ANTIBACTERIAL AND PHYTOCHEMICAL EVALUATION OF LEAF EXTRACT OF Microsorum punctatum (l.) Copel. TOWARDS BACTERIA INVOLVED IN CUTANEOUS DISEASES

Abstract

The work aimed to verify the antibacterial and phytochemical nature of the leaves of Microsorum punctatum. The leaves of the plant were especially evaluated for its antibacterial capability with respect to the bacteria involved in skin infections. Leaves of M. punctatum were checked for antibacterial potential and plant chemical constituents, especially in petroleum ether, acetone, ethanol and water in the manner of rising polarity. Antibacterial activity was confirmed by the standard disc diffusion experiment. Phytochemicals after being separated in silica gel Thin Layer Chromatography were identified by employing various standard spraying reagents. Pseudomonas aeruginosa was the most sensitive organism with respect to the extracts and therefore, its activity was further confirmed by minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) experiments. The phytochemicals obtained in acetone extracts of leaves the plant were active in terms of its antibacterial capability. No antibacterial activity was recorded for phytochemicals extracted in petroleum ether and water extracts. Detection of phenols, flavonoids, polyphenols and sterols in various extracts was noted in the experimentation. Presence of flavonoids, phenols, sterols and polyphenols in the ethanol extract of the plant accounted for its antibacterial activity. MIC and MBC values of 25 mg/ml and 50 mg/ml respectively were noted towards P. aeruginosa. Phytochemicals present in leaves of M. punctatum capable enough to react with the bacterium found in nosocomial infection.

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Toji Thomas: J. Pharm. Sci. Innov. 2018; 7(6)
228
Journal of Pharmaceutical and Scientific Innovation
www.jpsionline.com (ISSN : 2277 –4572)
Research Article
ANTIBACTERIAL AND PHYTOCHEMICAL EVALUATION OF LEAF EXTRACT OF Microsorum punctatum
(l.) Copel. TOWARDS BACTERIA INVOLVED IN CUTANEOUS DISEASES
Toji Thomas *
Post Graduate and Research Department of Botany, St. Thomas College Palai, Arunapuram P.O., Pala, Kerala, India
*Corresponding Author Email: tojidr@yahoo.com
DOI: 10.7897/2277-4572.076113
Received on: 15/11/18 Revised on: 25/12/18 Accepted on: 30/12/18
ABSTRACT
The work aimed to verify the antibacterial and phytochemical nature of the leaves of Microsorum punctatum. The leaves of the plant were especially
evaluated for its antibacterial capability with respect to the bacteria involved in skin infections. Leaves of M. punctatum were checked for antibacterial
potential and plant chemical constituents, especially in petroleum ether, acetone, ethanol and water in the manner of rising polarity. Antibacterial activity
was confirmed by the standard disc diffusion experiment. Phytochemicals after being separated in silica gel Thin Layer Chromatography were identified
by employing various standard spraying reagents. Pseudomonas aeruginosa was the most sensitive organism with respect to the extracts and therefore,
its activity was further confirmed by minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) experiments. The
phytochemicals obtained in acetone extracts of leaves the plant were active in terms of its antibacterial capability. No antibacterial activity was recorded
for phytochemicals extracted in petroleum ether and water extracts. Detection of phenols, flavonoids, polyphenols and sterols in various extracts was
noted in the experimentation. Presence of flavonoids, phenols, sterols and polyphenols in the ethanol extract of the plant accounted for its antibacterial
activity. MIC and MBC values of 25 mg/ml and 50 mg/ml respectively were noted towards P. aeruginosa. Phytochemicals present in leaves of M.
punctatum capable enough to react with the bacterium found in nosocomial infection.
Keywords: Microsorum punctatum, pteridophytes, fern, phytochemicals, disc diffusion, antibacterial activity
INTRODUCTION
Microsorum punctatum (L.) Copel. is a member of Pteridophyte
that belongs to the group fern. The plant is a common, medium
sized epiphytic herb, found in semi exposed as well as shaded
localities. This species is also cultivated as an ornamental plant.
M. punctatum belongs to Polypodiaceae, its synonym is
Acrostichum punctatum L.1. Medicinal uses of the plant include
its use as purgative, diuretic and wound healing2. Leaves of the
plant were utilized as medicine3,4. Wide spread application of
antibiotics in human being helped to develop new drug-resistant
bacteria. These types of drug-resistant bacteria are a larger
problem today5. Hence, people search for alternative natural
source of medicine to overcome the problem. Normally, human
beings depend on plants-based phytochemicals that can be
employed as a substitute medicine. Present study aimed to
evaluate the antibacterial capability and to detect the major
phytochemical compounds present in the plant, especially in
various solvents extracts of increasing polarity towards some
pathogenic bacteria observed in skin infections.
MATERIALS AND METHODS
Preparation of Plant Extract
Healthy specimens of M. punctatum were obtained in the month
of January from Pala, Kottayam District, Kerala. A voucher
specimen (TT 1572) was preserved and deposited at the
herbarium of St. Thomas College Palai. Leaves of the plant were
shade dried for three weeks and ground to fine powder by utilising
a mechanical grinder. The air-dried plant material (100g) was
used for preparing extracts. Soxhlet extractions or distillations
were successively performed in petroleum ether, acetone, ethanol
and water6 provided 0.37%, 3.4%, 4.1%, and 0.8% yield
respectively.
Bacterial Strains Used
The bacterial strains were procured from the culture collection of
Institute of Microbial Technology (IMTECH), Chandigarh.
These consist of Staphylococcus aureus subsp aureus (MTCC
96), Klebsiella pneumoniae subsp pneumoniae (MTCC-109),
Serratia marcescens (MTCC 6164), Pseudomomas aeruginosa
(MTCC 741) and Escherichia coli (MTCC 443). The bacteria
were later sub cultured on nutrient agar slants, further incubated
at 37oC for 12 hours and kept at 4oC in the refrigerator to maintain
the stock culture for future use.
In Vitro Antibacterial Assay
The standard procedure of disc diffusion method as explained by
Bauer et al. 7 was conducted to detect antibacterial activity. Petri-
dishes were sterilised before use and they were poured with sterile
liquid Mueller Hinton Agar media (pH 7.4  2). They were
allowed to solidify and the bacterial broth culture (1 ml broth of
approximately 105 CFU) was spread on the medium with a sterile
cotton plug needle in aseptic environment. Whatman No. 4 Filter
Paper was utilised to prepare sterile discs of 5-mm diameter. The
experimental control discs were also made with the original
solvents in which the extracts prepared. Phytochemicals were
then dissolved in the respective solvent to get a stock solution
with an accumulation of 150 mg/ml. Each disc was added with 10
L of the sample to receive an accumulation of about 1.5 mg/disc.
All the discs, including control were placed onto the medium only
after removing any trace of solvent from them. It was done by

