1 Abstract

2 The extraction of essential oil from rosemary leaves is of interest for industrial applications as it

3 is considered as a potential therapeutic agent to cure many diseases. Thus, the present study aims

4 to optimize the extraction conditions. The effects of processing parameters of a steam distillation

5 process on the extraction yield of rosemary essential oil are investigated using response surface

6 methodology. A central composite design is applied to evaluate three independent variables such

7 as extraction temperature (103-118 ºC), time (2-6 h) and feed mass (200-600 g) on the extraction

8 of rosemary essential oil. The maximum yield under the optimum condition is 3.10%. Further,

9 deviations between the experimental and predicted values are less, and they are statistically

10 insignificant. Furthermore, the predicted data from the mathematical model that involves

11 convection-diffusion processes show a good agreement with the experimental data of the

12 extraction of rosemary essential oil by steam distillation. The major chemical constituents are

13 determined using Gas Chromatography-Mass Spectroscopy. More importantly, rosemary

14 essential oil shows antibacterial activity against two microbes. Therefore, the present study

15 suggests that the optimum conditions for the extraction of rosemary essential oil and provide a

16 good alternative to synthetic antioxidant.

17 Key Words: Response surface methodology; Rosemary essential oil; Antibacterial; Antioxidant;

18 Optimization; Mathematical modeling

19

1
20 1. Introduction

21 Essential oils are mixtures of volatile and non-volatile compounds that are mostly derived from

22 different parts of an aromatic plant [1]. They can be extracted from different plants by several

23 techniques, such as steam distillation, supercritical fluid extraction, microwave extraction, hydro-

24 extraction, etc [2]. In recent years, there is a necessity to improve the quality of the essential oils

25 due to its use in food, pharma and perfumery products [3]. Particularly, in leaves and flowers, the

26 essential oil is secreted from glandular trichomes, and the highest yield is achieved from the

27 leaves [4]. Rosemary (Rosmarinus officinalis L.) is a well-known aromatic herb to the native of

28 the Mediterranean region and several parts of the world [5]. In the international market, among

29 various essential oils, the essential oil derived from Rosemary (Rosmarinus officinalis L.)

30 contributes mainly as an additive in the production of food and perfume products. It possesses

31 antibacterial, cytotoxic, antimutagenic, antioxidant, antiphlogistic, and chemopreventive

32 properties [6]. These properties have made rosemary as a potential therapeutic agent to cure

33 many diseases. The major chemical constituents present in rosemary are α-pinene, 1, 8-cineole,

34 and camphor [2].

35 In the literature, the antibacterial activity of different essential oils is tested

36 against pseudomonas fluorescens, and it is concluded that the essential oils derived from

37 cinnamon, pimento, clove, and rosemary are the most active against the microbes. The presence

38 of phenolic compounds in rosemary leaves such as carnosol, carnosic acid, rosmanol,

39 epirosmanol and isorosmanol rosmarinic and caffeic acids are responsible for antioxidant

40 properties. As a result, the extract from rosemary is accepted in the European Union as the

41 natural antioxidant [3,5]. In the literature, the extraction of rosemary essential oil is carried out

42 using two different techniques (hydrodistillation and microwave-assisted hydrodistillation), and

2
43 the maximum yield is found to be 1.35 %(w/w) [4]. In another report, the yield of essential oil

44 extracted from Algerian rosemary leaves is investigated with different parameters such as

45 temperature and pressure, and it is estimated in the range of 0.95- 3.52 % (w/w) [7]. In fact, the

46 extraction of rosemary essential oil using steam distillation is performed at different time

47 intervals for a fixed feed mass (225 g) and temperature (93º C), and the maximum yield is 0.51%

48 (w/w) [8]. Response Surface Methodology (RSM), a statistical technique, is used to determine

49 the effects of several variables and optimum process conditions [9]. Further, RSM reduces the

50 number of experimental runs needed to evaluate multiple parameters and their interactions.

