VIETNAM NATIONAL UNIVERSITY – HOCHIMINH CITY
INTERNATIONAL UNIVERSITY

EVALUATING THE ALPHA-GLUCOSIDASE INHIBITON OF THE
Artocarpus altilis Fosberg leaves

A thesis submitted to
The School of Biotechnology, International University
In partial fulfillment of the requirements for the degree of
B.S. inBiotechnology

Student name: Le Tran Thi Hai Yen – ID: BTIU08123
Supervisor: Dr. Le Thi Ly
Dr. Hoang Le Son
MSc. Phung Van Trung

February, 2013

1


EVALUATING THE ALPHA-GLUCOSIDASE INHIBITON OF THE
ARTOCARPUS ALTILIS FOSBERG LEAVES

Yen. T.T.H Lea, Ly. T. Lea, Son L. Hoanga, Trung V. Phungb

a

School of Biotechnology, International University – Vietnam National University in
HCMC
b

Department of Chemistry of Natural Products, Institute of Chemical Technology,
Vietnam Academy Science and Technology

Corresponding author’s email address:

2


ACKNOWLEDGEMENT
I would like to express my deepest gratitude to my supervisor, Dr. Le Thi Ly,
for her wide knowledge on Applied Chemistry. Her understanding,
encouragement and personal guidance have provided a good basis for this thesis.
I am also deeply grateful to my co-supervisors; Dr. Hoang Le Son and MSc. Phung
Van Trung for their support throughout this work.
L owe my most gratitude to these instrument officers in Department of Chemistry
of Natural Products, Institute of Chemical Technology, Vietnam Academy Science
and Technology; Mr. Minh, Mr. Phat, Miss Be, Miss Hien.
I wish to express my warm and sincere thanks to my former labmates for their
valuable advices and their friendship which I have treasured the most.
I cannot end thanking my family, and my dearest siblings on whose constant
encouragement and patience I have relied throughout my study. Friends and
colleagues for whom I have great regard, wish to extend my warmest thank
to all those who have helped me.

3


ABSTRACT
This study investigates the effect of Artocarpus altilis (Parkinson) Fosberg leaf

extracts on -glucosidase activity. Among the extracts tested, Ethyl Acetate extract
exhibited a potential -glucosidase inhibitory activity with an IC50 value of
11.38 1.074 (μg/mL). In contrast, the Hexane and Methanolic extracts showed
lowest inhibition with the IC50 value in turn 1,099 2.004 and 1,085 1.715
(μg/mL). Extracts were also tested for anti-oxidant activity by

1, 2- Diphenyl-2-

picrylhydrazyl (DPPH) assay. The E4 fraction of Ethyl Acetate extract had strongest
inhibitory activity with the lowest IC50 value 25.39

1.270 (μg/mL) while E17 and

E18 had low percentage inhibited DPPH with higher IC50 value 215,970 6.176 and
269,113 4.551 (μg/mL) respectively. The phytochemical analysis indicated the
varied distribution of tannins, glycosides, saponins, steroids, flavonoids and alkaloid
in crude extracts. The correlation between the phytochemical analysis and glucosidase activity and DPPH assay suggests that the high content of glycosides,
flavonoids and steroids compounds could be involved in exerting -glucosidase
activity and DPPH inhibition. This study supports the utilisation of Artocarpus altilis
(Parkinson) Fosberg leaf in the folk medicine for the better treatment of
antidiabeties.

Keyword: Artocarpus altilis (Parkinson) Fosberg leaves, -glucosidase inhibition,
DPPH inhibition.

4


I. INTRODUCTION
For centuries, people have been using herbs for medicinal purposes. Medicinal

plants have their own historical stake in every early civilization. Medicinal plants
have been used for centuries as traditionally to treat various types of diseases such
as infectious disease and malignancy.
Artocarpus altilis (Parkinson) Fosberg, originated in the western Pacific, is a
widely known food source, but is also commonly used as a folk medicine in Vietnam
where it is locally called Sake. The plant Artocarpus altilis (Parkinson) Fosberg is
belonging to the family Moraceae, 10-15m tall, evergreen. Bark grayish brown,
thick as showed in Figure 1. Branchlets are 0.5-1.5cm thick. Leaves broadly
obovate, almost entire with only slight lobing to deeply pinnately lobed with sinuses
from 2/3 to 4/5 of the distance from margin to midrib, or deeper. Fruits globose to
oblong, skin light green; surface smooth to slightly bumpy or spiny with individual
disks ranging from areolation [1].

