j o u r n a l o f p h a r m a c y r e s e a r c h 6 ( 2 0 1 3 ) 2 8 0 e2 8 3

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Original Article

Phytochemical screening and in vitro antimicrobial activity of

Typha angustifolia Linn leaves extract
d
against pathogenic
gram negative micro organisms

d Kirankumar Shivsharanappa,
Ramesh L. Londonkar*, Umesh Madire Kattegouga,
Jayashree V. Hanchinalmath
Department of P.G. Studies and Research in BioTechnology, Gulbarga University, Jnana Ganga Campus, Sedam Road, Gulbarga 585106,
Karnataka, India

article info abstract

Article history: Background: Typha is a monocot genus of the monotypic family Typhaceae with about 12
Received 24 January 2013 species distributed in the tropical and temperate regions of the world in marshes and
Accepted 13 February 2013 wetlands of varied depth. It a common plant of wetlands, is an unexploited taxon which
Available online 13 March 2013 can be used as a good source of food, medicines, and, fibres.
Methods: In the present study phytochemical evaluation and antimicrobial potency of
Keywords: Typha angustifolia has been investigated against 4 g negative standard reference bacterial
Antimicrobial strains i.e., Enterobacter aerogenes (MTCC111), Salmonella typhimurium (MTCC98), Klebsiella
Gram negative bacteria pneumonia (MTCC 109), Pseudomonas aeruginosa (MTCC 424) and Escherichia coli (clinical
Phytochemical isolates). The antibacterial activity was determined by agar well diffusion method and
Typha angustifolia broth dilution methods were used to determine the minimum inhibitory concentration
(MIC) of the above strains.
Results: It is estimated that total ash value in leaves is 10.83%, acid insoluble ash and water
soluble ash shows the value 4.66% and 3.16% respectively. Preliminary phytochemical
screening of the aerial part of the T. angustifolia reveals the presence of different secondary
metabolites in these extracts. The diameter of the zones of inhibition of the extracts ranged
from 13 to 17 mm for E. aerogenes, 0e19 for S. typhimurium, 12e16 for K. pneumoniae, 9-19 for
P. aeruginosa and 9e20 mm for E coli. The minimum inhibitory concentration (MIC) of the
different extracts ranged from 0.78 mg/ml to 12.5 mg/ml.
Conclusion: The results obtained with methanol extract was particularly significant as it
strongly inhibited the growth of S. typhimurium, P. aeruginosa and E. coli. The overall results
of this study provided informative data for the use of T. angustifolia for the treatment of
infections associated with the studied micro organisms.
Copyright ª 2013, JPR Solutions; Published by Reed Elsevier India Pvt. Ltd. All rights
reserved.

* Corresponding author. Tel.: þ91 (0) 9880416391.

E-mail addresses: [email protected], [email protected] (R.L. Londonkar).
0974-6943/$ e see front matter Copyright ª 2013, JPR Solutions; Published by Reed Elsevier India Pvt. Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.jopr.2013.02.010
 j o u r n a l o f p h a r m a c y r e s e a r c h 6 ( 2 0 1 3 ) 2 8 0 e2 8 3 281

