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Optimization of Microwave-Assisted
Extraction of Total Flavonoids
from China-Hemp Leaves and Evaluation
of Its Antioxidant Activities
Jie Cao, Limin Hao, Liming Zhang, Meng Xu, Huanhuan Ge,
Caicai Kang, Jianyong Yu and Zongzhen Wang
1 Introduction
Hemp (Cannabis sativa L.) is an annual herbaceous plant belongs to Cannabaceae
family and originated in Central Asia, grown in many countries such as China,
France, Chile, Russia, Turkey, United States and Canada. The plant has been
cultivated widely for the purposes of fiber, food and medicine [1]. The hemp leaves
is a by-product of hemp fiber and seed production, yield is rich. It is a rich source of
flavonoids [2].
Flavonoids, a non-cannabinoid phenols, have received more and more attention
by the biochemical and nutritional researchers because of their biological activities
and health function in health-care food or medicine, especially their antioxidant,
anti-ultraviolet radiation, and antibacterial effects [3]. Recently, natural antioxidants
are becoming an attractive option as they are abundant and high safety [4, 5].
Therefore, most of the researches are currently focused on the extraction and
antioxidant activities of flavonoids [6, 7].
Microwave assisted extraction (MAE) was relatively new method by which
microwave energy is used to heat polar solvent in contact with solid samples and to
partition compounds of interest between the sample and the solvent, reducing both
extraction time and solvent consumption [8]. In this paper, by MAE, the effect of
J. Cao L. Zhang (&) M. Xu H. Ge C. Kang
Key Laboratory of Industrial Fermentation Microbiology, Ministry of Education Tianjin
University of Science and Technology, Tianjin 300457, People’s Republic of China
e-mail: zhanglmd@126.com
L. Hao (&) J. Yu
The Quartermaster Equipment Institute of General Logistics Department of People’s
Liberation Army, Beijing 100010, People’s Republic of China
e-mail: hlm2005@163.com
Z. Wang
Ningbo Hemp Biotechnology Co. Ltd, Ningbo 315153, People’s Republic of China
© Springer Nature Singapore Pte Ltd. 2018
H. Liu et al. (eds.), Advances in Applied Biotechnology, Lecture Notes
in Electrical Engineering 444, https://doi.org/10.1007/978-981-10-4801-2_57
555
556
J. Cao et al.
ethanol concentration, solvent-to-solid ratio, extraction time and temperature on the
yield of total flavonoids from hemp leaves were investigated by RSM (response
surface methodology). RSM is an effective statistical technique, which is used to
find optimum processing parameters [9].The antioxidant activities of hemp leaves
extracts were evaluated, including 1,1-diphenyl-2-picryl-hydrazyl (DPPH) free
radical, reducing power and 2,2′-azino-di (3-ethylbenzthiazoline-6-sulfonate)
(ABTS) radical cation inhibition antioxidant test. We hope this study will be helpful
to further exploit and utilize this resource.
2 Materials and Methods
2.1
Materials and Chemical Reagents
China-hemp of leaves was collected from Mengwang County of Jinghong city,
Yunnan province, China, and was identified by Professor Wenbin Hou, Tianjin
Institute of Pharmaceutical Research, China. Collected leaves were dried in a
forced-air oven at 40 °C to constant weight, and then ground using an electric
grinder. The ground powder was passed through a standard 0.25 mm sieve and was
collected and stored at 40 °C in airtight bags for further use.
Rutin, ascorbic acid (Vc) and DPPH were purchased from Sigma-Aldrich (St.
Louis, MO, USA). Butylated hydroxytoluene (BHT), ABTS were from Merck
(Darmstadt, Germany). All other chemical reagents used in experiments were of
analytical grade and Millipore quality water was used throughout the experiment.
2.2
2.2.1
Extraction and Quantification Total Flavonoids Content
Microwave-Assisted Extraction
Flavonoids from powders of hemp leaves were extracted using a domestic microwave oven system. The apparatus was equipped with a digital control system for
irradiation time, microwave powder (the latter was linearly adjustable from 100 to
1000 W), magnetic stirrer speed and temperature.
Tow gram of hemp leaves powder was stirred into aqueous ethanol by stirring in
preparation for extraction using the MAE system. Parameters of the MAE extraction were ethanol proportion (40–80%), liquid-to-solid ratio (20–40 mL/g),
extraction time (5–40 min) and extraction temperature (40–80 °C). Table 1 provides the experimental conditions respectively, where the influence of each
parameter was in single-factor experiments. Each trial was carried out in triplicate.
