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Natural food that meet antibiotics resistance challenge: In
vitro synergistic antimicrobial activity of Azadirachta indica,
Terminalia chebula, piper nigrum
and photoactivated cow urine
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 new dawn was seen when antibiotics were Adiscovered
and we were assured to get rid of
deadly disease caused by microbes. It was Fleming who
[1]discovered Penicillin in year 1928 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
[2]Methicillin resistance S. aureus (MRSA) mocking at us in 1960
. 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
has resistance towards antibiotics. Sexually transmitted
bacteria responsible for gonorrhoea, Penicillin resistant
Streptococci causative agent for pneumonia, microbes responsible
for tuberculosis, influenza, HIV and malaria all have become
4antibiotics resistant. Indiscriminate use of antibiotics has
helped microbes to develop resistance and cause adverse effects
like hypersensitivity, allergic reactions and immune system
[5, 6]suppression to the user . Multiple antibiotic resistant
microbes are held responsible for nosocomial infections and
complicate treatment for conditions like cancer, joint replacement
and heart
[7]surgery . In the developing countries drugs are not only
[27]expensive but also cause various disorders during treatment
.
Resistance among microbes is the outcome of production of new
enzymes, which help them to resist the drug
[5]qualitatively and quantitatively . 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
[8]of healthcare . Herbs and spices have medicinal health
Olojo E.A.A., Awoniran R. ,Olurin K.B.
1Department of Biotechnology,Motilal Nehru National Institute of
Technology,Allahabad-211004, Uttar Pradesh, India.2Department of
Biotechnology,University Institute of Engineering And Technology,
Kurukshetra University, Kurukshetra-136119,
122
Abstract
Address for correspondence*Siddharth VatsDepartment of
Biotechnology,Motilal Nehru National Institute of Technology,
India.Email :[email protected]
Asian Journal of Pharmaceutical and Biological Research Original
Article
e ISSN: 2231-2218 2012 Asian J Pharm Biol Res
1 2 1Siddharth Vats* ,Rajesh Kumar ,Sangeeta Negi
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Asian J Pharm Biol Res |Apr-Jun 2012 | Vol-2 | Issue-2
benefiting properties with addition to their flavour and
fragrance 30qualities. 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
[9, 10]to India .
Numerous studies has been published on the antimicrobial
activities of plants extracts against different types
[28, 29] of microbes 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
[11]Neem . 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
[11]been isolated from Neem . 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].
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
[13]vitiligo . Trikatu which is a prepared from different kinds
of piper and ginger has shown to posses diverse biological
activity
[14]in mammalian systems . Black pepper aids in digestion, fight
tooth decay, powerful antioxidant, and have antimicrobial
[15]activity against pathogens . 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
[16, 17, 18, 19]vulgaris, scabies and allergies . Photo
activation of Cow urine produces metabolically active secondary
nitrogenous compounds, also a decrease in pH makes cow urine
more
[5, 20]antimicrobial . Sulfinol, Ketones, formaldehyde formation
[21]during photo-activation made it more antimicrobial . 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
[22]is well noted .
MATERIALS AND METHODSMicroorganisms
Bacterial strains used were Streptomyces aureofaciens (MTCC
325), E.coli (MTCC 448), Streptococcus mutans
(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 minutes at
7000 rpm. Supernatant was collected for antimicrobial
0activity test. This was stored at 4 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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Asian J Pharm Biol Res |Apr-Jun 2012 | Vol-2 | Issue-2
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 DISCUSSIONAmong 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, ternary
and quaternary combinations.
Figure: 1 The zone of inhibition in the case of P.
aeruginosa
Figure: 2 The zone of inhibition in the case of S. mutans
Figure: 3 The zone of inhibition in the case of C. albicans
Figure:4 The zone of inhibition in the case of S
aureofaciens
Figure: 6 The zone of inhibition in the case of C.
tropicalis
Figure: 7 The zone of inhibiton in the case of C.propsilosis
Figure: 8 The zone of inhibition in the case of E.coli.
124
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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
[5]Azadirachta indica . Neem oil and cow urine unary as well as
in synergistic combinations inhibited the growth of many
infection
[5]causing microbes . Antibacterial activity was also noted with
[23]the use of spice and medicinal plant Cinnamomum . 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
[5].Micrococcus luteus (ATCC 9341), E.Coli (ATCC 25922) It was
found that antimicrobial activity is due to presence of volatile
organic ingredients and when photo-activated they become more toxic
as CO , NH , CH , methanol, propanol and 2 3 4acetone etc.
Nitrosamines are also formed in cow urine on
[24].photoactivation
Phenolic extract of T. chebula is also found to have anti
[25]cancerous effects and antimicrobial . Antimicrobial agents
[26]from the plants of P. nigrum L. Were extracted and studied
.
CONCLUSIONResults 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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