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International Journal of Biosciences and Nanosciences
Volume 1 (1), 2014, pp. 12-18.
Original Article
Studies on Phytochemical composition, antibacterial and antioxidant potential
of methanolic stem extract of Cardiospermum halicacabum L (Sapindaceae)
Sagadevan.Pa, S. N. Suresh*b, S. Rathish Kumara and. Revathy Rajan A.Ra
a
PG & Research Department of Biotechnology, Kongunadu Arts and Science College, Coimbatore - 29.
Department of Biotechnology, Sree Narayana Guru College, K.G. Chavadi, Coimbatore.
*Corresponding author: [email protected]
b
Received 18 April 2014; Accepted 28 May 2014
Abstract
The present study reveals the medicinal properties of the Cardiospermum
halicacabum. The phytochemical screening revealed the presence of various
compounds such as Tannins, Flavonoids, Alkaloids, Polyphenols and Cardiac
glycosides. The GC MS analysis confirmed the presence of 20 compounds with
various area of percentage. This confirmed the presence compounds which are
responsible for good antibacterial and anti-oxidant properties of the study species.
The plant extract showed significant activity against the bacterial pathogens and
this may be due to the presence of various compounds. The antioxidant activity
also notably significant and anticancer study revealed the importance of the study
plant as a potential anticancer agent. The present study confirms the potentiality
of the plant rich source of therapeutic value. Extensive study will provide a good
source of medicinally important drugs in future.
Key words: Cardiospermum halicacabum, phytochemical screening, antioxidant,
antibacterial
apoptosis (Swammy, and Tan, 2000).
Microbial infections are an important health
problem throughout the world and plants are
possible sources of antimicrobial agents
(Burapadaja and Bunchoo, 1995). The interest
to evaluate plants possessing antibacterial
activity for various diseases is growing Clark
and Hufford1993).
INTRODUCTION
Nature has bestowed on us a very rich
botanical wealth and a large number of diverse
types of plants grow indifferent parts of the
country. India is rich in all the three levels of
biodiversity, namely species diversity, genetic
diversity and habitat diversity. In India
thousands of species are known to have
medicinal value and the use of different parts
of several medicinal plants to cure specific
aliments has been in vogue since ancient
times. Herbal medicine is still the mainstay of
about 75-80% of the whole population, mainly
in developing countries. These plants possess
various
phytochemicals
and
active
biomolecules, which play a major role in the
treatment of cancer. Many plants have been
examined to identify new and effective
anticancer compounds, as well as to elucidate
the mechanism of cancer prevention and
Extracts from plants used in allopathic
medicine are potential source of antiviral, antitumoral and antimicrobial agents Chung, et
al., 1995. Interest in large number of
traditional natural products has increased
Taylor et al., 1996.The study plant
Cardiospermum halicacabum Linn. belongs to
the family Sapindaceae, commonly known as
Balloon vine or Love in a puff.
Cardiospermum is the combination of the
Latin words cardio, meaning heart, and
sperma, meaning seed and refers to the white
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Int.J.Biosci.Nanosci. Vol (1)1,2014
heart-shaped
pattern
on
the
seed.
Halicacabum is derived from the Latin word
halicacabus, a plant with inflated fruits
(Brown, 1954). It is an annual or sometimes
perennaial climber, widely distributed
intropical and subtropical Africa and Asia. It
has been examined for anti diarrhoeal as well
as homoeopathic medicinal properties. C.
halicacabum has been used in the treatment of
rheumatism, nervous diseases, stiffness of the
limbs and snakebite. Young leaves can be
cooked as vegetables (Pieroni, et al., 2002).
Considering
the
above
features
of
Cardiospermum
halicacabum
was
investigated for their phytochemical and
antibacterial activity.
A loopful of strain was inoculated in 30ml of
nutrient broth in a conical flask and incubated
on a rotary shaker at 37º C for 24 hours to
activate the strain.
