Optimization of ultrasonic-assisted extraction

conditions for bioactive components from coffee

leaves using the Taguchi design and response
surface methodology
Xiumin Chen , Jian Ding, Dayi Ji, Suqun He, and Haile Ma

Abstract: Coffee leaves contain various bioactive compounds that are beneficial for human health. However, there
are very limited researches related to the extraction of the bioactive phytochemicals from coffee leaves. In the present
study, the extraction conditions for bioactive components from coffee leaves were optimized using Taguchi design and
response surface methodology (RSM). Taguchi design was used to screen significant factors that affected the yield of
phytochemicals including trigonelline, caffeine, chlorogenic acids, mangiferin, and rutin, total phenolic content (TPC)
and antioxidant activity. Sequentially, a Box-Behnken design (BBD) was used to optimize the extraction conditions.
Three factors including Liquid-to-solid (L:S) ratio, ethanol concentration, and extraction temperature that significantly
affected most of the phytochemical yields and antioxidant activity were selected from the six variables using Taguchi
design. The optimal extraction conditions obtained from RSM were 30.3:1 L:S ratio, 54.5% ethanol, and 80 °C when
simultaneously considered four responses, including TPC, the yields of mangiferin and 5-CQA and DPPH scavenging

Food Chemistry
capacity. Under the optimal conditions, the experimental results for the above four responses were 62.1 mg gallic acid/g,
4.1 mg/g, 11.4 mg/g, and 356.9 µmol Trolox/g, respectively, which were close to the predicted values. About 97% of
phytochemicals can be extracted in the first two times of extraction. In conclusion, the combination of Taguchi design
and response surface methodology can be successfully used to screen and optimize the significant factors that affected the
bioactive components extracted from coffee leaves.

Keywords: Coffee leaves, ultrasonic-assisted extraction, Box-Behnken design, Taguchi design, antioxidant

Practical Application: Coffee leaves, the byproducts of coffee plants, are considered no- or low-value although it has
a long history for using them as tea-like beverage and ethnomedicine by locals in the coffee plant growing countries.
Bioactive components extracted from coffee leaves can be used as ingredients in functional beverages, functional food,
and natural health products. These applications will add values to coffee leaves as well as increase the incomes of coffee
farmers and workers.

1. INTRODUCTION diabetics, anti-cardiovascular, and anti-cancer capacities (Campa

Coffee plants are widely grown in the developing countries such
as Brazil, Vietnam, Columbia, Indonesia, Ethiopia, and others that
are located in tropical or subtropical regions (FAOSTAT, 2017).
According to the statistics of the Food and Agriculture Organiza-
tion of the United Nations, the global coffee harvest area reaches
10.8Mha in 2017 (FAOSTAT, 2017). Coffee leaves are coffee
plant by-products that are generally considered non- or low-value
compared with the highly valuable coffee beans. However, coffee
leaves contain various phytochemicals such as chlorogenic acids,
mangiferin, rutin, caffeine, and trigonelline that possess different
bioactivities including antioxidant, anti-inflammatory, anti-
JFDS-2019-1972 Submitted 12/6/2019, Accepted 2/19/2020. Authors are with
School of Food and Biological Engineering, Jiangsu University, 301 Xuefu Road,
Jingkou District, Zhenjiang, Jiangsu, 212013, P.R. China. Authors Chen and Ma
are also with Institute of Food Physical Processing, Jiangsu University, 301 Xuefu
Road, Jingkou District, Zhenjiang, Jiangsu, 212013, P.R. China. Author Chen
is also with International Research Center for Food Nutrition and Safety, Jiangsu
University, Zhenjiang, 212013, China. Direct inquiries to author Chen (Email:
xmchen@ujs.edu.cn).
et al., 2012; Chen, 2019; Chen, Mu, & Kitts, 2019). Coffee leaves
have been used as ethnomedicine and tea-like beverage in coffee
plant growing countries such as South Sudan, Ethiopia, Indonesia,
et al. (Patay, Bencsik, & Papp, 2016; Ross, 2005). Recently,
industry applications of coffee leaves such as therapeutic agents for
hypertension, gastrointestinal inflammation, and diabetes; tobacco
substitute; animal feed; packaging material; personal hygienic
products; vehicle perfume; and antimicrobial agents et al. have
been discovered (Chen, 2019; Kenconojati, Ulkhaq, Budi, &
Azhar, 2019; Segheto et al., 2018). Due to the rich resources and
various applications, extraction of bioactive components from
coffee leaves is considered a promising way to produce value-
added products such as functional food, natural health products,
and beverage. However, to date, the information regarding the
extraction of bioactive components from coffee leaves are scant. In
our previous researches, hot water (Chen, Ma, & Kitts, 2018) and
80% methanol (Chen, Kitt, Ji, & Ding, 2019) were used to extract
phytochemicals from coffee leaves, however, both studies did not
focus on the extraction methods for phytochemicals. Marcheafave
et al. (2019) investigated the solvent effects on the pigments and

