Extraction Process of Polyphenols from Rosemary

(Rosmarinus officialis L.) : Optimization and Evaluation of

Antioxidant Activity
Quoc Cuong Truong 1, Xuan Tien Le 1,*, Minh Thuy Nguyen 2,
1 Department of Chemical Engineering, Ho Chi Minh City University of Technology,
Vietnam National University-Ho Chi Minh City, Ho Chi Minh City 700000, Vietnam
2 Vietnam Academy of Science and Technology, Graduate University of Science and Technology,
Hanoi 100000, Vietnam
3 Institute of Natural Products Chemistry, Vietnam Academy of Science and Technology,
Hanoi 100000, Vietnam
4 NTT Hi-Tech Institute, Nguyen Tat Thanh University, Ho Chi Minh City 700000, Vietnam
5 Center of Excellence for Biochemistry and Natural Products, Nguyen Tat Thanh University,
Ho Chi Minh City 700000, Vietnam
6 Food Technology Department, College of Agriculture, Can Tho University, Can Tho City 94000, Vietnam
7 Faculty of Biotechnology, Nguyen Tat Thanh University, Ho Chi Minh City 700000, Vietnam
* Correspondence: [email protected] (X.T.L.); [email protected] (P.T.H.H.)

Abstract: the present study optimized the conditions for solvent extraction in order to
maximized the antioxidant potential by the total polyphenol content (TPC) of extracts
from the spices of the Lamiaceae family; rosemary (Rosmarinus officialis L.). Optimized
parameters, including ethanol concentration (0-100% v/v),extraction temperature (50-80
o
C), extraction period (15-60 min), material-solvent ratio (1:5-1:10 g/mL), frequency of
extraction cycles (1,2 and 3 times). The outcomes obtained were used in the response
surface methodology, in combination with a central composite design, to construct the
total polyphenol content (TPC) with respect to the three most significant variables,
namely ethanol concentration, extraction temperature and material-solvent proportion.
The experimental conditions for optimal recovery of TPC consisted of ethanol
concentration of 65% (v/v), extraction temperature of 65 oC, material-solvent ratio of
1:7.5 g/mL, extraction time of 15 min, and 2 cycles of extraction. Based on the predicted
optimum conditions, the obtained and confirmed TPC was 88.64 mg acid gallic (GAE)/g
dry weight (d.w.). The estimated models were strongly significant (p<0.05) for TPC
values with significant regression coefficients (R2) of 0.9979.
Keywords: rosemary; antioxidant; polyphenol content; conditions; antioxidant activity

1. Introduction
Various examinations have shown that flavors have powerful antioxidant properties,
for the most part because of the amount and nature of polyphenolic compound quantity
present in them [1]. Rosemary (Rosmarinus officinalis L.) is a zest and restorative herb
broadly utilized the world over. Of the natural antioxidant, rosemary has been broadly
acknowledged as one of the flavors with the most noteworthy cancer prevention agent [2].
These properties are identified with the present of phenolic mixes, for the most part
rosmarinic acid (RA) and diterpenes, for example, carnosic acid (CA) and carnosol
(COH) [3-4].
In the United States and Europe, rosemary could be a unique interesting flavor
commercially accessible for use as an antioxidant [5]. Rosemary extracts have been
utilized within the treatment of maladies, due to its hepato-protective potential [6], helpful
potential for Alzheimer’s malady [7] and it’s anti-angiogenic impact [8]. By contrast, they
have been used in products conservation, since they anticipate oxidation and microbial
defilement [9-12]. In this manner, rosemary extract can be valuable for supplanting or
indeed diminishing engineered cancer prevention agents in foods. As additives, rosemary
extricates offer a few innovative points of interest and benefits to customers.
Antioxidant extraction from rosemary have been accounted for in the writing, for
example, an optimized handle for RA extraction form Melissa officinalis utilizing
methanol [13], upgraded the ultrasound assisted extraction of marjoram cancer prevention
agents counting RA, COH, and CA, utilizing methanol [14]. A few techniques have been
improved for antioxidant property from rosemary, for example, ultrasound assisted [15],
accelerated solvent extraction [16], pressurized green dissolvable extraction [17], And
CO2 supercritical liquid extraction [17-18]. Different investigations have revealed
rosemary extraction without reaction surface modeling and optimization [19-20].
The aim of this research is to apply a centered-central composite design (CCD)
subjected to response surface methodology (RSM) [21-24] to optimize the recoveries of
total polyphenol (TPC). Considered optimization parameters include ethanol
concentration, temperature of extraction, time of extraction, material-solvent ratio, and the
number of extraction cycles. The results of this study are expected to contribute to the
development of a new approach for large-scale production of phenolic compounds from
rosemary.
2. Materials and Methods
2.1 Plant Sample Preparation
The rosemary dried leaves were harvested at Lam Ha Commune, Ho Chi Minh City,
Vietnam, in February, 2019. Rosemary dried leaves were preserved in zip bags, placed in
a dry, ventilated place, and kept away from moisture during the study.
2.2 Extraction Method
 Rosemary extracted: Solvent extraction process was chosen. Ground dried rosemary
leaves (10 grams) were stored in closed flask, extracted in ethanol solvent with the
optimized operational condition showed in Figure 1. The sample was the filtrated under
vacuum and again extracted twice. After filtration, the combined liquid phases were
evaporated to dryness under vacuum.

