a-collection-of-research-articles-on-the-medical-potential-of-cow-urine

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 NON- 

PATHOGENIC BACTERIA 

S. Raad 1 , D.V. Deshmukh* 2 , S. N. Harke 2 and M.S. Kachole 1 

Department <strong>of</strong> Biochemistry, Dr. Babasaheb Amberdkar Marathwada University 1 , Aurangabad, Maharashtra, 

India 

MGM’s, Institute <strong>of</strong> Biosciences and Technology 2 , Auranagabad, Maharashtra, India 

Keywords: 

Cow urine, Antibacterial activity, Zone 

<strong>of</strong> inhibition, Antimicrobial Peptides 

Correspondence to Author: 

Dr. Devendra V. Deshmukh 

Assistant Pr<strong>of</strong>essor, MGM’s, Institute 

<strong>of</strong> Biosciences and Technology, N-6, 

CIDCO, Aurangabad, India 

E-mail: devcyano@gmail.com 

INTRODUCTION: It is widely accepted among 

clinicians, medical <strong>research</strong>ers, microbiologists and 

pharmacologists, that antibiotic resistance will, in the 

very near future, leave healthcare pr<strong>of</strong>essionals 

without effective therapies for bacterial infections. 

QUICK RESPONSE CODE 

ABSTRACT: Cow urine therapy and all traditional practices from 

Indian systems <strong>of</strong> medicine have a strong scientific base. The cow has 

proved to be a boon in the areas <strong>of</strong> agriculture, science and technology, 

industry, energy, medicine etc for the development <strong>of</strong> any nation, in 

addition being eco-friendly in nature. In the present study the 

antibacterial potentials <strong>of</strong> 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 <strong>of</strong> 

inhibition was shown by sample G against P aeruginosa NCIM 2945 

(1.8cm) while the smallest zone <strong>of</strong> 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 <strong>of</strong> 

inhibition was observed by sample G against P aeruginosa NCIM 2945 

as compared to that <strong>of</strong> 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 <strong>of</strong> 

proline which is considered as a major amino acid in antimicrobial 

peptides was also observed in the urine sample G. 

IJPSR: 

ICV (2011)- 5.07 

Article can be accessed 

online on: 

www.ijpsr.com 

As an example, it is now estimated that about half <strong>of</strong> 

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 <strong>of</strong> 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 <strong>of</strong> resistance to 

morbidity and mortality is quantitatively unclear. 

International Journal <strong>of</strong> Pharmaceutical Sciences and Research 1534

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

Cow, Bos indicus is a most valuable animal in all 

community. The cow urine is useful in number <strong>of</strong> 

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 <strong>of</strong> bacterial infection and 

fight against cancers. Medicinal usage <strong>of</strong> cow urine 

are extensively searched and scientifically endorsed 

8 . 

In the Present study the antibacterial potentials <strong>of</strong> the 

cow urine have been investigated against 14 different 

pathogenic and nonpathogenic bacteria. 

MATERIALS AND METHODS: 

Collection <strong>of</strong> 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 <strong>of</strong> choice for 

<strong>collection</strong> <strong>of</strong> the samples. Samples were collected in 

sterile containers, 20 ml <strong>of</strong> 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. 

Qualitative test for proteins: The qualitative test <strong>of</strong> 

protein was performed as according to Martin and 

Mittelman 9 . The urine Samples were centrifuged at 

3000 rpm for 10 mins for the removal <strong>of</strong> sediments. 

After centrifugation the supernatant was collected 

and heat test for proteins was performed to observe 

the presence <strong>of</strong> protein. 

Quantitative estimation <strong>of</strong> Protein: The Folin 

Lowry method was a method <strong>of</strong> choice for estimation 

<strong>of</strong> protein. Aliquots <strong>of</strong> protein standard solution were 

pipetted out as into a series <strong>of</strong> 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 <strong>of</strong> alkaline mix 

(reagent C) was pipetted out, mixed well and allowed 

to stand for 15 min, at room temperature. 0.5 ml <strong>of</strong> 

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 . 

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 <strong>of</strong> filter paper 

Whattman No. 1 were prepared. The discs were 

sterilized by autoclave at 121°C. After the 

sterilization the moisture discs were dried on hot air 

oven at 50°C. The sterile discs were kept in a 

presetrilized container until further use. 

Disc diffusion assay: Antibacterial activity <strong>of</strong> urine 

samples against the test organisms was done by disc 

diffusion assay 11 . Petri plate containing 15 ml <strong>of</strong> 

solidified nutrient agar was spread inoculated with 

100 μl <strong>of</strong> 12 h old test bacterial cultures. Presterilized 

Whatman No.1 paper discs (6 mm) were saturated 

with 50 µl <strong>of</strong> urine and dried to be used in assays. 

The plates were kept at 4 o C for 10 min before they 

were incubated at 37 o C for 24 h. Anti-bacterial was 

assessed by measuring the diameter <strong>of</strong> growth 

inhibition zone around the discs. Sensitivity <strong>of</strong> test 

organisms was also checked against commercial 

discs (Hi Media, India) containing standard 

antibiotics. 

Paper chromatography: The urine sample showing 

highest protein content and antibacterial activity was 

analyzed for the presence <strong>of</strong> amino acids using paper 

chromatography technique . A strip <strong>of</strong> wattman’s 

filter paper No. 1. was used, approximately 1 cm 

from one end <strong>of</strong> the length a line was drawn with the 

help <strong>of</strong> a pencil. At the centre <strong>of</strong> the line a tiny spot 

<strong>of</strong> 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 ¾ th the paper and then taken out and dried 

in an oven and then spayed with locating reagent 



(Ninhydrine). The Rf value <strong>of</strong> the spot that appeared 

was calculated. 

RESULTS: 

Qualitative test for proteins: All the urine samples 

tested for the presence <strong>of</strong> 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 <strong>of</strong> protein gave mixed results. 

Sample G gave the highest protein content 520 

µgm/ml while sample J showed the lowest 

concentration <strong>of</strong> protein (Table 1). 

Antibacterial assay: Antibacterial activities <strong>of</strong> all 

the urine samples were tested using disc diffusion 

method. On the basis <strong>of</strong> 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 

Sample 

Absorbance at Concentration <strong>of</strong> 

660 nm protein in µgm/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 

Bacterial 

Urine samples 

cultures A B C D E F G H I J 

E. coli 

NCIM 2345 

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) 

E. coli 

NCIM 2065 

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) 

E. coli 

NCIM 2015 

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 

B subtilis 

NCIM 2113 

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 

B licheniformis 

NCIM 2015 

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 

B megaterium 

0.9 

R R 

NCIM 2083 

(0.30) 

R R 1.0 (0.5) 1.1 (0.5) R 0.5 (0.34) 0.3 

S aureus 

0.5 

R R R R R 

NCIM 2124 

(0.75) 

0.9(0.11) R R R 

S aureus 

NCIM 2125 

R R R R 0.6(0.12) R 0.8(0.25) R R R 

S aureus 

NCIM 2079 

R R R R R R 0.5(0.45) R 0.6 (0.22) 0.6 

P aeruginosa 

NCIM 2945 

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 

P aeruginosa 

NCIM 2053 

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 

P vulgaris 

NCIM 2857 

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 

K pneumonie 

NCIM 2957 

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 

S typhimurium 

NCIM 2501 

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 

Cumulative 

Inhibition 

4.8 4.9 7.3 5.4 6.8 10 15 6 7.8 7 

* Zone <strong>of</strong> inhibitions in centimeters, Values in the parenthesis is standard deviations, R- Resistant. 



TABLE 3: ZONE OF INHIBITION SHOWN BY 14 BACTERIAL CULTUTRES AGAINST STANDARD ANTIBIOTICS 

Standard Antibiotics 

Bacterial cultures 

Methicillin 

(5mcg/disc). 