51 In the present work, the effects of extraction time, temperature, and feed mass on the

52 extraction yield of rosemary essential oils using steam distillation are analyzed by applying the

53 RSM. Further, the mathematical model developed from the differential mass balance for the

54 solid and fluid phases is correlated with the experimental data of the extraction of essential oil

55 from rosemary leaves. The composition of extracted oil under an optimum process condition was

56 analyzed by GC-MS. Further, antioxidant and antibacterial properties of the extract are also

57 investigated in this work.

58 2. Materials and Methods

59 2.1 Materials and chemicals

60 Fresh rosemary leaves were collected from wondo genet agricultural research center, Ethiopia

61 and removed any debris present in the sample for the process of extraction. The samples were

62 dried at room temperature for 10 days to remove moisture and stored in a moisture-free

63 environment for further processing. Ethanol, methanol, mueller-hinton broth (MHB), mueller-

3
64 hinton Agar (MHA), ciprofloxacin and 1,1-diphenyl-2- picrylhydrazyl (DPPH) were used for the

65 measurement of antioxidant and antibacterial activity.

66 2.2 Extraction of essential oil

67

68

69

70

71

72

73

74

75 Fig. 1 Experimental set-up for extraction of rosemary essential oil by steam distillation

76 The experimental set-up for the extraction of essential oils from Rosmarinus officinalis L. using

77 steam distillation is shown in fig. 1. It was composed of four elements such as boiler, extraction

78 chamber, condenser, and separator. The extraction chamber was made of a vessel in which the

79 steam interacts with leaves and vaporize the oils. First, leaves were fed into the extraction

80 chamber before starting the process. To avoid the channeling effect, the maximum packing was

81 ensured in the chamber. On the other hand, there could be a possibility of low yield if the effect

82 persists. A coil flow condenser was used to convert the steam and oil vapors into liquid. To

4
83 separate water from oil, the water layer was drained out by opening the tap till the meniscus was

84 just above the calibration mark.

85 The yield of essential oils derived from rosemary leaves in each experiment was calculated as

86 follows:

weight of essentialoil∗100
87 Yield ( % )=
weight of rosemary leaves

88 2.3 Physicochemical properties, Chemical analysis, Antioxidant and Antibacterial

89 activity

90 The physicochemical properties of essential oil extracted from rosemary leaves were measured

91 according to the procedure given in a report in the literature [10]. The chemical composition of

92 the extracted essential oil from rosemary leaves was determined using Gas Chromatography-

93 Mass Spectroscopy with HP 5890 series equipped with a mass selective detector. The

94 components were identified using helium gas with a volume of 1μl at a constant flow of 1ml/min

95 as the carrier gas, and injector temperature and ion-source temperature are 250º C and 280º C,

96 respectively. The compounds were detected by processing the raw data and comparing it with the

97 mass spectral database. The amount was calculated based on the peak area [11].

98 The antioxidant activity of the rosemary essential oil was evaluated as a free radical scavenging

99 activity [12]. The activity of phenolic compounds was done using 1,1-diphenyl-2-picrylhydrazyl

100 (DPPH). The total phenolic contents of the rosemary extracts were determined by the Folin-

101 Ciocalteu method at 760 nm using a UV-vis spectroscopy, as reported in the literature [6].

102 Further, total phenol values were expressed in terms of gallic acid equivalents (mg/g of dry

103 weight of the leaves). The total flavonoid content of extracts was calculated according to the

5
104 report in the literature [3]. Quercetin was used as standard, and the results were expressed as mg

105 of quercetin equivalents per gram of dry weight of the leaves. Antibacterial activity of the

106 rosemary essential oil was determined by disc diffusion method, as described previously in the

107 literature [13]. The selected microbes for the antibacterial activity are Stafilococ auriu and

108 Escherichia coli. Amoxicillin was used as a positive control for the inhibition of microbes.

109 Further, the experiments were repeated to measure the standard deviation.

110

111 2.4 Optimization

112 For process optimization, Design Expert software package was used for the experimental design

113 and analysis. Further, Linear, two factorial interaction (2FI), quadratic, and cubic models were

114 tried to fit the experimental data. The obtained results were compared with the predicted values.

