BMR Microbiology
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Research Article
ANTIBACTERIAL EVALUATION AND PHYTOCHEMICAL
SCREENING OF LEAVES AND FRUITS OF Passiflora subpeltata
ORTEGA
1*
2
3
2
Lingaraju D. P. , Sudarshana M. S. , Poornachandra Rao K. , Mahendra C.
1
Department of Botany, AVK College for women, Hassan, Karnataka, India
2
Department of studies in Botany, University of Mysore, Mysore, Karnataka, India
3
Department of studies in Microbiology, University of Mysore, Karnataka, India
Correspondence should be addressed to Lingaraju D. P.
Received January 17, 2015; Accepted March 10, 2015; Published March 20, 2015;
Copyright: © 2015 Lingaraju D. P. et al. This is an open access article distributed under the Creative Commons
Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the
original work is properly cited.
Cite This Article: D. P., Lingaraju, M. S., Sudarshana, Rao K., P., C., Mahendra, (2015). Antibacterial evaluation and
phytochemical screening of leaves and fruits of passiflora subpeltata ortega. BMR Microbiology. 1(1), 1-6
ABSTRACT
The purpose of present work is to study antimicrobial activity and medicinally active principles present in different solvent
extracts obtained from leaves and fruits of Passiflora subpeltata.The active principles were isolated by Soxhlet extractor and
identified by preliminary phytochemical test. The Soxhlet extraction was performed using petroleum ether, chloroform,
ethyl acetate and methanol. The results of analyses of solvent extract confirmed the active substances were sterols,
glycosides, flavonoids, tannins. The antibacterialia tests of isolated substances were performed with pathogenic bacteria like
– Escherichia coli , Klebsiella pneumoniae, Enterobacter aerogenes, Staphylococcus aureus, and Enterobacter faecalis. The
observation of microbial test of methanol and ethyl acetate extract of both leaves and fruits supports to antibacterial activity
to the greater extent than petroleum ether, and chloroform extract. In this study, both Gram positive and negative bacteria
tested were found to be sensitive to methanol and ethyl acetate extract of both leaves and fruits. P.subpeltata revealed the
highest antibacterial activity at a minimum inhibitory concentration against E.aerogenes, S.aureus, and E.faecalis. These
findings suggest the need for further research of this herb and its products. The results provide justification for the use of the
P.subpeltata in folk medicine to treat various infectious diseases.
KEY WORDS: Passiflora subpeltata, Phytochemical screening, Secondary metabolites, Antibacterial activity.
INTRODUCTION
N
ature provides everything for the wellbeing of human
being. For achieving this nature protects man from various
diseases. She stores her magical power for curing diseases
in plants and various organic and inorganic compounds. Of
these plants play a major role. The only thing is that we
have to identify and apply them in the proper way. WHO
estimates that about 80% of the global population still
relies on plant based medicines for primary health care,
and a major part of this therapy involves the use of plant
extract or their active principles. This estimate still shows
an increase day to day.
Health-care, which was a part of the traditional culture of
the people, has become a professional in the modern
industrial world. Synthetic drugs created by the affluent
and influential pharmaceutical industries have given rise to
effects which are more dangerous than the diseases they
claim to cure. The world’s attention has again turned to
traditional medical systems. The medicinal properties of a
crude drug depend on the presence of one or more
chemical constituents of physiological importance. They
may be glycosides, alkaloids, tannin, resins or enzymes.
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Plants are rich reservoir of antimicrobials. A single plant is
known to contain several active principles of biological
significance. Plant base antimicrobials have enormous
therapeutic potential as they can serve the purpose with
lesser side effects that are often associated with synthetic
antimicrobials. Biomolecules of plant origin appear to be
one of the alternatives for the control of these antibiotic
resistant human pathogens. The last two decades have
witnessed increased investigation on plants as a source of
human disease management due to the genetic variability
of microorganisms against the antibiotics.
The
development of multidrug resistance in the pathogenic
bacteria parasites has created major clinical problems in
the treatment of infectious diseases. In recent times, there
has been increasing interest in the study of bioactive
compounds from peels, seeds, leaves, flowers and stem
bark due to their antioxidative, antimicrobial and other
health promoting properties [1].
In this study we are reporting the results of preliminary
phytochemical screening and antibacterial activity of
different solvent extracts of leaves and fruits of P.subpeltata
(Fam: Passifloraceae) in order to orient future
investigations towards the finding of new potent and antiinfectious compounds.