C 2020 Institute of Food Technologists

 R

doi: 10.1111/1750-3841.15111 Vol. 0, Iss. 0, 2020 r Journal of Food Science 1

Further reproduction without permission is prohibited
 Optimizing extraction of coffee leaf . . .
antioxidant activities of coffee leave extract using experimental
mixture design. However, this study only focused on the solvent
effects and pigments instead of other extraction conditions and
beneficial phytochemicals such as chlorogenic acids, mangiferin,
rutin, et al.
Extraction conditions such as type of solvent, liquid-to-solid
(L:S) ratio, extraction temperature and time, and particle size
are known to affect the yield and bioactivities of plant extracts,
however, their impacts varied. Traditional one factor at a time
experiment is time-consuming and does not provide interaction
information. Taguchi design is very effective in the screening pro-
cedure to identify the significant factors while reduces the number
of experiments (Ashengroph, Nahvi, & Amini, 2013). Response
surface methodology (RSM) is an efficient mathematical and
statistical method that is generally used to improve parameters
in different procedures and to explore their probable interactions
using a mathematical model to predict the optimal conditions and
responses. RSM has been applied in the optimization of extraction
conditions (Ayim, Ma, & Alenyorege, 2018; Zhang & Ma, 2017).
The integration of Taguchi design and RSM approaches has been
widely applied in process optimization (Dash, Mohammed, &
Humaira, 2016; Thakur, Das, & Das, 2016). Ashengroph et al.
Food Chemistry
(2013) used Taguchi design to screen the significant influencing
components from ten ingredients that affect the conversion
of isoeugenol to vanillin by Psychrobacter sp. CSW4, followed
by optimization of the selected important factors using RSM.
Notwithstanding, to the best of our knowledge, there was no pre-
vious study regarding the extraction of bioactive compounds from
coffee leaves utilizing ultrasound-assisted extraction with Taguchi
design and RSM. Therefore, in the present study, the combination
of Taguchi design and RSM was used to screen the significant
factors and to the optimization of extraction conditions.
In the present study, a two-levels, six-factors, Taguchi design
was first used to screen the significant factors from six variables
including ethanol concentration, extraction temperature and
time, L:S ratio, age of coffee leaves, and ultrasound power. Twelve
responses that are the yield of trigonelline, caffeine, mangiferin,
rutin, chlorogenic acids including 3-caffeoylquinic acid (3-CQA),
4-caffeoylquinic acid (4-CQA), 5-caffeoylquinic acid (5-CQA),
3,4-dicaffeoylquinic acid (3,4-diCQA), 3,5-dicaffeoylquinic acid
(3,5-diCQA), 4,5-dicaffeoylquinic acid (4,5-diCQA), total phe-
nolic content (TPC), and 2,2-diphenyl-1-picrylhydrazyl (DPPH)
free radical scavenging capacity were measured. Sequentially,
the optimal extraction conditions for the three most significant
factors including L:S ratio, ethanol concentration, and extraction
temperature were optimized using Box-Behnken design (BBD)
when simultaneously optimized mangiferin, 5-CQA, TPC, and
DPPH scavenging capacity under ultrasonic condition. The
impacts of extraction times on the phytochemical content and
antioxidant activities of coffee leaf extracted with or without
ultrasound treatment were also compared.
2. MATERIALS AND METHODS
2.1 Coffee leaves and chemicals
Dry coffee leaves were donated by Wize Monkey Inc. (Vancou-
ver, BC, Canada). Young and mature Coffea Arabica var. Catimor
leaves were collected from Finca La Aurora coffee farm located in
the Matagalpa area of Nicaragua (13°03 35.5 N 85°54 19.0 W),
between August 18 and 25, 2016 and prepared with Chinese-style
green tea processed method (Chen et al., 2018). Trigonelline,
caffeine, mangiferin, rutin, and chlorogenic acids (CGAs) isoform
2 Journal of Food Science r Vol. 0, Iss. 0, 2020
standards comprising 3-CQA, 4-CQA, 5-CQA, 3,4-diCQA,
3,5-diCQA, 4,5-diCQA were obtained from Chengdu Must
Bio-Technology Co. Ltd. (Chengdu, China). DPPH and
(±)-6-hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid
(Trolox) were purchased from Shanghai Macklin Biochemical Co.
Ltd. (Shanghai, China). Folin-Ciocalteu reagent was purchased
from Beijing Solarbio Science & Technology Co. Ltd. (Beijing,
China). Trifluoroacetic acid (TFA) and ethanol were purchased
from Sinopharm Chemical Reagent Co. Ltd. (Shanghai, China).
HPLC grade acetonitrile (ACN) were purchased from Tedia
Company Inc. (Fairfield, OH, USA).
2.2 Extraction procedure
Approximately 1.0 g of coffee leaves were placed into a 50 mL
centrifuge tube that contained an appropriate volume of solvent.
After extraction, the resulting solution was filtered, centrifuged
(5,000 × g), and stored in the refrigerator for further study. All
experiments were performed in triplicate.
2.3 Analysis of total phenolic contents
Total phenolic contents of coffee leaf extracts were determined
using Folin–Ciocalteu method described by Chen, Tait, and Kitts
(2017) and the data were expressed as mg gallic acid (GA)/g
coffee leaves. Briefly, 20 µL appropriately diluted coffee leaf
extracts or gallic acid (0.1 mg/mL) were incubated with 100 µL,
ten times diluted Folin–Ciocalteu solution for 1 min followed by
reacting with 80 µL, 75 mg/mL Na2 CO3 for 30 min in a 96-well
plate. The absorbance was recorded at 765 nm with Spark  R
10M
multimode microplate reader (Tecan, MA, USA). Each sample
was measured three times.
2.4 Analysis of antioxidant activity
The antioxidant activity of coffee leaf extracts was determined
by the DPPH method, as described previously by Chen et al.
(2018). Briefly, coffee leaf extracts were diluted to 4 to 20 times
using methanol, then 5 to 50 µL diluted samples or Trolox
(0.2 mM) were added into 96-well plate that contained 20 µL,
1 mM DPPH. An appropriate amount of methanol was added
into each well to make up the final volume to 200 µL. The
mixtures were incubated at room temperature in darkness for
10 min followed by reading at 519 nm with Spark R
10M
multimode microplate reader (Tecan, MA, USA). The data were
expressed as µmol Trolox/g coffee leaves.
2.5 HPLC analysis of phytochemical content
The phytochemical contents of coffee leaves were analyzed and
quantified by the method that was described by Chen et al. (2018)
with some modification. Waters HPLC system including Waters
2707 autosampler, Waters 2998 photodiode array detector and
Waters 1525 binary HPLC Pump, equipped with a Phenomenex
Kinetex C18 (100 × 4.8 mm, 5 µm) column (Phenomenex,
Torrance, CA, USA) was used to quantify phytochemical contents
in the coffee leaf extract. The temperature was set at 25 °C
and the flow rate was 1.5 mL/min. Appropriately diluted coffee
leaf extracts (5 µl) were injected into the column and eluted
with solvent A (0.1% TFA in water) and solvent B (ACN). The
mobile phase gradient was as follows: 0 to 10 min, solvent B
linearly increased from 5% to 20%; 10 to 12 min, solvent B was
maintained at 20%; 12 to 15 min, solvent B decreased from 20%
to 5%; 15.1 to 16.5 min, solvent B was maintained at 95%; 16.6
to 19 min, solvent B was maintained at 5%. The compounds in-
cluding trigonelline, caffeine, CGAs (3-CQA, 4-CQA, 5-CQA,
Optimizing extraction of coffee leaf . . .

Figure 1–HPLC profile of coffee leaf extract

(monitored at 257 nm).