Drying rosemary
leaves

EtOH
Extracting

Solid

Filtering
Liquid
EtOH
Extracting

Filtering

Liquid
Rotary solvent
evaporating

Rosemary
oleoresin

Figure 1 Rosemary oleoresin extraction process.

2.3 Quantification of Total Polyphenol Content (TPC) Based on Folin-Ciocalteu (FC)
Methodology
Polyphenols (PPs) are reactive second metabolites widely contributed in plant and
categorized into several classes, which usually are phenolic acids, flavonoids, stilbenes
and lignans. They play important role in fruit quality as it contributes to create typical
taste, color and nutritional properties of fruit. To humans, PPs were found to possess
abilities of preventing oral diseases, neutralizing free radicals, againsting oral cancer,
inhibit periodontal pathogens and so on. Hence, TPC in each plant is an interesting factor
needed figuring out [25, 26].

Principle: The F-C assay depends on the transfer in alkaline medium from phenolic
compounds to phosphor-molybdic/phosphor-tungstic acid complexes, which creates a
blue complex solution measured at wavelength of 765 nm. As colorimetric reactions are
widely used in the UV/VIS spectrophotometric method because it is easy to perform,
rapid and applicable in routine laboratory use as well as low-cost but a reference subtance
is a need to measure the total concentration of phenolic hydroxyl groups in the plant
extract. Nonetheless, this reagent is extremely easily decomposed in alkaline solution.
After that, it needs an excess quantity for the reaction to complete. But, the excess causes
the turbidity and makes the spectrophotometric analysis impossible. Hence, lithium salt
was added into F-C reagent to prevent the turbidity. This assay provides accuracy and
specific data to several groups of PPs due to the difference in unit mass and reaction
kinetics that make many compounds change color differently [27, 28].
Procedure: 40 L solution of DMSO 100% and dissolved dry extract was added with
200 L FCR, covered by aluminium sheet before vibrated in 5 mins. Then 3160 L of
H2O and 600 L Na2CO3 were added into the mixture to dilute and support the reaction.
The samples were vibrated in 30 minutes at room temperature and measured at = 760
nm by Thermo Scientific GENESYS 10S Series UV-Visible Spectrophotometers. Both
dry extract were applied the same method.
The TPC was calculated as a gallic acid equivalent (GAE) from a calibration curve of
GA standard solution and expressed as mg of GAE per gram of dry extract. The amount
of TPC was calculated using the following equation:

( )

Where C1 is the concentration obtained from Genistein standard curve (mg GAE/ L
DMSO), C2 is the concentration of the dried extract (mg dried extract/L DMSO), m1 is the
weight of total dried extract (mg), and m is the weight of dried material (g).
2.4 Antioxidant evaluation with 1,1-Diphenyl-2-Picrylhydrazyl (DPPH)
DPPH estimations were utilized dependent on the technique of Brand-Williams and
co-workers and Lee and co-workers [29,30], in which the assurance of antioxidant
potency is depends on the scavenging activity of the stable DPPH free radical. DPPH was
first blended in with methanol (Sigma-Aldrich, St. Louis, MO, USA) 80% (with OD 517
nm = 0.80 ± 0.02) to form the solution with the concentration of 40 µg/mL. Vitamin C
(Sigma-Aldrich, St. Louis, MO, USA), blended in methanol 80% with concentrations of
0–100 µg/mL, was utilized as the positive control. To start the experiment, the sample
was first dissolved in methanol 80%. At that point, 180 µL of the readied DPPH solution
was included into 120 µL of the sample solution. The resultant solution was shaken, put
away in dimness at 30 oC for 30 min, and then measured at a wavelength at 517 nm. Each
test was repeated multiple times to compute the average value. Moreover, the color
solution was set up by including 180 µL of 80% MeOH (Sigma-Aldrich, St. Louis, MO,
USA) solution for 120 µL of the sample solution (120 µL). The absorbance of the color
solution was measured at 517 nm. The blank solution was prepared by adding 180 µL of
the prepared DPPH solution into 120 µL of 80% MeOH solution. The percentage of
DPPH radical scavenging is calculated according to the accompanying recipe:

DPPH (%)

Where Ab is the optical density of the blank sample; As is the optical density of
sample; Ac is the optical density of pigment; and IC50 value is calculated by the graph of
% inhibition.
2.5 Instruments
The following instruments were used: moisture meter (MA35, Sartorius, Göttingen,
Germany); UV-Vis spectrophotometer (Thermo Genesys 10S UV-Vis, Waltham, MA,
USA); ultrasonic bath (Elma S 100 H, Elma, Singen, Germany); Elisa microplate reader
(2100-C Optic Ivymen System, Biotech S.L., Madrid, Spain); rotary evaporator Buchi-
R210 (Marshall Scientific, Hampton, NH, USA).
2.6. Optimization of Extraction Process by Response Surface Methodology (RSM).
The classical method of optimization involves varying one parameter at a time and
keeping the other constant. But the method is inefficient as it fails to understand
relationships between the variables and the response. Response surface methodology
(RSM) is an effective statistical technique for the investigation of complex processes. The
main advantage of RSM is the reduced number of experimental runs needed to provide
sufficient information for statistically acceptable result. It is a faster and less expensive
method for gathering research result than the classical method.
RSM comprising three factors central composite design (CCD) was used in our work
to evaluate the interactive effects and to obtain the optimum conditions for total
polyphenol of rosemary extract. The factor levels for optimization process were selected
based on on-factor-at-one-time (OFTA) experimental method. The four variables were
ethanol concentration (X1), temperature (X2), material/solvent ratio (X3), extracting time
(X4).Each variable consisted of 3 different level from low (-1), to medium (0), and to high
(+1). The number of experiments performed was 30 times. The design consisted of 16
factorial points, 8 axial points and 6 center points . Distance from center to point α= =
1.682 (with k = 3 conditions). The data of Y obtained were fitted to a second-order
polynomial equation [31]:

∑ ∑

Where Y shows the response, o , i, ii and ij indicate the regression coefficient for
the intercept, linear, square and interaction respectively. xi represented the independent
variables.
Analysis of variance (ANOVA) was employed to evaluate the empirical mathematical
model at 5% significance level. The significant terms of the model were also obtained by
ANOVA. The adequacy of model was identified based on R-squared, adj-R-squared,
predicted-R-Squared, F-value and lack-of-fit.
The suitability of the model was checked by reproducing the Triplicate experiment
with the optimal parameters obtained. The results obtained were then checked by
comparing the expected value with the real values obtained from the experimental work.
Table 1. Experimental conditions for the rates used in the trial.

Level
Factor Symbol
-1 0 +1

EtOH concentration (%) X1 60 70 80

Temperature (oC) X2 50 60 70

Solvent-material ratio X3 6 8 10

Table 2. Central composite design of coded factors.

Run X1 X2 X3

1 (-1) (-1) (-1)

2 1 (-1) (-1)
 3 (-1) 1 (-1)

4 1 1 (-1)

5 (-1) (-1) 1

6 1 (-1) 1

7 (-1) 1 1

8 1 1 1

9 (-α) 0 0

10 (+α) 0 0

11 0 (-α) 0

12 0 (+α) 0

13 0 0 (-α)

14 0 0 (+α)