After MAE treatment, the extraction solution was separated from insoluble residue
Optimization of Microwave-Assisted Extraction …
557
Table 1 Experimental design with the observed responses of total flavonoids (TF) yield from
China-hemp leaves using MAE
Run
X1 ethanolm
concentration (%
v/v)
X2
liquid-to-solid
ratio (mL/g)
X3
extraction
time (min)
X4 extraction
temperature (°
C)
Y yield
of TF
(%)
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
60.00
60.00
60.00
60.00
80.00
60.00
80.00
80.00
60.00
40.00
60.00
80.00
60.00
40.00
60.00
40.00
60.00
60.00
60.00
40.00
40.00
60.00
60.00
40.00
80.00
80.00
60.00
60.00
60.00
35.00
35.00
30.00
35.00
35.00
40.00
35.00
35.00
35.00
35.00
35.00
40.00
30.00
40.00
30.00
35.00
35.00
40.00
35.00
35.00
30.00
30.00
40.00
35.00
35.00
30.00
40.00
35.00
35.00
10.00
20.00
20.00
20.00
10.00
20.00
30.00
20.00
20.00
30.00
30.00
20.00
10.00
20.00
20.00
20.00
10.00
10.00
20.00
20.00
20.00
30.00
30.00
10.00
20.00
20.00
20.00
20.00
30.00
70.00
60.00
50.00
60.00
60.00
70.00
60.00
50.00
60.00
60.00
70.00
60.00
60.00
60.00
70.00
70.00
50.00
60.00
60.00
50.00
60.00
60.00
60.00
60.00
70.00
60.00
50.00
60.00
50.00
2.66
2.77
2.43
2.81
2.33
2.85
2.95
2.28
2.64
1.88
2.88
2.50
2.42
2.10
2.88
2.52
2.11
2.33
2.76
1.63
2.33
2.68
2.56
2.05
3.01
2.65
2.22
2.66
2.34
by centrifugation (8000 rpm for 10 min), and then the supernatant was collected in
a volumetric flask for determination of FT content.
2.2.2
Determination of Flavonoids Contents
The flavonoid content of the extracts was estimated by the Al(NO3)3 method [10].
Briefly, 1 mL of 10-fold diluted supernatant was mixed with 0.3 mL of 5% NaNO2.
558
J. Cao et al.
The solutions were mixed thoroughly and incubated at room temperature for 6 min.
And then 0.3 mL of 10% Al(NO3)3 solution was added and mixed. 6 min later,
4 mL of 4% NaOH solution was added and used 60% ethanol diluted to 10 mL.
With 15 min standing, the absorbance of the solution was measured at 510 nm with
UV-2401 spectrophotometer against the same mixture, without the sample as a
blank. (The calibration curve: y = 9.4975 x + 0.0366, R2 = 0.9996).
2.3
Experimental Design and Statistical Analysis
Influence of the process parameter was investigate using a single-factor-test to
determine the preliminary range of the extraction variables including X1 (ethanol
concentration), X2 (liquid-to-solid ratio), X3 (extraction time), X4 (extraction temperature). As shown in Table 2, the four factors chosen for this study were designated as X1, X2, X3, X4, and prescribed into three levels, coded +1, 0, −1 for high,
intermediate and low value, respectively. The four variables were coded according
to the following equation
Xi ¼
xi x0
;i ¼ 1 4
Dx
ð1Þ
where Xi was a coded value of the variable; xi was the actual value of the variable;
x0 was the actual value of the independent variable at the center point; Dx and was
the step change of the variable.
Using a Box-Behnken design (BBD) [11], response surface methodology was
conducted to determine the MAE optimized extraction process variables for maximum recovery of TF yield. Tables 1 and 2 represent the non-coded values of the
experimental variables and 29 experimental points. On the basis of the experimental
date, a second-order polynominal model corresponding to the BBD was fitted to
correlate the relationship between the independent variables and the response to
predict the optimized condition. The nonlinear computer-generated quadratic model
was given as:
Y ¼ b0 þ
4
X
bi Xi þ
i¼0
Table 2 Variables and
experimental design levels for
response surface
4
X
j¼0
bii X2i þ
4 X
4
X
bij Xi Xj
ð2Þ
i¼0 j¼0
Independent
Coded symbols
Levels
−1 0
1
Ethanol concentration (%)
Liquid-to-solid ratio (mL/g)
Extraction time (min)
Extraction temperature (°C)
X1
X2
X3
X4
40
30
10
50
80
40
30
70
60
35
20
60
Optimization of Microwave-Assisted Extraction …
559
where Y was the response function; b0 was a constant; bi bii and bij were the linear,
quadratic and interactive coefficients, respectively; Xi was the coded levels of
independent variables. The terms XiXj and X2i represented the interaction and
quadratic terms, respectively.
