Untitled

Raad et al., IJPSR, 2013; Vol. 4(4): 1534-1539.
ISSN: 0975-8232
IJPSR (2013), Vol. 4, Issue 4
(Research Article)
Received on 04 June, 2012; received in revised form, 26 February, 2013; accepted, 13 March, 2013
ANTIBACTERIAL ACTIVITY OF COW URINE AGAINST SOME PATHOGENIC AND NONPATHOGENIC BACTERIA
S. Raad 1, D.V. Deshmukh*2, S. N. Harke 2 and M.S. Kachole 1
Department of Biochemistry, Dr. Babasaheb Amberdkar Marathwada University 1, Aurangabad, Maharashtra,
India
MGMs, Institute of Biosciences and Technology 2, Auranagabad, Maharashtra, India
Keywords:
Cow urine, Antibacterial activity, Zone
of inhibition, Antimicrobial Peptides
Correspondence to Author:
Dr. Devendra V. Deshmukh
Assistant Professor, MGMs, Institute
of Biosciences and Technology, N-6,
CIDCO, Aurangabad, India
E-mail: devcyano@gmail.com
ABSTRACT: Cow urine therapy and all traditional practices from

Indian systems of medicine have a strong scientific base. The cow has
proved to be a boon in the areas of agriculture, science and technology,
industry, energy, medicine etc for the development of any nation, in
addition being eco-friendly in nature. In the present study the
antibacterial potentials of cow urine were investigated. Total 14
pathogenic and non-pathogenic bacterial cultures were used as test
organism against 10 different cow urine samples. The highest zone of
inhibition was shown by sample G against P aeruginosa NCIM 2945
(1.8cm) while the smallest zone of inhibition was shown against E. coli
NCIM 2065(0.3 cm) by sample A. Based on cumulative effect against
the test organism, the urine sample G was found to be the most efficient
inhibiting all the 14 test cultures. The antibacterial activity reported by
sample G was comparable with standard antibiotics. A higher zone of
inhibition was observed by sample G against P aeruginosa NCIM 2945
as compared to that of Gentamicin, Oxacillin and vancomycin. Though
the urine sample G showed a strong antibacterial activity against all the
test organisms, but the activity was reported low against the entire Gram
positive bacteria compared to Gram negative bacteria. The presence of
proline which is considered as a major amino acid in antimicrobial
peptides was also observed in the urine sample G.
INTRODUCTION: It is widely accepted among

clinicians, medical researchers, microbiologists and
pharmacologists, that antibiotic resistance will, in the
very near future, leave healthcare professionals
without effective therapies for bacterial infections.
QUICK RESPONSE CODE
IJPSR:
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As an example, it is now estimated that about half of

all Staphylococcus aureus strains found in many
medical institutions are resistant to antibiotics such
as methicillin 1.
Presently we face a global public health crisis, as
infectious diseases top the list for causes of death
worldwide.
While it is likely that antibiotic resistance contributes
significantly to this problem, data on consumption
and resistance to antibiotics are limited for most
countries 2 and the relationship of resistance to
morbidity and mortality is quantitatively unclear.
International Journal of Pharmaceutical Sciences and Research
1534
Cow, Bos indicus is a most valuable animal in all

community. The cow urine is useful in number of
disease particularly in gulma, filaria, cancer ets. It is
also used with herbs to cure diseases like fever,
epilepsy, anemia, abdominal pain, constipation, etc
by the traditional healers 3 4. Immunomodulatory 5,
hypoglycemic 6 and cardio-respiratory effects 7.
Recently the cow urine has been granted U.S. Patents
(No. 6,896,907 and 6,410,059) for its medicinal
properties, particularly for its use along with
antibiotics for the control of bacterial infection and
fight against cancers. Medicinal usage of cow urine
are extensively searched and scientifically endorsed
8
.
In the Present study the antibacterial potentials of the
cow urine have been investigated against 14 different
pathogenic and nonpathogenic bacteria.
MATERIALS AND METHODS:
Collection of the urine sample: 10 urine samples
were collected from different cows from the farm; all
the samples were collected from milking cows.
Random sampling was a method of choice for
collection of the samples. Samples were collected in
sterile containers, 20 ml of middle stream urine was
collected and brought to the laboratory and stored in
fridge until further use. The samples were designated
as sample A, B, C to Sample J.
Antimicrobial activity:
Test bacterial cultures: Fourteen bacterial cultures
from laboratory repository viz. Escherichia coli
NCIM 2345, Escherichia coli NCIM 2065,
Escherichia coli NCIM 2310, Bacillus subtilis NCIM
2113, Bacillus licheniformis NCIM 2015, Bacillus
megaterium NCIM 2083, Staphylococcus aureus
NCIM 2124, Staphylococcus aureus NCIM 2079,
Staphylococcus aureus NCIM 2125, Pseudomonas
aeruginosa NCIM 2945, Pseudomonas aeruginosa
NCIM 2053, Proteus vulgaris NCIM 2857, Kebshella
pneumonie NCIM 2957 and Salmonella typhimurium
NCIM 2501 were used in the study. Freshly grown
12 h old cultures in nutrient broth were used as the
inoculum in antibacterial assays.
Disc Preparation: Paper disc of filter paper
Whattman No. 1 were prepared. The discs were
sterilized by autoclave at 121C. After the
sterilization the moisture discs were dried on hot air
oven at 50C. The sterile discs were kept in a
presetrilized container until further use.
Qualitative test for proteins: The qualitative test of

protein was performed as according to Martin and
Mittelman 9. The urine Samples were centrifuged at
3000 rpm for 10 mins for the removal of sediments.
After centrifugation the supernatant was collected
and heat test for proteins was performed to observe
the presence of protein.
Disc diffusion assay: Antibacterial activity of urine

samples against the test organisms was done by disc
diffusion assay 11. Petri plate containing 15 ml of
solidified nutrient agar was spread inoculated with
100 l of 12 h old test bacterial cultures. Presterilized
Whatman No.1 paper discs (6 mm) were saturated
with 50 l of urine and dried to be used in assays.
The plates were kept at 4oC for 10 min before they
were incubated at 37oC for 24 h. Anti-bacterial was
assessed by measuring the diameter of growth
inhibition zone around the discs. Sensitivity of test
organisms was also checked against commercial
discs (Hi Media, India) containing standard
antibiotics.
Quantitative estimation of Protein: The Folin

Lowry method was a method of choice for estimation
of protein. Aliquots of protein standard solution were
pipetted out as into a series of tubes as 0.1, 0.2.1.0
ml and the total volume was made to 4 ml with
distilled water. To each tube 5.5 ml of alkaline mix
(reagent C) was pipetted out, mixed well and allowed
to stand for 15 min, at room temperature. 0.5 ml of
FC reagent was pipetted out into each tube, mixed
thoroughly and kept in dark for 30 min. The blue
color formed was measured at 650 nm against a
proper blank. The same was conducted for the
samples 10.
Paper chromatography: The urine sample showing

highest protein content and antibacterial activity was
analyzed for the presence of amino acids using paper
chromatography technique . A strip of wattmans
filter paper No. 1. was used, approximately 1 cm
from one end of the length a line was drawn with the
help of a pencil. At the centre of the line a tiny spot
of the sample was placed. The spot was allowed to
dry and then placed in the chamber containing the
saturated solvent system (Butanol : Acetic acid :
Water, 4 : 1 : 5). The chromatogram was allowed to
run upto th the paper and then taken out and dried
in an oven and then spayed with locating reagent
1535
(Ninhydrine). The Rf value of the spot that appeared

was calculated.
RESULTS:
Qualitative test for proteins: All the urine samples
tested for the presence of protein gave a positive
result. All the test tubes contain the urine sample
showed cloudiness with granules which gave a
positive test for protein in the urine sample.
Protein estimation by Lowry method: The
qualitative estimation of protein gave mixed results.
Sample G gave the highest protein content 520
gm/ml while sample J showed the lowest
concentration of protein (Table 1).
Antibacterial assay: Antibacterial activities of all
the urine samples were tested using disc diffusion
method. On the basis of cumulative antibacterial
effect against all cultures under test, sample G

appeared as most effective. A highest cumulative
inhibition against all the fourteen bacterial cultures
was 15 cm for the urine sample G while the lowest
effect was shown by sample A (Table 2).
TABLE 1: PROTEIN ESTIMATED FROM ALL THE 10
URINE SAMPLES USING FOLIN LOWRY METHOD
Absorbance at
Concentration of
Sample
660 nm
protein in gm/ml
A
0.4420
250
B
0.7230
420
C
0.6830
400
D
0.7055
410
E
0.5882
330
F
0.8063
460
G
0.9490
550
H
0.8641
510
I
0.4990
290
J
0.4412
250
TABLE 2: ZONE OF INHIBITIONS (IN CM) OBSERVED AGAINST 14 BACTERIAL CULTURES FROM 10
DIFFERENT COW URINE SAMPLES
Urine samples
Bacterial
cultures
A
B
C
D
E
F
G
H
I
J
E. coli
0.5*(0.25) 1.2(0.30)
R
R
1.0(0.15)
1.5(0.5)
1.5(0.30) 1.0 (0.45)
1.5 (0.5)
0.5(0.12)
NCIM 2345
E. coli
0.3 (0.5)
1.0(0.45) 0.5(0.15) 1.5(0.15) 0.9(0.12) 0.5(0.15) 1.2(0.27)
1.0(0.5)
1.0(0.12)
1.2(0.35)
NCIM 2065
E. coli
0.8 (0.30) 0.7(0.15) 1.5(0.20) 0.5(0.20) 0.7(0.20) 0.5(0.75) 0.8(0.36) 0.7 (0.30) 0.7 (0.55)
R
NCIM 2015
B subtilis
R
0.5(1.0)
0.8(0.30)
R
0.5(0.15) 0.7(0.30) 1.0(0.15) 0.5 (0.12) 0.5 (0.35)
R
NCIM 2113
B licheniformis
0.5 (1.0)
R
0.8(0.15)
R
R
0.7(0.12) 1.2(0.30)
0.5 (0.5)
1.0 (0.36)
0.5
NCIM 2015
B megaterium
0.9
R
R
R
R
1.0 (0.5)
1.1 (0.5)
R
0.5 (0.34)
0.3
NCIM 2083
(0.30)
S aureus
0.5
R
R
R
R
R
0.9(0.11)
R
R
R
NCIM 2124
(0.75)
S aureus
R
R
R
R
0.6(0.12)
R
0.8(0.25)
R
R
R
NCIM 2125
S aureus
R
R
R
R
R
R
0.5(0.45)
R
0.6 (0.22)
0.6
NCIM 2079
P aeruginosa
R
1.0(0.30) 1.2(0.15) 0.6(0.12)
R
1.0(0.15) 1.8(0.12) 1.0 (0.15) 1.0 (0.45)
0.7
NCIM 2945
P aeruginosa
0.4(0.4)
0.5(0.12)
R
0.8(0.30)
0.5(0.5)
0.7(0.12) 1.0(0.36) 0.7 (0.25) 0.5 (0.12)
1.0
NCIM 2053
P vulgaris
0.8 (0.75)
R
1.1(0.25) 1.0(0.12) 1.0(0.12) 1.5 (0.5) 0.8(0.47) 0.6 (0.12)
R
0.4
NCIM 2857
K pneumonie
0.5 (1.0)
R
0.5(0.30) 0.5 (0.5)
1.1 (0.5) 0.8(0.30) 0.9(0.12)
R
0.5 (0.45)
1.2
NCIM 2957
S typhimurium
1.0 (0.5)
R
R
0.5(0.15) 0.5(0.12) 0.6(0.25) 1.5(0.12)
R
R
0.6
NCIM 2501
Cumulative
4.8
4.9
7.3
5.4
6.8
10
15
6
7.8
7
Inhibition
* Zone of inhibitions in centimeters, Values in the parenthesis is standard deviations, R- Resistant.
1536

TABLE 3: ZONE OF INHIBITION SHOWN BY 14 BACTERIAL CULTUTRES AGAINST STANDARD ANTIBIOTICS
Standard Antibiotics
Bacterial cultures
Methicillin
Gentamicin
Oxacillin
Vancomycin
(5mcg/disc).
(10 mcg/disc)
(5mcg/disc)
(30mcg/disc)
E. coli NCIM 2345
1.5*
1.2
1.5
1.0
E. coli NCIM 2065
1.3
1.5
1.2
1.5
E. coli NCIM 2015
2.8
1.6
1.5
1.5
B subtilis NCIM 2113
2.0
2.5
2.4
1.8
B licheniformis NCIM 2015
1.5
1.0
2.0
1.3
B megaterium NCIM 2083
1.5
2.8
1.7
1.7
S aureus NCIM 2124
1.3
1.5
1.5
1.3
S aureus NCIM 2125
1.4
1.4
1.9
2.0
S aureus NCIM 2079
1.9
1.8
1.7
2.3
P aeruginosa NCIM 2945
2.6
1.4
1.5
1.5
P aeruginosa NCIM 2053
2.3
1.5
1.8
1.5
P vulgaris NCIM 2857
1.8
2.0
2.5
1.0
K pneumonie NCIM 2957
1.5
1.8
2.0
1.7
S typhimurium NCIM 2501
1.0
2.8
2.7
1.5
Paper Chromatography: The chromatogram after

development was observed for the presence of spot
and the Rf value of the spot reveled the amino acid
present in the urine sample. From the calculated Rf
value it was clear that amino acid proline was
prominent amino acid and was confirmed with the Rf
value of standard proline.
DISCUSSION: Commonly, antibiotics are widely as
conservative treatment in various microbial
infections and diseases 12. Considering the enormous
quantity of antibiotics used, the situation should have
been that there would be no infectious diseases. But,
the fact is that the problems of infectious diseases are
increasing daybyday. Some of the major hindrances
are that bacteria have genetic ability to transmit and
acquire resistance towards the drugs 13 and there are
also adverse effects of drugs on the host.14 Therefore
to combat such problems many natural products have
been explored. The nature is an almost infinite
resource for drug development and discovery. It has
endowed with a complete repository of remedies to
cure all ailments of mankind, as it has always been a
first rate drug store with enormous range of plants,
micro organisms and animals.15
The ancient literature of cow urine has always
focused on prevention of disease and maintaining the
health and treatment of diseases. Cow urine acts like
a magical potion for the treatment of the disease like
cancer, asthma, chronic renal failure, hepatitis ABC,
urological disorders, respiratory diseases and also
plays its part as antimicrobial against disease like
Eczema, Psoriasis, acne vulgaris, scabies and other
various kinds of allergies. Urine contains volatile
salts which are beneficial to the human body because

these salts destroy acidity and get rid of pain in
kidney, intestine, and womb; furthermore urine, a
natural tonic, eliminates giddiness, tension in nerves,
lazy feeling, hemicrama, paralysis, common cold,
diseases of brain, nerves and joints.
In the present study the antibacterial potential of 10
different urine samples from cows at the MGMs
farm house was revealed. The variation in the color
of the urine samples may be due to the amount and
type of fodder consumed and the protein content in
them.
FIGURE 1: COLOR VARIATION IN THE 10 URINE

SAMPLES TAKEN FROM COWS.
According to Figure 3, 50% showed yellow color ,

weak yellow for 30% of the urine samples, while
20% of the urine samples showed deep yellow
coloration.
1537
Cow urine contains different constituents; it is rich in

potassium, chloride, calcium, estrogen, phosphorous,
urinary proteins 16. Various research have also found
different components like urea, uric acid, nitrogen,
sulfur, copper, iron, sodium, other salts, carbolic
acid, ammonia, sugar lactose, Vitamin-A,B,C,D,E,
gonadotropin, phenols and also some anticancer
substances.
All the cow urine samples showed the presence of
protein. Vats and Kanupriya 17 has reported that the
components of cow urine are responsible for
showing antimicrobial activity.
The gram negative bacteria were more efficiently

inhibited than gram positive bacteria. Sathasivam et
al 20 has also reported the antibacterial activity of the
cow urine distillate against 4 gram negative bacteria.
A synergistic effect of Azadirachta indica and cow
urine against some gram negative bacteria and yeast
was observed by Vats and Miglan 17. Though all the
urine samples showed the antibacterial activity,
sample G was a promising candidate showing
antibacterial activity against all given test organisms.
The presence of protein in all the samples was clear

evidence that all the samples do contain the presence
of bioactive compounds. Marshall and Arenas 18
pointed out the use of the importance of naturally
occurring peptides and their use as an alternative to
chemical antibiotics and their role as antimicrobials.
The antibacterial potentials of the cow urine was
tested against some pathogenic and non pathogenic
bacteria (Table 2). The highest zone of inhibition
was shown by sample G against P aeruginosa NCIM
2945 (1.8cm) while the smallest zone of inhibiton
was shown against E. coli NCIM 2065(0.3 cm) by
sample A.
FIGURE 3: ZONE OF INHIBITION (in cm) RECORDED

BY SAMPLE G AGAINST ALL THE 14 BACTERIAL
CULTURES
As according to figure 3, the sample G gave the

highest zone of inhibition against P aeruginosa
NCIM 2945(1.8 cm) followed by inhibition against
E.coli NCIM 2345 and S typhimurium NCIM 2501
(1.5). The antibacterial activity shown by the urine
sample G was comparable with the antibacterial
activity by standard antibiotics.
FIGURE 2: SENSITIVITY PATTERN OF THE TEST

CULTURES AGAINST 10 URINE SAMPLES
All the samples showed the presence of antibacterial

activity. From the Figure 2, it was observed that the
maximum activity of all the 10 urine samples was
against Gram negative bacteria than gram positive
bacteria. Similar results were obtained by Edwin et
al 19. Where they have reported the antibacterial
effect of cow urine against gram negative and gram
positive bacteria.
A higher zone of inhibition was observed by sample

G against P aeruginosa NCIM 2945 as compared to
that of Gentamicin, Oxacillin and vancomycin.
Though the urine sample G showed a strong
antibacterial activity against all the test organisms,
but the activity was reported low against all the S
aureus cultures, specifically S aureus NCIM 2079,
(0.5 cm).
The chromatography of the sample revealed the
presence of proline. The amino acid proline is
considered as a major amino acid in antimicrobial
peptides 21.
1538
CONCLUSION: The Antibacterial property of the

cow urine was reveiled using biological assay. Total
10 urine samples were tested against 14 different
strains of bacteria. The ability of the cow urine
sample was more to inhibit the gram negative
bacteria than that of gram positive bacteria. The
highest zone of inhibition that was observed was
1.8cm by sample G. Thus sample G was the only one
which has inhibited the growth of all the test
organism and when compared with standard
antibiotic proved to be more promising. The presence
of amino acid proline in the sample G has proved its
potential similar to peptide antibiotics.
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How to cite this article:

Raad S, Deshmukh DV, Harke SN and Kachole MS: Antibacterial activity of Cow urine against some Pathogenic and Nonpathogenic Bacteria. Int J Pharm Sci Res 2013; 4(4); 1534-1539.
1539
Pharmaceutical Biology
ISSN: 1388-0209 (Print) 1744-5116 (Online) Journal homepage: http://www.tandfonline.com/loi/iphb20
Antidiabetic Activity of Cow Urine and a Herbal

Preparation Prepared Using Cow Urine
E. Edwin Jarald, S. Edwin, V. Tiwari, R. Garg & E. Toppo
To cite this article: E. Edwin Jarald, S. Edwin, V. Tiwari, R. Garg & E. Toppo (2008) Antidiabetic
Activity of Cow Urine and a Herbal Preparation Prepared Using Cow Urine, Pharmaceutical
Biology, 46:10-11, 789-792
To link to this article: http://dx.doi.org/10.1080/13880200802315816
Published online: 05 Jan 2009.
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Download by: [University of Gothenburg]
Date: 17 September 2015, At: 21:45
Pharmaceutical Biology
2008, Vol. 46, Nos. 1011, pp. 789792
Antidiabetic Activity of Cow Urine and a Herbal Preparation

Prepared Using Cow Urine
E. Edwin Jarald,1 S. Edwin,1 V. Tiwari,1 R. Garg, and E. Toppo1
1
Herbal Drug Research Lab, B. R. Nahata College of Pharmacy and Research Centre, Mandsaur, Madhya Pradesh, India
Downloaded by [University of Gothenburg] at 21:45 17 September 2015
Abstract
An herbal preparation prepared by the traditional healers of Mandsaur using cow urine and Gymnema sylvestre
R. Br. (Asclepiadaceae), Momordica charantia L. (Cucurbitaceae), Eugenia jambolana Lam. (Myrtaceae), Aegle marmelos Correa (Rutaceae), Cinnamomum tamala
Buch.-Ham. (Lauraceae), Aloe barbadensis Linn. (Liliaceae), and Trigonella foenum-graecum L. (Leguminosae)
is being used in the treatment of diabetes. In order to scientifically appraise the claim, this preparation was studied for antidiabetic activity and also compared with the
herbal preparation prepared using water. Fresh cow urine
was also used in the study to identify the synergistic effect. The preparations were tested for antidiabetic activity
in alloxan-induced diabetic rats at two dose level, 200 and
400 mg/kg, respectively. The study was done for a period of
21 days. The activity was compared with reference standard,
insulin (1 unit/kg, i.p.) and control. The herbal preparations
significantly (P< 0.05, P < 0.01) lowered the blood sugar
level of hyperglycemic rats in a dose-dependent manner.
Comparatively, the cow urine preparation showed better activity than did the preparation prepared using water. Fresh
cow urine also exhibited significant antidiabetic effect. This
study supports the claim of the local traditional healers.
Keywords: Alloxan monohydrate, antidiabetic, cow urine,
herbal preparation.
Introduction
Diabetes mellitus is a group of metabolic diseases characterized by hyperglycemia, hypertriglyceridemia, and hypercholesterolemia, resulting from defects in insulin se-
cretion or action or both (Nyholm et al., 2000). Diabetes

mellitus is a metabolic disease as old as mankind, and its
incidence is considered to be high (45%) all over the
world. Oral hypoglycemic drugs, such as sulfonylureas and
biguanides, have been used in the treatment of diabetes
mellitus (Okinea et al., 2005). In spite of the introduction of hypoglycemic agents, diabetes and related complications continue to be a major medical problem. Since time
immemorial, patients with noninsulin-dependent diabetes
have been treated orally in folk medicine with a variety of
plant extracts. In India, a number of plants are mentioned in
ancient literature (Ayurveda) for the cure of diabetic conditions known as madhumeha, and some of them have been
experimentally evaluated and the active principles isolated
(Som et al., 2001).
Cow urine is used along with herbs to treat various diseases like fever, epilepsy, anemia, abdominal pain, constipation, and so forth, by traditional healers all over India
(Pathak & Kumar, 2003a; Krishnamurthi et al., 2004). The
traditional healers (Gayathri Parivar) in Mandsaur use an
herbal preparation prepared using cow urine for the treatment of diabetes. The traditional healers prepare a decoction
using cow urine instead of water that contains the following herbs: Gymnema sylvestre R. Br. (Asclepiadaceae), Momordica charantia L. (Cucurbitaceae), Eugenia jambolana
Lam. (Myrtaceae), Aegle marmelos Correa (Rutaceae),
Cinnamomum tamala Buch.-Ham. (Lauraceae), Aloe barbadensis Linn. (Liliaceae), and Trigonella foenum-graecum
L. (Leguminosae). The aim of this work was to validate the
folk claim. In order to create a logic base behind this treatment, the preparation using cow urine was compared with
the preparation using water. Fresh cow urine was also used
in this antidiabetic study to investigate the synergistic effect
if any.
Accepted: April 2, 2008

Address correspondence to: E. Edwin Jarald, Assistant Professor, Herbal Drug Research Lab, B. R. Nahata College of Pharmacy &
Contract Research Centre, Mhow Neemuch Road, Mandsaur 458001, Madhya Pradesh, India. E-mail: ejeru@rediffmail.com
DOI: 10.1080/13880200802315816

C
2008 Informa UK Ltd.
790
E.E. Jarald et al.
Materials and Methods

Procurement of materials
The urine of a 2-year-old virgin Gujarati Indian cow known

as Geer cow was used in the study. The study was performed after getting a certificate from the veterinary doctor
stating that the cow was free from diseases. Fresh cow urine
was collected daily and used after filtration. The plant drugs
were collected from the Gayathri Parivar (local traditional
healers) in order to minimize the variation in the claimed
therapeutic effect. The collected plant materials were positively identified by Dr. H.S. Chatree, Botanist, Govt. Arts
and Science College, Mandsaur, and the voucher specimens
were retained in our department for future reference.
Preparation of extracts
The herbal preparations using cow urine and distilled water were made using the above-mentioned different plant
species. Equal quantities of air-dried samples of each plant
species were ground and mixed with 10-times the equivalent volume of cow urine and water separately and boiled
for 4 h. The extracts were filtered and evaporated in a distillation assembly to get the residue. The percentage yield of
extracts prepared using cow urine and distilled water was
12.5% and 11.0%, w/w, respectively. Preliminary chemical investigation was carried out in the extracts to identify
the nature of constituents present in the extracts (Brain &
Turner, 1975; Khandelwal, 2005).
Animals and treatment

After getting approval from the institutional animal ethical
committee (reg. no. 918/ac/05/CPCSEA), male Wistar
strain rats (weighing between 150 and 200 g) procured
from the animal house of B. R. Nahata College of Pharmacy, Mandsaur, were used for the investigation. The animals were housed in standard environmental conditions of
temperature (21 2 C), humidity (55 10%), and a 12-h
light-dark cycle. Rats were supplied with standard pellet
diet and water ad libitum.
Acute toxicity studies

The acute toxicity test of the preparations and cow urine was
determined according to the OECD guidelines (No. 420,
Organization for Economic Cooperation and Development). Female albino mice (2025 g) were used for this
study. Dosing amounts for sample in liquid form were calculated with the help of density or specific gravity. After
the sighting study, a starting dose of 2000 mg/kg (p.o.) of
the test samples was given to various groups of five animals
each. The treated animals were monitored for 14 days for
mortality and general behavior. No deaths were observed
through the end of the study. The test samples were found
to be safe up to the dose of 2000 mg/kg, and doses of 200

and 400 mg/kg were chosen for further experimentation.
Antihyperglycemic activity
Diabetes was induced in rats by injecting 150 mg/kg
of alloxan monohydrate intraperitoneally in 0.9% w/v
NaCl (Ainapure et al., 1985; Porchezian et al., 2000).
Seventy-two hours after injection, blood glucose level was
measured, and the diabetic rats were divided into eight
groups of six animals each. Insulin [1 unit/kg (i.p.)] was
used as standard drug (Mukherjee, 2002). The first group
was kept as vehicle control, the second was treated with
insulin, and the third to eighth groups were treated with
herbal preparations prepared using cow urine, distilled
water, and pure cow urine at two dose levels, 200 and 400
mg/kg (p.o), respectively. One more group was included
in the study to determine the effects of fresh cow urine in
the blood glucose level of normal rats. Fresh cow urine at
a dose of 400 mg/kg was given to the rats in this group for
21 days. The treatment was given once daily for 21 days.
Blood samples were collected at regular intervals after
fasting overnight, before treatment, from rat-tail vein under
mild anesthesia and monitored. The blood sugar level was
monitored using Accu-chek Active Test strips in Accu-chek
Active Test meter (Roche Diagnostics, Germany).
Statistical analysis
Data were expressed as mean SEM, and the obtained data
were subjected to one-way ANOVA followed by Dunnets
test. The p values less than 0.05 were considered as significant.
Results
The phytochemical investigations performed in the extracts
revealed the presence of alkaloids, tannins, flavonoids, carbohydrates, and saponins in both the extracts. The results
of antidiabetic activity of cow urine and herbal preparations
prepared using cow urine and water are presented in Table 1.
The basal blood glucose levels of all the groups were
statistically not different from each other. Three days after
alloxan administration, blood glucose values were 5-fold
higher in all the groups and were not statistically different
from each other. After 21 days, values of blood glucose were
decreased in all the treatment groups (P < 0.05, P < 0.01).
The value in diabetic control group remained stable. The
preparations exhibited activity in a dose-dependent manner.
The activities of the preparations were found significant
from the 7th day onwards, whereas the activity of cow urine
was found significant only after 21 days of treatment. Normal rats treated with cow urine for 21 days did not show any
elevation in their blood glucose levels. Comparatively, the
preparations containing cow urine were found to be better
than the herbal preparation prepared using distilled water.
Antidiabetic activity of cow urine and cow urine preparation

Table 1.
791
Effect of various preparations in alloxan-induced diabetic rats.

