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Food & Function Accepted Manuscript This article can be cited before page numbers have been issued, to do this please use: Q. Zeng, X. Zhang, X. Xu, M. Jiang, K. Xu, J. Piao, L. Zhu, J. Chen and J. Jiang, Food Funct., 2013, DOI: 10.1039/C3FO60342C.
Food & Function Linking the chemistry and physics of food with health and nutrition www.rsc.org/foodfunction
Volume 2 | Number 5 | May 2011 | Pages 215–280
This is an Accepted Manuscript, which has been through the RSC Publishing peer review process and has been accepted for publication. Accepted Manuscripts are published online shortly after acceptance, which is prior to technical editing, formatting and proof reading. This free service from RSC Publishing allows authors to make their results available to the community, in citable form, before publication of the edited article. This Accepted Manuscript will be replaced by the edited and formatted Advance Article as soon as this is available. To cite this manuscript please use its permanent Digital Object Identifier (DOI®), which is identical for all formats of publication. More information about Accepted Manuscripts can be found in the Information for Authors.
ISSN 2042-6496
COVER ARTICLE Schini-Kerth et al. Evaluation of different fruit juices and purees and optimization of a red fruit juice blend
2042-6496(2011)2:5;1-7
Please note that technical editing may introduce minor changes to the text and/or graphics contained in the manuscript submitted by the author(s) which may alter content, and that the standard Terms & Conditions and the ethical guidelines that apply to the journal are still applicable. In no event shall the RSC be held responsible for any errors or omissions in these Accepted Manuscript manuscripts or any consequences arising from the use of any information contained in them.
www.rsc.org/foodfunction Registered Charity Number 207890
Page 1 of 18
Food & Function View Article Online
DOI: 10.1039/C3FO60342C
1
Antioxidant and anticomplement functions of flavonoids extracted
2
from Penthorum chinense Pursh
4
Qiao-Hui Zeng, Xue-Wu Zhang, Xi-Lin Xu, Ming-Hua Jiang, Kai-Peng Xu, Jin-Hua Piao, Liang
5
Zhu, Jian Chen, Jian-Guo Jiang*
6
College of Food and Bioengineering, South China University of Technology, Guangzhou, 510640,
7
China. *Author for correspondence (e-mail: [email protected]; phone +86-20-87113849; fax:
8
+86-20-87113843)
9 10
Abstract
11
Penthorum chinense Pursh is rich in flavonoids, which has strong antioxidant and anticomplement
12
activities. In order to optimize its extraction conditions, various extraction parameters were chosen
13
to identify their effects on flavonoids extraction. Single factor and Box-Behnken experimental
14
designs consisting of twenty four experimental runs and five replicates at zero point were applied
15
to obtain the optimal extraction yield. The optimization conditions for flavonoids extraction were
16
obtained as follow: ethanol concentration 60.89%, extraction time 68.15min, temperature 52.89°C
17
and liquid/solid ratio 19.70:1. Corresponding flavonoids content was 7.19%. The regression
18
equation was found to fit well with the actual situation. Furthermore, antioxidant activity (the free
19
radical scavenging ability and ferric reducing/antioxidant power) and anticomplement ability of
20
flavonoids from P. chinense were determined. Results showed that the flavonoids of P. chinense
21
displayed significant antioxidant and anticomplement activities. Its antioxidant activity can
22
compete with ascorbic acid (Vc), while its anticomplement activity (IC50=111.6µg/ml) surpassed
23
the effect of heparin (IC50=399.7µg/ml) that was used as the positive control, suggesting that P.
24
chinense flavonoids and its related products could potentially be used as a promising natural agent
25
in the treatment of humoral effector of inflammation.
26 27
Key words Penthorum chinense Pursh; flavonoids; anticomplement; antioxidant; extraction
1
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3
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28
1
Introduction
29
Penthorum chinense Pursh is a rooted vascular plant, its whole plant is used in China both for food
30
and folk medicine for the remedy of jaundice, oedema and traumatic injury, cholecystitis, adiposis
31
hepatica and infectious hepatitis.1 The dry unripe (or) ripe plant contains many functional
32
ingredients including alkaloids, flavonoids, volatile oils and glycosides. Abundant studies have
33
shown that its flavonoids are the most important active substances with jaundice-relieving effect,2
34
antiviral activity,3 and function of preventing alcoholic liver.4
35
The human complement system plays an important role in the host defense system in
36
resistance against foreign invasive organism through external wounds. Its effects are normally
37
beneficial to the host, but excessive activation of the system may induce pathologic reactions
38
causing a variety of inflammatory and degenerative diseases.5 As reported by Chung et al.,6 the
39
classical pathway is activated mainly by antigen-antibody complexes (mostly IgG or IgM) starting
40
with C1q, C1r, C1s, C4 and C2, eventually leading to the activation of C3 by cleavage into C3a
41
and C3b. However, the smaller molecules C3a, C4a, and C5a (anaphylatoxins) induce the release
42
of mediators from mast cells and lymphocytes, which cause a variety of inflammatory diseases,
43
and may be fatal if they occur after organ transplantation. Therefore, the modulation of
44
complement activity should be useful in the therapy of inflammatory diseases. It is reported that
45
some flavonoids showed significant anticomplement activities.7 In addition, A research showed
46
that flavonoids could alleviate inflammation caused by reactive oxygen species (ROS) system,
47
which is in close relationship with the antioxidant capacity of flavonoids.8 Therefore, the
48
identification of the potential anticomplement functions of flavonoids are often becomes one of
49
research goals, and the classical pathway of complement system is the commonly used model. In
50
addition, as we all know that flavonoids have extensively better antioxidant activity.