115 The P-value (probability of error value) was monitored for the significance of each regression

116 coefficient. The optimal process conditions were also validated by repeating the experiments.

117 2.5 Mathematical model

118 One dimensional two-phase model for extraction of essential oil from rosemary leaves has been

119 developed from the first principle of convection-diffusion-desorption equation. The assumption

120 of the model is follows: (a) the extractable oil is uniformly distributed and flat steam velocity

121 throughout the bed; (b) dispersion only in the axial direction with constant temperature and

122 pressure. The rate of oil fraction extract from solid to fluid assumed to be linear [14] and it can

123 be expressed as

124 [1]

6
125

126 Where, is volume fraction of essential oil extract from the plant (ml/gm), is extraction

127 time (s), is mass transfer coefficient (s-1).

128 The dynamics of oil extraction in a steam distillation process can be captured by writing

129 appropriate mass balance in fluid phase i.e.,

130 [2]

131

132 Where, is volume fraction of essential oil in fluid phase (ml/gm), is superficial steam

133 velocity (m/s), is effective diffusion coefficient (m2/s), and is void fraction. The mass

134 balance equation [1-2] for extraction of essential oil is subject to the following initial and

135 boundary conditions.

136

137 Initial conditions: and for all [3]

138 Boundary conditions: and for all [4]

139 Where, the initial fraction of essential oil present in plant ( ) and steam ( ) is 3.0 and 0.

140 The above equation [1-4] can be solved numerically using finite difference method. The model

141 equation was discretized into n equal intervals in axial coordinates resulting from systems of 2n

142 ordinary differential equations (ODEs). The kinetic parameters were estimated using Nelder-

143 Mead simplex direct search optimization algorithm, which is available in MATLAB fminsearch

144 subroutine. The calculated fractional oil extract from ODEs used to evaluate an error function

7
145 (sum of squares method) for error minimisation is . Where and are

146 the experimental and calculated fraction of oil extract, respectively and i is the number of

147 samples experimentally measured. The relative mean square of error is calculated based on the

148 following formula.

149 [5]

150 Where, N is number of experimental data.

151

152 3. Results and discussion

153 3.1 Regression analysis and model fitting

154 The influences of operating parameters such as feed mass, temperature, and extraction time on

155 the extraction of rosemary oil by steam distillation are studied using RSM. The experimental

156 values for the response (yield) under conditions are shown in Table. 1. The yield of rosemary

157 essential oil varies from 0.59 – 3.10 %. This is in good agreement with the findings of other

158 researchers in the literature [2].

