Antimicrobial and Antioxidant Activity of Leaf Extracts of Two Indigenous Angiosperm Species of ...

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Int.J.Curr.Microbiol.App.Sci (2015) 4(8): 643-655
ISSN: 2319-7706 Volume 4 Number 8 (2015) pp. 643-655
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Original Research Article
Antimicrobial and Antioxidant Activity of Leaf Extracts of Two
Indigenous Angiosperm Species of Tripura
Sunrit Basu Sarbadhikary1, Somnath Bhowmik2, Badal K Datta2 and Narayan C Mandal1*
1Mycology and Plant Pathology Laboratory, Department of Botany, Visva-Bharati,
Santiniketan 731235, West Bengal, India
2Department of Botany, Tripura University, Suryamani Nagar, Agartala, Tripura, India
*Corresponding author
A B S T R A C T
Antimicrobial study of plant extracts are done for determining the ability of plant
parts to kill or inhibit the growth of living pathogenic microorganisms by their
extractable metabolic power. Antioxidant study is done for observing the ability of
any sample that can scavenge the free radicals. In to-days stressful life role of
antioxidants from natural sources are very important to combat them. In this study
leaves of some indigenous angiosperm species were collected from Tripura and the
Keywords
ethanolic extracts were prepared to check their antimicrobial potential against a
number of pathogenic organisms. The antibacterial study was done using the leaf
Antimicrobial,
extracts of Schima wallichii, Phlogocanthus thyrsiflorus, Cissampelos pareira,
Antioxidant,
Smilax zeylanica, Hydnocarpus kurzii, and Melastoma malabathricum. Schima
Leaf extract,
wallichii and Melastoma malabathricum showed the significant activities. Both the
Schima
leaf extracts produced zones of inhibition in the range of 15mm to 20mm against
wallichii,
the bacteria used in the antibacterial study. The MIC value of the crude extracts of
Melastoma
Schima wallichii and Melastoma malabathricum were found to be 150µg/ml for
malabathricum,
one Gram positive and one Gram negative bacteria i.e. Staphylococcus aureus and
Indigenous
Escherichia coli respectively. The extracts showed bacteriostatic mode of action
plants
against both Staphylococcus aureus and Escherichia coli. Further, the antifungal
and the antioxidant activities were checked for these two indigenous angiosperms
and the results were remarkable. In Antifungal study both the leaf extracts
produced zone of inhibition ranging from 10 to 20 mm against the fungi used. In
antioxidant assay IC of Schima wallichii and Melastoma malabathricum were
50
found to be 14 g/ml and 16.5 g/ml respectively in comparison to the standard
control ascorbic acid where the value was found 8.5 g/ml.
Introduction
In many developing countries bacteria has Since the industrially made antibiotics are
become a big threat for mankind. Due to its becoming resistant by many bacteria and are
harmful effects many people are suffering of very high cost it is not possible for the
enormously as a consequence of infections. f o lk people to afford this. Now medicinal
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Int.J.Curr.Microbiol.App.Sci (2015) 4(8): 643-655
plants are used by the folk people as an a wide variety of secondary metabolites such
alternative source of medicine which is very as tannins, terpenoids, alkaloids and
economic to them (Jose et al., 2011). So in flavonoids which have been found in-vitro
this study the potential of some indigenous to have antimicrobial properties. The leaf
angiosperm against some human pathogenic extract of different plants have been used in
bacteria are studied and to show the path the present work to check their antibacterial
that they can be used in the treatment of effectiveness against animal bacterial
various infectious diseases caused by pathogens were Phlogocanthus thyrsiflorus,
pathogenic bacteria. Microbial Pathogens Schima wallichii, Smilax zeylanica,
such as Escherichia coli, Staphylococcus Hydnocarpus kurzii, Cissampelos pareira,
aureus, Salmonella typhimurium, Melastoma malabathricum.
Pseudomonas aeruginosa, Bacillus subtilis,
Listeria monocytogenes, Pantoea ananatis Little account of antimicrobial properties of
are widely distributed in nature responsible these plants are described below:
for mortality and morbidity in the
population. It has been reported that there Phlogocanthus thyrsiflorus Nees.
are many cases of human Salmonellosis
annually with 3 million deaths worldwide. Due to the presence of phytochemicals like
E.coli has been isolated from various saponins, diterpene lactone, flavonoids in
environments and is reported to cause the leaves the plant has medicinal value. It is
several deaths. Staphylococcus aureus effective in Whooping cough, fever etc. Leaf
causes a variety of suppurative (pus- part is beneficial in liver and spleen
forming) infections and more serious diseases. It has prominent free radical
infections such as pneumonia, mastitis, scavenging property so it may prove as a
meningitis, and urinary tract infections. S. very good medicinal herb (Upadhyay,
aureus is a major cause of hospital acquired 2009). It has antimicrobial activity also
infection of surgical wounds and infections (Singh and Singh, 2010).
associated with indwelling medical devices.