51
Although P. chinense and its flavonoids are widely used as a natural product in industries of
52
medicine and functional food, there are fewer reports on its optimisation extraction. In this
53
research we use ultrasonic-assisted extraction technique and response surface methodology (RSM)
54
to determine optimal extraction conditions for flavonoids. Additionally, the antioxidant and
55
anticomplement properties of total flavonoids from P. chinense were investigated for the first time.
2
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DOI: 10.1039/C3FO60342C
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56
2
Results and discussion
57
2.1 Single factor experiment
58
Main activity ingredients in P. chinense are flavonoids and other substances that can be dissolved
59
in ethanol since its safe and nontoxic, inexpensive, strong dissolving power characteristics. The
60
effect of different parameters (concentrations of alcohol, extraction time, extraction temperature
61
and liquid-solid ratio) on extraction yield of flavonoids is displayed in Fig. 1.
62
In the range of 40% to 60%, total flavonoids amount gradually increases. When the ethanol
63
concentration is increased to 60%, the extraction of flavonoids achieve maximum. Then, the
64
extraction rate declined while ethanol concentration continues to increase. Therefore, the ethanol
65
concentration being set at 60% was appropriate (Fig. 1 A). A similar tendency appeared in the rest
66
figures (Fig. 1 B, C, and D). In addition, in order to save energy and improve efficiency, the
67
extraction time 60 min, extraction temperature 60 °C and liquid-solid ratio 20:1 were the suitable
68
levels, which were chose as the central value for further optimization of RSM.
69
2.2
70
According to the method of Central Composite designed experiment and the levels of independent
71
variables that were chosen based on the values obtained in the single factor experiment. The
72
extraction rate of flavonoids (Y, %) was employed as response value, and the four factors and
73
three levels' RSM test was designed (Table 1). Model of flavonoids yield was significantly. The
74
quadratic regression equation was obtained as follows (formula 5):
75
R1 (yield of total flavonoids) =7.07+0.22A+0.39B+0.18C+0.12D+0.11AB-0.21AC
76
-0.37AD+0.062 BC -0.079BD -0.12CD -0.46A2-0.53 B2- 0.89C2-0.26 D2
77
Where A is extraction temperature (oC); B is extraction time (min); C is ethanol concentration (%);
78
D is liquid/solid ratio (ml/g).
Optimisation of extraction conditions
(5)
79
The p value of the model was less than 0.01 (p value C2 > A2 > D2 >
91
B > AD > A > AC > C > D.
92
The two-dimensional contour plots and three-dimensional response surface diagrams are
93
shown in Fig. 3. Each 3D plot represents the number of combinations of the two-test variable,
94
illustrating the three-dimension response surface curves of extraction yield for each pair of
95
parameters by keeping the third factor constant at its zero level.11 According to the best fitting
96
polynomial equation, the interactions between extraction temperature and ethanol concentration or
97
extraction temperature and liquid/solid ratio were significant in determining a higher extraction
98
yield (Table 2). There were non-significant interaction effects between any other factors.
99
From Fig. 3B, an increasing ethanol concentration resulted in a higher extraction yield
100
keeping extraction temperature in a constant value, while the flavonoids extraction yield reached a
101
maximum when was ethanol concentration up to a certain value, with no further significant
102
improvement. But when they exceeded a certain value, the extraction rate dropped. The best point
103
of balance should be sought for the maximum extraction rate of flavonoids between ethanol
104
concentration and extraction temperature. This could be explained by the fact that the high ethanol
105
concentration in the solvent would be easier to extract out low polar substances causing a low
106
flavonoids extraction yield.12
107
From Fig. 3C, a similar interaction between extraction temperature and liquid/solid ratio on
108
the extraction rate of flavonoids could be easily obtained. In another study which was conducted
109
by Yang et al.,9 they found a similar result about the effect of extraction temperature and
110
liquid/solid ratio on the flavonoid yield from Citrus aurantium L. var. amara Engl.
111
The optimal conditions (extraction temperature 52.89°C, time 68.15min, ethanol
112
concentration 60.89% and liquid/solid ratio of 19.70) were obtained from the regression equation.
113
Under the optimal conditions, the maximum response value of yield (7.19%) was predicted by the
4
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114
model. In order to validate the adequacy of the model, verification experiments were carried out
115
under adjusted conditions (extraction temperature 50°C, time 70min, ethanol concentration 60%
116
and liquid/solid ratio of 20). Flavonoids yield of 7.21% was obtained and was in good agreement
117
with the predicted one. The accuracy of the model was validated with triplicate experiments under
118
aforementioned optimal conditions. Therefore, the extraction conditions obtained by response
119
surface methodology were not only accurate and reliable, but also with practical value reflecting
120
the expected optimization.