3,4-diCQA, 3,5-diCQA, 4,5-diCQA), rutin, and mangiferin Where d1 . . . dm is the individual desirability for 1 . . . m
were detected at 264, 280, 330, 257, and 257 nm, respectively. number of responses; d is the overall desirability.
The HPLC profile of coffee leaf extract is shown in Figure 1.
2.7 Statistical analysis
2.6 Experimental Design

2.6.1 Taguchi design. A two-level Taguchi L8 (26 )
orthogonal design (Table 1) was used to screen the significant
factors out of six independent variables including L:S ratio,
ethanol concentration, extraction temperature, extraction time,
ultrasound power, and age of leaves. The twelve dependent
variables that were evaluated included the yields of caffeine,
trigonelline, chlorogenic acids (3-CQA, 4-CQA, 5-CQA, 3,4-
diCQA, 3,5-diCQA, and 4,5-diCQA), mangiferin, and rutin,
TPC, and DPPH scavenging capacity. The factors and levels are
as follows: ethanol concentration (0 & 60%), temperature (30 and
80 °C), ultrasound power (0 and 210 W), time (10 & 40 min),
coffee leaf age (young and mature), and L:S ratio (10:1 and 40:1).
2.6.2 Box–Behnken design. A Box–Behnken design
(BBD) comprised of three factors, three levels, and three center
points that includes 15 experiments (Table 2) was utilized to deter-
mine the optimal conditions of L:S ratio, ethanol concentration,
and extraction temperature on the responses when mature coffee
leaves were extracted for 10 min at ultrasound power of 210W
(ultrasonic cleaner KQ-300DE, Kunshan Ultrasonic Instrument
Co., Ltd). The 12 responses were analyzed by multiple regression
equation to fit into a second-order polynomial model shown as
follows:
 k K 
k−1
Y = β0 + βi Xi + βi i Xi2i + βi j Xi X j (1)
i =1 i =1 i the response variables, thus reduce the time and experimental cost.
where Y is the predicted response; Xi (i = 1, 2, 3) are independent
variables (L:S ratio, ethanol concentration (%), and temperature),
β 0 , βi, βii, and βij are the regression coefficients for the intercept,
linear, quadratic, and interaction coefficients of the model, respec-
tively. The optimal conditions for each response were predicted
based on the regression equation. In addition, the extraction con-
ditions when simultaneously optimized multi-responses including
TPC, DPPH scavenging capacity, 5-CQA, and mangiferin were
predicted based on desirability functions. The overall desirability
that represents the desirability of the multi-responses can be cal-
culated from the individual desirability values using the following
equation (Derringer & Suich, 1980).
d = (d1 · d2 · . . . · dm) 1/m
(2)
Food Chemistry
All experiments were carried out in triplicates. Taguchi and
BBD were designed and analyzed using Minitab version 17
software from Minitab Inc. (State College, PA, USA). Significant
differences were evaluated by analysis of variance (ANOVA) at P
< 0.05 and Tukey’s test was used to perform pairwise comparison.
3. RESULTS AND DISCUSSION
3.1 Results from the Taguchi design
Numerous factors such as solvent type and concentration, L:S
ratio, time, temperature, ultrasonic power, and particle size are
known to affect the extraction of bioactive components. How-
ever, the significance of those factors varied due to different plant
sources and responses that are evaluated. Therefore, screening of
factors that significantly affect the dependent variables before the
optimization of the extraction conditions is very critical in con-
sideration of reducing time and cost. The drawbacks of the single
factor experiment include that first, only one factor is varied at a
time while the rest of the factors remained the same; second, no in-
teraction information can be obtained, which results in difficulty
to correctly compare the relative significance of multi-factors.
Taguchi design is a robust parameter design that applies the orthog-
onal array for process optimization when a minimum number of
experiments are used. A two-level multi-factor Taguchi design can
be effectively used to screen the factors that significantly influence
The Taguchi experimental results are presented in Tables 1
and the main effects of each factor on different responses were
shown in Figure 2. The extraction yields of phytochemicals,
TPC, and DPPH free radical scavenging capacity of coffee leaf
extracts were increased as the extraction temperature, ethanol
concentration, L:S ratio, extraction time and ultrasound power
increased, whereas, in case of the age of leaves, the results were
vice versa. Our previous studies also showed that mature leaves
contained less chlorogenic acids, mangiferin, rutin, TPC and
antioxidant activities compared with their young counterparts
(Chen et al., 2019; Chen et al., 2018).
The ranking of the impact of each factor on the twelve
responses are shown in Table 1S (supplement data). L:S ra-
tio was the most significant factor that affected the yield of

Vol. 0, Iss. 0, 2020 r Journal of Food Science 3

Food Chemistry Optimizing extraction of coffee leaf . . .

Figure 2–Taguchi experimental results of the main effects of different factors on mean values of different responses. (A) Trigonelline; (B) Caffeine;
(C) 3-CQA; (D) 4-CQA; (E) 5-CQA; (F) 3,4-diCQA; (G) 3,5-diCQA; (H) 4,5-diCQA; (I) Mangiferin; (J) Rutin; (K) TPC; (L) DPPH scavenging capacity. T:
temperature (°C), L:S: liquid-to-solid ratio, UP: ultrasound power (W), t: time (min), Y: young coffee leaves, M: mature coffee leaves. TPC: total phenolic
content, expressed as mg gallic acid per g coffee leaf (dry weight). DPPH is expressed as µmol Trolox per g coffee leaf (dry weight).