15 0 0 0

16 0 0 0

17 0 0 0

18 0 0 0

19 0 0 0

20 0 0 0

3. Results and Discussion

3.1. Single-Factor Investigations with regard to TPC of the extracted.
3.1.1. Influence of Ethanol concentration
Ethanol has been broadly utilized for extraction of naturally active compounds from
plants due its low impact on human health. All the operate conditions except the solvent
concentration were kept fixed at 50 oC temperature, material-solvent ratio 1:5, operating
time 30 minutes and 2 extraction cycles. Figure 2 indicates the effect of different ethanol
concentrations ranging from 0% to 99.5% on the outcomes of TPC. The TPC recovered
from the study at 60% ethanol was 56.5 (mg GAE/g d.w.) significant higher than the
previous level. Further increase of ethanol concentration to 70% boost the yield of
recovery to 59.0 (mg GAE/g d.w.) which is the peak of the investigation. On the other
side of the peak, raising the concentration past 80% to 99.5% lowered the yield to 27.6
(mg GAE/g d.w.) which is the lowest outcome.

70
59.0
60 56.5
mg GAE/ g dried material

50 45.4
37.5 38.5
40
32.9
30 27.6

20

10

0
0 30 60 70 80 90 Absolute
EtOH concentration (%)

Figure 2. Quantity of polyphenols affected by concentration

According to Figure 2, from EtOH 80oC to absolute, the polarity reduced, which leads
the decrease of TPE. Additionally, at concentration of 70%, water still existed in solvent,
causing rosemary moderately swelled and solvent easily permeated into the material.
However, as the concentration increased, less water in solvent that leaded to weak
swelling making the decrease of the extracting efficiency. Therefore, Et-OH 70% was the
optimal concentration.
3.2.2. Influence of temperature
This report researched the polyphenol quantity in four temperatures 50 oC, 60 oC, 70
o
C and 80 oC with the same operating conditions of the previous part in order to figure out
the significant temperature for the highest polyphenol.
 50
45 42.4 41.9
40.5
mg GAE/ g dried material 40
35
28.8
30
25
20
15
10
5
0
50 60 70 80
Temperature (oC)

Figure 3. Quantity of polyphenols affected by temperature

Figure 3 outlines the TPC in connection to extraction temperature. As the temperature
was increased from 50 oC to 60 oC, the TPC expanded altogether from 28.8 to the top of
42.4 (mg GAE/g d.w.). The instrument through which temperature height moves forward
recuperation of extractant may be clarified by improved diffusivity of dissolvable and
progressed solvency of phenolic compounds in solvents. However, TPC diminished
marginally to the point of 40.5 (mg GAE/g d.w.) when the temperature expanded from 60
o
C to 80 oC. This diminish may be due to the thermal destruction of polyphenol. So, 60 oC
was chosen for the next condition investigating.

3.2.3. Influence of dry material – solvent ratio

The material-solvent ratio affects the extracting capacity. The minimum solvent in-use
must be enough to soak all the material to meet the requirement of contact between solid
and liquid phases. Accordingly, TPCs of ratios of 1:5, 1:6, 1:7, 1:8, 1:9, 1:10 and 1:15
respectively were evaluated. Results were shown as in Figure 4.
 120

mg GAE/ g dried material

100

80 69.9 72.2
67.7 69.2
63.4 61.2
60

40

20

0
1:5 1:6 1:7 1:8 1:9 1:10

Material - solvent ratio

Figure 4. Quantity of polyphenols affected by of solid/solvent ratio

Figure 4 appears that when the material-solvent proportion expanded from 1:5 to 1:9,
TPC increased from 63.4 to 69.9 (mg GAE/g d.w.) and come to the peak of 72.2 (mg
GAE/g d.w.) as the proportion come to 1:10. Typically since the expansion of dissolvable
raises the distinction of concentration slope, in this way keeping up the dissemination
until balance is come to. At the equilibrium state where the sum of polyphenols within the
material is depleted, the expansion of ethanol will not advance the TPC abdicate any
longer. Apparently, contrasts of yields that were accomplished at proportions of 1:8 , 1:9,
1:10 were unobtrusive and not factually noteworthy. As a result, the crude material-
solvent proportion of 1:8 was more temperate and chosen for consequent tests.