Gentamicin 

(10 mcg/disc) 

Oxacillin 

(5mcg/disc) 

Vancomycin 

(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 <strong>of</strong> spot 

and the Rf value <strong>of</strong> 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 <strong>of</strong> standard proline. 

DISCUSSION: Commonly, antibiotics are widely as 

conservative treatment in various microbial 

infections and diseases 12 . Considering the enormous 

quantity <strong>of</strong> antibiotics used, the situation should have 

been that there would be no infectious diseases. But, 

the fact is that the problems <strong>of</strong> infectious diseases are 

increasing day‐by‐day. Some <strong>of</strong> the major hindrances 

are that bacteria have genetic ability to transmit and 

acquire resistance towards the drugs 13 and there are 

also adverse effects <strong>of</strong> 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 <strong>of</strong> remedies to 

cure all ailments <strong>of</strong> mankind, as it has always been a 

first rate drug store with enormous range <strong>of</strong> plants, 

micro organisms and animals. 15 

The ancient literature <strong>of</strong> cow urine has always 

focused on prevention <strong>of</strong> disease and maintaining the 

health and treatment <strong>of</strong> diseases. Cow urine acts like 

a magical potion for the treatment <strong>of</strong> 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 <strong>of</strong> allergies. Urine contains volatile 

salts which are beneficial to the human body because 

these salts destroy acidity and get rid <strong>of</strong> pain in 

kidney, intestine, and womb; furthermore urine, a 

natural tonic, eliminates giddiness, tension in nerves, 

lazy feeling, hemicrama, paralysis, common cold, 

diseases <strong>of</strong> brain, nerves and joints. 

In the present study the antibacterial potential <strong>of</strong> 10 

different urine samples from cows at the MGM’s 

farm house was revealed. The variation in the color 

<strong>of</strong> the urine samples may be due to the amount and 

type <strong>of</strong> 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% <strong>of</strong> the urine samples, while 

20% <strong>of</strong> the urine samples showed deep yellow 

coloration. 



Cow urine contains different constituents; it is rich in 

potassium, chloride, calcium, estrogen, phosphorous, 

urinary proteins 16 . Various <strong>research</strong> 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 <strong>of</strong> 

protein. Vats and Kanupriya 17 has reported that the 

components <strong>of</strong> 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 <strong>of</strong> the 

cow urine distillate against 4 gram negative bacteria. 

A synergistic effect <strong>of</strong> 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 <strong>of</strong> protein in all the samples was clear 

evidence that all the samples do contain the presence 

<strong>of</strong> bioactive compounds. Marshall and Arenas 18 

pointed out the use <strong>of</strong> the importance <strong>of</strong> naturally 

occurring peptides and their use as an alternative to 

chemical antibiotics and their role as antimicrobials. 

The antibacterial potentials <strong>of</strong> the cow urine was 

tested against some pathogenic and non pathogenic 

bacteria (Table 2). The highest zone <strong>of</strong> inhibition 

was shown by sample G against P aeruginosa NCIM 

2945 (1.8cm) while the smallest zone <strong>of</strong> 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 <strong>of</strong> 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 <strong>of</strong> antibacterial 

activity. From the Figure 2, it was observed that the 

maximum activity <strong>of</strong> 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 <strong>of</strong> cow urine against gram negative and gram 

positive bacteria. 

A higher zone <strong>of</strong> inhibition was observed by sample 

G against P aeruginosa NCIM 2945 as compared to 

that <strong>of</strong> 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 <strong>of</strong> the sample revealed the 

presence <strong>of</strong> proline. The amino acid proline is 

considered as a major amino acid in antimicrobial 

peptides 21 . 



CONCLUSION: The Antibacterial property <strong>of</strong> the 

cow urine was reveiled using biological assay. Total 

10 urine samples were tested against 14 different 

strains <strong>of</strong> bacteria. The ability <strong>of</strong> the cow urine 

sample was more to inhibit the gram negative 

bacteria than that <strong>of</strong> gram positive bacteria. The 

highest zone <strong>of</strong> inhibition that was observed was 

1.8cm by sample G. Thus sample G was the only one 

which has inhibited the growth <strong>of</strong> all the test 

organism and when compared with standard 

antibiotic proved to be more promising. The presence 

<strong>of</strong> amino acid proline in the sample G has proved its 

potential similar to peptide antibiotics. 

REFERENCES: 

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

Skinhoj P, Rosdahl VT. Clinical features <strong>of</strong> Staphylococcus 

aureus endocarditis: a 10-year experience in Denmark. 

Archives Internal Medicine, 1999; 159(5):462-469. 

2. Col NF, O’Connor RW. Estimating worldwide current 

antibiotic usage: report <strong>of</strong> Task Force 1. Rev Infect Dis 

1987; 9: S232–243. 

3. Pathak ML, Kumar A. Cow praising and importance <strong>of</strong> 

anchyagavya as medicine. Sachitra Ayurveda 2003, 5: 56-59. 

4. Krishnamurthi, K, Dutta D, Devi SS, Chakrabarti T. 

Protective effect <strong>of</strong> diatillate and redistillate <strong>of</strong> cow,s urine in 

human polymorphonuclear leukocytes challenged with 

established genotoxic chemicals. Biomed. Environ. Sci 2001; 

17: 57-66. 

5. Chauhan RS, Singh BP, Singhal LK. Immunomodulation 

with kamdhenu Ark in mice. J. Immunol. Immunopathol 

2001; 71: 89-92. 

6. Ojewole JA, Olusi SO. Effects <strong>of</strong> cow’s urine concoction 

on plasma glucose concentration in fasted rats R. Soc. 

Trop. Med. Hyg 1976; 241-245. 

7. Elegbe RA, Oyebola DDO. Cow’s urine poisoning in 

Nigeria: the cardiotoxic effects <strong>of</strong> cow’s urine in dogs. 

Trans. R. Soc. Trop. Med. Hyg 1976; 127-132. 

8. Somvanshi R. Veterinary medicine and animal keeping in 

ancient India. Asian Agri-History 2006 ;10(2) :133–146. 

9. Martin AJP , Mittelman R Quantitative Micro-analysis <strong>of</strong> 

Amino-acid Mixtures on Paper Partition Chromatograms' 

1948. Biochem ; 43: 353- 358. 

10. Setsuro M, Nobuko I, Yuki N. Colorimetric estimation <strong>of</strong> 

amino acids and peptides with folin phenol reagent, 

Analytical Biochemistry 1996; 16(2): 365-371. 

11. Bauer AW, Kirby WMM, Sherris JC, Turck M. Antibiotic 

susceptibility testing by a standardized single disc method. 

American. Journal <strong>of</strong> Clinical. Pathology 1966; 45: 493-496. 

12. Daniel B, Alexander R, Ehud IA. Changing bacterial isolates 

and antibiotic sensitivities <strong>of</strong> purulent dacryocystitis. Orbit; 

2005: 24 (2): 95‐98. 

13. Cohen ML. Epidemiology <strong>of</strong> drug resistance: implications 

for a post‐antimicrobial era, Science 1992; 257: 1050‐1055. 

14. Ahmad I, Mehmood Z, Mohammad F. Screening <strong>of</strong> some 

Indian medicinal plants for their antimicrobial properties. J 

Ethnopharmacol 1998; 62: 183‐193. 

15. Sujata MB, Charles HB. New agents for Gram‐positive 

bacteria. Current Opinion in Microbiology. 2000; 3(5): 

528‐534. 

How to cite this article: 

Raad S, Deshmukh DV, Harke SN and Kachole MS: Antibacterial activity <strong>of</strong> Cow urine against some Pathogenic and Nonpathogenic 

Bacteria. Int J Pharm Sci Res 2013; 4(4); 1534-1539. 