phytochemicals and the antioxidant activity and ethanol concen-
tration was the second most important factor followed by the
extraction temperature. Extraction time, ultrasound power, and
age of leaves did not significantly affect most of the responses.
A studied conducted by Belwal, Dhyani, Bhatt, Rawal, and
Pande (2016) also showed that extraction temperature, L:S ratio,
and solvent concentration significantly affected the TPC, total
flavonoids, total anthocyanins, total tannins, and antioxidant
activities of the extracts derived from Berberis asiatica fruits,
whereas, extraction time and pH did not have significant effects.
Therefore, L:S ratio, ethanol concentration, and extraction
temperature were selected for further optimization using BBD.
3.2 Results for RSM
RSM is a combined mathematical and statistical techniques
that are used to model and analyze the response variables that
are affected by several independent variables and thus reach the
optimization purpose.
The results of RSM are shown in Table 2. The ranges of
mangiferin, 5-CQA, TPC, and DPPH scavenging capacity were
0.53 to 4.29 mg/g, 4.53 to 11.98 mg/g, 22.57 to 57.43 mg
GA/g, and 122.23 to 359.36 µmol Trolox/g, respectively. The
dependent variables and the independent variables were fitted
into the second-order polynomial equations that were shown in
supplement data. The ANOVA Tables 2S -12S (supplement data)
listed the P values of model, linear, quadratic, and interaction
terms for each response. After deleting the non-significant
quadratic and interaction terms, the variables were fitted into the
following equations:
Trigonelline = 2.560 + 0.4561 × 1 − 0.0236 × 2 + 0.0118 ×
3 − 0.007780 × 12 − 0.001038 × 1 ∗ X2 −
0.000848 × 1 ∗ X3 + 0.000735 × 2 ∗ X3

4 Journal of Food Science r Vol. 0, Iss. 0, 2020

 Table 1–Taguchi design and the results for the factor screening experiments.

Taguchi design Phytochemical content (mg/g)

DPPH
T EtOH L:S Time UP Age Trigon- 3- 4- 5- 3,4- 3,5- 4,5- Mangi- TPC (mg (µmol
No. (°C) (%) (w:v) (min) (W) (M) elline Caffeine CQA CQA CQA diCQA diCQA diCQA Rutin ferin GA/g) Trolox/g)

1 30 0 10 10 0 Y 3.7 ± 0.0 6.0 ± 0.1 0.1 ± 0.1 0.2 ± 0.0 4.0 ± 0.2 0.0 ± 0.0 0.4 ± 0.1 N.D. 1.0 ± 0.0 1.1 ± 0.0 19.1 ± 1.5 78.0 ± 6.0
2 30 0 10 40 210 M 4.0 ± 0.1 5.6 ± 0.1 0.1 ± 0.0 0.2 ± 0.0 5.0 ± 0.4 0.1 ± 0.0 0.5 ± 0.0 N.D. 1.4 ± 0.0 1.4 ± 0.0 25.3 ± 0.9 105.3 ± 6.6
3 30 60% 40 10 0 M 9.3 ± 0.7 8.7 ± 0.6 0.3 ± 0.0 0.4 ± 0.0 10.7 ± 0.7 0.3 ± 0.0 1.4 ± 0.1 0.5 ± 0.0 2.2 ± 0.3 2.2 ± 0.3 48.0 ± 2.6 272.4 ± 5.1
4 30 60% 40 40 210 Y 10.7 ± 0.1 12.2 ± 1.0 0.4 ± 0.0 0.5 ± 0.0 12.3 ± 0.8 0.4 ± 0.0 2.1 ± 0.2 0.5 ± 0.0 2.4 ± 0.4 2.4 ± 0.2 52.6 ± 3.2 295.0 ± 12.7
5 80 0 40 10 210 Y 8.5 ± 0.4 12.0 ± 0.1 0.3 ± 0.0 0.4 ± 0.0 9.8 ± 0.1 0.3 ± 0.0 1.7 ± 0.0 0.4 ± 0.0 2.2 ± 0.2 2.1 ± 0.0 47.2 ± 0.2 255.6 ± 11.2
Optimizing extraction of coffee leaf . . .

6 80 0 40 40 0 M 8.0 ± 0.5 9.0 ± 0.7 0.2 ± 0.0 0.4 ± 0.0 8.4 ± 0.1 0.2 ± 0.0 1.1 ± 0.1 0.4 ± 0.0 2.0 ± 0.2 2.2 ± 0.2 43.2 ± 0.9 242.8 ± 19.2
7 80 60% 10 10 210 M 6.8 ± 0.1 6.9 ± 0.4 0.2 ± 0.0 0.3 ± 0.1 7.7 ± 0.2 0.2 ± 0.0 0.9 ± 0.3 0.3 ± 0.1 1.7 ± 0.2 2.0 ± 0.2 40.9 ± 3.9 252.2 ± 10.6
8 80 60% 10 40 0 Y 8.2 ± 0.4 9.3 ± 0.1 0.3 ± 0.0 0.4 ± 0.0 9.4 ± 0.7 0.2 ± 0.0 1.7 ± 0.1 0.4 ± 0.0 2.1 ± 0.0 1.7 ± 0.0 42.1 ± 0.6 267.0 ± 19.7

Data are expressed as mean ± SD of triplicate experiments. T: temperature, L:S: liquid-to-solid ratio, UP: ultrasound power, Y: young coffee leaves, M: mature coffee leaves. TPC: total phenolic content, expressed as mg gallic acid per g coffee
leaf. DPPH is expressed as µmol Trolox per g coffee leaf.

Table 2–Box–Behnken design and results for optimization experiments.

Box-Behnken design Phytochemical content (mg/g)

L: S EtOH T Trigon- 3- 4- 5- 3,4- 3,5- 4,5- Mangi- TPC (mg DPPH (µmol
No. (w: v) (%) (°C) elline Caffeine CQA CQA CQA diCQA diCQA diCQA Rutin ferin GA/g) Trolox/g)