Blood glucose concentration (mg/dL)
Treatment
Insulin
Diabetic control
CUP
CUP
AP
AP
CU
CU
CU control
Daily dose (mg/kg)
0th day
3rd day (alloxan)
7th day
14th day
21st day
1 unit/kg
200
400
200
400
200
400
400
83.2 3.01
93.4 4.23
79.50 2.01
84.00 3.04
84.10 3.54
88.50 4.65
84.62 5.00
80.92 7.01
85.20 3.46
451.22 19.30
439.54 15.90
451.22 13.40
458.80 14.00
478.44 12.16
439.42 14.18
480.42 16.22
430.45 17.92
88.54 2.40
349.42 19.2
538.22 20.40
380.44 12.42
360.45 11.18
399.12 13.03
369.72 10.90
490.88 10.30
450.88 17.89
84.22 4.80
201.44 12.50
517.98 22.21
318.60 11.30
279.80 11.08
330.45 13.55
298.24 14.50
465.45 13.82
410.12 12.56
87.70 3.40
133.10 16.52
530.54 15.20
235.55 12.25
203.34 15.10
240.86 18.22
220.67 17.55
380.20 18.00
262.40 17.92
84.92 4.20
CUP, cow urine Preparation; AP, aqueous preparation; CU, fresh cow urine; CU control, non diabetic rats treated with fresh cow urine.
Values are expressed as mean SEM for six observations.
Statistical analysis was done by one-way ANOVA followed by Dunnets multiple comparison test. Significant at p < 0.05, p < 0.01,
p < 0.001 versus control.
Discussion
Cow, Bos indicus is a most valuable animal in all Veda;
it is called the Mother of all. A composition containing
cows excretionsurine, dung, milk, curd, and gheefive
ingredients together known as panchagawya, is given to
women after delivering a baby. Panchagawya is the main ingredient of many Ayurvedic preparations (Pathak & Kumar,
2003b). Cow urine, one of the ingredients in panchagawya,
is believed to have many therapeutic values. In India, cow
urine is used by the majority of the rural population as a
folklore remedy in almost all the states. Agencies in Gujarat
have been marketing cow urine preparations from multiple
outlets, advertising that they are sterilized and completely
fresh, with prices ranging from Rs. 20 to Rs. 30 per bottle. Keeping in view the enormous role of cows urine in
medicinal and veterinary medicine, a scientific experiment
was performed in rats to elucidate the effect of cow urine
and cow urine containing preparation as an antidiabetic.
Alloxan produces hyperglycemia by a selective cytotoxic
effect on pancreatic beta cells. One of the intracellular
phenomena for its cytotoxicity is through generation of
free radicals demonstrated both in vivo and in vitro (Yadav
et al., 2002). Our investigations indicate the efficiency of
the herbal preparations in maintaining blood glucose levels in alloxan-induced diabetic rats. The glucose-lowering
activity observed in diabetic animals may be due to stimulation of beta cells of pancreatic islets or stimulation of
glycogenesis (Miura et al., 2001). This may be due to the
presence of some hypoglycemic principles in the plants
used in these preparations because all these plants are well
known for their antidiabetic action (Grover et al., 2002;
Kar et al., 2003; Mohamed et al., 2006; Pulok et al., 2006),
and these plants have different types of mechanisms in
reducing blood glucose levels. Comparatively, the preparation using cow urine was found to exhibit better activity
than did the one prepared using distilled water. This could
not be correlated with the nature of the phytoconstituents

present in the extracts because both extracts contains the
same nature of constituents. The interesting observation in
our study was the antidiabetic activity of pure cow urine.
The hypoglycemic effect was not observed in the normal
rats treated with fresh cow urine, and this indicates that the
possible mechanism behind the antidiabetic effect of fresh
cow urine may be due to its stimulation in peripheral use of
glucose. According to literature, cow urine was found to exhibit an antioxidant effect (Krishnamurthi et al., 2004). Free
radicals are implicated in wide range of diseases including
diabetes; the antioxidant activity of cow urine also may be
one of the reasons for its observed antidiabetic effect.
Chemoprofiling of cow urine in our laboratory confirmed
the presence of protein, urea, uric acid, creatinine, phenol,
aromatic acids, enzymes such as acid phosphatase, alkaline phosphatase, amylase, and vitamins (Gowenlock &
McMurray, 1988). Along with these, there may be some
other constituents that may be responsible for the observed
activity. From these observations, it was clear that the better
activity of herbal preparation prepared using cow urine may
be due to its synergistic effect with cow urine or, according
to ancient literature, cow urine is a wonderful solvent for
extraction, and so it is the ability of cow urine to extract out
more active constituents from the herbal drugs and thereby
increase antidiabetic activity.
Further pharmacological investigations are needed to
elucidate the mechanism of the observed antihyperglycemic
effect. This study supports the claim of the traditional healers of Mandsaur.
Acknowledgement
The authors are thankful to Gayathri Parivar (local traditional healers) for providing the necessary information to
carry out this research work.
792
E.E. Jarald et al.
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AB (1985): Hypoglycemic activity of an indigenous preparation. Indian J Pharmacol 17: 238239.
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Phytopharmaceuticals. Bristol, Wright-Scientechnica,
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Gowenlock AH, McMurray RJ (1988): Varleys Practical Clinical
Biochemistry. New Dehli, CBS Publishers and Distributors,
pp. 149153.
Grover JK, Yadav S, Vats V (2002): Medicinal plants of India with
anti-diabetic potential. J Ethnopharmacol 81: 81100.
Kar A, Choudhary BK, Bandyopadhyay NG (2003): Comparative
evaluation of hypoglycaemic activity of some Indian medicinal plants in alloxan diabetic rats. J Ethnopharmacol 84:
105108.
Khandelwal KR (2005): Practical Pharmacognosy. Pune, Nirali
Prakashan, pp. 152154.
Krishnamurthi K, Dutta D, Devi SS, Chakrabarti T (2004): Protective effect of distillate and redistillate of cows urine in human
polymorphonuclear leukocytes challenged with established
genotoxic chemicals. Biomed Environ Sci 17: 5766.
Miura T, Itoh C, Iwamoto N, Aato M, Kawai M, Park SR, Suziki I
(2001): Hypoglycemic activity of the fruit of the Momordica
charantia in Type 2 diabetic mice. J Nutr Sci Vitaminol
(Tokyo) 47: 340344.
Mohamed B, Abderrahim Z, Hassane M, Abdelhafid T,
Abdelkhaleq L (2006): Medicinal plants with potential antidiabetic activityA review of ten years of herbal medicine
research (19902000): Int J Diabetes Metab 14: 125.
Mukherjee KP (2002): Quality Control of Herbal Drugs.

New Delhi, New Business Horrizons, p. 525.
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J, Veldhuis JD, Pincus S, Schmitz O (2000): Assessment
of insulin secretion in relatives of patients with type 2 (noninsulin-dependent) diabetes mellitus: evidence of early -cell
dysfunction. Metabolism 49: 896905.
Okinea LKN, Nyarkob AK, Osei-Kwabenaa N, Oppongc IV,
Barnesa F, Ofosuheneb M (2005): The antidiabetic activity
of the herbal preparation ADD-199 in mice: A comparative
study with two oral hypoglycaemic drugs. J Ethnopharmacol
97: 3138.
Pathak ML, Kumar A (2003a): Cow praising and importance
of Panchyagawya as medicine. Sachitra Ayurveda 5: 56
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Porchezian E, Ansari SH, Shreedharan NKK (2000): Antihyperglycemic activity of Euphrasia officinale leaves. Fitoterapia
71: 522526.
Pulok KM, Kuntal M, Kakali M, Peter JH (2006): Leads
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J Ethnopharmacol 106: 128.
Som NS, Praveen V, Shoba S, Radhey S, Kumria MML,
Ranganathan S, Sridharan K (2001): Effect of an antidiabetic extract of Catharanthus roseus on enzymic activities in
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Yadav S, Vats V, Dhunnoo Y, Grover JK (2002): Hypoglycemic
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Global Journal of Pharmacology 4 (1): 41-44, 2010

ISSN 1992-0075
IDOSI Publications, 2010
Antimicrobial Activities of Cow Urine Distillate Against Some Clinical Pathogens

1
Arunkumar Sathasivam, 2M. Muthuselvam and 1Rajasekran Rajendran
Muthaiyah Research Foundation, Thanjavur, Tamilnadu, India - 613 005

Department of Microbiology, Marudupandiyar College, Thanjavur, Tamilnadu, India
1
Abstract: From the ancient period cows urine has been used as a medicine. In India, drinking of cow urine has
been practiced for thousands of years. Panchagavya is a term used in Ayurveda to describe five important
substances obtained from cow namely Urine, Dung, Milk, Ghee and Curd. The present study analyzes the
antibacterial and antifungal activity of Cow Urine Distillate against the clinical pathogenic microorganisms.
Antibacterial activity of Cow Urine Distillate (5, 10 and 15l) was analyzed against the Bacillus subtilis,
Pseudomonas aeruginosa, Klebsiella pneumoniae and Salmonella typhi. Maximum antibacterial activity was
observed in Pseudomonas aeruginosa (7.060.05, 8.080.18 and 10.41.23, mm in diameter, respectively) and
Salmonella typhi (6.31.23, 8.060.17 and 10.41.2, mm in diameter, respectively). Antifungal activity of cow
urine distillate was analysed against Aspergillus niger and Aspergillus flavus. When the two fungal organisms
were compared, maximum growth suppression was observed in Aspergillus niger (30.14, 6.31.2 and 7.060.04,
mm in diameter) than Aspergillus flavus (2.030.25, 4.90.26 and 6.31.2, mm in diameter, respectively).
Finally concluded that the cow urine distillate has antibacterial and antifungal activities and the inhibitory
activity can be used in the control of bacteria and fungi of various origins.
Key words: Cow Urine Distillate
Antibacterial and Antifungal Activity
INTRODUCTION
In Veda, cows urine was compared to the nectar. In
substrata, several medicinal properties of cows urine
have been mentioned and are known to cause weight loss,
reversal of certain cardiac and kidney problems,
indigestion, stomach ache, edema, etc. Cow urine has a
unique place in Ayurveda and has been described in
Sushrita Sumhita and Ashtanga Sangraha to be the
most effective substance secretion of animal origin with
innumerable therapeutic values. It has been recognized as
water of life or Amrita (Beverages of immortality), the
nectar of the God. In India, drinking of cow urine has been
practiced for thousands of years. Panchagavya is a term
used in Ayurveda to describe five important substances
obtained from cow namely Urine, Dung, Milk, Ghee and
Curd. A number of formulations mentioned in Ayurveda
describe the use of Panchagavya components either alone
or in combination with drugs of herbal, animal or mineral
origin [1].
An exhaustive reference of cows urine having
curative properties in skin diseases, especially leprosy,
is referred to in Caraka samhita. Furthermore, in the
Corresponding Author:
treatment of falling body parts, discharging lymphs and

organism infested organs, use of cows urine (along with
some other ingredients) has been recommended for bath,
anointing and intake [2]. The cow urine distillate has been
patented as activity enhancer and availability facilitator
for bioactive molecules including anti- infective and
anti-cancer agents (US Patent No 6410 059/2002) [3].
Chakra pani mishra in vishva vallabba recommends using
extracts of cow urine in herbal insecticides [4]. Feeding of
cow urine increased the feed intake in white legborn
layers [5]. The present study was carried out to prepare
cow urine distillate and to determine the antibacterial and
antifungal activities.
MATERIALS AND METHODS
Collection of Sample: Cow urine sample was collected
from cow farm (at Avanam, Thanjavur Dt) using sterile
container and stored for further uses.
CUD Preparation: Cow urine was distilled at 100C using
distillation apparatus [6]. The single distilled cow urine
was acidified by lowering the pH below 2.0 with the
Arunkumar Sathasivam, Muthaiyah Research Foundation, Thanjavur, Tamilnadu, India-613 005.

Mob: 09486131235, Email: microbiologyarun@yahoo.com
41
Global J. Pharmacol., 4 (1): 41-44, 2010
addition of 85% orthophosphoric acid. The cow urine was

again distilled at 100C using a distillation apparatus to
remove ammonia. The distillate was stored in sterile glass
flask at refrigerator (4C).
plates with equal distance positive control disc was

also maintain. All the bacterial plates were incubated at
37C for 24hrs and fungal plates at 24C for 72 hrs.
After incubation the diameter of the minimum zone of
inhibition was measured in mm. For each test, three
replicates were performed.
Test Organisms: Bacterial and fungal cultures were

used as test organisms Bacillus subtilis (MTCC 7415)
Pseudomonas aeruginosa (MTCC 7436), Klebsiella
pneumoniae (MTCC 7407), Salmonella typhi, Aspergillus
niger and Aspergillus flavus were collected form microbial
type culture collection centre (MTCC) at Chandigarh.
Statistical Analysis: Mean and standard deviation were

calculated to facilitate the comparison of the data [8].
RESULTS AND DISCUSSION
Disc Preparation: 5 mm (diameter) discs were prepared

from whattman No.1 filter paper. The discs were
sterilized by autoclave at 121C. After the sterilization
the moisture discs were dried on hot air oven at 50C.
The sterile discs were rinsed in cow urine distillate at
different concentration (5, 10, 15l).
Antibacterial Activity: Antibacterial activity of cow urine

distillate was analyzed against the Bacillus subtilis,
Pseudomonas aeruginosa, Klebsiella pneumoniae and
Salmonella typhi (Table 1, Fig. 1 and Plate 1). 5, 10 and
15l concentrations of cow urine distillate discs were
taken for the study. Among the three concentrations
highest antibacterial activity was noted in 15l
concentration when compared with 5 and 10l. Maximum
antibacterial activity was observed in Pseudomonas
aeruginosa (12.60.05, 13.80.18 and 15.41.23, mm in
diameter, respectively) and Salmonella typhi (12.31.23,
13.60.17 and 15.41.23, mm in diameter, respectively)
when compared with other bacterial species and the
standard antibiotic (Ampicillin). US patent was obtained
by CSIR (Counsil for Scientific Industrial Research) India
which claimed a novel pharmaceutical composition
present in cow urine distillate and is effective as an
antifungal and antibacterial [6].
Antibacterial and Antifungal Activity of Cow Urine

Distillate: The antimicrobial and antifungal activity
studies were carried out by disc diffusion technique [7].
The sterile Mueller Hinton agar plates were prepared. The
test organisms like Bacillus subtilis, Pseudomonas
aeruginosa, Klebsiella pneumonia, Salmonella typhi,
Aspergillus niger and Aspergillus flavus were
spreaded over the Mueller Hinton ager plates by
using separate sterile cotton swabs. After the
spreading the different concentrated cow urine distillate
discs were placed separately on the organism inoculated
Table 1: Antibacterial activity of cow urine distillate
S.NO.
Bacteria
1.
2.
3.
4.
Bacillus subtilis
Pseudomonas aeruginosa
Klebsiella pneumoniae
Salmonella typhi
Zone of inhibition (mm in diameter) (MSD) (n=3)

----------------------------------------------------------------------------------------------------------------------------------------Concentration of CUD (l)
----------------------------------------------------------------------------------------------------------------------------------------5
10
15
(Amp*)
7.60.04
12.60.04
7.30.25
121.23
8.60.17
13.60.17
7.30.25
13.60.17
8.80.17
15.41.23
110.14
15.41.23
7.10.01
11.20.01
9.50.05
9.60.02
Values are triplicate mean Standard deviation

Amp* - Standard antibiotic disc Ampicillin (30mg/disc)
Table 2: Antifungal activity of cow urine distillate
S.NO.
Fungi
1.
2.
Aspergillus niger
Aspergillus flavus
Zone of inhibition (mm in diameter) (MSD) (n=3)

------------------------------------------------------------------------------------------------------------------------------------------Concentration of CUD (l)
------------------------------------------------------------------------------------------------------------------------------------------5
10
15
80.14
7.30.25
11.31.2
100.25
Values are triplicate mean Standard deviation
42
12.60.04
111.2
Zone of inhibition (mm in diameter)
16
14
12
10
5l
10l
15l
Amp*
2
0
Bacillus subtilis Pseudomonas

aeruginosa
Klebsiella
pneumoniae
Salmonella
typhi
Zone of inhibition (mm in diameter)
Fig. 1: Antibacterial activity of cow urine distillate

14
12
10
5l
10l
15l
4
2
0
Aspergillus niger
Aspergillus flavus
Fig. 2: Antifungal activity of cow urine distillate
Plate 1: Antibacterial activity of CUD against Pathogenic bacteria
Plate 2= Antifungal activity of CUD against Pathogenic fungi

43
Antifungal Activity: Antifungal activity of cow urine

distillate was analysed against Aspergillus niger and
Aspergillus flavus. The investigated results were
presented in Table 2, Fig. 2 and Plate 2. When the two
fungal organisms were compared, maximum growth
suppression was observed in Aspergillus niger
(80.14, 11.31.2 and 12.60.04, mm in diameter,
respectively) than Aspergillus flavus (7.30.25, 100.26
and 111.2, mm in diameter, respectively). A similar
result reported by Prashith Kekuda et al. [9] Cow Urine
Distillate at various concentrations was tested for
antifungal activity. The growth reduction in percentage
was taken into consideration and antifungal effect was
evaluated. 5% cow urine distillate was more effective
against Mucor sp. (37.1%) followed by A.oryzae (10.2%)
and A. niger (5.4%).
It was concluded that the cow urine distillate has
antibacterial and antifungal activities the inhibitory
activity can be used in the control of bacteria and fungi
of various origins. The test was done in vitro. Same
result may be obtained in vivo also. Now a days
urinotherapy treatment was developed in the medical
sectors. Further studies analyze which components are
responsible for antimicrobial activity and animal model
could reveal antibacterial and antifungal activity of cow
urine distillate in vivo.
ACKNOWLEDGMENTS
REFERENCES
1.
2.
3.
4.
5.
6.
7.
8.
9.
The authors are thankful to Muthaiyah Research

Foundation, Thanjavur for offering facilities to carry out
this study.
44
Shah, E., 1997. Herbal composition in cow urine

distillate. US 5693327. Dec 2.
Basham, A.L., 1998. Practices of medicine in ancient
India in Asian Medical systems: A Comparative
study. (Ed) by Charles leslie, Motilal Banarsidass,
Delhi, pp: 22.
Sarman Singh, 2001. Cow urine has anti Leshmania
donovani effect in vitro. International J. Cow Sci.,
1(2): 72-73.
Sadhale, N., 2004. Vishvallabba Agri History Bulletin
5. Asian agri-History Foundation, Secunderabad500009. pp: 134.
Garg, N., Ashok Kumar and R.S.Chauhan, 2005.
Effect of indigenous cow urine on nutrient utilization
of white leghorn layers. International J. Cow Sci.,
1: 36-38.
Khanuja, S.P.S., 2002. Pharmaceutical composition
containing cow urine distillate and an Antibiotic.
US patent 6410059. June 25.
Bauer, R.W., M.D.K. Kirby, J.C. Sherris and
M. Turck, 1966. Antibiotic susceptibility testing by
standard single disc diffusion method. American
J. Clinical Pathol., 45: 493-496.
Salil Bose, 1982. Biostatistics in Elementary
Biophysics. Jytoi Book, Madurai, pp: 127-128.
Prashith Kekuda, T.R., R. Kavya, R.M. Shrungashree
and S.V. Suchitra, 2007. Antifungal activity of cow
urine distillate. (http://www.microbiocare.com/).
Journal of Intercultural Ethnopharmacology
Review Article
www.jicep.com
DOI: 10.5455/jice.2015022210032
Chemotherapeutic potential of cow

urine: A review
Gurpreet Kaur Randhawa1, Rajiv Sharma2
Department of
Pharmacology,
Government Medical
College, Amritsar, Punjab,
India, 2Department of
Medicine, Guru Nanak
Dev Hospital, attached
to Government Medical
College, Amritsar, Punjab,
India
Address for correspondence:

Gurpreet Kaur Randhawa,
Department of
Pharmacology, Government
Medical College,
Amritsar, Punjab, India.
E-mail: kullar.g@gmail.com
Received: January 16, 2015
Accepted: February 22, 2015
Published: March 07, 2015
ABSTRACT
In the grim scenario where presently about 70% of pathogenic bacteria are resistant to at least one of the
drugs for the treatment, cue is to be taken from traditional/indigenous medicine to tackle it urgently. The Indian
traditional knowledge emanates from ayurveda, where Bos indicus is placed at a high pedestal for numerous
uses of its various products. Urine is one of the products of a cow with many benefits and without toxicity.
Various studies have found good antimicrobial activity of cows urine (CU) comparable with standard drugs such
as ofloxacin, cefpodoxime, and gentamycin, against a vast number of pathogenic bacteria, more so against
Gram-positive than negative bacteria. Interestingly antimicrobial activity has also been found against some
resistant strains such as multidrug-resistant (MDR) Escherichia coli and Klebsiella pneumoniae. Antimicrobial
action is enhanced still further by it being an immune-enhancer and bioenhancer of some antibiotic drugs.
Antifungal activity was comparable to amphotericin B. CU also has anthelmintic and antineoplastic action. CU
has, in addition, antioxidant properties, and it can prevent the damage to DNA caused by the environmental
stress. In the management of infectious diseases, CU can be used alone or as an adjunctive to prevent the
development of resistance and enhance the effect of standard antibiotics.
KEY WORDS:Antibiotic, antifungal, antineoplastic, bioenhancer, Bos indicus, immune-enhancer
INTRODUCTION
Infectious diseases remain a major threat to the public
health despite tremendous progress in human medicine.
Emergence of widespread drug resistance to the currently
available antimicrobials is a matter of deep concern. A high
percentage of nosocomial infections are caused by highly
resistant bacteria such as methicillin-resistant Staphylococcus
aureus or multidrug-resistant (MDR) Gram-negative bacteria.
Each year in the United States, about 2 million people
become infected with antibiotic resistant bacteria and at
least 23,000 people die every year as a consequence of these
infections. Many more people die from other conditions that
are complicated by an antibiotic-resistant infection [1]. In
2012, there were about 450000 new cases of MDR tuberculosis.
Extensively drug-resistant tuberculosis has been identified
in 92 countries. Development of resistance to oral drug of
choice fluoroquinolones, for urinary tract infections caused
by Escherichia coli is very widespread, often sensitivity
remains only for injectables [2]. Infections caused by
resistant microorganisms often fail to respond to the standard
treatment, resulting in prolonged illness, higher health care
expenditures, and a greater risk of death. There is a dire
need for the development of new antimicrobial agents with
sensitivity intact against microorganisms [3,4]. The rational
designing of novel drugs from traditional medicines to treat
180
these difficult to treat infections offers a new prospect for the

modern health-care system.
Ayurvedic texts (Sushruta Samhita, Ashtanga Sangrah and Bhav
Prakash Nighantu) describe cow urine (CU) (gomutra) as an
effective medicinal substance/secretion of animal origin with
innumerable therapeutic uses. Cow (Kamadhenu) has been
considered as a sacred animal in India. In Rigveda (10/15),
CU is compared to nectar. In Susruta (45/221) and in Charak
(sloka-100) several medicinal properties of CU have been
mentioned such as weight loss, reversal of certain cardiac and
renal diseases, indigestion, stomach ache, diarrhea, edema,
jaundice, anemia, hemorrhoids and skin diseases including
vitiligo. Gomutra is capable of removing all the imbalances in
the body, thus maintaining the general health [5]. CU contains
95% water, 2.5% urea, minerals, 24 types of salts, hormones,
and 2.5% enzymes. It also contains iron, calcium, phosphorus,
carbonic acid, potash, nitrogen, ammonia, manganese, iron,
sulfur, phosphates, potassium, urea, uric acid, amino acids,
enzymes, cytokine and lactose [6].
CU is an effective antibacterial agent against a broad spectrum
of Gram-negative and Gram-positive bacteria and also against
some drug-resistant bacteria. It acts as a bio-enhancer of
some antimicrobial drugs. It has antifungal, anthelmintic,
antineoplastic action, is useful in hypersensitivity reactions and
J Intercult Ethnopharmacol Apr-Jun 2015 Vol 4 Issue 2
Randhawa and Sharma: Chemotherapeutic potential of cow urine
in numerous other diseases including increasing the life-span of

a person. Recent researches have shown that CU is an immuneenhancer also [7-9]. Therapeutic properties of CU have been
validated by modern science also.
MECHANISM OF ACTION OF CU
Different fractions of CU possess antimicrobial activity due to
the presence of certain components like volatile and nonvolatile
ones [10-13]. Presence of urea, creatinine, swarn kshar (aurum
hydroxide), carbolic acid, phenols, calcium, and manganese has
strongly explained the antimicrobial and germicidal properties of
CU [14-16]. Presence of amino acids and urinary peptides may
enhance the bactericidal effect [17] by increasing the bacterial
cell surface hydrophobicity. CU enhances the phagocytic activity
of macrophages. Higher amounts of phenols in fresh CU than
CU distillate (CUD) makes it more effective against microbes.
After photo-activation, few biogenic volatile inorganic and
organic compounds such as CO 2, NH3, CH4, methanol,
propanol and acetone, and some metabolic secondary
nitrogenous products are also formed [18]. Photo-activated
CU (PhCU) becomes highly acidic in comparison to fresh CU.
An increase in bactericidal action may be due to a significant
decrease in pH [12], presence of inorganic phosphorus, chloride
and dimethylamine may also play an important role [19],
along with increased formation of some reactive compounds
like formaldehyde, sulfinol, ketones and some amines during
photo-activation and long term storage [20]. CU prevents the
development of antibacterial resistance by blocking the R-factor,
a part of plasmid genome of bacteria [21].
CU contains phenolic acids (gallic, caffeic, ferulic, o-coumaric,
cinnamic, and salicylic acids) which have antifungal
characteristics [22].
Antioxidant property of uric acid and allantoin present in CU
correlates with its anticancer effect. CU reduces apoptosis in
lymphocytes and helps them to survive better [5]. This action
may be due to the free radical scavenging activity of the urine
components, and these components may prevent the process of
aging [10]. It efficiently repairs the damaged DNA [5].
Daily consumption of CU improves immunity due to the presence
of swarn kshar and fastens the wound healing process, which
is due to allantoin [8]. CU enhances the immunocompetence
by facilitating the synthesis of interleukin-1 and -2 [23,24],
augments B - and T- lymphocyte blastogenesis, and IgA, IgM
and IgG antibody titers [25].
S. aureus, Bacillus cereus, Staphylococcus epidermidis, Klebsiella

pneumonia, Pseudomonas aeruginosa, Pseudomonas fragi,
Streptococcus agalactiae, Enterobacter aerogenes, Aeromonas
hydrophila, Micrococcus luteus, Streptococcus pyogenes,
Streptomyces aureofaciens, Lactobacillus acidophilus and
Bacillus subtilis, and Leishmania donovani has been observed
in various studies. In these studies the antimicrobial activity of
CU was found to be comparable with ofloxacin, ciprofloxacin,
ampicillin, chloramphenicol, nalidixic acid, rifampicin,
tetracycline, streptomycin, cefpodoxime and gentamycin in
different studies [27-36].
Studies with Indigenous Bos indicus Breeds of Cow

Fresh CU (FCU), Sterile, PhCU and CUD from a healthy
Geer cow, was used to assess the antibacterial effect against
different strains of bacteria. Against E. coli, FCU had the
bigger mean of inhibition zone (15 mm) than Sterile, PhCU,
and CUD (~10 mm). FCU had good activity of 15, 16 and
20 mm of inhibition against E. coli, B. subtilis, and S. typhi,
respectively. Other forms of CU showed activity against E. coli,
S. typhi, P. vulgaris, S. aureus and B. subtilis [27].
Rana and De [28] observed a greater activity against Grampositive than Gram-negative bacteria with CU obtained from
Geer cow. The minimum inhibitory concentration (MIC) in
all the four tested Gram-positive bacteria was 134 mg/ml.
Among Gram-negative organisms, P. aeruginosa was more
sensitive (MIC 134 mg/ml) than P. vulgaris (MIC 268 mg/ml).
Mean zone of inhibition (mm) standard error of the mean
against B. subtilis was found to be 18.671.15, which was less
than 27 for Gentamycin 10 mcg and cefpodoxime 10 mcg.
Activity (18.671.15) against B. cereus and was similar to
that of cefpodoxime (19) but less than with gentamycin (26).
Activity (16) against S. aureus and S. epidermidis was <25 for
Gentamycin and ~23 with Cefpodoxime. No inhibition against
P. aeruginosa was observed with Cefpodoxime while CU had
an inhibition of 19.33 1.15 mm and Gentamycin 35 mm.
Against P. vulgaris inhibition was comparable between CU
(16 1.73), gentamycin (21) and cefpodoxime (20). There
was comparable inhibition of P. vulagris by CU (16 1.73),
gentamycin (21) and cefpodoxime (20). Against K. pneumoniae,
inhibition observed with CU (15.67 0.57) was less than
gentamycin (34) and cefpodoxime (20).
Comparatively FCU obtained from Gujarati Geer cow was found
to have more antimicrobial activity than its distillate (Table 1).
AS ANTIMICROBIAL AGENT
Similar findings were reported by Jarald et al. [29]. Mean zone

of inhibition (mm), using Sahiwal CUD, was found to be 19.2
for S. aureus, 20.2 for P. fragi, 18.8 for E. coli, 23 for B. subtilis,
19 for S. agalactiae and 17 for P. vulgaris. There was a progressive
decrease in optical density (indicator of antimicrobial activity
and was measured by spectrophotometer at 600 nm) over 5 h
when FCU was added to the respective inoculums [29].
Antimicrobial activity of CU from both indigenous and hybrid

breeds against E. coli, Salmonella typhi, Proteus vulgaris,
The antibacterial efficacy (as mean zone of inhibition in mm) of

CU Concentrate (CUC) obtained from Karnataka breed, Amrit
Early morning first voided CU is more sterile and have more

macro and micronutrients along with other enzyme/urea
content could be more effective [26].
181
Table 1: Antimicrobial activity of CU, CUD (Gujrati Geer cow) in comparison to standard drug Ofloxacin [10]
FCU
CUD
Ofloxacin
E. coli
S. epidermidis
S. aureus
K. pneumoniae
P. vulgaris
B. subtilis
23
20
30
22
20
28
24
18
25
25
20
28
23
20
28
24
21
32
E. coli: Escherichia coli, K. pneumonia: Klebsiella pneumonia, P. vulgaris: Proteus vulgaris, B. subtilis: Bacillus subtilis, S. epidermidis: Staphylococcus
epidermidis, FCU: Fresh cow urine, CUD: Cow urine distillate, CU: Cow urine
mahal was comparable with Streptomycin on B subtilis (16:18),

S. aureus (16:19), E. coli (14:18) and E. aerogenes (15:18) using
Disc diffusion method [30].
In an in vitro study, 30 L of PhCU of Hariana breed was
found to be comparable in efficacy to Tetracycline (30 g mL).
Antimicrobial activity (mean zone of inhibition in mm) of
PhCU and Tetracycline, respectively against B. cereus was 17
and 22, S. aureus was 18 and 21, S. typhimurium was 21 and
22, A. hydrophila was 22 and 24, E. aerugenes was 13 and 18
and M. luteus was 15 and 17 [31]. Similar results were found
in another study with PhCU of Hariana breed against these
bacteria [32].
Studies where breed of cow is not mentioned

In an in-vitro test, activity of FCU was comparable to
Streptomycin. Similar mean zone of inhibition (mm) was
seen against gram positive organisms E. coli (16:16:13),
K. pneumonia (15:17:12) and P. aeruginosa (17:19:15) with
FCU and Streptomycin and lesser with PhCU (by keeping
urine in sunlight in sealed glass bottles for 72 h), respectively.
Comparatively lesser antibacterial activity against gram negative
organisms S. aureus (18:26:17), coagulase negative Staphylococci
(18:29:15), B. subtilis (20:29:15), and S. pyogenes (20:26:14)
was seen for FCU than streptomycin, and still lesser than with
PhCU [33]. No antibacterial activity was seen for CUD, which
is contradictory to some previous reports [34].
Vats et al. [35] studied the synergistic antimicrobial effect of
PhCU and herbs against bacterial and fungal strains. PhCU
and Azadirachta indica combination showed a remarkable
synergistic antimicrobial activity against Candida tropicalis,
Candida glabrata, P. aeruginosa, and S. aureofaciens. PhCU
and Terminalia chebula showed maximum activity against
S. auereofaciens (45 mm), and P. aeruginosa (zone of inhibition
of 40 mm). Piper nigrum, T. chebula and PhCU in combination
were most effective against C. glabrata (35 mm) and
C. tropicalis (45) mm.
Upadhyay et al. [18] found in in-vitro tests that PhCU has better
bactericidal activity against S. aureus, B. cereus, L. acidophilus,
M. luteus, K. pneumonia, S. pneumonia and E. coli, when
compared with Tetracycline, Ampicillin and Ciprofloxacin.
PhCU showed MIC value of 0.25 l/ml against S. aureus,
B. cereus, L. acidophilus and M. luteus, while it was found to
be 0.125 l/ml against E. coli, which was less than that for
antibiotics. A combination of CU with Neem (A. indica) oil and
Bavchi (Psoralea coryfolia) oil showed a synergistic effect (MIC
0.125-0.25 l/ml), which was less than that for antibiotics. Neem
182
oil and CU showed 33-35 mm inhibition zones against B. cereus,

L. acidophilus, M. luteus, K. pneumoniae and S. pneumonia.
Sathasivam et al. reported the antibacterial activity of CUD (5, 10
and 15 l) against the B. subtilis, P. aeruginosa, K. pneumoniae
and S. typhi. Antibacterial activity (mean zone of inhibition in
mm) was observed against B. subtilis (7.6 0.04, 8.6 0.17,
8.8 0.17, respectively) P. aeruginosa (12.6 0.04, 13.6
0.17, 15.4 1.23, respectively) and S. typhi (12 1.23, 13.6
0.17, 15.4 1.23, respectively). This antibacterial activity was
more than the positive control of ampicillin (30 mg/disc), which
was 7.1 0.01 mm against B. subtilis, 11.2 0.01 mm against
P. aeruginosa and 9.6 0.02 mm of inhibition zone against
S. typhi. Antibacterial activity against K. pneumonia was 11
0.14 mm with 15 l of CUD, which was more than the activity
(9.5 0.05 mm) with Ampicillin [34].
Yadav et al. reported the antimicrobial property of a herbal
formulation containing CU, Dalbergia sissoo, and Datura
stramonium. The antimicrobial activity of CU alone was also
found to be significant (P > 0.001). It was found that CU extract
showed the highest inhibition in gram-positive S. aureus (CI,
213%) and comparable activity in S. pneumoiae (95%) compared
to chloramphenicol (30 g), nalidixic acid (10 g), rifampicin
(30 g), and ampicillin (10 g). In gram-negative bacteria all
antibiotics were inactive, except chloramphenicol (30 g), while
CU extract showed significant (P < 0.05) activity (35% and 37%,
respectively) against E. coli and K. pneumonia as compared to
Chloramphenicol [36].
CU has anti-Leishmania donovani effect (Kala-azar) in an
in-vitro study [37]. This fact can be further validated by more
intensive studies.
PREVENTION OF ANTIBIOTIC RESISTANCE

Pathogenic bacteria are remarkably resilient and have developed
several ways to resist antimicrobial drugs. Due to increasing
use and rampant misuse of existing antibiotics in human
and veterinary medicine, and also in agriculture, threat from
antimicrobial resistance is increasing. Resistant strains like
Penicillin- and Methicillin- resistant S. aureus, vancomycin
resistant Enterococcus, and ciprofloxacin resistance P. aeruginosa
are an ever increasing global threat. After photoactivation and
purification, CU has been found to be effective against certain
drug resistant bacterial strains [38]. CU extract of A. indica
showed better MIC values than the organic fractions for MDR
E. coli (12.68 mm) and K. pneumonia (9 mm). CU extracts
of A. indica showed >8.66 mm zone of inhibition for MDR
S. aureus, P. aeruginosa and P. vulgaris [39].
FUNGICIDE AND BIOFUNGICIDE

Fungicidal effect against Aspergillus fumigatus, Aspergillus
flavus, Aspergillus niger, Aspergillus, Malassezia, C. tropicalis
and C. glabrata has been observed in various studies. CU was
highly stable and capable in inhibiting the growth of Malassezia
fungi (90-95%) responsible for causing dandruff for a longer time
(4-5 days), than rice water (due to B. cereus growth in rice water)
which was stably capable of inhibiting 85-90% of the growth for
3-4 days. Neem leaves extract and Lemon Juice extract were
comparatively less effective in this study [40].
15% CU was most active against Aspergillus, Rhizophus and
the percentage of inhibition obtained with it was 85% [41].
5% CUC showed maximum antifungal activity against A. niger
(93%), followed by A. oryzae (92.67%) and A. flavus (83%)[30].
CUD showed better antifungal activity against A. fumigatus
and C. albicans with mean zone of inhibition of 13 and 11 mm
than PhCU [27]. More fungal growth suppression (as mm in
diameter) was observed with CUD in A. niger (8 0.14, 11.3
1.2 and 12.6 0.04, respectively) than A. flavus (7.3 0.25,
10 0.26 and 11 1.2, respectively) with the use of 5, 10 and
15 l of CUD [34].
In vitro antifungal activity (in mm) of Geer CU against A. flavus
(17.33 0.57) was in between 50 g of amphotericin B (15)
and 10 g of clotrimazole (24) and against C. albicans, activity
was similar with CU (18.67 1.15) and amphotericin B (19),
but less than clotrimazole (30) [28]. Antifungal activity of
Geer CU is better than the others where source of CU is not
mentioned.
In an in vitro study, it was found that the urine samples of
outdoor feeding cow (OCU) was more effective and inhibited
growth of fungi more strongly as compared to indoor feeding
CU (ICU). This inhibition was concentration dependent.
No growth of Penicillium notatum, Trichoderma viridae, and
Alternaria solanii was observed with 10% OCU and with 20%
ICU and that of Claviceps purpurea, Rhizopus oligosporius, C.
albicans and A. candidus, no growth was observed with 20% of
OCU only [42].
ANTISEPTIC
Sanganal et al. observed the enhanced wound healing activity
of CU in Wistar albino rats [43]. On 4th day, the external
application of CU showed significant and progressive increase
in wound healing in rats compared to different concentrations
of CU and 1% w/w nitrofurazone ointment locally. Similar
findings were also observed by Maheshwary et al. [44].
ANTHELMINTIC ACTIVITY
CUC was found to be more effective than piperazine citrate
as anthelmintic agent at both 1% and 5% concentrations.
For anthelmintic activity, adult Indian earthworm Pheretima
posthuma was studied due to its anatomical and physiological
resemblance with the intestinal roundworm parasite of human
beings. Paralysis of earthworm occurred in 53 and 48 min

with 1% piperazine and CUC, respectively and 16 and 13 min
with 5% piperazine and CUC, respectively. Time taken for
the death of earthworms decreased from 72 min with 1%
piperazine to 60 min with 1% CUC, respectively. It further
decreased from 28 min with 5% piperazine to 18 min with 5%
CUC, respectively [30].
Different compositions of Panchgavya (five products of cow
namely milk, curd, ghee, urine and dung) alone and combination
of Panchgavya and ethanolic extract of Bauhinia variegata
Linn (10%, 50%, 75% in Panchgavya) were found to have
excellent anthelmintic activity against adult Indian earthworm
(P. posthuma) when we compared to control Piperazine (50 and
100 mg/ml). In combination, the anthelmintic activity was
synergistic and with increasing doses, time (in minutes) of onset
of paralysis and death in earthworm decreased [45].
BIOENHANCER
A bioenhancer/biopotentiator is an agent capable of
enhancing the bioavailability and efficacy of a drug with which
it is co-administered, without any pharmacological activity of
its own at the therapeutic dose used. In ayurveda, this concept
is known as yogvahi and is used to increase the effect of
medicines by increasing the oral bioavailability (especially of
medicines with poor oral bioavailability), decreasing their dose
and adverse effects, and were used to circumvent the parentral
routes of drug administration. We can develop more such useful
and economically viable drug combinations, by integrating the
knowledge of time tested ayurveda with modern methods of
research [8]. CU is the only agent of animal origin which acts
as bioenhancer of antimicrobial, antifungal, and anticancer
agents [30]. The indigenous CU contains Rasayana tatva,
which is responsible for modulation of the immune system and
also act as a bioenhancer [21].
CUD is more effective bioenhancer than CU [30,46].
CUD enhances the transport of antibiotics like rifampicin,
tetracycline and ampicillin across the gut wall by 2-7 folds
[47]. It also enhances the potency of taxol against MCF-7 cell
lines [48]. It enhances the bioavailability of rifampicin by 80
fold in 0.05 microgm/ml concentrations, ampicillin by 11.6
fold in 0.05 g/ml concentrations and clotrimazole by 5 fold
in 0.88 g/ml concentration [49]. The activity of rifampicin
increases by about 5-7 folds against E. coli and 3-11 folds against
Gram-positive bacteria, when used along with CU [50]. Potency
of paclitaxel has been observed to increase against MCF-7,
a human breast cancer cell line in in-vitro assays [49]. The
bioenhancing ability is by facilitating the absorption of drugs
across the cell membrane. The CU has been granted US Patents
for its medicinal properties, particularly as a bioenhancer along
with antibiotics, antifungal and anticancer drugs (6896907,
6410059).
CUD alone caused more inhibition of Gram-positive bacteria.
Inhibition caused by streptomycin (1 mg/ml) alone was higher
(31-34 mm) than that of CUD alone (19-22 mm). With the
183
combination of Pinguicula longifolia, CUD and streptomycin

together, S. aureus was inhibited to a more extent (38 mm)
followed by P. aeruginosa (37 mm) and an equal inhibition was
exhibited by B. subtilis and E. coli (36 mm) [51]. S. aureus and
P. aeuroginosa have been recognized as most common bacteria,
which have developed resistance against several antibiotics
and is a major hospital borne pathogen, which is particularly
dangerous to immunocompromised patients. This study is of
importance in this scenario.
This bioenhancing activity of CU has been aptly and
widely used in various ayurvedic formulations. It is one of
the constituents of Hingwadh ghrita, Lashunadh ghrita,
Sidhartak ghrita for psychiatric illness used in abdominal
tumors and in other formulations like Mandurvatak, Darvi
ghrita, and Punnarvamandur. CU is used as yogvahi along with
Hareetakyadi yog, Swarnkshiryad yog, Swarnmakshik bhasma
and Gvakshyadi churana. These preparations are commercially
available in the Indian market. Ghritas are available as semisolid
preparations while bhasms, yogs, and churans are in the powder
form.
ANTICANCER PROPERTIES
CU has antioxidant properties and is a free radical scavenger,
and thus it neutralizes the oxidative stress. Scientists proved
that the pesticides even at very low doses cause apoptosis
of lymphocytes and tissues through fragmentation of DNA
while CU helps the lymphocytes to survive by inhibiting their
apoptosis and by repairing the damaged DNA and is, therefore,
effective as anti-cancer therapy [52,53].
Chemopreventive potential of CU was observed in a study,
which was conducted on 70 Swiss albino mice for 16 weeks.
Papillomas were induced by 7, 12 dimethyl benzanthracene and
later promoted by repeated application of croton oil. In mice
treated with CU, the incidence of tumor (papillomas), tumor
yield, and its burden was statistically less than the untreated
group [54].
Jain et al. studied the effect of CU therapy on various types
of cancers in Mandsaur area. The severity of symptoms (pain,
inflammation, burning sensation, difficulty in swallowing, and
irritation) decreased from day 1 to day 8 with CU therapy.
Percent of patients with severe symptoms decreased from 82.16
to 7.9 on day 8, patients with moderate symptoms increased
from 15.8 to 55.3 and with mild symptoms, patients increased
from 1.58 to 36.34. The severity of symptoms decreased further
with continued CU therapy [15].
Dutta et al. reported the anti-clastogenic and anti-genotoxic
effect of redistilled CUD (RCUD) in human peripheral
lymphocytes, which have been challenged with manganese
dioxide (MnO2) and hexavalent chromium (Cr+6). Protection
in number of chromosomal aberrations and frequency of
micronuclei were more prominent when these cells were
pretreated with CU than simultaneous treatment with
CU [55].
184
IMMUNOSTIMULANT
The use of herbs and minerals (like chavanprash and
panchgavya) for improving the overall resistance of the body
against common infections and pathogens has been a guiding
principal of Ayurveda. Ancient books on Ayurveda state that
consuming CU daily increases the resistance to diseases by
up to 104%. CU enhances the humoral, and cell-mediated
immune response in mice [5]. CUD was found to augment
B- and T-lymphocyte blastogenesis; IgG, IgA and IgM antibody
titers in mice. It has been observed that CU also increases the
phagocytic activity of macrophages and is thus helpful in the
prevention and control of bacterial infections. The level of
both interleukins -1 and - 2 in mice was increased by 30.9%
and 11.0%, respectively, and in rats these levels were increased
significantly by 14.75% and 33.6%, respectively [52]. CUD
has been reported to be a potent and safe immunomodulator,
which increases both humoral, and cell-mediated immunity
in mice.
Cell-mediated immune response was evaluated on various
parameters in a study by Verma et al. using CU for 30 days.
There was a 55% increase in phagocytic index, and a significant
increase (16%) in neutrophil adhesion on regular use of whole
freeze dried CU. CU has the potential to boost the immune
functions by increasing the white blood cells counts and
subsequently reducing the red blood cells count to a certain
extent [56].
Traditional uses of CU
Some of the traditional uses of CU are in fever, where CU along
with pepper, curd and ghee is used; in leprosy, CU is used along
with dhruhardi and in deformities associated with leprosy, it is
used with Nimbuchal, whereas in chronic leprosy, CU is used
along with leaves of Vasaka and kanar, and bark of kuraila and
neem [11]. Local traditional healers in Mandsaur prescribe CU
for worm infestations, to develop immunity and to avoid aging.
They suggest 10-25 ml of CU to be taken on an empty stomach
for the same [15].
CONCLUSIONS
On analyzing the effect of different preparations of CU, FCU
had better activity than CUD [27-32]. Activity of FCU and
CUD from indigenous cows was similar to streptomycin and
tetracycline. Ayurveda also mentions that FCU of indigenous
cows is the best.
More well-planned studies in human subjects are required to
fully assess its potential as an effective antimicrobial agent as
most of the studies quoted are in vitro studies. Comparative
studies between CU obtained from indigenous breeds and of
inbred strains may be undertaken, as ayurveda was written when
inbred strains of cows were not present. Future development of
newer drugs can involve CU in its repository.
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SAGEYA. This is an open access article licensed under the terms

of the Creative Commons Attribution Non-Commercial License (http://
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Source of Support: Nil, Conict of Interest: None declared.
Mol Biol Rep (2014) 41:19671976

