Separation Science and Technology

ISSN: 0149-6395 (Print) 1520-5754 (Online) Journal homepage: http://www.tandfonline.com/loi/lsst20

OPTIMIZATION: MICROWAVE IRRADIATION EFFECT

ON POLYPHENOLIC COMPOUNDS EXTRACTION
FROM WINTER SAVORY (Satureja montana L.)

Zoran Zekovic, Aleksandra Gavaric, Branimir Pavlic, Senka Vidovic & Jelena
Vladic

To cite this article: Zoran Zekovic, Aleksandra Gavaric, Branimir Pavlic, Senka Vidovic & Jelena
Vladic (2017): OPTIMIZATION: MICROWAVE IRRADIATION EFFECT ON POLYPHENOLIC
COMPOUNDS EXTRACTION FROM WINTER SAVORY (Satureja montana L.), Separation
Science and Technology, DOI: 10.1080/01496395.2017.1288744

To link to this article: http://dx.doi.org/10.1080/01496395.2017.1288744

Accepted author version posted online: 06

Feb 2017.

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OPTIMIZATION: MICROWAVE IRRADIATION EFFECT ON

POLYPHENOLIC COMPOUNDS EXTRACTION FROM

WINTER SAVORY (Satureja montana L.)

Zoran Zekovic, Aleksandra Gavaric, Branimir Pavlic, Senka Vidovic, Jelena Vladic1

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

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Abstract an
Microwave-assisted extraction of polyphenols with elevated antioxidant activity from winter

savory was optimized by simultaneous maximization of total phenolics, total flavonoids

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yields, and antioxidant activity (measured by two assays - DPPH and reducing power assay).

For optimization of microwave assisted extraction, Box-Behnken experimental design

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coupled with response surface methodology was applied. Independent variables were

extraction time, ethanol concentration and irradiation power. Analysis of variance was used to
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evaluate model fitness and determine optimal conditions.

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Keywords: Satureja montana, microwave-assisted extraction, antioxidant activity, response

surface methodology, phenolics

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1
Corresponding author. E-mail: vladicjelena@gmail.com; Telephone: +38160 5957900, Bulevar
cara Lazara 1, 21000 Novi Sad, Serbia, +38121 485 3728
1. Introduction

Satureja montana L. (commonly known as winter savory) is a perennial herb of Lamiaceae

family which encompasses over 30 species, whose center of distribution is located in the

eastern part of the Mediterranean (1). This aromatic herb has often been used in

Mediterranean cooking as spice for food and teas, seasoning for meats and fish, and in

flavoring agents for soups, sausages, canned meats and spicy sauces (2-5). The species S.

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ip
montana L. show high variability, even within a single population, but especially in

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populations coming from distant habitats. This plant contains various biologically active

constituents such as essential oils (2), triterpenes (6) and phenolic compounds (4, 7). Different

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extracts and essential oil of winter savory possess antioxidant (8-11) and antimicrobial
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properties (12, 13). Moreover, winter savory elicits important biological activities such as

diuretic, anti-inflammatory, anti-HIV-1 and anticholinesterase activity (5, 14, 15, 16).
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Microwave assisted extraction (MAE) is becoming method of choice for recovery of phenols,
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essential oils, aromas, pesticides, phenols, dioxins and other organic compounds. One of the

main advantages of using MAE is the reduction of extraction time when applying microwaves
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(17). The other assets of using microwave energy, a noncontact heat source, for the extraction
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of bioactive compounds from plant matrices include more effective heating, faster energy

transfer, reduced thermal gradients, selective heating, faster response to process heating
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control, faster start-up, increased production and elimination of process steps (18). The

microwave heating leads to the expansion and ruptures of cell walls and is followed by the

releasing of chemicals into the solvent. Polar molecules and ionic solutions absorb microwave

energy strongly because they have a permanent dipole moment that will be affected by the

microwaves (17). This results in more uniform temperature and moisture profiles, improved

yields and enhanced product performance (19, 20).

The purpose of this study was to estimate effects of MAE conditions (extraction time, ethanol

concentration and irradiation power) and to optimize these conditions using response surface

methodology (RSM) in order to gain the highest polyphenolics content and antioxidant

activity of winter savory ethanol extracts.

