(Scco2) Extraction of Phenolic Compounds from Lavender (Lavandula Angustifolia) Flowers: a Box-Behnken Experimental Optimization

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Article Supercritical Carbon Dioxide (scCO2) Extraction of Phenolic Compounds from Lavender (Lavandula angustifolia) Flowers: A Box-Behnken Experimental Optimization

Katarzyna Ty´skiewicz* , Marcin Konkol and Edward Rój Supercritical Extraction Department, Sie´cBadawcza Łukasiewicz - Instytut Nowych Syntez Chemicznych, Al. Tysi ˛acleciaPa´nstwaPolskiego 13A, 24-110 Puławy, Poland; [email protected] (M.K.); [email protected] (E.R.) * Correspondence: [email protected]; Tel.: +48-814-731-452

Academic Editors: Robert Shellie and Francesco Cacciola Received: 17 August 2019; Accepted: 13 September 2019; Published: 15 September 2019 Abstract: Due to their numerous health benefits associated with various diseases and anti-oxidation properties, the phenolic compounds collectively referred to as phytochemicals have attracted a lot of interest, however, a single extraction method for polyphenols has not been developed yet. Supercritical fluid extraction, a green extraction method, provides the final product without organic solvent residues. In this work the extraction of lavender was performed using supercritical carbon dioxide. A statistical experimental design based on the Box-Behnken (B-B) method was planned, and the extraction yields and total phenolic contents were measured for three different variables: pressure, temperature and extraction time. The ranges were 200–300 bar, 40–60 ◦C and 15–45 min. The extracts yields from scCO2 extraction were in the range of 4.3–9.2 wt.%. The highest yield (9.2 wt.%) was achieved at a temperature of 60 ◦C under the pressure of 250 bar after 45 min. It also corresponded to the highest total phenolic content (10.17 mg GAE/g extract). Based on the study, the statistically generated optimal extraction conditions to obtain the highest total phenolic compounds concentration from flowers of Lavandula angustifolia were a temperature of 54.5 ◦C, pressure of 297.9 bar, and the time of 45 min. Based on the scavenging activity percentage (AA%) of scCO2 extracts, it is concluded that the increase of extraction pressure had a positive influence on the increase of AA% values.

Keywords: lavender; optimization; phenolic compounds; supercritical ﬂuid extraction; total phenolic content

1. Introduction Lavender (Lavandula angustifolia) is a perennial plant belonging to the family of Labiatae, widely used for essential oil production in Europe, Northern Africa, the Middle East and Asia. Due to its properties, lavender is commonly used by various industries, for instance, as a popular aromatic shrub for perfumes. The beneficial effects of lavender are attributed to the presence of a broad range of biologically active compounds (mainly phenolic and aromatic compounds) with antioxidant, antifungal and anti-carcinogenic activities. Besides this, lavender has been proven to exhibit antimicrobial, anti-inflammatory, antidepressive and other properties. Moreover, lavender has insecticidal activity attributed to the presence of aromatic and volatile compounds, including terpenes, linalool, pinenes, etc. [1,2]. According to Sytar et al. [3], the exact antioxidant properties of lavender are still unknown or only known to some extent. However, different studies are still being performed on lavender, especially in terms of its phenolic compounds content [4–6]. Figure1 shows the increasing tendency in the interest

Molecules 2019, 24, 3354; doi:10.3390/molecules24183354 www.mdpi.com/journal/molecules Molecules 2019, 24, x FOR PEER REVIEW 2 of 15

MoleculesAccording2019, 24, 3354 to Sytar et al. [3], the exact antioxidant properties of lavender are still unknown2 of or 15 only known to some extent. However, different studies are still being performed on lavender, especially in terms of its phenolic compounds content [4–6]. Figure 1 shows the increasing tendency inof Lavandulathe interest angustifolia of Lavandulabased angustifolia on bibliographic based search on bibliographic results with thesearch phrase results “Lavandula with angustifoliathe phrase” “andLavandula “Lavender” angustifolia in general.” and “Lavender” in general.

Figure 1.1.Evolution Evolution in in the the number number of papers of papers with the with keyword the keyword “Lavender” “Lavender” and “Lavandula and angustifolia“Lavandula” (Scienceangustifolia Direct,” (Science April Direct, 2019). April 2019).

