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VIETNAM NATIONAL UNIVERSITY – HOCHIMINH CITY INTERNATIONAL UNIVERSITY
EVALUATION OF THE ANTICANCER AND ANTIOXIDANT ACTIVITIES OF ASPARAGUS
COCHINCHINENSIS
A thesis submitted to The School of Biotechnology, International University
In partial fulfillment of the requirements for the degree of B.S. in Biotechnology
Student name: Nguyễn Phúc Anh - BTIU09340 Supervisor: Dr. Hoàng Lê Sơn
June/ 2013
i
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.
It’s 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.
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Table of Contents
1. Introduction ............................................................................................. 1
1.1. Introduction to Asparagus cochinchinensis ..................................................... 1
1.1.1. Morphology and distribution ............................................................. 1
1.1.2. Traditional uses .............................................................................. 2
1.2. Potential applications............................................................................................ 2
2. Materials and methods .............................................................................. 3
2.1. Research objective and location ........................................................................ 3
2.2. Fractionation from total extract ......................................................................... 3
2.2.1. Materials ....................................................................................... 3
2.2.2. Methods ........................................................................................ 3
2.3. Evaluation of anticancer activity of A. cochinchinensis tuber extract ...... 5
2.3.1. Materials ....................................................................................... 5
2.3.2. Methods ........................................................................................ 5
2.4 Evaluation of antioxidant activity of A. cochinchinensis tuber extract ...... 6
2.4.1. Materials ....................................................................................... 6
2.4.2. Methods ........................................................................................ 7
3. Results .................................................................................................... 8
3.1. Fractionation from total extract ......................................................................... 8
3.1.1 Extraction ...................................................................................... 8
3.1.2. Fractionation and purification ............................................................................. 9
3.2. Structural identification........................................................................................ 9
3.3. Evaluation of anticancer activity of A. cochinchinensis tuber .................... 12
3.3.1. Evaluation of anticancer activity against HeLa cell line ...................... 13
3.3.2. Evaluation of anticancer activity against NCI-H460 cell line ................ 14
3.4. Evaluation of antioxidant activity of A. cochinchinensis tuber .................. 15
3.4.1. Preparation .................................................................................. 15
3.4.2. Processes .................................................................................... 16
4. Discussion ............................................................................................. 21
4.1. Fractionation and purification ........................................................................... 21
4.2. Evaluation of anticancer activity of A. cochinchinensis tuber .................. 23
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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. Evaluation of antioxidant activity of A. cochinchinensis tuber .................. 24
5. Conclusion ............................................................................................. 25
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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
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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
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List of abbreviation
A. cochinchinensis : Asparagus cochinchinensis (Lour.) Merr.
EtOAc : Ethyl Acetate
MeOH : Methanol
MPLC : Medium Pressure Liquid Chromatography
NMR : Nuclear magnetic resonance spectroscopy
DMSO : Dimethyl sulfoxide
DPPH : 1,1-diphenyl-2-picrylhydrazyl
%E : Efficiency percentage
SD : Standard deviation
RSD : Relative standard deviation
HeLa : Ovary cancer cell line
NCI-H460 : Lung cancer cell line
IC50 : The half maximal inhibitory concentration
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EVALUATION OF THE ANTICANCER AND
ANTIOXIDANT ACTIVITIES OF ASPARAGUS
COCHINCHINENSIS
Anh P. Nguyena, Son L. Hoanga*, Trung V. Phungb
a School of Biotechnology, International University – Vietnam National University in
HCMC
b Institute of Chemical Technology – Vietnam Academy of Science and Technology
*Corresponding author’s email address:lshoang@gmail.com
Abstract
Five compounds including quercetin (AC01), asparagine (AC02), sucrose (AC03),
β- Sitosterol-3-O-β-glucopyranoside (AC04) and β- 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= 14.52±2.11 µg/mL (DPPH
method, compared to standard vitamin C with IC50 = 10.49±2.00 µg/mL).
Besides, quercetin (AC01) was evaluated cytotoxicity against the human ovary
HeLa cancer cell line with IC50 = 5.78±0.36 µg/mL (SRB method, compared to
standard Camptothecin with IC50 < 1 µg/mL)and lung NCI – H460 cancer cell
line with IC50 = 12.57±1.19 µg/mL (SRB method, compared to standard
Camptothecin with IC50 < 0.01 µg/mL).