3.2.4. Influence of extracting time

The longer the extracting time was, the higher the efficiency would be. However, until
at a certain time, lengthening the extracting time could not raise up the efficiency. On the
other hand, it caused waste on energy and solvent, which resulted in a necessity of
acknowledging the impact of stirring time on the TPC. Because of those, thesis studied
the polyphenols from samples extracted in 15, 30, 45 and 60 minutes.
 100
90

mg GAE/ g dried material

80
67.3 67.5 70
70 66.8

60
50
40
30
20
10
0
15 30 45 60
Extracting time (minute)

Figure 5. Polyphenols affected by extracting time

In accordance with Figure 5, if stirring the mixture from 15 to 45 minutes, the TPE
slightly increased. Over 45 minutes, due to the longer the retention time, the more heat
contact leading to small molecules decomposed. Yet, the small change in TPE between 15
and 45 minutes was not considerable but it would require more time-consumption and
energy. Therefore, 15 minutes should be applied for the extracting step.
3.2.5. Influence of number of extraction cycles
In order to minimize the volume of solvent and time used for the extraction but still
ensure the optimum TPC. Hence, the TPE of three samples extracted in 1, 2 and 3 times
respectively were investigated. From figure 6, it was shown that TPC was not thoroughly
extricated by only 1 cycle extraction. It can clearly be seen that the yield of the second-
cycle extraction was significantly expanded compared to the previous one, totaling 67.4
mg GAE/g d.w (increase over 52%). After the third extraction cycle, TPC value just
increase appropriate 8.8% from 67.4 to 76.2 mg GAE/g d.w. , proposing that the greater
part of TPC has been depleted after the second-cycle.
 100
90

mg GAE/ g dried maerial

80 76.2
67.4
70
60
50 44.2
40
30
20
10
0
1 2 3
Number of extraction

Figure 6. Quantity of polyphenols affected by number of extraction

Because of conducting the experiment once was not able to collect all the
TPC, but when conducting that of twice or more, the TPC detected were approximate, so
that, two-time repeating was considerated for application into the process.
3.2. Optimization Process with RSM
3.2.1 Model Fitting Using RSM
Variations of factors for optimization of processes were selected based on single factor
experiments. To be specific, the factor levels for optimization of TPC included ethanol
concentration X1 (60-90% v/v), extraction temperature X2 (50-70oC), and material:
solvent ratio X3 (1:6-1:10) g/mL). Extraction process time was fixed at 15 min and
number of extraction was 2 times. The obtained optimum response was 87.63 (mg GAE/g
d.w.), based on the average of center points (optimum condition). The results of
experimental design based on CCD and corresponding responses are shown in Table 3.
The second-order polynomial equation of optimized condition for TPC is as follows:
YTPC = 87.63 -3X1 +4.82X2 + 2.65X3 -1.27X1X3 -3.21X2X3 -3.31X12 -5.83X22-3.12X32
Where Y is the responses, X1, X2, and X3 are the factors, including ethanol concentration,
extraction temperature, and material-solvent ratio, respectively. Table 4 shows the result
of analysis of variance. Overall, most of model terms and interaction factors were
statistically significant (p < 0.05). The coefficient of determination (R2) was 0.9979,
which indicates a good correlation between predicted and actual (experimental) values.
The suitability of the model also relates to a good agreement between predicted R 2 and
adjusted R2. Furthermore, for a fitted model, a non-significant lack-of-fit and Adequate
precision value greater than 4 are desirable. Thus, the terms of experimental design
obtained in this study suggested that models are reliable and have good predictability.
This could be manually confirmed by examining the actual and predicted response in
Table 3.
Table 3. Central composite design of actual factors and responses based on actual an
predicted values

Factors Response
Run YTPC (Actual) YTPC (Predict)
X1 X2 X3
(mg GAE/g d.w.) (mg GAE/g d.w.)

1 60 50 6 66.58 66.59

2 80 50 6 63.01 63.02

3 60 70 6 82.61 82.30

4 80 70 6 79.62 79.19

5 60 50 10 80.89 80.86

6 80 50 10 72.12 71.96

7 60 70 10 83.98 83.74

8 80 70 10 76.01 75.53

9 53.2 60 8 83.21 83.32

10 86.8 60 8 72.68 73.22

11 70 43.2 8 63.01 62.79

12 70 76.8 8 78.59 79.24

13 70 60 4.6 74.01 74.35

14 70 60 11.3 82.95 83.27

15 70 60 8 88.12 87.63
 16 70 60 8 87.25 87.63

17 70 60 8 86.89 87.63

18 70 60 8 87.69 87.63

19 70 60 8 88.01 87.63

20 70 60 8 87.95 87.63

Table 4. Analysis of Variance (ANOVA) results for the quadratic model for optimization.

