Biological activity of methanolic extract derived from Ipomoea-pes-caprae (L.) collected in Xuan Thuy national park

JOURNAL OF SCIENCE OF HNUE
Chemical and Biological Sci., 2013, Vol. 58, No. 9, pp. 139-145
This paper is available online at

BIOLOGICAL ACTIVITY OF METHANOLIC EXTRACT DERIVED
FROM Ipomoea pes-caprae (L.) COLLECTED IN XUAN THUY NATIONAL PARK

Dao Thi Sen, Dao Van Tan and Hoang Thi La
Faculty of Biology, Hanoi National University of Education
Abstract. Ipomoea pes-caprae (L.) Sweet (Convolvulaceae), a common species in
Xuan Thuy National Park, Nam Dinh Province, is thought to be a potential source
of bioactive compounds. In this study, methanolic extracts from the stems, leaves
and flowers of Ipomoea pes-caprae that were collected in Xuan Thuy National Park
were evaluated for their antibacterial and antioxidative activities, and their ability to
inhibit plant growth. The DPPH free radical scavenging activity of crude methanol
extracts was evaluated. The extract of flower had the highest radical scavenging
activity (IC50 = 0.33 mg/mL) while stem extract showed the lowest activity (IC50
= 3.39 mg/mL). Antibacterial activity was tested using the agar diffusion method
and it was found that the extracts showed antibacterial activity against Bacillus
subtilis and Vibrio parahaemolyticus. The stem extract showed the highest activity
against V. parahaemolyticus (D, 14.7 mm) and was the only extract that inhibited
E. coli growth. However, no extract had an inhibitory effect on Vibrio spp. or
Staphyloccus aureus. Extracts of all parts of the plant inhibited the growth of the
roots of Raphanus salivus seedlings 44 - 73%.
Keywords: Ipomoea pes-caprae (L.) Sweet, DPPH, Antibacterial activity,
bioactivity.

1.

Introduction

Plants have been used as an important source of medicine for thousands of years.
Medicinal plants are the most important source of life saving drugs for the majority of
the world’s population. Recently, several distinct chemicals derived from plants have been
used as a component in important drugs. The World Health Organization (WHO) has
estimated that up to 80% of the human population has used traditional herbal remedies as
medicine [12].
The genus Ipomoea (Convolvulaceae) consists of over 200 species which are widely
distributed in tropical and subtropical countries. Some of them are used in folk medicine to
Received May 24, 2013. Accepted November 26, 2013.
Contact Dao Thi Sen, e-mail address:

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Dao Thi Sen, Dao Van Tan and Hoang Thi La

treat diseases [6,13]. Ipomoea pes-caprae grows profusely in low-lying broad coastal sand
dunes. It is known to inhibit sand erosion and it thrives in a marine environment of high
salinity, high temperature, continuous exposure to the sun, incessantly tidal fluctuation,
intense rain and wind, etc.
In Vietnam, I. pes-caprae is a commonly used traditional medicine. Dried leaves
and stems are used to treat stomach pain, fever and rheumatoid arthritis. This plant
has also been used to treat inflammation, skin disease, burn, wounds and ulcers [7]. I.
pes-caprae has been used as a herbal remedy in other countries to treat inflammations,
colic, diuretic disorders and gonorrhoea [8]. Preclinical and clinical investigations have
identified pharmacological properties of this plant [11]. It has been reported that I.
pes-caprae inhibited the contraction of guinea pig ileum relative to its concentration.
The same extract also showed considerable anti-inflammatory action when evaluating
in different experimental models [11].
In Vietnam, recent studies on I. pes-caprae focused primarily on identification and

ecological characterization. Therefore, the determination of antioxidant and antibacterial
activity, and inhibition of growth is necessary to characterize pharmacological properties
and possibly lead to future applications. In this study, the methanolic extract from various
parts including roots, stems, leaves and flowers of Ipomoea pes-caprae that were collected
in Xuan Thuy National Park, Nam Dinh Province, were evaluated for their antibacterial
and antioxidative activities and possible ability to inhibit plant growth.

2.
2.1.