Keywords: Asparagus cochinchinensis, quercetin, asparagine, sucrose, β-
Sitosterol-3-O-β-glucopyranoside, β- Sitosterol, DPPH, HeLa, NCI-H460, SRB.
1
1. Introduction
1.1. Introduction to Asparagus cochinchinensis
1.1.1. Morphology and distribution
Asparagus cochinchinensis (Lour.) Merr. or Cochinchinense asparagus (A.
cochinchinensis), belonging to Liliaceae family, has been known as a traditional medicinal
herb in China over thousand years [1].
This plant is a shrubby herb, typically between 1 to 1.5 metres in length. Roots are
rhombus with long stems and it grows in clusters (Fig. 1). There are many cylindrical and
intertwined branches such that it grows into thick bush. Leaves are 2-3 cm long,
crescent-shaped with pointed tip. The inflorescence consists of 1-2 white flowers; male
flowers with a perianth consist of 6 pieces, 6 binary and a stamen; and females with a
perianth, a shorter stamen and a reduced anther. The fruit is succulent, spherical, 5-6mm
in diameter and the colors are pale green, turning yellow then ivory white; seeds are
black. Flowering season is from March to May, fruiting season is between June and
September.
It is a perennial tuber that usually grows in abundance in eastern Asia including
China, Japan and Korea (Fig. 2). In Vietnam, the plants are grown mainly in the central
coastal provinces and islands such as Phu Quoc and Con Dao. In the northern provinces,
they are grown mostly for medicine, sometimes encountered naturally in some places
such as Cat Ba, Quang Ninh, Hai Phong and Thanh Hoa [1].
Fig. 2: A. cochinchinensis plant [4] Fig. 1: A. cochinchinensis tuber
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1.1.2. Traditional uses
A. cochinchinensis is distributed in many provinces of China. It is often used for the
treatment of fever, cough, hemoptysis, diabetes, constipation, swollen and throat pain
[1]. This plant is also used to treat lung cancer, tuberculosis, heart disease and
constipation [2]. Different morphological parts of A. cochinchinensis are used for different
traditional usages as summarized in Table 1 [3].
Table 1: Traditional usages of each morphological part of A. cochinchinensis
Morphological
parts Chemical compositions Traditional usages
Sprouts Wax and calcium, iron,
potassium and chlorophyll
Latte
Strong antioxidant activity
Root
Flavonoids
Antioxidant properties by neutralizing by-
products of metabolism named free radicals.
These substances enhance the immune system
and ease the absorption of vitamins
Rutin
Powerful antioxidant which is used for people
with low vision or eye conditions like macular
degeneration
Quercetin Boosting the effects of vitamins
Inulin Reducing cholesterol levels
1.2. Potential applications
In previous research, Hong-Jie Zhang et al. (2003) isolated a new spirostanol
saponin, asparacoside; two new C-27 spirosteroids, asparacosins A and B; a new
acetylenic derivative, 3’’-methoxyasparenydiol; and a new polyphenol, 3’-hydroxy-4’-
methoxy-4’- dehydroxynyasol, as well as five known phenolic compounds, asparenydiol,
nyasol, 3’’- methoxynyasol, 1,3-bis-di-p-hydroxyphenyl-4-penten-1-one, and trans-
coniferyl alcohol. These compounds showed the potential in cytotoxicities in a panel
comprised of KB, Col-2, LNCaP, Lu-1, and HUVEC cells [5].
In 2011, a new furostanol saponin, (25S)-26-O-β-d-glucopyranosyl-5β-furost-20
(22)–en - 3β, 15β, 26 – triol – 3 - O - [α – l – rhamnopyranosyl - (1 – 4)] – β - d -
glucopyranoside, namely, aspacochioside D was isolated from A. cochinchinensis. This
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compound was evaluated in cytotoxicity against the human tumor cell line, A549 and
showed IC50 value of 3.87 μg/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. Materials and methods
2.1. Research objective and location
The main objective of this research was to evaluate the anticancer and antioxidant
activities of A. cochinchinensis.