2.4
Antioxidant Activity Determinations
2.4.1
DPPH Radical Scavenging Ability Assay
Radical scavenging activity of the China-hemp leaves extract was evaluated using
DPPH radicals based on the method by Xu and Chang [12]. DPPH radical scavenging activity was calculated by the following equation: scavenging effect
(%) = [1 − (A1 − A2)/A0] 100, where A0 is the absorbance of the control, A1 is
the absorbance of the sample, and A2 is the background absorbance of the sample.
2.4.2
Reducing Power
The determination of reducing power was carried out according to the method of
Yen and Chen [13]. The reducing power was calculated as following: Reducing
power = A1 − A2, where A1 is the absorbance of the sample, A2 the absorbance of
the reagent blank without potassium ferricyanide.
2.4.3
ABTS Radical Cation Inhibition Antioxidant Assay
Determination of ABTS radical cation inhibition activity of sample extract was
performed according to the methods of Biglari et al. [14]. The percentage radical
inhibition activity was calculated as following: Radical inhibition activity
(%) = (1 − A1/A2) 100, where A1 was the absorbance of the sample and A2 was
the absorbance of the solvent control.
3 Results and Discussion
3.1
The Effect of Ethanol Concentration on the Total
Flavonoids Yield
In this study, the effect of ethanol concentration on the extraction yield of TF from
hemp leaves was investigated and prepared different concentrations of ethanol (40,
50, 60, 70, 80%), when other experimental parameters were set as follows: the ratio
560
J. Cao et al.
Fig. 1 Effect of different extraction parameters (a ethanol concentration, %; b liquid-to-solid
ratio, mL/g; c extraction time, min; d extraction temperature, °C)
of liquid to material 30:1 (mL/g), extraction time 20 min, extraction temperature
60 °C and number of extraction 1. As shown in Fig. 1a, extraction yield of TF was
highest when 70% ethanol and ethanol concentrations had important effects for it.
3.2
The Effect of Extraction of Liquid-to-Solid Ratio
on TF Yield
The choice of liquid-to-solid ratio to raw material was another important step [15].
In this study, effect of different ratio of liquid to material (20:1, 25:1, 30:1, 35:1 and
40:1) on the extraction yield was investigated. The results were displayed in
Fig. 1b. It was observed that the recovery of TF yield was maximized at a
liquid-to-solid ratio of 35:1 (mL/g). A ratio of 30–40 (mL/g) was further used in the
optimization of process parameters during MAE.
3.3
The Effect of Extraction Time on the Total
Flavonoids Yield
Generally, by increasing the extraction time, the quantity of analytes is increased,
although there is the risk of the degradation of extracted compounds. So the
selection of extraction time (5, 10, 20, 30, 40 min) was tested for extraction yield of
TF. The results were displayed in Fig. 1c. The yield of TF was increased markedly
with the increase of extraction time from 5 to 20 min. Over 20 min, the yield
decreased lightly. This might be due to the decomposition of active compounds
during the prolonged extraction time [16].
Optimization of Microwave-Assisted Extraction …
3.4
561
The Effect of Extraction Temperature on the Total
Flavonoids Yield
Extraction temperature was, respectively, set at 40, 50, 60, 70, 80 °C to examine the
influence of different temperature on the yield of TF. Figure 1d indicated that the
yield of TF rose gradually with the increase of temperature, reached the peak at 60 °
C, and finally dropped from 50 to 70 °C.
3.5
3.5.1
Optimization of the Procedure
Modeling and Fitting the Model Using Response Surface
Methodology
The experiment design and corresponding response date for TF content from
China-hemp leaves are presented in the Table 1.
All 29 of the designed experiments were conducted for optimizing the four
individual parameters in the current BBD. By applying multiple regression analysis
on the experimental date, the response variables and the best variables were related
by the following second-order polynomial equation:
Y ¼ 2:73 þ 0:27X1 0:069X2 þ 0:12X3 þ 0:32X4 þ 0:020X1 X2
þ 0:20X1 X3 0:040X1 X4 7:500E 033X2 X3 þ 0:045X2 X4
2:500E 033X3 X4 0:28X12 0:062X22 0:16X32 0:079X42
where Y, X1, X2, X3 and X4 were the coded valued of yield of TF, ethanol concentration, liquid solvent to solid ratio, extraction time and extraction temperature,
respectively.