Supercritical fluidfluid extraction (SFE) is a separation technique, the efficiency efficiency of which depends on several aspectsaspects such such as theas mobilethe mobile phase phase nature (purenature or (pure modified), or modified), the process the parameters process (temperature, parameters pressure(temperature, and time),pressure and and the typetime), of and raw the material, type of as raw well material, as its pretreatment as well as [ 7its]. Researcherspretreatment must [7]. Researcherschoose and optimizemust choose the extraction and optimize parameters the extraction properly parameters when it comes properly to the extractionwhen it comes of particular to the extractiongroups of compounds.of particular groups However, of compounds. in the case of However, lavender, in supercritical the case of lavender, fluid extraction supercritical is the mostfluid extractioncommon method is the most utilized common for extraction method utilized of the essential for extraction oil, which of the is essential a complex oil, matrix which consistingis a complex of matrixover one consisting hundred compoundsof over one [3 ,7hu–11ndred]. Jerkovićet compounds al. [ 11[3,7–11].] optimized Jerkovi supercriticalć et al. fluid[11] optimized extraction supercriticalof Lavandula angustifoliafluid extractionflowers of Lavandula in terms of angustifolia the content flowers of oxygenated in terms monoterpenes,of the content of coumarin oxygenated and monoterpenes,herniarin. The coumarin extraction and yield hernia was influencedrin. The extraction by the interaction yield was between influenced the by pressure the interaction and CO2 betweenflow rate, the as pressure well as between and CO2 the flow temperature rate, as well and as CObetween2 flow the rate. temperature The optimal and conditions CO2 flow basedrate. The on optimala Box-Behnken conditions design based were on w a temperatureBox-Behnken of design 41 ◦C, pressurewere w temperature of 100 bar and of a41 CO °C,2 flow pressure rate ofof 2.3100 kg bar/h. andSimilar a CO parameters2 flow rate (40 of◦ C,2.3 100 kg/h. bar) Similar were applied parameters by Nadalin (40 °C, et 100 al. [ 10bar)] for were the SFEapplied of lavender by Nadalin (Lavandula et al. [10]officinalis for the) flowers. SFE of Inlavender the study (Lavandula by Akgün officinalis et al. [8)], flowers. the SFE In was the conducted study by atAkgün relatively et al. low [8], pressurethe SFE (80–140was conducted bar) and at the relatively temperature low pressure used was (80–140 in the 35–50 bar) and◦C range. the temperature The application used ofwas supercritical in the 35–50 fluid °C range.extraction The to application phenolic compounds of supercritical has been fluid widely extraction developed to phenolic in a number compounds of studies has as been gathered widely in developeda recent review in a bynumber Ty´skiewiczet of studies al. [12as]. gathered Supercritical in a carbonrecent dioxidereview withby Ty a co-solventśkiewicz et was al. often[12]. Supercriticalused for the extractioncarbon dioxide of the phenolicwith a co-solvent compounds, was but often extraction used for with the pure extraction carbon dioxideof the phenolic was also compounds,employed [12 but]. In extraction some cases, with the pu SFEre providedcarbon dioxide higher was or similar also employed extraction [12]. efficiency In some values cases, compared the SFE providedto organic higher solvent or (methanol, similar extraction ethanol) extracts.efficiency Based values on compared the literature to reports,organic thesolvent methanolic (methanol, and ethanol)ethanolic extracts. extracts fromBasedBaccharis on the dracunculifolia literature reports,were characterizedthe methanolic by aand 2-fold ethanolic lower extraction extracts yieldfrom Baccharisthan the scCOdracunculifolia2 extract (60were◦C, characterized 400 bar) of this by plant. a 2-fold The lower analysis extraction of 3,5-diprenyl-4-hydroxycinnamic yield than the scCO2 extract (60acid °C, and 3-prenyl-4-hydroxycinnamic400 bar) of this plant. The acid analysis also indicated of 3,5-diprenyl-4-hydroxycinnamic a higher content in the scCO2 extractacid [and13]. 3-prenyl-4-hydroxycinnamicHowever, the literature data lacksacid also data indicated on the optimization a higher content of Lavandula in the scCO angustifolia2 extractextraction. [13]. However, the literatureThe aim ofdata the lacks current data study on the was optimization to investigate of the Lavandula extraction angustifolia of lavender extraction. flowers using supercritical CO2 (greenThe aim technology) of the current to obtain study polar was compounds to investigat and toe optimizethe extraction the main of extraction lavender parameters flowers using with supercriticalthe use of response CO2 (green surface technology) methodology to obtain (RSM) polar based compounds on the extraction and to yield optimize and total thephenolic main extraction content. parameters with the use of response surface methodology (RSM) based on the extraction yield and 2. Results and Discussion total phenolic content. 2.1. Supercritical Fluid Extraction of Lavender 2. Results and Discussion Supercritical fluid extraction has been applied to lavender extraction in a study by Av¸saret al. [14]. Lavandula officinalis flowers were extracted at 45 ◦C and 145 bar, resulting in an extraction yield of Molecules 2019, 24, 3354 3 of 15

4.68 wt.%. Similar yield values were observed in our study, although, different parameters were required. The yields of the Lavandula angustifolia flower extraction were 4.3 wt.% and 4.8 wt.%, respectively, for extraction at 50 ◦C/300 bar and 50 ◦C/200 bar. Costa et al. [15] performed SFE on Lavandula virdis extending the extraction with two separators. In comparison with the study by Av¸saret al. [14], lower extraction efficiency levels were observed (3.45 wt.% vs. 4.68 wt.%) for similar or not significantly different parameters (45 ◦C/145 bar vs. 40 ◦C/120 bar). The use of the second separator enabled achieving a higher yield of 9.27 wt.%. Increasing the pressure from 120 to 180 bar at the same extraction temperature (40 ◦C) did not influence the yield (3.45 wt.% vs. 3.41 wt.%). In the studies by Dahn et al. [16] volatile oils of Lavandula angustifolia were extracted in the range of 100–180 bar, 40–50 ◦C and 6.4–60 min. The highest extraction yield was obtained using the following parameters: 50 ◦C, 180 bar, and 60 min. The authors also succeeded in obtaining a yield of 9.0 wt.% at 53.4 ◦C and 140 bar. Our study resulted in obtaining the same yield (9.0 wt.%) at a higher temperature (60 ◦C) and over twice the pressure (300 bar). Lowering the pressure to 250 bar resulted in an even higher yield of up to 9.2 wt.%. Table1 presents the experimental results obtained in 15 runs of Lavandula angustifolia flower extraction.

Table 1. Responses of dependent variables using three levels-the factors Box-Behnken method to independent variables of the SFE method.

Total Phenolic Total Phenolic Temperature Antioxidant Activity Pressure (bar) Time (min) Observed Content (mg Content (mg ( C) (AA%) Exp ◦ Yield GAE/g Extract) GAE/g Dry Mass) (w/wt.%) C U C U C U Extracts Residues Extracts Residues 1 1 300 0 50 1 15 4.3 7.64 0.99 68.35 0.7 92.18 0.3 − ± ± 2 1 300 0 50 1 45 8.9 9.57 1.31 78.10 0.4 92.06 0.5 ± ± 3 0 250 0 50 0 30 7.3 8.84 1.28 61.27 1.1 92.09 0.4 ± ± 4 0 250 0 50 0 30 7.1 8.56 1.14 76.67 0.4 91.05 0.6 ± ± 5 0 250 1 60 1 15 6.5 8.25 1.20 52.39 0.5 89.88 0.9 − ± ± 6 1 200 1 40 0 30 7.1 8.75 1.32 50.55 0.7 93.44 1.1 − − ± ± 7 1 200 1 60 0 30 7.2 4.32 1.19 61.52 0.8 91.55 1.0 − ± ± 8 1 200 0 50 1 15 5.7 6.84 0.98 59.17 0.6 91.27 0.4 − − ± ± 9 0 250 1 40 1 15 6.2 9.81 1.19 78.83 1.3 91.39 0.6 − − ± ± 10 0 250 0 50 0 30 6.9 9.68 1.19 52.35 0.1 91.25 0.4 ± ± 11 0 250 1 40 1 45 6.9 6.91 1.08 63.89 0.5 90.97 1.2 − ± ± 12 1 200 0 50 1 45 4.8 5.9 1.08 60.65 0.3 91.36 0.4 − ± ± 13 1 300 1 60 0 30 9.0 9.95 1.23 65.86 0.7 91.18 0.8 ± ± 14 0 250 1 60 1 45 9.2 10.17 1.16 61.08 0.4 91.36 0.3 ± ± 15 1 300 1 40 0 30 7.4 5.82 1.30 57.00 0.5 91.35 0.4 − ± ± C—coded, U—uncoded.

