Asian Journal of Pharmaceutical and Biological Research Original Article

Natural food that meet antibiotics resistance challenge: In vitro synergistic

antimicrobial activity of Azadirachta indica, Terminalia chebula, piper nigrum
and photoactivated cow urine
Siddharth Vats*1,Rajesh Kumar2 ,Sangeeta Negi1
1
Department of Biotechnology,Motilal Nehru National Institute of Technology,Allahabad-211004, Uttar Pradesh, India.
2
Department of Biotechnology,University Institute of Engineering And Technology, Kurukshetra University, Kurukshetra-136119,

Olojo E.A.A., Awoniran R. ,Olurin K.B.

Abstract
Objective: Evolution, inappropriate and over use of antibiotics is responsible for antibiotics and drugs resistance among the microbes. Drug resistance
microbes can increase the chances of death, complications of illness, requires prolonged treatment and more hospital stays. This situation makes
immune-compromised patients more prone to nosocomial infections. Mother Nature has been inexhaustible and infinite source for medicinal
compounds. Asian communities are using spices, cow urine and medicinal plants extracts in their diets and naturopathy.
Methods: Antimicrobial activities of photo activated Cow Urine, Azadirachta indica, Terminalia chebula, and Piper nigrum were tested on the basis
of Minimum Inhibitory Concentration which was tested singularly as well as with all possible combinations by agar diffusion and broth dilution
methods against Streptomyces aureofaciens (MTCC 325), E.coli (MTCC 448), Streptococcus mutans (MTCC 497), Pseudomonas aeruginosa (MTCC
7093) Candida albicans (MTCC183), Candida paraopsilosis (MTCC 1965), Candida tropicalis (MTCC 184) and Candida glabrata (MTCC 3019).
Results: Motivating results were obtained. Azadirachta indica, Piper nigrum, terminalia chebula and Cow urine has shown remarkable antimicrobial
effects. On an average MIC for cow urine, Azadirachta indica Piper nigrum, terminalia chebula was 200l, 70-100l, 100l and 90l respectively. T.chebula
showed superb antimicrobial activity against C. propsiolosis, S. aureofaciens, S. mutans and C. albicans. It also has moderate antimicrobial activity
against P. aeruginosa, E.coli, C. tropicalis and C. glabrata. A. indica was antimicrobial against S. aerufaciens, C. albicans, E. coli, P. aeruginosa and C.
glabrata. Cow urine was antimicrobial agains E.coli, C. albicans and S. aeruginosa. Piper nigrum was antimicrobial against S. aerufaciens,
C.tropicalis and C. propsiolosis. Remarkable synergistic effects were observed where cow urine and T. chebula, A. indica and cow urine, T. chebula and
Piper nigrum, cow urine and Piper nigrum, A. indica and Piper nigrum, A. indica and T. chebula, and also when used in a combination of three and four
and the antimicrobicity against all microbes got enhanced.
Conclusion: The results from this study suggest the effects of these natural agents against pathogenic microbes were impressive but depend upon the
combination used and tested against which microbes. Overall the synergistic and singular use inhibited the growth of microbes. Results from this study
reconfirms and strengthen our position to naturally tackle the challenge of disease caused by pathogenic microbes and can be helpful in finding a
solution to outdating of antibiotics and decreases our dependence on them.

Key words: Antibiotics, antimicrobial resistance, pathogens, antimicrobial.