Factors Total Polyphenol Content (TPC)

Source Sum of squares F-value p-value -

Model 1308.19 524.60 <0.0001 Significant

X1 123.14 444.44 <0.0001 -

X2 317.25 1144.97 <0.0001 -

X3 96.04 346.60 <0.0001 -

X1 X2 0.2380 0.8591 0.3758 Not significant

X1 X3 12.95 46.75 <0.0001 Significant

X2 X3 82.30 297.04 <0.0001 -

X1 2 157.77 569.41 <0.0001 -

X2 2 490.64 1770.78 <0.0001 -

X3 2 140.25 506.17 <0.0001 -

Residual 2.77 - - -

Lack of Fit 1.59 1.35 0.3753 Not significant

Pure Error 1.18 - - -

Cor Total 1310.96 - - -

Coefficient of Variation 0.6641 - - -

 PRESS 13.74 - - -

R2 0.9979 - - -

R2 Adjusted 0.9960 - - -

R2 Predicted 0.9895 - - -

Adequate Precision 66.7504 - - -

3.2.2. Analysis of Response Surface

Figure 9 indicates the relationship between ethanol concentration and temperature on
the TPC at various material-solvent ratios. Apparently, both factors had a positive effect
on TPC growth. The increase in ratio often appeared to move the solution surface
upwards without altering the surface shape. The nature of the answer also suggested that
the yield of TPC was rapidly increased with an improvement in temperature extraction
and ethanol concentration. At 1:6, 1:8 and 1:10 g/mL ratios, further calculations showed
that the maximum yield was 84.819, 88.752 and 88.149 mg GAE/g d.w., respectively.

(a)
 (b)

(c)
Figure 9. Response surface plots showing effects of extraction temperature and ethanol
concentration on the yield of TPC. (a) Material-solvent ratio 1:6; (b) Material-solvent
ratio 1:8; (c) Material-solvent ratio 1:10.
Figure 10 demonstrates the surface response plot which resulted in the extraction yield
of TPC as a function of material: solvent ratio and ethanol concentration. Various plots
according to set temperature (50, 60 and 70oC) are displayed. The extraction yield of TPC
can be clearly seen to increase mechanistically with the temperature factor. However,
once the temperature started to reach 60oC, the response started to drop. At temperatures
of 50, 60 and 70oC, further analyses showed that the maximum yield was 81.213, 88.94
and 87.223 mg GAE/g d.w., respectively.

(a)

(b)
 (c)

Figure 10. Response surface plots showing effects of material: solvent ratio and ethanol
concentration on the yield of TPC. (a)Temperature at 50oC; (b)Temperature at 60oC;
(c)Temperature at 70oC.
TPC’s extraction yield was investigated at different concentrations of ethanol (60%,
70% and 80% v/v) with respect to two variables, namely materials: solvent ratio and
extraction temperature, as shown in Figure 11. In this case, the study response just
quietly altering the surface shape when the concentration factor increase from 60% to
70% v/v before fall down dramatically once the concentrations variable exceed 70% v/v.
The highest TPC value was 88.189 mg GAE/g d.w. , where the peak was detected at a
temperature of 65oC.
(a)

(b)
 (c)
Figure 11. Response surface plots showing effects of material: solvent ratio and
temperature on the yield of TPC. (a)Ethanol concentration of 60% v/v; (b) Ethanol
concentration of 70% v/v; (c) Ethanol concentration of 80% v/v.
3.2.3 Validation of the Model
Approvals of the models were completed dependent on ieal extraction cnditions
acquired from RSM examination. Table 5 shows the parameters and the aftereffects of
approval tests. Tripplicate endeavors of this analysis gave a TPC consequence of 87.42
mg GAE/g d.w. This showed there is an attractive understanding among anticipated and
test (real) values. By applying the matched t-test, no critical change among real and
anticipated qualities (p< 0.05) was watched. Hence, the created reaction model was
satisfactory in foreseeing the TPC yield.
Table 5. The result of optimum condition experiment.