2.2. Optimization of Operating Conditions—Extraction Yield A Box-Behnken statistical experimental design was utilized and the extraction yields were 2 measured for different variables such as pressure, temperature and time, coded as X1,X2,X3. The R adjusted of the extraction yield was 97.40% indicating that chosen models allowed one to fully predict the extraction yield, as presented in Figure2. The response surface model (RSM) obtained from the experimental design was evaluated using ANOVA and analysis of residuals. Multiple regression coefficients were determined by applying a least-squares technique in order to predict a second-order polynomial model. Both F-value and p-value were used to determine the significance of each coefficient. Corresponding p-values not higher than 0.1 suggest that, among the test variables used in this study X1 (pressure), X2 (temperature), X (time), X X (pressure temperature), X X (pressure time), X X (temperature time), (X )2 3 1 2 × 1 3 × 2 3 × 1 (pressure pressure), (X )2 (temperature temperature), (X )2 (time time) are all significant model × 2 × 3 × terms. The model F-value of 59.21 implies the model is significant. There is only a 0.02% chance that an F-value this large could occur due to noise. The results of variance and error for the response surface model analysis indicate that lack-of-fit is not significant relative to the pure error. There is a 49.87% chance that a lack-of-fit F-value this large could occur due to noise (Table2). Molecules 2019, 24, x FOR PEER REVIEW 4 of 15 Molecules 2019, 24, 3354 4 of 15

Predicted vs. Actual Residuals vs. Predicted

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X1: Actual X1: Predicted X2: Predicted X2: Residuals

Figure 2. Observed extraction yield versus predicted extraction yield and the analysis of residuals.

TableFigure 2. 2.Regression Observed coe extractionﬃcients ofyield predicated versus predicted second-order extr polynomialaction yield model and the for an thealysis response of residuals. variable.

TheSource response surface Sum ofmodel Squares (RSM) Degreeobtained of Freedomfrom the experimental Mean of Squares designF was-Value evaluatedp-Value using * ANOVAModel and analysis of residuals. 26.83 Multiple regressi 9on coefficients 2.98 were determined 59.21 by applying 0.0002 a least-squaresX1 (pressure) technique in 2.83order to predict a second-order 1 polynomial 2.83 model. 56.26 Both F-value 0.0007 and p-valueX2 (temperature) were used to determine 2.39 the significance 1 of each coefficient. 2.39 Corresponding 47.42 p-values 0.0010 not X3 (time) 6.00 1 6.00 119.26 0.0001 higher than 0.1 suggest that, among the test variables used in this study X1 (pressure), X2 X X 0.59 1 0.59 11.78 0.0186 1 × 2 (temperature),X X X3 (time), X17.29X2 (pressure × temperature), 1 X1X3 (pressure 7.29 × time), 144.82X2X3 (temperature<0.0001 × 1 × 3 time), (XX 1)2 X(pressure × pressure),1.01 (X2)2 (temperature 1 × temperature), 1.01 (X3)2 (time 20.06 × time) 0.0065 are all 2 × 3 X X 0.44 1 0.44 8.75 0.0316 significant1 × model1 terms. The model F-value of 59.21 implies the model is significant. There is only a X X 3.21 1 3.21 63.72 0.0005 0.02% chance2 × 2 that an F-value this large could occur due to noise. The results of variance and error for X X 2.56 1 2.56 50.89 0.0008 the response3 × 3surface model analysis indicate that lack-of-fit is not significant relative to the pure Residual 0.25 5 0.050 - - error. Lack-of-ﬁtThere is a 49.87% chance 0.16 that a lack-of-fit F 3-value this large 0.053could occur due 1.14 to noise 0.4987(Table 2). Pure error 0.093 2 0.046 - - Table 2. Regression coefficients* Signiﬁcant of predicated at p < 0.1. second-order “-” – not applicable. polynomial model for the response variable.

The plots of the quadraticSum of model withDegree three of variablesMean kept of at constant level and the other two Source F-value p-value * varying within the experimentalSquares ranges areFreedom shown in FigureSquares3. The ﬁnal equation in terms of coded variablesModel is as follows: 26.83 9 2.98 59.21 0.0002 X1 (pressure) 2.83 1 2.83 56.26 0.0007 2 2 2 YX2 =(temperature)7.09 + 0.59X + 0.55X2.39+ 0.87X + 0.39X1 X + 1.35X2.39X + 0.5 0.35X47.42+ 0.93X 0.83X0.0010 (1) 1 2 3 1 2 1 3 − 1 2 − 3 X3 (time) 6.00 1 6.00 119.26 0.0001 TheX1ﬁnal × X2 equation in terms0.59 of actual factors1 is as follows:0.59 11.78 0.0186 X1 × X3 7.29 1 7.29 144.82 <0.0001

X2 × X3 Y = 39.1395 1.010.0115X 1 1.1704X1 2 0.3376X31.01+ 0.0008X 1X2 +20.060.0018X 1X3 0.0065 X1 × X1 −0.44 − 1 − 2 0.442 2 8.75 0.0316 (2) + 0.0035X2X3 0.0001X1 + 0.0093X2 0.0037X3 X2 × X2 3.21 − 1 3.21 − 63.72 0.0005 X3 × X3 2.56 1 2.56 50.89 0.0008 Residual 0.25 5 0.050 - - Lack-of-fit 0.16 3 0.053 1.14 0.4987 Pure error 0.093 2 0.046 - - * Significant at p < 0.1. “-” – not applicable

Molecules 2019, 24, x FOR PEER REVIEW 5 of 15

The plots of the quadratic model with three variables kept at constant level and the other two varying within the experimental ranges are shown in Figure 3. The final equation in terms of coded variables is as follows:

Y = 7.09 0.59X 0.55X 0.87X 0.39XX 1.35XX 0.5−0.35X (1) 0.93X −0.83X

The final equation in terms of actual factors is as follows:

Y = 39.1395 − 0.0115X − 1.1704X − 0.3376X 0.0008XX 0.0018XX (2) Molecules 2019, 24, 3354 0.0035XX − 0.0001X 0.0093X − 0.0037X 5 of 15