DOI 10.1007/s11033-014-3044-6
Effect of treatment of cows urine Gomutra and antioxidants

in alleviating the lindane-induced oxidative stress in kidney
of Swiss mice (Mus musculus)
Girima Nagda Devendra Kumar Bhatt
Received: 5 December 2012 / Accepted: 4 January 2014 / Published online: 16 January 2014
Springer Science+Business Media Dordrecht 2014
Abstract The study aimed to evaluate the effect of cow

urine and combination of antioxidants against lindane
induced oxidative stress in Swiss mice. Male healthy mice,
810 weeks old, weighing 30 5 g were randomly selected
and divided into eight groups, namely, control (C); lindane
(L); antioxidant (A), antioxidant?lindane (A?L), cow urine
(U), cow urine?lindane (U?L), cow urine?antioxidants
(U?A) and cow urine?antioxidants?lindane (U?A?L).
Group C animals were administered only the vehicle (olive
oil); doses selected for other treatments were: lindane:
40 mg/kg b.w.; antioxidants: 125 mg/kg b.w. (vitamin C:
50 mg/kg b.w., vitamin E: 50 mg/kg b.w., a-lipoic acid:
25 mg/kg b.w.) and cow urine: 0.25 ml/kg b.w. In group
A?L and U?L antioxidants and cow urine were administered 1 h prior to lindane administration and in group U?A
and U?A?L cow urine was administered 10 min before
antioxidants. All treatments were administered orally continuously for 60 days. Lindane treated group showed
increased lipid peroxidation, whereas glutathione, glutathione peroxidase, superoxide dismutase, catalase, protein and
endogenous levels of vitamin C and E were significantly
decreased compared to control. Administration of cow urine
and antioxidants alleviated the levels of these biochemical
parameters.
Keywords Antioxidants Cow urine Kidney Lindane
Oxidative stress
G. Nagda (&) D. K. Bhatt

Cancer Biology and Toxicology Research Laboratory,
Department of Zoology, University College of Science,
M L Sukhadia University, Udaipur 313 001, India
e-mail: girima10@gmail.com
Introduction
Kidney is vital organ of the body responsible for segregating the metabolic waste products from the blood.
Accumulation of metabolites of xenobiotics contributes
significantly to its susceptibility to damage kidney [24].
Any nephrotoxic insult would result in accumulation of
waste materials in the blood which in turn may lead to
other toxic manifestations in the body. Toxic injury to the
kidney is known to occur as a result of exposures to halogenated hydrocarbons, such as lindane, carbon tetrachloride and trichloroethylene, and the heavy metals cadmium
and lead [3, 35, 36, 48, 59]. Some of these toxicants cause
acute injury to the kidney, while others produce chronic
changes that can lead to end-stage renal failure or cancer.
Lindane, the gamma isomer of HCH possesses the property of persistence, bioaccumulation and long term toxicity
[32] and fulfills the criteria of POPs i.e., persistent organochlorine pesticides. India has total installed capacity of lindane (technical) production of 1,300 tonnes per annum (tpa),
with two companies producing: Kanoria Chemicals and
Industries Ltd with a capacity of 1,000 tpa, and India Pesticides Limited with 300 tpa capacity. According to data
available from Department of Chemicals and Petrochemicals, Ministry of Chemicals and Fertilizers, between 1995
and 2005, India has produced 5,387 tonnes of technical grade
lindane. In India, lindane formulations are registered for use
in pharmaceutical products for control of head lice and
scabies on people, to control fly, flea, cockroach, mosquito,
bed bug, and beetle populations and for the control of pests in
cotton, sugarcane, pumpkin, cabbage, onion, apple, walnut,
maize, okhra, potato, tomato, cauliflower, radish, cucumber
and beans [15]. Lindane is highly lipophilic and absorbed by
the respiratory, digestive or cutaneous pathways. Its accumulation depends on the duration of the exposure and affect
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tissues in the following order: fat [ brain [ kidney [

muscle [ lung [ heart [ spleen [ liver [ blood [56]. Toxicity
induced by lindane is attributed to oxidative stress as it
induces the release of free radicals and generation of reactive
oxygen species (ROS) [67].
Oxidative stress is defined as a disruption of the prooxidantantioxidant balance in favor of the former, leading
to potential damage [37]. It is a result of one of three
factors: (i) an increase in ROS, (ii) an impairment of
antioxidant defence systems, or (iii) an incapacity to repair
oxidative damage.
A number of studies indicate the toxicity of lindane on
kidney [22, 48]. It has been considered that ROS play an
important role in the pathogenesis of renal injury. Since the
entire range of toxic metabolites in the body is excreted
mainly from the kidney, this organ is endowed with significant antioxidant defense system next only to liver. This
is understandable because ROS play a key intermediary
role in the pathophysiologic processes of a wide variety of
clinical and experimental renal diseases [50, 65].
The human body has a strong inherent synergistic and
multilevel defense mechanism, to combat and counteract
the damage caused by free radicals [41]. This defense is
mediated by endogenous antioxidant system which either
prevent these reactive species from being formed, or cause
their removal before they can damage vital components of
the cell [17]. The excessive free radicals or the suppression
of antioxidant defense of body results into toxicity. In such
conditions, though the body tissue is endowed with enzymatic and non-enzymatic protective systems, but it seems
that the homeostasis of the body fails. In such situations,
the use of exogenous substances with antioxidative
potential becomes important [64].
Antioxidants have gained immense importance in recent
years. The potency of various antioxidants on different
organ systems has been investigated against lindane toxicity [8, 9, 11, 42, 63].
Cow urine or Gomutra is considered sacred in Hindu
mythology and from ancient times it has been used as a
medicine in India. The medicinal properties of cows urine
have been mentioned in Sushrut (45/221) and Charak
(sloka-100) where it is considered useful in treating renal
colic, jaundice, anemia, diarrhea, gastric infection, piles
and skin diseases including vitiligo. It is also considered as
an appetizer and is known to reverse inflammation, a
diuretic as well as a nephroprotective agent. It also acts at
cellular level and generates bioenergy [31]. The analysis of
cow urine has shown that it contains nitrogen, sulphur,
phosphate, sodium, manganese, carbolic acid, iron, silicon,
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Mol Biol Rep (2014) 41:19671976
chlorine, magnesium, melci, citric, titric, succinic, calcium

salts, vitamin A, B, C, D, E, minerals, lactose, enzymes,
creatinine, hormones and gold acids. The cow urine contains those substances, which are present in the human
body and thus its consumption maintains the balance of
these substances and cures incurable diseases [49]. Cow
urine is also used along with herbs to treat various diseases
like fever, epilepsy, anemia, abdominal pain, constipation
etc. by the traditional healers [34].
There is a paucity of information regarding the role of
fresh cow urine and combination of vitamin E, vitamin C
and alpha lipoic acid against lindane toxicity in kidney.
Therefore, in the present study their role in alleviating the
oxidative stress induced by lindane intoxication in kidney
of mice has been investigated. Moreover, the use of cow
urine from ancient times has shown to be quite effective
but its efficiency against lindane induced toxicity and its
combined effect along with the various antioxidants is
hitherto unreported. Hence, present study was undertaken
to fill the lacuna in this regard.
Materials and methods

Chemicals
Lindane (c-HCH) was obtained from Sigma chemicals St.
Louis, Mo, USA (CAS No. 58-89-9 and purity 97 %). vitamin E, vitamin C, a-lipoic acid, sodium azide, thiobarbituric
acid, dinitrophenylhydrazine (DNPH), 2, 2 dipyridyl, and
phenazine methosulphate were obtained from Himedia,
India. Dithiobisnitro benzene (DTNB), reduced glutathione
and bovine serum albumin were purchased from Sisco
Research Laboratories, Mumbai, India. All other chemicals
and solvents used were of analytical grade. Cow urine: Urine
of young cow was collected from local cowshed and stored in
an air tight bottle for further use.
Animals and treatment
Male Swiss mice, weighing 30 5 g and 810 weeks old
were procured from Cadila Health Care Institute, Ahmedabad. Animals were maintained on sterilized rice husk
bedding in polypropylene cages and kept at a temperature
of about 23 3 C with 12 1 h L:D cycle. Animals
were fed on standard pelletal diet (Pranav Agro, Baroda).
Food and water were ad libitum. Experimental protocol
was approved by the Institutional Animal Ethics Committee. Handling of animals was according to the guidelines of
Mol Biol Rep (2014) 41:19671976
1969
Committee for the Purpose of Control and Supervision of

Experiments on Animals (CPCSEA), Ministry of Environment and Forests, Govt. of India.
Dose selection
Dose for lindane was selected after conducting pilot
experiments in our laboratory. LD50 for lindane was found
to be at 60 mg/kg body wt. considering this aspect, a dose
level which may show adverse effect on kidney was
selected as the dose for the present study. Duration of
treatment was based on occupational exposure of workers
i.e., for 2 months during active malaria vector control
programme. Dose selected for lindane was 40 mg/kg body
wt. and duration of treatment was for 2 months i.e.,
60 days. There was no mortality in exposure group during
the study.
Doses for antioxidants were calculated keeping the
doses prescribed for humans and also in accordance with
the previous reports [8, 9, 42]. The combined dose of
antioxidants selected was 125 mg/kg body wt. which
included vitamin C 50 mg/kg body wt., vitamin E 50 mg/kg
body wt. and a-lipoic acid 25 mg/kg body wt. Cow urine
was administered at a dose equivalent to the corresponding
dose for human in ml/kg b.w. i.e., 0.25 ml/kg b.w.
Doses of lindane, vitamin E and lipoic acid were prepared by dissolving in olive oil. Dose of vitamin C was
prepared in distilled water. Cow urine was administered
without any modification.
In the group IV and VIII the antioxidants and cow

urine were administered 1 h prior to lindane administration. In group VI and VII cow urine was administered
10 min before the antioxidants administration. All the
treatments were given continuously for a period of
60 days.
Mice were sacrificed by cervical dislocation at the
end of the scheduled period of 60 days and 24 h after
the last dose treatment. Both the kidneys were blotted
free of blood, weighed and to maintain uniformity in
all groups the right kidney was used for biochemical
analysis. The right kidney (just to maintain uniformity
amongst animals of all groups) was washed with ice
cold physiological saline and a 10 % w/v homogenate
was prepared in 0.1 M phosphate buffer (pH 7.4). The
homogenate was centrifuged at 6,0009g for 10 min
to obtain the supernatant. Supernatant was diluted
five times and used for estimating the biochemical
parameters.
Biochemical analysis
The kidney tissue homogenate was used for the estimation
of lipid peroxidation (LPO) [68], superoxide dismutase
(SOD) [30], catalase (CAT) [16], glutathione peroxidase
(GPx) [54], glutathione (GSH) [40], protein [38], vitamin E
(a-tocopherol) [18] and vitamin C (ascorbic acid) [45].
Statistical evaluation
Experimental protocol
A sub chronic study was done for 60 days and oral route of
dose administration was chosen for all treatments. Mice
were divided into eight groups with minimum of 810
animals in each group.
S. no.
Group no.
Group code
Treatment
1
2
II
III
Values are mean SD and the results obtained were

analyzed using one way ANOVA. Inter group comparisons
were performed by using the least significance difference
(LSD) test. A probability value of P \ 0.05, 0.01 was
considered as statistically significant.
Dose
Duration
Control: vehicle only
Olive oil only
60 days
Lindane
40 mg/kg b.w.
60 days
Antioxidants alone
Combined dose of 125 mg/kg b.w.
60 days
IV
A?L
Antioxidants?lindane
Antioxidant dose of 125 mg/kg b.w. followed

by lindane at 40 mg/kg b.w.
60 days
Cow urine alone
0.25 ml/kg b.w. cow urine
60 days
VI
U?L
Cow urine?lindane
0.25 ml/kg b.w. cow urine ?40 mg/kg b.w. lindane
60 days
VII
U?A
Cow urine?antioxidants
0.25 ml/kg b.w. cow urine ?125 mg/kg b.w. antioxidants
60 days
VIII
U?A?L
Cow urine?antioxidants?lindane
0.25 ml/kg b.w. cow urine ?125 mg/kg b.w.

antioxidants ?40 mg/kg b.w. lindane
60 days
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Mol Biol Rep (2014) 41:19671976
Results
The changes in various biochemical parameters in different
groups have been presented in Graphs 1, 2, 3, 4, 5, 6, 7, 8.
Effect on LPO (Graph 1)
A significant increase (P \ 0.01) of 32.21 % was observed in
the LPO levels after lindane intoxication as compared to control. All the pretreatment groups showed a significant decline
(P \ 0.01) in the LPO levels. 29.21, 27.66, and 29.47 %
decline was observed in the A?L, U?L, and U?A?L groups
respectively as compared to lindane group. The increasing
order of the LPO levels in various groups was as follow:
L [ C [ U?L [ A?L [ U?A?L [ U?A [ U [ A.
Graph. 3 Mean SD values of CAT (lmol of H2O2 decomposed/

min/mg protein) in various groups (a 60 day assessment). a compared
to control, b when compared to lindane, NS non significant,
* significant (P \ 0.05), ** highly significant (P \ 0.01)
Graph. 1 Mean SD values of LPO (nmoL TBARS/g tissue) in

various groups (a 60 day assessment). a when compared to control, b
when compared to lindane, NS non significant, * significant
(P \ 0.05), ** highly significant (P \ 0.01)
Graph. 4 Mean SD values of GPx (mg of GSH consumed/min/

mg protein) in various groups (A 60 day assessment). a compared to
control, b when compared to lindane, NS non significant, * significant
Graph. 2 Mean SD values of SOD (50 % inhibition of NBT/min/

mg protein) in various groups (a 60 day assessment). a compared to
Graph. 5 Mean SD values of glutathione (GSH) (mg/gm tissue)

in various groups (a 60 day assessment). a compared to control,
b when compared to lindane, NS non significant, * significant
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Mol Biol Rep (2014) 41:19671976
1971
control group. The pretreatment groups A?L, U?L and

U?A?L showed a significant increase (P \ 0.01) of about
57.74, 77.64, and 98.26 % respectively, as compared to
lindane group. The increasing levels of SOD in various
groups were as follow: L \ A?L \ U?L \ C \ U?A?
L \ A \ U?A \ U.
Effect on CAT (Graph 3)
Graph. 6 Mean SD values of total protein (mg/100 mg tissue

weight) in various groups (a 60 day assessment). a compared to
Lindane induced kidney toxicity showed a significant

decline (P \ 0.01) of 32.12 % in the CAT activity as
compared to control. A significant increase (P \ 0.01) of
58.91 % in A?L, 32.85 % in U?L and 51.97 % in
U?A?L was observed when compared to lindane. The
cow urine alone group also showed a decrease of 9.82 % as
compared to lindane but the decrease was not significant.
The maximum % of increase was observed in the A?L
group. The increasing order of the enzyme level in all the
eight groups was as follow: L \ U?L \ C \ U?A?L \
A?L \ U \ U?A \ A.
Effect on GPx (Graph 4)
Graph. 7 Mean SD values of endogenous vitamin E (mg/g tissue)

As compared to control animals, the lindane intoxicated

animals showed a significant decrease (P \ 0.01) of
45.19 % in the GPx levels. All the pretreatment groups
showed promising results and brought about a significant
rise of 101.89, 146.37, and 215.30 % in the GPx levels in
the groups A?L, U?L and U?A?L, respectively. The
increasing order of GPx levels in various groups was as
followed: L \ C \ A?L \ U?L \ U?A?L \ A \ U \
U?A.
Effect on GSH (Graph 5)
Graph. 8 Mean SD values of endogenous vitamin C (mg/g tissue)

Effect on SOD (Graph 2)

The SOD levels declined significantly (P \ 0.01) up to
45.96 % in lindane intoxicated mice as compared to
A significant decline (P \ 0.01) of 29.24 % was observed

in the GSH levels after lindane intoxication as compared to
control animals. The pretreatment groups showed a nonsignificant decrease in the levels of GSH as compared to
control which accounted to 0.10 % in the A?L group,
6.69 % in U?L group and 12.37 % in U?A?L group. But
when compared to lindane a significant increase (P \ 0.01)
of 41.18, 31.87, and 23.84 % was seen in the respective
groups A?L, U?L and U?A?L. The increasing order of
GSH levels in different groups was as follow: L \ U?
A?L \ U?L \ U?A \ A?L \ C \ U \ A.
Effect on protein (Graph 6)
A significant decrease (P \ 0.01) of 15.01 % was observed
in the total protein levels of lindane intoxicated animals as
compared to control. The pretreatment groups A?L, U?L
and U?A?L showed a significant rise (P \ 0.01) of 20.00,
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1972
17.08, and 11.95 % respectively, in the levels of total

protein as compared to lindane group. The best results were
shown by the pretreatment of combination of antioxidants
which resulted in about 20.00 % rise in the total protein
levels which had decreased to 15.01 % after lindane
intoxication. The increasing order of protein levels in
various groups is as follows: L \ U?A?L \ U?A \ U?
L \ C \ A?L \ U \ A.
Effect on endogenous vitamin E (Graph 7)
A significant decrease (P \ 0.01) of 34.94 % was registered in the endogenous levels of vitamin E in lindane
group as compared to control. A?L, U?L and U?A?L
groups significantly (P \ 0.01) alleviated the levels of
vitamin E by 86.65, 47.30, and 92.32 %, respectively in
comparison to lindane group. The increasing order of
vitamin E levels in all the groups was as follow:
L \ U?L \ C \ U \ A?L \ U?A?L \ A \ U?A.
Effect on endogenous vitamin C (Graph 8)
As compared to control, a significant decrease (P \ 0.01)
of 50.95 % was observed in the endogenous levels of
vitamin C in animals toxicated with lindane. This decrease
was alleviated significantly (P \ 0.01) when the different
pretreatments were given. In comparison to lindane treated
animals, the animals of A?L, U?L and U?A?L group
showed an increase of 140.95, 100.51, and 107.18 %,
respectively. The increasing order of the vitamin C levels
in all the eight groups was as follow: L \ U?L \ C \
U?A?L\ A?L \ U \ U?A \ A.
Discussion
The results of the present study clearly demonstrate that
LPO significantly increased in kidney after in vivo treatment of lindane. Thus, results of the present study are in
agreement with previous reports where increase in LPO
was also observed due to lindane in different tissues [11,
22, 42, 48, 67]. Moreover, renal LPO levels also increase
due to aflatoxin [61], CCl4 [7], chlorpryfos-ethyl [46], lead
[55], cisplatin [4] and gentamicin [1] toxicity. The increase
in LPO results owing to increase in ROS or alternatively
lindane might inhibit antioxidant molecules and antioxidant enzymes. The support for such an assumption comes
from the findings that lindane reduces antioxidant molecules and antioxidant enzymes [8, 42, 48] which is also
observed in the present study.
The pretreatment with vitamin C, E, lipoic acid and cow
urine in the groups A?L, U?L and U?A?L significantly
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Mol Biol Rep (2014) 41:19671976
lowered the LPO levels as compared to mice treated with

lindane alone. Earlier reports have also shown that supplementation with vitamin C and E attenuated the LPO
levels decreased due to cisplatin [4] and chlorpyrifos [46].
Lipoic acid also ameliorates renal oxidative stress [6].
Moreover, combination of lipoic acid and vitamin E [11],
vitamin C, E, lipoic acid and resveratrol [8] and vitamin C,
E and lipoic acid [42] ameliorated the lindane induced
increased LPO levels.
The results reveal that the pretreatment of combination
of antioxidants and cow urine (U?A?L) was the most
effective in lowering the LPO levels followed by combination of antioxidant (A?L) and cow urine pretreatment
(U?L).
Due to increase in LPO the amount of free radicals
crosses the threshold level. Nevertheless, decrease in the
activities of CAT, SOD, GPx and GSH further deteriorates
the situation and enhance the formation of lipid peroxides.
It could be assumed that lindane might have caused LPO
by inhibiting the antioxidant enzymes, molecule, vitamin E
and C. The mechanism by which lindane induces oxidative
stress involves the activity of cyt P450 system resulting in
the generation of superoxide radicals [21].
Similar to earlier reports, results of the present study
also show a decrease in SOD and CAT levels in the lindane
treated group and in all such cases the main culprit being
superoxide radicals [9, 42, 48]. Renal SOD and CAT levels
were also lowered due to chlorpryfos-ethyl [46], aflatoxin
[61], CCl4 [7], lead [55], cisplatin [4], alcohol [60] and
gentamicin [1] toxicity.
The decreased activity of SOD in kidney in lindane
treated mice may be due to the enhanced lipid peroxidation or inactivation of the antioxidative enzymes or
could result from inactivation by hydrogen peroxide or
glycation of the enzyme [62]. This would cause an
increased accumulation of superoxide radicals, which
could further stimulate lipid peroxidation. A decrease in
SOD activity favors the accumulation of superoxide
radicals, which in turn inhibit CAT [33] as also seen in
the present study.
All the pretreatment groups showed alleviation in the
levels of SOD and CAT. Earlier reports have shown that
vitamin C and E are capable of increasing the renal SOD
and CAT levels [4, 7, 46]. Combination of lipoic acid,
vitamin C, E and resveratrol [9] and lipoic acid and vitamin
C and E [42] attenuated the SOD and CAT levels in brain
and testis, respectively of lindane treated mice. Amongst
the three pretreatments the maximum amelioration in the
levels of SOD was observed in case of U?A?L group
followed by U?L group and was least in A?L group.
Moreover, in the present study the protection offered
by combination of antioxidants (A?L group) in ameliorating the CAT levels was the maximum followed by
Mol Biol Rep (2014) 41:19671976
combination of antioxidants and cow urine (U?A?L

group) and minimum in the only cow urine (U?L) group.
The reduction in the activity of SOD, CAT enzymes
may result in a number of deleterious effects due to the
accumulation of superoxide anion and hydrogen peroxide
[58]. The elimination of H2O2 is either effected by CAT or
GPx [69].
The level of GPx in the exposed group were lowered
which is in accordance with the earlier studies where lindane caused a similar decrease in the levels of GPx in
various tissues [42, 48]. Renal GPx levels were found to be
decreased due to chlorpryfos-ethyl [46], aflatoxin [61],
acetaminophen [27], cisplatin [4] and CCl4 [7] toxicity.
The decreased levels can be attributed to either increased
H2O2 generation or decreased GSH concentration because
GSH is one of the substrates for GPx [57]. A decreased
GSH concentration was observed in the present study.
Decreased activity of GPx may also result from radical
induced inactivation and glycation of the enzymes [26].
Because of the decreased GPx activity the accumulation
of H2O2 may cause inhibition of SOD activity [10]. During
oxidative stress, inactivation of GPx may occur, and on the
other hand superoxide anion (O2 ) itself can inhibit peroxidase function [12]. So GPx must be considered to be
complementary to SOD [43].
The pretreatment with vitamin C, E, lipoic acid and cow
urine in groups A?L, U?L and U?A?L significantly
improved the levels of GPx. It has been shown in earlier
reports that vitamin C and E are capable of increasing the
renal GPx levels [4, 7, 46] and combination of vitamin C, E
and lipoic acid ameliorated the testis GPx levels lowered
due to lindane [42]. It is also concluded that the significant
increase in the GPx levels was the maximum in case of
U?A?L group followed by U?L group and was least in
A?L group.
In the present study, the levels of GSH were decreased
significantly in lindane treated group indicating the oxidative
stress caused by lindane as also reported earlier [8, 22, 42, 48,
67]. Decrease in renal GSH levels has also been documented
in a case of alcohol [60], aflatoxin [61], gentamicin [1, 29],
acetaminophen [27] and cisplatin [4] toxicity.
The decrease in the GSH level as observed in the present
study can be due to increased utilization of GSH for
metabolism of lipid hydroperoxides by GPx or interaction
of GSH with free radicals. Similar analogy is being also
drawn in the earlier reports [5, 23].
The levels of GSH were significantly ameliorated after
the pretreatment with combination of antioxidants namely,
vitamin C, E and a- lipoic acid (A?L group), cow urine
(U?L group) and combination of antioxidants and cow
urine (U?A?L group). Earlier studies have shown that
vitamin C, E and lipoic acid attenuates the decreased renal
GSH levels [4, 29]. Lindane induced decreased GSH levels
1973
were ameliorated by combination of lipoic acid, vitamin C,