The passion flowers or passion vines (Passiflora) have a
genus of about 400 species of flowering plants and the
largest in the family of Passifloraceae [2][3]. Plants from
the genus Passiflora have been used in traditional medicine
by many cultures [4]. The genus Passiflora may be suitable
for the screening of bioactive molecules, since
ethnobotanical use, chemotaxonomic information, and
observation of the interaction of the plants with their
environment have been suggested as selection criteria for
potential sources of natural molecules of pharmacological
relevance [5][6]. Flavonoids, glycosides, alkaloids,
phenolic compounds and volatile constituents have been
reported as the major phyto-constituents of the Passiflora
species [7].
P.subpeltata (Syn. P. calcarata) or white passion flower, is
2
a type of climbing vine native to Brazil, Mexico, Central
America and more northern parts of South America but a
weed in New South Wales, Australia. Known to be
growing wild in California. It is also found as an escaped
ornamental to Zimbabwe and Africa. In India it is
distributed in southeren states mainly Karnataka
(Chikmagalur, Coorg, Hassan, Mysore dist.), Kerala
(Kottayam, Palakkad dist.) and Tamil Nadu (Dindigul,
Tiruvannamalai dist). This pale green climber has tendrils.
Leaves grow up to 10 cm long and are pale green and
usually three-lobed. The lobes have tips more or less
rounded. Between one and three glands are scattered along
the leaf stalk. Two leaf-like stipules occur where the leaf
stalk meets the stem. Flowers are white, tinged with green
and about 5 cm across. The fruit is oval-shaped and bluishgreen in color, a berry, about 4 cm long, spread by birds,
animal, water and humans. White passion flower will grow
over other vegetation and smother wanted plants and
shrubs. Their fruits and leaves are edible. In Western Ghats
of India, P. subpeltata leaves are widely consumed as a
green leafy vegetable [8]. In traditional medicine, the
leaves and fruits are used in the treatment of jaundice. A
coumarin derivative, cyanogenic glycosides- barterin have
been isolated in the P. subpeltata and the compound was
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found to possess sedative, anti-plasmodic and antimicrobial
properties [9][10]. Saravanan and Parimelazhaga reported
that the seeds of P. subpeltata have promising therapeutic
value for natural antioxidant and antimicrobial properties
[11]. They also reported that the acetone extract of P.
subpeltata leaves acquire substantial antioxidant,
analgesic, anti-inflammatory and antipyretic properties. No
reports are available revealing the antibacterial potential of
P. subpeltata leaves and fruits. Hence the present study
was undertaken.
MATERIALS AND METHODS
Collection of plant materials
P.subpeltata plants were collected from Aigoor village
(Somwarpet taluk, Kodagu district, Karnataka state),
identified using standard flora [12] and authenticated at the
Taxonomy Section of Department of studies in botany,
University of Mysore, Mysore. Fresh leaves and fruits of
P.subpeltata were used for the preparation of aqueous and
different organic solvent extracts.
Preparation of Solvent extracts
The leaves and fruits of P.subpeltata were separately
washed thoroughly 2-3 times with running water and once
with sterile water, chopped into small pieces, shade dried,
coarse powdered in a mechanical grinder, sieved and used
for extraction. The dried leaf and fruit materials (50gm)
were extracted separately with petroleum ether,
chloroform, ethyl acetate, and methanol in the increasing
order of their polarity by using Soxhlet apparatus [13]. The
extract was decanted, filtered with Whatman No. 1 filter
paper, concentrated and preserved at 5oC in air tight bottle
until further use. All the extracts were subjected to
antibacterial activity assay and preliminary phytochemical
analysis.
Test microorganisms
The test microorganisms used were Escherichia coli,
Klebsiella pneumonia, Enterobacter aerogenes (gramnegative), Staphylococcus aureus, and Enterobacter
faecalis (Gram- positive). All these microorganisms were
obtained from Herbal Drug Technology Laboratory,
Department of studies in Microbiology, University of
Mysore, Mysore, Karnataka.
Determination of antimicrobial activity
Antibacterial activity of the aqueous and organic extracts of
the plant sample was evaluated by the paper disc diffusion
method [14]. The bacterial cultures were adjusted to
0.5McFarland turbidiometric standard and inoculated onto
Nutrient agar plates (diameter: 15 cm) and incubated at 37
o
C for 18h.Sterile filter paper discs (diameter 6 mm)
impregnated with 100 μl of extract dilutions reconstituted
in minimum amount of solvent at concentrations of 20 to
100 mg/ml were applied over the culture plates. Paper
discs impregnated with 20 μl of a solution of 10 mg/ml of
streptomycin as standard antimicrobial was used for
comparison. Filter paper discs dipped into sterile distilled
water and allowed to dry were used as control. The plates
were then incubated at 37oC for 24 h. Antibacterial activity
was determined by measurement of zone of inhibition
BMR Microbiology
around each paper disc. For each extract three replicate
trials
were
conducted
against
the
test
organism.
