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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: Nguyn Phc Anh - BTIU09340


Supervisor: Dr. Hong L Sn

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.

Introduction to Asparagus cochinchinensis ..................................................... 1

1.1.1. Morphology and distribution ............................................................. 1


1.1.2. Traditional uses .............................................................................. 2
1.2.
2.

Potential applications............................................................................................ 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


ii

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.

Evaluation of antioxidant activity of A. cochinchinensis tuber .................. 24

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

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

vi

EVALUATION
ANTIOXIDANT

OF

THE

ANTICANCER

ACTIVITIES

OF

AND

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 authors email address:lshoang@gmail.com

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,

Sitosterol-3-O--glucopyranoside, - Sitosterol, DPPH, HeLa, NCI-H460, SRB.

vii

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

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
Sprouts

Chemical compositions

Traditional usages

Wax and calcium, iron,


potassium and chlorophyll

Latte

Strong antioxidant activity


Antioxidant properties by neutralizing byproducts of metabolism named free radicals.

Flavonoids

These substances enhance the immune system


and ease the absorption of vitamins

Root

Powerful antioxidant which is used for people


Rutin

with low vision or eye conditions like macular


degeneration

1.2.

Quercetin

Boosting the effects of vitamins

Inulin

Reducing cholesterol levels

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-4methoxy-4- dehydroxynyasol, as well as five known phenolic compounds, asparenydiol,
nyasol, 3- methoxynyasol, 1,3-bis-di-p-hydroxyphenyl-4-penten-1-one, and transconiferyl 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--d-glucopyranosyl-5-furost-20
(22)en - 3, 15, 26 triol 3 - O - [ l rhamnopyranosyl - (1 4)] - d glucopyranoside, namely, aspacochioside D was isolated from A. cochinchinensis. This

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.

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 (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

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.

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 C 18 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

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:

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.

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:

sample standard deviation

N:

size of the sample data set

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.
- Extract/10L MeOH

Crude powder (1.5 kg)

- Filtering
- Removing MeOH

Total extract (S) (1.2 kg)

- Partition/ n-hexane
- Filtering
n-hexan
extract

- Removing nhexane

Residue

EtOAc extract
- Partition/ EtOAc
- Filtering
- Removing EtOAc
Fig. 4: Extraction scheme

MeOH extract

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.

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.

Table 2a c summarizes the characteristics of those 5 pure components.

Table 2a: Characteristics of pure compounds


Components
Characteristics

AC01
1

H-NMR,

13

AC02

C-NMR, DEPT, HSQC,

H-NMR,

13

C-NMR, DEPT, HSQC,

HMBC

HMBC

(Appendix 1 - 5)

(Appendix 6 -10)

Common name

Quercetin

Asparagine [13, 14]

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

Table 2b: Characteristics of pure compounds


Components
Characteristics

AC03
1

Spectroscopy

H-NMR,

13

AC04

C-NMR, DEPT,

HMBC, HSQC, COSY


(Appendix 11 16)

Common name

IUPAC name

Sucrose [15, 16]


Hex-2-ulofuranosyl
hexopyranoside

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

Table 2c: Characteristics of pure compounds


Components
Characteristics
AC05
Spectroscopy

H-NMR,

Common name

13

C-NMR (Appendix 20, 21)

-Sitosterol [18, 19]


17-(5-Ethyl-6-methylheptan-2-yl)-10,13-dimethyl-

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.

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 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

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 g/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
Growth inhibition (%)
Samples (100 g/mL)
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


Growth inhibition (%)
Conc.

AC01

g/mL

AC02, AC03, AC04, AC05

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

5.78 0.36 g/mL

Insignificant

13

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 g/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
Growth inhibition (%)
Samples (100 g/mL)
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
Growth inhibition (%)
Conc.
g/mL

AC01

AC02, AC03, AC04, AC05

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

12.57 1.19 g/mL

Insignificant

14

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 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.

Control sample (Vitamin C) preparation:

Vitamin C stock solution at a concentration of 1000 M 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 M.
3.4.1.3.

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.

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

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

Fig. 8: Standard curve

16

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 (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

10.487 2.000 g/mL

Fig. 9: Positive control curve

17

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

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

Fig. 10: Antioxidant activity of A. cochinchinensis extract at different concentration

18

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


Fraction 1 (A)
Conc.
g/mL

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

Table 10b: Antioxidant activity of fraction 3 and fraction 4 at different concentrations


Fraction 3 (C)
Conc.
g/mL

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

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.
g/mL

AC01

AC02, AC03, AC04, AC05


%E

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

14.524 2.119 g/mL

20

Insignificant

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),

Sitosterol-3-O--

glucopyranoside (AC04) and - 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, antidiabetic, anti-inflammation

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.