Figure 1: the Artocarpus altilis (Parkinson) Fosberg (a) tree and (b) leaf
Traditionally, the leaves of Artocarpus altilis (Parkinson) Fosberg are used for
the treatment of various kinds of diseases such as liver cirrhosis, hypertension and
diabetes [2]. Scientifically, some biological activities of the extract of this plant have
been reported. The acetone extract of the leaves showed an inhibitory effect on 5αreductase activity which might make it useful for the selective treatment of benign
prostate hyperplasia and prostate cancer

[3]

. Recently, a study showed the

ethylacetate extract of the leaves had cytotoxic effects on some human cancer cell

5


lines, including human lung adenocarcinoma (SPC-A-1 cells), human colon

carcinoma (SW-480 cells), and human hepatocellular carcinoma (SMMC-7721 cells)
[4]

, thus indicating that the extract might be a potential anti-cancer agent.
In 2011, Vietnamese scientists have isolated three substances are Quercertin

(1), Rutin (2), 8-gerany-4,5,7-trihydroxyflavon (3) as can be seen from Figure 2.
The results indicated two of Quercetin and Rutin in yellow powder had the ability to
reduce the risk of cancer, help prevent heart disease, antioxidant, anti-inflammatory
activity, anti-virus, prevention and treatment of Depression bone [6].
OH
OH
HO

OH
HO

O

O
OH
O

OH
OH

OH

O


O

O

(1)

H 3C

(8)

O

HO

O

HO

HO
OH

HO
OH

(2)

(3)

(9)


Figure 2: The structure of (1) Quercertin, (2) Rutin,
(3) 8-gerany-4,5,7-trihydroxyflavon.
In 2007, Lotulung PDN. et al.
dihydroxyphenyl)

-3

-

[7]

isolated a yellow crystalline

[8-hydroxy-2-methyl-2

-

1 - (2,4-

(4-methyl-3-pentenyl)-2H-1-

benzopyran-5-yl]-1-propanone, also known as AC-GF (4) in CH2Cl2 leaves extract as
presented in Figure 3.
OH
O

Figure 3: The structure of AC-GF
O
HO


OH

(43)

(4)

Nowadays, the more modern living, the more diseases we have, once of them is
diabetes. That make we thought to find many ways to treat this disease. Carried out
this project can give one opportunity to treat it. Besides that, folk remedy already

6


using Artocarpus altilis Fosberg leaves for treating diabetes for a long time. So, after
this study, we can demonstrate the reliability of that folk remedy. The main purpose
of this study was to prepare the extract from Artocarpus altilis (Paskinson) Fosberg
leaves and then determined the -glucosidase inhibition of them.
II. MATERIALS AND METHODS
1. Preparation of extracts[9]
The leaves of Artocarpus altilis (Parkinson) Fosberg were collected from Ho Chi
Minh City. The leaves were washed thoroughly under tap water, shade dried,
powdered.
The powdered leaves were macerated with 96% Ethanol to produce crude
extract. The crude extract was partitioned with Hexane, Ethyl Acetate and Methanol
respectively. The Rotary evaporator R200 was used for eliminating solvents. The
procedure for extract of leaves of Artocarpus altilis (Parkinson) Fosberg can be
diagramed in Figure 4.

Figure 4: The process of extract leaves Artocarpus altilis (Parkinson) Fosberg
The extracts were fractionated by Medium pressure column with silicagel 60

d=0.04-0.063m.

