracts (produced by different extraction techniques) in which an

47

increase in concentration of certain constituents with desirable properties could be achieved.

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Special attention is given to essential oils and extracts containing essential oils, whose

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significant medicinal and nutritive features can improve nutritive and sensoric quality of food

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[10]. Many problems which occur during the application of classical methods of extractions

51

(use of toxic organic solvents, thermal degradation of thermolabile constituents, hydrolysis of

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certain components, extraction of heavy metals and inorganic salts, and so on) can be

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overcome by applying supercritical extraction [19]. The most frequently used extraction fluid

54

employed in supercritical extraction process is carbon dioxide. As carbon dioxide in

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supercritical state can be used at low temperatures, it can, moreover, as a non-oxidant medium

56

enable the extraction of thermolabile or easily oxidized compounds [20].

57

The high selectivity is one of the most important properties of supercritical carbon dioxide

58

(SC-CO2) extraction. The selectivity of this extraction process can be adjusted by tuning the

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process pressure and temperature. The increase of temperature reduces the density, thus

60

reducing the solvent power of the supercritical solvent, but it increases the vapor pressure of

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the compounds to be extracted [21]. Yet, the extraction pressure is the most dominant 2

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parameter which affects the SC-CO2 selectivity. The general rule is: the higher the pressure is,

63

the larger the solvent power, and lower the extraction selectivity are [21].

64

Regarding the moderate temperature extraction conditions and possibility of selective

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extraction, extraction by carbon dioxide (supercritical and/or liquid phase) is recognized as

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“method of choice” for extraction of essential oils and extracts containing constituents of

67

essential oils. Thus, properties of this extraction technique allows us to preserve the most

68

sensitive constituents of investigated material with important pharmacological activity.

69

Several published works report the usage of supercritical fluid extraction for isolation of

70

different groups of plant metabolites, among which supercritical extraction of S. montana was

71

also performed [19, 9, 22, 23, 13]. According to our previous research, S. montana extracts

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obtained by supercritical carbon dioxide at pressure of 100 bar and at 40°C contained as a

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dominant compound carvacrol (from 52.97 to 66.46% (w/w)), while the second most

74

dominant was p-cymene. Other less dominant components detected in obtained extracts were:

75

borneol, trans-caryophyllene, δ-cadinene, and caryophyllene oxide [13]. In a few reported

76

studies, authors worked on the comparison between supercritical and various conventional

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extracts of S. montana. The results of these studies clearly demonstrated improvements of the

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values of the final extracts which were obtained by supercritical fluid in relation to extracts

79

obtained by conventional methods of extraction (hydrodistillation and Soxhlet extraction).

80

Those improvements were related to the antioxidant activity [9], antimicrobial and

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anticholinesteraze activity [22].

82

Having in mind the results obtained in previous studies on S. montana extracts, the main idea

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of this investigation was to make a step forward by a thorough investigation of S.montana

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supercritical extraction process, with the main aim to define the set-up of best extraction

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process parameters (temperature and pressure), needed for the production of the highest

86

quality extract. Therefore, the production of extracts by supercritical carbon dioxide was

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performed under various process parameters (eleven different pressure values from 100 to 350

88

bar, and three different temperatures: 40, 50 and 60°C). By applying the GC-MS method of

89

analysis, determination of obtained extracts’ chemical composition was performed. Using the

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GC-FID method, quantification of dominant compound-carvacrol was done.

91 92

3

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2. Material and methods

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2.1. Chemicals

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Commercial carbon dioxide (purity of 99.9%) (Messer, Novi Sad, Serbia) was used for

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laboratory supercritical fluid extraction. All other chemicals were of analytical reagent grade.

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2.2. Plant material

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S. montana was cultivated at Institute of Field and Vegetable Crops, Bački Petrovac, Serbia,

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in year 2012. The collected plant material was air dried, milled and mean particle size (0.301

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mm) was determined by sieves set (Erweka, Germany).

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2.3. Soxhlet extraction

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Sample of S. montana (20.0 g) was extracted by methylene chloride using the Soxhlet

103

apparatus. After 12 changes of solvent (6h), the solvent was evaporated under vacuum, and

104

extraction yield was determined as % (w/w).

105

2.4. Supercritical carbon dioxide (SC-CO2) extraction

106

The extraction process was carried out on laboratory scale high pressure extraction plant

107

(HPEP, NOVA, Swiss, Effertikon, Switzerland). The main plant parts and properties, by

108

manufacturer specification, were: gas cylinder with CO2, the diaphragm type compressor

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(with pressure range up to 1000 bar), extractor (internal volume 200 ml, maximum operating

110

pressure of 700 bar, length 15.92 cm, diameter 4 cm), separator (internal volume 200 ml,

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maximum operating pressure of 250 bar), pressure control valve, temperature regulation

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system and regulation valves [24].

