Evaluation of Antimicrobial Activity of Ipomoea fistulosa, Furcreae foetida and Barleria mysorensis: An in vitro study

 

Akash Mazumdar, Neelam Gurung, Sumit Phulera, Jinsu Mathew, Gaurav Kumar, Loganathan Karthik, Kokati Venkata Bhaskara Rao*

Molecular and Microbiology Research Laboratory, Environmental Biotechnology Division,

School of Bio Sciences and Technology, VIT University, Vellore, Tamil Nadu - 632 014, India

*Corresponding Author E-mail: kokatibhaskar@yahoo.co.in

ABSTRACT:

The aim of this study was to screen the aqueous extract of leaves of Ipomoea fistulosa (Convolvulaceae), Furcraea foetida (Agavaceae), and Barleria mysorensis (Acanthaceae) for antimicrobial activity by in vitro methods. Antimicrobial activity was performed by agar well diffusion method on Muller Hinton agar and Potato Dextrose agar for bacteria and fungi respectively. Among the three plant I. fistulosa showed the highest activity, it showed broad spectrum activity against Escherichia coli,  Staphylococcus aureus, Proteus mirabilis, Micrococcus letues, Salmonella typhi, Klebsiella pneumonia, Candida albicans and Candida tropicalis. The aqueous extract of I. fistulosa showed maximum relative percentage inhibition against S. aureus (189.71%). Minimum inhibitory concentration was performed by modified agar well diffusion method on Muller Hinton agar and Potato Dextrose agar for bacteria and fungi respectively. Minimum inhibitory concentration values of aqueous extract of I. fistulosa varied from 0.05-50 mg/ml. With these results we conclude that aqueous extract of I. fistulosa leaves possess significant antimicrobial activity against a variety of microorganisms and could be used as a potential source of antimicrobial compounds.

 

 

1. INTRODUCTION:

Microbial infections are one of the major cause of health problem in humans, cause of 25% of death worldwide. ¹ An increased trend in the development of antibiotic resistance has been recorded in bacterial and fungal pathogens causing nosocomial infection.² These antibiotic resistant microorganisms are difficult to treat as of less drug options. Mortality rate is very high in immunocompromised patients, especially in patients suffering with Acquired Immuno Deficiency Syndrome (AIDS).³ Appearance of such microbial strain may be attributed to the indiscriminate use of broad spectrum antibiotics. ⁴ To encounter with such problems, there is a constant need to screen the alternative sources for the development of newer, safer and more potential drugs. Most of the synthetic drugs possess some toxic effects or side effects; hence there is a growing interest in the traditional system of medicine where plant parts and extracts are used as medicine.

 

The use of herbal medicine is well known and documented since Vedic period; traditional Ayurveda, Chinese, Siddha, Unani and Tibetian system of medicine is known worldwide and still in use in many parts of the world. Herbal medicines provide a safer and cost effective alternative to the synthetic antimicrobial drugs.

 

In this study Ipomoea fistulosa, Furcraea foetida and Barleria mysorensis were selected for the screening of antimicrobial activity. I. fistulosa commonly known as Morning glory belongs to a family Convolvulaceae. It is native to tropical America and widely distributed to South Asia. The plant parts are used to cure cold, leucoderma and other skin related diseases. Roots are use as laxative and to provoke menstruation. In the past few years I. fistulosa has been reported for anti-inflammatory activity, antimicrobial activity, neuromuscular blocking and antimicrobial activity.^5-8  F. foetida is an ornamental plant belonging to the family of Agavaceae. It is also commonly known as the giant cabuaya, green-aloe and Mauritius hemp. It is native of Caribbean and South America. Various preparations from F. foetida are used for treatment of rheumatism, paralysis, hair fall, swelling, uterus problems, hepatitis and stomach ache. Two new spirostanol glycosides (1, 2) and a new furostanol glycoside (3), from the leaves of F. foetida showed cytotoxic activities against human cell lines. ⁹ B. mysorensis is a xerophytic plant belonging to the family Acanthaceae, found mostly in India and Sri Lanka. The plant was first identified in a place called Barleria (South Africa) and hence got its genus name as Barleria. This plant is use as Febrifuge (lowering of temperature during fever), and relieving of stomach pain. Leaf decoction is given for the treatment of cough.¹⁰

 

There are very few scientific studies conducted to evaluate the antimicrobial potential of these plants hence the focus of this study was to determine the antimicrobial activity of the aqueous extract of leaves of I. fistulosa, F. foetida and B. mysorensis against a variety of pathogenic bacteria and fungi.

