全 文 ：天然产物研究与开发 Nat Prod Ｒes Dev 2014，26:750-760，805
文章编号:1001-6880(2014)5-0750-12
Ｒeceived:May 8，2013 Accepted:September 27，2013
* Corresponding author Tel:86-05926162302;E-mail:tangxuchong035022@
163． com
超声波辅助提取田基黄多酚类和
黄酮类化合物及其抗氧化活性研究
汤须崇* ，蔡婀娜
华侨大学化工学院，厦门 361021
摘 要:采用超声技术从田基黄中提取活性物质。运用响应面法确定超声提取田基黄的最佳条件。运用单因
素试验选取三个自变量。采用 Box-Behnken设计评价这三个自变量对总黄酮和总多酚提取、DPPH和 ABTS +活
性清除的影响。通过方差分析显示，二次模型对反应的贡献率具有统计学意义。运用响应面法进行最优化研
究，并用数学模型绘制出三维响应面。通过结合反应得出最佳试验条件:超声时间 48． 89 min，乙醇浓度
63. 72%，超声温度 66． 92 ℃。总黄酮值为 105． 06 mg ＲE /g DW，总多酚值为 51． 75 mg GAE /g DW，%DPPHsc为
58． 81%，%ABTSsc为 64． 99%。在最佳试验条件下，试验值与方差分析预测值一致。结果表明，采用四个模型
和响应面法优化总黄酮和总多酚的提取、DPPH和 ABTS +活性清除是切实可行的。
关键词:田基黄;总黄酮;总多酚;DPPH;ABTS;响应面法
中图分类号:Ｒ284. 1 文献标识码:A
Ultrasonic Assisted Extraction and Antioxidant Activity of
Hypericum japonicum Phenolic and Flavonoid Compounds
TANG Xu-chong* ，CAI E-nuo
College of Chemical Engineering，Huaqiao University，Xiamen 361021，China
Abstract:In this study，ultrasonication was employed to extract bioactive components from Hypericum． japonicum Thunb．
ex Murray (Hypericaceae)． The optimal conditions for ultrasonic extraction of Hypericaceae were determined by response
surface methodology． Three independent variables were chosen based on single factor tests． Box-Behnken design was ap-
plied to evaluate the effects of three independent variables (ultrasonic time，ethanol concentration，ultrasonic tempera-
ture)on the extraction yield of total flavonoid compound (TFC)，total phenolic compound (TPC)and their scavenging
activity to DPPH and ABTS + free radicals． The analysis of variance (ANOVA)showed that the contribution of quadratic
model was significant for the responses． The 3D response surfaces were plotted from the mathematical model． The opti-
mized extraction conditions based on combination responses were as follows:ultrasonic time of 48． 89 min，ethanol con-
centration of 63． 72% and ultrasonic temperature of 66． 92 ℃ ． Under the optimized condition，TFC，TPC，%DPPHsc and
%ABTSsc were determined to be 105． 06 mg ＲE /g DW，51． 75 mg GAE /g DW，58． 81% and 64． 99%，respectively．
The experimental values agreed with the predicted ones by analysis of variance (AVOVA)． It indicated high fitness of
four models used and the success of using response surface methodology for the optimization of the extraction conditions．
Key words:Hypericum japonicum;total flavonoid compound;total phenolic compound;DPPH;ABTS;response surface
methodology
Introduction
Hypericum japonicum，locally called‘Tian-ji-huang’，is
prepared from the entire herb of H． japonicum Thunb．
ex Murray (Hypericaceae)． It is one of traditional Chi-
nese medicines (TCM)widely distributed in the south
of the Yangtze Ｒiver，China ［1］． H． japonicum has been
used for the treatment of bacterial diseases，infectious
hepatitis，gastrointestinal disorder，internal hemorrhage
and tumors ［2-6］． As reported previously，H． japonicum
mainly contains xanthones ［6，7］，chromenes ［8］，fla-
vonoids ［9，10］，dipeptide derivatives ［11］，polyphenols
DOI:10.16333/j.1001-6880.2014.05.025
and phloroglucinol derivatives［12］． Some of these con-
stituents are known to exhibit pharmacological and bio-
logical activities［13］．
It has long been recognized that polyphenol and fla-
vonoid are an important class of antioxidants ［14］． An-
tioxidants play an indispensable role as health benefac-
tors in human life and are also added to food to prevent
or delay its oxidation ［15］． Synthetic antioxidants are
widely used since they are more effective and cheaper
than natural ones． However，the safety and toxicity of
synthetic antioxidants have brought great concerns．
Thus，it is essential to develop and utilize effective and
natural antioxidant to protect the body ［16］． In the pres-
ent study，the total phenol content (TPC)and total fla-
vonoid content (TFC)of H． japonicum were chosen to
determine their antioxidant activities．
Ｒecently，ultrasonic extraction method has been widely
employed to extract bioactive components from plant
material due to its high extraction efficiency［17］． Ｒe-
sponse surface methodology (ＲSM)is a relatively new