113

The extraction of S. montana herbal material (60 g) was conducted using supercritical carbon

114

dioxide at pressure in the range from 100 to 350 bar (with increase of 25 bar in every next

115

experiment), and at temperature of 40, 50 and 60°C. The extraction time was the same in all

116

cases (4.5 hours), and CO2 flow rate was 0.194 kg/h. The separator conditions were 15 bar

117

and 23°C. A total of 33 different extracts were produced. Total extraction yield was expressed

118

as g of extract/100 g of dry weight (DW). After extraction, obtained extracts were placed in

119

the glass bottles, sealed and stored at 4°C to prevent any possible degradation until further

120

analysis.

121 4

122

2.5. Mathematical modeling

123

The extraction curves of S. montana extracts were adjusted using models presented in the

124

literature.

125

The first model considered is based on the specific solution of Ficks low [25] and its

126

mathematical expression is given by the following equation:

127



YE  x0 1  e  kt

128



(1)

129

where YE is extraction yield; k is the rate constant and t is the extraction time; x0 is the initial

130

solute mass ratio in the solid phase obtained via Soxhlet (g/g).

131

Esquivel et al. [26] empirical model is represented by the following equation:

132

 t  mext  x0 F   bt 

(2)

133

where mext is mass of the extract (g); F is the mass of solid material (g); t is the extraction time

134

(s); x0 is the initial solute mass ratio in the solid phase (g/g); and b is an adjustable parameter

135

(s).

136

Empirical models for solubility determination are based on simple error minimization where

137

mainly there is no need for physical–chemical properties. In this investigation several models

138

were used. The first equation used was the one proposed by Chrastil [27] for correlating the

139

solubility behaviour:

140

a   S   k exp  a 2  3  T  

141

where S is solubility in g/L, ρ is the density of CO2 in g/L and T is the temperature in K.

142

Del Valle and Aquilera [28] gave the improved form of this equation in the following form:

143

a a   S   k exp  a 2  3  24  T T  

(3)

(4)

144

The units are the same as in the Chrastil equation.

145

Empirical equations proposed by Adachi and Lu [29] and Sparks et al. [30] were also used.

146

Adachi and Lu proposed empirical equation in the form:

147

2 a   S   ( a1  a 2   a 3  ) exp  a 4  5  T  

(5)

148 5

149

while Sparks et al. (2008) simplified Adachi and Lu equation into the following form:

150

a   S   ( a 1  a 2  ) exp  a 3  4  T  

151

(6)

152

where a1-a5 are the constants of solubility models and T is the temperature (K).

153

The parameters of all models were calculated by non-linear regression method using software

154

Mathcad 14.

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Statistical analysis

156

The concordance between the extraction yield experimental data and calculated value

157

obtained using different mathematical models was established by the average absolute relative

158

deviation (AARD) as follows:

AARD 

159

 S40i  scalci    100 n  S40  i  i1  1

n





(7)

160

The success of the approximation of applied mathematical model was analyzed based on

161

AARD (%) which is considered to be acceptable up to 5%, and above 10% indicates poor

162

approximation of experimental and model predicted values.

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2.6. Chemical analysis - chromatographic procedure

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GC/MS analysis was run on Agilent GC6890N system coupled to mass spectrometer model

165

Agilent MS 5795. An HP-5MS column (30 m length, 0.25 mm inner diameter and 0.25 μm

166

film thickness) was used. Injected volume of sample solution in methanol was 5 μl with split

167

ratio 30:1. The compounds were identified using the NIST 05 and Wiley 7n mass data base

168

and by comparing retention times to those in mass spectral libraries. The GC/MS operating

169

conditions were as follows: injector temperature 250°C, temperature program was: from 60°C

170

to 150°C (4°C/min), carrier gas He with flow rate 2 ml/min. Quantification of dominant

171

aromatic compound carvacrol was performed by GC/FID analysis and calibration curve for

172

carvacrol. The GC/FID operating conditions were: injector temperature 250°C, temperature

173

program from 60°C to 150°C (4°C/min).

174

2.7. Hierarchical cluster analysis

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Hierarchical cluster analysis (HCA) is a very simple and useful chemometric method, suitable

176

for division of a group of objects into different classes, so the similar objects are placed into

177

the same class [32]. The groups are not known prior to analysis and no assumptions can be

178

made regarding the distribution of the variables. The distance (D) between two points in n-

179

dimensional space with coordinates (x1, y1; x2, y2; … xn, yn) is called Euclidean distance

180

defined by the following equation:

181

D = ((x1 – y1)2 + (x2 – y2)2 +…+ (xn – yn)2)½

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There are different methods which can be used for clustering, such as Ward’s method, single

183

linkage, complete linkage, simple average method, etc. The graphical result of HCA is a

184

dendrogram. It represents the clustering of the objects in a form of a tree. A double

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dendrogram is a useful tool for representation of similarities or dissimilarities among both the

186

objects and objects’ variables. It clusters both the objects and the variables in a single graph.

