Separation Science and Technology

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Steviol glycosides and polyphenols extraction from

Stevia rebaudiana Bertoni leaves using maceration,
microwave-, and ultrasound-assisted techniques

Fatih Mehmet Yılmaz, Ahmet Görgüç, Özge Uygun & Cavit Bircan

To cite this article: Fatih Mehmet Yılmaz, Ahmet Görgüç, Özge Uygun & Cavit Bircan (2020):
Steviol glycosides and polyphenols extraction from Stevia�rebaudiana Bertoni leaves using
maceration, microwave-, and ultrasound-assisted techniques, Separation Science and Technology,
DOI: 10.1080/01496395.2020.1743311

To link to this article: https://doi.org/10.1080/01496395.2020.1743311

Published online: 09 Apr 2020.

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SEPARATION SCIENCE AND TECHNOLOGY
https://doi.org/10.1080/01496395.2020.1743311

Steviol glycosides and polyphenols extraction from Stevia rebaudiana Bertoni

leaves using maceration, microwave-, and ultrasound-assisted techniques
Fatih Mehmet Yılmaz, Ahmet Görgüç, Özge Uygun and Cavit Bircan
Engineering Faculty, Food Engineering Department, Aydın Adnan Menderes University, Efeler, Aydın, Turkey

ABSTRACT ARTICLE HISTORY

Optimization of steviol glycosides and polyphenols extraction from stevia leaves for maceration, Received 12 June 2019
microwave-assisted (MAE) and ultrasound-assisted (UAE) techniques were performed by investi- Accepted 10 March 2020
gating the effects of process variables using response surface methodology. Optimum extraction KEYWORDS
conditions for maceration were 50% EtOH concentration, 118 mL/g solvent ratio, 54°C tempera- Stevia rebaudiana; steviol
ture, and 112 min extraction time. Optimum operation temperatures and extraction times for glycosides; extraction;
MAE and UAE were estimated as 51°C & 16 min and 50°C & 43 min, respectively. MAE and UAE microwave; ultrasound;
significantly reduced processing time as compared to maceration. A total of 65 compounds were polyphenols; antioxidant
identified by LC/Q-TOF/MS analysis. compounds; LC/Q-TOF/MS

tevia rebaudiana Bertoni Harvesting Drying Grindin Stevia powder

Maceration Microwave Ultrasound Extracts

mV
Detector A:210nm
1000

750

500

250

0
4.0 5.0 6.0 7.0 8.0 9.0 10.0 min

Spectro hotometer HPLC RSM LC/Q-TOF/MS

Introduction sweetener[6] which contains at least eight different steviol

glycoside diterpenes of which stevioside and rebaudioside
There has been increasing interest to natural sweeteners
A are the most dominant ones accounting for 80–90% of
by the great demands of health conscious consumers.[1,2]
the total glycosides.[7,8] Stevioside and rebaudioside A are
In addition, natural sweeteners having been intensively
approximately 300 and 400 times sweeter than sucrose,
replaced synthetic counterparts mainly due to their more
respectively.[9] The industrial and scientific importance of
realistic sugar taste and healthier properties.[3,4]
stevia not only originate from its steviol glycosides but
Among the natural sweeteners, stevia (Stevia rebaudi-
also from phytochemical constituents, which have been
ana Bertoni) has outshined others in industrial and scien-
specifically reported to exhibit antioxidant, anti-
tific viewpoints especially in the last decade.[5] Stevia leaf
carcinogenic, anti-inflammatory, anti-hypertensive, and
is described as a reduced calorie (ca. 2.7 kcal/g)
anti-hyperglycemic activities.[10–12] Moreover, steviol