7


2. Testing -glucosidase inhibiton ability
a. Principles[10]

-Glucosidase Assay is designed to measure -glucosidase activity directly in
biological samples without pretreatment. The improved method utilizes pnitrophenyl-

-D-glucopyranoside

that

is

hydrolyzed

specifically

by

α-

glucosidase into a yellow colored product (maximal absorbance at 405nm). The
rate of the reaction is directly proportional to the enzyme activity.
p-nitrophenyl--D-glucopyranoside 

-D-glucopyranoside + p-nitrophenol


-glucosidase

Inhibitory activity of the extracts was calculated by the formula:
% inhibition =

{Absorbance (control) – Absorbance (sample)}*100%
Absorbance (control)

I%=

[A]o – [A]i

[A]o

*100 %

In which:
[A]o the average absorption of control
[A]i the average absorption of sample
IC50 value is defined as the concentration of extract inhibiting 50% of
-glucosidase activity under the stated assay conditions. In case of significant
inhibition, IC50 values were determined by linear regression by fitting to a
sigmoid dose-response equation with variable slope. All values are represented
as Mean ± Standard Deviation.

b. Procedure:
-

Preparation of test samples as can be seen in Figure 5:


8


Figure 5: The procedure of testing samples

-

The control (Acarbose) contained all reagents without the tested sample.

The reactions were conducted in triplicate.
3. Antioxidant assay[8]
The DPPH radical scavenging activities of the extracts were determined by
the method of Blois (1958) with slight modification. Initially, 4 mL of methanol
solution containing 0.1 mL each of the samples at different concentrations was
mixed with 1 mL of 0.15 mM DPPH (dissolved in methanol). The reaction
mixture was then incubated for 30 min at room temperature. The control
contained all reagents without the sample, whereas methanol was used as a
blank. All measurements were performed in triplicate. DPPH radical scavenging
activity was determined by measuring the absorbance at 490nm and expressed
as the inhibition percentage of free radicals by the sample after calculation using
the following formula:

% inhibition =

{Absorbance (control) – Absorbance (sample)}*100%
Absorbance (control)

I%=


[A]o – [A]i

[A]o

In which: [A]o the average absorption of control
[A]i the average absorption of sample

9

*100 %


4. Phytochemical screening[9]
The presence of phytochemical compounds in the extract of Artocarpus altilis
(Paskinson) Fosberg leaves was tested by the following methods:
-

Draggendorff’s reagent for Alkaloids

-

Fehling method for Glycosides

-

Shaking for Saponins

-

Ferric chloride for Tanins


-

Liebermann’s Burchard Test for Steroids

-

Shibata’s raction for Flavonoids

10


III.

RESULTS

1. Preparation of extracts
Initially, 8kg of Artocarpus altilis (Parkinson) Fosberg leaves were macerated
with 96% Ethanol to give 300g of crude extract. The crude extract partitioned with
different solvents to give 127g of Hexane extract, 85g of Ethyl Acetate extract and
14g of Methanol extract.
The extracts were fractionated by Medium Pressure Column. 12 fractions of
Hexane extract, 9 fractions of Ethyl Acetate extract and 8 fractions of Methanol
extract were collected. From the 8th fraction of Ethyl Acetate extract, after removing
the solvent, white powder was obtained. The powder was tested by TLC with the
solvent system Chloroform: Methanol (9:1) and sprayed 10% Sulfuric acid/ Ethanol.
The violet-brown color was observed. AA02 structure was elucidated by analysis of
1D, 2D – NMR spectra and compared with published data. AA02 was chemically
identified β-sitosterol-3-O-β-D-glucopyranoside. The structure of β-sitosterol-3-O-βD-glucopyranoside can be seen structure in Figure 6.


(a)

(b)

(c)
Figure 6: (a) TLC of AA02; (b) AA02 powder
(c)The structure of β-sitosterol-3-O-β-D-glucopyranoside

11


2. Testing -glucosidase inhibiton ability
Firstly, the absorption of positive control and 3 kinds of extracts were measured.
The Table 1 and

Figure 7 showed the inhibition of acarbose propotion to concentrations.

IC50 was determined as can be seen in the Table 2 and Figure 8.
Table 1: The inhibition of Acarbose
Concentration

12

24

36

30.814

44.089


52.130

48

60

(μg/mL)
Inhibition (%)

61.259

61.483

R2=0.9854

CONCENTRATION ( g/mL)

Figure 7: The inhibition of Acarbose
Table 2: IC50 of positive control (Acarbose) and 3 kinds of extract
Acarbose

Concentration

31.60

SE (μg/mL)

1.041


Hexane

Ethyl

extract

extract

extract

1,099 2.004

11.38 1.074

1,085 1.715

12

Acetate

Methanol


Figure 8: Comparing IC50 of positive control and extracts
Secondly, the various fractions of Ethyl Acetate extract were performed to test
the inhibition -glucosidase activity. The IC50 values of fractions were summarized
in Table 3 and Figure 9. In which, these fractions already obtained by MPLC.