E and resveratrol [8] and lipoic acid, vitamin C and E [42]
in brain and testis, respectively. The significant increase in
GSH levels in the pretreatment groups was least in
U?A?L group; intermediate in U?L group and maximum
in A?L group.
The present study reveals that lindane inhibits GPx and
CAT due to its capacity to generate ROS, which will result
in H2O2 accumulation. The increased H2O2 in turn could
cause SOD inhibition resulting in increased production of
superoxide radicals. Thus, increased production of superoxide radicals would inhibit both CAT and GPx. Treatment
with the adopted formulation reduced the level of lipid
peroxides indicating the effective antioxidant property of
the combination as well as cow urine alone in the moderation of tissue damage.
Antioxidant defense system protects the aerobic organism from the deleterious effects of reactive oxygen
metabolites. Vitamin E, a major lipophilic antioxidant and
vitamin C, play a vital role in the defense against oxidative
stress [53]. In our study, the levels of vitamin E and C were
decreased significantly during lindane intoxication. This is
in agreement with the previous reports that oxidative stress
results in deficiency in vitamin C and E [60, 61]. The
increased oxidative stress due to lindane intoxication might
have resulted in excess utilization of vitamin C and E,
consequently, depleting their levels. The observed decrease
in the level of kidney ascorbic acid and alpha tocopherol in
lindane treated group could be as a result of increased
utilization of these antioxidants in scavenging the free
radicals generated due to lindane.
Moreover, it is well established that GSH in blood keeps
up the cellular levels of the active forms of vitamin C and
vitamin E by neutralizing the free radicals. When there is a
reduction in the GSH the cellular levels of vitamin C is also
lowered, indicating that GSH, vitamin C, and vitamin E are
closely interlinked to each other [61]. In agreement with
these reports, the decreased levels of GSH, vitamin C and
vitamin E on lindane administration were observed in our
study.
The pretreatment with combination of antioxidants in
group A?L, with cow urine in group U?L and combination of antioxidants and cow urine both in group U?A?L,
resulted in significant increase in the endogenous levels of
vitamin C and E. The amelioration in the endogenous
levels of vitamin C was the maximum in A?L group,
followed by U?A?L group and minimum in U?L group.
In contrast, the significant increase in the levels of vitamin
E was the maximum in U?A?L group; intermediary in
A?L group and least in U?L group. The increase in levels
of vitamin C and E is obvious in the pretreatment groups
A?L and U?A?L as these were exogenously supplied
with both vitamin C and E, however, the cow urine alone
123
1974
(U?L) group also showed significant elevation in the

levels of the two vitamins. This could be attributed to the
composition of cow urine which is said to contain vitamins.
The analysis of total proteins is important for estimating
the degree of damage in the body. The protein profile of the
cells is indicative of the physiological status of animal and
there exists dynamic equilibrium between the synthetic and
degenerative pathways with these biomolecules. In the
present study a decline in the total proteins was observed
after lindane intoxication which can be due to decreased
protein synthesis or increased protein loss. Similar reduction in the levels of total proteins due to lindane have been
reported [9, 42, 48]. It is also reported that renal toxicity
due to CCl4 [7] and gentamicin [1] causes a similar
decrease in protein levels. Decreased protein levels could
be attributed to decreased feed consumption, maldigestion
or malabsorption, hepatic dysfunction [13, 47]. Reduced
protein levels can also be ascribed to increased urinary
excretion owing to kidney damage [66] and glomerular
apparatus or reduced protein synthesis [39]. Prabhakaran
and Devi [51] have proposed that a toxicant can affect the
protein content of the tissue either by inhibiting RNA
synthesis or inhibiting of amino acids into the polypeptide
chain.
The pretreatment groups showed an elevation in the
protein levels which is in accordance with earlier reports
where administration of either vitamin C, E or in combination and combination of vitamin C, E and lipoic acid and
resveratrol elevated the decreased protein levels [7, 9, 42].
The most effective pretreatment was that of combination of
antioxidants (A?L group); cow urine pretreatment group
(U?L) was moderately effective and least effective was
pretreatment with combination of antioxidants and cow
urine both (U?A?L group).
Hypoglycemic [44], cardio-respiratory effect [20],
immunomodulatory [14], antigenotoxic and antioxidant
properties in vitro [34], anticlastogenic [19] and chemoprotective [52] effects of distillate and redistillate of cow
urine have been reported. Attenuation of CCl4 induced
hepatotoxicity by Panchagavya ghrita (prepared by cow
milk, cow urine, cow dung, ghee and curd) [2] and cow
urine distillate [25] have also been reported. Efficacy of
cow urine therapy has been evaluated on cancer patients
[28]. The amelioration of oxidative stress by fresh cow
urine has not been reported so far and this work seems to be
the first report.
Thus it is inferred that the combination of antioxidants
taken in the present study are quiet helpful in mitigating and
modulating the oxidative stress in the kidney caused due to
lindane. Moreover, cow urine treatment also modulated the
oxidative stress parameters caused by lindane. From the
results it is apparent that the given combination of antioxidants and cow urine act synergistically in reducing lindane
123
Mol Biol Rep (2014) 41:19671976
induced dysfunction. The analysis of the results of the

present study reveals the efficacy of cow urine against
oxidative stress. It can be safely concluded that the suggested combination of vitamin C, vitamin E, alpha lipoic
acid and cow urine can prove to be beneficial in a number of
ailments. The highlight of the investigation is the efficiency
of cow urine against the pesticide toxicity which can open
new insights in the field of medicine. Cow urine can prove
to be an effective co-remedy for oxidative stress. This study
emphasizes the importance of antioxidants and cow urine
which could be beneficial in the therapeutic world for the
treatment of various disorders implicating oxidative stress.
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ISSN NO 2320-5407
International Journal of Advanced Research (2013), Volume 1, Issue 5, 71-78
Journal homepage: http://www.journalijar.com
INTERNATIONAL JOURNAL
OF ADVANCED RESEARCH
RESEARCH ARTICLE
Evaluation of Anticancer properties of Taxusbaccata and Badri cow urine in mice: Clinicohematological
study
Ankita Joshi, and R.S. Chauha
1. Post Doctoral Fellow, Cell Culture Laboratory, Institute of Biotechnology, G.B. Pant University of Agriculture
and Technology, Patwadangar-263128, Nainital, Uttarakhand, INDIA.
2. Campus Director, Institute of Biotechnology, G.B. Pant University of Agriculture and Technology, Patwadangar263128, Nainital, Uttarakhand, INDIA.
Manuscript Info
Abstract
Manuscript History:
In the present investigation, the anticancerous effect of Taxusbacaata and

distilled Badri cow urine was studied in mice for clinicohematology and
body weight for a period of six month at an interval of 15 days. The study
revealed that the values of total leucocyte count (TLC), absolute lymphocyte
count (ALC) and absolute neutrophil count (ANC) were significantly
increased in the treated groups of mice either by CUD alone and in
combination withTaxusbaccata extracts. At180th day, it was found that there
was an increase in body weight, Hemoglobin content (Hb), total erythrocyte
count (TEC), total leucocyte count (TLC), absolute lymphocyte count (ALC)
and absolute neutrophil count (ANC) levels in CUD +A treated group as
23%, 23.99%, 41%, 40%, 40.31%, and 40.13%, respectively. This clearly
indicate the, increase in vitality and defence mechanism of body which in
turn helps in further healing of cancer.
Received: 12 June 2013

Final Accepted: 23 June 2013
Published Online: July 2013
Key words:
Badricow urine,
Taxusbaccata, Mice,
Body weight, hematology.
Copy Right, IJAR, 2013,. All rights reserved.
Introduction
Cancer is a disease involving dynamic changes in
genome and is characterized by the uncontrolled,
uncoordinated and purposeless proliferation of
malignant cells and their ability to spread, either by
growth in the adjacent tissue through invasion or by
implantation at distant sites through metastasis.
World cancer report issued by International Agency
for Research on Cancer (IARC) reported in 2003 that
cancer rate is set to increase at an alarming rate
globally. The 5-year relative survival rate for all
cancers diagnosed between 1999-2005 is 68%, up
from 50% in 1975-1977. (Kinzler and Vogelstein,
2002)(Jemal et al., 2011).
In India about 70% of population obtains medical
help from private practitioners and half of those who
seek medicinal help obtain it from alternative and
traditional medicine (Kumar et al., 2004). Poverty
and socioeconomic status are other hurdles in
treatment (Pal and Mittal, 2004). American cancer
society defines complementary and alternative
medicines (CAM) simply as anything which is not
conventional (Zollman and Vickers, 1999; Park et al.,

2003). There are various CAM used for cancer
patient worldwide viz. Herbal medicine, acupuncture,
Ayurveda, biological agents, traditional Chinese
medicines, meditation and yoga etc. However, use of
herbs for cancer treatment is very popular throughout
the world.
Distilled cow urine protects DNA and
repairs it rapidly as observed after damage due to
pesticides. It protects chromosomal aberrations by
mitocycin in human leukocyte. Cow urine helps the
lymphocytes to survive and not to commit suicide
(apoptosis). Pathogenic effect of free radicals are
prevented through cow urine therapy. Use of cow
urine one can get the charm of a youth as it prevents
the free radicals formation. Taxus baccata,
commonly known as THUNER, which is mainly
found in high altitude area like, Patwadangar,
Nainital India also had anticancer and antiviral
properties. It is a small to medium-sized evergreen
tree, growing 10-20 m tall, exceptionally up to 28 m.
It is relatively slow growing, but can be very longlived, with the maximum recorded trunk diameter of
71
ISSN NO 2320-5407
4 m probably only being reached in around 2,0004,000 years. Thuner is the oldest plant at high altitude
region of Uttarakhand. Most parts of the tree are
toxic, except the bright aril surrounding the seed,
enabling ingestion and dispersal by birds. The major
toxin is the alkaloid taxane. Phytochemical analysis
of extracts of leaves and bark showed the presence of
lignans, flavonoid, glycosides, sterols, sugar, amino
acid, and triterpenoid, alkaloids, steroids, tannins,
mucilage, fixed oil, phenolic compounds and
protein.The leaves are the principal source of taxol;
the anti-cancer drug, but has not been widely
exploited in this connection (Hartzell, 2003).
Considering the severity of cancer as a
disease of man and animals and complexity of
therapeutic approaches and their harmful side effects,
it was planned to study the effect of Taxusbaccata
preparation along with cow urine distillate in mice as
measured through clinical haematology

1.
Extract preparation
Extracts of leaves and bark of T. baccata were

prepared by applying the standard methods with
different solvents like; Aqueous, ethanol, methanol
and ether as described by Govindachariet al., (1999)
and Udupaet al., (1995).
In-vivo study
2. Experimental design
Present study was performed in mice maintained in
the experimental animal house in Institute of
Biotechnology, G.B. Pant University of Agriculture
and Technology, Patwadangar, Nainital, Uttarakhand,
India. A total of 97 animals were equally divided into
11 groups. The mice were housed in clean
polypropylene cages and fed adlibitum with
commercially available feed and water. The
experiment was carried out in accordance with the
Institutional Animal Ethical Committee (IAEC), G.B.
Pant University of Agriculture and Technology,
Pantnagar, Uttarakhand, India. The 11 groups
were:Control group(9 mice), Negativecontrol(DEN
treated mice)(8 mice), CUD(without DEN)(8 mice),
A (Aqueous extract of leaves of Taxusbaccata)(8
mice),
(Ethanolic
extract
of
leaves
of
Taxusbaccata)(8 mice), G(Methanolic extract of bark
of Taxusbaccata)(8 mice), H(Ether extract of Bark of
Taxusbaccata)(8
mice),
CUD(Cow
Urine
Distillate)(with DEN)(8 mice), CUD+A(8 mice),
CUD+B(8 mice), CUD + G(8 mice), CUD+H (8
mice).
Single dose of diethyl nitrosamine (DEN) @ 200
l/kg body weight was given to each mice of
negative control group and tests groups. 500 ml of
each extract were made by adding 20% of extract in

500ml of distilled water (Kumar et al., 2004b). The
mice of 9 test groups were given different extracts of
taxusbaccata alone and in combination with CUD
(2ml/day/mice), daily p.o., from day 1 for 6 months;
however, the mice of negative control group were
maintained with routine feed and water.Body weight
of mice were taken regularly at an interval of 15 day
till the end of the experiment.Total leucocyte count
(TLC), absolute neutrophil count (ANC), absolute
leucocyte count (ALC), hemoglobin, total erythrocyte
count (TEC) and hemoglobin (Hb) content of all the
experimental animals in different groups were
determined regularly at an interval of 15 day till the
end of the experiment as per in standard procedures
(Chauhan, 2005).
Results
In-vivo study was carried out in mice using DEN as
carcinogen and plant extracts alone and/or
combination with CUD as test material for a period
six month.
Body Weight
Body weight of mice were taken in gram at an
interval of 15 days till the end of experiment. Data of
body weight change during experiment were givens
in table-1. Initially, the mean bodyweight of control
was 21.471.21 gm and after 6 month the mean body
weight of mice was 25.861.87 gm. In DEN
(negative control) treated group the initial mean body
weight at zero day of experiment was 22.731.33 gm,
which decreased to 19.161.81 gm at the end of
experiment. But in CUD treated group mean body
weight at zero day was 22.431.36 gm and at the end
of experiment it was 23.911.21 gm.CUD treated
group in which the carcinogen has been given, the
initial mean body weight at zero day was 21.421.56
gm. After the end of experiment, the mean body
weight was 21.061.91 gm. In test group A, the zero
day mean body weight 23.131.54 gm, which
marginally increased at the end of experiment to
23.521.01gm.In the group CUD+A the mean in
body weight at zero day was 21.361.47 gm which
was 26.480.902 at the end of experiment. In the
group CUD+B, the mean body weight was
22.761.44 gm at zero day and was 24.801.09 gm at
the end of experiment. In CUD+G and CUD+H
groups, the mean body weight at zero day was
22.971.37 gm and 22.811.21 gm which was
increased to 24.670.941 gm and 24.601.01 gm at
the end of the experiment. In group B, G and H, the
mean body weight at zero day was found 22.811.26
gm, 22.411.32 gm and 22.511.28 gm respectively
and at the end of experiment the body weight reaches
72
ISSN NO 2320-5407
to
23.401.02, 23.331.91 and 23.281.89,

respectively.
Table 1: Body weight in gm of experimental mice at an interval of 15 days (MeanSE).
Groups/Days
0 Day
15
30
45
60
75
90
105
120
135
150
165
180
Control
CUD (no
DEN)
DEN
21.471.21*
22.431.36*
22.521.32
22.481.29*
22.921.47
22.511.33
23.171.07
22.541.42*
23.191.36
22.611.62**
24.311.41
22.791.46
24.561.53
22.941.53
25.071.43
23.081.67*
25.190.947
23.481.82
25.371.12
23.541.29*
25.691.49
23.671.68
25.720.989
23.821.92
25.861.87
23.911.21**
22.731.33**
23.121.42
23.491.36*
23.791.21*
24.011.36
24.671.17**
24.921.07*
25.160.941
24.011.21*
23.721.06
22.091.09*
21.871.31*
19.161.81
CUD
21.421.56*
21.621.41*
21.711.52*
22.071.02
22.671.17**
22.891.19
23.121.23*
22.921.16
22.660.941
22.521.12
22.261.07
22.121.13*
21.061.91*
23.131.54*
23.631.71
24.761.23
24.931.32**
25.111.16*
25.721.21
25.911.47
24.371.41**
24.871.31
23.851.03**
23.711.07
23.681.13
23.521.01*
22.811.26
23.121.21**
23.711.28
23.911.17
24.111.24*
24.521.31**
25.031.28*
24.761.19*
24.101.07*
23.811.23
23.711.16*
23.671.09
23.401.02*
22.411.32
22.911.61*
23.141.10**
23.571.17
23.871.36*
24.101.30
24.471.28
24.321.21*
24.101.07
23.731.22*
23.640.940
23.501.11
23.331.91*
22.511.28*
22.681.31
22.911.12*
23.411.21**
23.621.31
23.821.40*
23.981.33
23.901.21
23.811.17
23.631.07
23.481.21
23.391.07**
23.281.89
A+CUD
21.361.47**
21.731.32*
22.071.12**
22.391.39
22.871.42*
23.011.25
23.931.61**
24.031.71*
24.180.981**
25.811.42*
25.911.07*
26.361.03
26.480.902**
B+CUD
22.761.44*
22.911.51*
23.571.07
23.911.40**
23.961.31
24.031.61
24.181.42*
24.431.27
24.891.19
24.971.36*
24.911.17
24.881.13
24.801.09
G+CUD
22.971.37
23.071.27
23.461.21*
23.711.41**
23.961.31*
24.101.31*
24.571.19
24.811.17**
24.951.16*
24.891.27*
24.841.11
24.721.08*
24.670.941
H+CUD
22.811.21*
22.901.20*
22.961.17
23.071.31
23.481.32**
23.691.20
23.841.23
23.961.19
24.971.23
24.901.21
24.781.31**
24.671.08
24.601.01*
Significant difference in comparison to control (*p0.5 and **p0.01)
Table 2: Total erythrocyte count (TEC) (x 106/cumm) of experimental mice at an interval of 15 days (MeanSE).
Groups/Days
15
30
45
60
75
90
105
120
135
150
165
180
Control
6.820.13
6.860.17
6.920.27
6.970.16
7.060.31
7.140.24
7.160.41
7.180.37
7.190.34
7.200.17
7.210.19
7.230.23
7.250.27
CUD (NO
DEN)
6.290.52*
6.360.41*
6.520.63
6.700.13*
6.830.46
6.940.32
7.030.21**
7.190.61*
7.220.73*
7.250.49*
7.290.36
7.340.51**
7.400.42
DEN
6.870.51
6.890.62**
6.960.71*
6.980.18*
7.080.42*
7.110.52*
7.010.63
6.940.87*
6.660.33*
6.060.61
5.040.12**
4.710.43
3.610.46*
CUD
6.420.83
6.480.17
6.630.21*
6.690.47*
6.780.82*
6.830.27*
6.910.16*
7.030.51*
7.140.21
7.010.32**
6.810.27
6.710.61
6.680.53
6.310.37**
6.380.41
6.480.28*
6.630.46*
6.890.72*
6.960.69
7.090.33*
7.470.38
7.340.28
7.120.48
7.190.59*
7.230.72
7.320.58
6.330.61
6.450.42*
6.610.47
6.750.51
6.950.80
7.080.72**
7.220.61
7.450.56
7.290.51*
7.090.62
7.150.68*
7.210.52*
7.290.57**
6.390.41
6.410.39
6.540.41*
6.710.43
6.900.53
7.050.62**
7.170.47
7.390.54**
7.060.57
6.890.62
7.050.41*
7.150.46
7.200.37
6.370.52*
6.390.59*
6.530.47
6.680.45
6.700.52**
6.720.58
6.960.43
7.150.48
7.410.51**
7.690.48
7.350.61
7.230.42*
7.180.41**
A+CUD
6.310.97**
6.460.818
6.790.17*
6.980.42**
7.090.78
7.350.18*
7.490.19**
7.830.63*
7.980.76
8.380.84
8.730.39**
8.830.36*
8.900.47
B+CUD
6.370.53
6.400.61**
6.660.59
6.770.41*
6.810.47*
6.960.53
7.090.12
7.180.35
7.210.61*
7.400.69**
7.770.63
8.150.71
8.590.62*
G+CUD
6.420.59*
6.440.53
6.790.49
6.830.50
6.930.60
7.070.59*
7.180.57
7.310.51
7.770.53
7.860.59
7.720.47*
8.190.44
8.510.42
H+CUD
6.350.61
6.400.57
6.550.64**
6.720.51*
7.130.57
7.450.63
7.680.53*
7.880.56*
8.040.47**
8.130.52
8.290.56
8.440.48**
8.480.43*
Haematological parameters
Data of TEC is expressed in number of
cellsx106/cumm and is mentioned in Table 2. Initially
the TEC of control was 6.820.13 x 106/cumm and
after 6 month, TEC of experimental mice was
7.250.27 x 106/cumm. In DEN treated (negative
control) group the initial TEC at zero day of
experiments was 6.870.51 x 106/cumm which
decreased to 3.610.46 x 106/cumm significantly at

the end of experiment. But in CUD treated group, the
TEC at zero day was 6.290.52 x 10 6/cumm and
7.400.42 x 106/cumm at the end of experiment.
CUD treated group in which the carcinogen had also
been given, the initial TEC at zero day was 6.420.83
x 106/cumm. After the end of experiment, it was
6.680.53x 106/cumm. In test group A, the zero day
73
ISSN NO 2320-5407
TEC was 6.310.37 x 106/cumm. The TEC decreased

to 7.320.58 x 106/cumm at 180 day of experiment.
In group CUD+A the total erythrocyte count, at zero
day was 6.310.97 x 106/cumm which was increased
to 8.900.47 x 106/cumm at the end of experiment.
Group CUD+B had 6.370.53 x 106/cumm TEC at
zero day and 8.590.62 x 106/cumm at the end of
experiment, respectively. In CUD+G and CUD+H
groups the TEC at zero day were 6.420.59 x
106/cumm and 6.350.61 x 106/cumm, respectively
and were 8.510.42 x 106/cumm and 8.480.43 x
106/cumm at the end of the experiment. In group B, G
and H, the TEC at zero day was found 6.330.61 x
106/cumm, 6.390.41 x 106/cumm and 6.370.52 x
106/cumm, respectively and at the end of experiment
increased to 7.290.57 x 106/cumm, 7.200.37 x
106/cumm and 7.180.41 x 106/cumm.
Data of TLC is expressed in no. of cells x 103/cumm
and is presented in Table-3.Initially the TLC count of
control group was 8.910.30 x 10 3/cumm and after 6
month the TLC was 12.110.04 x 103/cumm. In DEN
(negative control) group the initial TLC at zero day
of experiments was 9.123.20 x 103/cumm which
was decreased to 3.021.45 x 103/cumm. But in CUD
treated group, the TLC at zero day was 8.654.31 x
103/cumm and 9.534.67 x 103/cumm at the end of
experiment.
In CUD treated group, the initial
TLC at zero day was 9.025.31 x 103/cumm which
decreased to 7.813.11 x 103/cumm. In test group
like A, the zero day TLC was 9.085.68 x 10 3/cumm
which
was
decreased
to
8.643.08
x
103/cumm.CUD+A had the zero day mean TLC
count as 8.974.02 x 103/cumm which was increased
to 12.612.17 x 103/cumm, at the end of experiment.
Group CUD+B has 8.896.31 x 103/cumm at zero
day and was 10.553.18 x 103/cumm at the end of
experiment.In group B, G and H, the TLC at zero day
was observed as 9.016.81 x 103/cumm, 9.106.07 x
103/cumm and 9.077.03 x 103/cumm, respectively
and at the end of experiment 8.593.19 x 10 3/cumm,
8.333.61 x 103/cumm and 8.304.1 x 103/cumm
respectively. In groups CUD+G and CUD+H the
TLC at zero day was 9.115.98 x 103/cumm,
9.036.19 x 103/cumm and was 10.213.81 x
103/cumm and 10.053.14 x 103/cumm at the end of
the experiment, respectively.
Data of ALC is expressed in no. of cells x
103/cumm and is presented in Table-4.Initially the
ALC count of control group was 4.300.69 x
103/cumm and after 6 month the ALC was 5.920.98
x 103/cumm. In DEN (negative control) group the
initial ALC at zero day of experiments was 4.410.81
x 103/cumm which was decreased to 1.360.47 x