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Cluster analysis has become a very popular chemometric tool in many disciplines, such as

188

analytical chemistry, food engineering, microbiology, pharmaceutical engineering, etc. [33-

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36]. The HCA was carried out by using NCSS 2007 statistical software with double

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dendrogram tool and single linkage algorithm.

(8)

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3. Results and discussion

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3.1. Extraction yields

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The effects of operational parameters (pressure and temperature) on SC-CO2 extraction of S.

196

montana were investigated at ranges of 100 – 350 bar of pressure and 40 – 60°C of

197

temperature. Table 1 presents the results of the total extraction yield of S. montana by SC-

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CO2 after 4.5 h, with a constant CO2 flow rate (0.194 kg/h). Total extraction yields were in

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range from 1.88 to 3.09, from 1.54 to 4.04, and from 0.97 to 4.30 g/100 DW for extracts

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obtained at different pressures and at temperatures 40, 50 and 60°C, respectively. An increase

201

in pressure from 100 to 350 bar at constant temperature (40°C), corresponding to an increase

202

in CO2 density (from 628.7 to 934.9 kg/m3) caused a 1.64 times higher extraction yield. At

203

60°C CO2 density increased from 290 to 863.2 kg/m3, and the extraction yield was 4.43 times

204

higher by pressure increasing. The lowest extraction yield was achieved at 100 bar and 60°C,

205

while the highest yield was reached on 350 bar and 60°C. A similar observation was noticed

206

by Grosso et al. [19] who gained the extraction yield in the range from 0.9 to 1.5% by using

207

similar extraction parameters (pressure 90 and 100 bar; temperature 40 and 50°C).

208

Table 1. Extraction yields of SC-CO2 of S. montana (extraction time 4.5 h) p (bar) 100

125

150

175

200

225

250

T (°C) 40 50 60 40 50 60 40 50 60 40 50 60 40 50 60 40 50 60 40 50

Yield (g/100 g DW) 1.88 1.54 0.97 2.23 2.19 2.61 2.69 2.75 2.41 2.79 3.21 3.00 2.95 3.35 3.24 2.99 3.30 3.67 2.75 3.51 8

275

300

325

350

60 40 50 60 40 50 60 40 50 60 40 50 60

3.91 2.94 4.04 3.71 2.95 3.53 3.44 3.05 3.62 4.04 3.09 3.77 4.30

209 210

The extraction yield of 4.325% (w/w) was determined by the Soxhlet extraction method,

211

which is similar to that (4.30%) obtained by SC-CO2 (350 bar and 60°C).

212

3.2. Mathematical modelling of extraction system

213

As a result of mathematical modelling in this study, the parameters of different applied

214

models were obtained by minimizing the deviation value of the calculated yield and solubility

215

by model and experimental data. The average absolute relative deviations (AARD) were

216

calculated for each experiment.

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Table 2 shows the results of modeling of investigated extraction system using a specific

218

solution of Ficks low. This is the simplest model since it assumes that the extraction process

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occurs in one stage of diffusion controlled only by the internal mass transfer. Although

220

AARD values range from 1.486 to 18.079%, only for 325 bar, 600C, and 350 bar, 600C, these

221

values are under 5%, but most of these values exceed 10%.

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Table 2. Calculated parameters of the one stage diffision model for S. montana extraction Extraction conditions p (bar)

100

125

k

AARD

T (°C)

(1/h)

(%)

40

0.159

13.374

50

0.112

14.981

60

0.065

12.426

40

0.201

13.644

50

0.213

18.079 9

150

175

200

225

250

275

300

325

350

60

0.242

11.691

40

0.264

12.634

50

0.288

14.720

60

0.223

12.652

40

0.311

15.818

50

0.365

8.361

60

0.339

12.256

40

0.364

16.656

50

0.465

13.505

60

0.410

12.179

40

0.382

16.864

50

0.424

12.181

60

0.498

7.689

40

0.311

16.408

50

0.550

10.759

60

0.628

8.932

40

0.362

16.759

50

0.752

5.996

60

0.635

7.451

40

0.360

16.217

50

0.512

10.024

60

0.484

11.514

40

0.401

16.152

50

0.533

7.236

60

0.646

3.254

40

0.346

12.947

50

0.534

7.377

60

0.923

1.486

223 224

The extraction kinetics was investigated also by Esquivel et al. [26] model, used for the

225

modelling the extraction of olive oil by supercritical carbon dioxide. The values for the

226

adjustable model parameter k and AARD values for all the experiments are presented in Table

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3. AARD values range from 1.279 % to 11.152%. The best agreement between experimental 10

228

and model calculated yield was obtained at 250 bar and 50°C, and the worst agreement at 125

229

bar and 50°C. Also, it can be observed that for approximately 50% of experiments the

230

deviation between experimental data and model is up to 5%, and for 94% of experiments

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AARD values are lower than 10%. By comparing AARD values for models tested, it can be

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seen that the worst agreement with experimental data is obtained using the one stage diffusion

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model, and the best by applying the Esquivel et al. model.