CONTACT Fatih Mehmet Yılmaz fatih.yilmaz@adu.edu.tr Engineering Faculty, Food Engineering Department, Aydın Adnan Menderes University,
Efeler, Aydın 09010, Turkey
© 2020 Taylor & Francis
2 F. M. YILMAZ ET AL.
glycosides have been shown to exert significant stabilities
to thermal effects and pH changes[13] that make them
ideal in use as food ingredient.[14] Today, stevia extracts
are included in many food formulations such as nonalco-
holic beverages, jams, confectioneries, ice-creams, choco-
lates, yogurts, snacks, etc.[15–17]
Currently, China is the leader country in the pro-
duction and exportation of stevia whereas Brazil,
Japan, and Korea are the most utilizers in the
world.[18] However, stevia cultivation and production
is spreading very fast all over the world due to the
aforementioned positive technological and health
effects.[5] For example, in Turkey, a couple of stevia
gardens have launched in the last 3 years and more
recently a stevia extract production plant with 50 ton/
year capacity was established by General Directorate of
Tea Enterprises (Çay-Kur).
The edible part is generally the leaves of the plant since
steviol glycosides are mainly accumulated in this part.[19]
Concentrated or powdered form of stevia extracts are
utilized in food industry[5] and the first and foremost
important unit operation for the preparation is considered
as extraction step.[13] There have been published reports
on the extraction of steviol glycosides and bioactive com-
pounds from stevia leaves.[20] Novel technologies such as
microwave-assisted extraction (MAE) and ultrasound-
assisted extraction (UAE) techniques have also been stu-
died on this subject.[21] However, inconsistent results in
terms of extraction yields are available among literature
studies, and there has been no any unique report optimiz-
ing solvent, MAE and UAE parameters by considering
both steviol glycosides and antioxidant compounds of
stevia. In addition, only few studies demonstrated detailed
fractionation of compounds present in stevia.[22]
MAE and UAE have been demonstrated to be
convenient techniques in multiple fields, especially
for medicinal plants.[23] In MAE, due to the interac-
tion of microwaves with water molecules enclosed by
plant material, reciprocal heat and mass transfer take
place between inner section of the material and
extraction solvent.[24] In UAE, plant tissues are dis-
rupted by the acoustic cavitation thereby resulting
improved extraction.[25] Both of the extraction tech-
niques have been shown to increase extraction yield
with reducing extraction times.[25,26] However, high
installation costs and scale-up procedures could be
considered as challenges of these methods.
Based on the given information, the objective of this
study was to illuminate the effects of solvent, MAE, and
UAE techniques on the acquisition of steviol glycosides
and antioxidant compounds from stevia leaves. The
effectiveness of process parameters was investigated
and optimum extraction conditions were determined
for each of the technique using response surface meth-
odology (RSM). Additionally, detailed identification of
compounds present in stevia leaves extracts was per-
formed by LC/Q-TOF/MS analysis.
Materials and methods
Plant material
The plant material Stevia rebaudiana Bertoni was pro-
vided from Burhaniye Stevia Garden in Burhaniye,
Balıkesir, Turkey (39°30̍ 1̎ N; 26°58̍ 22̎ E; altitude of
33 m; seasonal mean rainfall 63 mm; and seasonal
average temperature 14.6 °C). The plant was grown in
calcimorphic soil within pots by breeding technique.
The plant was collected by pruning method, and leaves
were detached from the flower parts from the stems
toward the root side. Fresh leaves were immediately
transferred to laboratory within perforated boxes and
dried for 2 h using a tray dryer (Eksis Industrial Drying
Systems, TK-10, Turkey) which was operated at 65°C,
1.5 m/s air flow rate, <10% relative humidity. The
leaves after drying were separated from the impurities
and particle size was reduced using a grinder (Sinbo,
SCM-2934, Turkey). Dried and milled leaves were
stored in the zip lock bags at +4°C.
Chemicals
Stevioside and rebaudioside A standards were purchased
from Extrasynthese Co. (Genay, France). LC-grade water,
LC-grade acetonitrile, Folin-Ciocalteu reagent, sodium
carbonate, and aluminum chloride hexahydrate were pro-
vided from Merck Co. (Darmstadt, Germany). Ethanol,
2,2′-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)
diammonium salt, trolox, gallic acid, and catechin were
supplied from Sigma Chemical Co. (St. Louis, Mo, USA).
All other chemicals were of analytical grade.
Extraction procedures
Maceration (Solvent extraction)
Solvent extraction (maceration) was performed using
a water bath (Daihan Scientific, Maxturdy-30, Korea)
with an agitation speed fixed on 50 rpm. The extraction
parameters were EtOH concentration (0 – 100%), time
(10–120 min), temperature (25–55°C) and solvent ratio
(10–120 mL/g). The extraction solutions (different
volumes and proportions of EtOH, 0.1% HCl) within
covered glass vessels were kept for 7 min in the water
bath to reach experimental temperature and then a 2 g
dried powder stevia leaf was added into each vessel.
Operations were carried depending on the time and