159 The yield (response variable) is written in terms of independent variables as given below.

160 Yield ( % )=2.97−0.095 A+ 0.25 B−0.26 C+ 0.18 AB−0.061 AC −0.094 BC−0.47 A 2−0.63 B2−0.46 C 2

161 Where A is the temperature, B is the time and C is the feed mass.

162

163 Table 1 Design matrix using CCD with experimental data for rosemary leaves

8
 Run Factor1 Factor 2 Factor 3 Actual value Predicted

Temperature (oC) Time (h) Feed mass (g) Yield(%) value Yield%

1 117.00 6.00 400.00 2.09 2.16

2 113.00 4.00 331.82 2.03 2.10

3 113.00 4.00 500.00 2.88 2.97

4 109.00 2.00 600.00 1.4 1.33

5 106.00 4.00 500.00 1.72 1.80

6 117.00 2.00 400.00 1.18 1.12

7 113.00 4.00 500.00 3.04 2.97

8 113.00 4.00 500.00 3.10 2.97

9 117.00 6.00 600.00 1.27 1.33

10 120.00 4.00 500.00 1.56 1.48

11 109.00 2.00 400.00 1.6 1.54

12 109.00 6.00 600.00 1.22 1.28

13 113.00 4.00 500.00 2.80 2.97

14 117.00 2.00 600.00 0.59 0.66

15 109.00 6.00 400.00 1.94 1.82

16 113.00 7.36 500.00 1.69 1.62

17 113.00 4.00 500.00 3.00 2.97

18 113.00 4.00 500.00 3.00 2.97

19 113.00 0.64 500.00 0.71 0.78

20 113.00 4.00 668.18 1.30 1.23

164

165 Table 2 Analysis of variance (ANOVA) for the fitted quadratic model

9
 Source Sum D Mean F p-value Remark

of Squares Square Value Prob> F

Model 12.26 9 1.36 105.84 < 0.0001 Significant

A-temperature 0.12 1 0.12 9.60 0.0113 Significant

B-time 0.85 1 0.85 65.71 < 0.0001 Significant

C-feed mass 0.93 1 0.93 72.02 < 0.0001 Significant

AB 0.26 1 0.26 19.86 0.0012

AC 0.030 1 0.030 2.33 0.1577

BC 0.070 1 0.070 5.46 0.0415

A2 3.19 1 3.19 247.93 < 0.0001

B2 5.65 1 5.65 438.98 < 0.0001

C2 3.07 1 3.07 238.70 < 0.0001

Residual 0.13 10 0.013

Lack of Fit 0.068 5 0.014 1.12 0.4507 not

significant
166

167 In the above equation, the sign and magnitude of the coe fficient allow us to interpret the e ffect

168 of the independent variables. The negative sign indicates that when the level of the variable

169 increases the yield decreases, whereas the positive sign indicates an increase in the yield [15].

170 From the P-value of each model term in ANOVA (Table 2), it is clear that three linear terms are

171 significant. The coefficient of determination (R 2) is found to be 0.9896. This implies that the

172 variation (98.96%) in the yield due to the contribution of the independent variables. The

173 predicted model seems to represent the observed experimental values. To access the model, the

10
174 adjusted R2 is more appropriate if the value is more than 90%. The value of adjusted R 2 would be

175 smaller than the R2 If there are too many non-significant terms in the model. Thus, the adjusted

176 R2 (0.9896) is very close to R2 (0.9803), indicating that there is a correlation between observed

177 and predicted values [16].

178

179 The model is significant as the F-value is 105.84. Moreover, P-value (< 0.0001) for the model is

180 less than 0.05, indicating that the model is significant. Lack-of-fit is able to say to what extent the

181 model fits the data. From Table 2, the p-value of lack of fit (0.4570) is higher than 0.05 (non-

182 significant), indicating that the model predicts the relevant response accurately. The coefficient

183 of variation (CV) explains how the data are distributed in the sample. If the CV is less than 10 in

184 the model, it is reproducible. The smaller value of CV (5.95%) indicates that the experimental

185 values are reliable and precise. In the model, predicted residual sum of squares (PRESS) is a

186 measure of how well a particular regression model fits each point in the new experimental data.

187 A smaller value of PRESS is desirable, and it is found to be 0.62.

188

189

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191

192

193

194

195

196

197

198

199

200

201

202

203

204

205 Fig. 2 Response surface and contour plots showing interaction effect of different parameters on

206 the yield: Time and Temperature (a and b) ; Feed mass and Temperature (c and d); Feed mass

207 and Time (e and f).

12
208 3.2 Effect of independent variables on the yield

209 Adequate precision dictates the signal-to-noise ratio and found to be 28.786 for this model,

210 which is desirable as the ratio is greater than 4. The reliability of the model is explained through

211 these statistical parameters. The effects of independent variables (three) towards the yield of

212 Rosmarinus officinalis L. is expressed with the help of significant (p < 0.05) coe fficient of the

213 second-order polynomial equation. The effect of a parameter on the yield within the

214 experimental condition is the generation of response surface plots. The best way to correlate the

215 relationship between independent and dependent variables is from the plots (response surface

216 (3D) and contour (2D), as shown in fig. 2. Response surface plot demonstrates the influence of

217 the independent variables (temperature, time and feed mass) and their interaction on the yield,

218 whereas the contour plot is to observe the interaction effect on independent variables in the yield.