Received : 24 Mar 2012 Accepted : 12 Apr 2012 Published:10 Jun 2012

INTRODUCTION has resistance towards antibiotics. Sexually transmitted bacteria

A new dawn was seen when antibiotics were
discovered and we were assured to get rid of
deadly disease caused by microbes. It was Fleming who
discovered Penicillin in year 1928 [1] and Penicillin became open
to the market in year 1944. Within one year of launch we had our
first antibiotic resistance strain Penicillin Resistance S. aureus
(PRSA). To tackle the challenge, Methicillin a new drug effective
against PRSA was launched in the market in year 1959 but we had
Methicillin resistance S. aureus (MRSA) mocking at us in 1960[2].
Antibiotics have been losing their edge in fight against microbes
[3]
.Many antibiotic resistance microbes like Vancomycin resistant
Enterococcus (VRE), PRSA, MRSA, Quilone and Ciprofloxacin
resistance P. Aeruginosa (QCPRA) pose a challenge to our well
being. Many food borne pathogens such as E.coli, Salmonella
and Campylobacter responsible for diarrhoea and gastroenteritis
Address for correspondence*
Siddharth Vats
Department of Biotechnology,
Motilal Nehru National Institute of Technology, India.
Email :vatssidd@gmail.com
responsible for gonorrhoea, Penicillin resistant Streptococci
causative agent for pneumonia, microbes responsible for
tuberculosis, influenza, HIV and malaria all have become
antibiotics resistant.4 Indiscriminate use of antibiotics has helped
microbes to develop resistance and cause adverse effects like
hypersensitivity, allergic reactions and immune system
suppression to the user [5, 6]. Multiple antibiotic resistant microbes
are held responsible for nosocomial infections and complicate
treatment for conditions like cancer, joint replacement and heart
surgery [7]. In the developing countries drugs are not only
expensive but also cause various disorders during treatment [27].
Resistance among microbes is the outcome of
production of new enzymes, which help them to resist the drug
qualitatively and quantitatively [5]. A need has been felt to develop
new strategies to meet challenges posed by multidrug resistance
microbes. Nature has always been the foremost source of
medicinal and valuable agents. Old civilizations like Indian,
Chinese, Yunani, Egyptian and African have relied on herbal form
of healthcare [8]. Herbs and spices have medicinal health