121
2.3
122
Chemical methods, which are easy to execute and have high reproducibility, can be used to
123
measure the antioxidant capacity of drugs.13 One method for reducing power and three methods
124
based on the radical scavenging capacity are employed to evaluate the antioxidant activities of the
125
total flavonoids of P. chinense. The antioxidant and anticomplement activities are shown in Fig. 4.
126
From Fig. 4A, the DPPH radical scavenging capacity of the total flavonoids of P. chinense
127
showed the same tendency with Vc. From 25 to 100µg/ml, it gradually increases with the increase
128
of the concentration. When the concentration increases to 100µg/ml, the scavenging capacity of
129
flavonoids achieve maximum. Then, it keeps at a constant level with the concentration
130
continuously increases. When the concentration increased from 25 to 200µg/ml, the superoxide
131
anion radical scavenging activity was negligible (Fig. 4B). When it transcends 200 µg/ml, there is
132
an apparent increasing tendency, which was still much lower than Vc. From Fig. 4C, when the
133
concentration increased from 25 to 1000µg/ml, hydroxyl radical scavenging activity rises steadily,
134
showing potently scavenging capacity of hydroxyl free radical when compared to Vc. In Fig. 4D,
135
the total flavonoids of P. chinense showed strong reducing power, which has transcended that of
136
Vc when the concentration is over 400µg/ml.
Antioxidant and anticomplement activities
137
Inhibition of P. chinense flavonoids on the classical pathway of complement system can
138
compete with that of heparin (used as a reference) (Fig. 4E), at a concentration of 200 µg/ml, P.
139
chinense flavonoids almost inhibited all of the hemolytic activity of GPS (1:20), indicating a
140
strong inhibition effect on the complement activation through the classic pathway. Many scholars
141
have studied natural flavonoids with anti-complement activity. Flavonoids have strong
142
anti-complement activities, and there exists a certain relationship between the activities and
5
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143
structure. For instance, the inhibitory potencies of flavonoids from leaves of Litsea japonica
144
against complement activity increased in inverse proportion to number of free hydroxyls on B-ring
145
of
146
immunoregulatory functions, but complement activation can also lead to severe disturbances, such
147
as the pathogenesis of asthma, acquired haemolytic anaemia, and Alzheimer's disease.14 At the
148
same time, it is known that free radical scavengers can attenuate myocardial reperfusion injury by
149
quenching reactive oxygen species that are released on the reintroduction of blood flow. Lauver et
150
al.15 reported that carotenoids were a naturally occurring group of compounds that possess
151
antioxidant properties and anticomplement activities. Therefore, there exists potential
152
physiological relevance in the diseases caused by oxidative damage and over activation of the
153
complement system. In addition, abundant of researches showed that many kinds of flavonoid
154
monomers and mixtures exhibiting good anti-inflammatory effects, and this are consistent with
155
good anti-complement activity of P. chinense flavonoids.16, 17
5,7-dihydroxyflavone.4
Complement
system
has
important
immunoprotective
and
156
Considering that P. chinense is rich in flavonoids, isolation and purification of the total
157
flavonoids of P. chinense deserved further investigation to clarify the structure-activity
158
relationship. Flavonoids in P. chinense might be a good candidate as a compound for improving
159
the unwanted and excessive activation of the complement system.
160
3
161
3.1
162
The dried P. chinense were purchased from Qingping medicinal material market (Guangzhou
163
China). Samples were ground in a cutting mill to pass through 50-mesh sieve to obtain fine
164
powder, air-dried at 45℃ and stored in a well-closed container for further use. All chemical
165
reagents were of analytical grade.
166
3.2 Extraction of flavonoids
167
The pre-prepared powder (0.5g) was treated by ultrasonic for flavonoids under the designed
168
conditions. Extraction conditions were adjusted to ultrasonic power 100 W, and different ethanol
169
concentration (40%-95%), ratio of liquid/solid (ml/g) (6:1-30:1), extraction time (20min-120min),
170
water bath temperature 30°C-70°C. When one of the conditions was changed, other conditions
171
were adjusted accordingly. After that the extracts were combined, filtrated, and removed the
Experimental Materials
6
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172
solvent by vacuum concentration (40°C, vacuum degree 0.08 Pa). The obtained concentrated
173
solution was dissolved in ethanol (60%) to 25ml, then, 1ml was taken out to a defined volume of
174
50 ml for analysis. The experiment was repeated three times.
175
3.3 Determination of total flavonoids content
176
The total flavonoids content was estimated using NaNO2-Al(NO3)3-NaOH system described by
177
Choi et al. with minor modifications.18 The regression equation of rutin standard curve was
178
obtained as y=11.904x-0.0022 (R2=0.9997), exhibiting a good linear relationship within the range
179
of 0 ~ 0.04mg/ml.
180
As the method of standard curve preparation, 1 ml of prepared sample was added to
181
measure the flavonoids content of the extract. The absorbance of the mixture at 510 nm was
182
measured. The total flavonoids content was expressed as rutin (mg/ml).
183
Y (%) =
C 0 × V1 × V2 × 100% m × 1000 × V0
(1)
184
Where Y is the total flavonoids (%), C0 is the flavonoids content of prepared samples
185
(mg/ml), m is the weight of defatted powder of the P. chinense (g), V0 is the volume of solution to
186
be tested for constant volume (ml), V1 is the volume of the solution was diluted (ml), V2 is the
187
constant volume of the solvent to be measured.