Pharmaceutical Biology 

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

Antidiabetic Activity <strong>of</strong> 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 <strong>of</strong> 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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Pharmaceutical Biology 

2008, Vol. 46, Nos. 10–11, pp. 789–792 

Antidiabetic Activity <strong>of</strong> Cow Urine and a Herbal Preparation 

Prepared Using Cow Urine 

E. Edwin Jarald, 1 S. Edwin, 1 V. Tiwari, 1 R. Garg, and E. Toppo 1 

1 Herbal Drug Research Lab, B. R. Nahata College <strong>of</strong> Pharmacy and Research Centre, Mandsaur, Madhya Pradesh, India 

Downloaded by [University <strong>of</strong> Gothenburg] at 21:45 17 September 2015 

Abstract 

An herbal preparation prepared by the traditional healers 

<strong>of</strong> 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 <strong>of</strong> 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 <strong>of</strong> 

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 <strong>of</strong> 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 <strong>of</strong> the local traditional healers. 

Keywords: Alloxan monohydrate, antidiabetic, cow urine, 

herbal preparation. 

Introduction 

Diabetes mellitus is a group <strong>of</strong> 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 (4–5%) all over the 

world. Oral hypoglycemic drugs, such as sulfonylureas and 

biguanides, have been used in the treatment <strong>of</strong> diabetes 

mellitus (Okinea et al., 2005). In spite <strong>of</strong> the introduction 

<strong>of</strong> hypoglycemic agents, diabetes and related complications 

continue to be a major medical problem. Since time 

immemorial, patients with non–insulin-dependent diabetes 

have been treated orally in folk medicine with a variety <strong>of</strong> 

plant extracts. In India, a number <strong>of</strong> plants are mentioned in 

ancient literature (Ayurveda) for the cure <strong>of</strong> diabetic conditions 

known as “madhumeha,” and some <strong>of</strong> 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 

<strong>of</strong> diabetes. The traditional healers prepare a decoction 

using cow urine instead <strong>of</strong> 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 <strong>of</strong> 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 Pr<strong>of</strong>essor, Herbal Drug Research Lab, B. R. Nahata College <strong>of</strong> 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 <strong>of</strong> materials 

The urine <strong>of</strong> 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 <strong>of</strong> extracts 

The herbal preparations using cow urine and distilled water 

were made using the above-mentioned different plant 

species. Equal quantities <strong>of</strong> air-dried samples <strong>of</strong> each plant 

species were ground and mixed with 10-times the equivalent 

volume <strong>of</strong> 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 <strong>of</strong> 

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 <strong>of</strong> 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 <strong>of</strong> B. R. Nahata College <strong>of</strong> Pharmacy, 

Mandsaur, were used for the investigation. The animals 

were housed in standard environmental conditions <strong>of</strong> 

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 <strong>of</strong> the preparations and cow urine was 

determined according to the OECD guidelines (No. 420, 

Organization for Economic Cooperation and Development). 

Female albino mice (20–25 g) were used for this 

study. Dosing amounts for sample in liquid form were calculated 

with the help <strong>of</strong> density or specific gravity. After 

the sighting study, a starting dose <strong>of</strong> 2000 mg/kg (p.o.) <strong>of</strong> 

the test samples was given to various groups <strong>of</strong> five animals 

each. The treated animals were monitored for 14 days for 

mortality and general behavior. No deaths were observed 

through the end <strong>of</strong> the study. The test samples were found 

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

and 400 mg/kg were chosen for further experimentation. 

Antihyperglycemic activity 

Diabetes was induced in rats by injecting 150 mg/kg 

<strong>of</strong> 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 <strong>of</strong> 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 <strong>of</strong> fresh cow urine in 

the blood glucose level <strong>of</strong> normal rats. Fresh cow urine at 

a dose <strong>of</strong> 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 Dunnet’s 

test. The p values less than 0.05 were considered as significant. 

Results 

The phytochemical investigations performed in the extracts 

revealed the presence <strong>of</strong> alkaloids, tannins, flavonoids, carbohydrates, 

and saponins in both the extracts. The results 

<strong>of</strong> antidiabetic activity <strong>of</strong> cow urine and herbal preparations 

prepared using cow urine and water are presented in Table 1. 

The basal blood glucose levels <strong>of</strong> 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 <strong>of</strong> 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 <strong>of</strong> the preparations were found significant 

from the 7th day onwards, whereas the activity <strong>of</strong> cow urine 

was found significant only after 21 days <strong>of</strong> 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 <strong>of</strong> cow urine and cow urine preparation 791 

Table 1. 

Effect <strong>of</strong> various preparations in alloxan-induced diabetic rats. 

Blood glucose concentration (mg/dL) 

Treatment Daily dose (mg/kg) 0th day 3rd day (alloxan) 7th day 14th day 21st day 

Insulin 1 unit/kg 83.2 ± 3.01 451.22 ± 19.30 349.42 ± 19.2 ∗∗ 201.44 ± 12.50 ∗∗ 133.10 ± 16.52 ∗∗∗ 

Diabetic control — 93.4 ± 4.23 439.54 ± 15.90 538.22 ± 20.40 517.98 ± 22.21 530.54 ± 15.20 

CUP 200 79.50 ± 2.01 451.22 ± 13.40 380.44 ± 12.42 ∗∗ 318.60 ± 11.30 ∗∗ 235.55 ± 12.25 ∗ 

CUP 400 84.00 ± 3.04 458.80 ± 14.00 360.45 ± 11.18 ∗∗ 279.80 ± 11.08 ∗∗ 203.34 ± 15.10 ∗∗ 

AP 200 84.10 ± 3.54 478.44 ± 12.16 399.12 ± 13.03 ∗∗ 330.45 ± 13.55 ∗∗ 240.86 ± 18.22 ∗∗ 

AP 400 88.50 ± 4.65 439.42 ± 14.18 369.72 ± 10.90 ∗∗ 298.24 ± 14.50 ∗∗ 220.67 ± 17.55 ∗∗ 

CU 200 84.62 ± 5.00 480.42 ± 16.22 490.88 ± 10.30 465.45 ± 13.82 380.20 ± 18.00 ∗ 

CU 400 80.92 ± 7.01 430.45 ± 17.92 450.88 ± 17.89 410.12 ± 12.56 ∗ 262.40 ± 17.92 ∗∗ 

CU control 400 85.20 ± 3.46 88.54 ± 2.40 84.22 ± 4.80 87.70 ± 3.40 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 Dunnet’s 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 <strong>of</strong> all.” A composition containing 

cows excretions—urine, dung, milk, curd, and ghee—five 

ingredients together known as “panchagawya,” is given to 

women after delivering a baby. Panchagawya is the main ingredient 

<strong>of</strong> many Ayurvedic preparations (Pathak & Kumar, 

2003b). Cow urine, one <strong>of</strong> the ingredients in panchagawya, 

is believed to have many therapeutic values. In India, cow 

urine is used by the majority <strong>of</strong> 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 <strong>of</strong> cow’s urine in 

medicinal and veterinary medicine, a scientific experiment 

was performed in rats to elucidate the effect <strong>of</strong> cow urine 

and cow urine containing preparation as an antidiabetic. 

Alloxan produces hyperglycemia by a selective cytotoxic 

effect on pancreatic beta cells. One <strong>of</strong> the intracellular 

phenomena for its cytotoxicity is through generation <strong>of</strong> 

free radicals demonstrated both in vivo and in vitro (Yadav 

et al., 2002). Our investigations indicate the efficiency <strong>of</strong> 

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 

<strong>of</strong> beta cells <strong>of</strong> pancreatic islets or stimulation <strong>of</strong> 

glycogenesis (Miura et al., 2001). This may be due to the 

presence <strong>of</strong> 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 <strong>of</strong> 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 <strong>of</strong> the phytoconstituents 

present in the extracts because both extracts contains the 

same nature <strong>of</strong> constituents. The interesting observation in 

our study was the antidiabetic activity <strong>of</strong> 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 <strong>of</strong> fresh 

cow urine may be due to its stimulation in peripheral use <strong>of</strong> 

glucose. According to literature, cow urine was found to exhibit 

an antioxidant effect (Krishnamurthi et al., 2004). Free 

radicals are implicated in wide range <strong>of</strong> diseases including 

diabetes; the antioxidant activity <strong>of</strong> cow urine also may be 

one <strong>of</strong> the reasons for its observed antidiabetic effect. 

