Food Sci Biotechnol
https://doi.org/10.1007/s10068-019-00667-9
Optimizing extraction conditions for functional compounds
from ginger (Zingiber officinale Roscoe) using response surface
methodology
Jaeyoon Cha1 • Chong-Tai Kim2 • Yong-Jin Cho3
Received: 25 April 2019 / Revised: 23 July 2019 / Accepted: 6 August 2019
Ó The Korean Society of Food Science and Technology 2019
Abstract Extraction process was optimized for maximiz-
ing the contents of functional compounds from ginger
using response surface methodology which applied Box–
Behnken design. Ginger extracts were obtained at 3 levels
of ethanol concentration (0–70%) of solvent, extraction
time (30–90 min), and extraction temperature (50–70 °C)
as independent variables. The 6-shogaol and 6-gingerol of
the extracts were analyzed through HPLC. The significance
of each term in polynomial regression equations was
evaluated on functional compound contents and extraction
yield in extraction process. It was verified that the regres-
sion equations were accurate with high determination
coefficients over 0.892. The optimum ethanol concentra-
tion, extraction time, and extraction temperature for
extraction yield were determined as 41.38%, 78.16 min,
and 70 °C, respectively. The functional compound contents
predicted at optimal conditions were as follows: 39.55 mg/
g at 70%, 70 min, and 70 °C for 6-gingerol, 2.44 mg/g at
70%, 51.90 min, and 62.29 °C for 6-shogaol.
& Yong-Jin Cho
yjcho@kfri.re.kr
Jaeyoon Cha
chajaeyoon@dau.ac.kr
Chong-Tai Kim
ctkim@ieasthill.com
1
Department of Food Science and Nutrition, Dong-A
University, Busan 49315, Korea
2
R&D Center, EastHill Corporation, Suwon-si,
Gyeonggi-do 16642, Korea
3
Research Group of Process Engineering, Korea Food
Research Institute, Wanju-gun, Jeollabuk-do 55365, Korea
Keywords Extraction  Ginger  Optimization  Response
surface methodology  Regression equation
Introduction
With the economic growth and increasing average national
income, interest in securing safe food as well as in health
and food in general has been growing rapidly. Researchers
have focused on the search for active substances derived
from natural products which have useful physiological
activities without side effects. In particular, the studies on
antimicrobial, anti-aging, adult disease prevention, immu-
nity enhancement, and antioxidative effects are actively
conducted on natural plant resources (Chaovanalikit and
Wrolstad, 2004; Choi et al., 2002).
Ginger, the rhizome of Zingiber officinale Roscoe, has
been consumed as not only a spice and dietary supplement
but also medicine for asthma, cough, diabetes, stroke,
rheumatism, and stroke (Feng et al., 2011). Although gin-
ger has a few insignificant side effects and interacts with
some medications, Food and Drug Administration (FDA)
recognizes as a safe herbal medicine (Bilehal et al., 2011).
In addition, it has been used as a common additive in food
and medicine because of pungent components like gin-
gerol-related components (Bartley and Jacobs, 2000; Pawar
et al., 2011). The 6-gingerol was the most pungent among
the main pungent components such as 6-gingerol, 8-gin-
gerol, and 10-gingerol (Govindarajan, 1982). Lee and Ahn
(1985) reported that 6-gingerol exhibited anti-inflamma-
tory, antiseptic, and antioxidant activities. It has been
reported that oleoresin, gingerol, and shogaol activated
natural killer cells to enhance the effect of immunity in
studies related to the immunity effects of ginger (Zakaria-
Runkat et al., 2003; McCarthey et al., 1993). The 6-shogaol
123