Location: International University, HCMC
Institute of Chemical Technology
University of Science, HCMC
2.2. Fractionation from total extract
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 (MeOH-
extract) 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.
4
Fractionations, isolation, purification, ...
Testing bioactivities
- Fractionations
- Testing bioactivities
Testing bioactivities
Fractionation
Extract with MeOH solvent
Materials
Total extract
H- extract
(lower polar group)
EtOAc- extract
(higher polar group)
Potential group (s)
Active fraction (s)
Active compound (s)
MeOH-extract
(highest polar group)
2.2.2.1. Extraction methods
The plant powder was extracted by combination of two techniques: ultrasound
(sonication) and maceration [7, 8].
Ultrasound extraction (or sonication) involved the use of an ultrasound with
frequencies ranging from 20 - 2000 KHz; this increased the permeability of cell walls and
produced cavitation.
Maceration extraction involved putting the crude powder into a stoppered vessel with
the MeOH solvent at 37oC. The vessel was frequently agitated until the powder was
totally dissolved after which the mixture was strained and the damp solid material was
pressed.
The process was repeated many times until the constituents in the material were
extracted completely. The extract was filtered through filter paper then the solvent was
removed by a rotary evaporator.
Fig. 3: The scheme of experimental process
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2.2.2.2. Fractionation and purification
Two methods including open column chromatography (CC) and medium pressure
liquid chromatography (MPLC) were employed in this experiment.
Open column chromatography was used as a purification technique to isolate the
desired fractions from the total extract. In this chromatography, the stationary phase
involved packing the column with silica gel or C18 and then inserting the solvent and
sample, which was regarded as mobile phase. The eluent was passed through the column
by gravity and the fractions were collected separately based on the interactions between
those stationary and mobile phases.
The second method used was medium pressure liquid chromatography, MPLC. This
method differs from the open column method in the size of absorbent material. For MPLC,
the material is very fine requiring the use of a compressor or a pump to push the sample
through.
2.3. Evaluation of anticancer activity of A. cochinchinensis tuber extract
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
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gentle addition of 50 µL of cold 50% (w/v) TCA (final concentration of TCA per well was
10%) and incubated for 60 minutes at 4°C. The supernatant was discarded and the
plates were washed five times with tap water and air dried. Finally, SRB solution (100 µL)
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: OD value of the well which contains cells
ODblank: OD value of the well which is blank
ODTN: OD value of the well which contains samples calcuted from
the equation (1) and (2)
ODc: OD value of the well which contains the control solution
calculated from equation (1) and (2)
2.4 Evaluation of antioxidant activity of A. cochinchinensis tuber extract
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.
7
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 µL) of extracts was mixed with 150 µL 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 µL 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: sample standard deviation
N: size of the sample data set
8
- Partition/ EtOAc
- Filtering
- Removing EtOAc
- Partition/ n-hexane
- Filtering
- Removing n-hexane
- Extract/10L MeOH
- Filtering
- Removing MeOH
Crude powder (1.5 kg)
Total extract (S) (1.2 kg)
n-hexan extract Residue
EtOAc extract MeOH extract
Xi … xN : the sample data set
�̅�: mean value of the sample data set
CV: coefficient variance
3. Results
3.1. Fractionation from total extract
3.1.1 Extraction
Crude powder of A. cochinchinensis tuber (1.5 kilograms) was added to 10 liters of
methanol and the container was placed in an ultrasound bath. The whole extract was
filtered then the solvent was removed by rotary evaporator to get 1.2 kilograms of
methanol extract (named S). This methanol extract, S, was then partitioned by n-hexane
and EtOAC. After confirming by TLC analysis, the compositions in n-hexane (H-extract)
and EtOAC (EtOAc-extract) extracts were the same as each other and contained too small
amount of components compared to the total extract. The total extract used was,
therefore, the methanol extract (MeOH- extract), and this was used for the next step of
fractionation. The scheme is illustrated in Fig. 4.