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Table 3. Calculated parameters of the Esquivel et al. model for S. montana extraction Extraction conditions p (bar)

100

125

150

175

200

225

250

275

k

AARD

T (°C)

(1/h)

(%)

40

4.778

7.004

50

7.311

10.545

60

13.619

9.312

40

3.543

6.573

50

3.232

11.152

60

2.794

5.219

40

2.487

4.902

50

2.187

6.714

60

3.095

5.084

40

1.954

8.155

50

1.631

3.256

60

1.775

3.423

40

1.559

8.579

50

1.139

4.485

60

1.366

3.375

40

1.454

8.631

50

1.302

3.316

60

1.077

2.461

40

1.953

8.159

50

0.935

1.279

60

0.781

2.178

40

1.571

8.709

50

0.629

3.763

11

300

325

350

60

0.787

2.735

40

1.590

8.123

50

1.033

2.874

60

1.094

2.343

40

1.377

7.418

50

0.994

4.056

60

0.776

7.061

40

1.716

4.515

50

0.984

2.888

60

0.494

9.924

235 236

Extraction pressure had the most important influence on extraction yield of S. montana. Fig. 1

237

shows the extraction kinetics of S. montana obtained at different extraction pressures using

238

empirical model proposed by Esquivel et al [26]. This model shows the best agreement

239

between experimental and modelled results comparing models used. From extraction curves

240

at higher pressures (250, 300 and 350 bar) it can be seen that in the early stages of extraction

241

exist, the constant extraction rate period, controlled by the solubility of component in

242

supercritical carbon dioxide. As expected, the yield of SC-CO2 extraction of S. montana

243

increased with pressure. 2

mext (g)

1.5

1

0.5

0

244

0

1

2

3

4

5

t (h)

12

245

Fig. 1. Kinetic study of SC-CO2 extraction of S. montana at different process pressures and

246

time; experimental results at 400C and results obtained by the model equiation by Esquivel et

247

al.

248

The solubility data for S. montana were correlated using different empirical models. The

249

constants of the empirical equation (Eq. (3) to Eq. (6)) along with their AARD for all

250

investigated temperatures used are given in the Table 4. It can be seen that there are no

251

significant difference in the agreement of the model used with the experimental data i.e. for

252

all the model used similar AARD values were obtained.

253

Table 4. Calculated parameters and deviations for selected solubility models Chrastil

T (°C)

k

1.018

a2

-12.918

a3

235.567

AARD (%)

del Valle and Aguilera

40

29.677

50

4.194

60

14.204

T (°C)

k

1.018

a2

-12.610

a3

135.950

a4

-1.9109

AARD (%)

Adachi and Lu

40

28.406

50

4.194

60

13.731

T (°C)

a1

1.390

a2

5.25x10-5

a3

-9.30*10-8

a4

-15.631

a5

351.078

AARD (%)

40

30.367

50

4.353 13

60

12.555

T (°C)

Sparks et al. a1

1.683

a2

-0.00013

a3

-16.591

a4

214.173

AARD (%)

40

28.725

50

4.278

60

11.923

254 255

In Fig. 2, the accordance of experimental solubility data of S. montana extracts and Chrastil

256

model are present. The solubility of S. montana extracts in SC-CO2 at 313, 323 and 333 K is

257

indicated as a function of pressure. It can be seen that the solubility of extracts increased with

258

the increase of pressure and temperature.

259

Chrastil 0.01

313 K model 313 K 323 K model 323 K 333 K model 333 K

261

S (g/L)

260

0.005

262 263 0

264

0

200

400

600

800

1000

 (g/L)

265 266

Fig. 2. Solubility of S. montana extracts in SC-CO2; experimental results and results obtained

267

by Chrastil model

268

3.3. Chemical analysis of S. montana extracts obtained by SC-CO2

269 270

The extracts of S. montana obtained by SC-CO2 were subjected to GC-MS analyses in order

271

to determine chemical composition of aromatic constitutents. Applied GC-MS analyses

272

resulted in twenty-two identified compounds of the S. montana extracts. The percentage of

273

total identified compounds represents 90.00 – 95.83, 87.99 – 96.28, and 84.35 – 95.09% for 14

274

extracts obtained at different pressures and temperatures of 40, 50 and 60°C, respectively.

