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Life Science Archives (LSA)

ISSN: 2454-1354
Volume 1; Issue - 3; Year 2015; Page: 161 - 165

Research Article

In vitro STUDIES ON INHIBITORY EFFECT OF Tragia involucrata AGAINST

METHICILLIN RESISTANT Staphylococcus aureus
E. Gowthami* and G. Sudha supriya,
PG and Research Department of Microbiology, Sri Akilandeswari Womens College, Wandiwash,
Tiruvannamalai District, Tamil Nadu, India.
Abstract
Ayurveda is a medical system primarily practiced in India that has been known for nearly 5000 years.
It includes diet and herbal remedies, while emphasizing the body, mind and spirit in disease prevention and
treatment. India is one of the 12 mega biodiversity centers having over 45,000 plant species. Nowadays,
higher plants are playing a prominent role in the treatment of innumerable diseases including cancer,
lymphosarcoms, AIDS, senile dementia and auto-immune diseases. Classically, higher plants are occupying
a key position in the production of new therapeutic agents. Thus, the plant drugs are able to occupy an
important niche in modern medicine. MRSA has now become established outside the hospital environment
and is appearing in community populations without identifiable risk factor. Methicillin Resistant
Staphylococcus aureus (MRSA) strains that cause such infections are called community acquired MRSA.
This present study aims to determine the antibacterial properties of Tragia involucrata extract against
MRSA. The leaves of Tragia involucrata has been tested against MRSA. Its ethanolic extract shown higher
activity in comparison with other fractions of Ethyle acetate and Hexane. This is an encouraging result in
regards to the ability of MRSA to be resistant to most antibiotics.
Article History
Received : 28.04.2015
Revised : 06.05.2015
Accepted : 08.05.2015
1. Introduction

Ayurveda is a medical system primarily

practiced in India that has been known for nearly
5000 years. It includes diet and herbal remedies,
while emphasizing the body, mind and spirit in
disease prevention and treatment (Morgan, 2002).
India is one of the 12 mega biodiversity centers
having over 45,000 plant species (Anonymous,
1998). Nowadays, higher plants are playing a
*Corresponding author: E. Gowthami,
PG and Research Department of Microbiology, Sri
Akilandeswari Womens College, Wandiwash, Tamil
Nadu, India.

Key words: Ayurveda, Tragia Involucrata,

Antibacterial properties and Methicillin Resistant
Staphylococcus aureus (MRSA).
prominent role in the treatment of innumerable
diseases including cancer, lymphosarcoms, AIDS,
senile dementia and auto-immune diseases.
Classically higher plants are occupying a key
position in the production of new therapeutic
agents. Thus, the plant drugs are able to occupy an
important niche in modern medicine.
The Staphylococcus aureus is a Gram
positive, spherical shaped bacteria (0.5 - 1.5 m in
diameter), which occur singly, in pairs or tetrad.
They are short chained with three to four cells
forming irregular clusters (Brock et al., 2001;
Adejuwon et al., 2010). The Staphylococcus genus
includes at least forty species. Most are harmless
and reside normally on the skin and mucous

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E. Gowthami / Life Science Archives (LSA), Volume 1, Issue 3, Page 161 - 165, 2015

membranes of humans and other organisms

(Harris et al., 2002). It may also be found in many
parts of our environment, including dust, water air
and faeces and on clothing or utensils.
Staphylococcus aureus is considered to be
a poor competitor in complex microbial
populations and is frequently inhibited or
overgrown
by
other
faster
growing
microorganisms (spoilage organisms) in foods.
Therefore, foods that present the greatest risk of
Staphylococcal food poisoning are those where the
normal microflora has previously been destroyed
(e.g. cooked products) or inhibited (e.g. foods that
contain a high concentration of salt) but that have
subsequently been contaminated by S. aureus
(Bremer et al., 2004).
MRSA is known to be one of the most
prevalent nosocomial pathogens throughout the
world to be capable of causing a wide range of
hospital-linked infection. Some strains of MRSA
have been designated epidemic strains; these are
associated with a higher prevalence and have been
shown to spread within hospitals, between
hospitals, and between countries (Humphrevs et
al., 1990, Aires De souse et al., 1998). MRSA has
now become established outside the hospital
environment and is appearing in community
populations without identifiable risk factor.
MRSA strains that cause such infections are called
community acquired MRSA (Hreold et al., 1998).
Thus, this present study was planned to determine
if Tragia involucrate extract has antibacterial
properties against MRSA.

