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Antimicrobial activites of cow dung extracts against human pathogens

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Objective: For control of microbial infections and diseases, various synthetic drugs and chemical formulations are currently in use. But due to the problem of microbial drug resistance, new alternative synthetic drugs have been explored. Similarly, antimicrobial activities of so many natural products have also been explored.Methods: In this various study extracts of cow dung possessed antimicrobial property against human pathogens like Klebsiella pneumonia and Escherichia coli.Results: The Indian cow dung extracted possessed superior antimicrobial activity than other cow dung types and showed antimicrobial property against all the test microorganisms. Since cow dung and buffalo dung are abundant in nature, which make the process cost effective with processing ease and thus are a promising solution for a variety of health problems in the near future.Conclusion: The medicinal properties of these cow dung and buffalo dung can be exploited to formulate drugs for several diseases caused by antibiotic resistant pathogenic microorganisms.
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ANTIMICROBIAL ACTIVITES OF COW DUNG EXTRACTS AGAINST HUMAN PATHOGENS
Original Article
S. RAJESWARI, E. POONGOTHAI, N. HEMALATHA*
Applied Microbiology Laboratory, Department of Microbiology, Periyar University, Periyar Palkalai Nagar, Salem-11
Email: aarthihema2004@gmail.com
Received: 19 Jul 2016, Revised and Accepted: 20 Aug 2016
ABSTRACT
Objective: For control of microbial infections and diseases, various synthetic drugs and chemical formulations are currently in use. But due to the
problem of microbial drug resistance, new alternative synthetic drugs have been explored. Similarly, antimicrobial activities of so many natural
products have also been explored.
Methods: In this various study extracts of cow dung possessed antimicrobial property against human pathogens like Klebsiella pneumonia and
Escherichia coli.
Results: The Indian cow dung extracted possessed superior antimicrobial activity than other cow dung types and showed antimicrobial property
against all the test microorganisms. Since cow dung and buffalo dung are abundant in nature, which make the process cost effective with processing
ease and thus are a promising solution for a variety of health problems in the near future.
Conclusion: The medicinal properties of these cow dung and buffalo dung can be exploited to formulate drugs for several diseases caused by
antibiotic resistant pathogenic microorganisms.
Keywords: Cow dung, Klebsiella pneumonia, Escherichia coli, Antimicrobial activity
© 2016 The Authors. P ublished by Innovare Academic Sciences Pvt Ltd. This i s an open access article under th e CC BY license (http://creativecommons.org/licenses/by/4.0/)
DOI: http://dx.doi.org/10.22159/ijcpr.2016v8i4.15268
INTRODUCTION
In India, cattle’s rearing is a tradition in the country and intimately
limited to the agricultural economy. Different products obtained from
cow milk, ghee, curd, urine, and dung are used widely in a number of
ayurvedic formulations. Cow dung is traditionally used as organic
fertilizer in Indian sub-continental farming for centuries. The addition of
cow dung increases the mineral status of soil, enhances the resistance of
plant against pests and diseases; stimulate plant growth and other
beneficial activities such as sulpho oxidation and phosphorus
solubilization [1].
The Hindu Vedas say that the cow is holy and should be worshiped.
In India, cows are very important animal resources and are highly
useful in agriculture and dairy industry [2]. Panchagavya is a term
used to describe five major substances, obtained from cow, which
include cow's urine, milk, ghee, curd and dung. All the five products
possess medicinal properties against many disorders. This kind of
treatment is called Panchagavya therapy or cowpaths [3]. Cowpathy is
an old system of medicine mentioned in ancient Indian literature
(Ayurveda) as Panchagavya Chikitsa. The ayurvedic medicines of
animal origin are mainly prepared from Panchagavya which boost up
the body immune system and makes the body refractory to various
diseases [4]. Although some Indian literature mentioned the medicinal
property of cow excretion, only a few were proved. Several useful
properties of cow urine got confirmed by researchers patent also. But
there is no report available on antimicrobial activity of cow dung.
Cow dung is basically the rejects of herbivorous matter which is acted
upon by symbiotic bacteria residing within the animal's rumen. The
resultant faecal matter is rich in minerals. Cow dung is the undigested
residue of plant matter which has passed through the animal's gut. The
resultant faecal matter is rich in minerals. Cow dung is comprised of
organic matter including fibrous material that passed through the
cow's digestive system, among other liquid digesta that has been left
after the fermentation, absorption and filtration, then acidified, then
absorbed again. The chemical composition mostly carbon, nitrogen,
hydrogen, oxygen, phosphorus, etc. with salts, cells sloughed off as the
digest a went through the digestive tract, some urea, mucus, as well as
cellulose, lignin and hemicellulose.