The analysis of variance (ANOVE) for the experimental results of TF yield from
China-hemp leaves were given in Table 3. It shows that the model is significant at
F-value of 23.69. There is only a 0.01% chance that a “Model F-Value” this large
could occur due to noises. The determination coefficient (R2) was 0.9595, which
implied that the sample variations of 95.95% for the MAE efficiency of TF were
attributed to the independent variables, and only 4.05% of the total variations could
not be explained by the model. However, a large value of R2 does not always
indicate that the regression model is a sound one. Here, the Adj.R2 value was 0.919,
which meant most variation (>91.9%) of TF yield could be predicted by the models,
while only 8% variation could not be explained by the model.
562
J. Cao et al.
Table 3 Estimated regression coefficients for the quadratic polynomial model and the analysis of
variance (ANOVE) for the experimental results of total flavonoids yield from China-hemp leaves
Source
Sum of
squares
Degree of
freedom
Mean
square
F-Value
P-Value
(Prob>F)
Model
3.02
14
0.22
23.69
<0.0001
X1
0.86
1
0.86
94.41
<0.0001
X2
0.057
1
0.057
6.31
0.0249
X3
0.16
1
0.16
17.7
0.0009
X4
1.2
1
1.2
131.61
<0.0001
X1 X2
1.60E-03
1
1.60E-03
0.18
0.6813
X1 X3
0.16
1
0.16
17.16
0.001
X1 X4
6.40E-03
1
6.40E-03
0.7
0.4156
X2 X3
2.25E-04
1
2.25E-04
0.025
0.8773
X2 X4
8.10E-03
1
8.10E-03
0.89
0.3613
X3 X4
2.50E-05
1
2.50E-05
2.75E-03
0.9589
X21
0.5
1
0.5
54.53
<0.0001
X22
0.025
1
0.025
2.7
0.1228
X23
0.16
1
0.16
17.47
0.0009
4.45
0.0534
1.93
0.2757
X24
0.04
1
0.04
Residual
0.13
14
9.10E-03
Lack of fit
0.11
10
0.011
Pure error
0.022
4
5.470E-003
Cor total
3.14
28
R2
0.9595
Adj.R2
0.919
Pred.R2
0.7959
Adequate
precision
17.08
3.5.2
Analysis of Response Surface
The 3D response surface and 2D contour profiles for flavonoids extraction from
China-hemp leaves were presented in Figs. 2a–f and 3a–f. It has reported that the
3D response surface plots and 2D contour plots are able to reflect the effects of
multiple independent variables and sensitiveness of response value toward the
change of variables [17]. For example, if the response surface was more steeper, its
influence on response value is extremely significant. The elliptical shape of contour
plots was more significant than that of circular ones.
Figures 2a and 3a showed the combined effect of ethanol concentration (v/v) and
the ratio of solvent volume to solid on the extraction yield of TF. The yield of TF
increased with the increasing of ethanol concentration from 40 to 70%, then
decrease when ethanol concentration continues to increase. The reason for this is
that the appropriate concentration of ethanol could provide the most suitable
Optimization of Microwave-Assisted Extraction …
563
Fig. 2 Response surface plots showing the effect of X1 (ethanol concentration, %), X2
(solvent-to-solid ratio, mL/g), X3 (extraction time, min), X4 (extraction temperature, °C) to the
total flavonoids on the response yield
polarity for flavonoid glucosides. The result was in consistent with the previous
finding [18]. When the ratio of sovent to solid increased in the range from 34:1 to
40:1 mL/g, the yield of TF decreased. Therefore, the optimum ratio was 34:1 mL/L
for the extraction of TF, this value was in consistance with the preliminary
experimental result and could be determined for the accurate parameter.
Figures 2b and 3b showed the effects of ethanol concentration (v/v) and
extraction time on the flavonoid extraction yield under the fixed other conditions.
The extraction yield of TF increased with the increase of extraction time from 10 to
25 min, the yield was decreased when the extraction time was more than 25 min.
The yield of TF was increased with the increase of ethanol concentration from 40 to
72%, further enhancing ethanol concentration led to the decrease of TF yield.
Figures 2c and 3c presented the effects of ethanol concentration and extraction
temperature on the TF yield under the fixed other conditions. It could be seen that at
lower ethanol concentration (below 50%), the TF yield changed little as extraction
temperature improved. However, at higher ethanol concentration (50–60%), the TF
yield increased significantly with the increase of extraction temperature.
Figures 2d and 3d depicted the effects of the ratio of solvent to solid and
extraction time on the TF yield of TF under the fixed other conditions. It indicated
that variation of extraction time has a marked effect on the TF yield. However, at
low level of extraction time (from 10 to 15 min), the ratio of solvent to solid had
little influence on the TF yield.