Table 1: Results of Antibacterial activity of different solvent extracts of leaves and fruits of P. subpeltata by paper disc
diffusion method (zone of inhibition in mm)
Streptomycin
PE
CL
EA
MT
AQ
8 ± 2.121
8 ± 6.414
12 ± 2.828
10 ± 2.828
11 ± 1.212
15 ± 6.212
13 ± 4.121
12 ± 6.282
12 ± 2.121
13 ± 1.212
11 ± 1.414
8 ± 1.414
12 ± 2.828
9 ± 2.828
8 ± 1.414
FRUIT
E.coli
18±1.414
8 ± 2.121
6.4 ± 1.212
13 ± 1.414
K.pneumoniae
18 ± 1.414
11 ± 6.212
9 ± 1.414
14± 1.414
E.aerogenes
20 ± 2.828
15 ± 2.212
12 ± 1.414
8 ± 1.414
S.aureus
22 ± 2.828
12 ± 2.121
6 ± 2.828
12 ± 2.828
E. faecalis
20 ± 1.414
12± 2.121
5 ± 2.828
6 ± 2.212
PE- Petroleum ether CL- Chloroform, EA- Ethyl acetate, MT- Methanol, AQ- Aqueous
14 ± 1.412
15 ± 2.121
13 ± 2.121
8± 2.828
16 ± 2.121
12± 1.414
8 ± 1.414
11 ± 2.828
6 ± 1.414
13 ± 2.828
LEAVES
E.coli
K.pneumoniae
E.aerogenes
S.aureus
E. faecalis
18±1.414
18 ± 1.414
20 ± 2.828
22 ± 2.828
20 ± 1.414
9 ± 1.414
10 ± 2.828
13 ± 1.414
8 ± 1.414
15 ± 1.414
12 ± 2.121
10 ± 1.414
8 ± 1.414
14 ± 6.212
10.5 ± 2.121
Determination of minimum inhibitory concentration (MIC)
The minimum inhibitory concentration (MIC) of the methanol and ethyl acetate extracts was estimated for the test
organisms in triplicates, method described by Doughari et al [15]. To 0.5 ml of varying concentrations of the extracts (20.0,
18.0, 15.0, 10.0, 8.0, 5.0, 1.0 and 0.5 mg/ml), 2 ml of nutrient broth was added and then a loopful of the test organism
previously diluted to 0.5 McFarland turbidiometric standard was introduced to the tubes. The procedure was repeated on
the test organisms using the standard antibiotic -Streptomycin. A tube containing nutrient broth only was seeded with the
test organism as described above to serve as control. All the broth cultures were then incubated at 37 oC for 24 h. After
incubation the tubes were then examined for microbial growth by observing for turbidity. The lowest concentration showing
no turbidity in the tube was considered as the MIC.
Table 2: Minimum inhibitory concentration (in µg/ml) of ethyl acetate and methanol extracts of leaves and fruits of
P.subpeltata against selected bacteria
MIC(µg/mL)
Extract
Ethly acetate
Methanol
Ethyl acetate
Methanol
Streptomycin
Leaves
Fruits
Control
E.coli
2.5
2.0
2.5
2.0
2.0
K. pneumoniae
3.0
2.5
2.5
2.5
1.5
E.aerogenes
2.5
2.0
3.5
2.0
3.0
Preliminary phytochemical screening
The preliminary phytochemical tests were conducted to
find the presence of the active chemical constituents such
as alkaloids, glycosides, flavonoids, triterpenes, sterols and
tannins following method adapted by Harbone [16] and
Edeoga et al [17].
ii.
iii.
Alkaloids
The extract was dissolved in 1N Hcl, filtered and the
filtrate was used for alkaloid test.
i.
Mayer’s test (Potassium mercuric iodide
solution). The filtrate was treated with a few drops
S.aureus
2.5
2.0
3.5
2.0
2.5
E. faecalis
2.5
2.0
3.0
2.0
3.5
of Mayer’s reagent and observed for the presence
of yellow precipitation.
Dragendroff’s test (Potassium bismuth iodide
solution). The filtrate was treated with a few
drops of Dragendroff’s reagent and observed for
the presence of orange precipitation.
Wagner’s test (solution of iodine in Potassium
iodide). The filtrate was treated with a few drops
of Wagner’s reagent and observed for the presence
of reddish brown precipitation.
Terpenoids and sterols
i.