temperature specified in the experimental plan. The
slurries were then transferred to falcon tubes, centri-
fuged at 4800 x g for 5 min and finally supernatant
fractions were collected and stored at −20°C before
commencement of the analyzes.
Microwave-assisted extraction (MAE)
MAE was performed using a microwave extractor
system (Sineo Mas II Plus, Shanghai, China) equipped
with a digital control system for irradiation time,
temperature and microwave power. Stevia leaf powder
(2 g) and EtOH solution were mixed in a covered
vessel and kept in the microwave equipment with
stirring mode (250 rpm). Operations were carried at
different time (1–25 min) and temperature (20–55°C)
levels at 700 W constant microwave power. In the end
of the operations, the supernatant fractions were col-
lected after centrifugation at 4800 x g for 5 min and
stored at −20°C.
Ultrasound-assisted extraction (UAE)
An ultrasonic equipment (Ermaksan Ultrasonics, ULT
50-S, Turkey) operated at 35 kHz was used. The equip-
ment consisted of stainless steel cylindrical chamber,
piezoelectric crystals, heating – cooling system, digital
temperature controller and extraction vessel holder.
Temperature is controlled at target levels by the inte-
grated heating-cooling system. Briefly, 2 g of stevia leaf
powder was mixed with extraction solution in a glass
extraction vessel and the mixture was exposed to acous-
tic waves (540 W) for specified time (10–50 min) and
temperature (20–55°C) according to experimental plan.
After the UAE treatment, the supernatant was recov-
ered as described in MAE.
Experimental designs
Effect of each independent parameter was evaluated
using five level – central composite design (CCD) by
RSM. Solvent extraction had four independent para-
meters (EtOH concentration, solvent ratio, time, and
temperature) with 28 experimental runs while MAE
and UAE had two independent parameters (time and
temperature) with 11 runs for each treatment. The
ranges of the factors were selected by single factor tests
and preliminary experimental results (data not shown).
Analyzes
Determination of rebaudioside A and stevioside contents
All the extracts were filtered through 0.45 µm RC
filter (Sartorius, Germany) before being subjected to
HPLC analysis. The HPLC system (Shimadzu,
SEPARATION SCIENCE AND TECHNOLOGY 3
Prominence LC-20A, Japan) was equipped with UV/
VIS detector (Shimadzu, SPD-20A, Japan). The chro-
matographic separation was achieved using
a Hichrom ODS-3 column (250 mm × 4.6 mm × 5 µm)
at an oven temperature of 40 °C. The isocratic mobile
phase (1 mL/min) consisted acetonitrile and 0.1%
aqueous formic acid at a ratio of 31:69 (v:v). The
rebaudioside A and stevioside contents were quanti-
fied at 210 nm through standard calibration curves
(10–250 ppm) and results were expressed as mg per
g sample.[14] A sample HPLC chromatogram is pro-
vided in Fig. A1.
Determination of total phenolic compound (TPC)
TPC was determined according to Folin-Ciocalteu
method as described by Yılmaz & Ersus Bilek.[27]
Briefly, 30 μL extract, 2.37 mL dH2O and 150 μL
Folin-Ciocalteu reagent were mixed and kept in the
dark for 8 min. Then, 450 μL of saturated Na2CO3
was added and the mixture was incubated at 40°C
for 30 min. Subsequently, the absorbance value was
measured by a UV–VIS spectrophotometer
(Shimadzu, V-1800, Japan) at 750 nm. The results
were expressed as mg gallic acid equivalent per
gram of sample (mg GAE/g).
Determination of total flavonoids (TF)
Total flavonoids (TF) content was determined by
a colorimetric assay as described by Kim et al.[28]A 1 mL
of diluted sample extract (1:10; v:v) was added to a test
tube containing 0.3 mL 5% NaNO2 and kept at dark for
5 min. Then, 0.3 mL of 10% AlCl3 was added and waited
exactly for 1 min. Finally, 2 mL of 1 M NaOH and 2.4 mL
of deionized water were added consecutively to the mix-
ture. The mixture was vortex mixed and absorbance value
was read at 510 nm against prepared blank. The results
were expressed as mg catechin equivalent per gram sam-
ple (mg CE/g).
Determination of antioxidant capacity
Trolox equivalent antioxidant capacity (TEAC) values
were determined by ABTS method as described by
Görgüç et al.[29] Firstly, 7 mM ABTS radical solution
containing 2.45 mM K2S2O8 was diluted with PBS
(phosphate buffer saline, pH 7.4) until the absorbance
value reached 0.7 ± 0.02 at 734 nm. Then, 2.98 mL of
this solution was mixed with 20 μL extract and the
absorbance value was recorded after 0th and 6th min.
The results were expressed in terms of micromole tro-
lox equivalent per gram sample (μmol TE/g).
4 F. M. YILMAZ ET AL.
Liquid chromatography/Quadrupole-time of flight/
mass spectrometry (LC/Q-TOF/MS)
Analysis was performed using an UHPLC system (Agilent,
1260 Infinity, USA) and the chromatographic separation
was conducted by an EC-C18 (3.0 mm × 50 mm × 2.7 μm)
column (Agilent, Poroshell 120, USA). The binary mobile
phase system consisted of solvent A (0.1% formic acid in
water) and solvent B (acetonitrile) with linear gradient
program: 0th min (5% B), 8th min (15% B), 10th min
(20% B), 13th min (25% B), 18th min (30% B), 20th min
(45% B), 24th min (60% B), 27th min (80% B), 30th min (90%
B), 32nd min (5% B). The temperature of the column was
kept constant at 35°C and the injection volume was 3 μL.
Mass spectrometry was performed using a Q-TOF LC/MS
system (Agilent, 6550 iFunnel, USA) with electrospray
ionization in following conditions: Desiccant gas flow
14.0 L/min, nebulizer gas pressure 35 psi, drying gas tem-
perature 290°C, sheath gas temperature 400°C and sheath
gas flow 12 L/min. Mass spectra were recorded in both
positive and negative ionization modes in a mass range of
50–1700 m/z. Integration and data elaboration were per-
formed using MassHunter Workstation software using
Agilent METLIN Metabolomics database.[30]
Statistical analysis
Analysis of variance (ANOVA) of the data was con-
ducted using SPSS software (IBM, SPSS 15.0, United
States) at 95% confidence interval and the optimization
was performed by RSM using Design-Expert software
(Stat-Ease, Design-Expert v11.0, United States).
Experimental data were fitted to second order polyno-
mial model and regression coefficients were obtained
using multiple linear regression:
X
k X
k X
k1 X
k in EtOH concentration to a certain point accelerates
Y ¼β0 þ βi Xi þ βii Xi2 þ : βij Xi Xj
i¼1 i¼1 i¼1 j¼2
i<j
Here, Y is the predicted dependent variable; Xi and Xj are
the independent variables (i ≠ j); β0, βi, βii, βij are the
regression coefficients of the intersection, linear, second
order and interaction terms, respectively; and k is the num-
ber of variables. The validity of model was assessed by the
ANOVA outputs: Determination coefficient (R2), lack of fit
and Fisher’s test value (F-value).
Results and discussion
Maceration, MAE, and UAE techniques were compared
in terms of steviol glycoside and antioxidant compounds’
extraction from stevia leaf. Optimum parameters were
determined by evaluating effectiveness of each process
variables using rotatable CCD by RSM.
Maceration (solvent extraction) of steviol
glycosides and antioxidant compounds
The experimental design along with corresponding
results for solvent extraction is presented in Table 1.
Rebaudioside A content varied 32.99–40.77 mg/g and
stevioside content varied between 52.72 and 63.87 mg/g
depending on the experimental conditions. TPC, TF, and
TEAC (ABTS) values ranged 41.26–66.00 mg GAE/g,
27.21–40.68 mg CE/g and 469.80–737.40 µmol TE/g,
respectively.
The ANOVA results and regression coefficients of
the models are given in Table 2. The determination
coefficients (0.9782 < R2 < 0.9895) and adjusted deter-
mination coefficients (0.9548 < R2 < 0.9782) were rea-
sonably close to 1, indicating high degree of
correlations between the observed and predicted values.
In addition, lack of fit values was all insignificant
(P > 0.05), which verify adequacy of the fit. The linear
effects of process parameters except for EtOH concen-
tration on rebaudioside A and TPC were found signifi-
cant with varying confidence intervals. However,
the second order effects of EtOH concentration had
significant effect (P < 0.001) on these responses,
which indicate the direct relation of independent and
dependent variables.
Figs. A2 – A6 illustrate mutual effects of indepen-
dent variables of solvent extraction on the responses.
Rebaudioside A and stevisoside as well as TPC and TF
contents increased with increase in EtOH concentra-
tion up to 40–60% and then tend to decrease with
further increase in EtOH concentration. The increase
damages on the cellular matrix of plant materials and
thus increase extractability of the target compounds.
However, the higher concentrations of EtOH changes
solvent polarity and diffusion becomes more difficult,
which could be also linked to protein coagulation.[31]
When numerical optimization was performed by max-
imizing all of the responses, 50% EtOH was demon-
strated as the most convenient concentration. Previous
studies also showed that 50% is the favorable ethanol
concentration for the extraction of steviol glycosides
and phenolic compounds present in stevia leaf.[22] On
the other hand, Ameer et al.[9] reported that 75% as the
most efficient EtOH concentration.
Other parameters revealed uncertain dependency
to effect of EtOH concentration for different
responses. Solvent ratio and EtOH concentration
concomitantly affected the yields of the extracts.
 SEPARATION SCIENCE AND TECHNOLOGY 5

Table 1. RSM experimental design and responses belonging to solvent extraction.