2. Materials and methods

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2.1. Plant material

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S. montana samples were collected at the Institute of Field and Vegetable Crops, Backi

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Petrovac, Republic of Serbia, in July 2012. Collected plant material has been naturally dried

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and then stored in paper bags at the room temperature.

2.2. Chemicals
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Folin-Ciocalteu reagent, (±)-catechin and 1,1-diphenyl-2-picryl-hydrazyl-hydrate (DPPH)
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were purchased from Sigma (Sigma-Aldrich GmbH, Steinheim, Germany). Gallic acid was

purchased from Sigma (St. Luis, MO, USA). All other chemicals used were of analytical
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reagent grade.
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2.3. MAE procedure

Mono-mode microwave-assisted extraction was performed in home-made setup consisting of

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microwave oven (NN-E201W, Panasonic, Japan) and appropriate glass apparatus with round
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flask and condenser. Solid-liquid extraction process is influenced by various parameters such

as type and solvent concentration, solid-to-liquid ratio, extraction time, temperature, mean

particle size, etc. Since it is practically impossible to identify and control the small

contributions of each parameters, screening of variables must be performed that can provide

information about extraction parameters with major influence on response variables (21).

Therefore, kinetic of MAE was determined by measuring of total extract yield, total phenolics
(TP) and total flavonoids (TF) contents on different extraction times, while other MAE

parameters were fixed. Extractions were performed with 70% ethanol, 1:10 (w/v) of solid-to-

liquid ratio and 600 W of irradiation power and 1, 2, 3, 4, 5, 7.5, 10, 15, 20 and 25 min of

extraction time. After screening of extraction parameters, 17 experimental MAE runs on

different extraction time, ethanol concentration and irradiation power were performed. In each

experimental run, 5.0 g of sample were mixed with 50 mL of extraction solvent in 500 mL

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round flasks. Flasks were then placed in MAE apparatus and extractions were performed on

fixed frequency. Flasks were always positioned on the same distance from the magnetron and

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no additional agitation was applied. After the extraction, crude extracts were immediately

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filtered through filter paper under vacuum. Extracts were collected into glass flasks and stored

at 4 °C until the analysis.

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2.4. Total phenolics (TP) content
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Total phenolics content in obtained liquid extracts was determined using Folin-Ciocalteu

procedure (22, 23). Gallic acid was used as standard for standard diagram, and absorbance of
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the samples was measured at 750 nm (6300 Spectrophotometer, Jenway, UK). Content of

phenolic compounds was expressed as mg of gallic acid equivalents (GAE) per 100 g dry
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weight (DW). All experiments were performed in triplicate, and results are expressed as mean
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values.
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2.5. Total flavonoids (TF) content

Total flavonoids content was determined using aluminum chloride colorimetric assay (24).

Catechin was used for standard diagram and absorbance was measured at 510 nm. Results

were expressed as mg of catechin equivalents (CE) per 100 g DW. All experiments were

performed in triplicate, and results are expressed as mean values.

2.6. DPPH assay

Free radical scavenging activity of samples was determined using DPPH assay, previously

described by Espin et al. (25). A certain volume of diluted sample was mixed with 95%

methanol and 90 µM 1,1-diphenyl-2-picryl-hydrazyl (DPPH) in order to obtain different final

concentrations. After incubation on room temperature for 60 min, the absorbance was

measured at 515 nm and result was expressed as radical scavenging capacity (RSC, %) which

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was calculated using following equation:

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( )

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where Asample is the absorbance of sample solution and Ablank is the absorbance of blank probe.
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Antioxidant activity was further expressed as inhibition concentration at 50% of RSC value

(IC50). IC50 represents the concentration of test solution required to obtain 50% of radical
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scavenging capacity, expressed as mg per mL. All experiments were performed in triplicate,

and results are expressed as mean values.

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2.7. Reducing power assay

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Reducing power of the samples was determined according to assay based on the reduction of
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Fe3+ by polyphenol antioxidants, previously described by Oyaizu (26). Different dilutions of

liquid extract (1 mL) were mixed with phosphate buffer (1 mL, 0.2 M, pH 6.6) and 1%
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potassium ferricyanide (1 mL) in glass tubes. Tubes were incubated on 50 °C for 20 min.