Salkowski’s test. The extract was treated with few
drops of chloroform and con. Sulphuric acid,
shaken well, allowed to stand, appearance of
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golden yellow colour in the lower layer indicated
the presence of triterpenoids and red colour in the
lower layer indicated sterols.
Libermann-Burchard test. The extract was
treated with a few drops of chloroform and acetic
anhydride and mixed well. 1 ml of con. Sulphuric
acid was added from the sides of the test tube.
Appearance of red ring at the junction of two
layers indicated the presence of triterpenoids.
Appearance of a brown ring at the junction of two
layers and the upper layer turned green indicated
the presence of sterols.
ii.
ii.
ferric chloride solution and then 1 ml of con.
Sulphuric acid was added from the sides of the
test tube. Formation of reddish brown ring at the
junction of two layers indicated the presence of
glycosides.
Molisch’s test. The extract was mixed with 1 ml
of Molisch’s reagent and to which 1 ml of con.
Sulphuric acid was added from the sides of the
test tube. Formation of reddish violet ring at the
junction of two layers indicated the presence of
glycosides.
Tannins
Flavonoids
i.
ii.
iii.
i.
Shinoda test. Extract was dissolved in methanol
and to this a small piece of magnesium ribbon was
added. 1 ml of con. Hcl was added from the sides
of the test tube. Formation of magenta colour
indicated the presence of flavonoids.
Lead acetate test. Extract was treated with a few
drops of 10% lead acetate. formation of yellow
precipitate indicated the presence of flavonoids.
Ferric chloride test. Extract was treated with a
few drops of 1% neutral ferric chloride solution.
Formation of blackish green colour indicated the
presence of flavonoids.
Glycosides
i.
ii.
Saponins
i.
ii.
Kellar Killani test. The extract was mixed with a
few drops of glacial acetic acid, boiled for a
minute and cooled. To this solution, 2 drop of
Gelatin test. To the extract, 1% gelatin containing
10% sodium chloride was added. Formation of
White precipitate indicated the presence of
tannins.
Ferric chloride test. The extract was mixed with
few drops of 1% neutral ferric chloride solution.
Formation of blue-green or brownish colour
indicated the presence of tannins.
Foam test. The extract was shaken vigorously
with little quantity of distilled water. The
formation and persistence of honey comb like
foam indicated the presence of saponins.
Haemolysis test. The extract was mixed with a
drop of blood and observed under the microscope.
Occurrence of haemolysis (destruction of RBCs)
indicated the presence of saponins.
Table 3: Qualitative preliminary phytochemical analysis of leaf and fruit extracts of P.subpeltata
Phytocontituents
Alkaloids
Glycosides
Flavonoids
Triterpenes
Sterols
Tannins
Saponins
4
Tests
Dragendroff’s test
PE
--
CL
--
EA
--
MN
--
AQ
--
Wagner’s test
--
--
--
--
--
Mayer’s test
--
--
--
--
--
Keller Killani test
Molisch’s test
Shinoda test
Ferric chloride test
Lead acetate test
Libermann-Burchard test
Salkowski’s test
Libermann-Burchard test
Salkowski’s test
Gelatin test
Ferric chloride test
Foam test
Haemolysis test
-------++
++
-----
-------++
++
-----
++
++
++
++
++
--++
++
++
++
++
++
++
++
++
++
++
--++
++
++
++
---
--++
++
++
----++
++
++
++
++ Present, -- Absent
PE- Petroleum ether, CL- Chloroform, EA-Ethyl acetate, MN- Methanol, AQ- Aqueous
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RESULTS AND DISCUSSION
Antibacterial activity
Antibacterial activity of the all solvent extracts is presented
in Table 1. All the extracts showed broad spectrum activity
against the bacterial strains used. The ethyl acetate and
methanol extract showed significant antibacterial activity
as compared to chloroform and petroleum ether extracts of
leaves. The methanol extract of leaves showed highest
inhibitory activity against E. coli (15 ± 6.212 mm),
K.pneumoniae (13 ± 4.121mm). The Pet.ether extract of
leaves showed highest inhibitory activity against
E.aerogenes (13 ± 1.414) and E. faecalis (15 ± 1.414). The
chloroform extract showed highest inhibition against
S.aureus (14 ± 6.212).
The ethyl acetate and methanol extract of fruits showed
significant antibacterial activity. The methanol extract of
leaves showed highest inhibitory activity against E. coli
(14 ± 1.412 mm), K.pneumoniae (15 ± 2.121mm) and
E.faecalis (16 ± 2.121mm). The Pet.ether extract of leaves
showed highest inhibitory activity against E.aerogenes (15
± 2.212) and ethyl acetate extract against S.aureus (12 ±
2.828).