Independent variables Dependent variables
EtOH
Experiment concentration Time Temperature Solvent ratio Rebaudioside A Stevioside TPC TF TEAC (ABTS)
No (%) (min) (°C) (mL/g) (mg/g) (mg/g) (mg GAE/g) (mg CE/g) (µmol TE/g)
1 75 38 48 93 37.5 59.2 57.1 37.9 638.9
2 25 93 48 38 38.4 54.3 54.1 35.5 645.3
3 50 65 40 10 35.8 55.7 43.8 28.0 530.8
4 50 65 40 65 38.5 62.5 55.7 35.8 579.5
5 75 38 33 93 36.4 55.8 53.7 35.9 604.0
6 50 65 40 120 40.8 63.4 66.0 40.7 709.0
7 75 93 33 38 37.3 57.6 46.8 32.8 605.6
8 25 38 33 93 36.0 60.1 54.5 33.5 586.1
9 50 120 40 65 40.3 62.9 52.9 37.8 705.6
10 25 93 33 38 37.3 57.5 45.0 31.0 540.4
11 50 65 40 65 38.9 62.6 55.0 33.8 574.9
12 75 93 48 38 38.8 59.2 50.5 32.2 688.6
13 25 38 48 93 37.5 61.6 58.5 35.9 650.6
14 50 65 40 65 38.7 63.1 54.9 35.1 586.4
15 75 93 33 93 39.5 59.5 59.0 39.3 691.1
16 50 65 40 65 39.0 63.1 55.9 34.7 581.7
17 100 65 40 65 34.9 52.7 42.0 30.2 563.1
18 25 93 33 93 38.6 61.5 57.3 35.4 660.8
19 25 38 33 38 34.1 59.8 42.1 27.2 469.8
20 25 38 48 38 35.0 57.2 48.9 31.7 545.1
21 75 38 33 38 33.0 55.3 41.3 28.2 511.8
22 0 65 40 65 35.4 55.5 41.3 26.0 510.0
23 75 38 48 38 35.2 57.2 47.3 31.4 562.8
24 50 10 40 65 34.8 61.4 52.6 34.1 530.0
25 50 65 55 65 39.0 63.1 58.6 37.1 684.3
26 50 65 25 65 35.7 58.9 51.2 34.5 553.1
27 25 93 48 93 39.7 63.6 58.5 36.1 712.7
28 75 93 48 93 40.2 63.8 59.9 39.3 737.4

Table 2. Quadratic model coefficients and ANOVA results belonging to (maceration) solvent extraction.
Model parameters Coefficient Rebaudioside A Stevioside TPC TF TEAC (ABTS)
Intercept β0 +11.12*** +51.95*** −6.75*** +10.16*** +506.31***
Linear
EtOH concentration (%), A β1 +0.11 +0.12** +0.56 +0.29*** +3.00***
Time (min), B β2 +0.13*** −0.09** +0.11*** +0.11*** −1.30***
Temperature (°C), C β3 +0.64*** +0.29*** +0.88*** +0.07*** −10.93***
Solvent ratio (mL/g), D β4 +0.09*** +0.02*** +0.40*** +0.14*** +1.70***
Interaction
EtOH concentration x Time, A x B β12 +2.15x10−4 +1.31x10−3*** +5.55x10−4 +3.35x10−4 +8.87x10−3
EtOH concentration x Temperature, A x C β13 +2.28x10−4 +4.50x10−3*** −2.34x10−3 −1.53x10−3 −0.03
EtOH concentration x Solvent ratio, A x D β14 +2.03x10−4 −8.20x10−4** +4.67x10−4 +8.42x10−4** −9.73x10−3
Time x Temperature, B x C β23 −4.49 +2.20x10−4 −1.64x10−3 −3.21x−3*** +0.02
Time x Solvent ratio, B x D β24 −3.14x10−4* +1.03x10−3*** −4.86x10−4 −2.61x10−4 −5.62x10−3
Temperature x Solvent ratio, C x D β34 −4.10x10−4 +4.13x10−3*** −4.87x10−3*** −1.08x10−3 −0.04*
Second order
EtOH concentration x EtOH concentration, A2 β11 −1.44x10−3*** −3.55x10−3*** −5.38x10−3*** −2.66x10−3*** −0.01**
Time x Time, B2 β22 −3.96x10−4** −2.68x10−4 +1.19x10−4 +4.05x10−4* +0.02**
Temperature x Temperature, C2 β33 −6.31x10−3** −8.80x10−3* −8.26x10−4 +4.87x10−3* +0.022**
Solvent ratio x Solvent ratio, D2 β44 −1.58x10−4 −1.13x10−3*** −6.31x10−5 −1.32x10−4 +0.02**
R2 0.9858 0.9782 0.9895 0.9884 0.9858
Adjusted R2 0.9705 0.9548 0.9782 0.9760 0.9704
p-value <0.0001 <0.0001 <0.0001 <0.0001 <0.0001
F-value 64.5 41.7 87.7 79.4 64.3
Lack of fit 0.20 0.10 0.11 0.91 0.06
Significance levels: *P < 0.05; **P < 0.01; ***P < 0.001
Model equation: Y = β0 + β1A + β2B + β3 C + β4D + β12(AxB) +β13(AxC) + β14(AxD) + β23(BxC) + β24(BxD) + β34(CxD) + β11A2 + β22B2 + β33C2 + β44D2

The solvent ratio higher than 65 mL/g at which
EtOH concentration varies between 40 and 60%
yielded more rebaudioside A and stevisoside. The
dependency of other responses (TPC, TF, and
TEAC) to solvent ratio were higher than that of
stevioside glycosides with requirement of more than
95 mL/g for TPC and TF contents and more than
110 mL/g for TEAC value. This situation could be
explained by mass transfer phenomena since the con-
centration gradient, which is the driving force is
higher when higher solvent ratio used, leading to
a higher diffusion.[32] However, excessive use of sol-
vent is not reasonable because of economic and
environmental points of view. Therefore, the opti-
mum solvent ratio was estimated as 118 mL/g for
this study as considering all of the responses.
6 F. M. YILMAZ ET AL.

