International Journal of PharmTech Research coden (usa): ijprif issn : 0974-4304



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International Journal of PharmTech Research

CODEN (USA): IJPRIF        ISSN : 0974-4304

                                                                                                             Vol.4, No.1, pp 476-480,           Jan-Mar 2012

Evaluation of the Antimicrobial Activity of

Eugenia singampattiana

 Bedd. Endangered

Medicinal Plant leaves extract

 P. Pavendan

*

 and C. Sebastian Rajasekaran



Department of Botany, Bishop Heber College, Tiruchirappalli, South India.

*

Corres. author: pavendan2010@gmail.com



Abstract :

The antimicrobial activity of Eugenia singampattiana was evaluated on bacteria and fungal strains

like Escherichia coliSalmonella typhiKlebsiella pneumoniaePseudomonas aeruginosa, Bacillus subtilis,

Staphyllococcus aureus, Aspergillus flavas (moult), Aspergillus niger (moult), Penicillium notatum (moult) and

Candida albicans. Aqueous and methanol were used for the extraction. The antimicrobial activity was performed

by agar disc diffusion method. The antibacterial effect of methanol extract showed great activity against



Salmonella typhi (24 mm) and moderated activity were reported against Pseudomonas aeruginosa (20 mm),

Escherichia coli (19 mm) and Bacillus subtilis (19 mm). The same extract showed least activity against

Staphyllococcus aureus (17 mm) followed by Klebsiella pneumonia (14 mm). The aqueous extract exhibited

significant activity against Escherichia coli (21 mm), Pseudomonas aeruginosa (20 mm), Staphyllococcus aureus

(20 mm) followed by Salmonella typhi (19 mm)  and Bacillus subtilis (15 mm). The remaining bacterial pathogen

found to be least activity. Antifungal activity of Eugenia singampattiana methanol leaf extract showed great

activity against Candida albicans (37 mm) and moderated activity were reported against Penicillium notatum (20

mm). The same extract showed least activity against and Aspergillus flavas (17 mm) followed by Aspergillus niger

(15 mm). The aqueous extract exhibited significant activity against Candida albicans (35 mm) followed by

Penicillium notatum (19 mm) and Aspergillus flavas (16 mm). The remaining fungal pathogen found to be least

activity. Results of minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC)

showed that Staphylococcus aureus had the highest MIC (20 mg/ml) and MBC (20 mg/ml), while the lowest MIC

of  8  mg/ml  was  shown  by Bacillus subtilisSalmonella typhi had MIC and MBC values of 10 and 15 mg/ml

respectively. Further studies are needed to evaluate active compounds and probable medicinal benefits in

chemotherapy among humans.

Hence, the Eugenia singampattiana plant can be used to discover bioactive natural products that may serve

as leads in the development of new pharmaceuticals for therapeutic needs.



Keywords: pathogenic microorganisms, antimicrobial activity, disk diffusion method, Eugenia singampattiana,

natural products.



INTRODUCTION

Eugenia singampattiana Bedd. (Myrtaceae) is

a small tree found in evergreen forests, locally known

as  ‘Korandi’  and  “Jungle  Guava”  by  Kanni  tribes  in

Tirunelveli district, Tamil Nadu is one of the endemic,

threatened and aromatic tree species of the southern

Western Ghats in Peninsular India with medicinal

value

1

. The species is categorized as Endangered or



Possibly Extinct by the Botanical Survey of India.

They were used as edible in ripe fruits.

A large portion of the world’s population

depends on the traditional system of medicine for a

variety of diseases

2

.



 Indian flora and fauna consists of

P. Pavendan

 et al /Int.J.PharmTech Res.2012,4(1)

477


more than 2200 species of medicinal and aromatic

plants. The revival of interest in natural drugs started

in last decade mainly because of the wide spread belief

that green medicine is healthier than synthetic

products

3

.



In the Indian system of medicine, several

genera are used medicinally mainly as herbal

preparations in the indigenous system of medicine and

are sources of very potent and powerful drugs.