EtOH Extraction Material- TPC Model TPC Actual Error with

Concentration Temperature Solvent Ratio Model
(mg GAE/g (mg GAE/g
(% v/v) o
( C) (g/mL) d.w.) d.w.) (%)

88.12 0.41
65 65 1:7.5 88.64
87.25 1.10
 86.89 1.39

Average TPC ± Standard Deviation 87.42 ± 0.25

3.3 Evaluating the Antioxidant Ability.

References
1. Hossain, M. B., Brunton, N. P., Barry-Ryan, C., Martin-Diana, A. B., & Wilkinson, M. (2008). Antioxidant activity of
spice extracts and phenolics in comparison to synthetic antioxidants. Rasayan J. Chem, 1(4), 751-756
2. Shan, B., Cai, Y. Z., Sun, M., & Corke, H. (2005). Antioxidant capacity of 26 spice extracts and characterization of their
phenolic constituents. Journal of agricultural and food chemistry, 53(20), 7749-7759.
3. Collins, M. A., & Charles, H. P. (1987). Antimicrobial activity of Carnosol and Ursolic acid: two anti-oxidant
constituents of Rosmarinus officinalis L. Food Microbiology, 4(4), 311-315.
4. Moreno, S., Scheyer, T., Romano, C. S., & Vojnov, A. A. (2006). Antioxidant and antimicrobial activities of rosemary
extracts linked to their polyphenol composition. Free radical research, 40(2), 223-231.
5. Cuvelier, M.E.; Richard, H.; Berset, C. Antioxidative activity and phenolic composition of pilot-plant and commercial
extracts of sage and rosemary. J. Am. Oil Chem. Soc. 1996, 73, 645–652.
6. Raškovi´c, A.; Milanovi´c, I.; Pavlovi´c, N.; Cebovi´c, T.; Vukmirovi´c, S.; Mikov, M. Antioxidant activity ´ of
rosemary (Rosmarinus officinalis L.) essential oil and its hepatoprotective potential. BMC Complement. Altern. Med.
2014, 14, 225.
7. Habtemariam, S. The therapeutic potential of rosemary (Rosmarinus officinalis) diterpenes for Alzheimer’s disease.
Evid. Based Complement. Altern. Med. 2016, 2016, 2680409.
8. Kayashima, T.; Matsubara, K. Antiangiogenic effect of carnosic acid and carnosol, neuroprotective compounds in
rosemary leaves. Biosci. Biotechnol. Biochem. 2012, 76, 115–119
9. Djenane, D.; Sánchez-Escalante, A.; Beltrán, J.A.; Roncalés, P. Ability of α-tocopherol, taurine and rosemary, in
combination with vitamin C, to increase the oxidative stability of beef steaks displayed in modified atmosphere. Food
Chem. 2002, 76, 407 415
10. Nieto, G.; Díaz, P.; Bañón, S.; Garrido, M.D. Dietary administration of ewe diets with a distillate from rosemary leaves
(Rosmarinus officinalis L.): Influence on lamb meat quality. Meat Sci. 2010, 84, 23–29.
11. Nieto, G.; Bañon, S.; Garrido, M.D. Incorporation of thyme leaves in the diet of pregnant and lactating ewes: Effect on
the fatty acid profile of lamb. Small Rumin. Res. 2012, 105, 140–147.
12. Nieto, G.; Estrada, M.; Jordán, M.J.; Garrido, M.D.; Bañon, S. Effects in ewe diet of rosemary by-product on lipid
oxidation and the eating of cooked lamb under retail display conditions. Food Chem. 2011, 124, 1423–1429.
13. Kim, S., Yun, E. J., Bak, J. S., Lee, H., Lee, S. J., Kim, C. T., ... & Kim, K. H. (2010). Response surface optimised
extraction and chromatographic purification of rosmarinic acid from Melissa officinalis leaves. Food chemistry, 121(2),
521-526.
14. Hossain, M. B., Brunton, N. P., Patras, A., & Tiwari, B. (2012). O′ Donnell, CP, Martin-Diana, AB, Barry-Ryan,
C. Optimization of ultrasound assisted extraction of antioxidant compounds from marjoram (Origanum majorana L.)
using response surface methodology, Ultrason. Sonochem, 19, 582.
15. Paniwnyk, L., Cai, H., Albu, S., Mason, T. J., & Cole, R. (2009). The enhancement and scale up of the extraction of anti-
oxidants from Rosmarinus officinalis using ultrasound. Ultrasonics Sonochemistry, 16(2), 287-292.
16. Hossain, M. B., Barry-Ryan, C., Martin-Diana, A. B., & Brunton, N. P. (2011). Optimisation of accelerated solvent