162

2.3. Direct extraction

Direct extraction with hexane, ethyl acetate
and methanol was performed following the
method of Eloff (1998). In this method, finely
ground plant material was extracted with
chloroform, ethyl acetate and methanol in the ratio
of 1:10 in conical flask in shaking condition for
overnight. The extract was filtered through the
Whatman No. 1 filter paper in a separate
container. The process was repeated 3 times using
the same plant material and fresh solvent. The
solvent was removed by placing the extracts in
distillation unit in the respective temperature. The
extracted residues were weighed and re-dissolved
in different solvents to yield 10 mg/ml solutions
ready for further analysis.
2.4. Antibacterial activity
Antibacterial activity was performed by
Well diffusion assay (Eloff, 1998) using 4
different concentrations (250 g/ml, 500 g/ml,
750 g/ml and 1000 g/ml) of the plant extract,
and one negative control (DMSO).
2.5. Determination of Minimum Inhibitory
Concentration (MIC) (Broth dilution assay)
(Cos et al., 2006)
Broth Dilution assays is the standard
method used to compare the minimum inhibition
efficiency of the antimicrobial agents.
2.6. Detection of Phytochemical compounds
2.6.1. Detection of alkaloids

2. Materials and Methods

Detection of alkaloids was performed

using Mayers test

2.1. Plant collection

2.6.2. Detection of flavonoids

Fresh leaves of Tragia involucrata were

collected from the Podhigai hills, Western Ghats,
Tamil Nadu, India.

Three methods were used to test for the

detection of flavonoids. 0.5 g of extract was
dissolved in 5 ml of Distilled water and filtered.
Dilute ammonia (5 ml) was added to 1 ml of the
extract filtrate. Concentrated sulphuric acid (1 ml)
was added. Yellow colorations that disappear on
standing indicate the presence of flavonoids.

2.2. Preparation of Tragia involucrata plant

extract
The leaves were carefully washed with tap
water, rinsed with distilled water, and air-dried for
1 hour. Then leaves are separated and dried in
room temperature for one week. Then, they were
ground into powder and stored in room
temperature.

2.6.3. Detection of tannins

About 0.5 g of the extract was boiled in 10
ml of water in a test tube and then filtered. A few
drops of 0.1% ferric chloride was added and

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E. Gowthami / Life Science Archives (LSA), Volume 1, Issue 3, Page 161 - 165, 2015

observed for brownish green or a blue-black

coloration.
2.6.4. Detection of saponins
Detection of saponins was done by Foam

163

2.7.2. Estimation of Total flavonoid content

Estimation of Total flavonoid content was
determined using the Dowd method (Meda et al.,
2005).

test.

2.7.3. Thin layer chromatography

2.6.5. Detection of proteins

test.

Thin layer chromatography is used to

separate the compound present in the crude
extract.

2.6.6. Detection of glycosides

3. Results and Discussion

Detection of glycosides was done by

Borntragers test.

3.1. Extraction of plant material

Detection of proteins was done by Millons

2.6.7. Detection of reducing sugars

Detection of reducing sugars was done by
Fehlings test.
2.6.8. Detection of phenolic compounds
Detection of phenolic compounds was
done by Ferric chloride test.
2.7. Quantitative phytochemical analysis of T.
involucrata
2.7.1. Estimation of Total phenol content
Total phenol content in various solvent
extracts of leaves was determined by FolinCiocalteus reagent method (Mc Donald et al.,
2001).

The leaves of Tragia involucrata were

collected and as described earlier, extracts were
obtained.
3.2. Antibacterial activity - Well diffusion assay
The results obtained from well diffusion assay
suggest that the methanol extract of
T.
involucrata was efficient in inhibiting MRSA
strains with a maximum zone of inhibition (ZOI)
of 22 mm at a concentration of 1000 g/ml against
MRSA 1968. The ethyl acetate and hexane
extracts possessed less inhibitory action,
comparatively. Furthermore, the Minimum
Inhibitory Concentration (MIC) of methanol
extract of T. involucrata was studied to be
250 g/ml. Hence, the methanol extract was used
for further IC50 determination (Table 1).

Table 1: Effect of T. involucrata on MRSA strains

S.
No

Concentration
(g)
1

1.
2.
3.
4.
5.

250
500
750
1000
Std

9
10
25

Hexane
2
3
9
10
11
21 23

Zone of inhibition (mm)

Ethyl acetate
Methanol
4
1
2
3
4
1
2
3
9
15 15 17
10
10
17 17 18
11
11
9
19 18 20
13
12 11 23 20 21
19 21 21 22 19 25 23 25

4
16
17
19
21
25

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E. Gowthami / Life Science Archives (LSA), Volume 1, Issue 3, Page 161 - 165, 2015

3.3. Broth dilution assay

The data obtained from broth dilution
assay depicts that the methanol extract of
T. involucrata inhibited MRSA with IC50 value of
500 g/ml. Also, it was studied that the percentage
inhibition varied between 35 and 73% for
concentration range of 100 1000 g/ml (Table
2).
Table 2: IC50 of T. involucrata on MRSA
U1968
S.
No
1
2
3
4
5
6
7
8
9
10