and 6-gingerol were analyzed as functional compounds
from ginger in this study.
Extraction is the decisive process for recovering and
purifying functional compounds from medicinal plants
(Shouqin et al., 2007). Some extraction methods such as
cold extraction, heat reflux extraction, soxhlet extraction,
and extractions using ultrasound and microwave have been
used. These methods need a suitable agitation, power, and
solvent to increase the solubility and mass transfer rate
(Prasad et al., 2009). The antioxidant activity and extrac-
tion yields of the materials are changed depending on the
extraction solvent because of the different antioxidant
compounds with various polarities and chemical properties
(Peschel et al., 2006). A lot of studies have been carried out
to optimize the extraction conditions of functional or
bioactive components using response surface methodology
(Cha et al., 2019; Chuyen et al., 2017; Kadam et al., 2015;
Guglielmetti et al., 2017; Yan et al., 2015). Response
surface methodology (RSM) was designed to identify the
multiple effects of independent variables and their inter-
actions on dependent variables (Box and Wilson, 1951).
Although fewer experimental runs are conducted in RSM
than in full factorial design, statistically acceptable results
can still be provided. The RSM for optimization design is
applied so as to reduce high cost and numerical noise of
analysis methods (Tan et al., 2009). Aydar (2018) sum-
marized RSM application used to optimize extraction
process of plant materials.
In this study, the ethanol concentration, extraction time,
and extraction temperature were optimized for extraction
yield and functional compound contents from ginger. The
specific objectives were to identify the effects of extraction
conditions for 6-gingerol and 6-shogaol contents and
extraction yield and to develop the regression equations to
predict the functional compound contents and extraction
yield using RSM.
Materials and methods
Materials
The dried ginger (Zingiber officinale Roscoe) was culti-
vated in Korea and provided from Cheonho Bio Co. (Seoul,
Korea). The reagents were purchased from Sigma (Sigma
Aldrich Co., St. Louis, Mo., USA) as standard products of
6-gingerol and 6-shogaol, which were used for functional
compound analysis.
Proximate composition
According to AOAC (1995), moisture content was ana-
lyzed by drying method at 105 °C, crude fat by Soxhlet
123
J. Cha et al.
method, and ash by ash incineration at 550 °C. The crude
protein content of the sample was then measured using a
Tecator digestion system (2006 digestor, Foss, Denmark)
and Kjeltec auto sampler system (1035 analyzer, Foss,
Denmark). The amount of carbohydrate was calculated by
deduction of the amounts of ash, crude fat, crude protein,
and moisture from 100.
Extraction process
The dried ginger was extracted to determine the optimal
extraction conditions by using a reflux cooling extraction
system (HMO-F300, Hana Instrument, Seoul, Korea)
shown in Fig. 1. After the addition of 4 L of distilled water
(w/v) to the 150 g of dried ginger flake, the extracts were
obtained in 13 different combinations with 3 different
levels of ethanol concentration (%) of solvent, extraction
time, and extraction temperature (°C) according to exper-
iment design. After the extracts were centrifuged at 4 °C
and 11,0009g for 5 min, they were filtered through a filter
paper (Watman No. 4, Maidstone, England). The filtrate
was vaporized in a rotary vacuum evaporator at 40 °C. The
residue was lyophilized and used as the analysis sample.
Determination of functional compound contents
and extraction yield by HPLC
The extraction yield was determined by dividing the weight
of lyophilized extract by that of dried sample. Lyophilized
ginger extract was dissolved in methanol to a concentration
of 500 mg/mL, then sonicated for 30 min for complete
mixing and liberation of phenolic compounds with some
modification by Zhan et al. (2011) and Pawar et al. (2011).
The extract was used for functional compound analysis
after filtering through a 0.45 lm PVDF syringe filter. The
standard product of 6-gingerol was dissolved in methanol
to a concentration of 1 mg/mL and diluted to 200, 400,
Fig. 1 The schematic diagram of the reflux cooling extraction system
Optimal extraction condition for ginger

600, and 800 lg/mL. The diluted solution was filtered
through a 0.45 lm PVDF syringe filter, and then used for
analysis. In the case of 6-shogaol, the methanol solution of
1 mg/mL concentration was diluted to 20, 40, 60, 80, and
100 lg/mL. Prior to being used for the analysis, the diluted
solution of 6-shogaol was filtered using the same method as
that of 6-gingerol. (Baranowski 1985) reported that the best
method for the direct analysis of gingerols and shogaols
was HPLC analysis equipped with a reversed-phase col-
umn. As functional compounds for ginger, 6-gingerol and
6-shogaol were analyzed using HPLC (Agilent Technolo-
gies 1260 infinity, USA). The used column was ZORBAX
Eclipse XDB-C18 (250 mm 9 4 mm, 5 lm, Agilent
Technologies, USA), while water (A) and acetonitrile
(B) were used as mobile phases. The gradient program for
the HPLC was as follows: 65% B for 0–12 min, 60–80% B
for 12–18 min, 80% B for 18–25 min, 45% B for
25–30 min, and 65% B for 30–40 min, with a flow rate of
1 mL/min. The injection volume was 20 lL at the column
temperature of 25 °C. They were detected at 225 nm.
Statistical analysis
The experimental results in Table 1 were analyzed using
SPSS program (IBM SPSS 22 for windows, SPSS INC.,
Chicago, IL, USA). Data was analyzed by ANOVA and
Duncan’s multiple range test, which was used to resolve
the difference among treatment means. A value of p \ 0.05
was used to indicate significant difference.
Results and discussion
The contents of carbohydrate, crude ash, crude fat, crude
protein, and moisture of ginger used in this study were
72.74%, 6.81%, 3.46%, 10.52%, and 6.73%, respectively.
The general components of the ginger used in this study
were mostly composed of carbohydrates in a similar
composition to common ginger.
Effect of extraction conditions on extraction yield
As shown in Table 1, the extraction yield (Y1) of the
ginger extract ranged from 10.12 to 14.94%. The highest
extraction yield was obtained at 70 °C for 90 min using the
solvent with 35% ethanol concentration, and the lowest one
was obtained at 50 °C for 60 min using the solvent with
70% ethanol concentration. The extraction yield decreased
at 50 and 70 °C for 60 min as ethanol concentration of
solvent increased from 0 to 70%, whereas it increased at
60 °C for 90 min (Table 1). Kim et al. (1993) reported that
the extraction yield of cinnamon extract increased up to
70% of ethanol concentration, then decreased as with
ethanol concentration increased. Although it was not sig-
nificantly different at the extraction conditions of 30 min