Content
Material and methods

* Plant materials: Fresh stems, leaves, flowers of I. pes-caprae, free from disease,
were collected in Xuan Thuy National Park, Nam Dinh Province, in September, 2012,
and they were washed thoroughly 2 - 3 times under running tap water, shade-dried and
powdered before being used for extraction.
* Preparation of methanolic extract: Shade-dried powder of I. pes-caprae was
soaked in methanol for 24 h at room temperature and transferred to a Soxhlet apparatus
with methanol until a colorless extract was obtained on the top of the extractor (extracted
7 - 10 times). After complete methanol evaporation, each of the methanol extracts was
weighed and subjected to bioactivity assays.
* DPPH (1,1-diphenyl-2-picrylhydrazy) radical scavenging activity: The hydrogen
atom or electron donating ability of the extracts was measured from bleaching with a
purple colored ethanol solution of DPPH using the method of Hwang et al. (2001) [5]. The
degree of decoloration is directly proportional to the antioxidant activity of the extract.
A 10 µL aliquot of the extract was added to 190 µL of DPPH in ethanol (100 µM) in
a 96-well microtitre plate. After incubation at 30 ◦ C for 30 min, the absorbance was
measured at 517 nm using an Epoch microplate (Biotek, USA). Capability to scavenge
the DPPH radical was calculated using the following equation:

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Biological activity of methanolic extract derived from Ipomoea pes-caprae (L.) collected...

SC% (Scavenging capacity) = 100 - (Ac - At )/Ac × 100
where Ac is the absorbance of control and At is the absorbance of the sample.
Antioxidant activity of the extracts was expressed as IC50 . The IC50 value was
calculated based on the logarithmic regression equation of scavenging capacity against
sample concentration.
The quality standard used is 5 mM ascorbic acid ascorbic.
* Antibacterial activity: The bacterial strains Escherichia coli, Bacillus subtilis,
Staphylococcus aureus and Vibrio sp. including Vibrio parahaemolyticus were obtained
from the National Institute of Hygiene and Epidemiology.
Antibacterial activity was determined by cup diffusion method on an MPA medium.
The sterile medium (20 mL) was poured into a 9 cm petri plate. The medium was allowed
to cool in sterile conditions and plates were then incubated with 1 × 10 CFU cultures
of test bacteria. The concentration of bacterial cells in the suspension was adjusted to a
minimum of 1×108 CFU/mL in the MPA solution. Agar cups 10 mm in diameter were
made in the plates. The extracts were prepared by first being reconstituted in methanol
(30 g shade-dried powder), then diluted in 10 mL of sterile distilled water. An 100 µL
aliquot of each dilution was introduced in three wells into MPA plates. All test plates
were incubated at 37 ◦ C for 24 h [10].
Negative controls were prepared using sterile distilled water. Chloramphenicol
0.4% was used as the positive reference to determine the sensitivity of each bacterial
species tested.
* Inhibition of plant growth [3]: Seeds of Raphanus salivus L. were grown to
produce seedlings at 30 ◦ C for 1 day. The roots of seedlings were selected to be of the same
size. The roots were embedded in crude extracts for 1 hour (The extracts were prepared
by first being reconstituted in methanol (30 g shade-dried powder), then diluted in 10

mL of sterile distilled water). In negative controls, distilled water was prepared instead
of the crude extracts. The root length was measured after 24 hours. The inhibition of
root growth was evaluated through percentage of inhibition (I%) and calculated using the
following equation:
I% = (Lc – Ls ) × 100/Lc
where I%: Percent inhibition; Lc : Average length (cm) of roots in the control; Ls : Average
length (cm) of roots in the test.
* Data process: Each experiment was conducted in triplicate. Data were processed
using statistics generated by Microsoft Excel and SPSS software.

2.2.

Results and discussion

2.2.1. Antioxidant activity
The results of the antioxidant activity assay are given in Table 1. A DPPH radical
was used as a substrate to evaluate the free radical scavenging activity of the I. pes-caprae
extracts. A serial dilution of 4, 10, 20, 50 and 100 folds was used for each extract.
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Dao Thi Sen, Dao Van Tan and Hoang Thi La

Antioxidant activity of the extracts was expressed as IC50 . Antioxidant activity of the
extracts was compared with the standard quality (5 mM ascorbic acid).
Table 1. Antioxidant activity of Ipomoea pes-caprae extracts
Plant parts
IC50 (mg/mL)
% acid ascorbic
Stems

3.39
0.48
Leaves
2.99
0.49
Flowers
0.33
2.26
The IC50 value of all extracts ranged between 0.33 and 3.39 mg/mL. The highest
activity was observed for the extract from flowers (IC50 0.33 mg/mL) and lowest activity
was shown by the extract from stems (IC50 3.39 mg/mL). There is a tight relationship
between DPPH free radical scavenging capacity and extract concentration.