1. Introduction and stored in airtight container. The air dried leaf powders
(50 g) were successively extracted by soxhlet extraction with
Nature has been a source of medicinal agents since times im- solvents of increasing polarity i.e., petroleum ether (60e80  C),
memorial. Medicinal plants have been used for centuries as chloroform, methanol and distilled water. The extracts were
remedies for human diseases because they contain compo- dried and stored in a sterile container for further use.
nents of therapeutic value.1 It is estimated that there are about
250,000e500,000 species of plants are existing on Earth.2 The 2.2. Physicochemical analysis
traditional medicine still plays an important role in the pri-
mary health care in India. Approximately 60e80% of the The finely powdered leaves of T. angustifolia Linn was sub-
world’s population were relies on traditional medicines for the jected to various physicochemical studies for determination
treatment of common illnesses.3 Medicinal plants contain of ash value like total ash, acid insoluble ash and water soluble
large varieties of chemical substances which contain value ash.7 Extractive values like water soluble, methanol soluble,
added therapeutic properties that can be utilized in the treat- chloroform soluble and petroleum ether soluble were
ment of human diseases. The studies of medicinal plants used determined.
in folklore remedies have attracted the attention of many sci-
entists in finding solutions to the problems of multiple anti- 2.3. Phytochemical screening of crude extracts
biotics resistances organisms. Most of the synthetic antibiotics
now available in the market have major setback due to the The phytochemical components of the T. angustifolia leaves
multiple resistance developed by pathogenic micro organisms were screened for using the standard method described by
against these drugs. In addition to this problem, antibiotics are Harbone.8 The components analyzed are alkaloids, proteins,
sometimes associated with adverse effects on the host glycosides tannin, steroids, phenol, saponins, flavonoids,
including hypersensitivity, immune-suppression and allergic carbohydrates, oils and fats.
reactions. In present situation the development of microbial
resistance to antibiotics has lead the researchers to investigate 2.4. Source of micro organisms
the alternative source for treatment of resistant strains.4 Thus,
there is a need for search of new and more potent antimicrobial The micro organisms used for testing were Enterobacter aero-
compounds of natural origin to combat the activities of these genes (MTCC111), Salmonella typhimurium (MTCC 98), Klebsiella
pathogens which is the basis for this study. pneumonia (MTCC 109), Pseudomonas aeruginosa(MTCC 424),
Typha angustifolia are herbaceous, colonial, rhizomatous, Escherichia coli (Clinical strain). The above organisms were
perennial plant with long, slender, green stalks topped with obtained from the department of Microbiology and Biotech-
brown, fluffy, sausage-shaped flowering heads. It is a peren- nology, Gulbarga University, Gulbarga, Karnataka, India.
nial growing up to 3 m (9ft) often forming extensive colonies
along shores of shallow ponds, lakes and marshes. The results 2.5. Standardization of micro organisms
of Varpe SS reveals that the aqueous and 70% methanol ex-
tracts of T. angustifolia pollen grains exhibits anti- 200 ml of overnight cultures of each micro organisms was
inflammatory activity.5 In the present situation it has been dispensed into 20 ml of sterilized nutrient broth and incubated
proposed that Typha could be utilized as a biomass crop for at 37  C for 4e6 h to standardize the culture to 106 CFU/ml. A
renewable energy.6 The present study aimed to screen for loopful of the standard cultures was used for the antimicrobial
phytochemicals and to evaluate antibacterial activity of crude assay.9
extracts of T. angustifolia leaf and to find out minimum
inhibitory concentration (MIC) of different extracts against 2.6. Screening for antibacterial activity (agar well
Garm negative bacteria. diffusion assay)

In vitro antibacterial activities of all different extracts of T.

2. Materials and methods angustifolia were determined by standard agar well diffusion
assay.10 MullereHinton Agar (MHA) plates were seeded with
2.1. Collection of plant materials and extraction 18 h old culture of the isolates. Different extracts were dis-
solved in 1% Tween 80 in deionized water and made the final
Aerial part (leaves) of T .angustifolia was collected in and concentration of 50 mg/ml, from this 50 ml of different extracts
around the Gulbarga, Karnataka, India in the month of were added into the sterile 6 mm diameter well. 1% Tween 80
January 2012 and the plant was duly identified and authenti- and sterilized distilled water were used as negative controls
cated in the Herbarium of the Department of Post Graduates while chloramphenicol antibiotic disc (30 mcg, Hi-Media) was
Studies and Research in Botany, Gulbarga University, Gul- used as positive control. A loopful each of the standardized
barga, Karnataka, India. culture of test organisms was streaked on the solidified me-
The collected leaves were washed with running tap water dium and incubated for 24 h at 37  C. Antibacterial activity was
and allowed to air dry. The plant materials were dried in shade assayed by measuring the diameter of the zone of inhibition
for two to four weeks. Precaution was taken to avoid direct formed around the well using standard (Hi-Media) scale. The
sun light otherwise it will destroy the active compounds of experiment done in triplicate and the average values were
plant leaves. After drying, the plant leaves were grinded finely calculated for antibacterial activity.
282 j o u r n a l o f p h a r m a c y r e s e a r c h 6 ( 2 0 1 3 ) 2 8 0 e2 8 3

visible growth can be considered as the minimum inhibitory

concentration.

