Documenti di Didattica
Documenti di Professioni
Documenti di Cultura
INTERNATIONAL UNIVERSITY
A thesis submitted to
The School of Biotechnology, International University
In partial fulfillment of the requirements for the degree of
B.S. in Biotechnology
June/ 2013
Acknowledgement
I would like to express my gratitude to all those who gave me the possibility
to complete this thesis. I want to thank the Department of Chemistry and
Technology of Natural Products of Institute of Chemical Technology for giving me
permission to commence this thesis in the first instance, and for giving me to do
all the necessary work with their lab equipment.
I have furthermore to thank Dr. Phan Thanh Thao and Assoc. Prof. Dr.
Nguyen Ngoc Hanh and in Department of Chemistry and Technology of Natural
Products who gave and confirmed this permission and encouraged me to go
ahead with my research.
I am deeply indebted to MSc. Phung Van Trung from the Department of
Chemistry
and
Technology
of
Natural
Products
whose
help,
stimulating
suggestions and encouragement helped me in all the time of research for and
writing of this thesis.
I want to thank all the staffs in the Department of Chemistry and Technology
of Natural Products and the University of Science, HCMC for their help, support,
interest and valuable hints.
Its my pleased to be helped by Dr. Gay Marsden from the International
University who looked closely at the final version of the report for English
grammar and style, correcting both and offering suggestions for improvement.
It is also my duty to record my thankfulness to Dr. Hoang Le Son for sending
me to the institute, inspiring and helping me in undertaking this project.
I am particularly in indebted to the School of Biotechnology for opening the
thesis course, giving me a chance to work in a friendly as well as professional
environment of Department of Chemistry and Technology of Natural Products.
Table of Contents
1.
Introduction ............................................................................................. 1
1.1.
Potential applications............................................................................................ 2
2.2.
Results .................................................................................................... 8
3.1.
3.1.1
Extraction ...................................................................................... 8
Structural identification........................................................................................ 9
3.3.
Discussion ............................................................................................. 21
4.1.
4.2.
4.2.1. Evaluation of anticancer activity against ovary HeLa cancer cell line.... 23
4.2.2. Evaluation of anticancer activity against lung NCI-H460 cancer cell line
23
4.3.
5.
Conclusion ............................................................................................. 25
iii
List of tables
Table 1: Traditional usages of each morphological part of A. cochinchinensis ......... 2
Table 3: Anticancer activity of total extract and fractions against HeLa cancer cell
line ............................................................................................................. 13
Table 4: Anticancer activity of pure compounds against HeLa cell line ................. 13
Table 5: Growth inhibition percentage of total extract against NCI-H460 cell line .. 14
Table 6: Growth inhibition percentage of pure compounds against NCI-H460 cancer
cell line ....................................................................................................... 14
Table 7: Absorbance values of DPPH ............................................................... 16
Table 8: Absorbance values of vitamin C ......................................................... 17
Table 9: Absorbance values of MeOH extract .................................................... 18
Table 10: Antioxidant activity of fractions at different concentrations .................. 19
Table 11: Percentage of inhibition (%E) related to concentration of AC01 - AC05 . 20
Table 13: IC50 values of total extract and pure compounds against NCI-H460 cancer
cell line ....................................................................................................... 24
Table 14: IC50 values of samples in antioxidant assay. 24
iv
List of figures
Figure 1: A. cochinchinensis plant [4]................................................................ 1
Figure 2: A. cochinchinensis tuber .................................................................... 1
Figure 3: The scheme of experimental process ................................................... 4
Figure 4: Extraction scheme ............................................................................. 8
Figure 5: Fractionation and purification scheme .................................................. 9
Figure 6: Inhibition percentage of AC01 against HeLa cell line ............................ 14
Figure 7: Growth inhibition percentage of AC01 against NCI-H460 cell line .15
Figure 8: Standard curve (negative control curve) 16
Figure 9: Positive control curve . 17
Figure 10: Antioxidant activity of A. cochinchinensis extract at different
concentrations . 18
Figure 11: Antioxidant activity of fractions of A. cochinchinensis at different
concentrations . 20
Figure 12: Antioxidant activity of AC01 at different concentrations .. 21
List of abbreviation
A. cochinchinensis
EtOAc
Ethyl Acetate
MeOH
Methanol
MPLC
NMR
DMSO
Dimethyl sulfoxide
DPPH
1,1-diphenyl-2-picrylhydrazyl
%E
Efficiency percentage
SD
Standard deviation
RSD
HeLa
NCI-H460
IC50
vi
EVALUATION
ANTIOXIDANT
OF
THE
ANTICANCER
ACTIVITIES
OF
AND
ASPARAGUS
COCHINCHINENSIS
Anh P. Nguyena, Son L. Hoanga*, Trung V. Phungb
a
HCMC
b
Abstract
Five compounds including quercetin (AC01), asparagine (AC02), sucrose (AC03),
- Sitosterol-3-O--glucopyranoside (AC04) and - Sitosterol (AC05) were
isolated from the methanol extract from tuber of Asparagus cochinchinensis
(Lour.) Merr. collected in Ba Ria Vung Tau Province of Vietnam. Their
structures were elucidated by NMR (1D and 2D-NMR). Among them, quercetin
(AC01) had strong antioxidant activity with IC50= 14.522.11 g/mL (DPPH
method, compared to standard vitamin C with IC50 = 10.492.00 g/mL).