188
3.4 Antioxidant Activity
189
The scavenging activity was measured according to the modified method of our previous work.19
190
The superoxide radical scavenging activity was estimated according to the method of Pan et al.
191
with minor modifications.20 Each sample was run in triplicate, and ascorbic acid was used as a
192
positive control. A decrease of the absorbance was an indication of high superoxide radical
193
scavenging activity. Scavenging activity (SA) was calculated according to the following formula:
194
SA(%) =
195 196 197
A0 − ( A − Ab ) × 100% A0
(2)
Where A0 is the absorbance of solution without sample (control), A is the absorbance of sample mixed with pyrogallol, and Ab is the absorbance of sample without pyrogallol (blank). The hydroxyl radical is the most active and toxic free radical. The hydroxyl radical
7
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DOI: 10.1039/C3FO60342C
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198
scavenging activity was estimated according to the method of Smirnoff et al. with minor
199
modifications.21 The reaction mixture consisted of 1 ml 9 mM Fe2SO4, 1 ml 9 mM salicylic acid,
200
1 ml sample of different concentrations (25 to 1,000 µg/ml) diluted by 70% ethanol solution, and
201
1 ml 0.03% H2O2. The mixture was shaken vigorously and left to stand for 30 min in the dark at
202
room temperature. Absorbances of sample were measured at 510 nm, against a blank sample.
203
Ascorbic acid was used as a positive control. SA was calculated using the formula described as
204
follow:
205
SA(%) =
A0 − ( A − Ab ) × 100% A0
(3)
206
Where A0 is the absorbance of solution without sample (control), A is the absorbance of
207
reaction system with sample, and Ab is the absorbance of sample without salicylic acid (blank).
208
The reducing capacity assessment of each sample was determined using the modified procedure
209
by our previous work.19
210
3.5 Anticomplement activity.
211
Anti-complement activity through the classical pathway was examined referring to a modified
212
method of Kim et al.22 Sensitized erythrocytes (EAs) were prepared by incubation of sheep
213
erythrocytes (4.0×108 cells/ml) with equal volumes of rabbit anti-sheep erythrocyte antibody in
214
gelatin veronal buffer (GVB). Each sample was dissolved in DMSO (Percentage within 2%) and
215
used as a blank. Samples and heparin (used as positive control) were dissolved in GVB. Guinea
216
pig serum (GPS) was used as the complement source. The 1:20 diluted (GPS) was chosen to give
217
submaximal lysis in the absence of complement inhibitors. Various dilutions of tested samples
218
(50~400 µg/ml) 200µL were pre-incubated with 200 µL GPS at 37°C for 10min. Then 200 µL EAs
219
was added and then, the mixture was incubated at 37 °C for 30 min. Optical density of the
220
supernatant was measured at 405 nm with a spectrophotometer. Anti-complement activity was
221
determined as a mean of triplicate measurements and expressed as the hemolysis inhibition rate
222
(HIR) from complement-dependent hemolysis of the control. Anti-complement activity was
223
determined as a mean of triplicate measurements.
224
HIR(%) =
( A0 − Ab ) − ( A − Ab ) × 100% A0 − Ab
(4)
8
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225
Where A0 is the absorbance of solution without sample (control), namely, 200µL EAs and
226
200µL GVB and 200µL GPS; A is the absorbance of reaction system with sample, and Ab is the
227
absorbance of sample without sensitized erythrocytes (EAs) (blank),namely, 200µL EAs and
228
400µL GVB.
229
3.6 Statistical analysis
230
Values from all experiments were expresses as the means ± standard deviation (SD). DPS (Design
231
Expert 8.0) was used to establish mathematical model and obtain the optimum conditions of
232
technological. Statistical analyses were performed using SPSS statistical package 17.0 (SPSS Inc.,
233
Chicago, IL). One way analysis of variance was conducted to compare the yield of flavonoids
234
under different extraction conditions and values with p F
Model
10.20356
14
0.72882
37.0848
< 0.0001
significant
A (temperature)
0.569416
1
0.56941
28.9736
< 0.0001
**
1.808857
1
1.80885
92.0401
< 0.0001
**
0.408114
1
0.40811
20.7660
0.0004
** *
B (time) C (ethanol concentration) D (liquid/solid ratio)
0.1638
1
0.1638
8.33465
0.0119
AB
0.05085
1
0.05085
2.58741
0.1300
AC
0.171396
1
0.17139
8.72114
0.0105
*
AD
0.536556
1
0.53655
27.3016
0.0001
**
BC
0.015252
1
0.01525
0.77608
0.3932
BD
0.024806
1
0.02481
1.26222
0.2801
CD
0.060025
1
0.06003
3.05425
0.1024
A^2
1.385601
1
1.38560
70.5036
< 0.0001
**
B^2
1.815585
1
1.81559
92.3825
< 0.0001
**
C^2
5.132848
1
5.13285
261.175
< 0.0001
**
D^2
0.44164
1
0.44164
22.4720
0.0003
**
Residual
0.275141
14
0.01965
Lack of Fit
0.212962
10
0.02130
1.37000
0.4083
non-signifi cant
Pure Error
0.062179
4
0.01555
Cor Total 10.4787 28 306 Note: * significant (P
Food & Function Accepted Manuscript This article can be cited before page numbers have been issued, to do this please use: Q. Zeng, X. Zhang, X. Xu, M. Jiang, K. Xu, J. Piao, L. Zhu, J. Chen and J. Jiang, Food Funct., 2013, DOI: 10.1039/C3FO60342C.