Chemopr<strong>of</strong>iling <strong>of</strong> cow urine in our laboratory confirmed 

the presence <strong>of</strong> 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 <strong>of</strong> 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 <strong>of</strong> 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 <strong>of</strong> the observed antihyperglycemic 

effect. This study supports the claim <strong>of</strong> the traditional healers 

<strong>of</strong> Mandsaur. 

Acknowledgement 

The authors are thankful to Gayathri Parivar (local traditional 

healers) for providing the necessary information to 

carry out this <strong>research</strong> work.

792 E.E. Jarald et al. 


References 

Ainapure SS, Arjaria PD, Sawant VR, Baid PS, Maste SS, Varda 

AB (1985): Hypoglycemic activity <strong>of</strong> an indigenous preparation. 

Indian J Pharmacol 17: 238–239. 

Brain KR, Turner TD (1975): The Practical Evaluation <strong>of</strong> 

Phytopharmaceuticals. Bristol, Wright-Scientechnica, 

pp. 10–30. 

Gowenlock AH, McMurray RJ (1988): Varley’s Practical Clinical 

Biochemistry. New Dehli, CBS Publishers and Distributors, 

pp. 149–153. 

Grover JK, Yadav S, Vats V (2002): Medicinal plants <strong>of</strong> India with 

anti-diabetic potential. J Ethnopharmacol 81: 81–100. 

Kar A, Choudhary BK, Bandyopadhyay NG (2003): Comparative 

evaluation <strong>of</strong> hypoglycaemic activity <strong>of</strong> some Indian medicinal 

plants in alloxan diabetic rats. J Ethnopharmacol 84: 

105–108. 

Khandelwal KR (2005): Practical Pharmacognosy. Pune,Nirali 

Prakashan, pp. 152–154. 

Krishnamurthi K, Dutta D, Devi SS, Chakrabarti T (2004): Protective 

effect <strong>of</strong> distillate and redistillate <strong>of</strong> cow’s urine in human 

polymorphonuclear leukocytes challenged with established 

genotoxic chemicals. Biomed Environ Sci 17: 57–66. 

MiuraT,ItohC,IwamotoN,AatoM,KawaiM,ParkSR,SuzikiI 

(2001): Hypoglycemic activity <strong>of</strong> the fruit <strong>of</strong> the Momordica 

charantia in Type 2 diabetic mice. J Nutr Sci Vitaminol 

(Tokyo) 47: 340–344. 

Mohamed B, Abderrahim Z, Hassane M, Abdelhafid T, 

Abdelkhaleq L (2006): Medicinal plants with potential antidiabetic 

activity—A review <strong>of</strong> ten years <strong>of</strong> herbal medicine 

<strong>research</strong> (1990–2000): Int J Diabetes Metab 14: 1–25. 

Mukherjee KP (2002): Quality Control <strong>of</strong> Herbal Drugs. 

New Delhi, New Business Horrizons, p. 525. 

Nyholm B, Porksen N, Juhl CB, Gravholt CH, Butler PC, Weeke 

J, Veldhuis JD, Pincus S, Schmitz O (2000): Assessment 

<strong>of</strong> insulin secretion in relatives <strong>of</strong> patients with type 2 (noninsulin-dependent) 

diabetes mellitus: evidence <strong>of</strong> early β-cell 

dysfunction. Metabolism 49: 896–905. 

Okinea LKN, Nyarkob AK, Osei-Kwabenaa N, Oppongc IV, 

Barnesa F, Ofosuheneb M (2005): The antidiabetic activity 

<strong>of</strong> the herbal preparation ADD-199 in mice: A comparative 

study with two oral hypoglycaemic drugs. J Ethnopharmacol 

97: 31–38. 

Pathak ML, Kumar A (2003a): Cow praising and importance 

<strong>of</strong> Panchyagawya as medicine. Sachitra Ayurveda 5: 56– 

59. 

Pathak ML, Kumar A (2003b): Gomutra a descriptive study. SachitraAyurveda7: 

81–84. 

Porchezian E, Ansari SH, Shreedharan NKK (2000): Antihyperglycemic 

activity <strong>of</strong> Euphrasia <strong>of</strong>ficinale leaves. Fitoterapia 

71: 522–526. 

Pulok KM, Kuntal M, Kakali M, Peter JH (2006): Leads 

from Indian medicinal plants with hypoglycemic potentials. 

J Ethnopharmacol 106: 1–28. 

Som NS, Praveen V, Shoba S, Radhey S, Kumria MML, 

Ranganathan S, Sridharan K (2001): Effect <strong>of</strong> an antidiabetic 

extract <strong>of</strong> Catharanthus roseus on enzymic activities in 

streptozotocin induced diabetic rats. J Ethnopharmacol 76: 

269–277. 

Yadav S, Vats V, Dhunnoo Y, Grover JK (2002): Hypoglycemic 

and antihyperglycemic activity <strong>of</strong> Murraya koenigii leaves in 

diabetic rats. J Ethnopharmacol 82: 111–116.

Global Journal <strong>of</strong> Pharmacology 4 (1): 41-44, 2010 

ISSN 1992-0075 

© IDOSI Publications, 2010 

Antimicrobial Activities <strong>of</strong> Cow Urine Distillate Against Some Clinical Pathogens 

1 2 1 

Arunkumar Sathasivam, M. Muthuselvam and Rajasekran Rajendran 

1 

Muthaiyah Research Foundation, Thanjavur, Tamilnadu, India - 613 005 

2 

Department <strong>of</strong> Microbiology, Marudupandiyar College, Thanjavur, Tamilnadu, India 

Abstract: From the ancient period cow’s urine has been used as a medicine. In India, drinking <strong>of</strong> cow urine has 

been practiced for thousands <strong>of</strong> 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 <strong>of</strong> Cow Urine Distillate against the clinical pathogenic microorganisms. 

Antibacterial activity <strong>of</strong> Cow Urine Distillate (5, 10 and 15µl) was analyzed against the Bacillus subtilis, 

Pseudomonas aeruginosa, Klebsiella pneumoniae and Salmonella typhi. Maximum antibacterial activity was 

observed in Pseudomonas aeruginosa (7.06±0.05, 8.08±0.18 and 10.4±1.23, mm in diameter, respectively) and 

Salmonella typhi (6.3±1.23, 8.06±0.17 and 10.4±1.2, mm in diameter, respectively). Antifungal activity <strong>of</strong> 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 (3±0.14, 6.3±1.2 and 7.06±0.04, 

mm in diameter) than Aspergillus flavus (2.03±0.25, 4.9±0.26 and 6.3±1.2, 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 <strong>of</strong> bacteria and fungi <strong>of</strong> various origins. 