Toji Thomas: J. Pharm. Sci. Innov. 2018; 7(6)
229
keeping it in a hot air oven set at 40°C until all the solvent
removed from the disc. The finished plates were inserted in
incubator, kept at 37oC for 24 hours to detect any inhibition zones.
More than three replicates of the experiments were conducted,
and average inhibitory zone diameter was recorded.
Minimum inhibitory Concentration (MIC)
The MIC of the extracts was done by placing various amounts
(400–0.78mg/ml) of the extract prepared in sterile distilled water
into an array of test tubes with the culture media8. To each test
tube 50 l of the bacterial broth suspension culture was added9.
The cultures with plant extracts were kept in an incubator set at
37oC for 24 hours. Positive controls were also prepared, and this
consisted of growth medium and each bacterium. The minimum
inhibitory concentration was noticed as the lowest concentration
of the extracts that could not permit any visible growth as
compared to that of the control tubes.
Minimum Bactericidal Concentration (MBC)
Culture tubes from MIC studies, which didn’t give any visible
growth after a period of incubation, were again sub-cultured onto
a freshly prepared nutrient medium10. The minimum bactericidal
concentration was observed as the lowest concentration of the
extract that did not produce a single colony on a nutrient agar
plate after 24 hours incubation.
Preliminary Detection of Phytochemicals
Silica gel Thin Layer Chromatography (TLC) was performed to
separate various phytochemicals in the crude solvent extract of
the plant. This was done in appropriate solvent system. Then, the
plates were sprayed with different spraying reagents as pointed
out by Harborne11 and Stahl12 to detect phytochemical
components like alkaloids, phenolics, flavonoids, Triterpenoids,
and sterols.
RESULTS AND DISCUSSION
Phytochemicals from the leaves of M. punctatum were extracted
by gradient separation protocol and therefore, this technique
helped to separate nonpolar phytochemicals came before polar
compounds. If we perform single exaction in a solvent,
consequently, it can yield a mixture of polar and non-polar
compounds in solvents like ethanol or methanol. Gradient
separation may be advantageous to separate different
phytochemicals depending on their polarity. Phytochemicals
separated in water distillate of M. punctatum, did not provide any
antibacterial capability with respect to experimented organisms.
Phytochemicals eluted in ethanol distillate of M. punctatum gave
the greatest antibacterial action towards Pseudomonas
aeruginosa. Moderate grade of action was established towards
the rest of experimented bacteria. The ethanol extract of the plant
showed insensitivity to Klebsiella pneumonia in relation to other
bacteria. Pseudomonas aeruginosa could show the highest
performance in terms of its antibacterial activity with respect to
ethanol extract of the plant (Table 1). No control discs could give
positive results of antibacterial action. Results of the
phytochemical experimentation of M. punctatum are shown in the
Table 2. It was evident from the experiments that the antibacterial
power of leaves of the plant distillate could not be matched with
the efficiency of standard antibiotics (Table 3).
Table 1: Antibacterial Action of M. punctatum
Name of plant
Extract used
Zone diameter (in millimetre)
Pseudomonas
aeruginosa
(MTCC-741)
Staphylococcus
aureus
(MTCC-96)
Klebsiella
pneumoniae
(MTCC-109)
Escherichia coli
(MTCC-443)
Serratia
marcescens
(MTCC-97)
M. punctatum
Petroleum ether
Acetone
Ethanol
Water
8.4 ± 0.45
-
14.3 ± 0.35
-
7.4 ± 0.58
7.2 ± 0.53
6.1 ± 0.55
-
-
-
-
-
-
-
7.6 ± 0.39
-
7.5 ± 0.44
7.8 ± 0.41
6.4 ± 0.45
-
Value = no obvious growth inhibition (-)
Table 2: Results of Phytochemical Evaluation of M. punctatum
Name of plant
Plant extracts
Test for Flavonoids
Test for Alkaloids
Test for Phenols
Test for Sterols, steroid,
phenol and poly phenol
M. punctatum
Petroleum ether
+
-
-
+
Acetone
-
-
+
+
Ethanol
+
-
+
+
Water
-
-
+
-
Value = ‘+’ : Present ‘–’ : Absent
Table 3: Antibacterial Action of standard antibiotics
Name of Antibiotic
(Con. 25g/Disc)
Zone diameter (in millimetre)
MTCC – 6164
MTCC – 96
MTCC – 741
Streptomycin
Amoxicillin
Chloramphenicol
25.6± 0.28
-
-
19.7± 0.46
24.5± 0.45
23.5± 0.35
21.4 ± 0.36
-
-
Value = no obvious growth inhibition