Food & Function Linking the chemistry and physics of food with health and nutrition www.rsc.org/foodfunction
Volume 2 | Number 5 | May 2011 | Pages 215–280
This is an Accepted Manuscript, which has been through the RSC Publishing peer review process and has been accepted for publication. Accepted Manuscripts are published online shortly after acceptance, which is prior to technical editing, formatting and proof reading. This free service from RSC Publishing allows authors to make their results available to the community, in citable form, before publication of the edited article. This Accepted Manuscript will be replaced by the edited and formatted Advance Article as soon as this is available. To cite this manuscript please use its permanent Digital Object Identifier (DOI®), which is identical for all formats of publication. More information about Accepted Manuscripts can be found in the Information for Authors.
ISSN 2042-6496
COVER ARTICLE Schini-Kerth et al. Evaluation of different fruit juices and purees and optimization of a red fruit juice blend
2042-6496(2011)2:5;1-7
Please note that technical editing may introduce minor changes to the text and/or graphics contained in the manuscript submitted by the author(s) which may alter content, and that the standard Terms & Conditions and the ethical guidelines that apply to the journal are still applicable. In no event shall the RSC be held responsible for any errors or omissions in these Accepted Manuscript manuscripts or any consequences arising from the use of any information contained in them.
www.rsc.org/foodfunction Registered Charity Number 207890
Page 1 of 18
Food & Function View Article Online
DOI: 10.1039/C3FO60342C
1
Antioxidant and anticomplement functions of flavonoids extracted
2
from Penthorum chinense Pursh
4
Qiao-Hui Zeng, Xue-Wu Zhang, Xi-Lin Xu, Ming-Hua Jiang, Kai-Peng Xu, Jin-Hua Piao, Liang
5
Zhu, Jian Chen, Jian-Guo Jiang*
6
College of Food and Bioengineering, South China University of Technology, Guangzhou, 510640,
7
China. *Author for correspondence (e-mail: [email protected]; phone +86-20-87113849; fax:
8
+86-20-87113843)
9 10
Abstract
11
Penthorum chinense Pursh is rich in flavonoids, which has strong antioxidant and anticomplement
12
activities. In order to optimize its extraction conditions, various extraction parameters were chosen
13
to identify their effects on flavonoids extraction. Single factor and Box-Behnken experimental
14
designs consisting of twenty four experimental runs and five replicates at zero point were applied
15
to obtain the optimal extraction yield. The optimization conditions for flavonoids extraction were
16
obtained as follow: ethanol concentration 60.89%, extraction time 68.15min, temperature 52.89°C
17
and liquid/solid ratio 19.70:1. Corresponding flavonoids content was 7.19%. The regression
18
equation was found to fit well with the actual situation. Furthermore, antioxidant activity (the free
19
radical scavenging ability and ferric reducing/antioxidant power) and anticomplement ability of
20
flavonoids from P. chinense were determined. Results showed that the flavonoids of P. chinense
21
displayed significant antioxidant and anticomplement activities. Its antioxidant activity can
22
compete with ascorbic acid (Vc), while its anticomplement activity (IC50=111.6µg/ml) surpassed
23
the effect of heparin (IC50=399.7µg/ml) that was used as the positive control, suggesting that P.
24
chinense flavonoids and its related products could potentially be used as a promising natural agent
25
in the treatment of humoral effector of inflammation.
26 27
Key words Penthorum chinense Pursh; flavonoids; anticomplement; antioxidant; extraction
1
Food & Function Accepted Manuscript
Published on 24 September 2013. Downloaded by Purdue University on 28/09/2013 10:54:17.
3
Food & Function
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28
1
Introduction
29
Penthorum chinense Pursh is a rooted vascular plant, its whole plant is used in China both for food
30
and folk medicine for the remedy of jaundice, oedema and traumatic injury, cholecystitis, adiposis
31
hepatica and infectious hepatitis.1 The dry unripe (or) ripe plant contains many functional
32
ingredients including alkaloids, flavonoids, volatile oils and glycosides. Abundant studies have
33
shown that its flavonoids are the most important active substances with jaundice-relieving effect,2
34
antiviral activity,3 and function of preventing alcoholic liver.4
35
The human complement system plays an important role in the host defense system in
36
resistance against foreign invasive organism through external wounds. Its effects are normally
37
beneficial to the host, but excessive activation of the system may induce pathologic reactions
38
causing a variety of inflammatory and degenerative diseases.5 As reported by Chung et al.,6 the
39
classical pathway is activated mainly by antigen-antibody complexes (mostly IgG or IgM) starting
40
with C1q, C1r, C1s, C4 and C2, eventually leading to the activation of C3 by cleavage into C3a
41
and C3b. However, the smaller molecules C3a, C4a, and C5a (anaphylatoxins) induce the release
42
of mediators from mast cells and lymphocytes, which cause a variety of inflammatory diseases,
43
and may be fatal if they occur after organ transplantation. Therefore, the modulation of
44
complement activity should be useful in the therapy of inflammatory diseases. It is reported that
45
some flavonoids showed significant anticomplement activities.7 In addition, A research showed
46
that flavonoids could alleviate inflammation caused by reactive oxygen species (ROS) system,
47
which is in close relationship with the antioxidant capacity of flavonoids.8 Therefore, the
48
identification of the potential anticomplement functions of flavonoids are often becomes one of
49
research goals, and the classical pathway of complement system is the commonly used model. In
50
addition, as we all know that flavonoids have extensively better antioxidant activity.