FigureFigure 3.3. ContourContour plotsplots andand responseresponse surfacesurface ofof extractionextraction yieldyield asas aa functionfunction ofof supercriticalsupercritical COCO22 temperaturetemperature andand pressurepressure (first—15 (first—15 min; min; middle—30 middle—30 min; min; last—45 last—45 min). min). 2.3. Total Phenolic Content 2.3. Total Phenolic Content In their studies, Sytar et al. [3] highlighted the lack of sufficient knowledge about the antioxidant In their studies, Sytar et al. [3] highlighted the lack of sufficient knowledge about the activity of Lavandula angustifolia. Research on a methanolic extract from Lavandula officinalis leaves antioxidant activity of Lavandula angustifolia. Research on a methanolic extract from Lavandula indicated the presence of phenolic compounds at the level of 2.2 mg QE/DW (quercetin/dry weight). officinalis leaves indicated the presence of phenolic compounds at the level of 2.2 mg QE/DW In another study by Slavov et al. [17], SFE was applied to obtain polyphenol-rich extracts from (quercetin/dry weight). In another study by Slavov et al. [17], SFE was applied to obtain lavender waste, which gave 7.52 mg/DW as a total phenolic content. Adaszyńska-Skwirzyńskaet al. polyphenol-rich extracts from lavender waste, which gave 7.52 mg/DW as a total phenolic content. Reference [18] analyzed phenolic compounds in methanolic extracts of Lavandula angustifolia leaves and Adaszyńska-Skwirzyńska et al. Reference [18] analyzed phenolic compounds in methanolic extracts flowers. The contents were in the range of 3.71–4.06 mg/g DW and 1.13–1.14 mg/g DW, respectively. of Lavandula angustifolia leaves and flowers. The contents were in the range of 3.71–4.06 mg/g DW The flowers of Lavandula pubescens were characterized by a much lower content of polyphenols and 1.13–1.14 mg/g DW, respectively. The flowers of Lavandula pubescens were characterized by a (234.17 µg GAE/mg) in the ethanolic extract obtained by Mousa et al. [19]. Lavandula angustifolia was much lower content of polyphenols (234.17 μg GAE/mg) in the ethanolic extract obtained by Mousa also extracted with the use of subcritical fluid extraction, which resulted in a relatively low content of et al. [19]. Lavandula angustifolia was also extracted with the use of subcritical fluid extraction, which polyphenols (78.35 µg/mg total extract) in the flower extract [20]. Spiridon et al. [21] were the ones resulted in a relatively low content of polyphenols (78.35 μg/mg total extract) in the flower extract that obtained the Lavandula angustifolia extract with the highest level of phenolic compounds (50 mg [20]. Spiridon et al. [21] were the ones that obtained the Lavandula angustifolia extract with the highest GAE/g extract) among all performed studies described in the literature. However, methanol was used as a solvent for a whole plant extraction.

2.4. Optimization of Operating Conditions—Total Phenolic Content A statistical experimental design based on Box-Behnken method was utilized and extraction yields were obtained for diﬀerent variables such as pressure, temperature and time, coded as X1,X2, X3. Statistical analysis of results was performed with the use of Design Expert 9.0 (Stat-Ease, Inc., Minneapolis, MN, USA). The R2 adjusted of the total phenolic content was 90.65% indicating that chosen model enabled full prediction of TPC values (Figure4). Molecules 2019, 24, x FOR PEER REVIEW 6 of 15 level of phenolic compounds (50 mg GAE/g extract) among all performed studies described in the literature. However, methanol was used as a solvent for a whole plant extraction.

2.4. Optimization of Operating Conditions—Total Phenolic Content A statistical experimental design based on Box-Behnken method was utilized and extraction yields were obtained for different variables such as pressure, temperature and time, coded as X1, X2, X3. Statistical analysis of results was performed with the use of Design Expert 9.0 (Stat-Ease, Inc., 2 Minneapolis,Molecules 2019, 24 MN,, 3354 USA). The R adjusted of the total phenolic content was 90.65% indicating6 that of 15 chosen model enabled full prediction of TPC values (Figure 4).

TPC Predicted vs. Actual Residuals vs. Predicted

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Figure 4. Observed total phenolic content versus predicted total phenolic content and the analysis Figureof residuals. 4. Observed total phenolic content versus predicted total phenolic content and the analysis of residuals. Like for the extraction yield, ANOVA was used to evaluate the response surface model for the Like for the extraction yield, ANOVA was used to evaluate the response surface model for the total phenolic content. Among the tested variables, the significant ones were X1 (pressure), X3 (time), total phenolic content. Among the tested variables, the significant ones were X1 (pressure),2 X3 (time), X1X2 (pressure temperature), X1X3 (pressure time), X2X3 (temperature time), (X1) (pressure X1X2 (pressure ×× temperature),2 X1X3 (pressure × ×time), X2X3 (temperature × ×time), 2(X1)2 (pressure × pressure), (X3) (time time). Other variables, such as X2 (temperature), (X2) (temperature × 2 × 2 × pressure),temperature) (X3) are (time not significant.× time). Other The modelvariables, is significant, such as X as2 described(temperature), by the (XF2-value) (temperature (20.40) with × temperature)a 0.04% chance are that not an significant.F-value this The large model could is significant, occur due to as noise. described The resultsby the ofF-value a variance (20.40) and with error a 0.04%analysis chance for the that response an F-value surface this modellarge could indicate occur that due lack-of-fit to noise. is The not results significant of a relativevariance to and the error pure analysiserror. There for the is a response 63.29% chance surface that model a lack-of-fit indicateF that-value lack-of-fit this large is couldnot significant occur due relative to noise to (Table the pure3). error. There is a 63.29% chance that a lack-of-fit F-value this large could occur due to noise (Table 3). Table 3. Regression coefficients of predicated second-order polynomial model for the response variable. Table 3. Regression coefficients of predicated second-order polynomial model for the response variable.Source Sum of Squares Degree of Freedom Mean of Squares F-Value p-Value * Model 41.96 7 5.99 20.40 0.0004 Sum of Degree of Mean of X1Source(pressure) 6.43 1 6.43F-value 21.86p-value 0.0023 * Squares Freedom Squares X2 (temperature) 0.24 1 0.24 0.83 0.3916 XModelX 41.9618.32 7 1 5.99 18.3220.40 62.33 0.0004<0.0001 1 × 2 X1 (pressure)X X 6.432.06 1 16.43 2.0621.86 7.010.0023 0.0331 1 × 3 X2 (temperature)X X 0.245.81 1 10.24 5.810.83 19.760.3916 0.0030 2 × 3 X1 × XX2 18.329.02 1 118.32 9.0262.33 30.69<0.0001 0.0009 1 × 1 X1 × XX3 2.060.25 1 12.06 0.257.01 8.60.0331 0.0384 2 × 2 ResidualX2 × X3 5.81 2.06 1 75.81 0.2919.76 -0.0030 - Lack-of-fitX1 × X1 9.02 1.38 1 59.02 0.2830.69 0.810.0009 0.6329 Pure error 0.68 2 0.34 - - X2 × X2 0.25 1 0.25 8.6 0.0384 Residual 2.06 * Significant at7p < 0.1. “-” – not0.29 applicable. - - Lack-of-fit 1.38 5 0.28 0.81 0.6329

FigurePure error5 shows the e ﬀect0.68 of diﬀerent operating2 temperature0.34 and pressure- of supercritical - CO2 on total phenolic content in the extraction time in the range of 15–45 min.