Figure 1. The relationship between DPPH free radical scavenging capacity
and extract concentration
The generation of oxygen radicals such as superoxide radicals (O−
2 ), hydroxyl
radicals and non-free radical species such as hydrogen peroxide and singlet oxygen (1 O2 )
are associated with cellular and metabolic damage, aging and a number of illnesses
including cancer, cardiovascular disease, neurogenic disease and inflammation [9]. On
average, every cell in the body comes under the attack of free radicals once every
ten seconds. Although the body possesses defense mechanisms such as enzymes and
antioxidant nutrients, continuous exposure to chemicals and contaminants may lead to
an increase in the amount of free radicals in the body beyond its capacity to control them
and cause irreversible oxidative damage [4]. The free radical damage can be counteracted
by the consumption of antioxidants. Antioxidants act by donating an unpaired electron
to free radicals and thereby inhibiting the oxidation of macromolecules and cellular
structures [2]. Vitamin C, vitamin E, carotenoids and glutathione are some of the
basic antioxidants in the body. Plants produce lot of antioxidants to protect themselves
against oxidative stress caused by direct exposure to the sun and oxygen. Mangrove

trees and some halophytic herbs grow under such stressful conditions characteristic to
estuarine ecosystems. It has been found that phenolics are the major compounds present
in mangrove environments that are responsible for antioxidant activity and it might be that
the antioxidant activity of Ipomoea pes-caprae is due to a group of phenolic compounds.
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Biological activity of methanolic extract derived from Ipomoea pes-caprae (L.) collected...

2.2.2. Antibacterial activity
The methanolic extracts of plant stems, leaves, flowers was assayed in vitro using
the agar disc diffusion method against five bacterial species. All of the extracts had
antibacterial activity against Bacillus subtilis and Vibrio parahaemolyticus. The stem
extract showed the highest activity against V. parahaemolyticus (D, 14.7 mm) and was
the only extract to inhibit E. coli growth. However, none of the extracts had any effect on
Vibrio spp. and Staphylococcus aureus.
Table 2. Antibacterial activity of Ipomoea pes-caprae extracts
(Zone of inhibition, D mm)
Plant parts
Stems
Leaves
Flowers

E. coli
7.0 ± 1.4
-

B. subtilis
9.0 ± 1.4
7.0 ± 1.4

5.0 ± 1.4

V. parahaemolyticus
14.7 ± 1.2
10.0 ± 0.0
6.0 ± 0.0

Vibrio spp.
-

S. aureus
-

Note. (-) not having a zone of inhibition

Figure 2. Antibacterial activity of Ipomoea pes-caprae extracts
against Vibrio parahaemolyticus
Stems: extract from stems; leave:. extract from leaves; (+): Chloramphenicol 0,4%; (-): water

I. pes-caprae exhibits anti-inflammatory activities and is used as a traditional
medicinal plant in Vietnam for the treatment of various types of inflammation including
jellyfish sting and dermatitis. The results here confirm that the use of this species in
folklore has a scientific basis [7]. Pathogenic bacteria are considered to be a major cause
of morbidity and mortality in humans. Even though pharmaceutical companies have
produced a number of new antibiotics, resistance to these drugs has increased and this
has become a global concern [1]. Due to the increase of resistance to antibiotics, there is
a pressing need to develop new and innovative antimicrobial agents. Among the potential
sources of new agents, plants are an excellent candidate for investigation because they
contain many bioactive compounds that can be of interest in therapeutics. In this study, we
evaluated the preliminary antibacterial activity of the crude methanol extract of Ipomoea

pes-caprae with 5 strains of bacteria and found that it had an inhibitory effect on E. coli,
B. subtilis and V. parahaemolyticus. Therefore, a detail evaluation of antibacterial activity
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Dao Thi Sen, Dao Van Tan and Hoang Thi La

as well as the extraction of pure compounds and further investigation of other pathogenic
bacteria should be carried out.
2.2.3. Inhibition of plant growth
Seeds of Raphanus salivus were grown to produce seedlings at 30 ◦ C for 1 day. The
roots of seedlings were embedded in crude extracts for one hour then were returned to
the medium to germinate for 24 hours. The length of roots was measured. The inhibitory
effect of extracts on root growth of seedlings was assessed through the value I (%). The
results are shown in Table 3.
Table 3. Growth inhibition (%) of Ipomoea pes-caprae extracts
Plant parts
Stems
Leaves
Flowers
I%
73.60 ± 3.47
73.44 ± 2.54
44.56 ± 4.30

Figure 3. The inhibitory effect of methanol stem extract on the growth
of Raphanus salivus L. roots
All parts of the plant had an inhibitory effect on the growth of seedling roots,
ranging from 44.56 - 73.60%. The highest degree of inhibition was observed with the
extract from stems (I% 73.60) while the lowest value was found with the extract from

flowers (I% 44.56). The inhibition is generally an inhibition of mitotic activity. Further
studies are needed to prove anticancer properties of this plant.