219 In this study, the contour plot exhibits elliptical in nature, indicating that the interactions between

220 the variables are significant. In general, the contour plot around the stationary point is elliptical

221 and became elongated more while increasing the variable along the axis. This indicates that the

222 interaction effect of two variable exerts a slight effect on the response (volume of the yield).

223 From fig .2, Temperature range from 110 to 115 ºC and time range from 3 to 5 h are found to be

224 the condition where the maximum yield can be achieved. More importantly, the optimum

225 condition is achieved at temperature (113 ºC) and time (4h). Beyond this condition, the yield of

226 essential decreases. At lower temperature, steam travels through the leaves slowly, and the

227 pressure at this point is not adequate to extract essential oils from the leaves. However, as the

228 temperature increases, the essential oil will break out of the leaves and increases the yield.

229 Moreover, while increasing the extraction time at the optimum temperature (113 ºC), there could

230 be a possibility of some changes in the structure of leaves and turns out to be on the decreasing

13
231 side of the yield [9]. This is in good agreement with the findings of a report in the literature while

232 working on essential oil extraction by steam distillation. From fig. 2, feed mass is found to have

233 a significant effect on the yield. The yield of essential oil increases with an increase in the

234 quantity feed mass. The optimal condition is achieved at temperature (113 ºC) and feed mass

235 (500 g). Beyond this condition, the yield of essential oils decreases due to the channeling effect.

236 Fig 2 (e and f) shows the interactive effects of feed mass and time on the yield. As the extraction

237 time increases, the yield of essential oil increases. The optimal condition is achieved at the

238 extraction time (4 h) and feed mass (500 g). However, beyond the optimal condition, the yield

239 gradually decreases with extraction time. This could be due to the concentration gradient. The

240 curvature effect in extraction time, temperature and feed mass demonstrate the response of

241 extraction yield. All variables indicate a positive e ffect on the yield of rosemary essential oil.

242 From RSM, the optimal conditions to obtain the maximum yield from Rosmarinus officinalis L.

243 are feed mass (500g), extraction time (4h), and temperature (113 ºC).

244 3.3Mathematical Modeling

245 Mathematical model is able to predict the experimental condition that provides the maximum

246 yield of the essential oil derived from rosemary leaves (Fig 3a). The estimated parameters (Table

247 3) are optimized by minimizing the errors between the experimental and calculated data through

248 Nelder-Mead simplex optimization algorithm. The estimated diffusion coefficient and mass

249 transfer coefficient from the model are 3.13×10-8 m2/s and 3.33×10-1 s-1, respectively. The

250 computed residual norm is 0.084. To evaluate the agreement between experimental and

251 calculated data from model, the plot of experimental versus calculated data is construed (Fig 3b).

252 The calculated relative mean square of error (E) and regression coefficient (R2) are 0.081 and

253 0.998, respectively. Thus, the model can be considered to evaluate the kinetic process of the

254 extraction of essential oil from rosemary leaves.

14
255 (a) (b)

256 Fig. 3 a) Profile of essential oil extraction form rosemary leaves. using a steam distillation
257 process. O – Experimental data and -- - Calculated data from model. (b) Comparison of
258 Experimental and Calculated data from model.
259

260 Table 3. Value of parameter estimated for the mathematical model in extraction of essential oil

261 form Rosemary leaves using a steam distillation process.