method for optimizing experimental conditions． In the
present study，the ultrasonic technique was employed to
extract bioactive components from H． japonicum． Ｒe-
sponse surface methodology and Box-Behnken design
were used to evaluate the effects of ultrasonic time，
temperature and ethanol concentration，which were cho-
sen according to the single factor study，on the extrac-
tion of bioactive components and free radical scaven-
ging activity (determined by DPPH and ABTS + meth-
ods)of H． japonicum．
Materials and Instruments
Materials and chemicals
Hypericum japonicum was purchased from a drug store
in Fujian Province of China and stored at 4 ℃ till test-
ed． Folin-ciocalteu reagent，1，1-Diphenyl-2-picryl-
hydrazyl (DPPH)，2，2’-azinobis-3-ethylbenzothiazo-
line -6-sulfonic acid (ABTS)，butylated hydroxytoluene
(BHT)，6-hydroxy-2，5，7，8-tetramethylchroman-2-car-
boxylic acid (Trolox)were purchased from Sigma com-
pany． Deionized water was used throughout the experi-
ment． All other chemicals used were of analytical
grade．
Instruments
UV-Visible spectra were measured using Spectra
(Pharmaspec UV-2550，Shimadzu，Kyoto，Japan)spec-
trophotometer． The ultrasonic assisted extraction was
carried out in a KQ-600E ultrasonic device (Chang-
zhou Nuoji Instrument Company，China)with an ultra-
sonic power of 600 W，heating power of 800 W and fre-
quency of 40 kHz，equipped with a digital time and a
temperature controller．
Methods
Sample preparation
H． japonicum was dried at 60 ℃ till constant weight．
The dried material was pulverized to 100 meshes． The
powdered H． japonicum was accurately weighted and
then ultrasonically extracted once with ethanol (40
mL) for special time． The supernatant solution was
combined，filtrated and then cooled down to room tem-
perature． The solution was transferred to a 100 mL vol-
umetric flask and topped up to the volume with etha-
nol．
Single factor experiments for TFC extraction
The effects of five experimental variables on extraction
yield of TFC were investigated by single factor tests．
Five experimental variables were liquid-to-solid ratio
(10，15，20，25，30)，ultrasonic time (20，30，40，50，
60 min)，power (50%，60%，70%，80%，90%，
100%)，temperature (30，40，50，60，70 ℃)and etha-
nol concentration (50%，60%，70%，80%，90%)．
Determination of TFC content
The TFC was determined by the modified aluminum
chloride colorimetric method ［18］ with rutin as stand-
ard． Each of the plant extracts (2 mL，1． 6 mg /mL)or
rutin (0． 222 mg /mL)was added to 30% ethanol con-
centration (4 mL)and 5% NaNO2 solution (1 mL)．
After 6 min，10% Al(NO3)3 solution (1 mL)was add-
ed． After another 6 min，4% NaOH solution (10 mL)
was added，and the volume was made up with 30%
ethanol． The mixture was shaken thoroughly and meas-
ured at 510 nm． The results were expressed as milli-
gram Ｒutin equivalents /g of dry plant material．
The calibration curve of rutin was y = 12． 671x-
0. 0125，Ｒ2 = 0． 9998 (0． 00888-0． 05328 mg /mL)．
157
Vol. 26 TANG Xu-chong，et al:Ultrasonic Assisted Extraction and Antioxidant
Activity of Hypericum japonicum Phenolic and Flavonoid Compounds
Here，y = absorbance and x = concentration． All experi-
ments were done in triplicate．
Determination of TPC content
The TPC was determined by the modified Folin-Ciocal-
teu method［19］ with gallic acid as standard． Each of the
plant extracts (1 mL，1． 6 mg /mL)or gallic acid (150
μg /mL)was added to deionized water (10 mL)and
Folin-Ciocalteu reagent (1． 5 mL)． After 30s，10%
Na2CO3(6 mL)was added to the mixture． The mixture
was shaken thoroughly and allowed to stand at 30 ℃
for 2 h in the dark． The absorbance was measured at
765 nm． The results were expressed in mg Gallic acid
equivalent /g of dry plant material．
The calibration curve of gallic acid was y = 129． 34x-
0. 0125，Ｒ2 = 0． 9993 (0． 000616-0． 00616 mg /mL)．
Here，y = absorbance and x = concentration． All experi-
ments were done in triplicate．
DPPH and ABTS + free radical scavenging activity
assay
DPPH assay
The DPPH free radical scavenging activity of the ex-
tracts was determined using the reported method ［20］．