1 10 (-1) 0 (-1) 55 (0) 6.6 ± 0.1 5.5 ± 0.1 0.3 ± 0.0 0.4 ± 0.0 5.5 ± 0.3 0.4 ± 0.0 0.4 ± 0.0 0.3 ± 0.0 1.4 ± 0.1 1.6 ± 0.1 22.6 ± 2.0 122.2 ± 5.7
2 40 (+1) 0 (-1) 55 (0) 6.6 ± 0.5 7.1 ± 0.2 0.2 ± 0.0 0.2 ± 0.0 4.5 ± 0.5 0.2 ± 0.0 0.3 ± 0.0 N.D. 0.3 ± 0.0 0.5 ± 0.1 34.5 ± 1.4 144.4 ± 5.7
3 10 (-1) 60 (+1) 55 (0) 7.2 ± 0.0 7.1 ± 0.2 0.3 ± 0.0 0.5 ± 0.0 8.7 ± 0.1 0.4 ± 0.0 1.3 ± 0.0 0.5 ± 0.0 2.2 ± 0.0 3.1 ± 0.3 38.5 ± 1.0 286.2 ± 11.2
4 40 (+1) 60 (+1) 55 (0) 5.3 ± 0.3 7.0 ± 0.5 0.1 ± 0.0 0.4 ± 0.0 8.4 ± 0.5 0.3 ± 0.0 1.1 ± 0.0 0.4 ± 0.0 0.7 ± 0.0 2.7 ± 0.1 42.9 ± 0.3 340.3 ± 19.4
5 10 (-1) 30 (0) 30 (-1) 5.5 ± 0.1 6.0 ± 0.1 0.2 ± 0.0 0.3 ± 0.0 8.0 ± 0.1 0.2 ± 0.0 0.8 ± 0.0 0.2 ± 0.0 0.6 ± 0.0 2.2 ± 0.1 35.4 ± 1.2 229.4 ± 14.2
6 40 (+1) 30 (0) 30 (-1) 6.5 ± 0.1 7.6 ± 0.2 0.2 ± 0.0 0.4 ± 0.0 10.4 ± 0.2 0.3 ± 0.0 1.2 ± 0.0 0.4 ± 0.0 0.8 ± 0.0 3.0 ± 0.0 47.8 ± 2.0 263.5 ± 1.4
7 10 (-1) 30 (0) 80 (+1) 7.6 ± 0.2 6.7 ± 0.6 0.3 ± 0.0 0.6 ± 0.0 8.7 ± 0.8 0.4 ± 0.0 1.0 ± 0.0 0.4 ± 0.0 1.9 ± 0.0 2.7 ± 0.0 31.9 ± 3.1 207.4 ± 9.1
8 40 (+1) 30 (0) 80 (+1) 7.3 ± 0.1 8.2 ± 0.1 0.2 ± 0.0 0.4 ± 0.0 11.1 ± 0.3 0.4 ± 0.0 1.4 ± 0.1 0.4 ± 0.0 0.9 ± 0.0 3.4 ± 0.0 53.0 ± 1.0 336.0 ± 1.3
9 25 (0) 0 (-1) 30 (-1) 9.2 ± 0.9 7.4 ± 0.2 0.4 ± 0.0 0.4 ± 0.0 6.6 ± 0.2 0.4 ± 0.0 0.6 ± 0.0 0.3 ± 0.0 1.2 ± 0.0 2.0 ± 0.1 33.6 ± 3.5 132.3 ± 2.8
10 25 (0) 60 (+1) 30 (-1) 7.4 ± 0.1 8.9 ± 0.1 0.3 ± 0.0 0.6 ± 0.0 10.7 ± 0.5 0.4 ± 0.0 1.4 ± 0.1 0.5 ± 0.0 2.4 ± 0.0 3.6 ± 0.0 50.2 ± 1.8 318.2 ± 7.1
11 25 (0) 0 (-1) 80 (+1) 8.0 ± 0.2 7.9 ± 0.1 0.4 ± 0.0 0.6 ± 0.0 8.4 ± 0.1 0.5 ± 0.0 0.9 ± 0.0 0.3 ± 0.0 2.2 ± 0.1 2.7 ± 0.1 28.7 ± 0.6 161.8 ± 14.0
12 25 (0) 60 (+1) 80 (+1) 8.4 ± 0.0 9.5 ± 0.0 0.3 ± 0.0 0.8 ± 0.0 12.0 ± 0.2 0.5 ± 0.0 1.7 ± 0.0 0.5 ± 0.0 3.0 ± 0.0 4.3 ± 0.2 57.4 ± 3.7 359.4 ± 8.0
13 25 (0) 30 (0) 55 (0) 8.4 ± 0.5 7.9 ± 0.3 0.3 ± 0.0 0.6 ± 0.0 10. 5 ± 0.2 0.5 ± 0.0 1.3 ± 0.0 0.5 ± 0.0 2.3 ± 0.1 3.3 ± 0.1 44.5 ± 0.8 253.7 ± 0.1
14 25 (0) 30 (0) 55 (0) 9.0 ± 0.8 7.9 ± 0.2 0.3 ± 0.0 0.6 ± 0.0 10.4 ± 0.1 0.4 ± 0.0 1.2 ± 0.1 0.4 ± 0.0 2.3 ± 0.0 3.5 ± 0.0 47.0 ± 2.1 270.7 ± 11.7
15 25 (0) 30 (0) 55 (0) 7.8 ± 0.3 8.0 ± 0.2 0.3 ± 0.0 0.6 ± 0.1 10.2 ± 0.7 0.4 ± 0.0 1.2 ± 0.0 0.4 ± 0.0 2.3 ± 0.0 3.4 ± 0.2 48.8 ± 2.3 274.2 ± 17.2

Data are expressed as mean ± SD of triplicate experiments. T: temperature, L:S: liquid-to-solid ratio. TPC: total phenolic content, expressed as mg gallic acid per g coffee leaf. DPPH is expressed as µmol Trolox per g coffee leaf.