51
Although P. chinense and its flavonoids are widely used as a natural product in industries of
52
medicine and functional food, there are fewer reports on its optimisation extraction. In this
53
research we use ultrasonic-assisted extraction technique and response surface methodology (RSM)
54
to determine optimal extraction conditions for flavonoids. Additionally, the antioxidant and
55
anticomplement properties of total flavonoids from P. chinense were investigated for the first time.
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2
Results and discussion
57
2.1 Single factor experiment
58
Main activity ingredients in P. chinense are flavonoids and other substances that can be dissolved
59
in ethanol since its safe and nontoxic, inexpensive, strong dissolving power characteristics. The
60
effect of different parameters (concentrations of alcohol, extraction time, extraction temperature
61
and liquid-solid ratio) on extraction yield of flavonoids is displayed in Fig. 1.
62
In the range of 40% to 60%, total flavonoids amount gradually increases. When the ethanol
63
concentration is increased to 60%, the extraction of flavonoids achieve maximum. Then, the
64
extraction rate declined while ethanol concentration continues to increase. Therefore, the ethanol
65
concentration being set at 60% was appropriate (Fig. 1 A). A similar tendency appeared in the rest
66
figures (Fig. 1 B, C, and D). In addition, in order to save energy and improve efficiency, the
67
extraction time 60 min, extraction temperature 60 °C and liquid-solid ratio 20:1 were the suitable
68
levels, which were chose as the central value for further optimization of RSM.
69
2.2
70
According to the method of Central Composite designed experiment and the levels of independent
71
variables that were chosen based on the values obtained in the single factor experiment. The
72
extraction rate of flavonoids (Y, %) was employed as response value, and the four factors and
73
three levels' RSM test was designed (Table 1). Model of flavonoids yield was significantly. The
74
quadratic regression equation was obtained as follows (formula 5):
75
R1 (yield of total flavonoids) =7.07+0.22A+0.39B+0.18C+0.12D+0.11AB-0.21AC
76
-0.37AD+0.062 BC -0.079BD -0.12CD -0.46A2-0.53 B2- 0.89C2-0.26 D2
77
Where A is extraction temperature (oC); B is extraction time (min); C is ethanol concentration (%);
78
D is liquid/solid ratio (ml/g).
Optimisation of extraction conditions
(5)
79
The p value of the model was less than 0.01 (p value C2 > A2 > D2 >
91
B > AD > A > AC > C > D.
92
The two-dimensional contour plots and three-dimensional response surface diagrams are
93
shown in Fig. 3. Each 3D plot represents the number of combinations of the two-test variable,
94
illustrating the three-dimension response surface curves of extraction yield for each pair of
95
parameters by keeping the third factor constant at its zero level.11 According to the best fitting
96
polynomial equation, the interactions between extraction temperature and ethanol concentration or
97
extraction temperature and liquid/solid ratio were significant in determining a higher extraction
98
yield (Table 2). There were non-significant interaction effects between any other factors.
99
From Fig. 3B, an increasing ethanol concentration resulted in a higher extraction yield
100
keeping extraction temperature in a constant value, while the flavonoids extraction yield reached a
101
maximum when was ethanol concentration up to a certain value, with no further significant
102
improvement. But when they exceeded a certain value, the extraction rate dropped. The best point
103
of balance should be sought for the maximum extraction rate of flavonoids between ethanol
104
concentration and extraction temperature. This could be explained by the fact that the high ethanol
105
concentration in the solvent would be easier to extract out low polar substances causing a low
106
flavonoids extraction yield.12
107
From Fig. 3C, a similar interaction between extraction temperature and liquid/solid ratio on
108
the extraction rate of flavonoids could be easily obtained. In another study which was conducted
109
by Yang et al.,9 they found a similar result about the effect of extraction temperature and
110
liquid/solid ratio on the flavonoid yield from Citrus aurantium L. var. amara Engl.
111
The optimal conditions (extraction temperature 52.89°C, time 68.15min, ethanol
112
concentration 60.89% and liquid/solid ratio of 19.70) were obtained from the regression equation.
113
Under the optimal conditions, the maximum response value of yield (7.19%) was predicted by the
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114
model. In order to validate the adequacy of the model, verification experiments were carried out
115
under adjusted conditions (extraction temperature 50°C, time 70min, ethanol concentration 60%
116
and liquid/solid ratio of 20). Flavonoids yield of 7.21% was obtained and was in good agreement
117
with the predicted one. The accuracy of the model was validated with triplicate experiments under
118
aforementioned optimal conditions. Therefore, the extraction conditions obtained by response
119
surface methodology were not only accurate and reliable, but also with practical value reflecting
120
the expected optimization.