275

These findings are presented in the Tables 5-7. In all analyzed extracts the most abundant

276

were oxygenated monoterpenes (59.98 - 83.46, 61.61 – 82.23, and 62.77 – 83.33% for

277

extracts obtained at different pressures and temperatures of 40, 50 and 60°C, respectively),

278

followed by monoterpene hydrocarbons (4.62 – 13.34, 4.04 – 11.08, and 3.83 – 9.85% for

279

extracts obtained at different pressures and temperatures of 40, 50 and 60°C, respectively),

280

sesquiterpenes (4.99 – 8.28, 4.23 – 8.82 and 4.16 – 9.16% for extracts obtained at different

281

pressures and temperatures of 40, 50 and 60°C, respectively) and aliphatics (0 - 10.86, 0 –

282

11.03, and 0 – 9.87% for extracts obtained at different pressures and temperatures of 40, 50

283

and 60°C, respectively). The most dominant component in investigated extracts was

284

oxygenated monoterpene – carvacrol. Similar observations were noticed previous by authors

285

who investigated chemical composition of supercritical extracts of winter savory. According

286

to these studies, relative percentages of the most dominant component carvacrol were present

287

in supercritical extracts in the range: 52.20 - 62.00% [19]; 41.70 - 64.50% [23]; 52.70% [22],

288

and 53.00% [37]. In our study, content of this aromatic compound was 54.30 - 79.38% for

289

extracts obtained at different pressures and at temperature 40°C; 55.87-78.61% for extracts

290

obtained at temperature 50°C, and 57.09 – 79.61% for extracts obtained at temperature of

291

60°C. These contents are significantly higher in comparison to results presented by all other

292

authors. The results are of importance because carvacrol represents oxygenated monoterpene

293

with various important biological activities; therefore, the higher concentration of carvacrol

294

could imply the stronger biological activity of obtained extracts. This oxygenated

295

monoterpene

296

butyrylcholinesterase inhibitory activity [22, 39]. Also, carvacrol was described as the most

297

important compound responsible for antimicrobial activity of S. montana oil [22, 10, 40, 41].

298

Hulin et al. [42] reported bactericidal effect of carvacrol toward Salmonella in pieces of fish

299

stored at 4°C. It can be useful as flavoring compound for products associated with outbreaks

300

of B. cereus (e.g., rice, pasta, soup) as well [43]. Having in mind a potentially wide range of

301

carvacrol’s use in the food and pharmaceutical industry, it can be concluded that obtaining

302

extracts with a high carvacrol concentration is of the great importance.

303

The second most dominant component in obtained extracts was p-cymene. Content of this

304

monoterpene hydrocarbon was in ranges 3.64 - 10.24, 3.33 – 9.07, and 3.21 – 8.28%, for

305

extracts obtained at different pressures and temperatures 40, 50 and 60°C, respectively. These

306

results are in accordance with results of content of p-cymene in supercritical extracts reported

307

by other authors: 10.1% [22]; 6.0-17.8% [19]. Other main components of extracts were

shows

strong

antioxidant

properties

[38],

acetylcholinesterase

and

15

308

borneol (present in ranges 1.29 – 2.42, 1.14 - 2.70, 1.25 – 3.12% for extracts obtained at

309

different pressures and temperatures 40, 50 and 60°C, respectively) and sesquiterpene

310

hydrocarbon trans-caryophyllene (present in ranges 1.97 – 2.92, 1.56 – 3.19, and 1.77 –

311

3.68% for extracts obtained at different pressures and temperatures 40, 50 and 60°C,

312

respectively). The extracts also contained a lower percentage of caryophyllene oxide and γ-

313

terpinene. Other compounds as β-bisabolen, δ-cadinene, linalool and eucalyptol were present

314

as minor constituents.

315

According to Miyazawa and Yamafuji [44], compounds which are detected in lower

316

percentage than carvacrol (p-cymene, γ-terpinene) also possess acetylcholinesterase inhibitory

317

activity [22]. Also, some authors reported on antioxidant activity of constituents of S.

318

montana extract, such as α-terpinene, p-cymene, borneol and linalool [45-47]. In our previous

319

work, we presented the possibility that other less dominant components of the S. montana

320

extract can contribute the antioxidant properties of extract of this aromatic herb. This was

321

supported by observing that the extract with the highest content of carvacrol does not possess

322

the strongest antioxidant activity [13].

323

Relative percentage of carvacrol is a valuable for relative estimation of the content in

324

investigated extracts. For a more precise analysis and quantification of carvacrol, as the main

325

identified compound of obtained extracts, we applied the GC-FID. According to the GC-FID

326

analysis (Table 8), content of carvacrol in obtained extracts was in the range 41.51 – 60.17

327

g/100 g of extract for extracts obtained at different pressures and temperature 40°C; in the

328

range 46.44 – 60.82 g/100 g of extract for extracts obtained at different pressure and at

329

temperature 50°C and in the range 43.27 - 59.74 g/100 g of extract for extracts obtained at

330

temperature 60°C.