Equal volumes (800 μL)of different concentrations of
the extracts and ethanol (1200 μL)were added with
0． 1mM DPPH (2000 μL)． The mixture was measured
at 517 nm after 30 min of incubation at 37 ℃ in the
dark． The % DPPHsc was determined using the follow-
ing formula:%DPPHsc = ［1-(As-A0)/Ac］× 100
As:sample (800 μL)+ ethanol (1200 μL)+ 0． 1mM
DPPH (2000 μL)
A0:sample (800 μL)+ ethanol (3200 μL)
Ac:ethanol (2000 μL)+ 0． 1 mM DPPH (2000 μL)
The IC50 was calculated from the graph of scavenging
effect percentage against extract concentration． Synthet-
ic antioxidants (Trolox and BHT)were used as con-
trol，and all tests were performed in triplicate．
ABTS +· assay
ABTS + free radical was produced by reacting 2，2’-
azinobis ［3-ethylbenzothiazoline-6-sulphonic acid］
(ABTS)with potassium persulfate (K2S2O8)． ABTS
+
·
assay was carried out，according to the reported method
［21］ with slight modification． ABTS +· solution (2000
μL)was added to each of the samples (800 μL)，and
mixed vigorously． The reaction mixture was kept at
room temperature for 7 min before the absorbance was
measured at 734 nm． The %ABTSsc was determined u-
sing the following formula:% ABTSsc =［1-(As-A0)/
Ac］× 100
As:sample (800 μL)+ ethanol (1200 μL)+ ABTS
+
·
(2000 μL)
A0:sample (800 μL)+ ethanol (3200 μL)
Ac:ethanol (2000 μL)+ ABTS
+
·(2000 μL)
The IC50 was calculated from the graph of scavenging
effect percentage against extract concentration． Synthet-
ic antioxidants (Trolox and BHT)were used as con-
trol，and all tests were performed in triplicate．
Experimental design
The extraction parameters were optimized using re-
sponse surface methodology (ＲSM)． A Box-Behnken
design (BBD)was employed for experimental design，
data analysis and model building． Three variables used
in the study were ultrasonic time (X1)，ethanol con-
centration (X2)and temperature (X3)． The symbols
and levels presenting in Table 1 were based on single
factor pre-test． TFC，TPC，% DPPHsc，% ABTSsc were
selected as the responses for the combination of the in-
dependent variables given in Table 2． Three triplicate
experiments were carried out at each experimental de-
sign point and the mean values were stated as observed
responses． Experimental runs were randomized，to mini-
mize the effects of unexpected variability in the ob-
served responses． The variables were coded according
to the following equation:X =(Xi-Xo)/△X
Where X is the coded value，Xi is the corresponding
actual value，Xo is the actual value in the center of the
domain，and △X is the increment of Xi corresponding
to a variation of 1unit of X． The mathematical model
corresponding to the Box-Behnken design is: Y = β0
+ β1X1 + β2X2 + β3X3 + β11 X12 + β22 X22 + β33 X32 + β12
X1X2 + β13X1X3 + β23X2X3 + ε
Where Y is the dependent variable (TFC，TPC，%DP-
PHsc，%ABTSsc)，β is the model constant，βi，βii and
βij are the model coefficients，and ε is the error． They
represent the linear，quadratic and interaction effects of
the variables． Analysis of the experimental design data
and calculation of predicted responses were carried out
257 Nat Prod Ｒes Dev Vol. 26
using Design Expert software (version7． 0，stat-Ease，
Inc．，Minneapolis，MN)． Additional confirmation ex-
periments were subsequently conducted to verify the
validity of the statistical experimental design．
Table 1 Three factors and three levels design of ＲSM ex-
periment
Variable
Levels
-1 0 1
X1:Ultrasonic time (min) 40 50 60
X2:Ethanol concentration (%) 60 70 80
X3:Ultrasonic temperature 30 50 70
Ｒesults and Discussion
Single factor experiments for TFC extraction
Effect of liquid-to-solid ratio on the extraction yield of TFC
In order to evaluate the effect of liquid-to-solid ratio on
the extraction yield of TFC，different liquid-to-solid ra-
tios (10，15，20，25 and 30)were tested． Other experi-
mental parameters were set as follows:70% ethanol
concentration;60 ℃ ultrasonic temperature;100% ul-
trasonic power;30 min ultrasonic time． The results were
shown in Fig． 1．
120
110
100
90
80
70
60
50
40
30
0 10% 15% 20% 25% 30% 35
Liquid鄄to鄄solid%ratio(mL/g)
TF
C(
m
gR
E/
gD
W