extraction of antioxidant compounds from rosemary (Rosmarinus officinalis L.), marjoram (Origanum majorana L.) and
oregano (Origanum vulgare L.) using response surface methodology. Food Chemistry, 126(1), 339-346.
17. Herrero, M., Plaza, M., Cifuentes, A., & Ibáñez, E. (2010). Green processes for the extraction of bioactives from
Rosemary: Chemical and functional characterization via ultra-performance liquid chromatography-tandem mass
spectrometry and in-vitro assays. Journal of Chromatography A, 1217(16), 2512-2520.
18. Visentín, A., Cismondi, M., & Maestri, D. (2011). Supercritical CO2 fractionation of rosemary ethanolic oleoresins as a
method to improve carnosic acid recovery. Innovative Food Science & Emerging Technologies, 12(2), 142-145.
19. Babovic, N., Djilas, S., Jadranin, M., Vajs, V., Ivanovic, J., Petrovic, S., & Zizovic, I. (2010). Supercritical carbon
dioxide extraction of antioxidant fractions from selected Lamiaceae herbs and their antioxidant capacity. Innovative
Food Science & Emerging Technologies, 11(1), 98-107.
20. Couto, R. O., Conceição, E. C., Chaul, L. T., Oliveira, E. M., Martins, F. S., Bara, M. T. F., ... & Paula, J. R. (2012).
Spray-dried rosemary extracts: Physicochemical and antioxidant properties. Food chemistry, 131(1), 99-105.
21. Ye, Q.; Guo, L.; Liu, H.; Liu, Y.; Zhang, C.; Peng, C.; Liu, Z.; Huang, S.; Li, B. Optimization of ultrasound-assisted
extraction on antioxidative activity of Malus toringoides using response surface methodology. Processes 2019, 7, 270.
22. He, Y.; Chen, Y.; Shi, Y.; Zhao, K.; Tan, H.; Zeng, J.; Tang, Q.; Xie, H. Multiresponse optimization of ultrasonic-
assisted extraction for Aurantii Fructus to obtain high yield of antioxidant flavonoids using a response surface
methodology. Processes 2018, 6, 258.
23. Tran, T.H.; Nguyen, H.H.H.; Nguyen, D.C.; Nguyen, T.Q.; Tan, H.; Nhan, L.T.H.; Nguyen, D.H.; Tran, L.D.; Do, S.T.;
Nguyen, T.D. Optimization of microwave-assisted extraction of essential oil from Vietnamese Basil (Ocimum basilicum
L.) using response surface methodology. Processes 2018, 6, 206.
24. Dailey, A.; Vuong, Q.V. Optimum conditions for microwave assisted extraction for recovery of phenolic compounds and
antioxidant capacity from Macadamia (Macadamia tetraphylla) skin waste using water. Processes 2016, 4, 2.
25. Petti, S., & Scully, C. (2009). Polyphenols, oral health and disease: A review. Journal of dentistry, 37(6), 413-423.
26. El Gharras, H. (2009). Polyphenols: food sources, properties and applications–a review. International journal of food
science & technology, 44(12), 2512-2518.
27. Ainsworth, E. A., & Gillespie, K. M. (2007). Estimation of total phenolic content and other oxidation substrates in plant
tissues using Folin–Ciocalteu reagent. Nature protocols, 2(4), 875-877.
28. Blainski, A., Lopes, G. C., & De Mello, J. C. P. (2013). Application and analysis of the Folin Ciocalteu method for the
determination of the total phenolic content from Limonium Brasiliense L. Molecules, 18(6), 6852-6865.
29. Brand-Williams, W.; Cuvelier, M.E.; Berset, C. Use of a free radical method to evaluate antioxidant activity.
Lwt-Food Sci. Technol. 1995, 28, 25–30.
30. Lee, J.; Durst, R.W.; Wrolstad, R.E. Determination of total monomeric anthocyanin pigment content of fruit
juices, beverages, natural colorants, and wines by the ph differential method: Collaborative study. J. AOAC
Int. 2005, 88, 1269–1278.
31. Bezerra, M. A., Santelli, R. E., Oliveira, E. P., Villar, L. S., & Escaleira, L. A. (2008). Response
surface methodology (RSM) as a tool for optimization in analytical chemistry. Talanta, 76(5), 965-
977.