331

The lowest concentration of carvacrol was detected in extract obtained at pressure of 150 bar

332

and temperature of 40°C. The highest contet of carvacrol (60.82 g/100 g of extracts) was

333

obtained in S. montana extract produced using supercritical carbon dioxide at pressure of 350

334

bar and temperature of 50°C. A slightly lower concentration of carvacrol was measured in

335

several extracts obtained at lower pressures and temperatures: in extract obtained at pressure

336

of 300 bar and at temperature of 40°C (60.17 g/100 g of extract), extract obtained at 300 bar

337

and temperature of 50°C (60.02 g/100 g of extract) and in extract obtained at 275 bar and at

338

temperature of 60°C (59.74 g/100 g of extract). On the higher level of production, these

339

observations should be taken into consideration because price and value of obtained extracts

340

should be higher than the production costs.

341 16

342

Table 5. GC/MS analysis of S. montana extracts (relative percentage; %);

343

Extraction temperature 40°C Compound

Pressure (bar) 100*

125

150

175

200

225

250

275

300

325

350

Monoterpene hydrocarbons β-Myrcene

0.14

0.35

0.28

0.46

0.08

0.08

0.05

ni

0.06

ni

ni

α-Terpinene

0.40

0.47

0.47

0.60

0.21

0.17

0.21

0.17

0.13

ni

0.21

γ-Terpinene

0.48

1.11

1.37

2.04

1.20

1.24

1.01

1.01

0.79

1.12

0.78

p-Cymene

7.13

7.83

8.79

10.24

5.00

5.33

4.77

4.34

3.64

5.08

4.23

ni

ni

ni

0.44

0.52

0.32

ni

ni

ni

ni

0.70

0.68

0.74

0.83

0.33

ni

ni

0.23

0.24

0.31

ni

0.80

0.79

0.56

0.75

0.27

0.36

0.36

0.35

0.30

0.40

0.16

Cis-sabinene hydrate

0.26

0.29

0.33

0.25

0.12

0.16

0.16

0.19

0.19

0.24

0.19

Linalool

0.51

0.70

0.70

0.52

0.47

0.40

0.34

0.30

0.39

0.36

0.34

Borneol

2.42

2.00

1.99

2.14

1.34

1.29

1.42

1.29

1.44

1.52

1.42

Terpinene 4-ol

0.70

0.45

0.55

0.56

0.45

0.44

0.43

0.29

0.47

0.33

0.57

α-Terpineol

0.08

0.08

ni

ni

ni

0.08

0.08

0.16

0.18

0.17

ni

Carvone

0.82

0.87

1.01

0.63

0.69

0.85

0.54

ni

0.53

0.75

0.87

Carvacrol

67.58 60.70 57.43 54.30 71.34 72.69 73.21 77.36 76.67 79.38 76.10

m-Cymene

ni

a

Oxygenated monoterpenes Eucalyptol Trans-sabinene hydrate

Sesquiterpenes Trans-caryophyllene

2.80

2.45

2.79

2.92

2.03

2.04

2.26

2.10

1.97

2.06

2.08

α-Amorphen

0.55

0.60

0.67

0.66

0.32

0.37

0.43

0.38

0.31

0.37

0.36

β-Bisabolene

0.89

0.98

0.95

1.10

0.66

0.64

0.72

0.72

0.63

0.66

0.71

γ-Cadinene

0.59

0.57

0.61

0.68

0.39

0.35

0.42

0.45

0.44

0.39

0.33

δ-Cadinene

1.02

1.06

1.09

1.18

0.68

0.61

0.79

0.77

0.63

0.69

0.77

Caryophyllene oxide

1.58

1.63

1.54

1.74

0.95

1.07

1.14

1.31

1.01

1.12

1.58

Heptacosane

0.59

1.51

1.66

1.94

0.95

0.97

0.89

ni

ni

ni

0.27

Nonacosane

2.74

7.29

8.84

8.92

7.20

5.77

6.32

ni

ni

ni

ni

Total

92.77 92.40 92.39 92.44 95.10 95.43 95.83 91.41 90.00 94.93 90.96

Aliphatics

344

* These results (100 bar, 40°C) were used in another publishied paper (Vidovic et al., 2014).

345

a

not identified

346 347 17

348

Table 6. GC/MS analysis of S. montana extracts (relative percentage; %);