Key words: Cow Urine Distillate 

Antibacterial and Antifungal Activity 

INTRODUCTION 

treatment <strong>of</strong> falling body parts, discharging lymphs and 

organism infested organs, use <strong>of</strong> cow’s urine (along with 

In Veda, cow’s urine was compared to the nectar. In some other ingredients) has been recommended for bath, 

substrata, several medicinal properties <strong>of</strong> cow’s urine anointing and intake [2]. The cow urine distillate has been 

have been mentioned and are known to cause weight loss, patented as activity enhancer and availability facilitator 

reversal <strong>of</strong> certain cardiac and kidney problems, for bioactive molecules including anti- infective and 

indigestion, stomach ache, edema, etc. Cow urine has a anti-cancer agents (US Patent No 6410 059/2002) [3]. 

unique place in Ayurveda and has been described in Chakra pani mishra in vishva vallabba recommends using 

‘Sushrita Sumhita’ and Ashtanga Sangraha’ to be the extracts <strong>of</strong> cow urine in herbal insecticides [4]. Feeding <strong>of</strong> 

most effective substance secretion <strong>of</strong> animal origin with cow urine increased the feed intake in white legborn 

innumerable therapeutic values. It has been recognized as layers [5]. The present study was carried out to prepare 

water <strong>of</strong> life or “Amrita” (Beverages <strong>of</strong> immortality), the cow urine distillate and to determine the antibacterial and 

nectar <strong>of</strong> the God. In India, drinking <strong>of</strong> cow urine has been antifungal activities. 

practiced for thousands <strong>of</strong> years. Panchagavya is a term 

used in Ayurveda to describe five important substances 

MATERIALS AND METHODS 

obtained from cow namely Urine, Dung, Milk, Ghee and 

Curd. A number <strong>of</strong> formulations mentioned in Ayurveda Collection <strong>of</strong> Sample: Cow urine sample was collected 

describe the use <strong>of</strong> Panchagavya components either alone from cow farm (at Avanam, Thanjavur Dt) using sterile 

or in combination with drugs <strong>of</strong> herbal, animal or mineral container and stored for further uses. 

origin [1]. 

An exhaustive reference <strong>of</strong> cow’s urine having CUD Preparation: Cow urine was distilled at 100°C using 

curative properties in skin diseases, especially leprosy, distillation apparatus [6]. The single distilled cow urine 

is referred to in Caraka samhita. Furthermore, in the was acidified by lowering the pH below 2.0 with the 

Corresponding Author: 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 <strong>of</strong> 85% orthophosphoric acid. The cow urine was plates with equal distance positive control disc was 

again distilled at 100°C using a distillation apparatus to also maintain. All the bacterial plates were incubated at 

remove ammonia. The distillate was stored in sterile glass 37°C for 24hrs and fungal plates at 24°C for 72 hrs. 

flask at refrigerator (4°C). 

After incubation the diameter <strong>of</strong> the minimum zone <strong>of</strong> 

inhibition was measured in mm. For each test, three 

Test Organisms: Bacterial and fungal cultures were replicates were performed. 

used as test organisms Bacillus subtilis (MTCC 7415) 

Pseudomonas aeruginosa (MTCC 7436), Klebsiella Statistical Analysis: Mean and standard deviation were 

pneumoniae (MTCC 7407), Salmonella typhi, Aspergillus calculated to facilitate the comparison <strong>of</strong> the data [8]. 

niger and Aspergillus flavus were collected form microbial 

type culture <strong>collection</strong> centre (MTCC) at Chandigarh. 

RESULTS AND DISCUSSION 

Disc Preparation: 5 mm (diameter) discs were prepared Antibacterial Activity: Antibacterial activity <strong>of</strong> cow urine 

from whattman No.1 filter paper. The discs were distillate was analyzed against the Bacillus subtilis, 

sterilized by autoclave at 121°C. After the sterilization Pseudomonas aeruginosa, Klebsiella pneumoniae and 

the moisture discs were dried on hot air oven at 50°C. Salmonella typhi (Table 1, Fig. 1 and Plate 1). 5, 10 and 

The sterile discs were rinsed in cow urine distillate at 15µl concentrations <strong>of</strong> cow urine distillate discs were 

different concentration (5, 10, 15µl). 

taken for the study. Among the three concentrations 

highest antibacterial activity was noted in 15µl 

Antibacterial and Antifungal Activity <strong>of</strong> Cow Urine concentration when compared with 5 and 10µl. Maximum 

Distillate: The antimicrobial and antifungal activity antibacterial activity was observed in Pseudomonas 

studies were carried out by disc diffusion technique [7]. aeruginosa (12.6±0.05, 13.8±0.18 and 15.4±1.23, mm in 

The sterile Mueller Hinton agar plates were prepared. The diameter, respectively) and Salmonella typhi (12.3±1.23, 

test organisms like Bacillus subtilis, Pseudomonas 13.6±0.17 and 15.4±1.23, mm in diameter, respectively) 

aeruginosa, Klebsiella pneumonia, Salmonella typhi, when compared with other bacterial species and the 

Aspergillus niger and Aspergillus flavus were standard antibiotic (Ampicillin). US patent was obtained 

spreaded over the Mueller Hinton ager plates by by CSIR (Counsil for Scientific Industrial Research) India 

using separate sterile cotton swabs. After the which claimed a novel pharmaceutical composition 

spreading the different concentrated cow urine distillate present in cow urine distillate and is effective as an 

discs were placed separately on the organism inoculated antifungal and antibacterial [6]. 

Table 1: Antibacterial activity <strong>of</strong> cow urine distillate 

Zone <strong>of</strong> inhibition (mm in diameter) (M±SD) (n=3) 

----------------------------------------------------------------------------------------------------------------------------------------- 

Concentration <strong>of</strong> CUD (µl) 

----------------------------------------------------------------------------------------------------------------------------------------- 

S.NO. Bacteria 5 10 15 (Amp*) 

1. Bacillus subtilis 7.6±0.04 8.6±0.17 8.8±0.17 7.1±0.01 

2. Pseudomonas aeruginosa 12.6±0.04 13.6±0.17 15.4±1.23 11.2±0.01 

3. Klebsiella pneumoniae 7.3±0.25 7.3±0.25 11±0.14 9.5±0.05 

4. Salmonella typhi 12±1.23 13.6±0.17 15.4±1.23 9.6±0.02 

Values are triplicate mean ± Standard deviation 

Amp* - Standard antibiotic disc Ampicillin (30mg/disc) 

Table 2: Antifungal activity <strong>of</strong> cow urine distillate 

Zone <strong>of</strong> inhibition (mm in diameter) (M±SD) (n=3) 

------------------------------------------------------------------------------------------------------------------------------------------- 

Concentration <strong>of</strong> CUD (µl) 

------------------------------------------------------------------------------------------------------------------------------------------- 

S.NO. Fungi 5 10 15 

1. Aspergillus niger 8±0.14 11.3±1.2 12.6±0.04 

2. Aspergillus flavus 7.3±0.25 10±0.25 11±1.2 

Values are triplicate mean ± Standard deviation 

42


16 

Zone <strong>of</strong> inhibition (mm in diameter) 

14 

12 

10 

8 

6 

4 

2 

0 

Bacillus subtilis 

Pseudomonas 

aeruginosa 

Klebsiella 

pneumoniae 

Salmonella 

typhi 

5µl 

10µl 

15µl 

Amp* 

Fig. 1: Antibacterial activity <strong>of</strong> cow urine distillate 

14 

Zone <strong>of</strong> inhibition (mm in diameter) 

12 

10 

8 

6 

4 

2 

0 

Aspergillus niger 

Aspergillus flavus 

5µl 

10µl 

15µl 

Fig. 2: Antifungal activity <strong>of</strong> cow urine distillate 

Plate 1: Antibacterial activity <strong>of</strong> CUD against Pathogenic bacteria 

Plate 2= Antifungal activity <strong>of</strong> CUD against Pathogenic fungi 

43


Antifungal Activity: Antifungal activity <strong>of</strong> cow urine 

REFERENCES 

distillate was analysed against Aspergillus niger and 

Aspergillus flavus. The investigated results were 1. Shah, E., 1997. Herbal composition in cow urine 

presented in Table 2, Fig. 2 and Plate 2. When the two distillate. US 5693327. Dec 2. 

fungal organisms were compared, maximum growth 2. Basham, A.L., 1998. Practices <strong>of</strong> medicine in ancient 

suppression was observed in Aspergillus niger India in Asian Medical systems: A Comparative 

(8±0.14, 11.3±1.2 and 12.6±0.04, mm in diameter, study. (Ed) by Charles leslie, Motilal Banarsidass, 

respectively) than Aspergillus flavus (7.3±0.25, 10±0.26 Delhi, pp: 22. 

and 11±1.2, mm in diameter, respectively). A similar 3. Sarman Singh, 2001. Cow urine has anti Leshmania 

result reported by Prashith Kekuda et al. [9] Cow Urine donovani effect in vitro. International J. Cow Sci., 

Distillate at various concentrations was tested for 1(2): 72-73. 

antifungal activity. The growth reduction in percentage 4. Sadhale, N., 2004. Vishvallabba Agri History Bulletin 

was taken into consideration and antifungal effect was 5. Asian agri-History Foundation, Secunderabadevaluated. 