Table 1 The response of

Run no. Independent variables Dependent variables
extraction yield and 6-shogaol
and 6-gingerol contents from Coded values Uncoded values
ginger by extraction process
applied Box-Behnken design X1 X2 X3 X1 X2 X3 Y1 Y2 Y3

1 -1 -1 0 50 30 35 12.41d 11.20d 0.97b

cd h
2 1 -1 0 70 30 35 11.54 27.60 1.39c
3 -1 1 0 50 90 35 11.42c 16.42f 1.05b
h i
4 1 -1 0 70 90 35 14.94 30.68 1.47c
ef a
5 -1 1 -1 50 60 0 13.06 4.12 0.31a
f e
6 1 1 -1 70 60 0 13.75 14.09 0.53ab
a g
7 -1 0 1 50 60 70 10.12 22.22 1.58c
8 1 0 1 70 60 70 12.98e 35.17j 2.06d
9 0 -1 -1 60 30 0 12.49d 5.17b 0.35a
ab c
10 0 1 -1 60 90 0 10.54 6.95 0.35a
b h
11 0 -1 1 60 30 70 10.76 27.78 2.57e
c ih
12 0 1 1 60 90 70 11.79 33.15 2.43e
f g
13 0 0 0 60 60 35 14.04 21.15 1.67c
14 0 0 0 60 60 35 13.41f 20.88g 1.75c
f g
15 0 0 0 60 60 35 13.69 21.00 1.68c
X1 extraction temperature (°C), X2 extraction time (min), X3 ethanol concentration of solvent (%), Y1
extraction yield (%), Y2 content of 6-gingerol (mg/g), Y3 content of 6-shogaol (mg/g), Means with different
character superscripts in each column are significantly different (p \ 0.05)