Bioassay
The bioassay used was the standard Agar Disc
Diffusion assay adapted from (Nair, et al.,
2005). Mueller Hinton Agar was prepared for
the study. Mueller Hinton agar plates were
swabbed with a suspension of each bacterial
species, using a sterile cotton swab.
Subsequently, the sterilized filter paper discs
were completely saturated with the test
compound. The impregnated dried discs were
place the surface of each inoculated plate. The
plates were incubated overnight at 37ºC. Each
organism was tested against each organism in
triplicate. Methanol was used as negative
control. Standard discs of Ampicillin served as
positive antibacterial control. The test
materials having antimicrobial activity
inhibited the growth of the micro organisms
and a clear, distinct zone of inhibition was
visualized surrounding the disc. The
antimicrobial activity of the test agents’ was
determined by measuring the diameter of zone
of inhibition in mm.
MATERIALS METHODS
Collection of Plant Material
Fresh plant was collected from nearby places,
and the stem were separated and washed under
running tap water. Thoroughly washed stem
were allowed for shade drying under room
temperature in the laboratory. The dried leaves
were ground to fine powder using ablender.
The powder was preserved in an air tight
bottle for further studies.
Preparation of Solvent Extracts
10 grams of thoroughly grounded powder
were then soaked in 100ml of organic solvent
(methanol) in a conical flask, plugged with
cotton and kept on a rotary shaker at 190- 220
rpm for 24 hours at 40ºC. After 24hours, it
was filtered through a Whatmann filter paper
No 1 and the supernatant was collected and
the solvent was evaporated to make the final
volume one fourth of the original volume.
Antimicrobial Assay
Micro-organisms used
Five species of bacteria, two gram – positive
(Staphylococcus aureus & Bacillus subtilis)
and three gram negative (Esherichia coli,
Klebsialla pnuemoniae & Proteus vulgaris)
were obtained from KMCH, Coimbatore.
Chemical composition analysis
GC Analysis
Gas Chromatography (GC) analysis was
carried out using Varian 3800 gas
chromatography equipped with mass selective
detector coupled to front injector type 1079.
The chromatography was fit with VF 5 MS
capillary column (30 m × 0.25 mm).The
injector temperature was set at 240º C, and the
oven temperature was initially be at 70º C then
programmed to 300º C at the rate of 10 ºC /
minute and finally held at 300º C for 10 min.
Helium was used as carrier gas with the flow
rate of 1.51ml/min. The percentage of
composition of extract was calculated by GC
peak areas.
Preparation of Inoculum
GC- MS Analysis
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Int.J.Biosci.Nanosci. Vol (1)1,2014
Gas Chromatography coupled with mass
spectroscopy was performed using Varian
3800 gas chromatography equipped with
Varion 1200 L single quadruple mass
spectrometer. GC conditions were the same as
reported for GC analysis and the same column
was used. The mass spectrometer operated in
the electron impact mode at 70eV. Ion source
and transfer line temperature was maintained
at 250 º C. The compounds was identified
based on comparison of their retention indices
(RI), retention time (RT) and mass spectra.
Table.1:
The
phytochemical
and
Biochemical Analysis of Methanolic Stem
extract of Cardiospermum halicacabium
Compound
Observation
Presence of
precipitation
Formation of
coloured rings
Formation of
yellow colour
No colour
change
Result
Saponins
Frothing present
Present
Steroids
Presence of pink
Present
colour
Alkaloids
Cardiac
Glycosides
Flavanoids
Glycocides
Assessment of the activities of enzymatic
and non enzymatic antioxidants
The leaf samples of Artemisia niligirica were
analysed for the enzymatic and non enzymatic
antioxidants such as Catalase (Luck, 1974),
Superoxide dismutase (Misra and Fridovich,
1972), Glutathione reductase (David and
Richard, 1983), Glutathione-s-transferase
(Habig, et al., 1974), Glutathione peroxidase
(Rotruck, et al., 1973), Ascorbic acid (Roe
and Keuther 1953), α- Tocopherol (Rosenberg
1992), Reduced glutathione (Moron et al.,
1979), Polyphenols, Flavonoids (Moron et al.,
1979) and Carotenoids (Zakaria, et al., 1979).