According to WHO, 80% of the world’s population is

dependent on the traditional medicine and a major part

of the traditional therapies involves the use of plant

extracts or their active constituents. With the

continuous use of antibiotics, microorganisms have

become resistant. This has created immense clinical

problem in the treatment of infections diseases.

Therefore there is a need to develop alternative

antimicrobial drugs for the treatment of infectious

diseases. One approach is to screen local medicinal

plants for possible antibacterial and antifungal

properties.

There is no work done antimicrobial activity

of whole plant. Therefore, the present study has

investigated antimicrobial activity of

Eugenia

singampattiana leaf extracts against several pathogenic

microorganisms.



MATERIALS AND METHODS

Collection and Identification of Plant

Fresh disease free leaves of



Eugenia

singampattiana Bedd has been collected in the

Western Ghats area, Tirunelveli district, Tamil Nadu.

With  the  help  of  local  flora  the  plant  material  were

identified. The air dried leaf samples were powdered

and stored in screw cap bottles at room temperature for

further analysis.



Preparation of plant extracts

The powdered plant material was extracted in

soxhlet apparatus successively with methanol and

aqueous respectively due to their nature of polarity.

After extraction, the extracts were filtered through

Whatman No.1 filter paper and then concentrated in a

vacuum at 40°C using a rotary evaporator. Each

extract was transferred to glass vials and kept at 4°C

before use.

Test organisms used

Six bacterial strains were used namely



Escherichia coli,

Salmonella typhi,

Klebsiella

pneumoniae,

Pseudomonas aeruginosa, Bacillus

subtilis and Staphyllococcus aureus. Four Fungal

Strains were used namely Aspergillus flavas (moult),



Aspergillus niger (moult), Penicillium notatum (moult)

and Candida albicans.



Disc diffusion method

Antimicrobial activity of the leaf extracts was

tested using the disc diffusion method

4

. Sterile nutrient



agar plates were prepared for bacterial strains and

Sterile Sabouraud’s dextrose agar (SDA) were

prepared for fungal strains inoculated by a spread plate

method under aseptic conditions. The filter paper disc

of 5 mm diameter (Whatman’s No. 1 filter paper) was

prepared and sterilized. The leaf extracts to be tested

were prepared various concentrations of 25 μl, 50 μl,

75 μl, and 100μl and were added to each disc of

holding capacity 10 microlitres. The sterile

impregnated disc with plant extracts were placed on

the agar surface with framed forceps and gently

pressed down to ensure complete contact 2/4/2011of

the disc with the agar surface. Filter paper discs soaked

in solvent were used for negative controls. The

bacterial plates were incubated at 37

o

C for 24 hours

and fungal plates were incubated at 37

o

C for 72 h.

After incubation, the size (diameter) of the inhibition

zones was measured.



Determination of MIC and MBC

The minimum inhibitory concentration (MIC)

of  the  extracts  was  estimated  for  each  of  the  test

organisms in triplicates. To 0.5ml of varying

concentrations of the extracts (20.0, 18.0, 15.0, 10.0,

8.0, 5.0, 1.0 0.5, 0.05 and 0.005mg/ml), 2ml of nutrient

broth was added and then a loopful of the test

organism previously diluted to 0.5 McFarland turbidity

standard (0.5 McFarland standard is prepared by

mixing 0.05 mL of 1.175% barium chloride dihydrate,

with 9.95 mL of 1% sulfuric acid) for (bacterial

isolates) and 106 cfu /ml (for fungal isolates) was

introduced to the tubes. The procedure was repeated on

the test organisms using the standard antibiotics

(ciprofloxacin and cotrimoxazole for bacteria and

nystatin and amphoteracin B for fungal isolates). A

tube containing nutrient broth only was seeded with

the  test  organisms  as  described  above  to  serve  as

control. Tubes containing bacterial cultures were then

incubated at 37

o

C for 24 h while tubes containing



fungal spore cultures were incubated for 48 h at room

temperature (30 – 32

o

C). After incubation the tubes



were then examined for microbial growth by observing

for turbidity.