Parameter
(m/s)
(m2/s) (s-1)

Value 3.13×10-8 0.125 0.449 3.33×10-1

262

263 Table 4 physicochemical properties of essential oil derived from rosemary leaves

Property Rosemary essential oil

Color Light yellow
Odor Warm camphoraceous

15
 Solubility Insoluble in water
Specific Gravity 0.84
pH value 3.98
Refractive index + 1,47
@ 25℃
Boiling point (°C) 122
Dynamic viscosity 0.96
−6 2
Kinematic 10827 ×10 m
viscosity
264

265 3.3 Physicochemical properties and chemical analysis by GC-MS

266 The physicochemical properties of rosemary essential oil are presented in Table 4. The specific

267 gravity is found to be 0.84, which plays an important role in determining the purity of essential

268 oil. Particularly, the specific gravity values for the most of the essential oils are in the range of

269 0.696 - 1.88. The essential oil with a specific gravity of less than 1.0 contains oxygenated

270 aromatic compounds. The acid value is found to be 2.49 mg NaOH/g. Generally, essential oils

271 contain several aromatic compounds which are mostly free fatty acids. The low acid value

272 indicates the storage life of the essential oils. The saponification value is found to be 9.8 mg

273 KOH/g. It is inversely proportional to the molecular weight of the essential oil. The high

274 molecular weight indicates the presence of large proportion of shorter carbon chain lengths of

275 fatty acids, whereas the low molecular weight fatty acids have more number of glyceride

276 molecules per gram of fat, as compared to high molecular weight fatty acids.

277 For saponification, each glyceride molecule requires three KOH molecules. It gives information

278 about the type of glycerides present in the sample. The iodine value of the rosemary essential oil

16
279 is found to be 82 mg I2/ 100 mg. The degree of unsaturation in essential oils as well as the

280 number of double bonds, which eventually reflects the susceptibility of oil to oxidation [10].

281 From GC-MS analysis (Fig. 4), it is clear that sixteen components exist in the sample, which has

282 been eluted from the GC column, as shown in Table 5. Moreover, they are analyzed with an

283 electron impact mass spectroscopy voyager detector. The identification is based on their

284 retention time and mass spectral library. Table 5 shows the identified constituents of the essential

285 oils. The relative amount is calculated based on the peak area. It reveals that the essential oil

286 of Rosmarinus officinalis contains a mixture of terpenes that is eluted at different retention times

287 [2].

288

289

290

291

292

293

294

295

296

297

298 Fig. 4 GC-MS profile of optimized rosemary essential oil (Rosmarinus officinalis L.)

299

17
300 Table 5 Chemical composition of rosemary essential oil Identified by GC-MS

No. Components Library Retention Area

Match Time(min) (%)
Quality (%)

1 (1S)-2,6,6- 96 5.1192 29.7335

Trimethylbicyclo[3.1.1]hept-2-
ene(α-pinene)

2 D-Limonene 99 6.724 2.8909

3 Eucalyptol(1,8-Cineole) 97 7.0559 27.1433

4 Tricyclo[2.2.1.0(2,6)]heptane, 91 8.822 3.9337

5 (+)-2-Bornanone (camphor) 98 9.8657 2.8597

6 Terpinen-4-ol 93 9.9268 1.7702

7 Pinocarvone 90 10.0739 0.5168

8 endo-Borneol 97 10.2097 5.1151

9 α-Terpineol 94 10.3706 3.0238

10 (-)-Myrtenol 95 10.6721 0.6444

11 Bicyclo[2.2.1]heptane, 2- 53 10.7728 0.6055

methoxy-1,7,7-trimethyl-()

12 (+)-3-Carene 91 11.3239 4.6868

13 Bicyclo[3.1.1]hept-3-en-2-one, 98 11.3846 12.8102

4,6,6-trimethyl- (Verbenone)

14 2-Cyclohexen-1-one, 3,5- 47 12.2456 0.5736

dimethyl-

15 Caryophyllene 99 12.6375 3.1981

16 1,4,7,-Cycloundecatriene, 1,5,9,9- 97 13.1364 0.4942

tetramethyl-, Z,Z,Z-

301

302 Table 6 Antibacterial activities of rosemary essential oil

18
 Microorganisms Inhibition zone for Inhibition zone for control

rosemary oil (cm) (Amoxicillin ) (cm)