Fig. 4: Extraction scheme
9
MeOH extract
100% CHCl3
Fraction 1 (A)
Crystallization
AC04Residue
F1.1
Crystallization AC05
F1.2
90% CHCl3
Fraction 2 (B)
Crystallization AC01
80% CHCl3
Fraction 3(C)
F3.1
Crystallization AC02
F3.2
70% CHCl3
Fraction 4 (D)
Crystallization AC03
3.1.2. Fractionation and purification
The extract was fractionated by open column chromatography and MPLC so that the
extract was separated into small groups called fractions. Those potential fractions could
be purified by MPLC or crystallized to produce pure compounds.
The methanol extract (MeOH-extract) was fractionated by open chromatography with
silica gel, CHCl3-MeOH solvent. The ratio of CHCl3-MeOH solvent went from 100% down
to 70% CHCl3 and produced 4 fractions (F). Crystallization of three compounds, AC04,
AC01 and AC03, was observed directly from fraction 1, 2 and 4. Fraction 3 and 1 were
further fractionated to obtain further 2 compounds, AC02 and AC05 by MPLC. The process
is illustrated in Fig. 5.
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.
Table 2a – c summarizes the characteristics of those 5 pure components.
Fig. 5: Fractionation and purification scheme
10
Table 2a: Characteristics of pure compounds
Characteristics
Components
AC01 AC02
Spectroscopy
1H-NMR, 13C-NMR, DEPT, HSQC,
HMBC
(Appendix 1 - 5)
1H-NMR, 13C-NMR, DEPT, HSQC,
HMBC
(Appendix 6 -10)
Common name Quercetin Asparagine [13, 14]
IUPAC name 3,3',4',5,7-pentahydroxyflavone 2-amino-3-carbamoylpropanoic
acid
Chemical formula C15H10O7 C4H8N2O3
Molecular weight 302.236 g/mol 132.118 g/mol
Structure
Phytochemical Flavonoid Amino acid
Physical state Amorphos, yellow Crystal, white
Melting point 316oC 220oC
Rf (solvent)
0.22
CHCl3:MeOH=9:1
0.5
butanol acid
11
Table 2b: Characteristics of pure compounds
Characteristics
Components
AC03 AC04
Spectroscopy
1H-NMR, 13C-NMR, DEPT,
HMBC, HSQC, COSY
(Appendix 11 – 16)
1H-NMR, 13C-NMR, DEPT
(Appendix 17 – 19)
Common name Sucrose [15, 16] β-Sitosterol-3-O-β-D-glucopyranoside
[17, 18]
IUPAC name Hex-2-ulofuranosyl
hexopyranoside β-Sitosterol-3-O-β-D-glucopyranoside
Chemical formula C12H22O11 C35H60O6
Molecular weight 342.3 g/mol 576.85 g/mol
Structure
Phytochemical Sugar Steroid
Physical state Amorphos, white Amorphos, white
Melting point 160oC – 186oC 275–277°C
Rf (solvent)
0.54
CHCl3:MeOH=7:3
0.3
CHCl3:MeOH=9:1
12
Table 2c: Characteristics of pure compounds
Characteristics
Components
AC05
Spectroscopy 1H-NMR, 13C-NMR (Appendix 20, 21)
Common name β-Sitosterol [18, 19]
IUPAC name
17-(5-Ethyl-6-methylheptan-2-yl)-10,13-dimethyl-
2,3,4,7,8,9,11,12,14,15,16,17-dodecahydro-1H-
cyclopenta[a]phenanthren-3-ol
Chemical formula C29H50O
Molecular weight 414.71 g/mol
Structure
Phytochemical Steroid
Physical state Needle, colorless
Melting point 133–135°C
Rf (solvent)
0.86
CHCl3:MeOH=9:1
3.3. Evaluation of anticancer activity of A. cochinchinensis tuber
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 µg/mL, to
inhibite 51.22±3.43 % cell growth.
For NCI-H460 cancer cell line: the concentration of Camptothecin used was 0.01
µg/mL, to inhibite 78.04±1.75 % cell growth.
13
3.3.1. Evaluation of anticancer activity against HeLa cell line
Screening for anticancer activity: The absorbance values of total extract, fractions
(with the concentration of 100 µg/mL) were determined and transformed into percentage
of growth inhibition and summarized in Table 3.