 

MATERIALS AND METHODS:

Chemicals

Nutrient agar, Mueller Hinton broth (MHB), Mueller Hinton agar (MHA), Potato Dextrose broth (PDB), Potato Dextrose agar (PDA), Vancomycin, Chloroamphenicol, Erythromycin disc and Fluconazole disc were purchased from Himedia Pvt Ltd, Mumbai, India.

 

Plant material

I. fistulosa was collected from the natural population growing in Katpadi (12°58’47 N 79°07’47 E), Vellore, Tamil Nadu, India, during August 2011. F. foetida was collected from the VIT University (12°58’06N 79°09’20E), Vellore, Tamil Nadu, India, during August 2011 and B. mysorensis was collected from the natural population growing in Andhrahalli (13°00’32 N 77°28’51 E), Bangalore, Karnataka, India, during September 2011. Herbarium was maintained in our laboratory for the future reference.

 

Processing of plants

Leaves of the I. fistulosa, F. foetida and B. mysorensis were collected and washed properly under running tap water followed by distilled water. Leaves were dried in hot air oven at a temperature of 40°C. Dried leaves were powdered using a mechanical grinder. Pulverized leave material was extracted with distilled water using a Soxhlet extractor. These extracts were concentrated with a rotary evaporator and dried using lyophilizer. Dried extracts were collected in air tight container and stored at 4°C for further use.

 

Antimicrobial activity

Microorganisms

Clinical samples of bacteria and fungi were used in this present study. The bacterial isolates included four Gram negative bacteria (Escherichia coli, Proteus mirabilis, Salmonella typhi and Klebsiella pneumoniae) and two Gram positive bacteria (Staphylococcus aureus, Micrococcus luteus). The fungal isolates included Candida albicans and C. tropicalis.

 

Positive and negative control

Vancomycin (10 µg/disc) was used as positive control for S. aureus and S. typhi; Chloroamphenicol (30 µg/ disc) for P. mirabilis and K. pneumoniae; Erythromycin (10 µg/disc) for M. luteus and E. coli; Fluconazole (10 µg/disc) for fungi were used as positive control. Sterilized distilled water was used as negative control.

 

Preparation of the stock sample

The crude extract was dissolved in sterilized distilled water (5% w/v) as 50 mg/ml stock solution and sterilized by using 0.45μ syringe filter.

 

Determination of antimicrobial activity

Antimicrobial activity of the aqueous extracts of all the three plants was evaluated by the agar well diffusion method ^[13]. The bacterial and fungal cultures were uniformly spreaded using sterile cotton swabs on MHA and PDA plates for bacteria and fungi, respectively. In each of these plates, four wells were cut out using a standard cork borer (7 mm diameter). Using a micropipette, 100 μl of each extracts and negative control (Sterilized distilled water) was added in to different wells. A positive control disc was placed in the plate. Plates were incubated at 37°C for 24 hours. Antimicrobial activity was determined by measuring the zone of inhibition around each well. For each plant extract the experiment was performed in triplicates. ¹¹

 

Determination of relative percentage inhibition

The relative percentage inhibition of the test extract with respect to positive control was calculated by using the following formula ^{12, 13}

 

Relative percentage inhibition of the test extract

= [100 × (x-y)] / (z-y)

 

Where,

x: total area of inhibition of the test extract

y: total area of inhibition of the solvent

z: total area of inhibition of the standard drug

The total area of the inhibition was calculated by using area = πr²; where, r = radius of zone of inhibition.

 

Determination of minimum inhibitory concentration (MIC)

MIC of the plant extract was performed by modified agar well diffusion method. Two fold serial dilution of the stock solution was prepared in sterilized distilled water to make a concentration range from 0.01-50 mg/ml.

 

Test cultures were inoculated in MHB and PDB for bacteria and fungi respectively. Microbial suspensions were seeded on MHA and PDA for bacteria and fungi respectively using a sterilized cotton swab. In each of these plates four wells were cut out using a standard cork borer (7 mm). Using a micropipette, 100 µl of each dilution was added in to wells. All plates were incubated at 37ºC for 24 hours. The minimum concentration of each extract showing a clear zone of inhibition was considered to be MIC. ¹⁴

 

 

Table 1 Antimicrobial activity of Ipomoea fistulosa, Barleria mysorensis and Furcraea foetida

Test organism	Zone of inhibition (in mm)
	I. fistulosa	F. foetida	B. mysorensis	PC	NC
E. coli	24.6±1.52	n.a.	n.a.	32±2.0	n.a.
S. aureus	14.6±1.15	2.6±1.52	6±1.73	10.6±1.52	n.a.
P. mirabilis	8.3±0.57	15.3±2.08	15.6±1.52	12.6±2.08	n.a.
M. letues	13.6±1.15	n.a.	n.a.	32.3±1.52	n.a.
S. typhi	8.6±1.52	7.3±1.15	7.6±0.57	13.6±2.51	n.a.
K. pneumoniae	13.3±0.57	n.a.	n.a.	12.6±1.52	n.a.
C. albicans	14.3±1.52	n.a.	n.a.	21.3±0.52	n.a.
C. tropicalis	5.0±1.0	n.a.	n.a.	17.0±1.0	n.a.