S. aureus 1.5 1.6

E. coli 1.7 2.0

303

304 3.4 Antioxidant and Antibacterial activity

305 The scavenging activity of rosemary essential oil on DPPH radical expressed in terms of IC50

306 value, and it is found to be 20.2 µg/ml. Further, DPPH is decolorized by the release of electrons

307 from the antioxidant. The antioxidant activity is due to the presence of terpenes in the essential

308 oil, as confirmed in the chemical analysis by GC-MS. Total phenolic and flavonoid contents in

309 the rosemary extract are 288.8 GAE/mg and 62 QE/g, respectively. The presence of phenolic

310 contents is responsible for the antioxidant properties of the extract. Antibacterial activity of

311 rosemary essential oil is studied against microbes (S. aureus) and (E. coli). The antibacterial

312 activity is accessed by the presence of inhibition of zones. Moreover, Amoxicillin is used as

313 control and the antibacterial activity of the extract is shown in Table 6.

314 4. Conclusions

315 In the present work, RSM has been employed to find out the optimum process parameters that

316 could enhance the yield of rosemary essential oil during the steam distillation process. Further,

317 second-order polynomial predicts the yield of rosemary essential oil. The predicted and actual

318 values are in good agreement with each other (R 2 =0.9896). ANOVA has shown that the effects

319 of process parameters and their interactions influence significantly on the yield. The conditions

320 that provides maximum yield are extraction temperature (113ºC), time (4h), and feed mass (500

19
321 g). GC-MS analysis reveals that the extracted essential oil possesses bioactive compounds, which

322 are responsible for its antioxidant activity. Further, it is proved to have a strong antibacterial

323 activity that allows it to be used as a natural preservative in pharma and food industries. Thus,

324 the present work demonstrates that the extraction of rosemary essential oil can be improved by

325 adjusting the process parameters, and they show excellent antibacterial and antioxidant

326 properties.

327

328 Acknowledgements

329 Authors would like to thank AASTU for the financial support (ICBCE-2011/2012-10) to

330 carry out this work.

331 References

332 [1] P. Terpinc, M. Bezjak, H. Abramovič, A kinetic model for evaluation of the antioxidant

333 activity of several rosemary extracts, Food Chem. 115 (2009) 740–744.

334 https://doi.org/10.1016/j.foodchem.2008.12.033.

335 [2] A. Zermane, O. Larkeche, A.H. Meniai, C. Crampon, E. Badens, Optimization of Algerian

336 rosemary essential oil extraction yield by supercritical CO2 using response surface

337 methodology, Comptes Rendus Chim. 19 (2016) 538–543.

338 https://doi.org/10.1016/j.crci.2015.08.011.

339 [3] E. Choulitoudi, S. Ganiari, T. Tsironi, A. Ntzimani, D. Tsimogiannis, P. Taoukis, V.

340 Oreopoulou, Edible coating enriched with rosemary extracts to enhance oxidative and

341 microbial stability of smoked eel fillets, Food Packag. Shelf Life. 12 (2017) 107–113.

342 https://doi.org/10.1016/j.fpsl.2017.04.009.

343 [4] M. Elyemni, B. Louaste, I. Nechad, T. Elkamli, A. Bouia, M. Taleb, M. Chaouch, N.

20
344 Eloutassi, Extraction of Essential Oils of Rosmarinus officinalis L. by Two Different

345 Methods: Hydrodistillation and Microwave Assisted Hydrodistillation, Sci. World J. 2019

346 (2019). https://doi.org/10.1155/2019/3659432.

347 [5] H. Hosseini, S. Bolourian, E. Yaghoubi Hamgini, E. Ghanuni Mahababadi, Optimization

348 of heat- and ultrasound-assisted extraction of polyphenols from dried rosemary leaves

349 using response surface methodology, J. Food Process. Preserv. 42 (2018) 1–15.

350 https://doi.org/10.1111/jfpp.13778.