E.coli, B.subtilis are well known to causal Schima wallichii (DC.) Korth.
agents of food poisoning.
Schima wallichii has anti-cancer activities,
Tripura a state of India ( 56 to and have the ability to induce apoptotic
32 N latitude and 09 to 20 E mechanisms (Diantini et al., 2012). Several
longitude) encompasses the total area of phytoconstituents are present in this plant
10,498 sq km. 6292 sq km of the state is like flavonoids, alkaloids and triterpenoids
covered by the forest. Due to the suitable and shows antimicrobial activity against
annual rainfall and the temperature, Tripura wide range of microorganisms (Saikat et al.,
has a great plant bio-diversity. A large 2008).
number of ethnic people or tribe inhabited in
this part of India (Awadh et al., 2004). Some research on this plant reported that
There are several tribal people like Tripuri, ethanol extract of leaves has cytotoxic
Reang, Jamatia, Halam, Munda, Santhal, activity to leukemia cancer cells. Ethyl
Chakma, Bhutia, Lepcha, and Magh. They acetate fraction of leaves has cytotoxic and
have a vast knowledge of using plants, and pro-apoptosis activity to leukemia and breast
plant products for curing diseases in ethnic cancer cells leukemia.
mode (Toledo et al., 2009). Plants are rich in
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Smilax zeylanica L. M a t e r i a l s a n d M e t h o d s
Phytochemical analysis of the plant extract Collection of plant materials
revealed the presence of reducing sugars,
tannins, saponins, gums, steroids, alkaloids, A thorough survey was done on indigenous
and flavonoids. Traditionally, roots and angiosperm flora of Tripura near Udaipur
leaves of S. zeylancia are used as a area. Based on the survey we have selected
substitute for the official drug, Sarasparilla, seven plants and collected them carefully
in the treatment of venereal diseases; and were taken to the Mycology-Plant
decoction is applied for rheumatism, pain in Pathology laboratory Department of Botany,
the lower extremities, sores swellings, and Visva Bharati. Voucher specimens were
abscesses, and also used in the treatment of kept in the herbarium of Department of
dysentery. Leaves and roots contain Botany, Tripura University. The plant
diosgenin (Kar and Sen, 1984). members are as following: Schima wallichii
(Theaceae), Phlogacanthus thyrsiflorus
Hydnocarpus kurzii (King.) Warb. (Acanthaceae), Cissampelos pareira
(Menispermaceae), Smilax zeylanica
The seed oil is known as hydnocarpus oil, (Smilacaceae), Melastoma malabathricum
leprosy oil. The active principles of the oil (Melastomataceae), Hydnocarpus kurzii
are hydnocarpic and chaulmoogric acids (Achariaceae), Lepidagathis incurve
which are strongly antibacterial. It also (Acanthaceae). Fresh leaves were washed
shows the thrombolytic activity (Sikder et thoroughly 2-3 times with running tap water
al., 2011). and then with sterile water followed by
shade dried, powdered and used for
Cissampelos pareira L. extraction.
The phytochemical tests demonstrated the Solvent extraction of dried leaf
presence of phytochemicals like alkaloids,
flavonoids, tannins, terpenoids in root 2 grams of shade dried, powder of leaf of
extract (Ngoci et al., 2014). Cissampelos each plant materials were filled separately in
pareira root shows protective nature against the conical flask and extracted successively
gastric cancer. with 50 ml of ethanol. Then they were kept
in the rotary shaker for 36 hours at 28oC at
100 rpm. After shaking the contents of the
Melastoma malabathricum L.
conical flask were taken in the centrifuge
tube. Then the materials were centrifuged in
The phytochemical constituents of the dried
cooling centrifuge at 10,000rpm for 10
leaf extract contain flavonoids, triterpenes,
minutes. Supernatant from respective tube
tannins, saponins and steroids, but not
were taken and poured in the petridishes and
alkaloids. Based on the HPLC analysis
allowed to evaporate under the aseptic
several flavonoids, namely quercetin,
condition. Petridishes were kept open in the
quercitrin and rutin were found in methanol
open air for two days for evaporation of the
extract. Methanol extract of Melastoma
alcohol. After the reduction of volume it was
malabathricum leaves exerted antioxidant
passed through Cellulose Acetate membrane
and liver protective activity in rats (Siti et
filter (0.22µ pore size) and put inside
al., 2013).
laminar air flow for complete evaporation.