123

and 35% (run 1 and 2) and 60 min and 0% (run 5 and 6)
with the increase of extraction temperature from 50 to
70 °C (p [ 0.05), it significantly increased at the condition
of 90 min and 35% (run 3 and 4) and 60 min and 70% (run
7 and 8) (p \ 0.05). The change of extraction yield was not
constant with increasing extraction time at the same
extraction condition of ethanol concentration and extrac-
tion temperature. Among the first-order terms in the
regression equation for extraction yield, the p value of
extraction time was the highest at 0.526 (Table 3). As a
result, it was considered that extraction time did not affect
the extraction yield.
Effect of extraction conditions on the functional
compounds content
The contents of 6-shogaol and 6-gingerol at different
extraction conditions are shown in Table 1. The highest
content of 6-gingerol was 35.17 mg/g extracted at 70 °C
for 60 min using the solvent with 70% ethanol concentra-
tion, and the lowest content was 4.12 mg/g at 50 °C for
60 min using water. The content of 6-gingerol significantly
increased at the same ethanol concentration and extraction
time (run 1 and 2, 3 and 4, 5 and 6) with increasing
extraction temperature from 50 to 70 °C (p \ 0.05). It also
significantly increased at the same ethanol content and
extraction temperature (run 1 and 3, 2 and 4) with the
increase of extraction time from 30 to 90 min (p \ 0.05).
Dent et al. (2013) reported that the phenolic content
increased with increasing extraction time using a 70%
solution of acetone, ethanol, and water with maximum
values at 90 min. The content of 6-gingerol significantly
increased at the same extraction time and temperature (run
5–12) with the increase of ethanol concentration of solvent
from 0 to 70% (p \ 0.05). From these results, it can be
conjectured that each parameter independently affected the
content of 6-gingerol. The highest content of 6-shogaol was
2.57 mg/g extracted at 60 °C for 30 min using the solvent
with 70% ethanol concentration, and the lowest content
was 0.31 mg/g at 50 °C for 60 min using water (Table 1).
The content of 6-shogaol significantly increased in the
same ethanol concentration and extraction time (run 1 and
2, 3 and 4, 5 and 6) with increasing extraction temperature
from 50 to 70 °C (p \ 0.05). The content of 6-shogaol
significantly increased at the same extraction time and
extraction temperature (run 5–12) with increasing ethanol
concentration of solvent from 0 to 70% (p \ 0.05). From
these results, it can be guessed that ethanol concentration
and extraction temperature independently affected the
content of 6-shogaol.
123
J. Cha et al.
Regression modeling of extraction condition
The Box–Behnken design (BBD) was applied as an
experimental design used to fit the second-order response
surface based on the structure of balanced incomplete
block designs. For this experimental design, the ethanol
content (X3), extraction time (X2), and extraction tem-
perature (X1), which affected the extraction process, were
encoded as the independent variables. The extraction
conditions were determined by considering the results from
preliminary experiments with each independent variable,
extraction temperature (40–100 °C), extraction time (30–
120 min), and ethanol concentration (0–90%), respec-
tively. The extraction yield of ginger was a little increased
and then, decreased from 60 °C as with extraction tem-
perature increased. The response variables were the con-
tents of 6-gingerol and 6-shogaol and extraction yield. The
BBD applied in this study was composed of 12 different
combinations of the independent variables (1 to 12) and 3
central points (13 to 15). All experiments were conducted
with three replications. For regression analysis, mean val-
ues were used to identify mathematical relationship
between independent variables and response variables.
MiniTab (MiniTab 16, Minitab Inc., State College, PA)
was used to perform RSM. The significance of each term in
polynomial regression equations was statistically evaluated
at the significant level, a = 0.05. The dependent (Yn) and
independent variables (Yn) are shown in polynomial
regression equation below, and bn are the fixed constant
and regression coefficients of the equation.
Y ¼ b0 þ b1 X1 þ b2 X2 þ b3 X3 þ b11 X12 þ b22 X22 þ b33 X32
þ b12 X1 X2 þ b13 X1 X3 þ b23 X2 X3
Table 2 shows the p-value of each term in the regression
equations, which indicates the significance of each term to
the model.
For extraction yield, the first-order term of extraction
temperature was significant at p value 0.036, whereas those
of ethanol concentration and extraction time were not
significant at p values 0.114 and 0.525, respectively
(p [ 0.05). However, the significance of quadratic terms
was in opposition to the first-order terms (p \ 0.05). The
first-order terms of ethanol concentration and extraction
time and their reciprocal terms were not significant
(p [ 0.05) (Table 2). The coefficient of determination
(R2), which indicates the general validity and accuracy of
the polynomial regression equation, was 0.862 (Table 3).
The p value was 0.034 in the result of the error analysis,
which indicated a significant regression relationship
(p \ 0.05).
For 6-gingerol content, the first-order terms and the
quadratic term of ethanol concentration in the regression
Optimal extraction condition for ginger

Table 2 p value of each

Parameter p value of each parameter in polynomial regression equations
parameter in the polynomial
regression equations of Y1 Y2 Y3
extraction yield and 6-gingerol
and 6-shogaol contents from Constant 0.000 0.000 0.000
ginger X1 0.036 0.000 0.076
X2 0.526 0.039 0.978
X3 0.114 0.000 0.000
X21 0.950 0.613 0.027
X22 0.040 0.936 0.522
X23 0.030 0.048 0.200
X1X2 0.036 0.609 1.000
X1X3 0.220 0.482 0.618
X2X3 0.112 0.403 0.786
X1 extraction temperature (°C), X2 extraction time (min), X3 ethanol concentration of solvent (%), Y1
extraction yield (%), Y2 content of 6-gingerol (mg/g), Y3 content of 6-shogaol (mg/g)

Table 3 Polynomial regression equations of extraction yield and 6-gingerol and 6-shogaol contents from ginger
Response Polynomial regression equation R2 p value