Vol. 0, Iss. 0, 2020 r Journal of Food Science 5

Food Chemistry
 Optimizing extraction of coffee leaf . . .
Caffeine = 3.696 + 0.3492 × 1 + 0.04281 × 2 − 0.0673 ×
3 − 0.005647 × 12 + 0.000716 × 32 − 0.000939
× 1 ∗ X2
3 − CQA = 0.0184 + 0.02399 × 1 − 0.002378 × 2 +
0.002244 × 3 − 0.000469 × 12 + 0.000023
× 22 − 0.000059 × 1 ∗ X3
4 − CQA = −0.2094 + 0.05144 × 1 + 0.003696 × 2
+ 0.00118 × 3 − 0.000955 × 12 − 0.000047
× 22 + 0.000042 × 32 − 0.000140 × 1 ∗ X3
+ 0.000041 × 2 ∗ X3
5 − CQA = 4.36 + 0.4267 × 1 + 0.1903 × 2 − 0.1504
× 3 − 0.007894 × 12 − 0.002130 × 22
+ 0.001570 × 32
3, 4 − diCQA = −0.1890 + 0.03549 × 1 + 0.000958 × 2
Food Chemistry
+ 0.004962 × 3 − 0.000631 × 12
− 0.000111 × 1 ∗ X3
3, 5 − diCQA = 0.191 + 0.05786 × 1 + 0.02808 × 2
− 0.01771 × 3 − 0.001072 × 12 − 0.000229
× 22 + 0.000199 × 32
4, 5 − diCQA = −0.1962 + 0.02463 × 1 + 0.00655 × 2
+ 0.00488 × 3 − 0.000453 × 12 − 0.000078
× 22 + 0.000094 × 1 ∗ X2 − 0.000108
× 1 ∗ X3
Mangiferin = 0.550 + 0.1957 × 1 + 0.06737 × 2 − 0.0507
× 3 − 0.003853 × 12 − 0.000663 × 22
+ 0.000566 × 32
Rutin = −2.636 + 0.2757 × 1 + 0.01302 × 2 + 0.03589
× 3 − 0.005187 × 12 − 0.000817 × 1 ∗ X3
TPC = 18.39 + 1.526 × 1 + 0.464 × 2 − 0.2463 × 3
− 0.02856 × 12 − 0.00662 × 22 + 0.00577
× 1 ∗ X3 + 0.00405 × 2 ∗ X3
DPPH = 112.1 + 1.61 × 1 + 5.100 × 2 − 0.9760 × 3
− 0.00617 × 12 − 0.03337 × 22 + 0.0630.1 × 1 ∗ X3
where X1: L:S ratio, X2: EtOH concentration (%), and X3:
temperature.
Surface plots, contour plots, and optimal extraction conditions
for each response are shown in Figure 3, Figure 1S (supplementary
data) and Table 3, respectively. The coefficient of determination
(R2 ) greater than 0.8 for a chemical experiment indicates a
satisfy fitness of the regression model (Lundstedt et al., 1998).
In the present study, R2 for all the response models ranged from
6 Journal of Food Science r Vol. 0, Iss. 0, 2020
0.836 to 0.946, and the adjusted R2 s are in between 0.781 and
0.962 indicating that all the models adequately agree with the
experimental results. ANOVA analysis (Table 2S to 12S) showed
that all regression models are significant (P < 0.001) for all
these twelve responses. According to the P values of lack of fit
(Table 3), the models adequately fitted the experimental data for
trigonelline, caffeine, 3-CQA, 4-CQA, 3,4-diCQA, TPC, and
DPPH radical scavenging capacity, whereas, they did not fit the
experimental data of 5-CQA, 3,5-diCQA, 4,5-diCQA, rutin,
and mangiferin due to the small P-value (P < 0.05).
3.2.1 Effects of L:S ratio. According to the results of the
Taguchi design, L:S ratio was the most important factor that
affected the yield of phytochemicals and the antioxidant activity
of coffee leaf extract. In general, the higher of L:S ratio the
greater amounts of phenolic compounds dissolved in the solvent.
However, the increased solvent volume resulted in a waste of
organic solvent, therefore, an appropriate L:S ratio is crucial
for an efficient extraction process. The yield of phytochemicals
increased with increased L:S ratio followed by a decrease with
a further increase of L:S ratio. The optimal L:S ratio for the
twelve responses ranged from 19.7:1 to 40:1 and the results varied
depending on the responses. This is partly due to the different
polarities of those phytochemicals. Other researches also found
different optimal L:S ratio for phenolic compounds. For example,
Yang et al. (2009) found that the extraction rate of phenolic
compounds increased significantly when L:S ratio increased from
10:1 to 20:1, however, further increase of L:S ratio, could not
significantly increase the extraction rate. Rajha et al. (2014)
reported that an L:S ratio of 3:1 was the best to recover total
phenolics from grape pomace. However, a much higher L:S ratio,
50:1, was used by Belwal et al. (2016) to optimize the extraction of
phenolic compounds and antioxidant activity from Berberis asiatica
fruits.
3.2.2 Effects of ethanol concentration. Due to the varied
polarity of phytochemicals in plants, various solvents are used for
phytochemical extraction. Ethanol, dichloromethane, and hexane
were used to extract pigments including pheophytin α, zeaxan-
thin, crocetin, β-carotene and chlorophyll b from Coffea arabica L.
leaves and ethanol was found to be the best solvent for the yield and
the antioxidant activities of coffee leaf extract(Marcheafave et al.,
2019). Partition of methanol extract of coffee leaves with ethyl
acetate produced a fraction that contained the highest amount of
TPC, total flavonoids, 5-CQA, and antioxidant activities (Segheto
et al., 2018). Phenolic compounds are distributed in the cell
vacuoles or in the cell wall where they are bonded with proteins or
polysaccharides with hydrogen bond or hydrophobic interaction
(Yang et al., 2009). Alcohol and water mixtures have been found
to be more effective to extract phenolic compounds compared
with the mono-component solvent (Yilmaz & Toledo, 2006).
Water and low concentration of ethanol can easily penetrate into
the cell to dissolve the phenolic compounds, whereas, high con-
centration of ethanol was often found to decrease the extraction
rate because it can cause protein denaturation, thus prevents the
dissolving of phenolic compounds. Yang et al. (2009) found that
the extraction rate for polyphenols from the bark of Phyllanthus
emblica L. increased from 30 to 70% ethanol followed by a decrease
with a further increase of ethanol concentration. The optimal
ethanol concentration was 75% under microwave-assisted extrac-
tion condition. In the present study, for most of the responses,
the increased ethanol resulted in greater phytochemical content,
TPC and antioxidant activity, whereas, in the case of 3-CQA, a
decreasing trend was found. The optimal ethanol concentration
Optimizing extraction of coffee leaf . . .

Food Chemistry
Figure 3–Response surface plots for the effects of L:S ratio, ethanol concentration, and temperature on different responses. (A) Trigonelline; (B)
Caffeine; (C) 3-CQA; (D) 4-CQA; (E) 5-CQA; (F) 3,4-diCQA; (G) 3,5-diCQA; (H) 4,5-diCQA; (I) Mangiferin; (J) Rutin; (K) TPC; (L) DPPH scavenging capacity.
X1, L:S ratio; X2, ethanol concentration; X3, temperature.

Table 3–Optimal extraction conditions, P, R2 , R2 (adj.) and predicted values for RSM.