The addition of cow dung increases the mineral status of soil, enhances
the resistance of plant against pests and diseases; stimulate plant
growth and other beneficial activities such as sulphur oxidation and
phosphorus solubilization. Normally, Composition of cow dung is
about 80% water and supports a matrix of undigested plant material
that is rich in nutrients, micro-organisms, and their byproducts. Cow
dung microflora contains an abundant number of bacilli, lactobacilli
and cocci and some identified and unidentified fungi and yeasts [5].
Escherichia coli, is a Gram-negative, facultative anaerobic and non-
sporulating bacteria. Escherichia coli use mixed-acid fermentation in
anaerobic conditions, producing lactate, succinate, ethanol, acetate
and carbon dioxide [6]. It is a common kind of bacteria that lives in
the intestines of animals and humans and most are harmless. Eating
unwashed greens such as spinach, lettuce or green onions or
undercooked beef can cause the infection [7]. The spores are heat,
chemical and pH resistant, the extracts were passed through a
membrane filter (Millipore corp; 47 mm diameter; 0.2 µm pore size).
antibioticsBacterial infections are usually treated with . However,
the antibiotic sensitivities of different strains of Escherichia coli vary
widely. Gram-negative organisms, E. coli are resistant to many
antibiotics that are effective against Gram-positive organisms [8
Klebsiella pneumoniae is a
].
Gram-negative, non-motile, encapsulated,
lactose fermenting, facultative anaerobic, rod shaped bacterium found
in the normal flora of the mouth, skin, and intestines [9]. Klebsiella
pneumonia tends to affect people with underlying diseases, such as
alcoholism, diabetes and chronic lung disease. Multiple-resistant
Klebsiella pneumoniae have been killed in vivo via intra-peritoneal,
intravenous or intranasal administration of phages in laboratory tests.
While this treatment has been available for some time, a greater
danger of bacterial resistance exists to phages than to antibiotics [10].
With this view, this study focused on the
MATERIALS AND METHODS
antimicrobial activities of
dung extracts of Indian and imported cows and buffalos against
human pathogens.
Cow dung collection
Different cow dung (Indian cow, Jersey, Holstein) and buffalo dung
were collected in and around Dharmapuri District.
International Journal of Current Pharmaceutical Research
ISSN- 0975-7066 Vol 8, Issue 4, 2016
Hemalatha et al.
Int J Curr Pharm Res, Vol 8, Issue 4, 9-12
10
Culture collection
The two-gram negative pathogenic strains namely Escherichia coli
and Klebsiella pneumonia samples were collected from Gopi hospital;
Salem Were collected for this study.
Powdered cow dung
1000g of cow dung from Indian cow (Dharmapuri) was collected
and shadow dried for 5 d. The moisture content of the cow dung was
lower than the other types. The dried cow dung was powdered. The
powdered material had a net weight of 290g.
1000g of cow dung from Jersey was collected and shadow dried
for 5 d. The moisture content was high when compared to cow dung
from Indian cow. The dried cow dung was then powdered. The
powdered material had a net weight of 250g.
1000g of cow dung from Holstein was taken and shadow dried
for 5 d. The moisture content was high when compared to cow dung
from Sindhu. The dried cow dung was then powdered. The
powdered material had a net weight of 220g.
1000g of buffalo dung was taken and shadow dried for 5 d. The
moisture content was high when compared to cow dung from Jersey.
The dried buffalo dung was then powdered. The powdered material
had a net weight of 190g.
Preparation of cow dung extracts
100 ml of acetone and ethanol was added in 10 g of powdered different
cow dungs (Indian cow, Jersey, Holstein and buffalo dung) in a conical
flask and it was kept in a rotary shaker for 3 d. The extract was then
filtered using Whatman No 1 filter paper and stored in a vial for future
use.
Preparation of the disc containing cow dung extract
The empty discs were impregnated with 50μl (2 mg/disc) of acetone
extracts of cow dung from Indian cow, Jersey, Holstein and buffalo dung
separately and dried in the oven. Similarly, the empty discs were
impregnated with 50μl (2 mg/disc) of ethanol extracts of cow dung from
Indian cow, Jersey, Holstein and buffalo dung separately and dried in the
oven. This process was repeated until the disc was completely saturated
with the extract. The disc was then used to study the antimicrobial
activity of cow dung extracts against human pathogens [11].