Final equation in terms of coded variables is as follows:

TPC = 9.03 + 0.90X + 0.18X + 2.14X X + 0.72X X + 1.21X X 1.56X2 0.26X2 (3) 1 2 1 2 1 3 2 3 − 1 − 2 Final equation in terms of actual factors is as follows:

TPC = 31.04792 + 0.086642X 1.03392X + 0.004X X + 0.0009X X 1 − 2 1 2 1 3 (4) + 0.008X X 0.0006X2 + 0.003X2 2 3 − 1 2 Molecules 2019, 24, x FOR PEER REVIEW 7 of 15

* Significant at p < 0.1. “-” – not applicable.

Figure 5 shows the effect of different operating temperature and pressure of supercritical CO2 on total phenolic content in the extraction time in the range of 15–45 min. Final equation in terms of coded variables is as follows:

TPC = 9.03 0.90X 0.18X 2.14XX 0.72XX 1.21XX − 1.56X −0.26X (3) Final equation in terms of actual factors is as follows:

TPC = 31.04792 0.086642X − 1.03392X 0.004XX 0.0009XX 0.008X X − 0.0006X 0.003X (4) Molecules 2019, 24, 3354 7 of 15

Figure 5. Contour plots and response surface of total phenolic content as a function of supercritical Figure 5. Contour plots and response surface of total phenolic content as a function of supercritical CO2 temperature and pressure (first—15 min; middle—30 min; last—45 min). CO2 temperature and pressure (first—15 min; middle—30 min; last—45 min). 2.5. Variables Affecting Procedure 2.5. VariablesThe increase Affecting of theProcedure pressure to 300 bar in the 15-min-extraction resulted in a decrease of the extractionThe increase yield. On of thethe otherpressure hand, to the 300 extraction bar in the yield 15-min-extraction was not influenced resulted by pressure in a decrease (200–300 of bar)the extractionin longer extractionsyield. On the (30 other min) athand, the temperaturethe extraction in yield the 40–54 was not◦C range.influenced Significant by pressure changes (200–300 in the bar)yield in were longer observed extractions above (30 approx. min) at 54the◦ C.temperat With theure extraction in the 40–54 time °C of range. 45 min, Significant the extraction changes yield in theincreased yield were with observed the increase above of bothapprox. pressure 54 °C. and With temperature. the extraction The time lowest of 45 extraction min, the extraction yield (4.3 wt.%)yield increasedwas observed with when the increaseLavandula of angustifoliaboth pressureflowers and weretemperature. extracted The at the lowest temperature extraction of 50yield◦C, (4.3 under wt%) the waspressure observed of 300 when bar within Lavandula 15 min. angustifolia The highest flowers extraction were extracted yield (9.2 at wt.%) the temperature required the of increase 50 °C, under of the thetemperature pressure toof 60300◦ Cbar as within well as 15 the min. decrease The highest of the pressure extraction to 250yield bar (9.2 and wt%) three required times longer the increase extraction of thetime temperature (45 min). One to 60 trend °C as is well observed as the to decrease the yield of taking the pressure into consideration to 250 bar and parameters, three times especially longer extractiontemperature time with (45 corresponding min). One trend pressure is observed at the to highest the yield level taking in the into experiments consideration with parameters, a particular especiallyextraction temperature time. In other with words, corresp theonding highest pressure yield (6.5 at the wt.%) highest for the level conditions in the experiments with the shortest with a extraction time (15 min) was obtained with temperature of 60 ◦C and the pressure of 250 bar. The same parameters regarding temperature and pressure resulted in the highest extraction yield (9.2 wt.%) during 45 min. In the case of 30-min-extractions, the maximum parameters (60 ◦C, 300 bar) were the most appropriate to obtain the highest yield (9.0 wt.%) in these experiments. Another trend was noticed as the extraction time was relevant in determining the yield but lost importance regarding total phenolic content. In the case of 15-min-extraction, the significant influence on the TPC was observed in the temperature range 40–45 ◦C and pressure 200–260 bar. In a comparison with the shortest extraction time, 30-min-extraction resulted in the highest TPC above approx. 54 ◦C and 250 bar. The relatively high TPC values were obtained also at the lowest studied temperature (40 ◦C) with the pressure between 220–240 bar. With the increase of the extraction time to 45 min, the decrease of TPC was noticed in the region below approx. 46 ◦C along studied pressure values Molecules 2019, 24, x FOR PEER REVIEW 8 of 15

particular extraction time. In other words, the highest yield (6.5 wt%) for the conditions with the shortest extraction time (15 min) was obtained with temperature of 60 °C and the pressure of 250 bar. The same parameters regarding temperature and pressure resulted in the highest extraction yield (9.2 wt%) during 45 min. In the case of 30-min-extractions, the maximum parameters (60 °C, 300 bar) were the most appropriate to obtain the highest yield (9.0 wt%) in these experiments. Another trend was noticed as the extraction time was relevant in determining the yield but lost importance regarding total phenolic content. In the case of 15-min-extraction, the significant influence on the TPC was observed in the temperature range 40–45 °C and pressure 200–260 bar. In a comparison with the shortest extraction time, 30-min-extraction resulted in the highest TPC above approx. 54 °C and 250 bar. The relatively high TPC values were obtained also at the lowest studied Molecules 2019, 24, 3354 8 of 15 temperature (40 °C) with the pressure between 220–240 bar. With the increase of the extraction time to 45 min, the decrease of TPC was noticed in the region below approx. 46 °C along studied pressure