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2012 Asian J Pharm Biol Res

benefiting properties with addition to their flavour and fragrance
qualities.30 India is one of seven hot spots in terms of biodiversity
of flora and fauna. The land has provided habitat to rich
medicinal plants and 30 percent of the world's cattle. Out of
which 7500 plants species with proven medicinal value and 26
different breeds of cow the walking dispensary are indigenous
to India [9, 10].
Numerous studies has been published on the
antimicrobial activities of plants extracts against different types
of microbes [28, 29] but results reported out of those studies are very
difficult to compare directly and have never touched use of spices
and photo-activated cow urine synergistically against bacterial,
fungal and yeast strains. In the present study we have focused on
the synergistic effect of Azadirachta indica, Terminalia chebula,
Piper nigrum and cow urine.
In India, Azadirachta indica called as "Panacea for all
diseases. Neem tree was described as Azadirachta indica by De
Jussieu in 1830. He was also credited for taxonomic position of
Neem [11]. Sanskrit name of Neem is Arishtha means reliever of
sickness. Its value is recognized by U.S. National Academy of
Science, considering it as a tree for solving global problems. It
was Siddique who in 1942 isolated Nimbin the first compound
isolated from Neem oil. Till now more than 135 compounds have
been isolated from Neem [11]. Countries where professional dental
care is limited Neem is really a potion for dental care. Terminalia
chebula a native plant of South Asia, China and Vietnam
produces small sized, ribbed nut like fruits which is profoundly
used in pickles, spices and medicines. It has been said by
Ayurvedic geniuses that sometimes mother can harm a child
when she is angry but Terminalia chebula fruits can't harm when
it is taken as medicine. So it can be used even to the immune-
compromised patients. It has very high antioxidation activity too
[12]
. minutes at 7000 rpm. Supernatant was collected for antimicrobial
Piper nigrum has found its use in the treatment of cancer
by stimulating melanocytes proliferation and dendritic formation
and so was considered as the potent weapon against disorders like
vitiligo [13]. Trikatu which is a prepared from different kinds of
piper and ginger has shown to posses diverse biological activity
in mammalian systems [14]. Black pepper aids in digestion, fight
tooth decay, powerful antioxidant, and have antimicrobial
activity against pathogens [15]. Cow urine contains volatile salts of
potassium, chloride, calcium of which profoundly benefits
human body and acts like a magical potion for the treatment of
cancer, asthma, chronic renal failure, Eczema, Psoriasis, acne
vulgaris, scabies and allergies [16, 17, 18, 19]. Photo activation of Cow
urine produces metabolically active secondary nitrogenous
compounds, also a decrease in pH makes cow urine more
antimicrobial [5, 20]. Sulfinol, Ketones, formaldehyde formation
during photo-activation made it more antimicrobial [21]. Our
surrounding is full of microbes. Temperature and nutrients
present in the body provide a five star welcome to them. And the
use of Azadirachta indica and gou-Muttra as immune stimulants
is well noted [22].
MATERIALS AND METHODS
Microorganisms
Bacterial strains used were Streptomyces aureofaciens
(MTCC 325), E.coli (MTCC 448), Streptococcus mutans
Asian J Pharm Biol Res |Apr-Jun 2012 | Vol-2 | Issue-2
(MTCC 497) and Pseudomonas aeruginosa (MTCC 7093).
Fungal strains used were Candida albicans (MTCC183) and
yeasts like Candida paraopsilosis (MTCC 1965), Candida
tropicalis (MTCC 184), and Candida glabrata (MTCC 3019).
These were the pathogens used in the study. All the cultures were
obtained in pure form from the culture collection of Institute of
Microbial Technology (IMTECH), Chandigarh, India.
Preparation of herbal extracts of Azadirachata indica
Fresh leaves of Azadirachta indica were taken during the
monsoon season. Leaves were washed with boiling water to
remove microbes and dust present over the surface. Washed
leaves were air dried and grinded to juice with the pestle and
mortar. For each 10 gm of leaves 25ml of water was used to ease
the grinding. Juice was obtained and centrifuged @ 5000 rpm for
10 minutes at 4C. Super natant was taken and extract was
considered as 100 % pure.
Collection of Cow urine
Fresh cow urine was taken from Indian breed cow. Cow
urine used was photo-activated by keeping in a transparent bottle
in sunlight in the daytime and in artificial light at night for 48
hours. And then centrifuged at 10000 rpm for 15 minutes to
remove all debris and precipitated material. Super natant was
collected and filtered through 0-45 micron filter. Before
performing the test against microbes cow urine was tested for its
sterility. To check it was free of microbes it was inoculated in broth
and also nutrient agar.
Preparation of extracts of Terminalia chebula and Piper
nigrum
Dried fruits of plants are taken and crushed in to the
powder form. Then 10 gm powder / 25 ml of water are mixed and
formed a viscous solution. This juice is then centrifuged for 15
activity test. This was stored at 40 C for further use.
Determination of minimum inhibitory concentration
To find the minimum inhibitory concentration agar
diffusion method and broth dilution method were followed. For
agar diffusion method plates containing Muller Hinton agar
(MHA) were made and then spread with each of different strains
and well of 12 mm was made at centre. Different volume of extract
was added to find at which minimum volume of extracts growth of
microbe was inhibited.
Agar diffusion susceptibility test
Agar diffusion method was employed to find
antimicrobial activity of the Azadirachta indica, Piper nigrum,
Terminalia chebula extracts and Gou Muttra. Mueller Hinton agar
(MHA) from himedia was used. Agar plates were made as
mentioned in the instructions. Sterilize it by autoclaving at 121
degree Celsius under 15 lb psi (pounds per square inch) pressures.
Sterilized agar poured and allowed to solidify in Petri plates in
laminar flow. 100 l of 24 hours old microbial culture were then
spread on to it by sterile cotton buds. The 0/6 cm diameter of
punched hole were loaded with 150 micro liters of extracts and
cow urine unary as well as in all possible combination
combination and then incubated at 37C for 24 hours. Triplicate
tests were made for each experiment. The diameter in mm of
inhibition zones around each of the holes was recorded at the end
of the incubation time.
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Broth dilution susceptibility test
In case of broth dilution method, different volume of
plants extracts and cow urine were added in the Muller Hinton
broth. Microbes were inoculated into these tubes. The tubes were
incubated to allow microbial growth for 24 hours. The
interpretation of this MIC depends on the organism and
antimicrobial pair being tested.
RESULTS AND DISCUSSION
Among the three plant extracts and cow urine tested, all
has antimicrobial activity. The test was carried out at triplicates
and average results were represented in the graphs. The results
indicated that MIC for Cow urine was 200 ulfor Azadirachta
indica 70-100uland terminalia chebula 90ul respectuively.
Equilibrated amount of extract were used in unary, binary, Figure:4 The zone of inhibition in the case of S aureofaciens
ternary and quaternary combinations.