Streptomycin (standard antibacterial drug ) have shown
inhibitory activity ranged from 18 ± 1.414 mm to 22 ±
2..828 mm at a concentration of 20 μl of a solution of 10
mg/ml. The sensitive test result, showed that the extracts
were less potent than the standard antibiotic-streptomycin,
used in the study. Generally, the reduced efficacy of the
extracts, relative to the standard antibiotic, used in the
study may be due to the fact that, they are still crude and
require further purification.
The result of MIC assay of methanol and ethyl acetate
extracts is shown in Table 2. Both extracts showed broadspectrum activity against the bacterial strains used.
Methanol extract of both leaves and fruits exhibited the
highest antibacterial efficacy against S.aureus, E.faecalis
and E.aerogenes each at 2.0µg/ml concentration when
compare to Streptomycin with MIC values of 2.5µg/ml,
3.5µg/ml and 3.0µg/ml respectively. The ethyl acetate
extracts of leaves showed a good bactericidal activity at
2.5µg/ml against E.aerogenes and E.faecalis when compare
to standard antibiotic with MIC values of 3.0 µg/ml and
3.5µg/ml respectively.
In this study, E.aerogenes,
S.aureus, and E.faecalis were found to be sensitive to
methanol and ethyl acetate extract of both leaves and fruits
of P.subpeltata.
Preliminary phytochemical analysis
Information on chemical constituents of plants helps for the
discovery of novel drugs.The qualitative phytochemical
analysis of leaves and fruits of P.subpeltata is depicted in
Table 1. It is clear from the experimental data presented
that the substances like sterols, glycosides, flavonoids, and
tannins are medicinally active components of leaves and
fruits of P. subpeltata. These bioactive components are
naturally occurring in most plant materials, known to be
bactericidal and fungicidal, thus conferring the
antimicrobial property to plants [18]. All solvent extracts
confirmed the presence of sterols. The ethyl acetate and
methanol extract confirmed the presence of glycosides,
flavonoids, sterols, tannins and saponins. But alkaloids and
triterpenes were absent in all solvent extracts.
Flavonoids are hydroxylated phenolic substances. The
biological functions of flavonoids include: protection
against allergies, inflammation, free radicals, platelet
aggregation, microbes, ulcers, hepatotoxin, viruses and
tumours [19][20]. Their activity is probably due to their
ability to complex with bacterial cell walls. More lipophilic
flavonoids may also disrupt microbial membranes [21].
They exhibit activity against a wide range of gram positive
bacteria as well as fungi [22]. Flavonoids are found in
ethyl acetate, methanol and aqueous extracts.
Phenolic compounds are reported to act as antimicrobial
activities [23]. Tannin is a general descriptive name for a
group of polymeric phenolic substances capable of tanning
leather or precipitating gelatin from solution. They are
found in almost every plant part: bark, wood, leaves, fruits
and roots [24]. Many human physiological activities, such
as stimulation of phagocytic cell, host mediated tumor
activity and wide range of anti-infective actions, have been
assigned to tannins [25]. Their mode of antimicrobial
action may be related to their ability to inactivate microbial
adhesions, enzymes, cell envelope, transport-proteins etc.
[26] Ethyl acetate and methanol extracts of P.subpeltata
showed highest phenolic content. The presence of the
phenolic compounds in these studied samples proved that
they have antimicrobial activity.
The results show that antibacterial potency of P.subpeltata
may be due to the presence of some active principles like
flavonoids, sterols, cardiac glycosides, tannins and
saponins. This result agrees with the report of Trease and
Evans [27].
CONCLUSION
P.subpeltata,
a weed, contains medicinally useful
phytochemicals, such as flavonoids, sterols, tannins,
saponins, and cardiac glycosides. These substances are
antibacterial and could be extracted for bacterial
management, pharmaceutical exploits, research in
microbiology, biotechnology and general medicine.
Bioactive substances from P.subpeltata plant could be
employed in the formulation of antibacterial agents for the
treatment of various bacterial infections.The methanol leaf
extract of P.subpeltata can be used as the active constituent
of antibacterial agents. The presence of antibacterial
activity in P.subpeltata plant extracts give support to their
traditional use for treating conditions associated with
microorganisms in humans and consequently seems to
fight against multi-resistant microbes.
ACKNOWLEDGEMENT
Grateful thanks goes to the Department of Studies in
Botany, University of Mysore for providing the facilities
for research work. The authors also thank the Management
and Principal of AVK College, Hassan, for their invaluable
constant support during the research work.
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