(200–300values (200–300 bar). The bar). same The temperaturesame temperature (60 ◦C) (60 provided °C) provided the lowest the lowest (4.32 (4.32 mg GAEmg GAE/g/g extract) extract) and and the highestthe highest (10.17 (10.17 mg mg GAE GAE/g/g extract) extract) TPC TPC values values with with the the di differencefference in in pressure pressure and and time. time. AsAs forfor thethe lowestlowest TPC,TPC, thethe pressurepressure waswas 200200 barbar andand 3030 min,min, whereaswhereas thethe highesthighest totaltotal phenolicphenolic contentcontent waswas obtained withwith higherhigher pressurepressure (250(250 bar)bar) andand longerlonger extractionextraction timetime (45(45 min).min). The scavengingscavenging activity activity percentage percentage (AA%) (AA%) for scCO for2 extractsscCO2 fromextracts lavender from (Lavandulalavender angustifolia(Lavandula) flowersangustifolia were) flowers measured were at measured the level at of the 50.55–78.83%, level of 50.55–78.83%, whereas AA% whereas for residues AA% for was residues in the was range in ofthe 89.88–93.44%. range of 89.88–93.44%. Among extracts, Among theextracts, highest the antioxidant highest antioxidant activity (78.83%)activity (78.83%) was observed was observed for the extractfor the extract obtained obtained at 40 ◦ Cat and40 °C 250 and bar 250 (15 bar min) (15 andmin) the and lowest the lowest antioxidant antioxidant activity activity (50.55%) (50.55%) for thefor extractthe extract obtained obtained at 40 at ◦40C °C and and lower lower extraction extraction pressure pressure (200 (200 bar; bar; 30 30 min) min) (Table (Table1). 1). In In the the case case of of post-extractionpost-extraction residues, the the antioxidant antioxidant activity activity ranged ranged 89.88–93.44% 89.88–93.44% for for the the extraction extraction at 60 at °C, 60 250◦C, 250bar, bar,15 min 15 and min 40 and °C, 40 200◦C, bar, 200 30 bar, min. 30 According min. According to Dahn toet Dahnal. [16], et the al. supe [16],rcritical the supercritical fluid extraction fluid extractionof lavender of lavender(L. angustifolia (L. angustifolia) flowers) flowersin the inpressure the pressure range range 100–180 100–180 bar bar in interms terms of of essential essential oils resultedresulted inin anan antioxidantantioxidant activityactivity atat thethe levellevel ofof 32.3–73.8%.32.3–73.8%. The lowest pressure (100 bar)bar) providedprovided thethe lowestlowest AA%AA% (32.3%),(32.3%), whereaswhereas thethe highest pressure (180 bar) was requiredrequired toto obtainobtain thethe highesthighest AA%AA% (73.8%).(73.8%). The antioxidant activity (% inhibition of DPPH free radicals) of lavenderlavender extractsextracts atat variousvarious conditionsconditions ofof SFESFE isis presentedpresented inin TableTable1 .1. The antioxidantantioxidant activity of scCO2 extracts is shown in Figures 66 andand7 7as as a a function function of of pressure pressure and temperaturetemperature withwith anan extractionextraction timetime ofof 3030 min.min. In the case of extracts, pressure had a significantsignificant impactimpact onon thethe antioxidantantioxidant activity.activity. WithWith thethe increaseincrease ofof pressure,pressure, AA%AA% valuesvalues alsoalso increased.increased. OnOn thethe other hand,hand, temperaturetemperature hadhad aa negativenegative impactimpact onon AA%AA% values.values.

Predicted vs. Actual 80

50 55 60 65 70 75 80

X1: Actual X2: Predicted Figure 6. Observed antioxidant activity versus predictedpredicted antioxidant activity inin extracts.extracts. The ﬁnal equation in terms of coded variables is as follows:

AA% = 63.93 + 6.10X 2.60X + 0.62X (5) 1 − 2 3 The ﬁnal equation in terms of actual factors is as follows:

AA% = 45.19110 + 0.12191X 0.25966X + 0.041617X (6) 1 − 2 3 The antioxidant activity of post-extraction residues is shown in Figures8 and9 as a function of pressure and temperature with an extraction time of 30 min. In the case of residues, the observed activity values hardly depended on the pressure and temperature parameters, although, antioxidant values showed a slight downward trend as the extraction parameters increase. Time had a slight eﬀect on the increase of AA% values. Molecules 2019, 24, x FOR PEER REVIEW 9 of 15

Molecules 2019, 24, 3354 9 of 15 Molecules 2019, 24, x FOR PEER REVIEW 9 of 15

Figure 7. Response surface of antioxidant activity as a function of supercritical CO2 temperature and pressure (30 min) for extracts.

The final equation in terms of coded variables is as follows:

AA% = 63.93 6.10X −2.60X 0.62X (5) The final equation in terms of actual factors is as follows:

AA% = 45.19110 0.12191X − 0.25966X 0.041617X (6) The antioxidant activity of post-extraction residues is shown in Figures 8 and 9 as a function of pressure and temperature with an extraction time of 30 min. In the case of residues, the observed activity values hardly depended on the pressure and temperature parameters, although, antioxidant values showed a slight downward trend as the extraction parameters increase. Time had a slight Figure 7. Response surface of antioxidant activity as a function of supercritical CO2 temperature and Figure 7. Response surface of antioxidant activity as a function of supercritical CO2 temperature and effectpressure on the (30 increase min) for of extracts. AA% values. pressure (30 min) for extracts. Predicted vs. Actual The final equation in terms94 of coded variables is as follows:

AA% = 63.93 6.10X −2.60X 0.62X (5) 93 The final equation in terms of actual factors is as follows:

92 AA% = 45.19110 0.12191X − 0.25966X 0.041617X (6)

The antioxidant activity of 91post-extraction residues is shown in Figures 8 and 9 as a function of pressure and temperature with an extraction time of 30 min. In the case of residues, the observed activity values hardly depended90 on the pressure and temperature parameters, although, antioxidant values showed a slight downward trend as the extraction parameters increase. Time had a slight effect on the increase of AA% values.89

89 90Predicted 91 vs. Actual 92 93 94 94 X1: Actual X2: Predicted Molecules 2019, 24, x FOR PEER REVIEW 10 of 15 93 FigureFigure 8. 8.Observed Observed antioxidantantioxidant activityactivity versus versus predicted predicted antioxidant antioxidant activity activity in in residues. residues.

89 90 91 92 93 94

X1: Actual X2: Predicted Figure 8. Observed antioxidant activity versus predicted antioxidant activity in residues.

Figure 9. Response surface of antioxidant activity as a function of supercritical CO2 temperature and pressureFigure 9. (30 Response min) for surface residues. of antioxidant activity as a function of supercritical CO2 temperature and pressure (30 min) for residues.

The final equation in terms of coded variables is as follows:

AA% = 91.49 − 0.11X −0.40X 0.13X (7) The final equation in terms of actual factors is as follows:

AA% = 93.75031 − 0.0021335X − 0.039659X 0.00857083X (8)

3. The Cost of Manufacturing (COM) For the essential oils, various extraction methods are used, as described by Rassem et al. [22], including hydrodistillation, which is a process in which both water and plant material are boiled together in an extractor. The results are distillates, also known as floral waters, hydrosols, hydrolates, herbal waters, and essential waters [23]. Steam distillation is used in large-scale production of essential oils for commercial purposes. Steam distillation uses dry steam to vaporize and extract the oil. Solvent extraction uses organic solvents to extract both essential oils and oleoresins, which are then separated. Use of many of the organic solvents would not be compatible with certified organic production. The supercritical fluid extraction method uses carbon dioxide under high pressure to extract both essential oils and oleoresins. The mixture can then be fractionated by molecular distillation or steam distillation. Supercritical extraction products belong to the “green chemistry” group [22].