Research findings reported that elevated tempera-
tures beyond 60°C may degrade steviol glycosides,[1]
but on the other hand high yield extraction of phenolic
compounds present in stevia leaf could be achieved at
higher temperatures.[3,22] Actually, the time and tem-
perature of the operations mutually determine the
extraction yields as well as degradation rates of bioac-
tive compounds due to thermal effects.[33] For example,
the increase in extraction time increased rebaudioside
A content with increasing temperature to a certain
point, then reached a plateau and eventually decreased
the overall yield (Fig. A2). At a higher temperature, the
solubility of steviol glycosides and phenolic compounds
in stevia leaf could be enhanced, and the viscosity of
solution could be decreased. Similar observations were
reported for different plant materials.[34,35]
Microwave-assisted extraction (MAE) of steviol
glycosides and antioxidant compounds
The experimental design and corresponding results for
MAE are presented in Table 3. There were considerable
variations on the yields depending on design matrix.
The regression coefficients of the intercept, linear,
interaction, and second order terms of the models are
given in Table 4. Two linear parameters, temperature
(A) time (B) were significant on all of the responses,
also the interaction parameters (A x B) were found
significant on the extraction of rebaudioside A and
stevioside. The ANOVA results show that the models
reflect well for the prediction of responses as consider-
ing closest R2 and Adj-R2, significant F-values of the
model and insignificant lack of fit F-values.[36]
In fact, different microwave powers were also
desired to be included in the experimental plan, but
the conducted various microwave powers (100–1000 W)
had no significant effect (P > 0.05) on the responses
according to the preliminary results. That was because
the specific microwave extraction systems supply inter-
mittent power to keep constant temperature at the
target level. For this reason, process temperature
instead of microwave power was evaluated as indepen-
dent variable, which was reached by 700 W intermittent
microwave power. Similar approach was conducted by
other researchers.[22,37]
Three dimensional response surface plots (Fig. A7)
show the interactive effects of independent variables. As
can be seen, significant increases in rebaudioside A, TF,
and TEAC (ABTS) were observed between 1 and 13 min.
Other responses, i.e. stevioside and TPC exhibited highest
yields at less than 13 min depending on the process
temperature. The differences would arise from the differ-
ent nature of the extracting compounds and also their
different intracellular compartments. The yields of stevio-
side and TPC increased with the extraction time from 1 to
10 min and extraction temperature from 35 to 50°C. With
a further increase in the time and temperature, the
responses decreased slightly. It has been emphasized
that microwave irradiation interacts directly with cellular
matrix through in situ molecular interaction and thus
MAE offers quick penetration of solvent and the dissolu-
tion of the compounds to be extracted.[38,39] The conco-
mitant increase in time and temperature during MAE
decreases the viscosity of solution, increases molecular
movement and solubility which then accelerates recipro-
cal mass transfer between extraction solution and plant
matrix.[40] However, higher temperature is also associated
with degradation of thermo-sensitive compounds, caus-
ing decrease in yields.[41]
Ultrasound-assisted extraction (UAE) of steviol
glycosides and antioxidant compounds
Table 3 shows the process variables and experimental
data of UAE. The ANOVA results, F-tests of the mod-
els, regression coefficients and the individual

Table 3. RSM experimental design and responses belonging to microwave and ultrasound assisted extraction techniques.
Independent variables Dependent variables
Time Rebaudioside A Stevioside TPC TF TEAC (ABTS)
(min) (mg/g) (mg/g) (mg GAE/g) (mg CE/g) (µmol TE/g)
Temperature
(°C) MAE UAE MAE UAE MAE UAE MAE UAE MAE UAE MAE UAE
20 13 28 37.2 36.3 59.0 57.2 51.3 51.3 32.9 36.9 635.7 716.6
25 5 12 33.0 32.6 52.8 54.7 48.9 47.7 32.2 33.4 679.1 634.4
25 21 43 40.4 39.3 62.3 59.6 57.0 57.6 35.8 41.9 727.1 776.9
38 1 5 32.9 32.2 57.7 53.1 55.5 49.7 33.4 33.9 793.2 644.8
38 13 28 39.8 37.0 68.0 59.5 64.7 61.1 39.0 40.3 828.1 743.1
38 13 28 39.1 38.2 67.9 59.1 64.4 60.6 39.5 41.0 782.0 746.8
38 13 28 40.1 38.4 68.3 59.9 64.2 60.1 40.1 40.4 790.9 742.8
38 25 50 41.3 41.2 59.9 63.3 65.3 65.6 41.9 45.5 858.7 804.7
50 5 12 39.7 38.2 64.8 62.2 62.6 56.3 39.1 39.8 790.0 776.7
50 21 43 42.5 42.3 64.0 70.6 68.1 68.8 43.4 48.0 852.8 851.8
55 13 28 43.2 43.9 68.3 70.5 65.7 64.1 43.1 45.3 830.7 874.5
Table 4. Quadratic model coefficients and ANOVA results belonging to microwave assisted (MAE) and ultrasound assisted (UAE) extraction techniques.
Rebaudioside A Stevioside TPC TF TEAC (ABTS)
Model parameters Coefficient MAE UAE MAE UAE MAE UAE MAE UAE MAE UAE
Intercept β0 +23.87*** +29.62*** +11.44*** +64.30*** +6.30*** +21.79*** +15.77*** +25.99*** +256.25** +599.18***
Linear
Temperature (°C), A β1 +0.15*** −0.23*** +1.61*** −0.90*** +2.03*** +1.02*** +0.67*** +0.09*** +23.28*** −5.74***
Time (min), B β2 +1.17*** +0.45*** +3.04*** +0.15*** +1.38*** +0.56*** +0.59*** +0.35*** −1.38* +8.82***
Interaction
Temperature x Time, A x B β12 −0.01** −3.36x10−3 −0.02*** +4.35x10−3* −5.90x10−3 +3.27x10−3* +1.62x10−3 −4.38x10−4 0.04 −0.09***
Second order
Temperature x Temperature, A2 β11 +2.31x10−3 +6.86x10−3* −0.01*** −0.02*** −0.02*** −9.69x10−3*** −5.34x10−3* +2.30x10−3 −0.25** +0.17***
Time x Time, B2 β22 −0.02** −2.64x10−3 −0.07*** −1.70x10−3 −0.03** −5.90x10−3*** −0.01* −1.27x10−3 0.12 −0.04***
R2 0.9913 0.9812 0.9970 0.9947 0.9934 0.9981 0.9830 0.9965 0.9599 0.9996
Adjusted R2 0.9826 0.9624 0.9940 0.9894 0.9868 0.9963 0.9660 0.9931 0.9198 0.9992
p-value <0.0001 0.0003 <0.0001 <0.0001 <0.0001 <0.0001 0.0002 <0.0001 0.0017 <0.0001
F-value 113.9 52.2 332.5 187.4 150.8 534.9 57.8 287.9 23.9 2372.3
Lack of fit 0.17 0.65 0.13 0.27 0.08 0.73 0.34 0.60 0.74 0.55
Significance levels: *P < 0.05; **P < 0.01; ***P < 0.001
Model equation: Y = β0 + β1A + β2B + β12(AxB) + β11A2 + β22B2
SEPARATION SCIENCE AND TECHNOLOGY
7
8 F. M. YILMAZ ET AL.