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Int.J.Curr.Microbiol.App.Sci (2015) 4(8): 643-655
Each of these solvent extract was weighed and Melastoma malabathricum showed a
and dissolved in 2 ml DMSO. Finally they great antibacterial activity, MIC study of
were preserved at C in airtight bottles for these two extracts was studied against one
further use. Gram positive and one Gram negative
bacteria i.e. Staphylococcus aureus and
Antibacterial effect of the leaf extract Escherichia coli respectively. The MIC was
determined by counting the Colony Forming
Pathogenic bacteria such as Escherichia coli Units (CFU). Different concentration of
MTCC1667, Staphylococcus aureus these plant extracts including 25,
MTCC96, Salmonella typhimurium 50,100,150,200 µg/ml were used in this
MTCC98, Pseudomonas aeruginosa study. The ethanolic fraction of these plant
MTCC741, Bacillus subtilis MTCC121, materials were dissolved in DMSO and
Listeria monocytogenes MTCC657, and added to the Nutrient Broth in different
Pantoea ananatis MTCC2307 were used in concentration. After adding of fixed volume
the present study. These bacterial strains of bacterial culture, culture tubes were
were treated with the leaf extracts of incubated at 37ºC for overnight. Then 100 µl
Phlogocanthus thyrsiflorus, Schima of cultures were taken from each tube and
wallichii, Smilax zeylanica, Hydnocarpus spread on Nutrient Agar plates using glass
kurzii, Cissampelos pareira, Melastoma rod spreader after proper dilution. Then all
malabathricum. The assay for antibacterial the plates were incubated at 37ºC for
activity of each plant leaf extract was tested overnight. On the next day CFU were
by disc diffusion method (Kirby et al., counted and CFU/ml was calculated. MIC
1966). Bacterial suspensions (1.5× 10 ) value for each plant extract was also
were inoculated on Nutrient Agar plates calculated.
evenly using sterile swab. The Whatman No.
1 filter paper was used to prepare the paper Mode of action of the plant extracts
disc (6mm in diameter). Then the discs
impregnated with the leaf extracts were To determine the mode of action of the plant
placed individually on the Nutrient Agar extracts, time killed study with the leaf
plates with flamed forceps. A standard extracts of Schima wallichii and Melastoma
commercial disc of Ciprofloxacin 10 µg/ml malabathricum was performed against
was used as positive control and DMSO Staphylococcus aureus and Escherichia coli
impregnated disc was used as negative respectively. For this purpose the leaf
control in each case. The plates were extracts were added to the actively growing
incubated at 37 C for 24 hours in inverted liquid cultures of the bacteria at their
position. Zones of inhibition produced by minimum inhibitory concentration found
the sensitive organisms were observed by a previously. Activities of the leaf extracts in
clear circular area around the discs and were relation to time were measured by colony
compared with the zone of inhibition count method (Ray et al., 1999).
produced by the positive control
(Ciprofloxacin 10 µg/ml) and the negative Antifungal activity of the plant extracts
control (DMSO impregnated disc).
The ethanolic leaf extracts of Schima
MIC Study wallichii and Melastoma malabathricum
were prepared and their antifungal activity
Since the leaf extract of Schima wallichii was tested against Helminthosporium oryzae
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MTCC351, Alternaria alternata VBAV007, Percentage of inhibition of DPPH activity
Fusarium oxysporum MTCC2480, (%) = A-B/A×100
Colletotrichum acutatum MTCC281,
Candida albicans MTCC1644, Aspergillus Where A = optical density of the blank and
parasiticus MTCC2796 procured from B = optical density of the sample.
IMTech, Chandigarh. The assay for
antifungal activity of each plant leaf extract Results and Discussion
was tested by agar well diffusion method
(Fernandez-Garayzabal et al., 1992). Fungal Antibacterial effect of the leaf extract
suspensions were cultured in Malt Extract
Broth for 1-3 days depending on their All the bacteria were sensitive to the
growth pattern. 100µl of these cultures were positive control and resistant to the negative
spread on Malt Extract Agar in petri dishes. control. All the bacteria were also found
Then the wells were prepared on the petri sensitive to the leaf extracts of Schima
dishes with the help of the cork borer. In wallichii and Melastoma malabathricum.