Tannins
Extract change
into blue black
Present
Present
Present
Absent
Present
Antibacterial Activity
The antibacterial property of methanolic stem
extract of Cardiospermum halicacabum was
analysed against bacterial pathogens using
ampicillin (commercial antibiotic) as control.
Out of these five bacterial pathogens three
were found to be negative (Echerichia coli,
Kiebsiella pneumonia, Proteus vulgaris) and
two were positive (Bacillus subtilis,
Staphylococcus). Disc diffusion method was
used to evaluate the antibacterial activity of
taken samples. After twenty-four hours the
minimum inhibitory zones of methanolic stem
extract of Cardiospermum halicacabum and
control were measured. The maximum activity
(18±0.57) in extract was observed against
Staphylococcus aureus gram positive bacteria
(Table. 2). The control ampicillin showed
19±0.13. The least activity (10±0.03) was
observed against Proteus vulgaris gram
negative bacteria. The control against Proteus
vulgaris showed 12±0.89 with tested 10±0.0.
The treated disc shown zone of 17±1.15
against Bacillus subtilis, against E.coli and
Klebsilla pneumoniae showed a moderate
RESULT AND DISCUSSION
Preliminary Phytochemical Analysis
The phytochemical analysis of methanolic
stem extract of Cardiospermum halicacabum
was analysed for the compounds such as
Alkaloids, Cardiac Glycosides, Flavonoids,
Glycosides, Saponins, Steroids and Tannins.
The preliminary phytochemical analysis
revealed the presence of six compounds i.e.
Alkaloids, Cardiac Glycosides, Flavonoids,
Saponins, Steroids and Tannins and absence
of glycosides (Table. 1). Various tests has
been performed to find out the phytochemical
constituents.
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Int.J.Biosci.Nanosci. Vol (1)1,2014
activity. Similar results were also reported by
venkatesan et al.,(2006).
Table 2: Antibacterial Activity of Methanolic Stem Extract of Cardiospermum halicacabum
Bacteria
Bacillus subtilis (+ve)
Echerichia coli (-ve)
Klebsiella pneumoniae (-ve)
Proteus vulgaris (-ve)
Staphylococcus aureus (+ve)
Control Ampicillin (mm)
18 ± 0.57
14 ± 0.03
12 ± 0.01
12 ± 0.89
19 ± 0.13
Zone of Inhibition (mm)
17 ± 1.05
12 ± 0.03
10 ± 0.57
10 ± 0.03
18 ± 0.05
GC-MS ANALYSIS
In general, the plant sample has maximum
activity against gram positive bacterial
pathogens than that of gram negative bacterial
pathogens. Plant-based antibacterial have
enormous therapeutic potential as they can
serve the purpose with lesser side effects that
are often associated with synthetic
antibacterial (lwu et al., 1999). Natural
products, either as pure compounds or as
standardized plant extract, provide unlimited
opportunities for new drug leads because of
the unmatched availability of chemical
diversity. The increasing failure of
chemotherapeutics and antibiotic resistance
exhibited by pathogenic microbial infectious
agents has led to the screening of several
medicinal.
GC-MS analysis of methanolic stem extract of
Cardiospermum halicacabum showed the
presence of twenty different compounds with
various percentages is shown in Figure-1. Out
of these twenty compounds, Olean-12-en-3one was found to be maximum (22.98%) with
retention time of 28.438. The other important
constituents such as the n-Hexa decanoic acid
(15.33%) gamma-sitosterol (10.70%) were
found to be present. The presence of various
compounds with area of percentage and
retention time is given in Figure-1. The list of
twenty compounds which are present in the
samples were listed. The peak report indicates
the presence of compounds with various
intervals in peak forms was observed and
analysed.