351 [6] M.A. Pires, P.E.S. Munekata, N.D.M. Villanueva, F.G. Tonin, J.C. Baldin, Y.J.P. Rocha,

352 L.T. Carvalho, I. Rodrigues, M.A. Trindade, The antioxidant capacity of rosemary and

353 green tea extracts to replace the carcinogenic antioxidant (BHA) in chicken burgers, J.

354 Food Qual. 2017 (2017). https://doi.org/10.1155/2017/2409527.

355 [7] Z. Ahmed, M. Abdeslam-Hassan, L. Ouassila, B. Danielle, Extraction and modeling of

356 Algerian Rosemary essential oil using supercritical CO2: Effect of pressure and

357 temperature, Energy Procedia. 18 (2012) 1038–1046.

358 https://doi.org/10.1016/j.egypro.2012.05.118.

359 [8] E. Cassel, R.M.F. Vargas, N. Martinez, D. Lorenzo, E. Dellacassa, Steam distillation

360 modeling for essential oil extraction process, Ind. Crops Prod. 29 (2009) 171–176.

361 https://doi.org/10.1016/j.indcrop.2008.04.017.

362 [9] M. Akhbari, S. Masoum, F. Aghababaei, S. Hamedi, Optimization of microwave assisted

363 extraction of essential oils from Iranian Rosmarinus officinalis L. using RSM, J. Food Sci.

364 Technol. 55 (2018) 2197–2207. https://doi.org/10.1007/s13197-018-3137-7.

365 [10] O.A. Fakayode, K.E. Abobi, Optimization of oil and pectin extraction from orange (Citrus

366 sinensis) peels: a response surface approach, J. Anal. Sci. Technol. 9 (2018).

21
367 https://doi.org/10.1186/s40543-018-0151-3.

368 [11] H. Esmaeili, A. Karami, F. Maggi, Essential oil composition, total phenolic and

369 flavonoids contents, and antioxidant activity of Oliveria decumbens Vent. (Apiaceae) at

370 different phenological stages, J. Clean. Prod. 198 (2018) 91–95.

371 https://doi.org/10.1016/j.jclepro.2018.07.029.

372 [12] N. Erkan, G. Ayranci, E. Ayranci, Antioxidant activities of rosemary (Rosmarinus

373 Officinalis L.) extract, blackseed (Nigella sativa L.) essential oil, carnosic acid, rosmarinic

374 acid and sesamol, Food Chem. 110 (2008) 76–82.

375 https://doi.org/10.1016/j.foodchem.2008.01.058.

376 [13] A. Alizadeh, O. Alizadeh, G. Amari, M. Zare, Essential Oil Composition, Total Phenolic

377 Content, Antioxidant Activity and Antifungal Properties of Iranian Thymus daenensis

378 subsp. daenensis Celak. as in Influenced by Ontogenetical Variation, J. Essent. Oil-

379 Bearing Plants. 16 (2013) 59–70. https://doi.org/10.1080/0972060X.2013.764190.

380 [14] E. Reverchon, Supercritical fluid extraction and fractionation of essential oils and related

381 products, J. Supercrit. Fluids. 10 (1997) 1–37. https://doi.org/10.1016/S0896-

382 8446(97)00014-4.

383 [15] T. Baj, A. Baryluk, E. Sieniawska, Application of mixture design for optimum antioxidant

384 activity of mixtures of essential oils from Ocimum basilicum L., Origanum majorana L.

385 and Rosmarinus officinalis L., Ind. Crops Prod. 115 (2018) 52–61.

386 https://doi.org/10.1016/j.indcrop.2018.02.006.

387 [16] D. Belhachat, L. Mekimene, M. Belhachat, A. Ferradji, F. Aid, Application of response

388 surface methodology to optimize the extraction of essential oil from ripe berries of

389 Pistacia lentiscus using ultrasonic pretreatment, J. Appl. Res. Med. Aromat. Plants. 9

22
390 (2018) 132–140. https://doi.org/10.1016/j.jarmap.2018.04.003.

391

392

393

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