International Journal of Organic Chemistry, 2011, 1, 257-261
doi:10.4236/ijoc.2011.14037 Published Online December 2011 (http://www.SciRP.org/journal/ijoc)
257
In Vitr o Antibacterial and Antifungal Activity of Methanol,
Chloroform and Aqueous Extracts of Origanum vulgare
and Their Comparative Analysis
Zaman Ashraf1*, Aun Muhammad2, Muhammad Imran2, Ahmad Hussen Tareq2
1Chemistry Department, Allama Iqbal Open University, Islamabad, Pakistan
2Riphah Institute of Pharmaceutical Sciences, Riphah International University, Islamabad, Pakistan
Received July 23, 201 1; revised August 28, 2011; accepted September 8, 2011
Abstract
The work reports antibacterial and antifungal activity of different solvent extracts of Origanum vulgare. The
antimicrobial activity of methanol, chloroform and aqueous extracts were determined against nine different
gram negative and gram positive bacterial strains and three fungal stains. The bacterial strains were Bacillus
subtilis (ATCC 6633), Staphylococcus aureus (ATCC 29213), Micrococcus luteus (ATCC 9341), Pseudo-
monas aeruginosa (ATCC 33347), Escherichia coli (ATCC 25922), Salmonella typhi (ATCC 19430), Shig-
ella flexneri (ATCC 25929), Salmonella para typhi A (ATCC 9150) and Proteus mirabilis (ATCC 49565)
and fungal strains were Aspergillus flavus, Aspergillus nigar and Aspergillus pterus. Agar well diffusion
method was followed in this study. The comparative analysis of antibacterial activity reflects that among
these three extracts, chloroform and methanol extracts shows promising result by exhibiting maximum anti-
bacterial activity, whereas aqueous extract is not active against most of these strains. The analysis of anti-
fungal activity reveals chloroform extract as most efficacious unlike methanol and Aqueous extracts.
Keywords: Origanum vulgare, Antibacterial Activity, Antifungal Activity
1. Introduction
Medicinal plants deeply connected with our lives espe-
cially those who not only hold culinary importance but
also possess combative ability against number of mi-
crobes. They become cynosure in our homes, commer-
cial and research sector. Their natural origin paves expe-
dient outcomes, some in form of natural preservatives
along with aiding our immune system to fight against
pathogenic organisms. World Health Organization (W
HO) supported the use of herbal medicines as safe ther-
apy for the treatment of different diseases. Medicinal
plants would be the paramount basis to find a range of
drugs [1].
New drugs must be needed to eliminate the resistant
microorganisms because a number of well known antibi-
otics cannot fight with resistant strains and become su-
perseded [2]. Literature bear witness that this problem
has proliferated to whole globe. As, the occurrence of
fluoroquinolone resistant gram negative Bacilli colo-
nizing community dwelling people with spinal cord dys-
function [3], ciprofloxacin-resistant gram negative Ba-
cilli association with serious infections in ch ild ren [4] are
common examples. The resistant gram negative and
gram positive species are associated with increased mor-
tality and morbidity, prolonged hospitalization and in-
creased costs. Screening of synthetic compounds and
plant extracts determining their antibacterial and anti-
fungal potential holds immense worth, as they may serve
as a solution to eradicate antibiotic resistant microbes.
Antibacterial derived from plants are not related with
undesired effects as the synthetic drugs and can be suc-
cessfully employed to heal many infectious diseases [5].
About 80% populations of the developed countries use
herbal medicines. A number of important drugs like qui-
nine (antimalarial), vincristine (antitumor drug) and di-
gitalis (heart regulator) were extracted from medicinal
plants. The utilization of plant extracts and phytochemi-
cals with identified biological activity may be of huge
significance in therapeutic treatments [6-10].
The plant selected in this study is Origanum vulgare
also commonly known as oregano belongs from genus

Z. ASHRAF ET AL.
258
Origanum of lamiaceae family (mint family). This plant
is located in hunza valley lies in karakoram ranges. Few
of the important medicinal plants of this region are Ar-
temisia maritime, Chenopodium ambrosioides, Ephedra
gerardianam, Astragalus macropterus, Corydalis Adian-
tifolia, Sonchus asper (L)Hill, Hippophae rhamnoides L,
Tamarix arceuthoides Bge, Salix acmophylla Boiss,
Atriplex crossifolia C.A.Mey, Aquilegia pubiflora Wall.
ex Royle, Primula veris L, Lonicera periclymenum L,
Galium boreale L, Lactuca decipiens (H. and T) Clark
[11]. The plant selected for antibacterial assay may have
great potential for industrial applications [12-15].