5% cow urine distillate was more effective 500009. pp: 134. 

against Mucor sp. (37.1%) followed by A.oryzae (10.2%) 5. Garg, N., Ashok Kumar and R.S.Chauhan, 2005. 

and A. niger (5.4%). 

Effect <strong>of</strong> indigenous cow urine on nutrient utilization 

It was concluded that the cow urine distillate has <strong>of</strong> white leghorn layers. International J. Cow Sci., 

antibacterial and antifungal activities the inhibitory 1: 36-38. 

activity can be used in the control <strong>of</strong> bacteria and fungi 6. Khanuja, S.P.S., 2002. Pharmaceutical composition 

<strong>of</strong> various origins. The test was done in vitro. Same containing cow urine distillate and an Antibiotic. 

result may be obtained in vivo also. Now a day’s US patent 6410059. June 25. 

urinotherapy treatment was developed in the medical 7. Bauer, R.W., M.D.K. Kirby, J.C. Sherris and 

sectors. Further studies analyze which components are M. Turck, 1966. Antibiotic susceptibility testing by 

responsible for antimicrobial activity and animal model standard single disc diffusion method. American 

could reveal antibacterial and antifungal activity <strong>of</strong> cow J. Clinical Pathol., 45: 493-496. 

urine distillate in vivo. 8. Salil Bose, 1982. Biostatistics in Elementary 

ACKNOWLEDGMENTS 9. 

Biophysics. Jytoi Book, Madurai, pp: 127-128. 

Prashith Kekuda, T.R., R. Kavya, R.M. Shrungashree 

and S.V. Suchitra, 2007. Antifungal activity <strong>of</strong> cow 

The authors are thankful to Muthaiyah Research urine distillate. (http://www.microbiocare.com/). 

Foundation, Thanjavur for <strong>of</strong>fering facilities to carry out 

this study. 

44

Review Article 

Journal <strong>of</strong> Intercultural Ethnopharmacology 

www.jicep.com 

DOI: 10.5455/jice.2015022210032 

Chemotherapeutic potential <strong>of</strong> cow 

urine: A review 

Gurpreet Kaur Randhawa 1 , Rajiv Sharma 2 

1 

Department <strong>of</strong> 

Pharmacology, 

Government Medical 

College, Amritsar, Punjab, 

India, 2 Department <strong>of</strong> 

Medicine, Guru Nanak 

Dev Hospital, attached 

to Government Medical 

College, Amritsar, Punjab, 

India 

Address for correspondence: 

Gurpreet Kaur Randhawa, 

Department <strong>of</strong> 

Pharmacology, Government 

Medical College, 

Amritsar, Punjab, India. 

E-mail: kullar.g@gmail.com 

ABSTRACT 

In the grim scenario where presently about 70% <strong>of</strong> pathogenic bacteria are resistant to at least one <strong>of</strong> 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 <strong>of</strong> its various products. Urine is one <strong>of</strong> the products <strong>of</strong> a cow with many benefits and without toxicity. 

Various studies have found good antimicrobial activity <strong>of</strong> cow’s urine (CU) comparable with standard drugs such 

as <strong>of</strong>loxacin, cefpodoxime, and gentamycin, against a vast number <strong>of</strong> 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 <strong>of</strong> 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 <strong>of</strong> infectious diseases, CU can be used alone or as an adjunctive to prevent the 

development <strong>of</strong> resistance and enhance the effect <strong>of</strong> standard antibiotics. 

Received: January 16, 2015 

Accepted: February 22, 2015 

Published: March 07, 2015 

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 <strong>of</strong> widespread drug resistance to the currently 

available antimicrobials is a matter <strong>of</strong> deep concern. A high 

percentage <strong>of</strong> 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 <strong>of</strong> 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 <strong>of</strong> MDR tuberculosis. 

Extensively drug-resistant tuberculosis has been identified 

in 92 countries. Development <strong>of</strong> resistance to oral drug <strong>of</strong> 

choice fluoroquinolones, for urinary tract infections caused 

by Escherichia coli is very widespread, <strong>of</strong>ten sensitivity 

remains only for injectables [2]. Infections caused by 

resistant microorganisms <strong>of</strong>ten fail to respond to the standard 

treatment, resulting in prolonged illness, higher health care 

expenditures, and a greater risk <strong>of</strong> death. There is a dire 

need for the development <strong>of</strong> new antimicrobial agents with 

sensitivity intact against microorganisms [3,4]. The rational 

designing <strong>of</strong> novel drugs from traditional medicines to treat 

these difficult to treat infections <strong>of</strong>fers 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 <strong>of</strong> 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 <strong>of</strong> CU have been 

mentioned such as weight loss, reversal <strong>of</strong> certain cardiac and 

renal diseases, indigestion, stomach ache, diarrhea, edema, 

jaundice, anemia, hemorrhoids and skin diseases including 

vitiligo. Gomutra is capable <strong>of</strong> removing all the imbalances in 

the body, thus maintaining the general health [5]. CU contains 

95% water, 2.5% urea, minerals, 24 types <strong>of</strong> 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 

<strong>of</strong> Gram-negative and Gram-positive bacteria and also against 

some drug-resistant bacteria. It acts as a bio-enhancer <strong>of</strong> 

some antimicrobial drugs. It has antifungal, anthelmintic, 

antineoplastic action, is useful in hypersensitivity reactions and 

180 J Intercult Ethnopharmacol ● Apr-Jun 2015 ● Vol 4 ● Issue 2

Randhawa and Sharma: Chemotherapeutic potential <strong>of</strong> cow urine 

in numerous other diseases including increasing the life-span <strong>of</strong> 

a person. Recent <strong>research</strong>es have shown that CU is an immuneenhancer 

also [7-9]. Therapeutic properties <strong>of</strong> CU have been 

validated by modern science also. 

MECHANISM OF ACTION OF CU 

Different fractions <strong>of</strong> CU possess antimicrobial activity due to 

the presence <strong>of</strong> certain components like volatile and nonvolatile 

ones [10-13]. Presence <strong>of</strong> urea, creatinine, swarn kshar (aurum 

hydroxide), carbolic acid, phenols, calcium, and manganese has 

strongly explained the antimicrobial and germicidal properties <strong>of</strong> 

CU [14-16]. Presence <strong>of</strong> amino acids and urinary peptides may 

enhance the bactericidal effect [17] by increasing the bacterial 

cell surface hydrophobicity. CU enhances the phagocytic activity 

<strong>of</strong> macrophages. Higher amounts <strong>of</strong> 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 <strong>of</strong> inorganic phosphorus, chloride 

and dimethylamine may also play an important role [19], 

along with increased formation <strong>of</strong> some reactive compounds 

like formaldehyde, sulfinol, ketones and some amines during 

photo-activation and long term storage [20]. CU prevents the 

development <strong>of</strong> antibacterial resistance by blocking the R-factor, 

a part <strong>of</strong> plasmid genome <strong>of</strong> bacteria [21]. 

