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

iii

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

vii

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

2

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

3

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

5

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

6

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

References

[1] Bich, H.D., 2007, Cây thuốc và động vật làm thuốc ở Việt Nam, 863-

866.

[2] Jiang, X.H., Zeng, G.P., Ou, L.J. and She, C.W., 2010, An efficient

system for the production of the medicinally important plant: Asparagus

cochinchinensis (Lour.) Merr., African Journal of Biotechnology, 9(37):

6207-6212.

[3] Loi, D.T., 2004, Những cây thuốc và vị thuốc Việt Nam, Nhà xuất bản Y

học, 12: 713-714.

[4] Dan, N.V., 1989, Medicinal plants in Viet Nam, Institute of Materia

Media HANOI, 3ed, 57.

[5] Zhang, H.J., Sydara, K., Tan, G.T., Ma, C., Southavong,

B., Soejarto, D.D., Pezzuto, J.M., Fong, H.H., 2004 Feb, Bioactive

Constituents from Asparagus cochinchinensis,J Nat Prod, 67(2):194-200.

[6] Yang, S., Cong, L.X., Wei, D.X., Hui, L.L., Run, H.L., Xi, K.X., Hai,

S.C., 2011, A New Furostanol Saponin from Asparagus cochinchinensis,

Arch Pharm Res, 34(10): 1587-1591.

[7] Handa, S.S., Khanuja, S.P. S., Longo, G., Rakesh, D.D., 2008,

Extraction Technologies for Medicinal and Aromatic Plants, International

Centre for Science and High Technology, 1: 22, 25.

[8] Atkins, P., Jones, L., 2009, Chemical Principles, 5: 702.

[9] Vichai, V., Kanyawim, K., 2006, Sulforhodamine B colorimetric assay

for cytotoxicity screening, Nature protocol, 1(3): 1112-1116.

[10] Skehen, P., Storeng, R., Scudiero, D., Monks, A., McMahon, J.,

Vistica, D., Warren, J.T., Bokesch, H., Kenney, S., Boyd, M.R.,

1990, New colorimetric cytotoxicity assay for anticancer-drug sceening, J.

Natl Cancer Inst., 82(13): 1107-1112.

ii

[11] Amin, I., Norazaidah, Y., Emmy, H.K., 2006, Antioxidant activity and

phenolic content of raw and blanched Amaranthus species, Food Chem,

94: 47-52.

[12] Satyendra, S.B., Nikhil, S., Rajendra, S.B., Preeti, A., Sarlesh, R.,

May 2012, A review of quercetin: Antioxidant and anticancer properties,

World Journal of Pharmacy and Pharmaceutical Sciences, 1(1): 146-160.

[13] Laura, C., David, J. C., 2010, Naturally occurring circular proteins:

distribution, biosynthesis and evolution, Org. Biomol. Chem., 8: 5035.

[14] Matthew, S. L., Bryan, S. D., Sabato, D., Gabriella, P., Luisa, I.,

Daniela, M., Jonathan, D. D., 2010, Amino acid transport in

thermophiles: Characterization of an arginine-binding protein in

Thermotoga maritima, Mol. Biosyst., 6: 142.

[15] Mitchell, J. M., Karen, K. F., 2012, Dietary Sugars:

Chemistry, CHAPTER 30 Analysis of Sucrose from Sugar Beet, 526-545.

[16] Leonardo, M. M., Fabio, V. S., Juliana, P. L., Patrícia, L., Vanessa,

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

Fabrício, L. S., Landulfo, S., 2012, Dietary Sugars:

Chemistry, CHAPTER 29 Sucrose Determination by Raman Spectroscopy,

503-525.

[17] Firouz, M.M., Mahdi, M. F., Sabah, S., Gholamreza, A.,

2007, Chemical Constituents of Dichloromethane Extract of

Cultivated Satureja khuzistanica, Evid Based Complement Alternat

Med., March 4(1):95–98.

[18] Martin, H., Kristine, A. K., Bent, H.S., Erland, B., 2011, Determination

of ten steroid hormones in animal waste manure and agricultural soil

using inverse and integrated clean-up pressurized liquid extraction and

gas chromatography-tandem mass spectrometry, Anal. Methods, 3:

1087.

[19] Nanako, I., Hideki, H., Fumiyo, K., 2010, Simultaneous determination

of β-sitosterol, campesterol, stigmasterol, and brassicasterol in serum

iii

by high-performance liquid chromatography with electrochemical

detection, Anal. Methods, 2: 174.

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

glycosides in 6 onion cultivars and comparisons of hydrolysis-HPLC and

spectrophotometric methods in measuring total quercetin concentrations,

J Food Sci.

[21] Vauquelin, L.N., Robiquet, P.J., 1806, La découverte d'un nouveau

principe végétal dans le suc des asperges, Annales de Chimie, 57: 88–

93.

[22] Tomoda Masashi et al., 1974, Chem Pharm Bull, 22(10): 2306.

[23] Ivorra, M.D., D'Ocon, M.P., Paya, M., Villar. A., 1988,

Antihyperglycemic and insulin-releasing effects of beta-sitosterol 3-beta-

D-glucoside and its aglycone, beta-sitosterol, Arch Int Pharmacodyn

Ther., 296:224-31.

[24] Oja Vahur et al., 2009, Sublimation Thermodynamic Parameters for

Cholesterol, Ergosterol, β-Sitosterol, and Stigmasterol, Journal of

Chemical & Engineering Data, 54 (3): 730–734.

[25] Matsuoka, K., Nakazawa, T., Nakamura, A., Honda, C., Endo, K.,

Tsukada, M., 2008, Study of Thermodynamic Parameters for

Solubilization of Plant Sterol and Stanol in Bile Salt Micelles, Chem. Phys.

Lipids, 154 (2): 87–93.

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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