significance of independent variables are shown in temperatures. Moreover, corresponding responses

Table 4. All of the intercept and linear effects of UAE
had lowest probability values (P < 0.001) suggesting
high model significance. As being determined in sol-
vent extraction and MAE, R2 (0.9812–0.9996) and Adj-
R2 (0.9624–0.9992) values were fairly high and were
close to each other. The interaction effect of tempera-
ture and time for stevioside, TPC and TEAC were also
found significant (P < 0.05) in UAE. These were also
verified by non-significant lack of fit values suggesting
that the quadratic model was valid with better reliabil-
ity and precision. The mutual effects of temperature
and time in UAE were demonstrated as 3D response
surface plots (Fig. A8). Linear increases were observed
in the responses as temperature and time increased to
specific points, then reached steady points. Increasing
the temperature to 40–55°C and time to 35–50 min
gave almost highest yields for each response.
Optimization of extraction procedures
Table 5 summarizes optimum extraction conditions
with their predicted responses. In the solvent (macera-
tion) extraction, optimum EtOH concentration (50%),
and solvent ratio (118 mL/g) were estimated and fixed
for MAE and UAE techniques as considering perturba-
tion graphs and further pre-trials. Optimum time and
temperature were found as 112 min and 54 °C for
solvent extraction, 16 min and 51 °C for MAE, and
43 min and 50 °C for UAE, respectively. Results imply
that MAE and UAE decreased optimum operation time
significantly with no large reduction in optimum
were higher in both MAE and UAE. Despite MAE
provided the lowest operation time, the highest
responses were observed in UAE. The advantage of
MAE over maceration is ascribed to the disruption of
weak hydrogen bounds promoted by the dipole rota-
tion of the molecules.[42] On the other hand, the effec-
tiveness of UAE is explained by disturbing cellular
structures of plant tissues by acoustic streaming.[43]
At optimum UAE condition, 42.9 and 70.4 mg/g
rebaudioside A and stevioside could be extracted,
respectively. The resulting TPC, TF and TEAC values
were 68.6 mg GAE/g, 47.7 mg CE/g and 853.7 µmol
TE/g, respectively. Yildiz-Ozturk et al.[3] compared
MAE and UAE techniques in terms of steviol glyco-
sides. They found MAE more effective than UAE with
respect to higher glycoside yield with shorter process
time and also reported that 21.21 and 14.90 mg steviol
glycosides and 80.13 and 86.47 mg GAE TPC per gram
leaf could be extracted by MAE and UAE, respectively.
Jaitak et al.[10] also showed MAE as the most efficient
method compared to UAE and solvent extraction. They
reported 2.34 and 8.64% rebaudioside A and stevioside
yields for MAE, while 6.54 and 1.20%, and 4.20 and
1.98% for UAE and solvent extraction, respectively.
Bursać Kovačević et al.[5] emphasized that UAE and
MAE were economical and promising extraction tech-
niques that preserved the time and energy, and suitable
for obtaining a high-quality extract from stevia leaf.
Periche et al.[7] compared MAE, UAE and solvent
extraction and showed MAE was most efficient for
steviol glycosides whereas solvent extraction for

Table 5. Optimum process conditions and experimental results of extraction procedures.

Maceration (Solvent Extraction)
Time Temperature Solvent ratio
EtOH conc. (%) (min) (°C) (mL/g) Desirability Parameter Result
50 112 54 118 1.000 Rebaudioside A 41.1 mg/g
Stevioside 64.9 mg/g
TPC 66.6 mg GAE/g
TF 42.1 mg CE/g
TEAC (ABTS) 786.5 µmol TE/g
Microwave Assisted Extraction
Time Temperature
(min) (°C) Desirability Parameter Result
Rebaudioside A 42.8 mg/g
Stevioside 68.0 mg/g
16 51 0.967 TPC 67.8 mg GAE/g
TF 43.4 mg CE/g
TEAC (ABTS) 838.6 µmol TE/g
Ultrasound Assisted Extraction
Time Temperature
(min) (°C) Desirability Parameter Result
Rebaudioside A 42.9 mg/g
Stevioside 70.4 mg/g
43 50 0.957 TPC 68.6 mg GAE/g
TF 47.7 mg CE/g
TEAC (ABTS) 853.7 µmol TE/g