After incubation, 10% trichloroacetic acid solution (1 mL) was added to the reaction mixture.

Tubes were then centrifuged at 3000 rpm for 10 min and supernatant (2 mL) was further

mixed with double distilled water (2 mL) and 0.1% FeCl3 solution (0.4 mL). Absorbance was

measured at 700 nm. The blank probe was prepared by using proper extraction solvent instead

of sample. Antioxidant activity was further expressed as EC50 value (mg/mL), which causes
reduction of 50% Fe3+ ions in reaction mixture. All experiments were performed in triplicate,

and results are expressed as mean values.

2.8. Box-Behnken design and statistical analysis

The extraction efficiency of MAE is affected by irradiation power, temperature, extraction

time and cycle of heating, therefore, all parameters must be adequately optimized. In the

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current study RSM coupled with Box-Behnken design (BBD) was applied in order to

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optimize MAE process. Design consisted of seventeen randomized runs with five replicates at

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the central point. Independent variables used in experimental design were extraction time

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(t, 12.5 – 27.5min), ethanol concentration (c, 50 – 90%) and irradiation power (P, 400 – 800

W). In order to normalize parameters, each of the coded variables was forced to range from
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−1 to 1, so that they all affect the response more evenly, and so the units of the parameters are

irrelevant (27). The natural and coded values of independent variables used in BBD are
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presented in Table 1.
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The response variables were fitted to the following second-order polynomial model (Eq. (2))
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which is generally able to describe relationship between the responses and the independent

variables (21):
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∑ ∑ ∑ ∑
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where Y represents the response variable, Xi and Xj are the independent variables affecting the

response, and β0, βi, βii, and βij are the regression coefficients for intercept, linear, quadratic

and interaction terms. Optimal extraction conditions were determined considering total

phenols and total flavonoids content, and antioxidant activity simultaneously as responses.
Treatment of multiple responses and selection of optimal conditions were based on

desirability function D (28). The experimental design and multiple linear regression analysis

were performed using Design-Expert v.7 Trial (Stat-Ease, Minneapolis, Minnesota, USA).

The fitness of the polynomial model equation is expressed by the coefficient of determination

(R2) and its statistical significance was confirmed by F-test at a probability (p) of 0.10 or 0.05.

3. Results and Discussion

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3.1. Extraction kinetics

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Unlike the classical conductive heating methods, microwaves can heat the entire sample

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almost simultaneously and at a higher rate (29). This improves efficacy and decreases time

necessary for extraction. Numerous factors influence MAE such as nature and particle size of
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plant material, type and concentration of solvent, sample to solvent ratio, extraction time and

temperature. One of the most important parameter that affects MAE process is solvent
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selection. Solvent selection depends on the solubility of the compounds of interest, solvent

penetration, its interaction with the sample matrix, its dielectric constant [30] and the mass
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transfer kinetic of the process (31). Solvents such as ethanol, methanol and water are

sufficiently polar to be heated by microwave energy (32). Ethanol is especially convenient

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solvent, which provides satisfying yield of polyphenols, due to its moderately polarity (ε=
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25.5 at 20 °C) (33). Therefore, ethanol concentration in the range from 50 to 90% (w/w) was

chosen as independent variable.

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Rezvanpanah et al. (34) reported winter savory yield of 0.7% (w/w), obtained by microwave

assisted hydrodistillation at power of 660 W for 90 min. This yield was similar to one

obtained via conventional hydrodistillation, but necessary extraction time was decreased by

50%. Prolonged irradiation time can lead to a thermal degradation of the simple flavonoids

(35). Dahmoune et al. (36) found that the extraction time of phenolic compounds from M.
communis leaves extracted using MAE was about 14 and 15 times lower than the ultrasound

assisted and traditional extraction methods, respectively. Thus, preliminary kinetic

experiments were performed in order to select interval of irradiation time. Increased

temperature as a consequence of microwave irradiation could also potentially cause

degradation of target compounds. TP and TF yields, as well as total extract yields obtained by

MAE with 70% ethanol, 1:10 (w/v) of solid-to-liquid ratio and 600 W of irradiation power are

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presented on Fig. 1a and 1b, respectively. Increasing the irradiation time from 1 to 5 min,

increases the TP and TF yields rapidly, after that extraction reaches stationary phase in both

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cases. However, increasing the irradiation time from 20 to 25 min, yield rises for 8.33%.