Toji Thomas: J. Pharm. Sci. Innov. 2018; 7(6)
230
The exaction technique initiated with non-polar solvent like
petroleum ether. The extract contained non-polar molecules and
these chemicals provided moderate level of antibacterial activity
with respect to ethanol distillate. Medium polar chemicals were
dissolved in acetone distillate and they showed more or less
similar intensity of antibacterial activity as petroleum ether
extract; while, ethanol distillate contained mostly polar
compounds and they gave better results of antibacterial action.
Ethanol distillate of leaves of M. punctatum provided good
antibacterial action to Pseudomonas aeruginosa, a gram-negative
bacterium. Ethanol distillate of leaves of the plant showed
Minimum Inhibitory Concentration (MIC) of 25 mg/ml and
Minimum Bactericidal Concentration (MBC) of 50 mg/ml against
P. aeruginosa. P. aeruginosa is invariably detected in nosocomial
bacterial contaminations and its infection is almost frequent in-
patients receiving medication of serious burns or other dreadful
skin injury and in patients affected with cystic fibrosis. This
pathogen can spread lungs of diseased individuals and accelerate
the death rate13. Almost entire polar phytochemical compounds
were carried away with ethanol distillate and there might be very
less phytochemicals compounds left after ethanolic distillation.
This would be one of the reasons for decline in antibacterial
action of water extract. Flavonoids were found in various extracts
of leaves of the plant. Not any of the extracts gave positive
indication of alkaloids. The present antibacterial analysis of the
plant reinforces the medicinal and ethnobotanical usefulness of
M. punctatum 2-4.
CONCLUSION
Ethanol distillate of leaves of M. punctatum exhibited
antibacterial action. Antibacterial activity detected as gradient
exaction was done. The ethanol distillate of leaves the plant gave
better result of action against P. aeruginosa. Water extracts of the
plant did not provide antibacterial action against the tested
bacteria. Phytochemicals like flavonoids, phenols, Sterols,
steroids, phenol and poly phenol were detected in ethanol extract
of the plant. The effective ethanol distillate demonstrated a
minimum inhibitory concentration of 25 mg/ml and minimum
bactericidal concentration of 50 mg/ml with respect to P.
aeruginosa.
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How to cite this article:
Toji Thomas. Antibacterial and phytochemical evaluation of leaf
extract of Microsorum punctatum (L.) Copel. towards bacteria
involved in cutaneous diseases. J Pharm Sci Innov.
2018;7(6):228-230.
http://dx.doi.org/10.7897/2277-4572.076113
Source of support: Nil, Conflict of interest: None Declared
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