each well 50µl of leaf extracts were poured. Both Schima wallichii and Melastoma
Then the plates were kept in incubation at malabathricum showed the maximum
28ºC for 1 to 3 days. The fungi which activity against E.coli, Pseudomonas
showed clear zone were considered sensitive aeuruginosa, Staphylococcus aureus and
and those not as resistant. Listeria monocytogens. Hydnocarpus kurzii
also exhibited good activity against Listeria
Antioxidant activity (DPPH free radical monocytogens and moderate activity against
scavenging activity) of the plant extracts E.coli. Phlogocanthus thyrsiflorus and
Smilax zeylanica showed morderate
Antioxidant activity of the plant extracts was potential against Bacillus subtilis and E.coli
done on the basis of the radical scavenging respectively. The measurement of inhibition
effect of the stable DPPH (2 2-diphenyl-2- zones (in mm) produced by the leaf extracts
picrilhydrazyl) radical (Braca et al., 2002). and the positive control are presented in the
The solutions of the plant extracts were Table 1 and Figures 1 and 2. From the above
prepared in methanol as 0.01 gm of plant study it was observed that leaf extracts of
extracts were dissolved in 1 ml of methanol. Schima wallichii and Melastoma
0.004% of DPPH was prepared. Different malabathricum were most effective against
concentrations from 5 to 50 µg/ml were all the selected microorganisms.
prepared by mixing the DPPH stock solution
and respective amount of the methanolic Minimum Inhibitory Concentration
solution of the plant extracts. Ascorbic acid (MIC) of the plant extracts
was used as standard in the same
concentrations. These solution mixtures The Minimum Inhibitory Concentration
were kept in dark for 30 min and optical (MIC) of the leaf extracts against
density was measured at 517 nm using Staphylococcus aureus and Escherichia coli
Spectrophotometer. Methanol with DPPH were determined by counting the Colony
solution (0.004%) was used as blank. The Forming Units (CFU). The MIC values of
optical density was recorded and % both Schima wallichii and Melastoma
inhibition was calculated using the formula malabathricum against both the bacteria
given below: were found to be 150µg/ml (Table 3 and 4).
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Table.1 Zone of inhibitions (in mm) as shown by ethanolic extracts (50mg/ml) of selected plant
species of Tripura against bacteria
E. coli Bacillus Pantoea Salmonella Pseudomonas Staphylococcus Listeria
subtilis ananatis typhimurium aeruginosa aureus monocytogens
Phlogocanthus -- 8 -- -- -- -- --
Thyrsiflorus
Schima 20 17 17 8 19 19 18
Wallichii
Smilax 10 -- -- -- -- -- --
Zeylanica
Hydnocarpus 8 -- -- -- -- -- 18
Kurzii
Cissampelos -- -- -- -- -- -- --
Pareira
Melastoma 20 18 17 17 18 18 20
malabathricum
Ciprofloxacin 20 20 20 20 21 21 20
(10µg/ml)
Table.3 MIC of Schima wallichii against Staphylococcus aureus and Escherichia coli
CFU/ml
Staphylococcus aureus Escherichia coli
Concentration (µg/ml)
Control 1.2× 10
3.3× 10
25 1.5× 108 6.0× 108
50 1.2× 108 1.5× 108
100 1.0× 108 1.2× 107
150 2.0× 106 8.3× 105
200 1.5× 104 3.1× 103
Table.4 MIC of Melastoma malabathricum against Staphylococcus aureus and Escherichia coli
CFU/ml
Staphylococcus aureus Escherichia coli
Concentration (µg/ml)
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Control 1.0× 10
2.0× 10
25 1.8× 108 3.0× 108
50 1.5× 108 2.5× 108
100 1.2× 108 1.6× 107
150 1.0× 106 4.2× 105
200 3.0× 103 1.2× 103
Figure.1 Antibacterial effects of the leaf extracts (50mg/ml)
25
20
)
m E. coli
m
(
n B. subtilis
o 15
i
t
bi P.ananatis
hi
n
f i 10 S.typhimurium
o
e P. aeruginosa
n
o
Z 5
S.aureus
L.monocytogenes
0
LEPT LESW LESZ LEHK LECP LEMM CIPRO
Leaf extracts
Figure.2 Antibacterial effects of the leaf extracts (25mg/ml)
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Figure.3 Mode of action of leaf extracts of Schima wallichii on Staphylococcus aureus
Figure.4 Mode of action of leaf extracts of Schima wallichii on Escherichia coli
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Figure.5 Mode of action of leaf extracts of Melastoma malabathricum on Staphylococcus aureus
Figure.6 Mode of action of leaf extracts of Melastoma malabathricum on Escherichia coli
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Figure.7 Antifungal activity of the leaf extracts (50mg/ml)
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