Table 3: The inhibition of fractions from Ethyl Acetate extract
Concentration


10

40

80

120

160

(μg/mL)

IC50 SE
(μg/mL)

Inhibition

E2

23.24

87.74

93.98

97.97

52.36


1.029

(%)

E4

2.05

37.44

70.93

79.19

94.98

1.047

E5

5.18

41.68

44.08

81.82

106.10


1.144

E7

24.51

33.19

34.05

35.79

696.20

1.639

E9

8.89

63.55

74.83

88.72

73.37

1.091


E10

22.87

72.66

83.09

98.92

22.10

1.324

E12

40.35

62.94

94.61

97.84

16.03

1.357

E17


37.56

55.56

68.89

91.11

23.08

E18

47.13

85.93

96.67

97.26

25.86

13

1.132
1.030


Figure 9: IC50 values of fractions of Ethyl Acetate extract (μg/mL)
3. Antioxidant assay

The antioxidant was tested by using DPPH assay. Ascorbic acid seemed to be the
positive control. Extracts were tested firstly. Then, fractions of Ethyl Acetate extract
were tested. The tested results were summarized in Table 4. Figure 10 showed the
IC50 value of Ethyl Acetate extract and its fraction and positive control, except E17
and E18 fraction.
Table 4: the inhibition of extracts and fractions of Ethyl Acetate extract of
Artocarpus altilis (Parkinson) Fosberg leaves
Concentration (μg/mL)

20

40

60

80

IC50

SE

(μg/mL)
Inhibition

Ascorbic acid

51.93

83.04


85.41

87.12

15.77

1.100

Hexane

10.35

12.37

14.12

14.72

27,664 1.606

25.17

32.67

45.70

60.54

63.79 1.127


10.57

12.15

14.20

15.74

11,626

(%)
extract
Ethyl Acetate
extract
Methanol
extract

14

1.935


E2

45.08

47.91

57.93


67.73

32.95

1.211

E4

40.45

68.87

68.65

69.52

25.39

1.270

E5

39.61

68.43

67.37

72.45


26.32

1.213

E7

36.16

66.26

67.12

69.51

29.29

1.203

E9

13.58

31.38

47.18

62.97

61.58


1.023

E10

14.32

28.24

41.58

59.24

68.43

1.061

E12

12.80

21.33

32.78

46.35

93.98

1.094


E17

11.34

12.49

13.26

15.02

215,970 6.176

E18

11.46

12.27

13.91

14.72

269,113 4.551

Figure 10: IC50 value of Ethyl Acetate extract and its fractions of Artocarpus altilis
(Parkinson) Fosberg leaves in antioxidant test.
4. Phytochemical screening
The presence of phytochemical compounds in the crude extract of Artocarpus
altilis (Paskinson) Fosberg leaves and its Ethyl Acetate extract were summarized in
Table 5.


15


Table 5: The phytochemical screening of Artocarpus altilis (Paskinson) Fosberg
leaves.
Type
compounds

of

Result

Observation

Crude

Ethyl Acetate

extract

extract

Alkaloids

+

-

Reddish precipitate


Glycosides

+

+

Brick red precipitate

Saponins

+

-

Foaming/frothing

Tannins

+

-

Greenish black precipitate

Steroids

+

+


Reddish brown ring color

Flavonoids

+

+

Orange–pink coloration

(+): present
(-): absent

16


IV. DISSCUSION
The Artocarpus altilis (Paskinson) Fosberg leaves were fractionated into 12
fractions of Hexane extract, 9 fractions of Ethyl Acetate extract and 8 fractions of
Methanol extract by using different solvents with different polarity. From the 8th
fraction

of

Ethyl

Acetate

extract,


β-sitosterol-3-O-β-D-glucopyranoside,

was

obtained. That compound was white amorphous, with melting point is 269 – 270oC,
crystallized in Methanol

[13]

. β-sitosterol-3-O-β-D-glucopyranoside did not show any

antidiabetics and DPPH scavenging effect[14] but showed selective activity with the
antimicrobial spectra to 95 %[15].