121
2.3
122
Chemical methods, which are easy to execute and have high reproducibility, can be used to
123
measure the antioxidant capacity of drugs.13 One method for reducing power and three methods
124
based on the radical scavenging capacity are employed to evaluate the antioxidant activities of the
125
total flavonoids of P. chinense. The antioxidant and anticomplement activities are shown in Fig. 4.
126
From Fig. 4A, the DPPH radical scavenging capacity of the total flavonoids of P. chinense
127
showed the same tendency with Vc. From 25 to 100µg/ml, it gradually increases with the increase
128
of the concentration. When the concentration increases to 100µg/ml, the scavenging capacity of
129
flavonoids achieve maximum. Then, it keeps at a constant level with the concentration
130
continuously increases. When the concentration increased from 25 to 200µg/ml, the superoxide
131
anion radical scavenging activity was negligible (Fig. 4B). When it transcends 200 µg/ml, there is
132
an apparent increasing tendency, which was still much lower than Vc. From Fig. 4C, when the
133
concentration increased from 25 to 1000µg/ml, hydroxyl radical scavenging activity rises steadily,
134
showing potently scavenging capacity of hydroxyl free radical when compared to Vc. In Fig. 4D,
135
the total flavonoids of P. chinense showed strong reducing power, which has transcended that of
136
Vc when the concentration is over 400µg/ml.
Antioxidant and anticomplement activities
137
Inhibition of P. chinense flavonoids on the classical pathway of complement system can
138
compete with that of heparin (used as a reference) (Fig. 4E), at a concentration of 200 µg/ml, P.
139
chinense flavonoids almost inhibited all of the hemolytic activity of GPS (1:20), indicating a
140
strong inhibition effect on the complement activation through the classic pathway. Many scholars
141
have studied natural flavonoids with anti-complement activity. Flavonoids have strong
142
anti-complement activities, and there exists a certain relationship between the activities and
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structure. For instance, the inhibitory potencies of flavonoids from leaves of Litsea japonica
144
against complement activity increased in inverse proportion to number of free hydroxyls on B-ring
145
of
146
immunoregulatory functions, but complement activation can also lead to severe disturbances, such
147
as the pathogenesis of asthma, acquired haemolytic anaemia, and Alzheimer's disease.14 At the
148
same time, it is known that free radical scavengers can attenuate myocardial reperfusion injury by
149
quenching reactive oxygen species that are released on the reintroduction of blood flow. Lauver et
150
al.15 reported that carotenoids were a naturally occurring group of compounds that possess
151
antioxidant properties and anticomplement activities. Therefore, there exists potential
152
physiological relevance in the diseases caused by oxidative damage and over activation of the
153
complement system. In addition, abundant of researches showed that many kinds of flavonoid
154
monomers and mixtures exhibiting good anti-inflammatory effects, and this are consistent with
155
good anti-complement activity of P. chinense flavonoids.16, 17
5,7-dihydroxyflavone.4
Complement
system
has
important
immunoprotective
and
156
Considering that P. chinense is rich in flavonoids, isolation and purification of the total
157
flavonoids of P. chinense deserved further investigation to clarify the structure-activity
158
relationship. Flavonoids in P. chinense might be a good candidate as a compound for improving
159
the unwanted and excessive activation of the complement system.
160
3
161
3.1
162
The dried P. chinense were purchased from Qingping medicinal material market (Guangzhou
163
China). Samples were ground in a cutting mill to pass through 50-mesh sieve to obtain fine
164
powder, air-dried at 45℃ and stored in a well-closed container for further use. All chemical
165
reagents were of analytical grade.
166
3.2 Extraction of flavonoids
167
The pre-prepared powder (0.5g) was treated by ultrasonic for flavonoids under the designed
168
conditions. Extraction conditions were adjusted to ultrasonic power 100 W, and different ethanol
169
concentration (40%-95%), ratio of liquid/solid (ml/g) (6:1-30:1), extraction time (20min-120min),
170
water bath temperature 30°C-70°C. When one of the conditions was changed, other conditions
171
were adjusted accordingly. After that the extracts were combined, filtrated, and removed the
Experimental Materials
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solvent by vacuum concentration (40°C, vacuum degree 0.08 Pa). The obtained concentrated
173
solution was dissolved in ethanol (60%) to 25ml, then, 1ml was taken out to a defined volume of
174
50 ml for analysis. The experiment was repeated three times.
175
3.3 Determination of total flavonoids content
176
The total flavonoids content was estimated using NaNO2-Al(NO3)3-NaOH system described by
177
Choi et al. with minor modifications.18 The regression equation of rutin standard curve was
178
obtained as y=11.904x-0.0022 (R2=0.9997), exhibiting a good linear relationship within the range
179
of 0 ~ 0.04mg/ml.
180
As the method of standard curve preparation, 1 ml of prepared sample was added to
181
measure the flavonoids content of the extract. The absorbance of the mixture at 510 nm was
182
measured. The total flavonoids content was expressed as rutin (mg/ml).
183
Y (%) =
C 0 × V1 × V2 × 100% m × 1000 × V0
(1)
184
Where Y is the total flavonoids (%), C0 is the flavonoids content of prepared samples
185
(mg/ml), m is the weight of defatted powder of the P. chinense (g), V0 is the volume of solution to
186
be tested for constant volume (ml), V1 is the volume of the solution was diluted (ml), V2 is the
187
constant volume of the solvent to be measured.