Antibiotic sensitivity test
Kirby-Bauer method also known as disc diffusion antibiotic
sensitivity testing is a test which uses antibiotic-impregnated wafers
to test whether particular bacteria are susceptible to specific
antibiotics. It is based on the observation that the degree of
inhibition of bacterial growth on agar medium surrounding an
antimicrobial compound containing disc correlates with
susceptibility to the agent. The zone of inhibition determines
whether the organism is sensitive, resistant or intermediate to a
particular antibiotic or the antimicrobial compound.
Four to five similar colonies of identified organism from pure culture
plates were transferred into the nutrient broth and incubated at 37
°C for 24 h. To determine the antimicrobial sensitivity, the
inoculums was spread on the entire surface of the Mueller-Hinton
agar plates with the sterile cotton swab. The commercially available
antibiotic discs and one disc containing the antimicrobial compound
were gently pressed onto the microbe carpeted plate at a distance
15 mm away from the edge and 24 mm apart from each other and
incubated at 37 °C overnight (18-24 h). The diameter of the zone of
bacterial growth inhibition around each disc was measured and the
susceptibility or resistance to the agent in each disc was determined
according to the standardized table provided by the Hi-media
Laboratories, Mumbai. Antibiotics used were Ampicillin, Amikacin,
Chloramphenicol, Gentamycin, Ofloxacin, Vancomycin, Methicillin
and Penicillin-G (5).
Phytochemical screening of cow dung extract
The ethanol and acetone extract of cow dung were used for
phytochemical screening. [12, 13]
Test for flavonoids
Lead acetate test
To 0.5 ml of the extract, a few drops of lead acetate solution were added.
The yellow color precipitate was formed in the presence of flavonoids.
Test for glycosides
A small amount of the cow dung extract was dissolved in 1 ml of
water, and then aqueous 10% Sodium hydroxide solution was
added. Formation of yellow color indicated the presence of
glycosides.
Test for steroids
Salkowski test
To 2 ml of extract, added 2 ml of chloroform and 2 ml of concentrated
H2SO4 and shaken well. Chloroform layer appeared red and acid layer
showed greenish yellow fluorescence, in the presence of steroids.
Test for tannins
To 5 ml of the extract, 1 ml of 10% lead acetate solution was added.
Formation of yellow precipitate showed the presence of tannins.
Test for phenols
A small quantity of the extract was dissolved in 0.5 ml of 20% Sulphuric
acid solution. Followed by addition of a few drops of 2% Sodium
hydroxide solution, it turned blue in the presence of phenols.
RESULTS AND DISCUSSION
Antimicrobial activity
Antimicrobial activity of cow dung from Indian cow
Indian cow dung had possessed superior antimicrobial activity than
cow dung. All the test microorganisms were sensitive to the Indian
cow dung. Ethanol extract of the Indian cow dung had shown
antimicrobial activity against the entire test organism, while acetone
extract had shown antimicrobial activity against Klebsiella pneumonia
and Escherichia coli only. The test microorganisms were resistant to
cow dung extract from Jersey. Only the Klebsiella pneumonia was
sensitive to acetone extract of cow dung. Other extract had no
antimicrobial activity against test microorganisms. Cow dung extract
from Holstein had shown antimicrobial activity against Klebsiella
pneumonia only. The other two test microorganisms were resistant to
cow dung extracts. Both acetone and methanol extract had shown
antimicrobial activity against Klebsiella pneumonia. Cow dung extract
from buffalo dung had shown partial antimicrobial activity against test
microorganisms. The Klebsiella pneumonia was sensitive to acetone
extract of buffalo dung, but the same microorganisms were resistant to
ethanol extract of the buffalo dung. The Escherichia coli were resistant
to ethanol and acetone extract. This shows that the buffalo dung had
partial antimicrobial activity (table 1).
Fig. 1: Antimicrobial sensitivity test
Hemalatha et al.
Int J Curr Pharm Res, Vol 8, Issue 4, 9-12
11
Table 1: Antimicrobial activities of acetone and ethanol extracts of various cows and buffalo dung
S.
No.
Test
organisms
Antibiotics Zone of
inhibition(mm)
Inhibitory
pattern
Acetone Inhibitory
pattern
Ethanol Inhibitory
pattern
1.
E. coli
20
S
CAE(I) 20 mm
S
CEE(I) 16 mm
S
24
S
CAE(J) No zone
R
CEE(J) No Zone
R
22
S
CAE(H) No
Zone
R
CEE(H) No
Zone
R
BAE 16 mm
S
BEE 18 mm
S
2.