Figure: 1 The zone of inhibition in the case of P. aeruginosa

Figure: 6 The zone of inhibition in the case of C. tropicalis

Figure: 2 The zone of inhibition in the case of S. mutans

Figure: 7 The zone of inhibiton in the case of C.propsilosis

Figure: 3 The zone of inhibition in the case of C. albicans

Figure: 8 The zone of inhibition in the case of E.coli.

124

Figure :9 Synergistic activity of various extracts
In case of P. Aeruginosa the most effective combination
was cow urine and Terminalia chebula with a zone of inhibition
40mm. For Candida albicans maximum inhibition was observed
for the ternary combination of Azadirachta indica, Terminalia
chebula and Piper nigrum with zone of inhibition 37 mm. For S.
mutans binary combination of Azadirachta indica and Piper
nigrum was found to be inhibitory with a zone of 45 mm.
Similarly for S auereofaciens maximum inhibition was observed
of 45mm by the application of cow urine and Terminalia chebula.
Piper nigrum, Terminalia chebula and cow urine in combination
were most effective against the C. glabrata.with a zone of 35mm.
For inhibition of C. tropicalis all extracts and cow urine
combination was most effective with a zone of 45mm. Growth of
C. propsilosis was inhibited by the use of Terminalia chebula
with a zone of 35 mm. Combination of Azadirachta indica and
Terminalia chebula was inhibitory against E.coli with 25mm
zone of inhibition.
From this study it is clear that natural extracts of spices,
cow urine and medicinal plants can be used to treat infection
caused by microbes. Similar results were obtained in the case of
Azadirachta indica [5]. Neem oil and cow urine unary as well as in
synergistic combinations inhibited the growth of many infection
causing microbes [5]. Antibacterial activity was also noted with
the use of spice and medicinal plant Cinnamomum [23]. Neem is
the diverse and versatile medicinal plant and is the unique source
of various types of compounds. Antimicrobial activity of photo-
activated cow urine against certain pathogenic bacterial strains
like Staphylococcus aureus (ATCC 25923), Bacillus cereus
(ATCC 11778), Lactobacillus acidophilus (ATCC 53103) and
Micrococcus luteus (ATCC 9341), E.Coli (ATCC 25922) [5]. It
was found that antimicrobial activity is due to presence of
volatile organic ingredients and when photo-activated they
become more toxic as CO2, NH3, CH4, methanol, propanol and
acetone etc. Nitrosamines are also formed in cow urine on
photoactivation [24].
Phenolic extract of T. chebula is also found to have anti
cancerous effects and antimicrobial [25]. Antimicrobial agents
from the plants of P. nigrum L. Were extracted and studied [26].
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CONCLUSION
Results reported from this one of kind study confirm that
nature is the best healer. Synergistics effects resulting from the
combinations of Terminalia chebula, Piper nigrum, Azadirachta
indica and of cow urine have never been documented earlier.
Combined effect of natural substances i.e. drug synergism is a
novel concept. Azadirachta indica was found to be most effective
against S aureofaciens, Terminalia chebula against C. propsilosis,
cow urine against C. albicans and Piper nigrum against C.
tropicalis. Phytochemicals from T. chebula, Piper nigrum, A.
indica and cow urine in different combinations can be of great
significance in therapeutic treatments. Antimicrobial agents
present in these Indian spices, medicinal plants and cow urine can
be effective against plants, human and animal pathogens.
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