103/cumm. But in CUD treated group, the ALC at
zero day was 4.130.70 x 103/cumm and 4.630.43 x
103/cumm at the end of experiment.In CUD treated
group, the initial ALC at zero day was 4.430.84 x
103/cumm which decreased to 3.760.53 x
103/cumm. In test group like A, the zero day ALC
was 4.420.91 x 103/cumm which was decreased to
4.190.27 x 103/cumm.CUD+A had the zero day
mean ALC count as 4.390.87 x 103/cumm which
was increased to 6.160.42 x 103/cumm, at the end of
experiment. Group CUD+B has 4.360.89 x
103/cumm at zero day and was 5.160.80 x
103/cumm at the end of experiment.In group B, G and
H, the ALC at zero day was observed as 4.400.94 x
103/cumm, 4.480.73 x 103/cumm and 4.440.84 x
4.180.94 x 103/cumm, 4.010.37 x 103/cumm and
3.980.77 x 103/cumm respectively. In groups
CUD+G and CUD+H the ALC at zero day was
4.410.80 x 103/cumm, 4.420.79 x 103/cumm and
was 4.900.28 x 103/cumm and 4.890.11 x
103/cumm at the end of the experiment, respectively.
Data of ANC is expressed in no. of cells x
103/cumm and is presented in Table-5.Initially the
ANC count of control group was 4.570.72 x
103/cumm and after 6 month the ANC was 6.000.99
x 103/cumm. In DEN (negative control) group the
initial ANC at zero day of experiments was
4.690.83 x 103/cumm which was decreased to
1.490.52 x 103/cumm. But in CUD treated group,
the ANC at zero day was 4.380.78 x 10 3/cumm and
4.710.47 x 103/cumm at the end of experiment.
In CUD treated group, the initial ANC at
zero day was 4.510.97 x 103/cumm which decreased
to 3.880.56 x 103/cumm. In test group like A, the
zero day ANC was 4.600.97 x 103/cumm which was
decreased to 4.300.31 x 103/cumm.CUD+A had the
zero day mean ANC count as 4.460.89 x 10 3/cumm
which was increased to 6.250.45 x 10 3/cumm, at the
end of experiment. Group CUD+B has 4.410.92 x
103/cumm at zero day and was 5.240.82 x
103/cumm at the end of experiment.In group B, G and
H, the ANC at zero day was observed as 4.580.99 x
103/cumm, 4.560.75 x 103/cumm and 4.510.87 x
4.260.96 x 103/cumm, 4.110.38 x 103/cumm and
4.090.79 x 103/cumm respectively. In groups
CUD+G and CUD+H the ANC at zero day was
4.520.84 x 103/cumm, 4.500.85 x 103/cumm and
was 5.010.29 x 103/cumm and 4.960.12 x
103/cumm at the end of the experiment, respectively.
74
ISSN NO 2320-5407
Table 3: Total leucocyte count (TLC) (x 103/cumm) count of experimental mice at an interval of 15 days (MeanSE).
Groups/Days
15
30
45
60
75
90
105
120
135
150
165
180
Control
8.910.30
9.060.17
9.380.32
9.910.37
10.080.51
10.210.62
10.530.02
10.730.12
11.050.81
11.320.91
11.510.07
11.870.47
12.110.04
CUD (NO
DEN)
DEN
8.654.31
8.714.36**
8.864.51*
8.934.70
9.085.01*
9.135.21
9.183.24*
9.204.22
9.274.56**
9.324.61
9.384.28
9.464.39*
9.534.67
9.123.20**
9.213.52*
9.434.07**
9.893.81
8.073.61
7.423.82
6.412.01
6.123.10*
5.872.12*
4.313.19
4.162.16
4.081.87*
3.021.45*
CUD
9.025.31*
9.135.27
9.315.17
9.674.81**
9.894.72
10.074.55
9.914.31**
9.734.24
9.414.27*
9.013.69
8.233.16**
8.113.08*
7.813.11
9.085.68*
9.155.51*
9.285.42
9.575.23
9.795.05**
9.984.93
9.784.61*
9.614.34
9.324.21*
8.914.07
8.833.21
8.793.12
8.643.08
9.016.81*
9.116.72
9.196.61**
9.416.52
9.616.37*
9.775.83*
9.585.61
8.915.37
8.875.21*
8.815.08
8.794.61*
8.684.52
8.593.19**
9.106.07
9.165.91*
9.245.82
9.335.74**
9.515.41*
9.675.34*
9.485.28
8.875.05
8.764.81
8.694.23*
8.594.07
8.413.82
8.333.61
9.077.03
9.116.87
9.156.67**
9.216.41
9.385.91
9.475.80
9.315.47*
8.835.32**
8.735.16
8.624.77*
8.574.41*
8.464.21*
8.304.10
A+CUD
8.974.02**
9.214.47
9.493.32
9.893.77*
10.082.01*
10.332.61*
10.693.16*
10.934.03
11.312.12**
11.673.41*
11.914.91*
12.253.53*
12.612.17**
B+CUD
8.896.31*
9.126.27*
9.256.08
9.495.61*
9.685.32*
9.875.17*
9.675.03
9.474.71
9.734.57
9.844.31
10.084.17
10.674.08*
10.553.18
G+CUD
9.115.98
9.185.81*
9.275.61
9.395.47*
9.595.27
9.715.17
9.515.09**
9.304.81
9.264.67
9.424.51
10.024.41**
10.174.32*
10.213.81*
H+CUD
9.036.19
9.136.01
9.195.81**
9.275.72
9.445.51
9.595.42*
9.405.31
9.315.06*
9.124.87*
9.094.62*
9.514.41
9.884.17
10.053.14
Significant difference in comparison to control(*p0.5 and **p0.01)

Table 4: Absolute lymphocyte count (ALC) (x 106/cumm) of experimental mice at an interval of 15 days (MeanSE).
Groups/Days
15
30
45
60
75
90
105
120
135
150
165
180
Control
4.300.69
4.410.42
4.590.24
4.810.49
4.920.19
4.980.33
5.170.54
5.230.68
5.410.57
5.530.28
5.620.31
5.810.73
5.920.98
CUD (NO
DEN)
DEN
4.130.70*
4.240.72
4.310.69*
4.370.58*
4.430.42
4.450.39*
4.480.69**
4.490.84
4.520.92*
4.540.14
4.550.28*
4.610.63*
4.630.43
4.410.81*
4.430.61
4.560.71
4.860.24*
3.920.92**
3.590.43*
3.050.64
2.940.59
2.810.36
2.080.31*
1.920.27*
1.900.17
1.360.47*
CUD
4.430.84*
4.410.64
4.540.84
4.740.18
4.830.29
4.850.36
4.850.47*
4.720.53*
4.590.61
4.350.74*
3.960.89
3.890.92
3.760.53
4.420.91
4.440.738
4.510.42**
4.670.75
4.780.94*
4.860.86
4.780.73
4.660.21**
4.530.28*
4.360.69
4.280.74**
4.250.18**
4.190.27*
4.400.94
4.420.57
4.440.55
4.600.85
4.690.69
4.770.54**
4.650.22*
4.330.51
4.340.49*
4.260.16
4.240.21*
4.220.86
4.180.94*
4.480.73*
4.410.82**
4.510.98*
4.560.37
4.640.76
4.760.41
4.620.47*
4.320.53
4.270.84
4.230.61
4.150.65
4.100.21*
4.010.37*
4.440.84
4.400.34*
4.480.76
4.500.26**
4.540.86*
4.630.16
4.560.36
4.300.61*
4.240.42*
4.150.69**
4.160.48
4.090.59*
3.980.77
A+CUD
4.390.87
4.490.29
4.620.59
4.840.43*
4.900.33
5.020.77
5.210.61
5.330.98
4.550.71
5.710.14
5.830.21*
5.980.63
6.160.42**
B+CUD
4.360.89**
4.470.19*
4.510.41**
4.620.82*
4.740.27
4.830.87*
4.720.91**
4.610.84**
4.730.16**
4.760.72
4.910.18
5.210.68*
5.160.80
G+CUD
4.410.80*
4.480.47
4.530.63
4.580.92
4.650.46**
4.740.18*
4.660.78
4.560.20
4.520.91
4.570.39*
4.870.65*
4.910.21
4.900.28
H+CUD
4.420.79
4.450.77
4.480.27*
4.520.39
4.610.65
4.690.23
4.590.88
4.540.49
4.460.64*
4.430.72*
4.630.47
4.860.30*
4.890.11*

Table 5: Absolute Neutrophil count (ANC) (x 106/cumm) of experimental mice at an interval of 15 days (MeanSE).
Groups/Days
15
30
45
60
75
90
105
120
135
150
165
180
Control
4.570.72
4.500.43
4.670.29
4.900.51
5.000.23
5.070.38
5.220.55
5.320.69
5.500.59
5.610.37
5.710.37
5.900.77
6.000.99
CUD (NO
DEN)
DEN
4.380.78**
4.330.74**
4.400.72
4.440.60
4.520.45*
4.530.43
4.560.72
4.570.86*
4.600.97
4.620.19
4.670.34
4.700.65
4.710.47*
4.690.83
4.560.65**
4.680.75
4.920.26
4.010.96
3.680.46
3.140.67**
3.000.61*
2.900.41
2.130.34
2.000.31
1.990.23*
1.490.52
CUD
4.510.87
4.500.69*
4.600.89*
4.810.21
4.920.31
4.990.37
4.930.49
4.830.57
4.670.66**
4.470.77
4.070.92**
3.990.95*
3.880.56**
4.600.97*
4.530.79
4.610.46
4.750.77
4.870.98
4.960.89*
4.860.74**
4.780.26
4.610.32
4.420.75*
4.390.77
4.360.23
4.300.31
4.580.99*
4.510.58**
4.560.57**
4.680.87*
4.780.69*
4.850.56
4.770.26
4.400.55
4.400.51
4.380.19*
4.350.26
4.310.89
4.260.96
4.560.75
4.540.86
4.620.99
4.640.39*
4.730.79*
4.800.43**
4.710.49
4.410.54**
4.350.85*
4.310.66*
4.260.66
4.180.24
4.110.38
4.510.87**
4.520.37
4.530.80*
4.580.29
4.660.88
4.710.17*
4.610.38
4.390.63
4.320.46
4.270.74
4.250.53*
4.200.61
4.090.79
A+CUD
4.460.89*
4.580.31*
4.700.61**
4.920.45*
4.990.35**
5.110.78*
5.320.64*
5.410.99*
5.620.74*
5.800.15**
5.910.23
6.100.65**
6.250.45*
B+CUD
4.410.92
4.510.23
4.630.46
4.730.86
4.800.29*
4.910.89
4.800.93
4.700.86
4.810.18
4.890.74*
5.000.20*
5.300.69
5.240.82
75
ISSN NO 2320-5407
G+CUD
4.520.84
4.560.49
4.610.66*
4.670.95*
4.770.48
4.800.20
4.730.79**
4.620.23
4.600.95*
4.680.44
4.980.67
5.000.24*
5.010.29*
H+CUD
4.500.85*
4.540.81*
4.560.29
4.610.40
4.700.66*
4.750.25
4.670.90
4.610.50*
4.530.65
4.510.73
4.720.49**
4.950.32
4.960.12
Table 6: Haemoglobin content (Hb) of experimental mice at regular interval of 15 days (gm%, meanSE).
Groups/Days
15
30
45
60
75
90
105
120
135
150
165
180
Control
12.451.42
12.471.51
12.531.52
12.511.42
12.551.47
12.501.44
12.521.41
12.551.37
12.561.35
12.581.39
12.621.34
12.661.21
12.681.36
CUD(NO
DEN)
12.191.31*
12.211.42
12.271.51*
12.491.36
12.621.47*
12.851.51
12.991.62*
13.411.73*
13.531.81*
13.661.74*
13.701.32
13.761.21*
13.781.19
DEN
12.401.36*
12.421.38
12.431.29
12.451.31**
12.461.33
12.251.36*
12.081.39
11.821.36*
11.651.30
10.521.32*
10.091.21
9.361.19*
8.021.07
CUD
12.321.31
12.451.28**
12.861.33
12.901.37
13.061.29*
13.401.42
13.871.47
13.671.40*
13.461.33**
13.221.28*
12.791.21*
12.091.19**
12.271.17**
12.431.27
12.841.29
12.871.32*
13.071.33
13.381.38*
13.851.41
14.041.43
13.951.47
13.801.27
13.761.23*
12.341.28
13.711.19
12.311.40
12.391.35*
12.791.25
12.831.38**
13.031.27
13.301.30
13.791.37**
14.021.39*
13.951.41*
13.751.26
13.711.19**
13.671.21**
13.561.18
12.341.28*
12.371.21
12.721.24**
12.791.41
13.041.32**
13.251.19**
13.721.29
13.961.26
13.911.25*
13.701.32*
13.651.27
13.611.21
13.491.29
12.341.42
12.361.40
12.681.23**
12.741.21
12.951.31*
13.101.22
13.671.28
13.871.27
13.821.30
13.651.33*
13.591.24**
13.551.32*
13.401.25
A+CUD
12.411.48*
12.481.47
12.891.36**
12.941.39
13.081.33*
13.421.41*
13.891.52*
14.061.47**
14.641.37
14.821.42
15.071.37
15.211.27
15.381.31*
B+CUD
12.291.42*
12.401.37
12.811.27
12.851.36**
13.051.31
13.351.32
13.811.39
14.011.41
14.221.42
14.481.25
14.741.21*
14.891.22*
14.901.17*
G+CUD
12.271.41
12.361.39**
12.761.21
12.801.29*
13.011.25
13.271.28
13.761.31*
13.981.30*
14.031.27*
14.331.21**
14.691.23
14.741.17
14.861.14
H+CUD
12.301.39*
12.351.35
12.701.27*
12.761.23
12.981.33**
13.121.29*
13.701.20
13.901.24
13.971.26
14.081.31
14.621.28*
14.701.22
14.781.11**
Data of Hemoglobin is expressed in gm% and is

presented in Table-6. Initially the Hb content of
control was 12.451.42gm% and after 6 month the
Hb content of mice was 12.681.36gm%. In DEN
(negative control) group the initial Hb content at zero
day of experiment was 12.401.36gm%, which
decreased to 8.021.07gm%, at the end of
experiment. But in CUD treated group, their Hb
content at zero day was 12.191.31gm% and
13.781.19gm% at the end of experiment. In CUD
treated group in which the carcinogen has been given,
the initial Hb content at zero day was
12.321.31gm%, after the end of experiment the Hb
content was 12.091.19gm%. In test group A, the
zero day Hb content was 12.271.17gm%. The Hb
content increased to 13.631.13gm% at the end of
experiment. In group B, G and H, the Hb content at
zero
day
was
found
12.311.40gm%,
12.341.28gm% and 12.341.42gm%, respectively
and at the end of experiment the Hb content reached
to
13.561.18gm%,
13.491.29gm%
and
13.401.25gm%, respectivelyCUD+A had Hb
content at zero day 12.411.48gm% which was
increased to 15.381.31gm%, at the end of
experiment. Group CUD+B had 12.291.42gm% Hb
content on zero day which was increased to
14.901.17gm% at the end of experiment. In
CUD+G and CUD+H, the Hb content at zero day was
12.271.41gm%,
12.301.39gm%
and
was
14.861.14gm% and 14.781.11gm% at the end of
the experiment, respectively.
Total Leucocyte count (TLC) in experimental mice
76
13.631.13
ISSN NO 2320-5407
Absolute lymphocyte count (AlC) of experiment of mice.
Haemoglobin content (Hb) in experimental mice
Absolute neutrophil count (AlC) of experiment of mice.
Total erythrocyte count (TEC) in experimental mice
Discussion
In-vivo study with T. baccata leaves and bark extracts
alone and in combination with CUD were carried out
in experimental mice for a period of six months. With
an observation at 15 days interval in mice, an attempt
was made to produce cancer using DEN and various
clinicohematological parameters were observed. The
body weight of mice was decreased substantially in
DEN treated mice indicating in the development of
cancer, due to DEN.Ramji and You, (1992) reported
that aflatoxin has been directly related to under
weight status in children in Benin and Togo. Bedi et
al.,(1996) reported decreased in body weight in
Guinea fowl fed on aflatoxin B1. In present study
body weight in DEN treated mice was decreased at
the end of experiment. However, there was increase
in body weight in other test groups. This study
showed that the weight loss in DEN treated group
may be due to the carcinogenic effect of DEN;
however, herbal formulations of extracts and CUD
were found to be a preventive agent against the
carcinogenic effects of DEN. DEN is already known
chemical carcinogen.Increased immunocompetence
of an individual is a very essential parameter to
prevent the development of cancers by several
mechanisms, of which the upregulation of
lymphocyte proliferation and stimulation activity,
increased macrophage activity, higher antibody
production and increased synthesis and secretion of
cytokines (IL-1, Il-2) plays significant role by
enhancing the recognition of tumor cells by the
immune cells of the body and cytotoxic activities of
the tumor killing cells, the lymphocytes. Using herbs
for cancer treatment can help the body to support its
77
ISSN NO 2320-5407
healing power. In the present investigation, both

doses of QC (5 and 25 mg/kg) led to a significant
decrease in the number as well as the mean area of
GST-P positive foci, TUNEL positive apoptotic cells,
p53 positive hepatocytes, and restoration of cellular
morphology. These results clearly indicate that
quercetin inhibits diethylnitrosamine-induced hepatic
preneoplastic lesions in medium-term rat liver
bioassay. In the mice given T. baccataalone and
along with CUD. The body weight either remain
constant or enhanced substantially.
These
preparations as shown in the in-vitro study were
having anti-carcinogenic effect, which might be
altering the clinoco effects of the cancer caused by
DEN.
Jemal A., Bray F., Center M.M., Ferlay J., Ward E.

and Forman D. Global cancer statistics. CA: a cancer
journal for clinicians, 2011, 61 (2): 6990.
Various hematological parameters indicated the

leukocytosis, erythrocytosis higher hemoglobin
content in treated mice with T. baccata products
along with indigenous cow urine. While, in DEN
treated mice there was leucopenia, erythropenia and
decreased heamoglobin content. These findings are
further supported by the fact that CUD had the
immunomodulatory
property
which
caused
leukocytosis leading to the control of the highly
proliferating cells through their destruction by the
white blood cells. Erythrocytosis and increased
hemoglobin content are the indication of good health
and recovery and neutralization of the effect of DEN
by T. baccata and CUD. Joshi et al., 2013
investigated that the immunomodulatory effect of
distilled
Gir
cow
urine
in
rabbits
throughhaematological parameters. The study
revealed that the values of total leucocyte count
(TLC), absolute lymphocyte count (ALC) and
absolute neutrophil count (ANC) were significantly
increased in Group II, in which the rabbits were
given Gir cow urine distillate alone and Gir cow
urine distillate with citric acid, respectively. In the
present study the ALC and ANC increased 40.31%
and 40.13% inn extracts and/or CUD treated mice in
comparison to control or DEN treated mice. Increase
in TEC and Hb content is an indication of enhanced
vitality
of
mice.
Similarly
leukocytosis,
lymphocytosis and neutrophilia are the immune cell
showing immunopoturtration, which is considered
protective against cancer and an indication of a good
prognosis (chauhan, 2005). It further needs a detailed
study for further confirmation.
Udupa A.L.,Kulkarni D.R. and Udupa S.L. Effect of

Tridaxprocumbens extract on wound healing, Int. J.
Pharmacognosy, 1995, 1: 37-40.
References
Joshi A.,Bankoti K.,Bisht T. and Chauhan R.S.

Immunomodulatory effect of Gir cow urine distillate
in Rabbits. Journal of Immunology and
Immunopathology, 2012, 14(1): 57-61.
Kumar A.,Kumar P.,Singh L.K and Agrawal D.K.
Pathogenic effect of free radicals and their prevention
throughCowpathy. The Indian Cow, 2004, 2:27-34.
Zollman A. and Vickers G. Dervan.Understanding
Cancer. Jefferson, NC: McFarland (1999).
Ramji C and You W. Differential sensitivity of

human mammary epithelial and breast carcinoma cell
lines to curcumin .Br. Canc. Res. Treat., 1999,
54(1):269-79.
ParkI.K.,Qian D., KielM.,Becker M.W, Pihalja M.,
Weissman I.L., Morrison S.J and Clarke M.F. "Bmi-1
is required for maintenance of adult self-renewing
haematopoietic stem cells". Nature. 2003, 423
(6937): 3025.
Kinzler K.W and Vogelstein B.The genetic basis of
human cancer.Medical Pub. Division, 2002, 2:1-5.
IARC.Cancer an entropic disease, 2003, 143: 112131.
Bedi P.S, Singh H.,Johri H.S and Agarwal R.N.
Effect of aflatoxin on growth and feed consumption
in Guinea fowl. XX World Poultry Congress, 1996,
4(3):665-667.
Chauhan R.S. Cowpathy: A new version of Ancient
Science. Employment News, 2005, 30(15):1-2.
Pal S and Mittal K. Use of alternative cancer
medicine in India. Lancetoncol.2004, 3: 394-395.
Govindachari T.R., Suresh G. and Masailmani S.
Antifungal activity of Azadirachtaindica leaf hexane
extract fitoterpia, 1999, 70: 427-420.
Hartzell M. Carcinogenic effect of Taxusbacata in

diabetic condition., 2003, 45:147-168.
***********
78
Original Research Paper
Lipid-lowering activity of Cow urine ark in guinea pigs fed with a high
cholesterol diet
Chawda Hiren Manubhai1, Mandavia Divyesh Rasiklal1, Baxi Seema Natvarlal2, Vadgama
Vishalkumar Kishorbhai1, Tripathi Chandrabhanu Rajkishor1*
1
2
Department of Pharmacology, Government Medical College, Bhavnagar-364001, Gujarat, India

Department of Pathology, Government Medical College, Bhavnagar-364001, Gujarat, India
Article history:
Received: Sep 28, 2013
Received in revised form:
Apr 5, 2014
Accepted: May 14, 2014
Vol. 4, No. 5, Sep-Oct 2014,
354-363.
* Corresponding Author:
Tel: +919825951678
Fax: +9102782422011
cbrtripathi@yahoo.co.in
Keywords:
Antioxidant activity
Cow urine ark
Dyslipidemia
Hypolipidemia
Guinea pig
Statin
Abstract
Objectives: Cow urine ark (CUA), known as Amrita as
mentioned in Ayurveda, contains anti-hyperglycemic and
antioxidant effects. Therefore, we designed the present study to
evaluate the lipid lowering activity of CUA and its possible
implication in metabolic syndrome.
Materials and Methods: Thirty guinea pigs of either sex were
divided into five groups: Group 1 and 2 serving as a vehicle and
sham control, received normal and high fat diet for 60 days
respectively; Group 3, 4 and 5 received high fat diet for 60 days
with CUA 0.8 ml/kg, 1.6 ml/kg and rosuvastatin (1.5 mg/kg) on
thelast 30 days of study period, respectively. Serum lipid profile
(total cholesterol, triglycerides, LDL-C, VLDL-C, HDL-C, total
Cholesterol/HDL-C) and serum enzymes (ALT, AST, ALP, LDH
and CK-MB) were performed in each group at the beginning and
end of the study. Histological study of liver and kidney was done
in each group.
Results: CUA (0.8 ml/kg) significantly decreased the serum
triglycerides and VLDL-C, but CUA (1.6 ml/kg) decreased the
total serum Cholesterol, triglycerides and VLDL-C (p < 0.05).
Higher dose (1.6 ml/kg) of CUA also increased HDL-C level,
significantly (p < 0.05). CUA reduced serum AST, ALP and LDH
level, which was statistically significant as well, while it also
decreased the accumulation of lipid in hepatocytes as compared to
sham control.
Conclusions: CUA reduced triglycerides, increased HDL-C and
found to be hepatoprotective in animals that are on a high fat diet.
Please cite this paper as:

Chawda Hiren M, Mandavia DR, Baxi SN, Vadgama VK, Tripathi CR.Lipid-lowering activity of Cow urine ark
in guinea pigs fed with a high cholesterol Diet. Avicenna J Phytomed, 2014; 4 (5): 354-363.
Introduction
Cardiovascular diseases (CVD) are
emerging problem in developing countries
(Freedman, 2003; Badimon et al., 2010).
CVD may become the reason for the loss
of 17.9 million Potentially Productive

Years of Life Lost (PPYLL) in India, by
2030 (Anchala et al., 2012). Dyslipidaemia
is well recognized risk factor for the
development of cardiovascular diseases
AJP, Vol. 4, No. 5, Sep-Oct 2014
354
Lipid-lowering activity of Cow urine ark
(Freedman, 2003; Badimon et al., 2010).

Reactive oxygen species induce the
oxidative stress, which play significant
role in the development of CVD and
atherosclerosis. Liver, kidney and heart are
also under oxidative stress due to
hyperlipidemia in CVD (Kwiterovich,
1997; Vijayakumar et al., 2004). Other
crucial factors regarding CVD, associated
morbidity and mortality, are the insulin
resistance, diabetes, elevated blood
pressure, obesity and dyslipidaemia that
are included in metabolic syndrome (Li et
al., 2013). Modern life style like smoking,
alcohol consumption and junk food diet
are major obstacles in the management of
dyslipidaemia. Elevated liver enzymes are
the major risk factors associated with the
current
allopathic
treatment
for
dyslipidaemia and thus, the alternative
medicines from Ayurveda, have been
getting attention in the management of
dyslipidaemia (Suanarunsawat et al.,
2011).
Cow urine, knownasAmritaorwater
of life as mentioned in Ayurveda (Dhama
et al., 2005), is one of the ingredients of
Panchagavya Ghrita. Panchagavyapathy
was proved to be effective in life
threatening diseases such as diabetes,
cancer and AIDS (Dhama et al., 2005;
Achliya et al., 2003; Jarald et al., 2008).
Cow urine has medicinal properties like
antimicrobial, antifungal and anticancer
which granted US Patents (No. 6,896,907
and 6,410,059) (Randhawa, 2010). Several
studies revealed that cow urine has
antidiabetic and antioxidant activity
(Sachdev et al., 2012; Krishnamurthi et al.,
2004). Therefore, the present study was
planned to evaluate the lipid lowering
effect of Cow urine ark (CUA) and also to
know its possible implication in metabolic
syndrome.