) TFC
Fig. 1 Effect of liquid-to-solid ratio on the extraction
yield of TFC
The extraction yield of TFC increased from 35． 14 mg
ＲE /g DW to 110． 06 mg ＲE /g DW as the liquid-to-
solid ratio increased within the range of 10 ～ 25 (V /
W)． When the liquid-to-solid ratio increased to 30 (V /
W)，the yield of TFC increased to 88． 64 mg ＲE /g
DW． The results indicated that 1∶ 25 was more suitable
for the extraction of TFC．
Effect of ethanol concentration on the extraction yield of
TFC
Ethanol concentration was the most important step to-
wards parameter optimization，which had a strong im-
pact on extraction yield of TFC． Different ethanol con-
centrations (50%，60%，70%，80% and 90%)were
tested in the experiment． Other experimental parameters
were set as follows∶ 1∶ 20 liquid-to-solid ratio;60 ℃ ul-
trasonic temperature;100% ultrasonic power;30min
ultrasonic time． The results were shown in Fig． 2．
120
110
100
90
80
70
60
50
40
30
50% 60% 70% 80% 90
Ethanol%concentration(%)
TF
C(
m
gR
E/
gD
W
)
TFC
Fig. 2 Effect of ethanol concentration on the extraction
yield of TFC
The extraction yield of TFC increased from 85． 09 mg
ＲE /g DW to 87． 55 mg ＲE /g DW as the ethanol con-
centration increased from 50% to 60% ． When the eth-
anol concentration continued to increase up to 90%，
the yield of TFC decreased to 61． 90 mg ＲE /g DW．
The results indicated that 60% ethanol concentration
was suitable for the extraction of TFC．
Effect of ultrasonic power on the extraction yield of TFC
The yield from the TFC extraction could be influenced
by the ultrasonic power． Different ultrasonic powers
(100%，90%，80%，70%，60% and 50%)were test-
ed in the experiment． Other experimental parameters
were set as follows∶ 1∶ 25 liquid-to-solid ratio;70% eth-
anol concentration;60 ℃ ultrasonic temperature;30
min ultrasonic time． The results were shown in Fig． 3．
120
110
100
90
80
70
60
50
40
30
90% 80% 70% 60% 50
Ultrasonic%temperature(%)
TF
C(
m
gR
E/
gD
W
) TFC
100
Fig. 3 Effect of ultrasonic power on the extraction yield
of TFC
The extraction yield of TFC changed within the range of
85． 87 mg ＲE /g DW to 91． 22 mg ＲE /g DW as the ul-
357
Vol. 26 TANG Xu-chong，et al:Ultrasonic Assisted Extraction and Antioxidant
Activity of Hypericum japonicum Phenolic and Flavonoid Compounds
trasonic power increased from 50% to 100% ． The ul-
trasonic power had little impact on the extraction yield
of TFC．
Effect of ultrasonic temperature on the extraction yield of
TFC
Ultrasonic temperature was a factor that would signifi-
cantly influence the extraction efficiency of TFC． Differ-
ent ultrasonic temperatures (30，40，50，60 and 70 ℃)
were tested in the experiment． Other experimental parame-
ters were set as follows∶ 1 ∶ 25 liquid-to-solid ratio;60%
ethanol concentration;100%ultrasonic power;30min ultra-
sonic time． The results were shown in Fig． 4．
120
110
100
90
80
70
60
50
40
30
30% 40% 50% 60% 70
Ultrasonic%temperature(℃)
TF
C(
m
gR
E/
gD
W
)
TFC
Fig. 4 Effect of ultrasonic temperature on the extraction
yield of TFC
The extraction yield of TFC increased from 86． 63 mg
ＲE / g DW to 97． 11mg ＲE /g DW as ultrasonic temper-
ature increased from 30 ℃ to 70 ℃ ． It indicated ultra-
sonic temperature had a significant impact on the ex-
traction yield of TFC． The results showed that 70 ℃
was suitable for the extraction of TFC．
Effect of ultrasonic time on the extraction yield of TFC
Different ultrasonic time (20，30，40，50，60 min)was
tested in the experiment． Other experimental parameters
were set as follows∶ 1∶ 25 liquid-to-solid ratio;60% eth-
anol concentration;100% ultrasonic power;60 ℃ ultra-
sonic temperature． The results were shown in Fig． 5．
120
110
100
90
80
70
60
50
40
30
20% 30% 40% 50% 60
Ultrasonic%time(min)
TF
C(
m
gR
E/
gD
W
) TFC
Fig. 5 Effect of ultrasonic time on the extraction yield
of TFC
The extraction yield of TFC significantly increased from
97． 73 mg ＲE /g DW to 102． 66 mg ＲE /g DW as ultra-
sonic time increased from 20 min to 50 min． However，
as ultrasonic time increased up to 60 min，the yield of
TFC decreased to 94． 64 mg ＲE /g DW． The result