Responses L:S ratio (w/v) EtOH conc. (%) Temperature (°C) P-value R2 R2 (adj.) Predicted values (mg/g)
Trigonelline 21.2 55.8 80 0.743 0.836 0.781 8.98
Caffeine 25.8 60.0 80 0.302 0.925 0.899 9.28
3-CQA 20.9 4.0 80 0.116 0.928 0.904 0.39
4-CQA 21.0 60.0 80 0.561 0.971 0.962 0.82
5-CQA 27.2 43.6 80 0.033 0.906 0.875 12.33
3,4-diCQA 21.5 60.0 80 0.067 0.842 0.789 0.55
3,5-diCQA 26.4 60.0 80 0.185 0.907 0.875 1.70
4,5-diCQA 23.6 57.0 80 0.000 0.846 0.794 0.58
Rutin 19.7 60.0 80 0.000 0.951 0.934 2.99
Mangiferin 26.1 51.5 80 0.007 0.899 0.865 4.34
TPC 33.0 57.6 80 0.195 0.910 0.880 56.4 mg gallic acid/g
DPPH 40.0 60.0 80 0.966 0.964 0.952 402.0 µmol trolox/g
P, significant (P < 0.05) of lack of fit; R2 is the coefficient of determination of each model; R2 (adj.) is the adjusted R2 .

(Table 3) was close to 60% for most of the responses, except for
3-CQA.
3.2.3 Effects of temperature. Our results found that in
the range of temperature that was tested in the present study, the
increase of temperature resulted in the greater phytochemical con-
tent, TPC and antioxidant activity, whereas, it showed an opposite
tend in case of 3-CQA. The optimal temperature in the range of
present study was 80 °C, indicating that increased temperature ac-
celerated the yield of phytochemicals, which positively correlated
with the increased DPPH free radical scavenging capacity.
Rajha et al. (2014) also found that the increase of temperature
caused the increases of TPC, flavonoid, tannin, anthocyanin, and
antioxidant activity of grape pomace extract and the optimum
extraction temperature was 93 °C in consideration of the above

Vol. 0, Iss. 0, 2020 r Journal of Food Science 7

 Optimizing extraction of coffee leaf . . .

DPPH (µmol
multiple responses. Other researches reported that the extraction

2.7a∗
3.2b
0.3b

4.5b
0.8c
2.8a
Trolox/g)
yield of phenolic compounds and antioxidant activities was

±
±
±
±
±
±
enhanced with increasing temperature (Santos et al., 2016). High

Data are expressed as mean ± SD of triplicate experiments. TPC is expressed as mg gallic acid per g coffee leaf. DPPH is expressed as µmol Trolox per g coffee leaf. The same group data in the same column with different letters denote
356.9
42.2
4.9
325.8
49.3
5.1
temperature accelerates the mass transfer, increases the solubility
of solute as well as reduces the surface tension and viscosity
(Rajha et al., 2014). However, heat-labile phenolic compounds

(mg GA/g)

1.3b

0.8b
0.1c

0.3c
4.8a

1.5a
can be degraded under high-temperature long-time extraction,

TPC

±
±
±
±
±
±
thus resulting in the low TPC. Yang et al. (2009) found that the

62.1
11.1
1.9
62.3
12.8
2.4
extraction rate gradually increased when temperature increased

significantly different at P < 0.05. N.D.: not detectable. Asterisk denotes significantly different when compared phytochemical contents extracted with or without ultrasound treatment at the same extraction time
from 20 °C to 50 °C followed by a decrease when further
increased the temperature. In the present study, the optimum
Mangiferin

0.1b

0.1b
0.0b
0.0c
0.1a

0.9a
temperature for all the responses was 80 °C, which was similar to
±
±
±
±
±
±
the results obtained by Belwal et al. (2016).
4.1
1.0
0.1
3.5
1.1
0.2
3.2.4 Optimal conditions when considered multi-
responses. In some experiments, researchers are interested in
0.1b

0.0b
0.0b
0.0c
0.1a

0.3a

several responses rather than a single response. In the case of

Rutin

extraction bioactive components from plant, both yield and

±
±
±
±
±
±

bioactivities are the target responses because a single response

2.5
0.6
0.1
2.1
0.7
0.1

does not adequately represent the quality of the plant extract.

3,4-diCQA 3,5-diCQA 4,5-diCQA

Moreover, due to the different polarity of phytochemicals, their

0.1 ± 0.0b

0.1 ± 0.0b
0.1 ± 0.0b
0.4 ± 0.0a

0.6 ± 0.1a

solubility varied depending on different solvents and extraction

N.D.

conditions. Therefore, the optimal extraction conditions for

Food Chemistry

different phytochemicals and the bioactivities are different. To

reach the goal of simultaneous optimization of multiple responses,
0.5 ± 0.1b

0.5 ± 0.0b
0.1 ± 0.0b
1.5 ± 0.1a

1.3 ± 0.2a

an optimal response surface model for each response is built first,

N.D.

then the condition that keeps all responses in the desired range is
assessed later. Derringer’s desirability function is one of the tech-
Phytochemical content(mg/g)

niques that is used for simultaneous optimizing multiple responses

0.1 ± 0.0b

0.1 ± 0.0b
0.4 ± 0.0a

0.4 ± 0.0a

(Montgomery, 2013). The closer of the d value to 1, the more

N.D.

N.D.

desirable response. In the present study, the appropriate response

surface models for twelve responses including trigonelline, caf-
feine, chlorogenic acids, mangiferin, rutin, TPC and antioxidant
1.3a∗
0.4b

0.2b
0.0b
0.1c
1.2a

activities were built. 5-CQA is the most abundant phenolic that

5-CQA

exists in coffee leaves. Mangiferin is a xanthone that is unique in

±
±
±
±
±
±

Coffea Arabica leaves but not detectable in coffee beans. It is well

12.4
3.2
0.4
9.9
3.5
0.5

known that mangiferin possesses various bioactivities that are able

to prevent or inhibit inflammation, diabetics, hyperlipidemia,
0.1 ± 0.0b

0.1 ± 0.0b
0.5 ± 0.2a

0.7 ± 0.1a
4-CQA

oxidation, microbial, and neuro-dysfunction (Jyotshna, Khare &

N.D.

N.D.

Shanker, 2016). The choice of the extraction conditions must

consider both the chemical compositions of the extracts and the
antioxidant activity; therefore, the aim of this research was to
0.1 ± 0.0b

0.1 ± 0.0b
0.4 ± 0.0a

0.5 ± 0.2a

establish the extraction conditions that mangiferin, 5-CQA, TPC,

3-CQA

N.D.