Table 2: Anticancer activity of total extract and fractions against HeLa cancer cell line
Samples (100 µg/mL)
Growth inhibition (%)
Mean ± SD % RSD %E
AC-S - - - Undefined
AC-A - - - Undefined
AC-B - - - Undefined
AC-C - - - Undefined
AC-D - - - Undefined
The absorbance values of different concentrations of pure compounds (AC01-AC05)
were determined and transformed into percentage of growth inhibition as illustrated in
Table 4 and Fig. 6 describing the anticancer activity of AC01.
Table 3: Anticancer activity of pure compounds against HeLa cell line
Conc.
µg/mL
Growth inhibition (%)
AC01 AC02, AC03, AC04, AC05
Mean ± SD % RSD Mean ± SD % RSD
1 - - - -
2.5 14.777 ± 5.873 39.747 - -
5 46.986 ± 2.283 4.859 - -
10 65.452 ± 3.827 5.847 - -
20 76.222 ± 1.886 2.475 - -
IC50 5.78 ± 0.36 µg/mL Insignificant
14
Fig. 6: Inhibition percentage of AC01 against HeLa cell line
3.3.2. Evaluation of anticancer activity against NCI-H460 cell line
Screening for anticancer activity: The absorbance values of total extract
(concentrated as 100 µg/mL) were determined and transformed into percentage of
inhibition as illustrated in Table 5.
Table 4: Growth inhibition percentage of total extract against NCI-H460 cell line
Samples (100 µg/mL)
Growth inhibition (%)
Mean ± SD % RSD %E
AC-S - - - Undefined
The absorbance values of different concentrations of pure compounds (AC01-AC05)
were determined and transformed into percentage of inhibition as illustrated in Table 6
and Fig. 7 descrbing the percentage of growth inhibition of AC01.
Table 5: Growth inhibition percentage of pure compounds against NCI-H460 cancer cell line
Conc.
µg/mL
Growth inhibition (%)
AC01 AC02, AC03, AC04, AC05
Mean ± SD % RSD Mean ± SD % RSD
5 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 12.57 ± 1.19 µg/mL Insignificant
15
Fig. 7: Growth inhibition percentage of AC01 against NCI-H460 cell line
3.4. Evaluation of antioxidant activity of A. cochinchinensis tuber
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 µM. This DPPH stock solution was then diluted
with absolute ethanol to get solutions with concentrations of 50, 100, 150 and 200 µM.
3.4.1.2. Control sample (Vitamin C) preparation:
Vitamin C stock solution at a concentration of 1000 µM was made by dissolving 3 mg
of vitamin C in 3 mL of DMSO. DMSO was then added to stock solution to produce
concentrations of 20, 80, 160, 240, 320 and 400 µM.
3.4.1.3. Sample preparation:
Percentage of moisture: 1g of sample was weighed and heated at 1050C for 6 hours
to calculate the percentage of humidity.
Three mg of each sample was dissolved in 3 mL of DMSO to get 1000 µM stock
solution. DMSO was added to the stock to yield concentrations of 20, 80, 160, 240, 320
and 400 µM.
16
3.4.2. Processes
3.4.2.1. Negative control curve (DPPH curve)
The absorbance values of different concentrations DPPH solutions are illustrated in
Table 7 and Fig. 8 demonstrating standard curve based on those values.
Table 6: Absorbance values of DPPH
[DPPH] (µM)
Absorbance
Mean ± SD %RSD
0 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
Fig. 8: Standard curve
17
3.4.2.2. Positive control curve (vitamin C curve)
The absorbance values of the different concentrations of vitamin C solutions are
illustrated in Table 8. Fig. 9 demostrates the standard curve based on those values.
Table 7: Absorbance values of vitamin C
Concentration of
Vitamin C (µg/mL)
Absorbance
%E
Mean ± SD % RSD
0 0.44 ± 0.01 2.273 0.000
5 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 10.487 ± 2.000 µg/mL
Fig. 9: Positive control curve
18
3.4.2.3. Sample curves
The absorbance values of different concentrations of methanol extract are illustrated
in Table 9. Fig. 10 describes the antioxidant activity of A. cochinchinensis at different
concentrations.