3.

Conclusion

Methanolic extracts from stems, leaves and flowers of Ipomoea pes-caprae
collected in Xuan Thuy National Park, Nam Dinh Province, exhibited antibacterial and
antioxidative activities and the ability to inhibit plant growth. The results of the present
study support the use of Ipomoea pes-caprae in traditional medicine.
Acknowledgement. This work was supported by the project: The Study of Antibacterial
and Antioxidative Activities and the Inhibitory Effect on Plant Growth of some Plants in
Xuan Thuy National Park, Nam Dinh Province. Implementing institution: Hanoi National
University of Education (Code number: SPHN-12 - 129).
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REFERENCES
[1] G. Adwan, M. Mhanna, 2008. Synergistic effects of plant extracts and antibiotics on
staphylococcus aureus strains isolated from clinical specimens. Journal of Scientific
Research,Vol. 3, pp.134-139.
[2] J. C. Dekkers, L. J. Van Doornen and H. C. Kemper, 1996. The role of antioxidant
vitamins and enzymes in the prevention of exercise-induced muscle damage. Sports
Med., Vol. 21, pp. 213-238.
[3] T. T. Dat, B. T. Lien, T. V. Thanh, 1999. Preliminary studies on anti-mitotic effects
of some medicinal plants used as anti-cancer drugs by experienced folk. Journal of
Medicine, Vol. 11, pp. 6-7.

[4] B. Halliwell, 1994. Free radicals and antioxidants: A personal view. Nutr. Res., 52:
253-265.
[5] B. Y. Hwang, H. S. Kim, J. H. Lee, Y. S. Hong, J. S. Ro, K. S. Lee and J. J. Lee,
2001. Antioxidant benzoylated flavan-3-ol glycoside from Celastrus orbiculatus. J.
Nat. Prod., Vol. 64, pp. 82-84.
[6] C. P. Locher, M. Burch, H. F.Mower, J. Berestecky, H. Davis, B. Van-Poel, A.
Lasure, D.A. Vanden-Berghe, D.A and Vlietinck, A.J. 1995. Anti-microbial activity
of extracts obtained from selected Hawaiian medicinal plants. J. Ethnopharmacol,
Vol. 49, pp. 23-32.
[7] D.T. Loi, 1977. The medicinal plants and remedies in Viet Nam. Publisher of
scientific and technical, Ha Noi.
[8] M. M. Khan, F. Ahmad, A. K. Rastogi and J. R. Kidwai, 1994. Insulinogenicand
hypoglycaemic activities of Ipomoea pes-caprae. Fitoterapia, Vol. 65, pp. 231-234.
[9] J. Mimi-Oka, D. V. Simz and T. P. Simi, 1999. Free radicals in cardiovascular
diseases. Sci. J. Facta Univ. Ser. Med. Biol., Vol. 6, pp. 11-22.
[10] P. R. Murray, E. J. Baron, M. A. Pfaller, F. C. Tenover and H. R. Yolken, 1995.
Manual of clinical microbiology. ASM Press.
[11] U. Pongprayoon, P. Baeckstrom, U. Jacobsson, M. Lindstrom and L. Bohlin 1991a.
Compounds inhibiting prostaglandin synthesis isolated from Ipomoea pes-caprae.
Planta Med, Vol. 57, pp. 516-518.
[12] R. Shanmugapriya, T. Ramanathan, G. Renugadevi, 2012. Invitro studies on the
production of secondary metabolites in Ipomoea pes-caprae (L.) R. Br.. International
Journal of Advances in Pharmaceutical Research, Vol. 1, No. 1, pp. 9-11.
[13] M. M. Souza, A. M. Oliveira, F. V. Cechinel, C. Berti, R. A. Yunes and R. Krogh,
1998. Estudos preliminaries dapropriedate antinociceptiva de Ipomoea pes-caprae
emmodelos farmacologicos especificos. Alcance (UNIVALI) Vol. 5, pp. 53-57.

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