349

Extraction temperature 50°C Compound

Pressure (bar) 100

125

150

175

200

225

250

275

300

325

350

Monoterpene hydrocarbons β-Myrcene

0.61

0.44

0.47

0.05

ni

ni

0.05

ni

0.11

0.08

ni

α-Terpinene

0.28

0.50

0.51

0.16

0.23

0.21

0.19

0.20

0.16

0.20

0.15

p-Cymene

5.17

9.07

8.85

3.33

5.14

4.69

4.77

5.24

4.73

4.97

3.96

γ-Terpinene

ni

0.83

1.25

0.50

1.20

0.99

0.67

0.90

0.89

0.90

0.80

m-Cymene

ni

ni

ni

ni

0.54

0.51

0.35

ni

ni

ni

ni

0.58

0.75

0.72

0.24

0.21

ni

ni

0.38

0.32

0.41

0.42

0.71

0.67

0.62

0.24

0.26

0.24

0.22

0.21

0.22

0.29

0.33

0.31

0.32

0.30

0.11

0.20

0.19

0.18

0.20

0.17

0.22

0.15

Linalool

0.80

0.82

0.69

0.29

0.37

0.36

0.43

0.32

0.30

0.40

0.29

Borneol

2.70

2.20

2.06

1.14

1.26

1.26

1.44

1.41

1.60

1.49

1.56

Terpinene 4-ol

0.92

0.79

0.64

0.33

0.31

0.30

0.53

0.54

0.68

0.43

0.84

α-Terpineol

0.05

0.18

0.22

0.09

ni

0.07

0.09

ni

0.07

0.10

ni

Carvone

1.77

0.90

0.49

0.89

0.27

0.67

1.02

0.50

0.51

0.60

ni

Carvacrol

65.55 57.43 55.87 69.06 69.91 68.85 73.31 74.67 74.80 78.29 78.61

Oxygenated monoterpenes Eucalyptol Trans-sabinene hydrate Cis-sabinene hydrate

Sesquiterpenes Trans-

3.19

2.76

2.65

1.56

2.31

2.21

2.16

2.11

2.22

2.32

2.40

α-Amorphen

0.62

0.57

0.62

0.26

0.38

0.36

0.33

0.34

0.39

0.43

0.46

β-Bisabolen

1.24

1.08

0.98

0.51

0.66

0.70

0.63

0.59

0.70

0.71

0.74

γ-Cadinene

0.63

0.63

0.53

0.30

0.32

0.34

0.32

0.47

0.45

0.40

0.53

δ-Cadinene

1.30

1.11

1.02

0.59

0.75

0.71

0.61

0.61

0.81

0.67

0.78

1.84

1.75

1.64

1.01

1.25

1.13

0.94

1.24

1.41

1.35

1.26

Heptacosane

0.51

1.09

2.02

0.44

0.81

0.95

0.90

ni

ni

ni

0.17

Nonacosane

2.05

5.25

9.01

6.92

9.65

5.20

7.14

ni

ni

ni

0.19

Total

90.84 89.12 91.15 87.99 96.05 89.91 96.28 89.93 90.54 94.25 93.64

caryophyllene

Caryophyllene oxide Aliphatics

350

18

351

Table 7. GC/MS analysis of S. montana extracts (relative percentage; %);