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Therefore, in optimization study, extraction time in range from 12.5 to 27.5 min was

investigated as independent variable. Irradiation power was investigated in range from 400 to
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800 W, due to limitation of instrument which maximum working power is 800 W.

3.2. Model fitting

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Several authors investigated MAE of less familiar species of genus Satureja. Memarzadeh et
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al. (37) confirmed that the most convenient results were obtained with the MAE of S.

bachtiarica L., which provided 8-fold reduced extraction time in comparison with
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conventional distillation methods and the highest percentage of oxygenated monoterpenes, i.e.
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thymol (34.53 ± 1.48%) and carvacrol (31.66 ± 0.61%). Furthermore, MAE was superior

method in terms of energy saving since only 0.3–0.5 kWh was spent during microwave
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procedure. Damyeh et al. (38) also found that application of microwaves in the extraction

process of aerial parts of S. macrosiphonia L. may result in a significant and to some extent

more selective separation of oxygenated components. The recovery of cis-sabinene hydrate

was more successful (for 33%) by using MAE than hydrodistillation.

Different MAE conditions (extraction time, ethanol concentration and irradiation power) were

applied in order to experimentally obtain responses (TP, TF, IC50 and EC50 values).
Experimental values were fitted to a second-order polynomial model (Eq. 2) and multiple

regression coefficients were provided for all responses using the method of least square. The

regression coefficients of the model for each response are presented in Table 2, while results

of the analysis of variance (ANOVA) are presented in Table 3. Coefficient of determination

(R2) is defined as the ratio of the explained variation to the total variation and is a

measurement of the degree of fitness (39). The low value of R2 indicates the poor relevance of

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the dependent variables in the model. According to particularly high R2 for TP, TF, IC50 and

EC50 (0.9826, 0.9271, 0.8340 and 0.8319, respectively), all applied polynomial models were

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in accordance with experimental results. Moreover, satisfying reproducibility was confirmed

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by relatively low CV (<10%) for all responses (Table 3) of the investigated systems since CV

describes dispersion of the data and low values indicates low variation in the mean value (40).
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Mathematical models, for all four responses, were statistically acceptable due to significant

regression for the model (p<0.05). Lack of fit testing confirmed adequacy of fitting
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experimental data to a second order polynomial model. Obtained experimental data were used
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for providing response surface contour plots and regression equations which could predict

response values at investigated experimental domain.

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3.3. Total phenols content

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Experimental results of TP in ethanol extracts of winter savory are presented in Table 4.

Experimentally obtained values for TP varied from 2.95 g GAE/100 g DW to 7.44 g GAE/100
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g DW. The lowest value was obtained at middle level of extraction time (20 min), higher level

of both ethanol concentration and irradiation power (90%, 800 W), while the highest value

was achieved at higher level of extraction time (27.5 min), lower level of ethanol

concentration (50%) and middle level of irradiation power (600 W). Generally, low TP yields

(2.95 – 3.45 g GAE/100 g DW) were obtained using 90% ethanol, while higher TP yields

(6.85–7.44 g GAE/100 g DW) were obtained using 50% ethanol as extraction solvent. This
implies that ethanol concentration was the most significant factor influencing TP yield.

Furthermore, ethanol concentration of 50% proved to be the most convenient for recovery of

TP, TF and antioxidant capacity obtained by MAE of Ocimum basilicum, another

representative of family Lamiaceae (41).