- Sitosterol-3-O--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 (-sitosterol) [23].

- Sitosterol (AC05) is a type of phytosterol which is regarded as the main


constituent

in

A.

cochinchinensis

tuber

[1].

In

some

researches,

Sitosterol

reduces levels of cholesterol in blood and can be used in treating hypercholesterolemia


[24]. - Sitosterol also inhibits cholesterol absorption in the intestine [25].

22

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 (g/mL)

Total extract

Insignificant

Fraction 1 (A)

Insignificant

Fraction 2 (B)

Insignificant

Fraction 3 (C)

Insignificant

Fraction 4 (D)

Insignificant

Quercetin

5.78 0.36 g/mL

Asparagine

Insignificant

Sucrose

Insignificant

- Sitosterol -3-O-- glucopyranoside

Insignificant

- Sitosterol

Insignificant

Camptothecin

< 1.00 g/mL

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.

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.

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

12.57 1.19 g/mL

Asparagine

Insignificant

Sucrose

Insignificant

- Sitosterol -3-O-- glucopyranoside

Insignificant

- Sitosterol

Insignificant

Camptothecin

< 0.01 g/mL

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.

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 (g/mL)

Fraction 1 (A)

Insignificant

Fraction 2 (B)

Insignificant

Fraction 3 (C)

Insignificant

Fraction 4 (D)

Insignificant

Quercetin

14.524 2.119 g/mL

Asparagine

Insignificant

Sucrose

Insignificant

- Sitosterol -3-O-- glucopyranoside

Insignificant

- Sitosterol

Insignificant

Vitamin C

10.487 2.000 g/mL

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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Satyendra, S.B., Nikhil, S., Rajendra, S.B., Preeti, A., Sarlesh, R.,
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Jonathan,

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J. S. V. S., Pedro, C. G., Jos, P. R. F., Valmar, C. B., Carlos, J.,
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L.

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Landulfo,

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503-525.
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2007, Chemical

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Nanako, I., Hideki, H., Fumiyo, K., 2010, Simultaneous determination


of -sitosterol, campesterol, stigmasterol,
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detection, Anal. Methods, 2: 174.


[20] Yoo, K.S., Lee, E.J., Patil, B.S., 2010, Quantification of quercetin
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M.D., D'Ocon,

M.P., Paya,

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1988,

Antihyperglycemic and insulin-releasing effects of beta-sitosterol 3-betaD-glucoside and its aglycone, beta-sitosterol, Arch Int Pharmacodyn
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iii

Appendix
Appendix 1a: 1H-NMR spectrum of AC01

Appendix 1b: 1H-NMR spectrum of AC01 (cont.)

iv

Appendix 2:

C-NMR spectrum of AC01

13

Appendix 3a: DEPT spectrum of AC01

Appendix 3b: DEPT spectrum of AC01

Appendix 4: HSQC spectrum of AC01

vi

Appendix 5: HMBC spectrum of AC01

Appendix 6a: 1H-NMR spectrum of AC02

vii

Appendix 6b: 1H-NMR spectrum of AC02 (cont)

Appendix 7:

C-NMR spectrum of AC02

13

viii

Appendix 8: DEPT spectrum of AC02

Appendix 9a: HMBC spectrum of AC02

Appendix 9b: HMBC spectrum of AC02 (cont)

ix

Appendix 9c: HMBC spectrum of AC02 (cont)

Appendix 10a: HSQC spectrum of AC02

Appendix 10b: HSQC spectrum of AC02 (cont)

Appendix 11a: 1H-NMR spectrum of AC03

Appendix 11b: 1H-NMR spectrum of AC03

Appendix 11c: 1H-NMR spectrum of AC03

xi

Appendix 12:

C-NMR spectrum of AC03

13

Appendix 13: DEPT spectrum of AC03

xii

Appendix 14: HMBC spectrum of AC03

Appendix 16: COSY spectrum of AC03

xiii

Appendix 15: HSQC spectrum of AC03

Appendix 17: 1H-NMR spectrum of AC04

Appendix 18:

C-NMR spectrum of AC04

13

Appendix 19: DEPT spectrum of AC04

xiv

Appendix 20: 1H-NMR spectrum of AC05

Appendix 21:

C-NMR spectrum of AC05

13

xv

The evaluation of anticancer activity received from University of


Science, HCMC.

Appendix 22: The positive control for anticancer activity test:

KT QU XC NH C TNH T BO

n v: Trng H Quc T
M s: 87

Phn trm gy c t bo (%)


Mu
Ln 1

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

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_____________________________
Date Signed
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