CU contains phenolic acids (gallic, caffeic, ferulic, o-coumaric, 

cinnamic, and salicylic acids) which have antifungal 

characteristics [22]. 

Antioxidant property <strong>of</strong> 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 <strong>of</strong> the urine 

components, and these components may prevent the process <strong>of</strong> 

aging [10]. It efficiently repairs the damaged DNA [5]. 

Daily consumption <strong>of</strong> CU improves immunity due to the presence 

<strong>of</strong> swarn kshar and fastens the wound healing process, which 

is due to allantoin [8]. CU enhances the immunocompetence 

by facilitating the synthesis <strong>of</strong> interleukin-1 and -2 [23,24], 

augments B - and T- lymphocyte blastogenesis, and IgA, IgM 

and IgG antibody titers [25]. 

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]. 

AS ANTIMICROBIAL AGENT 

Antimicrobial activity <strong>of</strong> CU from both indigenous and hybrid 

breeds against E. coli, Salmonella typhi, Proteus vulgaris, 

S. aureus, Bacillus cereus, Staphylococcus epidermidis, Klebsiella 

pneumonia, Pseudomonas aeruginosa, Pseudomonas fragi, 

Streptococcus agalactiae, Enterobacter aerogenes, Aeromonas 

hydrophila, Micrococcus luteus, Streptococcus pyogenes, 

Streptomyces aure<strong>of</strong>aciens, Lactobacillus acidophilus and 

Bacillus subtilis, and Leishmania donovani has been observed 

in various studies. In these studies the antimicrobial activity <strong>of</strong> 

CU was found to be comparable with <strong>of</strong>loxacin, cipr<strong>of</strong>loxacin, 

ampicillin, chloramphenicol, nalidixic acid, rifampicin, 

tetracycline, streptomycin, cefpodoxime and gentamycin in 

different studies [27-36]. 

Studies with Indigenous Bos indicus Breeds <strong>of</strong> Cow 

Fresh CU (FCU), Sterile, PhCU and CUD from a healthy 

Geer cow, was used to assess the antibacterial effect against 

different strains <strong>of</strong> bacteria. Against E. coli, FCU had the 

bigger mean <strong>of</strong> inhibition zone (15 mm) than Sterile, PhCU, 

and CUD (~10 mm). FCU had good activity <strong>of</strong> 15, 16 and 

20 mm <strong>of</strong> inhibition against E. coli, B. subtilis, and S. typhi, 

respectively. Other forms <strong>of</strong> 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 <strong>of</strong> inhibition (mm)± standard error <strong>of</strong> the mean 

against B. subtilis was found to be 18.67±1.15, which was less 

than 27 for Gentamycin 10 mcg and cefpodoxime 10 mcg. 

Activity (18.67±1.15) against B. cereus and was similar to 

that <strong>of</strong> cefpodoxime (19) but less than with gentamycin (26). 

Activity (16) against S. aureus and S. epidermidis was


Table 1: Antimicrobial activity <strong>of</strong> CU, CUD (Gujrati Geer cow) in comparison to standard drug Ofloxacin [10] 

E. coli S. epidermidis S. aureus K. pneumoniae P. vulgaris B. subtilis 

FCU 23 22 24 25 23 24 

CUD 20 20 18 20 20 21 

Ofloxacin 30 28 25 28 28 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 μL <strong>of</strong> PhCU <strong>of</strong> Hariana breed was 

found to be comparable in efficacy to Tetracycline (30 μg mL). 

Antimicrobial activity (mean zone <strong>of</strong> inhibition in mm) <strong>of</strong> 

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 <strong>of</strong> Hariana breed against these 

bacteria [32]. 

Studies where breed <strong>of</strong> cow is not mentioned 

In an in-vitro test, activity <strong>of</strong> FCU was comparable to 

Streptomycin. Similar mean zone <strong>of</strong> 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 <strong>of</strong> 

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. aure<strong>of</strong>aciens. PhCU 

and Terminalia chebula showed maximum activity against 

S. auere<strong>of</strong>aciens (45 mm), and P. aeruginosa (zone <strong>of</strong> inhibition 

<strong>of</strong> 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 Cipr<strong>of</strong>loxacin. 

PhCU showed MIC value <strong>of</strong> 0.25 μl/ml against S. aureus, 

B. cereus, L. acidophilus and M. luteus, while it was found to 

be 0.125 μl/ml against E. coli, which was less than that for 

antibiotics. A combination <strong>of</strong> CU with Neem (A. indica) oil and 

Bavchi (Psoralea coryfolia) oil showed a synergistic effect (MIC 

0.125-0.25 μl/ml), which was less than that for antibiotics. Neem 

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 <strong>of</strong> CUD (5, 10 

and 15 μl) against the B. subtilis, P. aeruginosa, K. pneumoniae 

and S. typhi. Antibacterial activity (mean zone <strong>of</strong> 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 <strong>of</strong> 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 <strong>of</strong> inhibition zone against 

S. typhi. Antibacterial activity against K. pneumonia was 11 ± 

0.14 mm with 15 μl <strong>of</strong> CUD, which was more than the activity 

(9.5 ± 0.05 mm) with Ampicillin [34]. 

Yadav et al. reported the antimicrobial property <strong>of</strong> a herbal 

formulation containing CU, Dalbergia sissoo, and Datura 

stramonium. The antimicrobial activity <strong>of</strong> 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 μg), nalidixic acid (10 μg), rifampicin 

(30 μg), and ampicillin (10 μg). In gram-negative bacteria all 

antibiotics were inactive, except chloramphenicol (30 μg), 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 <strong>of</strong> 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 cipr<strong>of</strong>loxacin 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 <strong>of</strong> A. indica 

showed better MIC values than the organic fractions for MDR 

E. coli (12.68 mm) and K. pneumonia (9 mm). CU extracts 

<strong>of</strong> A. indica showed >8.66 mm zone <strong>of</strong> 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 <strong>of</strong> 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 <strong>of</strong> inhibiting 85-90% <strong>of</strong> 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 <strong>of</strong> 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 <strong>of</strong> inhibition <strong>of</strong> 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 <strong>of</strong> 5, 10 and 

15 μl <strong>of</strong> CUD [34]. 

In vitro antifungal activity (in mm) <strong>of</strong> Geer CU against A. flavus 

(17.33 ± 0.57) was in between 50 μg <strong>of</strong> amphotericin B (15) 

and 10 μg <strong>of</strong> 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 <strong>of</strong> 

Geer CU is better than the others where source <strong>of</strong> CU is not 

mentioned. 

In an in vitro study, it was found that the urine samples <strong>of</strong> 

outdoor feeding cow (OCU) was more effective and inhibited 

growth <strong>of</strong> fungi more strongly as compared to indoor feeding 

CU (ICU). This inhibition was concentration dependent. 

No growth <strong>of</strong> Penicillium notatum, Trichoderma viridae, and 

Alternaria solanii was observed with 10% OCU and with 20% 

ICU and that <strong>of</strong> Claviceps purpurea, Rhizopus oligosporius, C. 

albicans and A. candidus, no growth was observed with 20% <strong>of</strong> 

OCU only [42]. 

ANTISEPTIC 

Sanganal et al. observed the enhanced wound healing activity 

<strong>of</strong> CU in Wistar albino rats [43]. On 4 th day, the external 

application <strong>of</strong> CU showed significant and progressive increase 

in wound healing in rats compared to different concentrations 

<strong>of</strong> CU and 1% w/w nitr<strong>of</strong>urazone 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 <strong>of</strong> human 

beings. Paralysis <strong>of</strong> 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 <strong>of</strong> 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 <strong>of</strong> Panchgavya (five products <strong>of</strong> cow 

namely milk, curd, ghee, urine and dung) alone and combination 

<strong>of</strong> Panchgavya and ethanolic extract <strong>of</strong> 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) <strong>of</strong> onset 

<strong>of</strong> paralysis and death in earthworm decreased [45]. 