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Cetkovic et al. [4] determined total phenolic content (expressed as chlorogenic acid equivalent

(CAE) per g dry weight of plant material) of different extracts from S. montana. They

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macerated winter savory with 70% methanol, evaporated under reduced pressure and then

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extracted sequentially with chloroform, ethyl acetate and n-butanol. The chloroform, ethyl

acetate, n-butanol and remaining water extract were evaporated to dryness under reduced
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pressure. The results obtained by Folin Ciocalteu method showed that the quantities of

phenolic compounds in ethyl acetate (9.69 mg CAE/g DW) and n-butanol (1.36 mg CAE/g
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DW) extracts were significantly higher (p<0.001) compared to other investigated extracts
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(0.0963 mg CAE/g DW for water extract and 0.0084 mg CAE/g DW for chloroform extract).

Maximum TP yield obtained in MAE winter savory extract (7.44 g GAE/100 g DW) wasat
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least seven times higher than maximum TP yield obtained in ethyl acetate extract (0.97 g

CAE/100 g DW). In these work, methanol demonstrated to be suitable extraction solvent to

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reach satisfying yields of the above-mentioned phenolic compounds. However,

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environmentally benign and non-toxic food grade organic solvents like ethanol, n-butanol and

isopropanol, recommended by the US Food and Drug Administration, might be used for MAE

purposes (42).

Seranno et al. (43) found that essential oil derived from winter savory possesses the highest

phenolic content (22.10 g GAE/100 g DW) followed by hot aqueous (18.75 g GAE/100 g

DW), cold aqueous (16.42 g GAE/100 g DW) and ethanol extracts (11.12 g GAE/100 g DW).
Value of TP yield (7.44 g GAE/100 g DW) obtained for 27.5 min of MAE in present study is

lower than TP yield obtained for 72h of ethanol maceration. However, taking into account

that extraction time was less than 30 min in case of MAE, tremendous reduction in time

consumption and energy is evident. Maximum TP yield (7.30 g GAE/100 g DW), obtained

using 38.6% ethanol for 2h of maceration (9), is in agreement with higher TP yields (6.85 –

7.44 g GAE/100 g DW) achieved using 50% ethanol by MAE for less than 30 min.

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3.4. Total flavonoids content

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Experimentally obtained values for TF varied from 2.40 to 4.81 g CE/100 g DW. Maximum

recovery of total flavonoids (4.81 g CE/100 g DW) was recorded under the experimental

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conditions: extraction time of 12.5 min, ethanol concentration of 70% and microwave power
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of 800 W. Minimum recovery of TF (2.40 g CE/100 g DW) was obtained under the

experimental conditions: extraction time of 20 min, ethanol concentration of 90% and

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irradiation power of 800 W. Generally, low TF yields (2.40 – 2.84 g CE/100 g DW) were

obtained using 90% ethanol, while higher TF yields (3.68 –4.81 g CE/100 g DW) were
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obtained using 70% ethanol as extraction solvent.

Vladic et al. (9) concluded that maximum TF yield (6.3 g CE/100g DW) was recovered using
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33.3% ethanol for 2h of maceration. This maximum TF yield is for 23.8% higher than
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maximum TF content (4.81 g CE/100 g DW) recorded for 12.5 min using 70% ethanol.

Bearing in mind that higher TP and TF yields were obtained using 50% and 70% ethanol,
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respectively, indicating that added water, the most polar solvent used, caused further

extraction of polar components.

3.5. Antioxidant activity

The antioxidant properties of MAE winter savory extracts were evaluated by DPPH and

reducing power assays. Experimentally obtained values for antioxidant activity varied from

3.23 to 4.60 µg/mL. The strongest antioxidant capacity (3.23 µg/mL) was achieved for 12.5

min, at ethanol concentration of 70% and irradiation power of 800 W. The lowest antioxidant

capacity was obtained at extraction time of 20 min, ethanol concentration of 90% and

irradiation power of 400 W. Range of IC50 values (3.21 –3.95 µg/mL) obtained using 70%

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ethanol overlapped with range of IC50 values (3.40 –4.01 µg/mL) obtained using 50% ethanol

as extraction solvent. Previous reported results showed that IC50 values ranged from 9.8 to

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508.5 μg/mL. The hot aqueous winter savory extract showed the strongest antioxidant