The fractions were used to test inhibit -

glucosidase activity and DPPH assay.
Recently, Lee et al.[12] reported that natural products with wide structural
diversity were a good source of

-glucosidase inhibitors. Thus, it is necessary to

determine the -glucosidase inhibitory activity of Artocarpus altilis (Paskinson)
Fosberg. In this study, to determine the effects of the Artocarpus altilis (Paskinson)
Fosberg leaves extracts on -glucosidase activity, we calculated the IC50 values, as
shown in Table 2 and 3. Almost fractions of the Ethyl Acetate extracts seemed to
have potent inhibitory activities towards -glucosidase. The E12 was the strong
inhibit fraction with the IC50 value of 16.03


1.357 μg/mL. However, the -

glucosidase inhibitory activities of the different extracts varied greatly. The Hexane
and Methanol extract had a higher IC50 value 1,099 2.004 and 1,085 1.715
μg/mL, respectively, as compared to the Ethyl Acetate extract with the lowest IC50
value (11.38 1.074 μg/mL). This may be due to the fact that Ethyl Acetate extract
contained more effective -glucosidase inhibitory compounds, including glycosides,
steroids and flavonoids. Acarbose, known to be an effective microbial -glucosidase
inhibitor (IC50 =31.6

1.041 μg/mL), is used for diabetes therapy. The Ethyl

Acetate extract and fractions had lower IC50 value than Acarbose. So, this herb
may be a promising drug for antidiabeties therapy.
DPPH, a stable free radical is purple in color, but changes into a stable yellow
compound upon reaction with an antioxidant. It is common to measure the
concentration of antioxidant required to reduce or inhibit 50% of the DPPH radicals
present. The inhibition abilities of various extracts against DPPH radical are
illustrated in Table 4, and the results are expressed as IC50 values (μg/mL) for
comparison purposes. In experiments, all of the extracts tested possessed DPPH

17


radical scavenging activity. The Ethyl Acetate extract was the most active DPPH
inhibition with IC50 value of 63.79 1.127 μg/mL whereas the lowest activity was
found in Hexane and Methanol extract with IC50 value in turn 27,664 1.606 and
11626

1.935 μg/mL. Overall, the DPPH inhibition activities of all the extracts were


lower to that of the positive control, Ascorbic acid (IC50 value is 15.77
1.10μg/mL). Extracts obtained with different solvents varied in their DPPH
inhibition activities, indicating that the extraction medium significantly affected the
strength of antioxidants in the extracts. In general, the most active DPPH inhibition
was fractions of Ethyl Acetate extract with IC50 value varies from 25.39
93.98

1.270 to

1.094 μg/mL, possibly due to a higher content of glycosides, steroids and

flavonoids compounds. E17 and E18 fractions did not inhibit. This observation is
consistent with a report that flavonoid compounds contribute directly to the antioxidative action [11].
A number of different classes of compounds with antidiabeties and antioxidant
activity have been isolated from plants including tannins, glycosides, saponins,
steroids, flavonoids and alkaloid. Therefore, the phytochemical analysis of leaf
extracts was carried out to locate the possible classes of compounds responsible for
antidiabeties and antioxidant activity. Among these phytoconstituents, glycosides,
steroids and flavonoids of Ethyl Acetate extract showed significant correlation with
antidiabeties and antioxidant activity.
V. CONCLUSION:
In this study, the antioxidative, -glucosidase inhibitory activities of Artocarpus
altilis (Paskinson) Fosberg leaves extracts were investigated. β-sitosterol-3-O-β-Dglucopyranoside was collected from Ethyl Acetate extract. The -glucosidase
inhibitory potential of Artocarpus altilis (Paskinson) Fosberg leaf extracts shore up
its utilisation in the folk medicine for the better treatment of antidiabeties. The
result of DPPH assay indicated Artocarpus altilis (Paskinson) Fosberg leaf extracts
had lower ability to inhibit DPPH activity. The correlation between the phytochemical
analysis and


-glucosidase and DPPH inhibitory activity in this investigation

suggests that the high content of glycosides, steroids and flavonoids compounds in
the leaf extracts could be involved in exerting the -glucosidase and DPPH inhibitory
activity. Further studies on isolation and characterization from Ethyl Acetate extract
of Artocarpus altilis leaf would be highly recommended.

18


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