antioxidant compounds. Optimizing extraction condi-
tions is upmost important especially for MAE, because
studies showed that longer time and higher tempera-
ture in MAE results considerable degradation of target
compounds.[44] In the present study, the suitability of
the models for predicting the optimum conditions were
tested by real experiments (quadruple repetitions) and
no significant differences were evidenced between pre-
dicted and experimental values as a result of applied
t-tests (P > 0.05). The results confirmed that the quad-
ratic models were adequate for optimization.
Identification of compounds present in stevia
leaves
LC/Q-TOF/MS analysis was performed to identify
compounds present in stevia leaf extracts. Table 6
shows the molecular formulas, polarities, retention
times, m/z values, score values, and errors (ppm) of
identified compounds. A total of 65 compounds includ-
ing diterpene glycosides, phenolic compounds, flavo-
noids, amino acids, and fatty acids were tentatively
identified in stevia leaf extracts regardless of extraction
techniques. The diterpene glycosides, phenolic com-
pounds and flavonoids were generally identified in
negative polarity. Diterpene glycosides were identified
between 15 and 23 min retention times on the chro-
matogram. Isorebaudioside A, stevioside and steviol-
bioside were identified in positive polarity, but the
rest of the steviol glycosides were identified in negative
polarity. Of the diterpene glycosides in stevia leaves,
identification of stevioside, steviolbioside, rebaudioside
A, C, D, E, and F were previously reported by other
studies.[45] Sucrose was also identified in this study and
similar to our results, Molina-Calle et al.[46] also
reported the presence of disaccharide in stevia.
Chlorogenic acid, caffeic acid, and 1,3-dicaffeoylqui-
nic acid (derivative of caffeic acid) were identified as
hydroxycinnamic acids in stevia leaf extracts. These
compounds have essential roles in blood circulation of
human body. These compounds were also shown to
inhibit oxidation of LDL in vitro and might therefore
protect against cardiovascular diseases. Moreover they
inhibit the formation of mutagenic and carcinogenic
N-nitroso compounds.[47] Muanda et al.[48] and Lemus-
Mondaca et al.[49] reported the presence of those com-
pounds in stevia leaf, but the latter study could identify
in dried forms, not in fresh leaves. Quercetin and its
derivatives, i.e. quercetin 3-galactoside and herbacetin
were identified as important members of flavonoids.
Mendes et al.[50] also identified those compounds in
stevia leaves and Boots et al.[51] reported significant
protective effects of these compounds on osteoporosis,
SEPARATION SCIENCE AND TECHNOLOGY 9
cardiovascular diseases and also certain forms of
cancer.
Esculetin and 3-Hydroxycoumarin were identified as
coumarin derivatives in this study and to the best of
our knowledge, there has been no any report showing
presence of these compounds in stevia leaf to date.
Apart from these compounds, diosmetin, casticin,
dioonflavone and kaempferol-7-o-glucoside, which
were identified flavonoids in this study have not been
reported in the literature.
The presence of L-Tyrosine, D-Tryptophan as amino
acids were observed in the stevia leaf extracts.
Tryptophan derivative kynurenic acid and histidine
derivative urocanic acid were also detected in stevia
leaf extracts. Contrary to identification in this study,
previous studies specifically reported that stevia do not
contain tryptophan.[21]
Prostaglandins (physiologically active lipid com-
pounds) have been rarely identified in plant sources.[52]
In this study, prostaglandin derivatives such as 16-phe-
noxy tetranor Prostaglandin E2, 19(R)-hydroxy-PGF1α,
11β-Prostaglandin F1β, 11-deoxy-PGF1β, 11-deoxy-
PGE1 and 11-Deoxy-16,16-dimethyl-PGE2 were identi-
fied in the extracts. For this viewpoint, only Melis[53]
reported that stevia leave extracts would exhibit prosta-
glandin activity on rats.
Stearidonic acid, stearic acid, stearamide and melis-
sic acid were the identified fatty acids. There was no
any report detecting melissic acid in stevia leaves in the
literature to date. Apart from all these compounds,
butaprost (a free acid), glycerophosphocholine (a gly-
coprotein) and trigonelline (plant hormone) were
detected in stevia leaf extracts.
Conclusions
The highest steviol glycosides, phenolic, flavonoid con-
tents as well as antioxidant capacity values were
obtained by UAE, followed by microwave-assisted
and maceration extraction. The extraction times were
16, 43 and 112 min for MAE, UAE, and maceration
extraction, respectively. The novel technologies, i.e.
MAE and UAE methods significantly reduced the pro-
cess time. A total of 65 metabolites were identified as
a result of LC/Q-TOF/MS analysis and some of the
compounds present in stevia leaf were reported for
the first time with this study. Future studies should
be implemented to evaluate stabilities and functions
of the obtained stevia extracts when used within var-
ious food materials. Results of this study would be
useful for industrial and scientific communities in sev-
eral disciplines such as food, pharmacology, biology,
and chemistry.
10 F. M. YILMAZ ET AL.

Table 6. Identified compounds present in stevia leaf extract by LC/Q-TOF/MS.