showed that 50 min was suitable for the extraction of
TFC．
Optimization of bioactive components and antioxi-
dant activities by Ｒesponse Surface Methodology
(ＲSM)
Three variables［ultrasonic time (X1)，ethanol concen-
tration (X2)and temperature(X3)］，which had higher
impact on the extraction yield of TFC，were selected ac-
cording to single factor tests． The four responses varia-
bles were TFC，TPC，% DPPHsc，% ABTSsc． The re-
sults of 15 runs using BBD design were shown in Table
2．
Table 2 BBD with observed responses for TFC，TPC，%DPPHsc and %ABTSsc
Ｒun
Coded variable levels Observed Predicted
X1 X2 X3
TPC
(mg GAE
/g DW)
TFC
(mgＲutin
/g DW)
DPPHsc
(%)
ABTSsc
(%)
TPC
(mg GAE
/g DW)
TFC
(mgＲutin
/g DW)
DPPHsc
(%)
ABTSsc
(%)
1 50 80 30 42． 58 82． 31 45． 16 53． 35 42． 58 82． 69 45． 33 53． 53
2 50 70 50 49． 32 106． 97 55． 31 62． 22 48． 89 107． 18 54． 4 61． 94
3 60 70 30 46． 33 88． 48 48． 53 54． 15 46． 01 86． 94 48． 41 54． 67
4 50 60 70 52． 49 102． 66 59． 14 63． 33 52． 49 102． 28 58． 97 63． 15
5 50 80 70 45． 97 92． 79 58． 11 60． 17 45． 55 92． 37 57． 55 60． 46
6 60 80 50 45． 12 90． 33 51． 26 59． 72 45． 45 91． 49 51． 2 59． 02
7 40 60 50 50． 5 100． 9 54． 23 61． 46 50． 17 99． 74 54． 29 62． 16
8 40 70 30 44． 7 84． 9 47． 12 54． 21 44． 61 85． 64 46． 5 53． 8
457 Nat Prod Ｒes Dev Vol. 26
9 60 70 70 49． 7 97． 73 56． 43 59． 89 49． 79 96． 99 57． 05 60． 3
10 50 70 50 50． 07 108． 21 54． 49 62． 5 48． 89 107． 18 54． 4 61． 94
11 40 80 50 42． 95 93 50． 11 55． 21 43． 05 91． 88 50． 56 55． 44
12 60 60 50 51． 2 100． 5 55． 15 58． 59 51． 1 101． 62 54． 7 58． 36
13 50 70 50 47． 54 106． 36 53． 39 61． 11 48． 89 107． 18 54． 4 61． 94
14 40 70 70 47． 54 95． 26 57． 79 61． 9 47． 86 96． 8 57． 91 61． 38
15 50 60 30 48 90． 33 50． 57 57． 18 48． 42 90． 75 51． 14 56． 89
aMean of triplicate determinations．
Table 3 ANOVA for response surface quadratic model:estimated regression model of relationship between response varia-
bles and independent variables (X1，X2 and X3)
Source Sum of squares DF Mean square F-value p-Value
TFC(mgＲutin /g DW)a
Model 906． 2 9 100． 69 37． 41 0． 0005 significant
A 1． 11 1 1． 11 0． 41 0． 549
B 161． 64 1 161． 64 60． 05 0． 0006
C 224． 93 1 224． 93 83． 57 0． 0003
AB 1． 29 1 1． 29 0． 48 0． 5199
AC 0． 31 1 0． 31 0． 11 0． 7489
BC 0． 86 1 0． 86 0． 32 0． 5972
A2 120． 54 1 120． 54 44． 78 0． 0011
B2 103． 08 1 103． 08 38． 3 0． 0016
C2 359． 97 1 359． 97 133． 73 ＜ 0． 0001
Ｒesidual 13． 46 5 2． 69
Lack of Fit 11． 68 3 3． 89 4． 38 0． 1914 not significant
Pure Error 1． 78 2 0． 89
Core Total 919． 65 14
TPC(mg GAE /g DW)b
Model 120． 38 9 13． 38 115． 97 0． 0035 significant
A 5． 54 1 5． 54 6． 62 0． 0499
B 81． 73 1 81． 73 97． 56 0． 0002
C 24． 82 1 24． 82 29． 62 0． 0028
AB 0． 54 1 0． 54 0． 64 0． 4584
AC 0． 07 1 0． 07 0． 084 0． 7838
BC 0． 3 1 0． 3 0． 36 0． 5741
A2 2． 75 1 2． 75 3． 29 0． 1297
B2 1． 66 1 1． 66 1． 98 0． 2181
C2 4． 04 1 4． 04 4． 82 0． 0795
Ｒesidual 4． 19 5 0． 84
Lack of Fit 0． 81 3 0． 27 0． 16 0． 9147 not significant
Pure Error 3． 38 2 1． 69
Core Total 124． 57 14
557
Vol. 26 TANG Xu-chong，et al:Ultrasonic Assisted Extraction and Antioxidant
Activity of Hypericum japonicum Phenolic and Flavonoid Compounds
%DPPHscc
Model 241． 69 9 26． 85 35． 69 0． 0005 significant
A 0． 56 1 0． 56 0． 75 0． 427
B 26． 1 1 26． 1 34． 69 0． 002
C 200． 9 1 200． 9 267 ＜ 0． 0001
AB 0． 013 1 0． 013 0． 018 0． 8997
AC 1． 92 1 1． 92 2． 55 0． 1712
BC 4． 8 1 4． 8 6． 37 0． 0529
A2 5． 71 1 5． 71 7． 59 0． 0401
B2 0． 8 1 0． 8 1． 06 0． 3494
C2 1． 74 1 1． 74 2． 31 0． 1892
Ｒesidual 3． 76 5 0． 75
Lack of Fit 1． 91 3 0． 64 0． 68 0． 6394 not significant
Pure Error 1． 86 2 0． 93
Core Total 245． 46 14
%ABTSscd
Model 155． 93 9 17． 33 26． 53 0． 0011 significant
A 0． 023 1 0． 023 0． 035 0． 8582
B 18． 33 1 18． 33 28． 07 0． 0032
C 87． 12 1 87． 12 133． 42 ＜ 0． 0001
AB 13． 62 1 13． 62 20． 85 0． 006
AC 0． 95 1 0． 95 1． 46 0． 2816
BC 0． 11 1 0． 11 0． 17 0． 6956
A2 16． 04 1 16． 04 24． 56 0． 0043
B2 4． 58 1 4． 58 7． 02 0． 0455
C2 19． 9 1 19． 9 30． 48 0． 0027
Ｒesidual 3． 26 5 0． 65
Lack of Fit 2． 18 3 0． 73 1． 35 0． 4529 not significant
Pure Error 1． 08 2 0． 54
Core Total 159． 19 14