N.D.

and DPPH scavenging capacity were simultaneously optimized

to keep in a desirable range. Our results showed that the optimal
conditions were L:S ratio, 30.3:1; ethanol concentration, 54.5%;
temperature, 80 °C when considered those four responses
0.5a∗
0.3b

0.1b
0.0c

0.0c
1.0a
Caffeine
Table 4–Results of confirmation experiment.

simultaneously. The predicted results for the above responses were

±
±
±
±
±
±

12.01 mg/g, 4.27 mg/g, 56.1 mg GA/g, and 365.8 µmol Trolox/g,
8.5
2.1
0.2
7.1
2.3
0.3

respectively. The overall desirability for these four responses is

0.957, which is close to 1, indicating that the optimal extraction
Trigonelline
0.4 a
0.3b

0.1b
0.0c

0.0c
0.8a

conditions were overall desirable for these four responses.

±
±
±
±
±
±
7.0
2.2
0.19
6.3
2.4
0.3

3.3 Conformation experiment

The confirmation experiments were done under the above opti-
mal conditions with and without ultrasound treatment. The leaves
1
2
3
1
2
3

were also extracted three times sequentially to compare the differ-

Without ultrasound

ent extraction times on the yield of phytochemicals and antioxi-

Extraction times
With ultrasound

dant activity. Under the optimal conditions, the yield of 5-CQA,

mangiferin, and TPC were 12.4 mg/g and 4.1 mg/g, 62.1 GA/g,
respectively, and DPPH scavenging capacity reached 356.9 µmol
Trolox/g (Table 4). The experimental results were similar to the
predicted values. The first time extraction obtained 74.5 to 83.3%

8 Journal of Food Science r Vol. 0, Iss. 0, 2020

Optimizing extraction of coffee leaf . . .
and 68.4 to 87.5% phytochemicals under ultrasonic condition and
without ultrasound treatment, respectively. About 12.4 mg/g 5-
CQA was extracted from coffee leaves under ultrasound condition
at the first time extraction, which is significantly higher than the
yield of 9.9 mg/g that was obtained under the condition without
ultrasound treatment. Similar results were also found in the case
of DPPH scavenging capacity, which was 356.9 and 325.8 µmol
Trolox/g, respectively. The phytochemicals in the first two times
extraction accounted for 96.9% of the total three times extraction,
indicating most of the phytochemicals were extracted at the first
two times extraction. A greater amount of 5-CQA (16.0 mg/g vs
13.9 mg/g) and caffeine (10.8 mg/g vs 9.7 mg/g) were extracted
under ultrasonic condition compared with no ultrasonic treatment
(P < 0.05), which was associated with the higher DPPH scav-
enging capacity (404.0 µmol Trolox/g vs 379.4 µmol Trolox/g).
Green extraction techniques such as accelerated solvent,
ultrasound-assisted, microwave-assisted, high hydrostatic pressure,
subcritical fluid, supercritical fluid, and enzyme-assisted extraction
have been developed for the extraction of bioactive compounds
(Engmann, Ma, Tchabo, Ma, & Zhang, 2014; Luo et al., 2018a;
Luo, Cui, Zhang, & Duan, 2018b; Tchabo, Ma, Engmann, &
Zhang, 2015). Among these, ultrasound-assisted extraction (UAE)
is considered as a more economical, and competent extraction
method because of its simplicity, low-cost, rapidity, and low
solvent volumes (Vilkhu, Mawson, Simons, & Bates, 2008). UAE
has been widely utilized for the extraction of antioxidant phenolic
compounds from various teas (Both, Chemat, & Strube, 2014;
Li, Raza, Wang, Xu, & Chen, 2017); phenolic compounds from
red sorghum barn (Luo et al., 2018a), and phytochemicals from
mulberry (Tchabo et al., 2015).
Acoustic cavitation and the mechanical effect produced by
ultrasound wave can accelerate solvent penetrating into the
plant tissue and increase the contact surface area between
sample and solvent, thus enhance extraction efficiency (Ghafoor,
Choi, Jeon, & Jo, 2009; Zou, Jia, Li, Wang, & Wu, 2013).
Although the ultrasonic treatment did not significantly enhance
the phytochemical yield and antioxidant activity of coffee leaf
extract, it did increase 5-CQA and DPPH scavenging capacity
in the confirmation experiment. Therefore, in further study,
more ultrasonic conditions such as power, frequency and the
combination of different frequency can be investigated to further
increase the yield under ultrasound treatment.
4. CONCLUSION
An ultrasound-assisted extraction method has been developed
for extracting phytochemicals from coffee leaves using the
combination of Taguchi design and Box–Behnken Design.
Taguchi design was effectively employed to screen the significant
factors including L:S ratio, ethanol concentration and extraction
temperature from the non-significant factors such as ultrasound
power, extraction time and age of leaves. The RSM was success-
fully used to optimize the extraction conditions and the optimum
included L:S ratio = 30.3:1, ethanol concentration = 54.5%, and
temperature = 80 °C coupling with ultrasound power of 210W
and extraction time for 10 min. Under the optimal conditions,
TPC, mangiferin, rutin, and DPPH scavenging capacity were
62.1 mg GA/g, 4.1 mg/g, 11.4 mg/g, and 356.9 µmol Trolox/g,
respectively. The results indicated that combination of Taguchi
design and RSM is an effective method for the extraction of
bioactive components from coffee leaves.
ACKNOWLEDGMENTS
This study was supported by Jiangsu Specially-Appointed
Professor Program (1711360016) and Senior Talent Startup
Fund of Jiangsu University (4111360002) to X. Chen, Graduate
Student Innovative Program of Jiangsu University (5561360022)
to J. Ding and X. Chen. We thank Dr. David D Kitts from
University of British Columbia, Arnaud Petitvallet and Max
Rivest from Wize Monkey Inc. (Vancouver, BC, Canada) for the
kind donation of coffee leaves, as well as the Finca La Aurora
coffee farm in Nicaragua for providing samples of coffee leaves.
AUTHOR CONTRIBUTIONS
X. Chen conceived and designed the study, collected and
interpreted the test data, drafted and revised the manuscript. J.
Ding, D. Ji, and S. He collected and analyzed the test data. H.
Ma gave critical suggestions.
CONFLICT Of INTEREST
The authors have no conflicts of interest to declare.
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10 Journal of Food Science r Vol. 0, Iss. 0, 2020