Extraction yield (%) Y1 = 13.7133 ? 0.7750X1 - 1.1092X22 - 1.2092X23 ? 1.0975X1X2 0.862 0.034
Content of 6-gingerol (mg/g) Y2 = 21.010 ? 6.6975X1 ? 1.9312X2 ? 10.9987X3 - 2.6613X23 0.982 0.000
Content of 6-shogaol (mg/g) Y3 = 1.7000 ? 0.8875X3 - 0.3925X21 0.955 0.005
X1 extraction temperature (°C), X2 extraction time (min), X3 ethanol concentration of solvent (%), Y1 extraction yield (%), Y2 content of
6-gingerol (mg/g), Y3 content of 6-shogaol (mg/g)

equation were significantly affected to the content of
6-gingerol, whereas the other quadratic terms and recip-
rocal terms were not (p \ 0.05). The p values of ethanol
concentration and extraction temperature were 0.000
(Table 2), which indicate a great importance for 6-gingerol
content. The regression equation to predict 6-gingerol
content is shown in Table 3. It is considered that this
equation fit well due to high determination coefficient of
0.982 (Table 3). The p value was 0.000 in the result of
error analysis, which indicated a significant relationship.
For 6-shogaol content, the significance in the first-order
term of ethanol concentration and the quadratic term of
extraction temperature was indicated for this equation,
whereas that in the other terms was not statistically indi-
cated (p \ 0.05). The p value of ethanol concentration was
0.000 (Table 2), which explains that this term was the most
important in the regression equation for 6-shogaol content.
The regression equation for 6-shogaol content in the sam-
ple extracted at different conditions is shown in Table 3.
The determination coefficient of 0.955 indicated that this
equation fit well. The p value of 0.005 was in the result of
error analysis, which indicated a significant relationship.
Therefore, these responses are thoroughly explained by
these regression equations. It is also reasonable to predict
the effects of extraction conditions on extraction yield and
6-gingerol and 6-shogaol contents and extraction yield
from ginger.
Optimal extraction condition using RSM
The optimal ethanol concentration of solvent, extraction
time, and extraction temperature were determined by
polynomial regression equations using MiniTab. Figure 2
shows optimum values and interaction between the vari-
ables which were analyzed and expressed as a three-di-
mensional surface response graph. Extraction yield (Y1)
increased as extraction temperature increased in the range
of this study, while it increased up to 78.16 min of
extraction time and 41.38% of ethanol concentration and
thereafter decreased with the increase of ethanol concen-
tration and extraction time. Kim et al. (2014) reported that
the extraction yield of kirenol stayed constant below 60%
of ethanol concentration, while it decreased 13.5 times with
an increase in ethanol concentration from 60 to 100%.
Durling et al. (2007) reported that a high extraction yield of
phenolics extracted from Salvia officinal was obtained
using 55-75% ethanol concentration of solvent. In high
hydrostatic pressure extraction for propolis, a high
extraction yield was obtained at 75% ethanol concentration
(Zhang et al., 2005). For the highest extraction yield using

123

Fig. 2 Response surface plots for the effects of the extraction
temperature (°C, X1), extraction time (min, X2), and ethanol
concentration of solvent (%, X3) on the extraction yield (%, Y1)
RSM, the optimal ethanol concentration, extraction time,
and extraction temperature were determined as 41.38%,
78.16 min, and 70 °C, respectively. For 6-gingerol (Y2), its
content increased with increasing ethanol concentration,
extraction time, and extraction temperature in the range of
this study. The optimum conditions using the regression
equation were 70 °C, 90 min, and 70%. Dvorackova et al.
(2015) reported that the most efficient ethanol concentra-
tion of solvent was 60% for phenolic compounds in the
classic extraction of cinnamon. The content of 6-shogaol
(Y3) increased with increasing ethanol concentration in the
range of this study. As extraction temperature and time
increase up to 62.29 °C and 51.90 min, the content of
6-shogaol tended to increase and then decreased after that.
The optimum ethanol concentration, extraction time, and
extraction temperature using the regression equations were
determined as 62.29 °C, 51.90 min, and 70%, respectively.
123
J. Cha et al.
and the contents of 6-gingerol (mg/g, Y2) and 6-shogaol (mg/g, Y3)
from ginger by extraction process
The experiment at the optimal conditions was conducted
to validate the regression model for predicting the response
value. The experimental and predicted values for extraction
yield were 16.10 ± 1.2 and 14.88%, respectively. The
experimental and predicted values for 6-gingerol content
were 41.58 ± 2.7 and 39.55 mg/g, respectively. Those
values for 6-shogaol content were 2.63 ± 0.5 and 2.44 mg/
g in this optimal extraction condition. The experimental
values were good agreed with the predicted values using
regression models obtained by RSM. Therefore, it is con-
sidered that the model could accurately predict the
response.
Acknowledgements This research was supported by the research
grant of the Ministry for Agriculture, Food and Rural Affairs for the
2016 joint research and development of industry-academy-research
cooperation technology. This support is appreciated.
Optimal extraction condition for ginger
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