Klebsiella
pneumonia
No zone
R
CAE(I) 22 mm
S
CEE(I) 18 mm
S
No zone
R
CAE(J) 16 mm
S
CEE(J) No zone
R
20
S
CAE(H) 20 mm
S
CEE(H) 18 mm
S
BAE 20 mm
S
BAE No zone
R
Table 2: Phytochemical analysis of acetone and ethanol extract of different cow dung
S. No.
Phytochemical
Extract
Acetone
Ethanol
I
J
H
B
I
J
H
B
1.
Flavanoids
+
+
+
+
+
+
+
+
2.
Glycosides
+
+
+
+
+
+
+
+
3.
Steroids
+
+
+
+
+
+
+
+
4.
Tannins
+
+
+
+
+
+
+
+
5.
Phenols
+
-
-
-
+
+
-
+
Phytochemical analysis of acetone extraction
Fig. 2: Phytochemical analysis of ethanol extraction
DISCUSSION
In Indian Vedas, the cow is considered the most valuable and
religious animal of Hindus. In India, cows are a very important
animal and useful in agriculture and dairy industry. The cow dung
has been used as organic fertilizer and in the production of biogas.
The evaporated extract of cow dung is called “Dalangor “Dalam” in
northeast Nigeria and in some part of Northem Cameroun and has
been used as soup condiment and in the treatment of infections.
Indian cow, jersey, Holstein and buffalo a different healthy cows Cow
dung was collected in the early morning from cattle breed of
dharmapuri. Cow dung was also put for shed dried. 1000g of
different cow dung was collected and shadow dried for 5 d. The
dried cow dung was powdered. The powdered material 100 ml of
acetone and ethanol was added in 10 g of powdered different cow
dungs (Indian cow, Jersey, Holstein and buffalo dung) in a conical
flask and it was kept in rotary shaker for 3 d. The extract was then
filtered using What man No 1 filter paper and stored in vial for
future use the cow dung extraction procedure was followed by [14]
Phytochemical analysis was performed by each cow dung extract
present the flavonoids, Glycosides, tannins, saponins and phenols
this result was similar to [15] which reports these phytochemical
compound are present the cow urine. The antimicrobial activities of
disc diffusion technique at different cow dung extract against K.
pneumonia and E. coli. Indian cow dung extracts were activity
against the both pathogens in this result are similar to [16] which
report that the cow dung extract was inhibition zone of
Staphylococcus aureus, Bacillus subtilis and Escherichia coli.
CONCLUSION
From the experiment conducted it was concluded that the various
extracts of cow dung possessed partial antimicrobial property
against human pathogens. The cow dung from various cow had
antimicrobial property against klebsiella pneumonia. Besides the
Indian cow dung extracts possess superior antimicrobial activity
than other cow dung and that shown antimicrobial property against
all the test microorganisms. Since cow dung and buffalo dung are
abundant in nature, cost effective and easy to be processed, they are
a promising solution for a variety of health problems in the near
future. The medicinal properties of these cow dung and buffalo dung
can be exploited to formulate drugs for several diseases caused by
antibiotic resistant pathogenic microorganisms.
CONFLICT OF INTERESTS
Declare none
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How to cite this article
S Rajeswari, E Poongothai, N Hemalatha. Antimicrobial
activities of cow dung extracts against human pathogens. Int J
Curr Pharm Res 2016;8(4):9-12.
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The present study includes the collection, isolation and characterization of microorganisms from the cow dung of local varieties from different places of Dehradun, Uttrakhand, India. Four different strains of bacteria B1, B3, B4, B5 were isolated in which three are gram positive (cocci form) and one is gram-negative (bacillus form). Only one strain shows (B4) the formation of endospore. The enzymatic activity of four isolates revealed that strains B3, B5 and both control1 (E. coli) and control 2(B. cereus) showed amylase activity whereas none of the strains showed protease and lipase activity. To test the susceptibility of isolated strains against chemotherapeutic agents, eight antimicrobial drugs were used to treat the susceptibility patterns of isolated bacteria. Among four isolates (B1, B3, B4, B5) strain B3 and control 2 (B. cereus) shows resistance to penicillin and rest of the strains were sensitive to all these antibiotics and also shows antagonistic activity against different human pathogens. The strains B1 and B3 shows moderate inhibition zone against Listeria monocytogenes ATCC 657, Klebsiella pneumonia MTCC5615 and Bacillus pumilis MTCC 1607 whereas strain B4 and B5 shows maximum zone of inhibition against Listeria monocytogenes ATCC657and Bacillus pumilis MTCC 1607.Therefore, intensive efforts must be initiated to identify and preserve all the indigenous breeds of cows for comparative chemical, microbiological and immunological analysis of milk, urine and dung with special reference to their agricultural, medicinal and nutritional significance.
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