Here, PC: positive control, NC: negative control, n.a.: no activity; Values are expressed as mean ± standard deviation of the three replicates

Zone of inhibition not include the diameter of the well

 

Statistical analysis

The values of antimicrobial activity of the extracts are expressed as mean ± standard deviation of the response of 3 replicates determinations per sample. Results were analyzed statically by using Microsoft Excel 2007 (Roselle, IL, USA).

 

RESULT AND DISCUSSION:

Microbial infections are one of the major threats to human population. Limitations of the existing drugs lead scientists to develop new drugs from alternative sources such as plants, algae etc. Hence in this study, the aqueous extracts of the three plants I. fistulosa, F. foetida and B. mysorensis were evaluated for the antimicrobial activity.

 

Antimicrobial assay

Antimicrobial activity of I. fistulosa, B. mysorensis and F. foetida was screened against Gram positive bacteria, Gram negative bacteria and fungal cultures. Among all three plants I. fistulosa exhibited broad spectrum antimicrobial activity as it inhibited E. coli, S. aureus, P. mirabilis, M. leuteus, S. typhi, K. pneumoniae, C. albicans and C. tropicalis. B. mysorensis and F. foetida exhibited high activity against P. mirabilis and S. typhi. Antimicrobial activity of the extracts are expressed as zone of inhibition and reported as mean ± standard deviation (n= 3). Results are summarized in Table 1.

 

Determination of relative percentage inhibition

Antimicrobial activity of aqueous extract of I. fistulosa, B. mysorensis and F. foetida leaves was compared with the antimicrobial activity of standard drugs for evaluating relative percentage inhibition (Table 2). The aqueous extract of I. fistulosa leaves exhibited maximum relative percentage inhibition against S. aureus (189.71) and C. albicans (45.07) for bacteria and fungi respectively.

 

Table 2 Relative percentage inhibition of I. fistulosa, F. foetida, B. mysorensis

Test organism	Relative percentage inhibition (in %)
	I. fistulosa	F. foetida	B. mysorensis
E. coli	59.09	n.a.	n.a.
S. aureus	189.71	6.01	32.03
P. mirabilis	43.39	147.44	153.28
M. letues	17.72	n.a.	n.a.
S. typhi	39.98	28.81	31.22
K. pneumonia	111.41	n.a.	n.a.
C. albicans	45.07	n.a.	n.a.
C. tropicalis	8.65	n.a.	n.a.

Here, n.a.: no activity

Minimum inhibitory concentration (MIC)

Above mention results clearly reflects the high antimicrobial activity of the aqueous extract of I. fistulosa leaves, hence only aqueous extract of I. fistulosa was screened for MIC analysis. MIC values of the aqueous extract of I. fistulosa leaves against bacterial and fungal strains were varied from 0.05-500 mg/ml. I. fistulosa exhibited 25 mg/ml, 50 mg/ml, 10 mg/ml, 1 mg/ml, 0.05 mg/ml MIC values against E. coli, S. aureus, P. mirabili, M. letues and C. tropicalis respectively. Results are summarized in Table 3.

 

Table 3 MIC value of Ipomoea fistulosa

Test organism	MIC (in mg/ml)
Escherichia coli	25
Staphylococcus aureus	50
Proteus mirabilis	10
Micrococcus letues	1
Salmonella typhi	50
Klebsiella pneumoniae	50
Candida albicans	25
Candida tropicalis	0.05

 

CONCLUSION:

I. fistulosa, F. foetida and B. mysorensis L. are important medicinal plants with several medicinal uses in folk and traditional medicinal system. Results of our study revealed the antimicrobial potential of aqueous extract of I. fistulosa against the clinical isolates of bacteria and fungi. Further, the bioactive compounds can be isolated and characterize by using advance analytical techniques. We conclude that the aqueous extract of I. fistulosa can be used for the development of safe and potent antimicrobial compounds.

 

ACKNOWLEDGEMENTS:

The authors wish to thank the Management and Staff of VIT University, Vellore, TN, India for providing necessary facilities to carry out this study.

 

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Received on 19.06.2012       Modified on 26.07.2012

Accepted on 29.07.2012      © RJPT All right reserved