Drugs and Chemicals
Cholesterol powder (analytical grade): was
purchased from High Purity Laboratory
Chemicals Pvt. Ltd., Mumbai, India. Cow

urine ark (CUA) was obtained from Go
VigyanAnushandhan Kendra, Sevadhanm,
Devalpur, Nagpur, Maharashtra, India and
(US Patent No 6410 059/2002).
Rosuvastatin Calcium powder: (Gift
sample from Torrent Pharmaceuticals Ltd.,
Torrent research center, Ahmedabad.
(Batch no: ARD2110109)
Animal preparation
Thirty guinea pigs weighing 520 860
g of either sex were procured from the
Central Animal House of Government
Medical College, Bhavnagar, which is
registered in the Committee for the
Purpose of Control and Supervision of the
Experiments on Animals (CPCSEA), New
Delhi, India. CPCSEA guidelines were
followed during animal experiments of our
study. The guinea pigs were housed in
stainless steel cages under 12 hour lightdark cycle room with controlled
temperature at 232C, being fed with
standard laboratory food and water ad
libitum. After the proper acclimatization
for 15 days, they were divided into five
groups of six guinea pigs.
Group I: Normal diet plus distilled
water (Vehicle control); Group II: High fat
diet plus distilled water (Sham control);
Group III: High fat diet plus a lower dose
of CUA (0.8 ml/kg); Group IV: High fat
diet plus a higher dose of CUA (1.6
ml/kg); Group V: High fat diet plus
rosuvastatin (1.5 mg/kg)
Diet composition
Normal diet: in the morning, mixtures
of cereals and pulses (60% wheat plus
35% Bengal gram plus 15% peanuts), total
50 g/animal. In the evening: green leafy
vegetables, 30 g/ animal. High fat diet: in
the morning, cholesterol powder (500
mg/kg) mixed with 10 g of wheat and
Bengal gram flour, followed by 40 g of the
mixtures mentioned above, in the normal
diet/animal. In the evening: green leafy
vegetables, 30 g/animal.
355
Chawda Hiren et al.
Methodology
The study was conducted in Animal
room, Department of Pharmacology,
Government Medical College, Bhavnagar,
Gujarat, after approval from the
Institutional Animal Ethics Committee of
the same institute. The baseline blood
sample was collected from a lateral
saphenous vein of the hind paw of each
guinea pig in overnight fasting state.
Blood samples were sent to Clinical
Biochemistry Laboratory of our institute
which is accredited by National
Accreditation Board for Testing and
Calibration Laboratories (NABL), for the
serum lipid profile, liver and cardiac
enzymes. Animals were separated into
groups as mentioned above. Throughout
the study period in each group, diet was
given according to the respective group
diet plan. During the last 30 days of the
experiment, animals of group I and II were
given distilled water daily, animals of
group III, IV and IV were given a lower
dose of CUA, higher dose of CUA and
rosuvastatin (1.5 mg/kg), respectively.
Distilled water, CUA and rosuvastatin
calcium were given orally by gavages
feeding tube in a daily basis on mornings
in fasting state to ensure maximum
absorption. Animals of all groups were
sacrificed after blood collection from the
lateral saphenous vein in the overnight
fasting stat at the end of 60 days.
Bloodsmples were sent to labratory for the
analysis of the serum lipid profile, liver
and cardiac enzymes. We obtained the
liver and kidney from each animal of the
five groups for histopathological analysis,
which was done by senior faculty from the
Pathology department of our institute.
Serum lipid profile
The serum levels of triglycerides, total
cholesterol and high density lipoprotein
cholesterol (HDL-C) and Low density
lipoprotein cholesterol (LDL-C) were
analyzed by GPO PAP METHOD, CHOD
PAP METHOD, IMMUNOINHIBITION
and ENZYME SELECTIVE methods,

respectively. Very low density lipoprotein
cholesterol (VLDL-C) was calculated by
the Friedwald method (Friedewald et al.,
1972) as well as the ratio of the total
cholesterol and HDL-C.
Evaluation of liver and cardiac enzymes
Liver function was evaluated by serum
alanine aminotransferase (ALT), aspartate
aminotransferase (AST), and alkaline
phosphatase (AP) levels. Cardiac injury
was assessed by measuring the serum level
of creatine kinase MB subunit (CK-MB)
and lactate dehydrogenase (LDH). ALT
and AST were examined by UV KINETIC
method and serum alkaline phosphatase
was determinedby PNP AMP KINETIC
method. LDH and CK-MB were analyzed
by IMMUNO-INHIBITION and UV
KINETIC, respectively.
Effect of CUA on the weight of the
animals
Weighing of each animal in all groups
was done before the start of the study and
also at the end of 60 days to rule out any
effect of CUA on the weight of the guinea
pig.
Histological analysis
The liver and kidney were isolated,
cleaned, dried, and fixed at 10% neutral
buffer formalin followed by paraffin
embedding and stained with haematoxylin
and
eosin
(H&E)
dye.
All
histopathological slides were coded and
evaluated by a pathologist blindly without
knowledge of the groups. Scalinggrades 0,
1, 2, 3 and 4 were given for no change,
slight, mild, moderate and severe changes,
respectively
regarding
theseverity
assessment of histopathologicalresults.
Statistical analysis:
All parameters were expressed as
Meanstandard error of mean (S.E.M.).
One-way Analysis of Variance (ANOVA)
followed by Tukey-Kramer Multiple
comparison test was used to compare the
356
inter group differences of lipid profile,

liver enzymes, cardiac enzymes and extent
of body weight gain at the end of 60 days.
Paired t-test was used to compare intra
group differences of lipid profile, liver
enzymes, cardiac enzymes and extent of
body weight gain. Value of p<0.05 was
considered significant. The statistical
calculations
were
done
using
GraphPadInStat, Demo version 3.06.
Table 1]. The percentages of increment in

serum level of the total cholesterol,
triglyceride, HDL-C, LDL-C and VLDL-C
were 92 22.8 (%), 29.8 7.5 (%), 63.2
45.4 (%), 161.93 45.5 (%) and 29.8 7.5
(%) in sham control, as showed in Table 2.
A significant reduction in serum
triglyceride and VLDL-C was observed in
high fat diet treated group with the lower
dose of cow urine ark (CUA), in
comparison to sham control. The serum
total cholesterol, serum triglyceride and
VLDL-C were significantly decreased in
the higher dose of CUA as compared to
sham control [p<0.05; (Table 1)].
However, the HDL-C level was
significantly increased with higher dose of
CUA (p < 0.05) but, percentage of
increment in HDL-C with rosuvastatin
treatment was significantly greater than the
lower and higher dose of CUA treatment.
The percentage of increment in
triglyceride
and
VLDL-C
with
rosuvastatin treatment were- 8.82 2.3
and - 8.8 2.3, respectively at 60 days,
while with lower dose of CUA and higher
dose of CUA, the percentage results were 26.5 12.7 and - 37.1 8.2, (Table 2).
Result
Serum lipid profile
The baseline serum level of total
cholesterol, triglyceride, HDL-C, LDL-C
and VLDL-C are 46.33 4.04, 92 5.24,
4.5 0.9, 23.56 2.6 and 19.2 1.16,
respectively in vehicle control (Table 1).
There are not statistically significant
differences in the baseline values of each
variable in different treatment groups. The
serum level of the total cholesterol,
triglyceride, HDL-C, LDL-C and VLDL-C
were 82.6 4.65, 109.2 7.63, 3.9 0.8,
54.38 7.1 and 22.5 3.5, respectively at
the end of 60 days, which were statistically
significant as compared to the baseline
lipid profile, in Sham control [p<0.05;
Table 1. Effect of each treatment strategy on serum lipid profile in guinea pigs
Treatment Groups
(n = 6)
Time
Period
Total
Cholesterol
(mg/dl)
Triglycerides
(mg/dl)
HDL
Cholesterol
(mg/dl)
LDL
Cholesterol
(mg/dl)
VLDL
Cholesterol
(mg/dl)
Total
Cholesterol/
HDL-C
Vehicle control
(Group 1)
Base line
46.33 4.04
92 5.24
4.5 0.9
23.56 2.6
19.2 1.16
11.4 0.8
60 Days
Base line
60 Days
Base line
46.53 5.02
46.2 7.03
82.6 4.65**
47.8 3.43
88.7 6.65
87.38 6.2
109.2 7.63**
75 9.25
4.4 0.5
4.1 0.8
3.9 0.8
4.5 0.6
24.17 2.13
23.26 3.47
54.38 7.1**
28.33 2.7
18.7 1.22
17.3 1.24
22.5 3.5**
15 1.85
12.02 0.6
9.78 1.5
15.39 7.48
12.53 3.19
60 Days
64.9 7.92
49.5 2.65*
7.16 1.6
47.6 6.4
9.9 0.5*
10.44 1.86
Base line
46.23 2.24
75.3 6.01
4.3 0.42
26.83 4.26
15.06 1.2
Sham control
(Group 2)
High fat diet plus
CUA (0.8 ml/kg)
(Group 3)
High fat diet plus
CUA (1.6 ml/kg)
(Group 4)
High fat diet plus
rosuvastatin
(1.5 mg/kg)
( (Group 5)
45.3 3.21
**
11.2 1.45
*
60 Days
57.53 2.9
6.3 0.6
42.1 1.9
9.06 0.64
Base line
42.09 8.64
86.83 2.14
5.8 0.7
26 2.57
15.57 2.3
9.29 0.52
8.8 0.6
60 Days
46.32 5.21*
76.42 4.6*
9.6 0.9*
28 5.15*
15.08 2.3*
4.55 0.65*
Values are expressed as Mean standard error of mean; LDL: low density lipoprotein, VLDL: very low density
lipoprotein, HDL: high density lipoprotein; CUA: Cow urine ark; * p< 0.05 as compared to sham control,
ANOVA followed by Tukey-Kramer Multiple comparison test; ** p< 0.05 as compared to baseline level, paired
t-test.
357
Chawda Hiren et al.

Table 2. The effects of each treatment strategy on serum lipid profile (% increment) on guinea pigsat the end of
60 days treatment
Treatment Groups(n = 6)
Time
Period
Total
Cholesterol
(mg/dl)
Triglycerides
(mg/dl)
HDL
Cholesterol
(mg/dl)
LDL
Cholesterol
(mg/dl)
VLDL
Cholesterol
(mg/dl)
Total
Cholesterol/
HDL-C
Vehicle control (Group 1)
60
Days
- 2.4 1.8
- 2.4 1.8
- 2.4 2.4
- 2.8 3.8
- 2.3 1.8
0.3 3.4
Sham control (Group 2)
60
Days
92 22.8*
29.8 7.5*
63.2 45.4
161.93 45.5* 29.8 7.5*
High fat diet plus CUA

(0.8 ml/kg) (Group 3)
60
Days
42.9 25.3
- 26. 5 12.7#
70.8 36##
78.9 34.9
- 26.5 12.7#
5.9 30
High fat diet plus CUA

(1.6 ml/kg) (Group 4)
60
Days
27.2 8.5
- 37.1 8.2#
50.5 16##
72 21.7
- 37.1 8.2#
- 8.85 13.2
High fat diet plus

Rosuvastatin
(1.5 mg/kg) (Group 5)
60
Days
4.5 4.9**
- 8.82 2.3**
234 31**
- 18.9 3.9**
- 8.8 2.3**
- 67.9 2.1**
54.2 30.7
Values are expressed as Mean standard error of mean; LDL: low density lipoprotein, VLDL: very low density
lipoprotein, HDL: high density lipoprotein; CUA: Cow urine ark; * p< 0.05 as compared to vehicle control,
ANOVA followed by Tukey-Kramer Multiple comparison test; ** p< 0.05 as compared to sham control,
ANOVA followed by Tukey-Kramer Multiple comparison test;# p< 0.001 as compared to sham control,
ANOVA followed by Tukey-Kramer Multiple comparison test; ## p< 0.05 as compared to rosuvastatin
treatment group, ANOVA followed by Tukey-Kramer Multiple comparison test.
The baseline and 60 days values of

serum enzymes in each diet treatment
group were shown (Table3).In sham
control, there is a significant increase in
ALT, AST, ALP, LDH and CK-MB level
(p<0.05) at the end of 60 days, compared
to the baseline.ALT, AST and LDH levels
were significantly decreased in the high fat
diet group treated with low dose of CUA.
However, LDH level was significantly
decreased with the higher dose of CUA
compared to sham control [p<0.05;(Table
3)].High fat diet treated group with
rosuvastatin
(1.5
mg/kg)
showed
significant reduction in the serum total
cholesterol, LDL-C and ratio of total
cholesterol: HDL-C level and an increase
in HDL-C level as compared to sham
control [p<0.05; (Table 1)]. Compared to
the baseline level, there was an increase in
AST and ALP level in the rosuvastatin
treated group at the end of 60 days
period[p<0.05; (Table 3)].
The normal histological structure of
liver and kidney was shown in Figure 1 A
and 2 A, respectively. In sham control,

histological study of the liver showed
diffuse areas ballooning degeneration of
hepatocytes (grade 3+, 4+), Steatosis (up
to grade 2.5), fatty changes (midzone and
periportal) and congestion of central vein
and hepatic sinusoids (Figure 1 B) in all
animals, while the kidneys showed a
normal structure (Figure 2 B). In the group
treated with CUA and high fat diet, liver
histology showed no fatty changes, with
only ballooning degeneration (Grade 1+)
with repaired and regeneration process
(Figure 1 C) and the kidney examination
of this group were normal (Figure 2 C). In
rosuvastatin
treated
group,
no
morphological changes in the liver and
kidney was detected (Figure 1 D and
Figure: 2 D).
Increase in mean body weight of all
groups at the end of 60 days was shown in
Table 4 and the extent of weight gain was
not statistically significant between the
groups (p<0.05).
Table 3.Effect of each treatment strategy on serum enzymes in guinea pigs
358
Treatment Groups
(n = 6)
Time Period
ALT
(U/L)
AST
(U/L)
ALP
(U/L)
LDH
(U/L)
CKMB
(U/L)
Base line
53.46 1.86
61.33 7.95
94.8 12.67
372.83 44.03
268.8 11.5
60 Days
55.81 2.86
63.33 7.6
97.5 9.5
377.16 46.12
267.7 17.6
Base line
61.17 6.21
50.33 5.3
85.83 11.7
311.5 39.05
285 34.74
60 Days
104 12.72**
162.4 25.4**
145.6 9.6**
456.5 56.8**
360.23 27.1**
High fat diet plus

CUA (0.8 ml/kg)
(Group 3)
Base line
57.14 4.3
63.76 14.9
126.5 18.8
345.3 22.6
433.56 22.1
60 Days
50.83 9.9*
72.66 22.1*
139.9 9.07
194.16 28.12*
353.8 36.5
High fat diet plus

CUA (1.6 ml/kg)
(Group 4)
Base line
51 1.3
73 14.7
86.6 15
283.6 27.2
317.33 22.5
60 Days
63.8 6.5
96.8 14.6
124.3 12.73
243.5 37.4*
297.6 17.5
High fat diet plus

rosuvastatin
(1.5 mg/kg)
(Group 5)
Base line
63.26 13.2
62.26 9.65
103.6 12.3
241 46.64
374.5 42.25
60 Days
64.34 5.5
169 12.68**
161.5 10.91** 301.8 31.12
Vehicle control
(Group 1)
Sham control
(Group 2)
392.19 28.3
Values are expressed as Mean standard error of mean; ALT: Alanine aminotransferase, AST: Aspartate
aminotransferase,AP: Akaline phosphatase , LDH: Lactate dehyrogenase, CK-MB: Creatine kinase-MB, CUA:
Cow urine ark; *p<0.05 as compared to sham control, ANOVA followed by Tukey-Kramer Multiple
comparison test;**p< 0.05 as compared to baseline level, paired t-test.
B
C
A
Figure 1.Histological photograph of liver of guinea

pig (H&E 40x). (A) C, H and S indicates
normal structure of central vein, hepatocytes with
round nucleus and sinusoid, respectively; (B) Fatty
changes (Grade 4+) (arrow) and diffuse areas of
ballooning degeneration were shown in high fat
diet fed guinea pig; (C) Encircled area shown no
fatty changes, only ballooning degeneration (Grade
1+) with repaired and regeneration process in CUA
(1.6 ml/kg) treated group; (D) Normal
morphological changes seen in rosuvastatin (1.5
mg/kg) treated group.
Figure 2.Histological photograph of kidney of

guinea pig (H&E 40x). (A) G, T and I indicate
normal structure of glomeruli, tubule and
interstitum, respectively; (B) No fatty changes were
seen in high fat diet fed guinea pig; (C) and (D)
indicate normal histological appearance of kidney
in CUA (1.6 ml/kg) and rosuvastatin (1.5 mg/kg)
treated groups, respectively.
359
Chawda Hiren et al.

Table 4. Effect of each treatment strategy on weight of guinea pigs
Weight of animal in grams
Treatment group
Baseline
60 days
Vehicle control
540.66 6.14
554.16 15.97
Sham control
632.33 44.3
667.82 45.32*
High fat diet plus CUA (0.8 ml/kg)
602.3 29.3
637.6 17.8*
High fat diet plus CUA (1.6 ml/kg)
604.3 9.2
630.1 4.6*
High fat diet plus rosuvastatin(1.5mg/kg)
658 36.8
682.5 22.02*
Values are expressed as Mean standard error of mean; CUA: Cow urine ark; *p< 0.05 as compared to baseline
value, paired t-test.
Discussion
In the present study, we selected guinea
pig as the experimental animal for the
evaluation of lipid lowering activity of
Cow urine ark (CUA). Lipoprotein
metabolism of guinea pigs is closest to
human and several lines of evidence
proved that guinea pigs are admirable
models to assess hypolipidemic activity
and lipoprotein metabolism of drugs
(Fernandez and Volek, 2006).
The present study showed that 60 days
of high cholesterol diet feeding, raised the
serum lipid profile (total cholesterol,
triglyceride, LDL-C and VLDL-C) and
induced the histopathological changes in
liver (Table 1, Figure 1 B). The liver plays
a major role in equilibrium cholesterol
homeostasis (Suanarunsawat et al., 2011).
High cholesterol diet increases the hepatic
cholesterol content and is responsible for
the elevated triglyceride synthesis and
cholesteryl ester-rich VLDL-C production
(Patel Y et al., 2011; Goldstein et al.,
1983; Demacker et al., 1991). The
reduction in the amount ofhepatic LDL-C
receptors is caused by high fat diet which
diminishes cholesterol removal rate from it
(Patel et al., 2011; Goldstein et al., 1983;
Demacker et al., 1991). We opted a study
period, 60 days, which is sufficient to
produce fatty changes in guinea pigs, also
supported by previous studies (Patel et al.,
2011; Ahmad-Raus et al., 2001).
In the present study, CUA therapy for 30
days was found to be highly effective to
reduce the total serum cholesterol,

triglycerides, VLDL-C [p<0.05; (Table 1
and Figure 1 C)]. Elevated triglyceride
level is an independent risk factor for
atherosclerosis (Beatriz et al., 2011).
Triglyceride rich lipoproteins (TRLs) have
apo C-III that stimulates activation of proinflammatory transcription factors nuclear
factor-B, processes ultimately leads to
atherosclerosis (Kawakami et al., 2006).
Remnant species of TRLs can easily
accumulate in endothelial cells and taken
up by macrophage to form foam cells
similar to oxidized LDL-C (Gianturco et
al., 1998; Botham et al., 2007). Foam cell
promotes the formation of fatty streak in
blood vessels that is an early marker of
atherosclerosis (Beatriz et al., 2011). TRLs
block sterol efflux from monocytes and
macrophages which may diminish the
protective effect of HDL-C (Palmer et al.,
2004; Patel et al., 2009).
Biochemical analysis of Cow urine
proved to have many constituents; copper,
kallikrein, urokinase, nitrogen, uric acid,
hippuric acid, phosphate and others (Jain
et al., 2010). Both, dietary and serum
copper were inversely associated with
fasting glucose, total cholesterol, and
LDL-C according to Bo S et al (Bo et al.,
2008). Previous study also indicated that
the supplementation of moderate dietary
copper inhibits atherogenesis in the
cholesterol-fed rabbit (Lamb et al., 2001),
thus the copper in CUA may be
responsible for its lipid lowering activity.
360
Oxidative stress and free radical

induced injuries play a major role in the
pathogenesis of a number of diseases.
Hyperlipidemia produces lots of free
radicals and oxidative stress in blood
vessels
along
with
atherosclerosis
progression, and it endangers vital organs
such as liver, kidney, heart and brain
(Suanarunsawat
et
al.,
2011).
Augmentation of serum levels of AST,
ALT, AP, LDH, and CK-MB suggested
suppressed cardiac and hepatic functions,
due to the retention of lipid in liver and
heart in sham control (Table 3 and Figure
1 B). CUA treatment decreased serum
level of ALT, AST and LDH (p < 0.05),
and
improvement
in
hepatocytes
histopathologically also seen (Table 3 and
Figure 1 C). CUA has many volatile fatty
acids; acetic acid 2 propenyl ester, acetic
acid methyl ester, 2, 2, 3 trichloro
propionic acid, butanoic acid-3methyl,
propyl ester, 1H indol-3-acetate, acetic
acid phenyl ester and quinoline. They are
responsible for its antioxidant action
which is confirmed by the estimation of
thiobarbituric acid, ascorbic acid, DPPH
radical scavenging activity and ABTS
assay (Sachdev et al., 2012). Hence, the
antioxidant activity of CUA might be
responsible for the cytoprotective action
that was found in our study.
Several studies reported that residual
cardiovascular risk is still apparent in spite
of intensive statin therapy. Residual
cardiovascular risk with 80 mg
atorvastatin was 22.4%, 12% and 8.7%,
reported in the PROVE IT-TIMI study, the
IDEAL study and the TNT study,
respectively (Cannon et al., 2004; LaRosa
et al., 2005; Pedersen et al., 2005).
Residual cardiovascular risk in patients
that were treated with statins is attributed
to the triglycerides and low HDL-C which
is predominantly higher among diabetics
than non diabetics (Sampson et al., 2012).
Diabetes has a triad of lipid abnormality,
including high levels of triglycerides, low
levels of HDL-C and small, dense LDL-C
(Fruchart, 2013). CUA increases HDL-C
as well as reducing triglycerides (Table 1).