Table 8: Absorbance values of MeOH extract
Concentrations
(µg/mL)
Absorbance
% E
Mean ± SD % RSD
0 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
Fig. 10: Antioxidant activity of A. cochinchinensis extract at different concentration
19
The absorbance values of different concentrations of fractions are illustrated in Table
10 and Fig. 11 demonstrating the antioxidant activity of fractions of A. cochinchinensis
at different concentratrions.
Table 9a: Antioxidant activity of fraction 1 and fraction 2 at different concentrations
Conc.
µg/mL
Fraction 1 (A) Fraction 2 (B)
Absorbance
% E
Absorbance
% E
Mean ± SD % RSD Mean ± SD % RSD
0 0.738 ± 0.001 0.136 0.000 1.006 ± 0.001 0.099 0.000
20 0.724 ± 0.008 1.043 1.898 0.956 ± 0.015 1.583 4.970
40 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
Table 10b: Antioxidant activity of fraction 3 and fraction 4 at different concentrations
Conc.
µg/mL
Fraction 3 (C) Fraction 4 (D)
Absorbance
% E
Absorbance
% E
Mean ± SD % RSD Mean ± SD % RSD
0 0.367 ± 0.019 5.134 0.000 0.395 ± 0.006 1.395 0.000
20 0.368 ± 0.012 3.925 2.902 0.381 ± 0.004 0.946 4.750
40 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
20
Fig. 11: Antioxidant activity of fractions of A. cochinchinensis at different concentrations
The absorbance values of different concentrations of pure compounds (AC01 – AC05)
are illustrated in Table 11. Fig. 12 demonstrates the curve based on those values.
Table 10: Percentage of inhibition (%E) related to concentration of AC01 - AC05
Conc.
µg/mL
AC01
% E
AC02, AC03, AC04, AC05
% E
Mean ± SD % RSD Mean ± SD % RSD
0 0.604 ± 0.004 0.662 0.000 - - -
20 0.207 ± 0.010 4.903 65.728 - - -
40 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 14.524 ± 2.119 µg/mL Insignificant
21
Fig. 12: Antioxidant activity of AC01 at different concentrations
4. Discussion
4.1. Fractionation and purification
Five pure compounds were isolated from A. cochinchinensis tuber extract including
quercetin (AC01), asparagine (AC02), sucrose (AC03), β- Sitosterol-3-O-β-
glucopyranoside (AC04) and β- Sitosterol (AC05).
Quercetin (AC01) was the first time isolated from A. cochinchinensis tuber growing in
Viet Nam. Quercetin is yellow and highly water-soluble, it is one of the most prominent
bioflavonoid compounds in plants and the highest content of quercetin could be found in
onions with 60 – 100 mg/ 100g fresh weight [20]. It is one of many flavonoids which
play an important role in many activities of this plant, such as: anti-oxidant, anti-
diabetic, anti-inflammation …
22
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.
β- Sitosterol-3-O-β-glucopyranoside (AC04) is a popular steroid that it appears in
nearly all of researched plant. This compound is regarded as an antihyperglycemic
reagent due to its aglycone (β-sitosterol) [23].
β- Sitosterol (AC05) is a type of phytosterol which is regarded as the main
constituent in A. cochinchinensis tuber [1]. In some researches, β- Sitosterol
reduces levels of cholesterol in blood and can be used in treating hypercholesterolemia
[24]. β- Sitosterol also inhibits cholesterol absorption in the intestine [25].
23
4.2. Evaluation of anticancer activity of A. cochinchinensis tuber
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 (µg/mL)
Total extract Insignificant
Fractions
Fraction 1 (A) Insignificant
Fraction 2 (B) Insignificant
Fraction 3 (C) Insignificant
Fraction 4 (D) Insignificant
Pure compounds
Quercetin 5.78 ± 0.36 µg/mL
Asparagine Insignificant
Sucrose Insignificant
β- Sitosterol -3-O-β- glucopyranoside Insignificant
β- Sitosterol Insignificant
Positive control Camptothecin < 1.00 µg/mL
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 µg/mL.