The Cost of Manufacturing Using Supercritical Fluid Extraction Supercritical extraction is widely thought to be expensive. It is true that supercritical fluid extraction is expensive at the equipment investment stage [24]. Operating costs are however not high and are comparable to other extraction technologies. It also often happens that the cost of supercritical extraction is lower than the cost of liquid extraction. The analysis of COM dependence on the pressure (100–300 bar) and the temperature of the extraction (40–50 °C), and capacity of the extractors (0.1; 0.4 and 1.0 m3) is presented using Turton et al.’s formula [25]:

COM = 0.304FCI 2.73COL 1.23(CRM CWT CUT) (9) where FCI—fixed cost of investment, COL—cost of operational labor, CRM—cost of raw material, CWT—cost of waste treatment, CUT—cost of waste utilization (extraction residues) Comparing the calculated COM with the real COM or market price of the same or similar product with literature data allows one to assess the analyzed project, whether it is and to what

Molecules 2019, 24, 3354 10 of 15

The ﬁnal equation in terms of coded variables is as follows:

AA% = 91.49 0.11X 0.40X + 0.13X (7) − 1 − 2 3 The ﬁnal equation in terms of actual factors is as follows:

AA% = 93.75031 0.0021335X 0.039659X + 0.00857083X (8) − 1 − 2 3 3. The Cost of Manufacturing (COM) For the essential oils, various extraction methods are used, as described by Rassem et al. [22], including hydrodistillation, which is a process in which both water and plant material are boiled together in an extractor. The results are distillates, also known as floral waters, hydrosols, hydrolates, herbal waters, and essential waters [23]. Steam distillation is used in large-scale production of essential oils for commercial purposes. Steam distillation uses dry steam to vaporize and extract the oil. Solvent extraction uses organic solvents to extract both essential oils and oleoresins, which are then separated. Use of many of the organic solvents would not be compatible with certified organic production. The supercritical fluid extraction method uses carbon dioxide under high pressure to extract both essential oils and oleoresins. The mixture can then be fractionated by molecular distillation or steam distillation. Supercritical extraction products belong to the “green chemistry” group [22].

The Cost of Manufacturing Using Supercritical Fluid Extraction Supercritical extraction is widely thought to be expensive. It is true that supercritical ﬂuid extraction is expensive at the equipment investment stage [24]. Operating costs are however not high and are comparable to other extraction technologies. It also often happens that the cost of supercritical extraction is lower than the cost of liquid extraction. The analysis of COM dependence on the pressure (100–300 bar) and the temperature of the extraction (40–50 ◦C), and capacity of the extractors (0.1; 0.4 and 1.0 m3) is presented using Turton et al.’s formula [25]:

COM = 0.304FCI + 2.73COL + 1.23(CRM + CWT + CUT) (9) where FCI—fixed cost of investment, COL—cost of operational labor, CRM—cost of raw material, CWT—cost of waste treatment, CUT—cost of waste utilization (extraction residues) Comparing the calculated COM with the real COM or market price of the same or similar product with literature data allows one to assess the analyzed project, whether it is and to what extent competitive and economically justified. This comparison also allows one to estimate possible profits. The structure of COM shows what influence on this cost particular unit costs have, which of them are crucial and which are worth looking at for savings, as well as those which have small secondary importance. The examples of COM in US$/kg of the extract are listed in Table4.

Table 4. The examples of COM in US$/kg of the extract [26–28].

Extract COM (US$/kg Extract) Clove (Dianthus) 9.15 Cloves 4.70 Lavender 66.50 Retail value of lavender oil 2400 $/dm3 Ginger 99.80 Buriti palm 22.81 Pupunha palm 17.15 Pomegranate leaves 114.36 Habanero pepper 540.19 Molecules 2019, 24, 3354 11 of 15

A Rowan University group in the US compared peanut extraction with supercritical and hexane methods. The results are shown in Table5. It is evident that the cost of extraction using CO 2 in a supercritical state is two times lower than the cost of extraction with hexane.

Table 5. Comparison of the extraction cost of peanut with carbon dioxide and hexane [29].

Factor Carbon Dioxide Extraction Hexane Extraction Solvent 0.07 $/lb 0.07 $/lb Max solubility 38 mg/g 80 mg/g CO2 ﬂow 87 million lb/yr 38 million lb/yr Energy input 1.8 GWh/y 4.6 GWh/yr COM 6.2 million $/yr 14 million $/yr

4. Materials and Methods The flowers of Bulgarian lavender (Lavandula angustifolia) were obtained from the INCO Company (Warszawa, Poland). As for the extraction process, dried flowers were milled and subjected to SFE. The size of the lavender flowers after milling (1.5 mm sieve) based on the granulometric analysis is provided in Table6. An authentic analytical standard of gallic acid as well as Folin-Ciocalteau reagent, were purchased from Sigma-Aldrich (Darmstadt, Germany). HPLC grade ethanol (Baker), as well as anhydrous sodium carbonate (Chemsolve) were purchased from Witko (Łód´z,Poland). Carbon dioxide (99.9%, v/v), which was used as the mobile phase in SFE, was stored in a CO2 installation tank.

Table 6. The size of milled lavender ﬂowers for the SFE.

Fraction Size (mm) Fraction Mass (g) Fraction Percentage (%) 2.50 0.01 0.01 2.00 0.08 0.10 1.60 0.11 0.14 1.00 6.22 7.93 0.80 7.32 9.33 0.60 17.26 21.99 0.40 16.68 21.25 0.30 11.05 14.08 0.10 17.82 22.71 <0.1 1.93 2.46 SUM 78.48 100

4.1. Supercritical Fluid Extraction The dynamic SFE process was performed on a laboratory scale installation, equipped with a 1 L extractor (SITEC-Sieber Engineering AG, Switzerland) operating at temperatures up to 80 ◦C and pressures up to 450 bar (Figure 10). Dried and milled Lavandula angustifolia flowers (100 g) were extracted with pure carbon dioxide. The temperature and pressure were set in the range of 40–60 ◦C and 200–300 bar, and the extraction time in the range of 15–45 min. The CO2 flow (10 kg/h) was constant for all experiments. The complete design included fifteen experiments at different conditions with the use of three center points. In order to evaluate the influence of these factors, the extraction yield (Y, % dry mass) and total phenolic content (TPC, mg GAE/g extract) were determined and used to optimize the extraction conditions. The statistical analysis of the results was performed with the Design Expert 9.0 software. Table7 lists the ranges chosen for the three independent variables at three levels. Molecules 2019, 24, x FOR PEER REVIEW 12 of 15