564
J. Cao et al.
Fig. 3 Contour plots showing the effect of X1 (ethanol concentration, %), X2 (solvent-to-solid
ratio, mL/g), X3 (extraction time, min), X4 (extraction temperature, °C) to the total flavonoids on
the response yield
Figures 2e and 3e showed effect of the ratio of solvent to solid and extraction
temperature on the yield of TF under the fixed other conditions. It revealed that the
ratio of solvent to solid had little influence on the TF yield. However, the TF yield
was linearly increased with the extraction temperature increment.
Figures 2f and 3f showed the effects of extraction temperature and extraction
time on the yield of TF under the fixed other conditions. It can be seen that
extraction time and extraction temperature exhibited a significant effect on TF yield.
The maximum yield of TF was achieved when the process operated at extraction
temperature of 70 °C for 20 min of extraction time.
3.5.3
Optimization of Extracting Parameters and Validation
of the Model
In this study, the optimal microware extraction condition for obtain maximal yield
of TF predicted by the quadratic model was as follows: ethanol concentration of
69.15%, solvent-to-solid ratio of 31.69 mL/g, extraction time of 25.14 min and
extraction temperature of 69.96 °C. The predicted extraction yield of TF was
3.06%, which was consistent with the experimental yield of 3.04 ± 0.62%.
Optimization of Microwave-Assisted Extraction …
565
Fig. 4 Antioxidant activity assay: a scavenging effects on DPPH; b reducing power; c ABTS
radical cation inhibition activity. a Vc; b BHT; c HL-95E; d HL-60E; e HL-30E. Each value is the
mean ± SD of triplicate measurements
3.6
3.6.1
Antioxidant Activity of Different Concentration
of Ethanol Extract
Scavenging Effects on DPPH Radicals
The hemp leaves powder (10 g) was respectively extracted by different concentration aqueous ethanol (95, 60, 30%) at 60 °C for 2 h. After filtration, the
supernatants were concentrated and vacuum dried to obtain HL-95E (95% ethanol
extraction), HL-60E (60% ethanol extraction), HL-30E (30% ethanol extraction),
respectively.
The DPPH radical-scavenging activity of HL-95E, HL-60E, HL-30E were
investigated at different concentrations (0.2–1.0 mg/mL) and the results were presented in Fig. 4a. In the results, Vc showed obvious scavenging activity and BHT,
HL-95E, HL-60E, HL-30E showed scavenging activity on DPPH radical in a
concentration-dependent manner. HL-95E, HL-60E and HL-30E showed a linear
increase in scavenging activity with increase sample concentration, and exhibited
under Vc and BHT. It was found that the ability to scavenge DPPH radical were in
order of HL-95E > HL-60E > HL-30E.
3.6.2
Reducing Power
The reducing capacity of HL-95E, HL-60E and HL-30E always showed lower than
Vc and BHT and indicated relatively low of its potential antioxidant activity. In
Fig. 4b, the absorbance at 700 nm increased with the concentration of HL-95E,
HL-60E and HL-30E. At 1.0 mg/mL, the reducing power of HL-95E, HL-60E and
HL-30E was only 0.504, 1.079 and 1.146 respectively.
566
3.6.3
J. Cao et al.
ABTS Radical Cation Inhibition Activity
ABTS inhibition activity of HL-95E, HL-60E, HL-30E were carried out and
showed in Fig. 4c. These samples exhibited an interesting ABTS radical cation
inhibition activity and were found active close to each other. It was found that
the ability to inhibit ABTS radical cation were in order of HL-60E >
HL-30E > HL-95E.
4 Conclusion
In this work, the microwave-assisted extraction (MAE) method was used to extract
TF from China-hemp leaves. Based on single factor experiment and RSM analysis,
the optimum technology parameters of MAE for extracting TF were obtained. The
optimized results are as follows: ethanol concentration of 69.15%, solvent-to-solid
ratio of 31.69 mL/g, extraction time of 25.14 min and extraction temperature of
69.96 °C. Under these conditions, a maximum TF yield was 3.04 ± 0.62%.
The RSM could successfully model and optimize extraction process of TF from
China-hemp leaves.
Additionally, the antioxidant activity of TF obtained from different concentration
ethanol was evaluated in vitro by scavenging capacity of DPPH, ABTS, and
reducing power. It is revealed that the ethanol concentration has significant effects
on the antioxidant activity of TF. This result should be useful to further develop and
apply China-hemp leaves resource.
Acknowledgements This research was financially supported by the Research Project of People’s
Liberation Army (No. AX110C002 and BX115C007).
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