Oregano has been recognized as one of the most used
vegetable all over the world with abundant occurrence in
East Europe, in the Middle Asia and South and North
America [16,17]. The volatile oil of oregano has been
used traditionally for respiratory disorders, indigestion,
dental caries, rheumatoid arthritis and urinary tract dis-
orders [18]. Carvacrol is a major active component of
oregano and has potential uses as a food preservative
[19]. Other chemical constituents include limonene, ga-
mmacariofilene, rho-cymenene, canfor, linalol, al-pha-
pinene and thymol [20].
The present work has been designed to evaluate the
potential of methanol, chloroform and aqueous extracts
of the Origanum vulgare against nine different gram
positive and gram negative pathogenic bacterial strains
and three different fungal strains. The results obtained in
this bioassay were compared and it was found that chlo-
roform extract has high potential against tested bacterial
and fungal strains.
2. Experimental
The part used are leaves of Origanum vulgare collected
from hunza valley in August 2009. The leaves were then
dried, homogenized and further subjected for extraction.
The hot extraction method was followed to obtain metha-
nol, chloroform and aqueous extracts of leaves of the
plant.
2.1. Methanol Extract
The soxhlet extractor was used to afford different solvent
extracts of the Origanum vulgare. The crude methanol
extract was achieved by putting 10 g of the dried, ho-
mogenized plant in the porous thimble (made of tough
filter paper) and 100 ml of the methanol in the bolt head
flask of the extraction apparatus. The solvent was then
boiled for one hour and methanol was rotary evaporated.
The extract was then vacuum dried and yield of the
methanol extract was 71.30%. The extract was then
stored in refrigerator at 4˚C for further study.
2.2. Chloroform and Aqueous Extracts
The same method was adopted for the preparation of
chloroform and aqueous extracts. The yield of chloro-
form extract was 6.20% and that of aqueous extract was
53.31%.
2.3. Antibacterial Activity
The antimicrobial activity of these crude extracts was
determined against nine different gram positive and gram
negative bacteria. Agar well diffusion assay was used to
evaluate the antibacterial activity of these extracts [21].
The selected bacterial strains are Bacillus subtilis (ATCC
6633), Staphylococcus aureus (ATCC 29213), Micro-
coccus luteus (ATCC 9341), Pseudomonas aeruginosa
(ATCC 33347), Escherichia coli (ATCC 25922), Sal-
monella typhi (ATCC 19430), Shigella flexneri (ATCC
25929), Sa lmonella para typhi A (ATCC 9150) and Pro-
teus mirabilis (ATCC 49565).
Assay was conducted by using the Nutrient Agar. The
fresh inoculum of these strains were prepared and diluted
with sterilized normal saline. The turbidity of these cul-
tures was adjusted by using 0.5 Mc-Farland. A uniform
bacterial lawn was developed by sterile cotton swabs. 8
mm sized borer was used to make the wells in the inocu-
lated plates. Various dilutions of the dried plant extracts
including 1000 ug/ml, 500 ug/ml, 250 ug/ml, 125 ug/ml
and 62.5 ug/ml were prepared by using dimethyl sulfox-
ide (DMSO) as solvent. 200 ul of test extract and stan-
dard were then delivered to each well. DMSO was used
as control in this antimicrobial study. Levofloxacin (125
ug/ml) a broad spectrum antibiotic effective against a
number of gram positive and gram negative bacterial
strains was used as standard. These plates were incubated
at 37˚C for 24 hours. Antibacterial activity of the plant
extracts was determined by measuring the diameter of
zone of inhibition (mm) and presented by subtracting the
activity of the control.
2.4. Antibfungal Activity
In vitro antifungal activity of the solvent extracts was
tested against three fungi; Aspergillus flavus, Aspergillu s
nigar and Aspergillus pterus using poison plate method
[22]. Potato dextrose agar (PDA) plates were prepared by
using pour plate technique for each extract. A 2% con-
centration of each extract in DMSO as a solvent was
used. A 2% solution of fluconazole was used as standard.
A drug free control was included and plates were ob-
served for growth after 48 h of static incubation at 30˚C
and results are presented in Table 4. All of the plant ex-
tracts showed good to excellent antifungal activity.

Z. ASHRAF ET AL.
259
3. Results and Discussion
The results of antibacterial activity of the methanol ex-
tract are presented in Table 1. The gram positive bacteria
showed more susceptibility than gram negative to the
methanol extract. The methanol extract is more active
against Staphylococcus aureus, Micrococcus luteus as
compared to all other bacterial strains. The extract has
also shown pronounced effects against Bacillus subtilis.
Among gram negative strains Shigella flexneri have
shown competitive results. The findings are interesting
for Micrococcus luteus as it is resistant towards the
standard.
The Table 2 presented the antibacterial activity of
chloroform extract. The antibacterial activity was ob-
served to be in dose dependent manner i.e., 125 ug/ml
showed more level of activity than 62.5 ug/ml agains t all
the tested strains. Chloroform extract of Origanum vul-
gare was most active against gram negative bacteria,
although it has shown significant effects against gram
positive strains. The excellent results were shown ag ainst
Pseudomonas aeruginosa and Bacillus subtilis in com-
parison to all the microorganisms tested. The extract has
demonstrated considerable effect over Mircococcus lu-
teus and Salmonella typhi by producing adequate zone of
inhibition.