Enzymatic and non enzymatic antioxidants
Activities of various Enzymatic antioxidants
like
Catalase,
Superoxide
dismutase,
Glutathione peroxidase, Glutathione reductase
and Glutathione-s-transferase and nonenzymic
antioxidant a such as Ascorbic acid. αTocopherol,
Reduced
glutathione,
Polyphenols, Flavonoids and Carotenoids in
the leaves of C.helicacabam was tested and
results obtained are depicted in table II and III.
From the table II, it is clear that the leaves of
C.helicacabam exhibited the maximum
activities of Glutathione reductase and
Superoxide dismutase when compared to other
enzymatic antioxidants. Catalase found to be
considerable source in the leaves. Others like
Glutathione s- transferase and Glutathione
GC-MS ANALYSIS
GC-MS analysis of methanolic stem extract of
Cardiospermum halicacabum showed the
presence of twenty different compounds with
various percentages is shown in Figure-1. Out
of these twenty compounds, Olean-12-en-3one was found to be maximum (22.98%) with
retention time of 28.438. The other important
constituents such as the n-Hexa decanoic acid
(15.33%) gamma-sitosterol (10.70%) were
found to be present. The presence of various
compounds with area of percentage and
retention time is given in Figure-1. The list of
twenty compounds which are present in the
samples were listed. The peak report indicates
the presence of compounds with various
intervals in peak forms was observed and
analysed.
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Int.J.Biosci.Nanosci. Vol (1)1,2014
peroxidase were found to negligible amount in
the leaves.
Fig.-1GC-MS analysis
of all the enzymatic and non enzymatic
antioxidants analysed it may prevent the risk
factor serious disease caused by free radicals
such as cancer, heart diseases, diabetes, aging
and cataract.
The table III reveals that the leaves of
Artemisia niligirica contain maximum activity
of Flavonoids and Carotenoids. Ascorbic acid,
α – Tocopherol, Polyphenols and Reduced
glutathione were present in minimum levels.
Since, C.helicacabam contains good amount
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Int.J.Biosci.Nanosci. Vol (1)1,2014
Table 3: Enzymatic Antioxidants Present In
Methanolic Stem Extract of Cardiospermum
halicacabum
Enzymatic Antioxidants
David, M and Richard, J.S. (1983), In
methods of enzymatic analysis – 3,
(Ed. BergmeyerJ and Marianna,
Gra.B), Verlag chemic wein hein dein,
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Habig, W.H., Pabst, M.J and Jacoby, W.B.
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The first enzymatic step in mercapturic
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HPLC analysis for the determination of
provitamin carotenoids in tomatoes,
Journal of Chromatography, 176; 109
– 117.
Luck, H. (1974), Methods of enzymatic
analysis, Academic Press, 78; 885 –
894. Misra, H.P. and Fridovich, A.
(1972),
Assay
of
superoxide
dismutase, Journal of Biol.Chem.,
247:3170-3171.
Lwu MW, Duncan AR, Okunji CO (1999).
New antimicrobials of plant origin.In
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Malick, C.P., and Singh, M.B. (1980) Plant
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Moron, M.S., De, P. J.N. and Manervik, V.
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Nair, R., Kalariya, T., Chanda, S., 2005.
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mg/ ml
Catalase
26.2 ± 0.05
8.2 ± 0.16
Glutathione S Transferase
Values are mean ± SD of triplicates.
Table 4: Non- Enzymatic Antioxidants Present in
Methanolic Stem Extract of Cardiospermum
halicacabum
Non Enzymatic
Antioxidants
Ascorbic acid
Flavonoids
Polyphenols
Reduced glutathione
Total carotenoids
mg/ml
0.726 ± 0.0208
0.53± 36.53
24.83 ± 0.62
0.0056 ± 0.00015
72.03± 0.03
Values are mean ± SD of triplicates.
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