3. Results

It is estimated that total ash value in leaves is 10.83%, acid

insoluble ash and water soluble ash shows the value 4.66% and
3.16% respectively. The extractive value of methanol is more
followed by aqueous, chloroform and petroleum ether with
20.12%, 6.98%, 4.36% and 2.14% respectively. Phytochemical
screening of crude extracts of the aerial part of the T. angusti-
Fig. 1 e Effect of different extracts of Typha angustifolia
folia reveals the presence of alkaloids, tannin, steroids, phenol,
leaves on five different gram negative bacterial strains.
saponins, flavonoids in aqueous and methanolic extracts
Dve Control represents the chloramphenicol antibiotic disc
where as carbohydrates, tannins, oils and fats were present in
(30 mcg, Hi-Media). Dve Control represents 1% tween 80 in
Petroleum ether and chloroform extract. In addition to this
sterilized distilled water. The methanol and aqueous
chloroform extract also contains flavonoids and phenols.
extract of T angustifolia shows the maximum zone of
The antimicrobial activity of different extracts against the
inhibition compared to chloroform extract.
test organisms with varying zones of inhibition ranging from
09 to 20 mm (Fig. 1) has revealed the antimicrobial potency of
this plant. Methanolic extract showed highest zone of inhi-
2.7. Determination of minimum inhibitory concentration bition against E. coli (20 mm) followed by P aeruginosa, S typhi-
murium, E aerogenes and K pneumonia. The aqueous extract
Minimum inhibitory concentration (MIC) was defined as the showed greater potential against E. coli > E. aerogenes > P.
lowest concentration where no visible turbidity was observed aeruginosa > K. pneumonia > S. typhimurium. Chloroform
in the test tubes. The concentrations were determined by the extract shows moderate inhibitory effect on these organisms.
method described by Vollekova11 with minor modification The result of MIC assay is shown in Table 1 Methanol extract
was employed. The MIC was determined for the micro or- of T. angustifolia exhibited the highest antibacterial efficacy
ganisms that showed maximum sensitivity to the test ex- against E. coli at 0.78 mg/ml and least efficacy was shown by
tracts. In this method the broth dilution technique was used, chloroform against S. typhimurium, P. aeruginosa and E. coli
where the leaf extract was prepared to the highest concen- which was inhibited at 12.5 mg/ml concentration.
tration of 25 mg/ml (stock concentration). By adding sterile
distilled water serially diluted (two fold dilutions) using the
nutrient broth and it is later inoculated with 0.2 ml stan- 4. Discussion
dardized suspension of the test organisms. After 18 h of in-
cubation at 37  C, the test tubes were observed for turbidity Plants are important source of potentially bioactive constitu-
.The lowest concentration of the tube that did not show any ents for the development of new chemotherapeutic agents. It

Table 1 e Minimum inhibitory concentration (Mic in mg/ml) of plant extracts on different bacterial strains.
Organisms Concentration (mg/ml)

EXT 0.098 0.195 0.39 0.78 1.563 3.125 6.25 12.50 25.00

Enterobacter aerogenes CE þ þ þ þ þ þ ***  

ME þ þ þ þ þ ***   
AE þ þ þ þ þ ***   
Salmonella typhimurium CE þ þ þ þ þ   *** 
ME þ þ þ þ ***    
AE þ þ þ þ þ þ ***  
Klebsiella pneumonia CE þ þ þ þ þ þ ***  
ME þ þ þ þ þ ***   
AE þ þ þ þ þ ***   
Pseudomonas aeruginosa CE þ þ þ þ þ þ þ *** e
ME þ þ þ þ ***    
AE þ þ þ þ þ ***   
E. coli CE þ þ þ þ þ þ þ *** e
ME þ þ þ ***     
AE þ þ þ þ ***    

CE is a chloroform extract, ME is a methanol extract, AE is an aqueous extract þ indicates turbidity is observed,  indicates turbidity is not
observed, *** represents the MIC value. Methanol extract significantly inhibit the growth of micro organisms at a low concentration followed by
aqueous extract and chloroform extract is least.
 j o u r n a l o f p h a r m a c y r e s e a r c h 6 ( 2 0 1 3 ) 2 8 0 e2 8 3 283

has been well documented that the antimicrobial compound carrying out this work. The author (M.K. Umesh) acknowl-
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The authors gratefully acknowledge the financial support screening and antimicrobial investigation of Typha
from the University Grant Commission (UGC), New Delhi for angustifolia Linn. Int J Chem Sci. 2009;7(3):1905e1910.