Besides, quercetin (AC01) was evaluated cytotoxicity against the human ovary
HeLa cancer cell line with IC 50 = 5.780.36 g/mL (SRB method, compared to
standard Camptothecin with IC50 < 1 g/mL)and lung NCI H460 cancer cell
line with IC50 = 12.571.19 g/mL (SRB method, compared to standard
Camptothecin with IC50 < 0.01 g/mL).
Keywords:
Asparagus
cochinchinensis,
quercetin,
asparagine,
sucrose,
vii
1.
Introduction
1.1.
cochinchinensis
(Lour.)
Merr.
or
Cochinchinense
asparagus
(A.
Chemical compositions
Traditional usages
Latte
Flavonoids
Root
1.2.
Quercetin
Inulin
Potential applications
compound was evaluated in cytotoxicity against the human tumor cell line, A549 and
showed IC50 value of 3.87 g/mL [6].
The aim of this research is to isolate more compounds and to test bioactivities
including anticancer and antioxidant activities of total extract, fractions and pure
compounds in Asparagus cochinchinensis.
2.
2.1.
The main objective of this research was to evaluate the anticancer and antioxidant
activities of A. cochinchinensis.
Location:
2.2.
2.2.1. Materials
The fresh A. cochinchinensis tuber was collected in Ba Ria Vung Tau province of
Vietnam in January, 2013. The tuber was washed with water and cut into thin slices
before being heat dried at 600C. The sample was then ground into fine powder by a
mechanical grinder. The chemicals used were n-hexan, EtOAC, chloroform and methanol.
TLC Silica gel F254 and Silica gel60 (diameter: 0.006-0.2 mm) were purchased from
MERCK.
2.2.2. Methods
The plant powder was extracted by MeOH. The extract was then separated into three
groups based on the polarization of constituents. The first group (H- extract) consisted of
the lower polarized constituents and was separated using the n-hexan. EtOAC was used
to isolate the higher polarized constituents within EtOAc-extract. The last group (MeOHextract) contained the highest polarized constituents that dissolved in the methanol. All
three groups were tested for anticancer and antioxidant activities to evaluate their
potential for further experiments. Chromatography was used to separate compounds that
were then subjected to bioassays. The fractionation process is diagramed in Fig. 3.
Materials
Extract with MeOH
solvent
Total extract
Fractionation
H- extract
EtOAc- extract
(lower polar
group)
(higher polar
group)
MeOHextract
(highest polar
group)
Testing bioactivities
Potential
group (s)
- Fractionations
- Testing bioactivities
Active
fraction (s)
Fractionations, isolation,
purification, ...
Testing bioactivities
Active
compound (s)
Fig. 3: The scheme of experimental process
2.2.2.1.
Extraction methods
2.2.2.2.
medium pressure
2.3.1. Materials
The cervical cancer HeLa cell line and lung cancer NCI-H460 cell line were supplied
from the National Cancer Institute of the United States (NCI - Frederick, MD, USA).
The samples tested were the total extract, fractions and pure compounds. The
fractions were tested at different concentrations after dilution with DMSO.