4.1. Supercritical Fluid Extraction The dynamic SFE process was performed on a laboratory scale installation, equipped with a 1 L extractor (SITEC-Sieber Engineering AG, Switzerland) operating at temperatures up to 80 °C and pressures up to 450 bar (Figure 10). Dried and milled Lavandula angustifolia flowers (100 g) were extracted with pure carbon dioxide. The temperature and pressure were set in the range of 40–60 °C and 200–300 bar, and the extraction time in the range of 15–45 min. The CO2 flow (10 kg/h) was constant for all experiments. The complete design included fifteen experiments at different conditions with the use of three center points. In order to evaluate the influence of these factors, the extraction yield (Y, % dry mass) and total phenolic content (TPC, mg GAE/g extract) were determined and used to optimize the extraction conditions. The statistical analysis of the results was performed with the Design Expert 9.0 software. Table 7 lists the ranges chosen for the three Molecules 2019, 24, 3354 12 of 15 independent variables at three levels.

Figure 10. Technological scheme of the supercritical extraction unit in a lab scale. Figure 10. Technological scheme of the supercritical extraction unit in a lab scale. Table 7. Ranges of three independent variables in the Box-Behnken method. Table 7. Ranges of three independent variables in the Box-Behnken method. Variables Unit 1 0 1 − Temperature C 40 50 60 Variables Unit ◦ −1 0 1 Pressure bar 200 250 300 Temperature Time°C min 1540 30 4550 60 Pressure bar 200 250 300 4.2. Total Phenolic ContentTime min 15 30 45 The uniqueness of the phenolic compounds is attributed to their over twelve studied properties, 4.2. Total Phenolic Content antioxidant, medical (antidiabetic, gastroprotective, cardioprotective), antibacterial, antifungal and otherThe [30]. uniqueness The nutraceutical of the phenolic value of lavender compounds flowers is attrib probablyuted to results their from over thetwelve presence studied of biologically properties, activeantioxidant, substances medical from (antidiabetic, the group of gastroprotective coumarins, phytosterols, cardioprotective), with the antibacterial, main focus on antifungal essential and oil componentsother [30]. The and phenolicnutraceutical compounds value of (tannins, lavender flavonoids, flowers probably anthocyanins) results [31 from]. The the literature presence data of reportsbiologically the activity active ofsubstances these compounds from the ingroup neoplasm of coumarins, treatment phytos [32]. Theterols total with phenolic the main content focus was on determinedessential oil spectrophotometrically components and phenolic using Folin-Ciocalteaucompounds (tannins, reagent flavonoids, according toanthocyanins) the method described[31]. The inliterature the literature data [reports33,34]. Thethe activity extracts wereof these dissolved compounds in ethanol in neoplasm (50 µL; approximately treatment [32]. 10 The mg/ mL),total filteredphenolic through content a 0.45was µdeterminedm filter and spectrophotometric mixed with distilledally water using (1.58 Folin-Ciocalteau mL) and Folin-Ciocalteu reagent according reagent (100to theµL). method After 30described min, an aqueousin the literature solution [33,34]. of sodium The carbonateextracts were (300 µdissolvedL) was added. in ethanol After (50 1 h μ atL; 25approximately◦C in the dark 10 absorbance mg/mL), filtered was measured through at a 7650.45 nm μm using filter aand UV-Vis mixed spectrophotometer with distilled water (V-650, (1.58 Jasco, mL) Pfungstadt,and Folin-Ciocalteu Germany). reagent Gallic acid(100 wasμL). used After to 30 build min, a calibrationan aqueous curve solu (0.1-0.5tion of mgsodium/mL; Rcarbonate2 = 0.999) and(300 theμL) results was added. were expressed After 1 h in at milligrams 25 °C in the of gallicdark acidabsorbance equivalent was (mg measured GAE/g extract).at 765 nm The using stock a solution UV-Vis of gallic acid (10 mg) was prepared in ethanol (2 mL). The post-extraction residues (approx. 100 mg) were extracted with 2 mL of ethanol and subjected to ultrasounds for 5 min and left for decantation. Similarly to the extracts, the residues were prepared for the reaction with Folin-Ciocalteau reagent.

4.3. Radical Scavenging Activity Using DPPH Method The scavenging activity percentage (AA%) of extracts and residues was assessed by DPPH free radical assay according to the methodology described by Brand-Williams et al. [35] and Garcia et al. [36]. Each extract (approx. 50 mg) was weighed and dissolved in ethanol (1 mL). The DPPH was prepared by dissolving 19.71 mg of DPPH in 100 mL of ethanol to obtain a concentration of 0.5 mM. A volume of 0.5 mL of extract solution was transferred to a separate flask and 3 mL of ethanol and 0.3 mL of DPPH were also added. The post-extraction residues (approx. 100 mg) were extracted with 2 mL of Molecules 2019, 24, 3354 13 of 15 ethanol and subjected to ultrasound for 5 min and left for decantation. The extracts from residues (0.5 mL) were filtered through a 0.45 µm filter and mixed with DPPH solution (0.3 mL) and ethanol (3 mL). The reaction mixtures were left for 30 min in the dark and the absorbance was measured at 517 nm using a UV-Vis spectrophotometer. The analyses were performed at triplicate. The percentage of inhibition was calculated according to Equation (10):

AA% = 100(A Aav)/A (10) 0 − 0 where: A0—the absorbance of DPPH solution, Aav—the average absorbance of samples.

5. Conclusions

Optimization of Lavandula angustifolia flowers scCO2 extraction shows that all three variables affect the yield of the extraction individually, as well as in combinations with each other. The effect of temperature and pressure on the extraction yield is highly pronounced. The predictive model fits well with the experimental results. The temperature, 54.5 ◦C; pressure, 297.9 bar; extraction time, 45 min were found to be the optimum conditions to achieve the maximum yield and maximum polyphenols content in scCO2 extract from Lavandula angustifolia flowers.

Author Contributions: Conceptualization, K.T., M.K., E.R.; Formal analysis, M.K. and E.R.; Investigation, K.T. and M.K.; Methodology, M.K. and E.R.; Project administration, Supervision, E.R.; Validation, K.T., M.K., E.R.; Visualization, K.T., M.K., E.R.; Writing—original draft, K.T.; Writing—review & editing—M.K. Funding: This research received no external funding. Conﬂicts of Interest: The authors declare no conﬂict of interest

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Sample Availability: Samples of the compounds are not available from the authors.

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