These findings are in accordance with the
previous studies of cow urine distillate in
diabetic wistar albino rats (Gururaja et al.,
2011). Thus, CUA may be useful as an add
on therapy to reduce statin related residual
CV risk in diabetic patients.
Metabolic syndrome is a group of
interrelated metabolic abnormalities that
includes insulin resistance, diabetes,
elevated blood pressure, obesity and
dyslipidaemia (Li et al., 2013). CUA has
lipid lowering, antidiabetic and antioxidant
activities (Sachdev et al., 2012). Cow
urine contains copper (Jain et al., 2010);
while previous studies revealed an inverse
association between diastolic blood
pressure and dietary copper intake (Bo et
al., 2008). Cow urine has a diuretic action
which might be due to nitrogen, uric acid,
phosphates and hippuricacid (Jain et al.,
2010), Therefore cow urine may be
effective as an antihypertensive that has
the potential to decrease all the metabolic
abnormalities and also a possible
implication in metabolic syndrome.
Hence, the present study revealed that
CUA has lipid lowering and cytoprotective
effects that may be implicated in metabolic
syndrome. It is vital to mention that this
study has several limitations since we did
not evaluate molecular mechanism,
objective evidence for atherosclerosis and
metabolic syndrome.
From the current study, we concluded
that CUA reduces triglycerides, improves
HDL-C and hepatoprotective in animals
with high fat diet; therefore it may be
useful in diabetic dyslipidemic patients.
Acknowledgement
We would like to thank Torrent
Pharmaceuticals Ltd., Torrent research
center,
Ahmedabad
for
providing
rosuvastatin calcium powder as a free gift
sample.
Conflict of interest
There is not any clash of attentiveness
in this study.
361
Chawda Hiren et al.
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INTERNATIONAL RESEARCH JOURNAL OF PHARMACY

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Research Article
PROTECTIVE ROLE OF PUNGANUR COW URINE ON STREPTOZOTOCIN INDUCED DIABETES IN RATS

M. Vijaya Bhaskara Reddy1*, A. Karthik2, P. Sasikala1
1
Department of LPM, College of Veterinary University, Sri Venkateswara Veterinary University, Tirupati, India
2
Department of Microbiology, College of Veterinary University, Sri Venkateswara Veterinary University, Tirupati, India
*Corresponding Author Email: vijaybhaskar24@gmail.com
Article Received on: 16/06/13 Revised on: 08/07/13 Approved for publication: 11/08/13
DOI: 10.7897/2230-8407.04832
IRJP is an official publication of Moksha Publishing House. Website: www.mokshaph.com
All rights reserved.
ABSTRACT
Punganur Cattle is the worlds smallest Bos indicus cattle originated in Punganur town in Chittoor district of Andhra Pradesh, India. This breed is known for
its short stature, high milk production efficiency and efficient reproductive characters. In Ancient Ayurvedic scriptures such as Charaka samhita, Shushruta
samhita and Brahad-Wagbhatt mention various medicinal properties of cow urine. It is used as an insecticide and in disorders like intestinal gas, acidity and
cough. Although Indian Ayurvedic literature cites many medicinal properties of cow urine, there is no scientific evidence to support this. Hence, the present
study was aimed to evaluate the antidiabetic activity of cow urine was undertaken. The effect of a distillate of cow urine was studied in vivo in rats given
streptozotocin. Diabetes was induced by administration of streptozotocin (50 mg / kg body wt., i.p). The antidiabetic effect of three different doses and a
standard drug, Glibenclamide (0.25 mg/kg, p.o), was studied in diabetic rats. Fasting blood glucose levels, serum lipid profiles, liver glycogen levels and initial
and final changes in body weight were assessed. The cow urine distillate produced a significant (P < 0.05) reduction of the elevated blood glucose, serum
cholesterol and serum triglycerides levels when compared with the diabetic control. The diabetic animals treated with cow urine distillate also showed a
significant increase in HDL levels and a gain in body weight when compared with the diabetic control. Earlier studies have revealed the presence of
antioxidants and free radical scavengers in cow urine which might be responsible for antidiabetic effects. The present communication records the first ever
report on protective role of cow urine on diabetic in a bizarre situation of tropical climate of Andhra Pradesh (AP), India.
Keywords: Punganur Cow, anti diabetes, cow urine distillate, streptozotocin, glibenclamide.
INTRODUCTION
Nature has provided us with all Natural Amenities like Air,
Water, Sun light etc., which are essential for our Body. It has
also provided us with Divine Nectar known as Urine
which flows from our Body. Urine has a Natural Healing
Power to Control and Cure all kinds of Diseases. Just like
Nature has provided milk in the mothers breast for
nourishment of the infant child, similarly Nature has also
provided Urine in human body for preservations of their
health and cure of various diseases. Urine Therapy is the
Most Effective Natural Remedy and the safest method of
treatment which does not have any side effects. It can
Prevent, Control and Cure all kinds of chronic diseases such
as Cancer, Diabetes, Blood Pressure HIV / AIDS, Kidney
failure, Muscular Dystrophy, Arthritis, Psoriasis, Hair loss,
Mental Retardation and Cerebral Palsy etc. It can boost the
Immune System, Improve Nervous Disorder, Dissolves and
Removes the Toxins Accumulated in our body. It can revive
dead Tissues, Rebuilds Resistance Power of the Vital Organs
like, Brain, Heart, Lungs, Pancreas, Liver and Intestine etc. It
rejuvenates our body and safe guards the general Health of
the people. The whole world at large can get rid of dreadful
diseases and blessed with the Natural healing Power by
Urine Therapy. The very feeling that the Devine Panacea
the solution for all Ailments is within us can fill your life
with immense pleasure. Persons self confidence and positive
attitude can solve all their problems and they will be able to
maintain healthy and happy life. Lord Shiva has himself
narrated the Benefits of Urine Therapy to Mother Parvati
which has been referred in the ancient book Dammar
Tantra in Vedas. In Ancient Books and Vedas Urine is
referred as Shivambu (Auto Urine) meaning Water of
Shiva. Urine Therapy is the ancient method of treatment. The
Powerful practice for healing Self-Urine Therapy has been
referred in Shivambu Kalpa Vidhi part of 5000 years old
document called Damar Tantra linking this practice to Vedas
the sacred Hindu texts. Reference of Urine Therapy is also

found in almost all the volume of Ayurveda and in one of the
volumes Bhavprakasha Urine is termed as Vishaghna killer
of all poisons and Rasayana which can rejuvenate even old
person and Raktapamaharam which purifies blood and
cures all skin diseases. In Tantrik Yoga culture this practice is
termed as Amroli. Amroli comes from the root word
Amar. They termed Shivambu as Holy Liquid.
According to them Urine is more nutritious than even milk as
you are not only physically benefited by the practice, but you
become spiritually advanced because it is an Elixir for body,
mind and spirit1. God has given us this precious Gift (Urine)
right from our very birth. The proverb 5:15 have also been
referred in the Holy Bible: Drink water out of thane own
cistern. In India, Cowpathy is known as an old system of
traditional medicine mentioned in ancient Indian literature
(Ayurveda) as Panchgavya Chikitsa. The Ayurvedic
medicines of animal origin are mainly prepared from
Panchgavya (five things from Indian cow viz., urine, dung,
milk, butter oil and curd), which boost up the body immune
system and makes body refractory to various diseases2. The
specificity of immune system depends upon the number and
activity of lymphocytes.3 studied the immunomodulatory
effect of cow urine in mice and found that cow urine
enhances both T- and B-cell blastogenesis and also increases
the level of IgG. Kumar and Chauhan et al., reported increase
in both cellular and humoral immune responses due to cow
urine2,4. The present study was planned to investigate the
blastogenic activity of lymphocytes and effect of in-vivo
cow urine treatment on it so as to find out their potential to
mount protective immune response against diseases.
Oxidative stress defined as an imbalance between oxidants
and anti-oxidants leads to many biochemical changes and is
an important causative factor in several human chronic
diseases, such as atherosclerosis and cardiovascular diseases,
mutagenesis and cancer, several neurodegenerative disorders
Page 164

and the aging process. Diabetes mellitus is one such disease,
mostly spread over worldwide and the diabetic patients will
continue to increase worldwide in the future. It has been
postulated that the etiology of the complications of diabetes
involves oxidative stress perhaps as a result of
hyperglycemia. The elevated level of blood glucose in
diabetes produces oxygen free radicals which cause
membrane damage due to peroxidation of membrane lipids
and protein glycation. It has been suggested that free radical
activity is increased in diabetes. Under physiological
conditions, glucose produces oxidants that exhibit reactivity
similar to that of hydroxyl free radicals5. In recent years,
there has been a renewed interest in variety of natural
products with antioxidant potential which can play a major
role in protecting against the molecular damage induced by
reactive oxygen species. The present study was aimed to
study the protective role of cow urine as an anti-diabetic and
potentiality. Punganur cow the sacred Indian cow, Bos
indicus, is believed to be a mobile hospital for the
treatment of many diseases. A number of diseases can be
cured by the use of medicines derived from the cow. Cow
urine is described in detail in ancient Ayurvedic scriptures,
such as Charaka samhita, Shushruta samhita and BrahadWagbhatt, as being bitter, pungent, spicy and warm. Now a
days Cow urine is used as an insecticide and as a regulator
for various disorders like intestinal gas, acidity and cough
and is claimed to make humans wiser and can be used as a
universal easily digestible medicine6. In classical texts Vedas
like Ayurveda, like Charaka samhita and Shushruta samhita,
several medicinal properties of cow urine are described very
much with examples. Cow urine is known to cause weight
loss, and reverse certain cardiac and kidney problems, as well
as indigestion, stomach ache and edema. Cow urine is
considered useful in treating renal colic, jaundice, anemia,
diarrhea, gastric infection, piles and skin diseases including
vitilago and considered as an appetizer and is known to
reverse inflammation, and acts as a diuretic as well as a
nephro-protective agent. However the anti-diabetic properties
of cow urine have not been described in the literature.
Further, although Indian Ayurvedic literature cites various
medicinal properties of cow urine, there is very little
scientific evidence to support7. Hence, the present study was
undertaken.
MATERIALS AND METHODS
Cow Urine Distillate Preparation
The first early morning voided urine of punganur cow (Bos
indicus) was collected from the cow sheds Livestock
Research Station, Palamaner, under Sri Venkateswara
Veterinary University, immediately distilled by using double
distillation unit (at 10000C using a temperature- controlled
distillation apparatus and then stored below 1000C) used for
further studies.
Chemicals
All chemicals and reagents used were of analytical grade and
obtained from Sigma Chemical Company (St. Louis, MO,
USA). The kits for the estimation of blood glucose levels and
serum lipid profiles were obtained from Ranbaxy Diagnostics
and Reckon Diagnostics Pvt. Ltd., India. The standard drug
glibenclamide was purchased from a local pharmacy named
Hetiro Pharamcy, Tirupati. Chittoor Dist, India.
Dose Selection
Evaluations of the anti-diabetic activity of the cow urine
distillate, three dose levels were selected. The rat dose was
calculated from the human dose (60 ml per day), multiplied
by a factor of 0.018 5 which is equal to 5.4 ml / kg body
weight (first dose)8. The second dose selected was twice that
of the first dose, i.e. 10.8 ml / kg body weight and the third
dose were selected as 50 % of the first dose i.e. 2.7 ml / kg
body weight.
Animal Treatment
Healthy Wistar albino rats (150 to 180 g) Animals were
housed in a room with temperature maintained at 22 20C
and humidity 55 4 %. They were fed with standard
laboratory diet (SKS Feed, India). Rats were divided into 6
groups each containing 8 animals and allowed food and water
ad libitum throughout the investigation. All the procedures
are approved by the institutional animal ethics committee.
Preparation of Streptozotocin Solution
Preparation of 0.1 M citrate buffer solution of pH = 4.5: An
accurately weighed quantity of Trisodium citrate (14.9 g) was
dissolved in sufficient distilled water to produce 1000 ml and
the pH was adjusted to 4.5 using conc. HCl. The solution of
streptozotocin was prepared by dissolving the weighed
quantity of streptozotocin in 0.1 M freshly prepared ice -cold
citrate buffer (pH 4.5).
Experimental Induction of Diabetes
Diabetes was induced in rats by streptozotocin
intraperitoneally injection at a dose level of 50 mg / kg b.wt.
It is dissolved in citrate buffer (0.1M, pH 4.5) in the volume
of 1 ml / kg. In order to prevent hypoglycemia during the first
day after the streptozotocin administration, the diabetic rats
were given 5 % w/v glucose solution orally. Three days after
the injection, the blood glucose levels were measured and the
animals with blood glucose levels above 300 mg/dl were
considered to be diabetic and were used in the subsequent
experiments. In all the experiments, rats were fasted for 16 h
prior to streptozotocin injection. Animals were divided into
six groups of 8 rats per group. The test samples were
administered orally for 2 weeks9.
Group I: Normal control group-Animals received only
vehicle
Group II: Diabetic control group (streptozotocin treated) Animals received only vehicle.
Group III: Standard drug group-Diabetic animals received
daily a single oral dose of the reference drug glibenclamide
(0.25 mg / kg) from day 1 to14.
Group IV: Diabetic animals received daily a single oral dose
of Cow urine distillate 2.7 ml / kg body weight from day 1
to14.
Group V: Diabetic animals received daily a single oral dose
of Cow urine distillate 5.4 ml / kg body weight from day 1 to
14.
Group VI: Diabetic animals received daily a single oral dose
of Cow urine distillate 10.8 ml / kg body weight from day 1
to 14.
The effects of administration of cow urine distillate to
diabetic rats were determined by measuring the fasting blood
glucose levels, serum lipid profiles, liver glycogen levels and
initial and final changes in body weight. Day 3 of induction
was designated as day 1 for administration of the test sample
Page 165

to diabetic rats. Fasting blood glucose levels were measured
on day 1, 5, 10 and 14 of the test sample administration
period. Other parameters were determined on 15th day of
experimentation, after the animals were sacrificed by
decapitation.
Blood Sampling
Blood samples were collected retro-orbitally from the inner
canthus of the eye under light ether anesthesia using capillary
tubes. Blood was transferred into fresh vials and serum was
separated by centrifuging at 2000 rpm for 2 minutes. Blood

glucose levels were measured using glucose kit.
Statistical Analysis
The data were expressed as Mean SEM and analyzed using
one way analysis of variance (Anova), followed by a post hoc
Sheffes multiple comparison tests using SPSS computer
software version 10. The values were considered significant
when P < 0.05.
Table 1: Effect of Cow Urine Distillate on Blood Glucose Levels in Streptozotocin-treated Diabetic Rats
Group
Dose
Glucose levels in blood (mg / dl)

Day 1
Day 5
Day 10
Day 14
Group I
-85.89 0.96bc
84.88 1.44bc
84.89 0.89bc
85.66 0.73bc
-336.10 4.88a
Group II Diabetic control
359.54 2.78ac
367.78 1.65ac
374.84 8.11ac
0.25
340.52 0.99a
III- Standard glibenclamide
261.47 1.01ab
194.88 1.00ab
141.19 2.87ab
2.70
336.011 2.89a
250.89 1.77ab
231.11 1.76ab
200.15 1.70ab
Group IV
a
ab
ab
Group V
5.40
341.44 1.88
250.11 0.89
209.78 2.88
173.96 1.83ab
Group VI
10.80
339.91 2.81a
230.96 2.11ab
199.16 1.79ab
145.59 1.78ab
All the values are expressed as mean SEM (n = 8), values are statistically significant at P < 0.05 a P<0.05 when compared with the normal control group b
P < 0.05 when compared with the diabetic control group c P < 0.05 when compared with the standard group
Table 2: Effect of Cow Urine Distillate on the Glycogen Content, Body Weight and Lipid Profiles in Streptozotocin-treated Diabetic Rats
Group
Glycogen content mg / g
Body weight % change
Cholesterol mg / dl
Triglycerides mg / dl
HDL mg / dl
Group I
35.26 88bc
+8.99 1.89bc
62.019 2.44bc
79.09 1.16bc
59.16 0.67bc
ac
ac
ac
ac
Group II Diabetic control
16.57 1.01
-13.87 1.08
126.74 0.71
189.24 0.69
36.09 1.11ac
30.89 2.09ab
III- Standard glibenclamide
-3.88 0.79ab
68.45 1.21ab
89.99 1.10ab
49.71 1.19ab
23.88 2.12ab
Group IV
-7.01 2.03ab
89.03 1.08ab
125.81 1.006ab
38.21 1.10ab
Group V
24.11 0.99ab
-7.10 0.28ab
87.88 0.10ab
120.19 2.01ab
40.65 1.11ab
25.72 2.71ab
Group VI
-5.12 0.19ab
82.81 1.11ab
134.11 1.91ab
44.32 1.011ab
All the values are expressed as mean SEM (n = 8), values are statistically significant at P < 0.05 a P < 0.05 when compared with the normal control group b
P < 0.05 when compared with the diabetic control group c P < 0.05 when compared with the standard group
RESULTS AND DISCUSSION

Streptozotocin administration to experimental animals
resulted in a significant (P < 0.05) rise in blood glucose
levels. The changes in body weights and fasting blood
glucose levels, before and after treatment with the test drug in
streptozotocin -induced diabetic animals are shown in Table
1 and 2. Fasting blood glucose levels of untreated diabetic
rats were significantly higher and the body weights were
lower than those in normal rats. Diabetic animals treated with
cow urine distillate showed significant lowering of blood
glucose levels and a significant increase in body weights (P <
0.05). Serum cholesterol, triglycerides and HDL levels in all
the groups of streptozotocin-treated diabetic animals are
given in Table 2. The cholesterol and triglyceride levels were
significantly higher and the HDL levels were significantly
lower in the untreated diabetic rats compared with the values
in normal rats. The treated diabetic rats had lower levels of
cholesterol, and triglycerides and a higher level of HDL
compared with those in the untreated diabetic group. The
treatment with cow urine distillate produced almost normal
levels of cholesterol, triglyceride and HDL. Table 2 shows
the hepatic glycogen levels in all the animal groups. The liver
glycogen levels in streptozotocin- treated diabetic rats were
significantly lower than those in normal rats. Treatment with
cow urine distillate improved the liver glycogen significantly,
as indicated by the higher levels of hepatic glycogen in the
treated diabetic group compared with those in the untreated
diabetic group. Cow urine is one of a number of traditional
remedies that have several pharmacological actions. The use
of cow urine as an anti-diabetic agent has been described in
various Ayurvedic texts. However, there is no information
about its activity in experimental diabetes. The present study
indicates that cow urine was able to provide significant
protection against diabetes in streptozotocin treated diabetic

rats. Streptozotocin is widely used to induce experimental
diabetes in animals. Streptozocin or Streptozotocin is a
cytotoxic nitrosoureidogluco pyranose obtained from the
fermentation of Streptomyces achcromogenesis and it
produces diabetes in a number of animals such as rats,
rabbits, and mice. The mechanism of their action on
pancreatic cells has been intensively investigated. The
cytotoxic action of this diabetogenic agent is mediated by
reactive oxygen species. Streptozotocin enters cells via a
glucose transporter (GLUT2) and causes alkylation of DNA.
DNA damage induces activation of poly ADP-ribosylation, a
process that is more important for the diabetogenic effect of
streptozotocin than DNA damage itself. Poly ADPribosylation leads to depletion of cellular NAD+ and ATP.
Enhanced ATP dephosphorylation after streptozotocin
treatment supplies a substrate for xanthine oxidase resulting
in the formation of superoxide radicals. Consequently,
hydrogen peroxide and hydroxyl radicals are also generated.
Furthermore, streptozotocin liberates toxic amounts of nitric
oxide that inhibits aconitase activity and participates in DNA
damage. As a result of the streptozotocin action, cells are
destroyed by necrosis10. The fundamental mechanism
underlying hyperglycemia in diabetes mellitus involves the
over-production (excessive hepatic glycogenolysis and
gluconeogenesis) and decreased use of glucose by the
tissues11. Studies in animals with streptozotocin-induced
diabetes treated with cow urine distillate revealed a
significant reduction in the blood glucose level when
compared with diabetic control groups at the end of the
experimental period. Induction of diabetes with
streptozotocin is associated with a characteristic loss of body
weight, which is due to increased muscle wasting in
Page 166

12
diabetes . Diabetic rats treated with the cow urine showed an

improvement in weight gain compared with the diabetic
control. The marked increase observed in serum triglycerides
and cholesterol and the decrease in HDL in untreated diabetic
rats is in agreement with the findings of Nikkila and Kekki13.
Diabetic rats treated with the cow urine exhibited a
significant decrease in cholesterol and triglycerides and an
increase in HDL compared with the diabetic control.
Glycogen syntheses in the rat liver and skeletal muscles are
impaired during diabetes14. The decreased glycogen levels
may probably be due to the lack of insulin in the diabetic
state, which results in the inactivation of the glycogen
synthase systems. In the present investigation, a significant
increase in glycogen levels was observed in the treated
groups, which might be due to the reactivation of the
glycogen synthase system. Oxidative stress has been shown
to play an important role in the etiology of diabetes15.
Streptozotocin produces oxygen radicals in the body, which
causes pancreatic injury and could be responsible for the
increased blood glucose16. The compounds responsible for
the anti-diabetic activity of cow urine are at presently not
known. Studies have been carried out to examine the antioxidant potential of cow urine. For example17 described the
anti oxidant properties of cow urine using two in vitro
models, DPPH radical scavenging activity and superoxide
scavenging activity using ascorbic acid as a reference
standard.7 have described the antioxidant action of cow urine
using an ABTS assay model and the antioxidant effect of cow
urine was b due to the presence of volatile fatty acids. Further
research in this field can be carried out by assessing the anti
diabetic protective role of Punganur cow urine. Presence of
antioxidants, free radical scavengers in cow urine could be
responsible for its antidiabetic action. There are estimation
that there are over 5.8 crore people who are diabetic in India.
Diabetes is more common almost everywhere in the world. It
is considered to be the root cause of many chronic diseases.
Urine Therapy is the safest and easiest method of treatment to
prevent, Control and cure Diabetes. It can safe guard all other
complications arising from diabetes include heart disease,
hypertension, and diabetic retinopathy.
ACKNOWLEDGEMENT
It is with most profound feelings of respect, sincerity and high regards that I
express my indebtedness and deep sense of gratitude to my venerable
brother, Pioneer and teacher M. Chandrasekhara Reddy and family K.
Bharani, Master M. Druvin Reddy, SWE, Ohio, USA, for his meticulous
guidance, Stimulating discussion, Awe inspiring Encouragement and
suggestions to complete this work with confidence and for providing
necessary financial asset facilities to carry out this study. I will ever remain
grateful to him for his inspiring guidance, wise counsel unfailing attention.
He has been a source of inspiration and confidence in my research and in my
whole life. The authors are thankful to Dept. of LPM, SVVU for providing
the facilities to carry out this study.
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2. Chauhan RS, Singh DD, Singhal LK and Kumar R. Effect of cow Urine
on IL-1 and IL-2. Journal of immunology and Immunopathology 2004;
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3. Chauhan RS, Singh BP and Singhal LK. Immunomodulation with
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5. Vijayakumar M, Govindarajan R, Rao GMM, Shirwaikar A, Rao Ch V,
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/BF00405106 PMid:2210118
13. Nikkila EA, Kekki M. Plasma Transport kinetics in diabetes mellitus.
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14. Huang X, Vaag A, Hanson M, Weng J, Goop L. Impaired insulin
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Cite this article as:
M. Vijaya Bhaskara Reddy, A. Karthik, P. Sasikala. Protective role of
Punganur cow urine on streptozotocin induced diabetes in rats. Int. Res. J.
Pharm. 2013; 4(8):164-167 http://dx.doi.org/10.7897/2230-8407.04832
Source of support: Nil, Conflict of interest: None Declared
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Raad et al., IJPSR, 2013; Vol. 4(4): 1534-1539.

IJPSR (2013), Vol. 4, Issue 4

ABSTRACT: Cow urine therapy and all traditional practices from

INTRODUCTION: It is widely accepted among

As an example, it is now estimated that about half of

International Journal of Pharmaceutical Sciences and Research

Raad et al., IJPSR, 2013; Vol. 4(4): 1534-1539.

Cow, Bos indicus is a most valuable animal in all

Qualitative test for proteins: The qualitative test of

Disc diffusion assay: Antibacterial activity of urine

Quantitative estimation of Protein: The Folin

Paper chromatography: The urine sample showing

International Journal of Pharmaceutical Sciences and Research

Raad et al., IJPSR, 2013; Vol. 4(4): 1534-1539.

(Ninhydrine). The Rf value of the spot that appeared

effect against all cultures under test, sample G

International Journal of Pharmaceutical Sciences and Research

Raad et al., IJPSR, 2013; Vol. 4(4): 1534-1539.

Paper Chromatography: The chromatogram after

salts which are beneficial to the human body because

FIGURE 1: COLOR VARIATION IN THE 10 URINE

According to Figure 3, 50% showed yellow color ,

International Journal of Pharmaceutical Sciences and Research

Raad et al., IJPSR, 2013; Vol. 4(4): 1534-1539.

Cow urine contains different constituents; it is rich in

The gram negative bacteria were more efficiently

The presence of protein in all the samples was clear

FIGURE 3: ZONE OF INHIBITION (in cm) RECORDED

As according to figure 3, the sample G gave the

FIGURE 2: SENSITIVITY PATTERN OF THE TEST

All the samples showed the presence of antibacterial

A higher zone of inhibition was observed by sample

International Journal of Pharmaceutical Sciences and Research

Raad et al., IJPSR, 2013; Vol. 4(4): 1534-1539.

CONCLUSION: The Antibacterial property of the

Roder BL, Wandall DA, Frimodt-Moller N, Espersen F,

established genotoxic chemicals. Biomed. Environ. Sci 2001;

How to cite this article:

International Journal of Pharmaceutical Sciences and Research

ISSN: 1388-0209 (Print) 1744-5116 (Online) Journal homepage: http://www.tandfonline.com/loi/iphb20

Antidiabetic Activity of Cow Urine and a Herbal

Published online: 05 Jan 2009.

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Antidiabetic Activity of Cow Urine and a Herbal Preparation

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cretion or action or both (Nyholm et al., 2000). Diabetes

Accepted: April 2, 2008

2008 Informa UK Ltd.

E.E. Jarald et al.

Materials and Methods

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The urine of a 2-year-old virgin Gujarati Indian cow known

Animals and treatment

Acute toxicity studies

to be safe up to the dose of 2000 mg/kg, and doses of 200

Antidiabetic activity of cow urine and cow urine preparation

Effect of various preparations in alloxan-induced diabetic rats.

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Daily dose (mg/kg)

3rd day (alloxan)