The chemicals used for this experiment were DMSO, trichloroacetic acid (TCA),
sulforhodamine B (SRB) solution, acetic acid and trizma base supplied by the University
of Science.
2.3.2. Methods
This experiment was performed following two procedures of Vanicha V. and Skehan P.
with minor modifications [9, 10] .
First, the cells were inoculated and incubated in the 96-well plates at 37oC with 5%
CO2, 95% air and 100% relative humidity for 24 hours. Next, the plates were fixed with
TCA, to represent a measurement of the cell population for each cell line at the time of
drug addition. The samples were prepared to double concentration of initial sample
concentration and loaded into wells before being incubated for further 48 hours at 37oC
with 5% CO2, 95% air and 100% relative humidity. The cells were then fixed by the
gentle addition of 50 L of cold 50% (w/v) TCA (final concentration of TCA per well was
10%) and incubated for 60 minutes at 4C. The supernatant was discarded and the
plates were washed five times with tap water and air dried. Finally, SRB solution (100 L)
at 0.4% (w/v) in 1% acetic acid, was added to each well and plates were incubated for
10 minutes at room temperature. After staining, unbound dye was removed by washing
five times with 1% acetic acid and the plates were air dried. Bound stain was
subsequently solubilized with 10 mM trizma base, and the absorbance was read on an
automated plate reader.
By measuring the absorbance at 492 and 620 nm, the percentage of growth was
calculated using the following formulas:
OD492 (or OD620) = ODcell ODblank (1)
OD = OD492 OD620 (2)
The concentration of cytotoxic inhibition was calculated as follows:
With:
ODcell:
ODblank:
ODTN:
ODc:
2.4
2.4.1. Materials
The chemicals used in this experiment were DPPH (1,1-diphenyl, 2-picrylhydrazyl),
absolute ethanol, DMSO and ascorbic acid (vitamin C), supplied by Institute of Chemical
Technology.
Sample tested: purified compounds and fractions from tuber extract were diluted by
DMSO in different concentrations.
2.4.2. Methods
This DPPH assay was carried out as described by Amin et al. (2006) with slight
modifications [11, 12]. This assay measured hydrogen atom (or one electron) donating
activity and hence provided a measure of free-radical scavenging antioxidant activity.
DPPH was a purple-coloured stable free radical that formed a yellow color when it was
reduced as diphenylpicrylhydrazine complex.
The initial absorbance of ethanolic DPPH was measured at 517 nm without sample.
An aliquot (50 L) of extracts was mixed with 150 L of ethanolic DPPH solution. The
change in absorbance at 517 nm was measured after 30 minutes of incubation at room
temperature. Ascorbic acid was served as positive control. The experiment was replicated
three times.
Three types of curves were generated; standard curve (DPPH negative control
curve), positive control curve (vitamin C curve) and sample curve. With different
prepared concentrations, 200 L of each solution was loaded into wells in one row in a
micro plate, 3 replicates were loaded into 3 adjacent wells. Each test took 30 minutes to
complete (not including the negative control test). The absorbance of the plate was
measured at 517 nm.
The aim of this experiment was to compare the mean (average value) and the
relative standard deviation (%RSD) of optical density values within samples and between
samples and controls.
Where:
s:
N:
Xi xN :
CV:
coefficient variance
3.
Results
3.1.
3.1.1
Extraction
- Filtering
- Removing MeOH
- Partition/ n-hexane
- Filtering
n-hexan
extract
- Removing nhexane
Residue
EtOAc extract
- Partition/ EtOAc
- Filtering
- Removing EtOAc
Fig. 4: Extraction scheme
MeOH extract
MeOH extract
100% CHCl3
90% CHCl3
80% CHCl3
70% CHCl3
Fraction 1 (A)
Fraction 2 (B)
Fraction 3(C)
Fraction 4 (D)
Residue
Crystallization
F3.1
F3.2
AC04
Crystallization
AC02
F1.2
F1.1
Crystallization
AC01
Crystallization
AC03
Crystallization
AC05
Fig. 5: Fractionation and purification scheme
3.2.
Structural identification
The IUPAC name and structure of the compounds (AC01 AC05) were determined by
1H, 13C-NMR,
DEPT, HSQC, HMBC and COSY at the University of Natural Science, HCMC.