188
3.4 Antioxidant Activity
189
The scavenging activity was measured according to the modified method of our previous work.19
190
The superoxide radical scavenging activity was estimated according to the method of Pan et al.
191
with minor modifications.20 Each sample was run in triplicate, and ascorbic acid was used as a
192
positive control. A decrease of the absorbance was an indication of high superoxide radical
193
scavenging activity. Scavenging activity (SA) was calculated according to the following formula:
194
SA(%) =
195 196 197
A0 − ( A − Ab ) × 100% A0
(2)
Where A0 is the absorbance of solution without sample (control), A is the absorbance of sample mixed with pyrogallol, and Ab is the absorbance of sample without pyrogallol (blank). The hydroxyl radical is the most active and toxic free radical. The hydroxyl radical
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198
scavenging activity was estimated according to the method of Smirnoff et al. with minor
199
modifications.21 The reaction mixture consisted of 1 ml 9 mM Fe2SO4, 1 ml 9 mM salicylic acid,
200
1 ml sample of different concentrations (25 to 1,000 µg/ml) diluted by 70% ethanol solution, and
201
1 ml 0.03% H2O2. The mixture was shaken vigorously and left to stand for 30 min in the dark at
202
room temperature. Absorbances of sample were measured at 510 nm, against a blank sample.
203
Ascorbic acid was used as a positive control. SA was calculated using the formula described as
204
follow:
205
SA(%) =
A0 − ( A − Ab ) × 100% A0
(3)
206
Where A0 is the absorbance of solution without sample (control), A is the absorbance of
207
reaction system with sample, and Ab is the absorbance of sample without salicylic acid (blank).
208
The reducing capacity assessment of each sample was determined using the modified procedure
209
by our previous work.19
210
3.5 Anticomplement activity.
211
Anti-complement activity through the classical pathway was examined referring to a modified
212
method of Kim et al.22 Sensitized erythrocytes (EAs) were prepared by incubation of sheep
213
erythrocytes (4.0×108 cells/ml) with equal volumes of rabbit anti-sheep erythrocyte antibody in
214
gelatin veronal buffer (GVB). Each sample was dissolved in DMSO (Percentage within 2%) and
215
used as a blank. Samples and heparin (used as positive control) were dissolved in GVB. Guinea
216
pig serum (GPS) was used as the complement source. The 1:20 diluted (GPS) was chosen to give
217
submaximal lysis in the absence of complement inhibitors. Various dilutions of tested samples
218
(50~400 µg/ml) 200µL were pre-incubated with 200 µL GPS at 37°C for 10min. Then 200 µL EAs
219
was added and then, the mixture was incubated at 37 °C for 30 min. Optical density of the
220
supernatant was measured at 405 nm with a spectrophotometer. Anti-complement activity was
221
determined as a mean of triplicate measurements and expressed as the hemolysis inhibition rate
222
(HIR) from complement-dependent hemolysis of the control. Anti-complement activity was
223
determined as a mean of triplicate measurements.
224
HIR(%) =
( A0 − Ab ) − ( A − Ab ) × 100% A0 − Ab
(4)
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225
Where A0 is the absorbance of solution without sample (control), namely, 200µL EAs and
226
200µL GVB and 200µL GPS; A is the absorbance of reaction system with sample, and Ab is the
227
absorbance of sample without sensitized erythrocytes (EAs) (blank),namely, 200µL EAs and
228
400µL GVB.
229
3.6 Statistical analysis
230
Values from all experiments were expresses as the means ± standard deviation (SD). DPS (Design
231
Expert 8.0) was used to establish mathematical model and obtain the optimum conditions of
232
technological. Statistical analyses were performed using SPSS statistical package 17.0 (SPSS Inc.,
233
Chicago, IL). One way analysis of variance was conducted to compare the yield of flavonoids
234
under different extraction conditions and values with p F
Model
10.20356
14
0.72882
37.0848
< 0.0001
significant
A (temperature)
0.569416
1
0.56941
28.9736
< 0.0001
**
1.808857
1
1.80885
92.0401
< 0.0001
**
0.408114
1
0.40811
20.7660
0.0004
** *
B (time) C (ethanol concentration) D (liquid/solid ratio)
0.1638
1
0.1638
8.33465
0.0119
AB
0.05085
1
0.05085
2.58741
0.1300
AC
0.171396
1
0.17139
8.72114
0.0105
*
AD
0.536556
1
0.53655
27.3016
0.0001
**
BC
0.015252
1
0.01525
0.77608
0.3932
BD
0.024806
1
0.02481
1.26222
0.2801
CD
0.060025
1
0.06003
3.05425
0.1024
A^2
1.385601
1
1.38560
70.5036
< 0.0001
**
B^2
1.815585
1
1.81559
92.3825
< 0.0001
**
C^2
5.132848
1
5.13285
261.175
< 0.0001
**
D^2
0.44164
1
0.44164
22.4720
0.0003
**
Residual
0.275141
14
0.01965
Lack of Fit
0.212962
10
0.02130
1.37000
0.4083
non-signifi cant
Pure Error
0.062179
4
0.01555
Cor Total 10.4787 28 306 Note: * significant (P