In Table 3 it is obvious that the aqueous extract
showed interesting activity again st Sa lmon ella para typh i
A as the chloroform and methanol extract are inactive
against this strain. It possesses activity against Mirco-
coccus luteus, Shigella flexneri. The remaining tested
bacterial strains are resistant towards the aqueous extract.
Table 4 represent the antifungal studies. In Antifun gal
analysis, the chloroform, Aqueous and Methanol extracts
activity were compared with standard Fluconazole. The
chloroform extract exhibits most efficacious results
against Aspergillus flavus and Aspergillus pterus. Its
activity is against Aspergillus niger is quite low. The
Aqueous and Methanol ex tracts do not exhibit reportable
activity against these stains.
In this study the preparation of the solvent extracts
was made by using both the organic solvent and water.
From our investigation, it is concluded that the active
antibacterial present in the leaves of Origanum vulgare
are chloroform and methanol soluble. The active ingre-
dients contained in extract of chloroform are quite effi-
cacious against Pseudomonas aeruginosa and Bacillus
subtilis along with activity against other strains. Whereas
the active in Methanol extract was worthwhile against
Staphylococcus aureus, Micrococcus luteus, Shigella
flexnari along with other strains, which have shown con-
siderable zone of inhibition. In antifungal analysis, the
chloroform extract showed efficacious results.
Table 1. In vitro antibacterial activity of methanol extract of Origanum vulgare.
Methanol Extract Levofloxacin
Bacterial Strains 1000 ug/ml500 ug/ ml250 ug/ml125 ug/ml62.5 ug/ml 125 ug/ml
Escherichia coli 13 12 10 Nil Nil 22
Shigella flexneri 20 15 11 10 Nil 29
Salmonella para typhi A Nil Nil Nil Nil Nil 17
Salmonella typhi Nil Nil Nil Nil Nil 28
Pseudomonas aeruginosa 15 12 11 10 Nil 21
Micrococcus luteus 24 19 15 14 12 Nil
Proteus mirabilis Nil Nil Nil Nil Nil Nil
Bacillus subtilis 17 15 12 Nil Nil 24
Staphylococcus aureus 25 20 18 16 14 22
Table 2. In vitro antibacterial activity of chloroform extract of Origanum vulgare.
Chloroform Extract Levofloxacin
Bacterial Strains 1000 ug/ml500 ug/ml250 ug/ml125 ug/ml62.5 ug/ml 125 ug/ml
Escherichia coli Nil Nil Nil Nil Nil 22
Shigella flexneri 13 12 11 10 9 29
Salmonella para typhi A Nil Nil Nil Nil Nil 17
Salmonella typhi 16 13 12 11 10 28
Pseudomonas aeruginosa 23 20 18 17 13 21
Micrococcus luteus 17 14 13 10 10 Nil
Proteus mirabilis Nil Nil Nil Nil Nil Nil
Bacillus subtilis 22 16 15 14 11 24
Staphylococcus aureus Nil Nil Nil Nil Nil 22

Z. ASHRAF ET AL.
260
Table 3. In vitro antibacterial activity of aqueous extract of Origanum vulgare.
Aqueous Extract Levofloxacin
Bacterial Strains 1000 ug/ml500 ug/ml250 ug/ml125 ug/ml62.5 ug/ml 125 ug/ml
Escherichia coli Nil Nil Nil Nil Nil 22
Shigella flexneri 14 13 12 11 10 29
Salmonella para typhi A 13 12 10 Nil Nil 17
Salmonella typhi Nil Nil Nil Nil Nil 28
Pseudomonas aeruginosa Nil Nil Nil Nil Nil 21
Micrococcus luteus 14 12 10 9 Nil Nil
Proteus mirabilis Nil Nil Nil Nil Nil Nil
Bacillus subtilis Nil Nil Nil Nil Nil 24
Staphylococcus aureus Nil Nil Nil Nil Nil 22
Table 4. In vitro antifungal activity of the solvent extracts of Origanum vulgare.
Plant Extracts
Sr. No Fungal Strains Aqeous Chloroform Methanol Fluconazole
1 Aspergillus flavus 10 mm 23 mm 10 mm 37 mm
2 Aspergillus niger 9 mm 11 mm - 23 mm
3 Aspergillus pterus - 30 mm - 36 mm
4. Conclusions
The constituents contained in Chloroform exhibit sub-
stantial activity against Aspergillus flavus and Aspergil-
lus pterus. The results obtained confirm the therapeutic
potency of Origanum vulgare used in traditional medi-
cine. In addition, these results form a good basis for se-
lection of the plant for further phytochemical and phar-
macological investig ation.
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