352

Extraction temperature 60°C Compound

Pressure (bar) 100

125

150

175

200

225

250

275

300

325

350

Monoterpene hydrocarbons β-Myrcene

0.40

0.35

0.44

0.04

0.05

0.03

ni

ni

ni

ni

ni

α-Terpinene

0.21

0.41

0.44

0.17

0.18

0.12

0.10

0.24

ni

0.20

0.25

γ-Terpinene

ni

0.32

0.69

0.46

0.56

0.51

0.52

0.83

0.63

0.63

0.73

p-Cymene

3.68

7.71

8.28

4.01

5.01

3.95

3.21

5.47

3.37

4.96

5.02

m-cymene

ni

ni

ni

0.41

0.60

0.29

ni

ni

ni

ni

ni

0.55

0.65

0.63

0.28

ni

ni

0.17

0.36

ni

0.62

0.77

0.95

0.48

0.72

0.28

0.27

0.11

0.33

0.25

0.43

0.31

0.27

Cis-sabinene hydrate

0.41

0.26

0.23

0.14

0.15

0.12

ni

0.14

0.26

0.23

0.15

Linalool

1.32

0.86

0.66

0.32

0.30

0.29

0.56

0.44

0.37

0.46

0.37

Borneol

3.12

2.27

1.86

1.25

1.38

1.25

1.44

1.59

1.52

1.75

1.56

Terpinene 4-ol

1.08

1.14

0.55

0.34

0.33

0.54

0.41

0.66

0.30

0.69

0.87

α-Terpineol

0.29

0.20

0.20

0.07

0.09

0.11

ni

0.10

0.15

ni

ni

Carvon

2.21

1.46

0.83

0.52

1.10

1.49

0.47

0.85

0.69

1.16

ni

Carvacrol

67.91 61.62 57.09 64.96 73.13 65.95 71.83 77.58 79.61 76.41 76.31

Oxygenated monoterpenes Eucalyptol Trans-sabinene hydrate

Sesquiterpenes Trans-caryophyllene

3.68

2.70

2.66

1.77

2.02

1.77

2.13

2.09

2.25

2.59

2.60

α-Amorphen

0.80

0.63

0.62

0.27

0.41

0.34

0.37

0.41

0.36

0.36

0.52

β-Bisabolene

1.37

1.08

0.95

0.52

0.63

0.515

0.61

0.66

0.73

0.68

0.75

0.61

0.52

0.34

0.37

0.28

0.47

0.36

0.52

0.40

0.44

γ-Cadinene δ-Cadinene

1.48

1.16

1.06

0.53

0.71

0.52

0.63

0.61

0.85

0.67

0.67

Caryophyllene oxide

1.83

1.66

1.58

0.73

0.95

0.95

1.10

1.01

1.38

1.44

1.29

Heptacosane

0.18

1.17

1.48

0.63

0.84

0.75

ni

ni

ni

ni

0.20

Nonacosane

0.66

4.77

8.39

7.33

6.03

4.99

ni

ni

ni

ni

0.25

Total

92.10 91.51 89.86 85.36 95.09 84.88 84.35 93.65 93.40 93.56 93.02

Aliphatics

353 354 355

19

356

Table 8. GC/FID analysis of carvacrol content (g/100 g of extract); Temperature 40, 50 and

357

60°C Pressure (bar)

Temperature (°C)

100

125

150

175

200

225

250

275

300

325

350

40

52.97 52.60 41.51 45.42 57.20 55.25 56.13 52.87 60.17 52.56 55.72

50

50.36 49.70 47.63 55.35 51.99 46.44 52.60 59.10 60.02 51.81 60.82

60

59.14 56.49 52.94 55.49 55.46 43.27 52.05 59.74 58.63 59.50 55.88

358 359 360

3.4. Hierarchical cluster analysis of compounds detected in S. montana extracts

361

Chemometric analysis is undoubtedly of great importance in modern sciences also including

362

plant extracts. It means performing calculations on measurements of chemical data.

363

Hierarchical cluster analysis (HCA), as a chemometric classification tool, was used in order to

364

reveal possible similarities and dissimilarities among the obtained samples obtained at

365

different extraction conditions. Also, the purpose of the HCA was to present the content of

366

different constituents of the samples in a simple way.

367

The obtained dendrograms were formed on the basis of the data given in Tables 5-7, including

368

only the compounds which are present in all extracts. Therefore, clustering of the extracts was

369

achieved based on 12 detected compounds, listed in description of Fig. 3. Every dendrogram

370

refers to the extracts obtained at the specific extraction temperature (40, 50 and 60°C).

371

Vertical dendrograms present the clustering of extracted compounds regarding their quantity.

372

The highest quantities of carvacrol, followed by p-cymene, trans-caryophyllene, borneol and

373

caryophyllene oxide, can be observed at each extraction temperature. The horizontal

374

dendrograms describe the clustering of the extraction procedures at different pressures based

375

on the quantity of the extracted compounds. The dendrogram based on the extracts obtained at

376

40°C indicate the similarity among the extractions at 100, 125, 150 and 175 bar. The increase

377

of the pressure over 175 bar provides a significant change in quantities of the analyzed

378

compounds in the extracts (the pressures 100, 125, 150 and 175 bar are placed in the same

379

cluster). However, at the 50 and 60°C, according to the components quantities, the differences

380

between the extraction procedures become significant with increase in pressure from 150 to

20

381

175 bar (the pressures 100, 125 and 150 bar are placed in the same cluster or are outside the

382

main cluster with pressures of 200, 225, 250, 275, 300, 325 and 350 bar).

383

A)

384 385

B)

386 387

C)

388

21

389

Fig. 3. Double dendrograms as a result of HCA of the extracts obatined by the extraction

390

procedure at 40°C (A), 50°C (B) and 60°C (C) and different pressures (100–350 bar)

391

Compounds: 1 – p-Cymene, 2 – Trans-sabinene hydrate, 3 – Linalool, 4 – Borneol, 5 -

392

Terpinene 4-ol, 6 – Carvacrol, 7 – Trans-caryophyllene, 8 – α-Amorphen, 9 – β-Bisabolene,

393

10 – γ-Cadinene, 11 – δ-Cadinene, 12 – Caryophyllene oxide. Colour representation: blue -

394

high similarity, green - slightly lower similarity, red - lowest similarity.

395

4. Conclusions

396

This work presents successful modelling of the extraction kinetic of the S. montana extracts

397

obtained under different process parameters using two models. We have concluded that

398

Esquivel et al. model best describes the agreement between experimental and model

399

calculated data. The entire investigation is necessary if the possibility of industrial application

400

is considered. The solubility data for S. montana extracts were correlated using different

401

empirical models. All models used show similar deviation from experimental data.

402

Moreover, the outcome of this work is the production of extracts by using SC-CO2 with a

403

significantly high content of carvacrol which was greater than in all previously published

404

works. Taking into account many important biological activities of the S. montana extracts

405

and the possibility to use them as natural food preservatives or as potential sources of

406

nutritional and medicinal benefits, it resulted in a considerably high research interest.

407 408 409

Acknowledgements

410

The authors gratefully acknowledge the financial support of this work by the Ministry of

411

Education

and

Science,

Republic

of

Serbia

(Project

No.

TR3101)

22

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