4.2.2. Evaluation of anticancer activity against lung NCI-H460 cancer cell
line
The anticancer activity against the NCI-H460 cancer cell line of samples including
total extract and pure compounds were determined and showed in Table 13.
24
Table 11: IC50 values of total extract and pure compounds against NCI-H460 cancer cell line
Samples IC50 (µg/mL)
Total extract Insignificant
Pure compounds
Quercetin 12.57 ± 1.19 µg/mL
Asparagine Insignificant
Sucrose Insignificant
β- Sitosterol -3-O-β- glucopyranoside Insignificant
β- Sitosterol Insignificant
Positve control Camptothecin < 0.01 µg/mL
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
µg/mL. In contrast, the total extract has low anticancer activity against that cell line.
4.3. Evaluation of antioxidant activity of A. cochinchinensis tuber
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 (µg/mL)
Fractions
Fraction 1 (A) Insignificant
Fraction 2 (B) Insignificant
Fraction 3 (C) Insignificant
Fraction 4 (D) Insignificant
Pure compounds
Quercetin 14.524 ± 2.119 µg/mL
Asparagine Insignificant
Sucrose Insignificant
β- Sitosterol -3-O-β- glucopyranoside Insignificant
β- Sitosterol Insignificant
Positive control Vitamin C 10.487 ± 2.000 µg/mL
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 µg/mL compared to vitamin C with IC50 value of 10.487 ± 2.000 µg/mL.
25
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), β- Sitosterol
(AC04), β- Sitosterol-3-O-β-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.
i
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iv
Appendix
Appendix 1a: 1H-NMR spectrum of AC01
Appendix 1b: 1H-NMR spectrum of AC01 (cont.)
v
Appendix 2: 13C-NMR spectrum of AC01
Appendix 3a: DEPT spectrum of AC01
vi
Appendix 3b: DEPT spectrum of AC01
Appendix 4: HSQC spectrum of AC01
vii
Appendix 5: HMBC spectrum of AC01
Appendix 6a: 1H-NMR spectrum of AC02
viii
Appendix 6b: 1H-NMR spectrum of AC02 (cont)
Appendix 7: 13C-NMR spectrum of AC02
ix
Appendix 8: DEPT spectrum of AC02
Appendix 9a: HMBC spectrum of AC02
Appendix 9b: HMBC spectrum of AC02 (cont)
x
Appendix 9c: HMBC spectrum of AC02 (cont)
Appendix 10a: HSQC spectrum of AC02
Appendix 10b: HSQC spectrum of AC02 (cont)
xi
Appendix 11a: 1H-NMR spectrum of AC03
Appendix 11b: 1H-NMR spectrum of AC03
Appendix 11c: 1H-NMR spectrum of AC03
xii
Appendix 12: 13C-NMR spectrum of AC03
Appendix 13: DEPT spectrum of AC03
xiii
Appendix 14: HMBC spectrum of AC03 Appendix 15: HSQC spectrum of AC03
Appendix 16: COSY spectrum of AC03
xiv
Appendix 17: 1H-NMR spectrum of AC04
Appendix 18: 13C-NMR spectrum of AC04
Appendix 19: DEPT spectrum of AC04
xv
Appendix 20: 1H-NMR spectrum of AC05
Appendix 21: 13C-NMR spectrum of AC05
xvi
The evaluation of anticancer activity received from University of
Science, HCMC.
Appendix 22: The positive control for anticancer activity test:
KẾT QUẢ XÁC ĐỊNH ĐỘC TÍNH TẾ BÀO
Đơn vị: Trường ĐH Quốc Tế
Mã số: 87
Mẫu Phần trăm gây độc tế bào (%)
Lần 1 Lần 2 Lần 3 TB ± ĐLC
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
Chứng dương sử dụng là Camptothecin. Ở dòng tế bào MCF 7 và NCI H460 sử dụng nồng độ
0.01 µg/ml, ở dòng Hep G2 là 0.07 µg/ml và HeLa là 1 µg/ml.
xvii
Appendix 23: Evaluation of anticancer activity against HeLa cancer cell line
xviii
xix
Appendix 24: Evaluation of anticancer activity against NCI-H460 cancer cell line
xx
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