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capacity (9.8 μg/mL) followed by cold aqueous winter savory extract (27.9 μg/mL), ethanol

winter savory extract (108.8 μg/mL) and essential oil (508.5 μg/mL). The difference in
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antioxidant activity between the water and ethanol extracts might be related to the high

content of phenolics acids, such as rosmarinic acid and derivatives in winter savory, which
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possess a strong ability to scavenge radicals due to their phenolic hydroxyl groups (44, 45).
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Vladic et al. (9) investigated antioxidant activity of winter savory extracts obtained using

different extraction solvents, in terms of ethanol concentration (21.7% - 78.3%). The solid-to-
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liquid ratio was 1:10 (w/v) and extraction time was 120 min. The lowest IC50value (12.33

µg/mL) was achieved at 74.9°C using ethanol concentration of 34.6%. The antioxidant
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capacity (3.23 µg/mL) of MAE winter savory extract achieved for 12.5 min and at ethanol
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concentration of 70% was at least 3 times stronger than IC50value (12.33 µg/mL) obtained via

2h of maceration.

Results of reducing power towards Fe3+ ions, expressed as EC50 value, in winter savory

ethanol extracts obtained by MAE are presented in Table 4. The lowest EC50 value, that is, the

highest reducing power (9.53µg/mL), was obtained for 27.5 min using 70% ethanol and

irradiation power of 800 W. However, the highest EC50 was reached for 20 min using 90%
ethanol and irradiation power of 800 W, suggesting that increase of ethanol concentration

cause decrease of reducing power. Generally, lower EC50 values (9.53 – 11.87µg/mL) were

obtained using 70% ethanol, while higher EC50 values (11.68 – 15.10µg/mL) were obtained

using 90% ethanol as extraction solvent. This is in agreement with higher TF yields obtained

using 70% ethanol, suggesting that flavonoid compounds are the major contributor to

antioxidant activity. The trend for the ferric ion reducing activities of winter savory extracts

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showed a similar pattern to the DPPH scavenging activities. In both applied assays (DPPH

and reducing power assay), the strongest antioxidant capacity was achieved using 70%

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ethanol, while the weakest antioxidant capacity was obtained using 90% ethanol. Generally,

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moderate negative correlation (r = -0.709) was observed between experimentally obtained TF

and EC50, which means that EC50 decrease, i.e. antioxidant activity increases, with increasing
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TF. Also, there is a poor negative correlation (r = -0.576) between experimentally obtained

TP and EC50.
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3.6. Influence of independent variables on investigated responses

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The influence of extraction conditions toward investigated responses was reported through

significant (p<0.05) and moderately significant (p<0.10) regression coefficients of the second-
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order polynomial regression equation. Unlike previously reported experiment where both
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linear and quadratic terms of ethanol concentration showed significant negative effect on TP

yield (9), in our study both interaction between ethanol concentration and irradiation power

and quadratic term of irradiation power displayed significant effect (p<0.05) on the yield of

TP presented in the contour plots in Fig. 2A and 2C. Linear term of extraction time showed

moderately significant effect (p<0.10) on TP yield. Determination of regression coefficients

in Eq. (2), using method of least square, provided model equation (Eq. (3)) for MAE of winter
savory polyphenols, which is able to predict TP yield within investigated experimental

domain:

(3)

In the case of TF content, linear term of extraction time displayed highly significant (p<0.005)

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negative effect on the yield of TF presented in the contour plots in Fig. 2E and 2F. This is not

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in compliance with results obtained in previous study, in which both linear and quadratic

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terms of ethanol concentration had significant negative influence (p<0.05) on second

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investigated response–TF content (9). Model equation (Eq. (4)) for MAE of winter savory TF,

which is able to predict TF yield within investigated experimental domain:

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(4)

In the case of IC50 value, both interaction between ethanol concentration and irradiation
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power and interaction between extraction time and ethanol concentration exhibited significant

(p<0.05) negative effect on IC50 value, presented in the contour plots in Fig. 2G. Extraction
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time exhibited moderately significant (p<0.10) negative effect on antioxidant activity.