Concentration
(g)
100
200
300
400
500
600
700
800
900
1000

The preliminary phytochemical screening

of methanol extract T. involucrata showed the
presence of alkaloids, terpenoids, glycosides,
flavonoids, tannins, saponin, reducing sugar
whereas the proteins were completely absent
(Table 3 and Table 4).
Table 3: Qualitative phytochemical screening
of methanol extract of T. involucrata
Results
++
++
++
+
+++
++
+
+++

S.
No

Phytochemical

Total Phenols

Total Flavonoids

Composition
(mg/ml)
25.271 GAE/g
97.32 QE/g

The thin layer chromatography was carried

out using the methanol extract of Tragia
involucrata was loaded on per-coated (silica). The
TLC plate which has ethyl acetate: hexane solvent
(1.8:0.2) as a solvent system. Spots were observed
under UV light and in iodine chamber. The result
revealed that the ratio 1.8:0.2 was significant in
separating compounds where presence of 6 major
compounds at the Rf values of 0.45, 0.34, 0.32,
0.31, 0.24 and 0.09.

Inhibition
(%)
35.826
37.381
38.527
43.219
49.692
55.346
62.93
66.84
69.18
73.34

Phytochemicals
Alkaloids
Terpenoids
Tannins
Saponins
Flavonoids
Proteins
Glycosides
Reducing Sugar
Phenols

Table 4: Quantitative estimation of

phytochemicals

3.4. Thin Layer Chromatography (TLC)

3.4. Phytochemical screening of crude extracts

of T. involucrata

S.No
1
2
3
4
5
6
7
8
9

164

Methicillin-resistant S. aureus (MRSA)

has become increasingly widespread as a major
cause of both nosocomial and community
infections. The recent increase in the methicillinresistant and multiple resistant strains at large
hospitals have started. Hence, an accurate and
rapid detection of methicillin resistance in
Staphylococci is therefore important, not only for
choosing the appropriate antibiotic therapy for the
individual patient, but also for the control of the
endemicity of the MRSA. This situation has
placed limits on our options to treat infections by
this organism. Glycopeptide derivatives, such as
vancomycin and teicoplanin, are now considered
to be agents of last resort for the treatment of
MRSA infections (Sucilathangam et al., 2012).
However, there are increasing numbers of reports
indicating the emergence of vancomycin-resistant
S. aureus (VRSA) strains exhibiting two different
resistance mechanisms. A similar study showed
inhibition zones ranging from 1117 mm by the
ethanolic extract of Quercus infectoria
demonstrated the largest inhibition zone. Similar
result was observed in a study conducted by
Chusri and Voravuthikunchai (2007).
The phytochemical profile of T.
involucrata is well corroborating with previous

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E. Gowthami / Life Science Archives (LSA), Volume 1, Issue 3, Page 161 - 165, 2015

literature and hence it can be derived that this

plant has bioactive compounds like Terpenoides,
Glycosides, Carbohydrate, Alkaloids, Phenolic
compound, Flavanoids, Saponins, and Tannins
form the characteristic nature for its medicinal
property (Satish et al., 2013).
4. Conclusion

165

1.Srikanth K, T Murugesan et al., 2002. Effect of

Trichodesma indicum extract on cough reflex
induced by sulphur dioxide in mice.
Phytomedicine, Volume 9, Issue 1, Pages 75-77.
2.Vedavathy. S., 2003. Scope and importance of
traditional medicine. Indian J Traditional
Knowledge 2(20), 236-241.

In conclusion, the results of the present

study reveal that the medicinal plant Tragia
involucrata could be considered an effective
source of Methicillin-resistant Staphylococcus
aureus (MRSA) therapeutics, however further
mechanistic studies are required to study the
bioactive components present in the medicinal
plant Tragia involucrata.
5. References
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and Olutiola PO, 2010. Antibiotics
resistance and susceptibility pattern of a
strain of Staphylococus aureus associated
with acne. International Journal of
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2) Asima Chatterjee, Satish Chandraprakash,
1997. The treatise of Indian medicinal
plants. New Delhi: Publications and
Information Directorate; 3(2): 120 170.
3) Harborne, J.B., 1984. Phytochemical
Methods: A Guide to Modern Techniques
of Plant Analysis, 2nd edition, Chapman
and Hall, London.
4) Heidanadia Zulkefli, Jamaludin Mohamad
& Nurhayati Zainal Abidin,2013.
Antioxidant Activity of Methanol Extract
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Tinospora
crispa
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Tabernaemontana corymbosa. Sains
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5) McDonald, S., Prenzler, P.D., Autolovich,
M., Robards K. (2001). Phenolic content
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6) Morgan, K., 2002. Medicine of the Gods:
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yurveda.htm].

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