AC01
1
H-NMR,
13
AC02
H-NMR,
13
HMBC
HMBC
(Appendix 1 - 5)
(Appendix 6 -10)
Common name
Quercetin
IUPAC name
3,3',4',5,7-pentahydroxyflavone
Chemical formula
C15H10O7
C4H8N2O3
Molecular weight
302.236 g/mol
132.118 g/mol
Phytochemical
Flavonoid
Amino acid
Physical state
Amorphos, yellow
Crystal, white
Melting point
316oC
220oC
0.22
0.5
CHCl3:MeOH=9:1
butanol acid
Spectroscopy
2-amino-3-carbamoylpropanoic
acid
Structure
Rf (solvent)
10
AC03
1
Spectroscopy
H-NMR,
13
AC04
C-NMR, DEPT,
Common name
IUPAC name
H-NMR,
13
C-NMR, DEPT
(Appendix 17 19)
-Sitosterol-3-O--D-glucopyranoside
[17, 18]
-Sitosterol-3-O--D-glucopyranoside
Chemical formula
C12H22O11
C35H60O6
Molecular weight
342.3 g/mol
576.85 g/mol
Phytochemical
Sugar
Steroid
Physical state
Amorphos, white
Amorphos, white
Melting point
160oC 186oC
275277C
0.54
0.3
CHCl3:MeOH=7:3
CHCl3:MeOH=9:1
Structure
Rf (solvent)
11
H-NMR,
Common name
13
IUPAC name
2,3,4,7,8,9,11,12,14,15,16,17-dodecahydro-1Hcyclopenta[a]phenanthren-3-ol
Chemical formula
C29H50O
Molecular weight
414.71 g/mol
Structure
Phytochemical
Steroid
Physical state
Needle, colorless
Melting point
133135C
0.86
Rf (solvent)
CHCl3:MeOH=9:1
3.3.
Positive control: Camptothecin was served as the positive control of this method. This
drug was tested to evaluate the growth inhibition percentage against HeLa and NCI-H460
cell lines (Appendix 22).
For HeLa cancer cell line: the concentration of Camptothecin used was 1 g/mL, to
inhibite 51.223.43 % cell growth.
For NCI-H460 cancer cell line: the concentration of Camptothecin used was 0.01
g/mL, to inhibite 78.041.75 % cell growth.
12
% RSD
%E
AC-S
Undefined
AC-A
Undefined
AC-B
Undefined
AC-C
Undefined
AC-D
Undefined
AC01
g/mL
Mean SD
% RSD
Mean SD
% RSD
2.5
14.777 5.873
39.747
46.986 2.283
4.859
10
65.452 3.827
5.847
20
76.222 1.886
2.475
IC50
Insignificant
13
for
anticancer
activity:
The
absorbance
values
of
total
extract
% RSD
%E
AC-S
Undefined
AC01
Mean SD
% RSD
Mean SD
% RSD
3.110 6.344
203.956
10
43.310 3.464
7.998
20
68.970 2.129
3.087
40
74.471 2.085
2.799
60
77.923 1.592
2.044
IC50
Insignificant
14
3.4.
3.4.1. Preparation
3.4.1.1.
DPPH preparation:
DPPH stock solution was prepared by adding 2.46 mg of DPPH in 25mL of absolute
ethanol to make the concentration of 250 M. This DPPH stock solution was then diluted
with absolute ethanol to get solutions with concentrations of 50, 100, 150 and 200 M.
3.4.1.2.
Sample preparation:
Percentage of moisture: 1g of sample was weighed and heated at 105 0C for 6 hours
to calculate the percentage of humidity.
Three mg of each sample was dissolved in 3 mL of DMSO to get 1000 M stock
solution. DMSO was added to the stock to yield concentrations of 20, 80, 160, 240, 320
and 400 M.
15
3.4.2. Processes
3.4.2.1.
Absorbance
[DPPH] (M)
Mean SD
%RSD
0.000 0.002
27.713
50
0.266 0.002
0.651
100
0.473 0.001
0.244
50
0.680 0.001
0.170
200
0.877 0.007
0.801
250
1.059 0.005
0.491
16
3.4.2.2.