a． The coefficient of determination (Ｒ2)of the model was 0． 9723．
b． The coefficient of determination (Ｒ2)of the model was 0． 9358．
c． The coefficient of determination (Ｒ2)of the model was 0． 9812．
d． The coefficient of determination (Ｒ2)of the model was 0． 9767．
Model fitting
Table 3 showed the results of fitting quadratic models to
the data． The results of analysis of variance (ANOVA)
indicated that the contribution of the quadratic model
was significant． The significance of each coefficient was
determined using the F-test and P-value in Table 3．
The lack of fit was also used to verify the adequacy of
the model． ANOVA for the lack of fit was not signifi-
cant (P ＞ 0． 05) for the model，indicating that the
model can adequately fit the experiment data． Coeffi-
cient (Ｒ2)of determination was defined as the ratio of
the explained variation to the total variation and was a
measurement of the degree of fitness． The small value of
Ｒ2 indicated the poor relevance of the dependent varia-
bles in the model． The model can fit well with the actu-
al data approaches unity．
The mathematical models representing TFC，TPC，%
DPPHsc，%ABTSsc as a function of the response varia-
657 Nat Prod Ｒes Dev Vol. 26
bles within the region under investigation were ex-
pressed by the following equation:
TFC = 105． 53 + 1． 14X1-4． 12X2 + 5． 70X3 +
0. 060X1X2-1. 08X1X3-0. 46X2X3-6. 36X
2
1-5. 12X
2
2-
8. 39X23
TPC = 49. 71 + 1. 42X1-1. 89X2 + 2. 55X3 +
0. 028X1X2-1. 60X1X3-1. 02X2X3-2. 66X
2
1-1. 48X
2
2-
1. 72X23
% DPPHsc = 54. 40 + 1. 14X1-0. 68X2 + 6. 26X3-
0. 69X1X2-1. 69X1X3 + 2. 60X2X3-2. 87X
2
1 + 0. 41X
2
2-
0. 061X23
% ABTSsc = 62. 22-0. 10X1-1. 80X2 + 4. 40X3 +
2. 10X1X2-1. 64X1X3-0. 15X2X3-2. 34X
2
1-0. 89X
2
2-
2. 50X23
TFC
It can be seen that the variable with the largest effect
on TFC extraction was the quadratic term of (X23)fol-
lowed by the linear terms of ultrasonic temperature
(X3)and the quadratic terms of ultrasonic temperature
(X23)and ultrasonic time (X
2
1) (P ＜ 0. 01)，the linear
term of ethanol concentration (X2)and the quadratic
term of (X22)were not significant，indicating that the
relationship between response variable (TFC)and the
process variables was not simply a linear one. The Mod-
el F-value of 19. 50 implied the model was signifi-
cant. The Lack of Fit F-value of 0. 64 implied that
the Lack of Fit was not significantly relative to the pure
error. Non-significant lack of fit was good. Ｒ2 value of
the model for TFC was determined to be 0. 9723. These
values gave a relative good fit to the mathematic model
in Fig. 6(A-C).
TPC
It can be seen the variable with the largest effect on
TPC extraction was the linear terms of ultrasonic tem-
perature(X3)(P ＜ 0. 01)and ethanol concentration
(X2)followed by the quadratic term of ultrasonic time
(X21)，the linear of (X1) (P ＜ 0. 05)，while the inter-
action terms were not significant (P ＞ 0. 05)，indica-
ting that the change of X1，X2，X3，X
2
1 had significant
effects on TPC extraction. The Model F-value of 8. 09
implied the model was significant. The Lack of Fit F-
value of 0. 18 implied the Lack of Fit was not sig-
nificant relative to the pure error. Non-significant
lack of fit was good. Ｒ2 value of the model for TFC was
determined to be 0. 9358. These values would give a
relative good fit to the mathematic model in Fig. 6(D-
F).
%DPPHsc
It can be seen the variable with the largest effect on
DPPH scavenging activity was the linear terms of ultra-
sonic temperature (X3)and the quadratic term of ul-
trasonic time (X21)，the interaction (X2 X3) (P ＜
0. 01)，and (X1 X3)，the linear term of (X1). It indi-
cated that the relationship between response variable
(%DPPHsc)and the process variables was not simply
a linear one. The Model F-value of 29. 01 implied the
model was significant. The Lack of Fit F-value of
2. 08 implied the Lack of Fit was not significant relative
to the pure error. Non-significant lack of fit was
good. Ｒ2 value of the model for % DPPHsc was deter-
mined to be 0. 9812. These values gave a relative good
fit to the mathematic model in Fig. 6(G-I).