To  determine  the  MBC,  for  each  set  of  test

tubes in the MIC determination, a loopful of broth was

collected from those tubes which did not show any

growth and inoculated on sterile nutrient agar (for

bacteria) and saboraud dextrose agar (for fungi) by

streaking. Nutrient agar and saboraud agar only were

streaked with the test organisms respectively to serve

as control. Plates inoculated with bacteria were then

incubated at 37

o

C for 24 hours while those inoculated



with fungi were incubated at room temperature (30 –

P. Pavendan

 et al /Int.J.PharmTech Res.2012,4(1)

478


32

o

C) for 48 h. After incubation the concentration at



which no visible growth was seen was noted as the

minimum bactericidal concentration.



RESULTS

The increase of antibiotic resistance of

microorganism to conventional drugs has necessitated

the search for new efficient and cost effective ways for

the control of infectious diseases, the result of different

studies provide evidence that some medicinal plants

might indeed be potential source of new antibacterial

agents


5

. Plants are important source of potentially

useful structures for the development of new

chemotherapeutic agents, the first step towards this

goal is in vitro antibacterial activity

6

. The extracts of



higher plant can be very good source of antibiotics

against various bacterial pathogen

7

. Plant based



antimicrobial compounds have enormous therapeutics

potential as they can serve the purpose without any

side effects that are often associated with synthetic

antibacterial compounds.

In the present study, the antimicrobial

activities were performed with methanol and aqueous

extracts of the leaf of Eugenia singampattiana. The

study was made against six pathogenic bacteria and

four fungal strains using the standard disc diffusion

method. Antimicrobial activity of the extracts of



Eugenia singampattiana was first time investigated

against Escherichia coliSalmonella typhiKlebsiella



pneumoniae,

Pseudomonas aeruginosa, Bacillus

subtilisStaphyllococcus aureus, Aspergillus flavas

(moult),


Aspergillus niger (moult),

Penicillium

notatum (moult) and Candida albicans.

All the extracts were inhibited growth of

almost  all  the  selected  bacteria  in  the  range  of      7-24

mm and selected fungi in the range 0f 7-37 mm.

Among them methanol extract showed great activity

against Salmonella typhi (24 mm) and moderated

activity were reported against

Pseudomonas

aeruginosa (20 mm), Escherichia coli (19  mm)    and

Bacillus subtilis (19 mm). The same extract showed

least activity against Staphyllococcus aureus (17 mm)

followed by Klebsiella pneumonia (14 mm). The

aqueous extract exhibited significant activity against



Escherichia coli (21 mm), Pseudomonas aeruginosa

(20 mm), Staphyllococcus aureus (20 mm) followed

by Salmonella typhi (19 mm)  and Bacillus subtilis (15

mm). The remaining bacterial pathogen found to be

least activity.

Antifungal activity of Eugenia singampattiana

methanol leaf extract showed great activity against

Candida albicans (37 mm) and moderated activity

were reported against Penicillium notatum (20 mm).

The same extract showed least activity against and

Aspergillus flavas (17 mm) followed by Aspergillus

niger (15 mm). The aqueous extract exhibited

significant activity against Candida albicans (35 mm)

followed by Penicillium notatum  (19  mm)  and

Aspergillus flavas (16 mm). The remaining fungal

pathogen found to be least activity.

Results of minimum inhibitory concentration

(MIC) and minimum bactericidal concentration (MBC)

are shown in Table 3. The result showed that

Staphylococcus aureus had the highest MIC (20

mg/ml)  and  MBC  (20  mg/ml),  while  the  lowest  MIC

of 8 mg/ml was shown by Bacillus subtilisSalmonella

typhi  had  MIC  and  MBC  values  of  10  and  15  mg/ml

respectively.