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However, regarding antioxidant activity of winter savory extracts, it was reported that both

linear and quadratic terms of ethanol concentration had a significant positive effect on IC50
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value (9). Model equation (Eq. (5)) for MAE of winter savory IC50 value, which represents

concentration of extract required for 50% inhibition in vitro, able to predict IC50value within

investigated experimental domain:

(5)
In the case of EC50, only interaction between extraction time and irradiation power exhibited

moderately significant (p<0.1) negative influence on reducing power, presented in Fig. 2J and

2L. Predictive second-order polynomial model equation for MAE with EC50 as target

response is:

(6)

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3.7. Optimization of MAE process

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Optimization of MAE conditions has been reported in many studies so far (17, 46, 47). RSM

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was chosen as approach to process optimization. The ultimate aim of RSM is to determine the

optimum operating conditions for the system or to determine a region of the factor space in
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which operating requirements are satisfied (48).

In order to optimize all four responses at the same time, desirability function was employed
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and optimized conditions were extraction time of 27.5 min, ethanol concentration of 55.8%
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and irradiation power of 632 W. The optimal values of TP, TF, IC50 and EC50 value would be

6.87 g GAE/100g DW, 4.48 g CAT/100g DW, 3.20 µg/mL and 9.83 µg/mL, respectively,
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while desirability was 0.918. These predicted data were experimentally confirmed with a

deviation of ±5%. Since the experiment was conducted on the home-made setup constructed
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from a kitchen microwave oven, the irradiation power of 632 W was set with the precision of
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±15 W.

Finally, by applying suggested optimal input parameters, production of winter savory extracts

with a high value of polyphenolics and low values of IC50 and EC50 can be accomplished in a

very short time compared to conventional extraction techniques. Furthermore, it increases the

economic feasibility of the entire process which is highly beneficial for the industrial plant

production.
4. Conclusions

MAE proved to be suitable for fast and effective extraction of polyphenols from winter

savory. The high correlation of the mathematical model indicated that quadratic polynomial

model could be employed to properly define the true behavior of the system. Among three

factors (extraction time, ethanol concentration and irradiation power), linear term of

extraction time was the most dominant, highly significantly (p<0.005) influencing total

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flavonoids content. Apart from linear term of extraction time, the second most dominant

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factor was interaction between ethanol concentration and irradiation power. This cross

product term displayed significant effect (p<0.05) on the yield of TP and significant (p<0.05)

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negative effect on IC50 value. The starting premise that response surface methodology coupled
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with Box-Behnken design could be applied in order to optimize MAE process by

simultaneous maximization of four responses was confirmed. Under these optimal conditions,
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the experimental values were in compliance with the predicted values by analysis of variance.
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Acknowledgements

The financial support provided by the Ministry of Education, Science and Technological
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Development, Republic of Serbia (Project No. TR31013) was appreciated.

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1. Silic, C. (1979) The Monograph of genus Satureja L., Calamintha Miller, Micromeria

Bentham, Acinos Miller and Clinopodium L. in Flora of Yugoslavia, Svijetlost: Sarajevo,

BiH.

2. Slavkovska, V.; Jancic, R.; Bojović, S.; Milosavljević, S.; Djoković, D. (2001) Variability

of essential oils of Satureja montana L. and Satureja kitaibelii wierzb. ex Heuff. from the

central part of the balkan peninsula. Phytochemistry, 57(1): 71-76.

3. Mastelic, J.; Jerkovic, I. (2003) Gas chromatography–mass spectrometry analysis of free

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4. Cetkovic, G.S.; Mandic, A.I.; Canadanovic‐Brunet, J.M.; Djilas, S.M.; Tumbas, V.T.

(2007) HPLC screening of phenolic compounds in winter savory (Satureja montana L.)

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Table 1. Experimental domain with natural and coded values of independent variables used in

BBD

Factor levels
Independent variable
-1 0 1

Extraction time [min] 12.5 20 27.5

Concentration [%] 50 70 90

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Irradiation power [W] 400 600 800

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Table 2. Corresponding p-values of linear, interaction and quadratic terms of regression

coefficients, obtained for selected response variables

Response
Term
TP TF IC50 EC50

Linear

X1 0.068 -3.135∙10-3 -0.081 -0.189

t
X2 -1.933 -0.767 0.113 1.143

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X3 -0.100 -0.068 -0.154 -0.350