Concentration of
Vitamin C (g/mL)
Absorbance
%E
Mean SD
% RSD
0.44 0.01
2.273
0.000
0.346 0.033
9.551
21.364
20
0.097 0.008
7.833
78.030
40
0.090 0.029
32.129
79.470
60
0.082 0.010
12.287
81.288
80
0.068 0.006
8.139
84.621
100
0.069 0.008
11.123
84.394
IC50
17
3.4.2.3.
Sample curves
Absorbance
%E
(g/mL)
Mean SD
% RSD
0.39 0.01
2.564
0.000
20
0.367 0.003
0.944
5.897
40
0.362 0.005
1.419
7.265
60
0.361 0.005
1.439
7.436
80
0.357 0.014
3.817
8.547
100
0.352 0.003
0.752
9.744
IC50
Undefined
18
Fraction 2 (B)
Absorbance
Absorbance
%E
Mean SD
% RSD
0.738 0.001
0.136
20
0.724 0.008
40
%E
Mean SD
% RSD
0.000
1.006 0.001
0.099
0.000
1.043
1.898
0.956 0.015
1.583
4.970
0.709 0.013
1.866
3.930
0.940 0.007
0.745
6.560
60
0.688 0.018
2.553
6.820
0.934 0.004
0.386
7.157
80
0.684 0.007
1.023
7.317
0.922 0.006
0.660
8.350
100
0.670 0.008
1.121
9.259
0.903 0.012
1.294
10.272
IC50
Insignificant
Insignificant
Fraction 4 (D)
Absorbance
Absorbance
%E
Mean SD
% RSD
0.367 0.019
5.134
20
0.368 0.012
40
%E
Mean SD
% RSD
0.000
0.395 0.006
1.395
0.000
3.925
2.902
0.381 0.004
0.946
4.750
0.355 0.018
5.163
6.332
0.370 0.005
1.249
7.583
60
0.353 0.005
1.279
6.948
0.372 0.009
2.464
7.000
80
0.343 0.030
8.781
9.587
0.366 0.010
2.773
8.500
100
0.338 0.019
5.479
10.818
0.360 0.002
0.425
10.083
IC50
Insignificant
Insignificant
19
Conc.
g/mL
AC01
Mean SD
% RSD
0.604 0.004
0.662
20
0.207 0.010
40
%E
Mean SD
% RSD
0.000
4.903
65.728
0.180 0.039
21.565
70.143
60
0.141 0.008
5.354
76.656
80
0.177 0.047
26.247
70.640
100
0.144 0.012
8.127
76.214
IC50
20
Insignificant
4.
Discussion
4.1.
Five pure compounds were isolated from A. cochinchinensis tuber extract including
quercetin
(AC01),
asparagine
(AC02),
sucrose
(AC03),
Sitosterol-3-O--
21
Asparagine (AC02) is one of non-polared amino acid which occupies the large
percentage among components in A. cochinchinensis tuber [1]. Asparagine was first
isolated by Louis Nicolas Vauquelin and Pierre Jean Robiquet, (1806) under a crystallize
form from asparagus juice and became the first amino acid to be isolated [21].
Sucrose (AC03) is a white, crystallized and ordorless table sugar. Sucrose was
isolated from A. cochinchinensis tuber by Tomoda Masashi et al. (1974) [22]. There are,
by now, two important sugar crops predominate. They are sugarcane and sugar beets in
which sucrose can account for 12 to 20% of the plant's dry weight.
in
A.
cochinchinensis
tuber
[1].
In
some
researches,
Sitosterol
22
4.2.
4.2.1. Evaluation of anticancer activity against ovary HeLa cancer cell line
The anticancer activity against the HeLa cancer cell line of samples including total
extract, fractions and pure compounds were determined and showed in Table 12.
Table 12: IC50 values of samples against HeLa cancer cell line
Samples
IC50 (g/mL)
Total extract
Insignificant
Fraction 1 (A)
Insignificant
Fraction 2 (B)
Insignificant
Fraction 3 (C)
Insignificant
Fraction 4 (D)
Insignificant
Quercetin
Asparagine
Insignificant
Sucrose
Insignificant
Insignificant
- Sitosterol
Insignificant
Camptothecin
Fractions
Pure compounds
Positive control
The result showed the low anticancer activity against Hela cancer cell line of total
extract and 4 fractions of A. cochinchinensis tuber; whereas the pure compound AC01
showed the strong activity with IC50 = 5.78 0.36 g/mL.