%ABTSsc
It can be seen the variable with the largest effect on
ABTS scavenging activity was the linear terms of (X3)
and (X2)followed by the quadratic terms of ultrasonic
temperature (X23)and ethanol concentration(X
2
1) (P
＜ 0. 01)，the interaction (X1，X2)and (X1，X3) (P
＜ 0. 05)，indicating that the relationship between re-
sponse variable (%ABTSsc)and the process variables
was not simply a linear one. The Model F-value of
23. 29 implied the model was significant. The Lack of
Fit F-value of 0. 79 impliesd the Lack of Fit was not
significant relative to the pure error. Non-significant
lack of fit was good . Ｒ2 value of the model for %
ABTSsc was determined to be 0. 9767. These values
gave a relative good fit to the mathematic model in
Fig. 6(J-L).
Interpretation of response surface method
Graphs of ＲSM directly reflected the impact of factors on
the response value，which the extraction yield was corre-
sponding to the factor X1，X2，X3 consisting of a Three-di-
mensional response surface plot and two-dimensional con-
tour plot. Its interactions during the procedure can be
found from the response surface plot. The contour plot and
response surface graph of TFC，TPC，% DPPHsc，%
ABTSsc were shown in Fig. 6.
757
Vol. 26 TANG Xu-chong，et al:Ultrasonic Assisted Extraction and Antioxidant
Activity of Hypericum japonicum Phenolic and Flavonoid Compounds
110
105
100
95
90
TF
C
110
105
100
95
90
85
80
TF
C
110
105
100
95
90
85
80
TF
C
52
50
48
46
44
42
TP
C
52
50
48
46
44
42
TP
C
52
50
48
46
44
42
TP
C
60
55
50
45
DP
PH
sc（
%
）
60
55
50
45
DP
PH
sc（
%
）
60
55
50
45
DP
PH
sc（
%
）
64
62
60
58
56
54
AB
TS
sc（
%
）
64
62
60
58
56
54
52
AB
TS
sc（
%
）
64
62
60
58
56
54
52
AB
TS
sc（
%
）
-1.0
-0.5
0
0.5
1.0 -1.0 -0.5
0 0.5
1.0
A:Ultrasonic%time
B：Ethanol%concentration
70
62
54 46
38 40
45
50
55
60
C：Ultrasonice%
temperature A:Ultrasonic%time
70
62
54 46
38C：Ultrasonice%
temperature
60 65
70
75
80
B：Ethanol%concentration
60
65 70
75
80
B：Ethanol%concentration
70
62
54 46
38C：Ultrasonice%
temperature
70
62
54 46
38C：Ultrasonice%
temperature
40
45
50
55
60
A:Ultrasonic%time
40
45
50
55
60
A:Ultrasonic%time
80
75
70
65
60B：Ethanol%concentration
80
75
70
65
60B：Ethanol%concentration
80
75
70
65
60B：Ethanol%concentration
70
62
54 46
38C：Ultrasonice%
temperature
70
62
54 46
38C：Ultrasonice%
temperature
70
62
54 46
38C：Ultrasonice%
temperature
70
62
54 46
38C：Ultrasonice%
temperature
40
45
50
55
60
A:Ultrasonic%time 40
45
50
55
60
A:Ultrasonic%time
60
65 70
75
80
B：Ethanol%concentration
60
65 70
75
80
B：Ethanol%concentration40
45
50
55
60
A:Ultrasonic%time
40
45
50
55
60
A:Ultrasonic%time
A B C
D E F
H I J
K L M
Fig. 6 Ｒesponse surface plots for extraction yield of TFC (A-C)，extraction yield of TPC (D-F)，%DPPH (G-I)and %
ABTS (J-L)
Optimization ultrasonic conditon by ＲSM
Table 4 showed the optimal conditions for each individ-
ual response with the predicted and experimental val-
ues. Optimal conditions for TFC were:ultrasonic time of
50. 58 min，ethanol concentration of 65. 83% and ultra-
sonic temperature of 59. 96 ℃ . Optimal conditions for
TPC were ultrasonic time of 49. 59 min，ethanol con-
centration of 60. 15% and ultrasonic temperature of
70. 00 ℃. Optimal condition for % DPPHsc was ultra-
sonic time of 47. 84 min，ethanol concentration of
80. 00% and ultrasonic temperature of 70. 00 ℃ ． Opti-
mal condition for % ABTSsc was ultrasonic time of
41. 78 min，ethanol concentration of 60. 00% and ultra-
sonic temperature of 70. 00 ℃. The conditions gave
857 Nat Prod Ｒes Dev Vol. 26
Table 4 Predicted and experimental values under optimal conditions based on individual response (TFC，TPC，%DP-
PHsc，%ABTSsc)
Ｒesponse variables
Optimal ultrasonic condition Maximum values
Ultrasonic
time (min)
Ethanol
concentration (%)
Ultrasonic
temperature (℃) Predicted value
Experimental
valuea
TFC 50. 58 65. 83 59. 96 107. 42 106. 55
TPC 49. 59 60. 15 70. 00 51. 98 52. 00
%DPPHsc 47. 84 80. 00 70. 00 63. 06 63. 14
%ABTSsc 41. 78 60. 00 70. 00 66. 40 65. 40
a:Analysis results were mean value.