Table - 1. Antibacterial activity of Eugenia singampattiana leaves using Disc diffusion method

Antibacterial Activity of

plant Extracts (μl)*

Extracts

Study of indicator Test bacteria

Control


25

50

75 100



Methanol extracts

Escherichia coli

5

8



14

16 19


Salmonella typhi

5

12



17

20 24


Klebsiella pneumoniae

5

7



9

12 14


Pseudomonas aeruginosa

5

9



13

17 20


Bacillus subtilis

5

8



13

16 19


Staphyllococcus aureus

5

7



10

13 17


Escherichia coli

5

10



13

16 21


Salmonella typhi

5

7



15

16 19


Klebsiella pneumoniae

5

7



10

12 13


Pseudomonas aeruginosa

5

8



14

16 20


Bacillus subtilis

5

6



7

7

15



Aqueous extracts

Staphyllococcus aureus

7

12



15

17 20


* Each experiment was repeated thrice

P. Pavendan

 et al /Int.J.PharmTech Res.2012,4(1)

479


Table - 2. Antibacterial activity of Eugenia singampattiana leaves using Disc diffusion method

Antibacterial Activity of plant Extracts (μl)*

Extracts

Study of indicator Test fungi

Control


25

50

75 100



Aspergillus flavas (moult)

5

8



13

15

17



Aspergillus niger (moult)

5

7



11

13

15



Penicillium notatum (moult)

5

12



14

16

20



Methanol extracts

Candida albicans

5

11



16

25

37



Aspergillus flavas (moult)

5

7



11

13

16



Aspergillus niger (moult)

5

7



8

10

13



Penicillium notatum (moult)

5

9



12

16

19



Aqueous extracts

Candida albicans

5

15



19

26

35



* Each experiment was repeated thrice

Table - 3. Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration

(MBC) of Methanol extracts of Eugenia singampattiana

Leaf extracts

Organism

MIC (mg/ml)

MIC (mg/ml)

MBC (mg/ml)

Escherichia coli

15.5


15

18

Salmonella typhi

10

10

15



Klebsiella pneumoniae

10

10



15

Pseudomonas aeruginosa

14

14



20

Bacillus subtilis

8

8



18

Staphyllococcus aureus

8

20



20

Aspergillus flavas (moult)

-

-



-

Aspergillus niger (moult)

-

-



-

Penicillium notatum (moult)

-

-



-

Candida albicans

-

-



-

DISCUSSION

The use of medicinal plants play a vital role in

covering the basic health needs in developing countries

and these plants may offer a new source of

antibacterial, antifungal and antiviral agents with

significant activity against infective microorganisms

8

.

The results of present investigation showed broad



spectrum antibacterial, anti fungal activity against the

tested bacteria and fungi. Generally, antimicrobials

provide the main basis for the therapy of microbial

infections, and their effectiveness depends largely on

the ability of such antimicrobial compound to stop or

inhibit the growth of any microorganism in the body

system they infect. However the high genetic

variability of microorganisms enables them to rapidly

evade the action of antimicrobials by developing

resistance

9

.

Oliveira, et al.,



10

have been studied

antimicrobial activity of Syzygium cumini leaves

extracts belonging to the family Myrtaceae. The leaves

extract of

Syzygium cumini showed highest

antibacterial activity among the extracts tested. The

present observation reveals that the aqueous leaves

extract of Eugenia singampattiana showed the

maximum antibacterial properties against all the tested

bacteria. The similar observations were also studied by

the4,

11 12


.

There are several investigators have proved the

antimicrobial potentiality of many plants. Mishra et

al.,

 13


studied extracts of Carambola, Guava. Kiwi,

Papaya and Strawberry

were tested against

Escherichia coli by the agar well diffusion method.

Likewise, Panda et al.,

14

have reported the



antibacterial activity of various extracts of V. negundo

plant and showed zone of inhibitions were maximum

for E. coli followed by Staphylococcus epidermidis

followed by Pseudomonas  aeruginosa.

The discovery of a potent remedy from plant

origin will be a great advancement in bacterial

infection therapies. The result of present investigation

highlights that the antibacterial, antifungal potentiality

of the extracts of Eugenia singampattiana. This study

encourages the use of herbal extracts demonstrated that

folk medicine can be used as effective modern

medicine to combat pathogenic microorganisms.

This study is extendable with other major

pathogenic bacteria to develop a novel broad spectrum

antibacterial formulation in future. Now, our research

will be focused to develop a broad spectrum

antimicrobial combined herbal formulation with these

plants.


P. Pavendan

 et al /Int.J.PharmTech Res.2012,4(1)

480


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