23
Table 11: IC50 values of total extract and pure compounds against NCI-H460 cancer cell line
Samples
IC50 (g/mL)
Total extract
Insignificant
Quercetin
Asparagine
Insignificant
Sucrose
Insignificant
Insignificant
- Sitosterol
Insignificant
Camptothecin
Pure compounds
Positve control
Among five pure compounds isolated from the methanol extract, AC01 showed the
strong anticancer activity against the NCI-H460 cancer cell line with IC50 = 12.57 1.19
g/mL. In contrast, the total extract has low anticancer activity against that cell line.
4.3.
Table 14 is the summary of samples including fractions and pure compounds tested
for the antioxidant activity.
Table 14: IC50 values of samples in antioxidant assay
Samples
IC50 (g/mL)
Fraction 1 (A)
Insignificant
Fraction 2 (B)
Insignificant
Fraction 3 (C)
Insignificant
Fraction 4 (D)
Insignificant
Quercetin
Asparagine
Insignificant
Sucrose
Insignificant
Insignificant
- Sitosterol
Insignificant
Vitamin C
Fractions
Pure compounds
Positive control
The results showed that all tested fractions had no antioxidant activity at all.
Meanwhile, the pure compound AC01 exhibited the potent antioxidant activity with IC50 =
14.524 2.119 g/mL compared to vitamin C with IC50 value of 10.487 2.000 g/mL.
24
In general, A. cochinchinensis tuber extract has low antioxidant activity whereas the
isolated quercetin has very strong antioxidant activity.
5. Conclusion
Five compounds isolated from Asparagus cochinchinensis tuber were identified their
structures including quercetin (AC01), asparagine (AC02), sucrose (AC03), - Sitosterol
(AC04), - Sitosterol-3-O--glucopyranoside (AC05). The total tuber extract showed low
antioxidant activity and anticancer activity in general. However, quercetin (AC01) has
been proven to be a potent anticancer agent from the A. cochinchinensis tuber. This
showed that A. cochinchinensis is medicinal plant which can be taken into consideration
for further study.
Besides five isolated compounds, it is necessary to isolate more pure compounds and
to assay other pharmacological activities of this plant.
25
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Alternat
and brassicasterol in
serum
by high-performance
liquid
M.D., D'Ocon,
M.P., Paya,
M., Villar.
A.,
1988,
Antihyperglycemic and insulin-releasing effects of beta-sitosterol 3-betaD-glucoside and its aglycone, beta-sitosterol, Arch Int Pharmacodyn
Ther., 296:224-31.
Ergosterol,
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and
Stigmasterol,
Journal
of
M.,
2008,
Study
of
Thermodynamic
Parameters
for
Solubilization of Plant Sterol and Stanol in Bile Salt Micelles, Chem. Phys.
Lipids, 154 (2): 8793.
iii
Appendix
Appendix 1a: 1H-NMR spectrum of AC01
iv
Appendix 2:
13
vi
vii
Appendix 7:
13
viii
ix
xi
Appendix 12:
13
xii
xiii
Appendix 18:
13
xiv
Appendix 21:
13
xv
KT QU XC NH C TNH T BO
n v: Trng H Quc T
M s: 87
Ln 2
Ln 3
TB LC
MCF-7
57.44
54.87
58.58
56.97 1.90
Hep G2
65.26
64.33
60.38
63.32 2.59
NCI-H460
78.25
79.68
76.19
78.04 1.75
HeLa
49.89
48.66
55.11
51.22 3.43
Chng dng s dng l Camptothecin. dng t bo MCF 7 v NCI H460 s dng nng
0.01 g/ml, dng Hep G2 l 0.07 g/ml v HeLa l 1 g/ml.
xvi
Appendix 23: Evaluation of anticancer activity against HeLa cancer cell line
xvii
xviii
Appendix 24: Evaluation of anticancer activity against NCI-H460 cancer cell line
xix
Form BT03
INTERNATIONAL UNIVERSITY
School of Biotechnology
Evaluation Form
Academic Adviser
Criteria
Maximum marks
Independence in work
10
Creativity
10
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20
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20
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Total
100
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