Compound Molecular formula Polarity RT (min) m/z Error (ppm) Score
Sucrose C12H22O11 - 1.22 341.1097 −2.35 95.71
Glycerophosphocholine C8H21NO6P + 1.28 258.1099 0.80 91.91
Trigonelline CH8NO2 + 1.39 138.0553 −2.59 85.48
L-Tyrosine C9H11NO3 + 1.73 182.0818 −3.33 90.46
Urocanic acid C6H6N2O2 + 1.85 139.0499 1.89 88.95
Guanosine C10H13N5O5 - 2.19 282.0848 −1.36 97.55
Adenosine C10H13N5O4 + 2.22 268.1044 −1.39 100.0
Salidroside C14H20O7 + 3.19 323.1107 −1.98 72.28
D-Tryptophan C11H12N2O2 - 3.31 203.0830 −2.14 97.42
Chlorogenic acid C16H18O9 - 3.60 353.0889 −3.08 99.27
Esculetin C9H6O4 - 6.02 177.0201 −4.23 95.52
Esculin C15H16O9 - 6.29 339.0734 −3.60 97.52
Kynurenic acid C10H7NO3 + 6.31 190.0503 −2.48 95.75
Dioonflavone C36H30O10 + 6.64 623.1937 −4.00 38.74
Caffeic acid C9H8O4 - 7.05 179.0357 −4.19 99.91
3-Hydroxycoumarin C9H6O3 - 7.38 161.0249 −3.27 85.08
Prim-o-glucosylcimifugin C22H28O11 + 9.11 469.1684 4.30 83.34
Herbacetin C15H10O7 + 12.22 303.0503 −1.00 92.92
Quercetin 3-galactoside C21H20O12 - 12.57 463.0898 −3.35 98.82
Quercetin C15H10O7 + 12.63 303.0503 −1.00 92.74
Isoquercitrin C21H20O12 + 12.67 465.1029 −0.33 93.02
16-phenoxy tetranor prostaglandin E2 C22H28O6 + 13.35 411.1783 −1.22 68.89
Kaempferol-7-o-glucoside C21H20O11 - 13.43 447.0953 −4.50 83.38
19(R)-hydroxy-PGF1α C20H36O6 + 14.38 395.2403 0.14 83.71
Genistein C21H20O10 + 14.50 433.1126 −0.40 100.0
1,3-Dicaffeoylquinic acid C25H24O12 + 14.66 517.1345 0.27 98.54
Rebaudioside D C50H80O28 - 15.18 1127.484 −6.78 100.0
11β-Prostaglandin F1β C20H36O5 + 16.37 379.2453 0.63 89.20
Bromhexine C14H20Br2N2 + 16.54 375.0056 2.20 91.53
Diosmetin C16H12O6 - 16.71 299.0574 −4.20 94.98
Hieracin C15H10O7 - 16.88 301.0364 3.31 99.72
Rebaudioside B C38H60O18 - 17.22 803.3774 −8.30 28.34
Izorebaudioside A C44H70O23 + 17.37 989.4188 1.28 100.0
Rebaudioside A C44H70O23 - 17.42 965.4322 −9.00 82.76
15(R)-PGE1 C20H34O5 + 17.54 377.2294 0.87 100.0
8-izo-PGF2β C20H34O5 + 17.58 377.2294 1.28 100.0
Isostevioside C38H60O18 - 17.78 803.3774 −8.30 86.09
11-deoxy-PGF1β C20H36O4 + 18.05 363.2501 1.39 84.60
Rebaudioside C C44H70O22 - 18.55 949.4379 −9.82 88.58
Rebaudioside F C43H68O22 - 18.62 935.4207 −8.32 81.85
8-iso Prostaglandin F1β C20H36O5 + 18.91 379.2453 0.66 100.0
15(R),19(R)-hidroxyprostaglandin E1 C20H34O6 + 18.94 393.2235 3.50 100.0
Stevioside C38H60O18 + 19.51 827.3671 −0.02 100.0
8-iso Prostaglandin F1α C20H36O5 + 20.09 379.2452 0.78 100.0
16,16-dimethyl-6-ketoprostaglandin E1 C22H36O6 + 21.09 419.2400 1.03 100.0
11-deoxy-PGE1 C20H34O4 + 21.20 361.2348 −0.28 100.0
Steviolbioside C32H50O13 + 21.26 665.3143 0.11 65.81
PGF2β C20H34O5 - 22.17 353.2348 −4.19 87.62
1a,1b-dihomo-PGE1 C22H38O5 + 22.34 405.2607 1.05 100.0
Limaprost C22H36O5 + 22.44 403.2457 −0.41 100.0
5-trans Prostaglandin D2 C20H32O5 + 22.68 375.2136 1.80 89.62
Isosteviol C20H30O3 - 22.69 317.2138 −5.08 50.88
16,16-dimethyl-PGD2 C22H36O5 + 22.73 403.2457 −0.41 89.89
Phytosphingosine C18H39NO3 + 22.92 318.3001 0.60 84.43
Casticin C19H18O8 - 22.97 373.0943 −3.68 94.35
20-ethyl-PGE2 C22H36O5 + 23.57 403.2457 −0.59 100.0
15-F2 t-IsoP C20H34O5 + 23.63 377.2294 0.87 89.06
Butaprost C24H40O5 + 24.56 431.2768 −0.07 41.07
Stearidonic Asit C18H28O2 + 24.76 277.2161 −0.70 80.30
11-Deoxy-16,16-dimethyl-PGE2 C22H36O4 + 24.93 387.2506 0.07 100.0
1-Palmitoyllysophosphatidylcholine C24H51NO7P + 25.22 496.3394 0.52 99.78
3b,12a-Dihidroxy-5b-cholanic acid C24H40O4 + 26.93 415.2814 0.99 59.28
Stearic acid C18H36O2 - 28.95 283.2657 −4.93 68.58
Stearamide C18H37NO + 29.01 284.2948 −0.03 93.05
Melissic acid C30H60O2 - 31.28 451.4542 −4.79 64.57

Acknowledgements Funding
Authors would like to give special thanks to Mr. Selahattin This study was financially supported by Aydın Adnan
Güvenç (Burhaniye Stevia Garden, Balıkesir, Turkey) for his Menderes University Scientific Research Projects Unit
kind and outstanding supports. [ADÜ – BAP; Project#: MF18019].

Novelty statement
The global growing interests to stevia extracts have led to
more technological and scientific efforts on the subject espe-
cially in the last decade. The plantation and marketing of
stevia are tremendously increasing all over the world. Stevia
is not only recommended due to its diterpene glycosides, but
also recognized with its phytochemical contents which have
approved positive health effects. This study for the first time
compared maceration, microwave assisted and ultrasound
assisted extraction techniques by optimizing for steviol glyco-
sides and polyphenols all together. In addition, a couple of
unexploited compounds present in stevia were for the first
time identified by LC/Q-TOF/MS. This study could be an
insight for several technologists and scientists in several dis-
ciplines such as food, pharmacology, biology and chemistry.
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