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Cross product

X12 -0.229 -0.171 -0.023 -0.404

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X13 -0.135 -0.199 0.196 -0.077

X23 -0.021 -0.107 -6.108∙10-3 0.638

Quadratic
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X11 0.153 0.103 -0.408 -0.455

X22 -0.177 -0.968 0.249 1.682

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X33 0.024 0.200 0.291 0.835
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Table 3. Analysis of variance (ANOVA) of the fitted model for TP, TF, IC50 and EC50

Sum of
Source DF Mean square F – value p – value
squares

Total phenolics content

t
Model 30.50 9 3.39 43.93 < 0.0001

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Residual 0.54 7 0.08

Lack of fit 0.06 3 0.02 0.15 0.9220

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Pure error 0.48 4 0.12

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Total 31.04 16

R2 = 0.9826

CV = 5.30
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Total flavonoids content

Model 9.11 9 1.01 9.88 0.0032

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Residual 0.72 7 0.10

Lack of fit 0.14 3 0.05 0.33 0.8031

Pure error 0.57 4 0.14

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Total 9.83 16

R2 = 0.9271
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CV = 8.17

IC50 value
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Model 1.75 9 0.19 3.91 0.0430

Residual 0.35 7 0.05

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Lack of fit 0.23 3 0.08 2.71 0.1795

Pure error 0.12 4 0.03

Total 2.10 16

R2 = 0.8340

CV = 6.07

EC50 value

Model 29.94 9 3.33 3.85 0.0446

Residual 6.05 7 0.86

 Lack of fit 5.03 3 1.68 6.56 0.0504

Pure error 1.02 4 0.26

Total 35.99 16

R2 = 0.8319

CV = 8.27

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Table 4. Box-Behnken experimental design with natural and coded MAE parameters and

experimentally obtained values of investigated responses (TP, TF, IC50 and EC50)

Independent variables Response variables

Extraction Ethanol Irradiation TP TF

Run IC50 EC50
time concentration power [g GAE/100 [g CE/100 g

t
[µg/mL] [µg/mL]

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[min] [%] [W] g DW] DW]

1 27.5 (1) 70 (0) 400 (-1) 5.64 4.65 3.38 11.87

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2 12.5 (-1) 70 (0) 400 (-1) 5.23 4.46 3.95 11.63

3 12.5 (-1) 90 (1) 600 (0) 3.45 2.75 3.50 13.33

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4 20 (0) 50 (-1) 400 (-1) 7.19 4.31 4.01 11.76

5 12.5 (-1) 70 (0) 800 (1) 5.47 4.81 3.21 9.59

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6 20 (0) 70 (0) 600 (0) 4.65 4.00 3.44 10.58

7 27.5(1) 70 (0) 800 (1) 5.33 4.21 3.45 9.53

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8 20 (0) 70 (0) 600 (0) 5.56 4.58 3.52 10.18

9 20 (0) 70 (0) 600 (0) 5.30 4.46 3.53 9.97

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10 20 (0) 90 (1) 800 (1) 2.95 2.40 4.29 15.10

11 20 (0) 50 (-1) 800 (1) 6.86 4.30 3.73 11.27

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12 27.5 (1) 50 (-1) 600 (0) 7.44 4.32 3.46 10.48

13 20 (0) 70 (0) 600 (0) 5.30 4.43 3.83 10.96

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14 20 (0) 90 (1) 400 (-1) 3.36 2.84 4.59 13.03

15 12.5 (-1) 50 (-1) 600 (0) 6.85 3.79 3.57 10.51

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16 27.5 (1) 90 (1) 600 (0) 3.13 2.60 3.30 11.68

17 20 (0) 70 (0) 600 (0) 5.40 3.68 3.76 9.68

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Fig. 1. Extraction kinetic of (a) TP, TF and (b) total extractable compounds obtained by MAE

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with 70% ethanol, 1:10 (w/v) of solid-to-liquid ratio and 600 W of irradiation power
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Fig. 2. Response surface contour plots showing combined effects of MAE parameters

(extraction time, ethanol concentration and irradiation power) on: A-C) TP content, D-F) TF
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content, G-I) IC50 value and J-L) EC50 value

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