TFC，TPC，%DPPHsc and %ABTSsc values of 107. 42
mg ＲE /g DW，51. 98 mg GAE /g DW，63. 06% and
66. 40%，respectively.
Table 5 showed that the three optimal conditions were
based on combination of all responses. The optimal con-
dition was ultrasonic time of 48. 89 min，ethanol con-
centration of 63. 72% and ultrasonic temperature of
66. 92 ℃ . The condition gave TFC，TPC，% DPPHsc
and %ABTSsc values of 105. 06 mg ＲE /g DW，51. 75
mg GAE /g DW，58. 81% and 64. 99%.
Table 5 Predicted and experimental values under optimal conditions based on combination of responses (TFC，TPC，%DP-
PHsc and %ABTSsc)
Optimal ultrasonic condition Maximum values
Ultrasonic
time
(min)
Ethanol
concentration
(%)
Ultrasonic
temperature
(℃)
Predicted values Experimental valuesa
TFC TPC DPPHsc(%)ABTSsc(%) TFC TPC DPPHsc(%)ABTSsc(%)
48. 89 63. 72 66. 92 105. 06 51. 75 58. 81 64. 99 106. 52 50. 60 59. 08 65. 87
a:Analysis results were mean values.
Antioxidant activity
The extract of H. japonicum was chosen for the DPPH
and ABTS +· scavenging assay，Trolox and BHT were
used as positive control. The results were shown in
Fig. 7 and Fig. 8. The IC50 values of H. japonicum，BHT
and Trolox in DPPH scavenging assays were 34. 07 μg /
mL，22. 79 μg /mL and 2. 78 μg /mL，respectively. The
IC50values of H. japonicum，BHT and Trolox in ABTS
+
·
scavenging assays were 25. 48 μg /mL，6. 59 μg /mL
and 1. 39 μg /mL. The results showed that the DPPH
and ABTS free radical scavenging activities of
H. japonicum were lower than the positive control
(BHT and Trolox).
100
90
80
70
60
50
40
30
20
10
0
20% 40% 60% 80
Sa
m
pl
e(
ug
/m
L)
Sample
BHT
Trolox
1000
DPPH%Scavenging%activity（%）
Fig. 7 DPPH free radical scavenging activity of
H. japonicum，BHT and Trolox
Conclusion
The response surface methodology and Box-Behnken
design were applied to evaluate the effects of three in-
dependent variables (ultrasonic time，ethanol concen-
tration，ultrasonic temperature) on the extraction of
TFC，TPC and the scavenging activities to DPPH and
ABTS + free radicals. The analysis of variance (ANO-
VA)indicated that the relationship between response
variable (TFC，TPC，% DPPHsc，% ABTSsc)and the
process variables was not simply linear one.
100
90
80
70
60
50
40
30
20
10
0
20%30%40%50%60%70%80%90
Sa
m
pl
e(
ug
/m
L)
Sample
BHT
Trolox
100
ABTS%Scavenging%activity（%）
Fig. 8 ABTS + free radical scavenging activity of
H. japonicum，BHT and Trolox
957
Vol. 26 TANG Xu-chong，et al:Ultrasonic Assisted Extraction and Antioxidant
Activity of Hypericum japonicum Phenolic and Flavonoid Compounds
From the data of the 3D response plots and model e-
quations of TFC，TPC，%DPPHsc，% ABTSsc，the opti-
mal conditions of each individual response and all re-
sponses were determined to be:
The optimal conditions of each individual response:Op-
timal conditions for TFC were:ultrasonic time of 50. 58
min，ethanol concentration of 65. 83% and ultrasonic
temperature of 59. 96 ℃ . Optimal conditions for TPC
were ultrasonic time of 49. 59 min，ethanol concentra-
tion of 60. 15% and ultrasonic temperature of 70. 00
℃ ． Optimal conditions for % DPPHsc were ultrasonic
time of 47. 84 min，ethanol concentration of 80. 00%
and ultrasonic temperature of 70. 00 ℃. Optimal condi-
tions for %ABTSsc were ultrasonic time of 41. 78 min，
ethanol concentration of 60. 00% and ultrasonic tem-
perature of 70. 00 ℃ . Under these optimized condi-
tions，The yields of TFC and TPC were 107. 42 mg ＲE /
g DW and 51. 98 mg GAE /g DW. The % DPPHsc and
%ABTSsc values were 63. 06% and 66. 40%.
The optimal conditions of all responses:ultrasonic time
of 48. 89 min，ethanol concentration of 63. 72% and ul-
trasonic temperature of 66. 92 ℃ . The optimal condi-
tions gave TFC，TPC，%DPPHsc and % ABTSsc values
of 105. 06 mg ＲE /g DW，51. 75 mg GAE /g DW，
58. 81% and 64. 99%，respectively.
Under these optical conditions，the experimental values
agreed with the predicted values. It indicated the high
fitness of four models used and the success of response
surface methodology for optimizing the extraction of
TFC and TPC，for maximizing scavenging activities of
H. japonicum on DPPH and ABTS + free radicals.
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