BIOENHANCER 

A ‘bioenhancer’/‘biopotentiator’ is an agent capable <strong>of</strong> 

enhancing the bioavailability and efficacy <strong>of</strong> a drug with which 

it is co-administered, without any pharmacological activity <strong>of</strong> 

its own at the therapeutic dose used. In ayurveda, this concept 

is known as ‘yogvahi’ and is used to increase the effect <strong>of</strong> 

medicines by increasing the oral bioavailability (especially <strong>of</strong> 

medicines with poor oral bioavailability), decreasing their dose 

and adverse effects, and were used to circumvent the parentral 

routes <strong>of</strong> drug administration. We can develop more such useful 

and economically viable drug combinations, by integrating the 

knowledge <strong>of</strong> time tested ayurveda with modern methods <strong>of</strong> 

<strong>research</strong> [8]. CU is the only agent <strong>of</strong> animal origin which acts 

as bioenhancer <strong>of</strong> antimicrobial, antifungal, and anticancer 

agents [30]. The indigenous CU contains ‘Rasayana’ tatva, 

which is responsible for modulation <strong>of</strong> the immune system and 

also act as a bioenhancer [21]. 

CUD is more effective bioenhancer than CU [30,46]. 

CUD enhances the transport <strong>of</strong> antibiotics like rifampicin, 

tetracycline and ampicillin across the gut wall by 2-7 folds 

[47]. It also enhances the potency <strong>of</strong> taxol against MCF-7 cell 

lines [48]. It enhances the bioavailability <strong>of</strong> rifampicin by 80 

fold in 0.05 microgm/ml concentrations, ampicillin by 11.6 

fold in 0.05 μ g/ml concentrations and clotrimazole by 5 fold 

in 0.88 μ g/ml concentration [49]. The activity <strong>of</strong> 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 

<strong>of</strong> 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 <strong>of</strong> 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 <strong>of</strong> Gram-positive bacteria. 

Inhibition caused by streptomycin (1 mg/ml) alone was higher 

(31-34 mm) than that <strong>of</strong> CUD alone (19-22 mm). With the 

J Intercult Ethnopharmacol ● Apr-Jun 2015 ● Vol 4 ● Issue 2 183


combination <strong>of</strong> 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 <strong>of</strong> 

importance in this scenario. 

This bioenhancing activity <strong>of</strong> CU has been aptly and 

widely used in various ayurvedic formulations. It is one <strong>of</strong> 

the constituents <strong>of</strong> 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 

<strong>of</strong> lymphocytes and tissues through fragmentation <strong>of</strong> 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 <strong>of</strong> 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 <strong>of</strong> croton oil. In mice 

treated with CU, the incidence <strong>of</strong> tumor (papillomas), tumor 

yield, and its burden was statistically less than the untreated 

group [54]. 

Jain et al. studied the effect <strong>of</strong> CU therapy on various types 

<strong>of</strong> cancers in Mandsaur area. The severity <strong>of</strong> symptoms (pain, 

inflammation, burning sensation, difficulty in swallowing, and 

irritation) decreased from day 1 to day 8 with CU therapy. 

Percent <strong>of</strong> 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 <strong>of</strong> symptoms decreased further 

with continued CU therapy [15]. 

Dutta et al. reported the anti-clastogenic and anti-genotoxic 

effect <strong>of</strong> redistilled CUD (RCUD) in human peripheral 

lymphocytes, which have been challenged with manganese 

dioxide (MnO 2 

) and hexavalent chromium (Cr+6). Protection 

in number <strong>of</strong> chromosomal aberrations and frequency <strong>of</strong> 

micronuclei were more prominent when these cells were 

pretreated with CU than simultaneous treatment with 

CU [55]. 

IMMUNOSTIMULANT 

The use <strong>of</strong> herbs and minerals (like chavanprash and 

panchgavya) for improving the overall resistance <strong>of</strong> the body 

against common infections and pathogens has been a guiding 

principal <strong>of</strong> 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 <strong>of</strong> macrophages and is thus helpful in the 

prevention and control <strong>of</strong> bacterial infections. The level <strong>of</strong> 

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 <strong>of</strong> 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 <strong>of</strong> CU 

Some <strong>of</strong> the traditional uses <strong>of</strong> 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 <strong>of</strong> Vasaka and kanar, and bark <strong>of</strong> 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 <strong>of</strong> CU to be taken on an empty stomach 

for the same [15]. 

CONCLUSIONS 

On analyzing the effect <strong>of</strong> different preparations <strong>of</strong> CU, FCU 

had better activity than CUD [27-32]. Activity <strong>of</strong> FCU and 

CUD from indigenous cows was similar to streptomycin and 

tetracycline. Ayurveda also mentions that FCU <strong>of</strong> 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 <strong>of</strong> the studies quoted are in vitro studies. Comparative 

studies between CU obtained from indigenous breeds and <strong>of</strong> 

inbred strains may be undertaken, as ayurveda was written when 

inbred strains <strong>of</strong> cows were not present. Future development <strong>of</strong> 

newer drugs can involve CU in its repository. 



REFERENCES 

1. Available from: http://www.cdc.gov/drugresistance/threatreport-2013/pdf/ar-threats-2013-508.pdf#page=11. 

[Last accessed 

on 2015 Jan 15]. 

2. Available from: http://www.who.int/mediacentre/factsheets/fs194/ 

en/. [Last accessed on 2015 Jan 15]. 

3. Murray CK. Infectious disease complications <strong>of</strong> combat-related 

injuries. Crit Care Med 2008;36:S358-64. 

4. Shafer RW, Rhee SY, Bennett DE. Consensus drug resistance 

mutations for epidemiological surveillance: Basic principles and 

potential controversies. Antivir Ther 2008;13 Suppl 2:59-68. 

5. Chauhan RS, Singh BP, Singhal LK. Immunomodulation with 

Kamdhenu ark in mice. J Immunol Immunopathol 2001;3:74-7. 

6. Bhadauria H. Cow urine- A magical therapy. Int J Cow Sci 2002;1:32-6. 

7. Chauhan RS, Garg N. Cow Therapy as an Alternative to Antibiotic. 

Banglore, Karnataka: Indian Science Congress; 2003. 

8. Randhawa GK. Cow urine distillate as bioenhancer. J Ayurveda Integr 

Med 2010;1:240-1. 

9. Randhawa GK, Kullar JS, Rajkumar. Bioenhancers from mother nature 

and their applicability in modern medicine. Int J Appl Basic Med Res 

2011;1:5-10. 

10. Jarald E, Edwin S, Tiwari V, Garg R, Toppo E. Antioxidant and 

antimicrobial activities <strong>of</strong> cow urine. Glob J Pharmacol 2008;2:20-2. 

11. Mohanty I, Senapati MR, Jena D, Pallai S. Diversified uses <strong>of</strong> cow 

urine. Int J Pharm Pharm Sci 2014;6:20-2. 

12. Hu W, Murphy MR, Constable PD, Block E. Dietary cation-anion 

difference effects on performance and acid-base status <strong>of</strong> dairy 

cows postpartum. J Dairy Sci 2007;90:3367-75. 

13. Shaw SL, Mitloehner FM, Jackson W, Depeters EJ, Fadel JG, 

Robinson PH, et al. Volatile organic compound emissions from dairy 

cows and their waste as measured by proton-transfer-reaction mass 

spectrometry. Environ Sci Technol 2007;41:1310-6. 

14. Achliya GS, Meghre VS, Wadodkar SG, Dorle AK. Antimicrobial activity 

<strong>of</strong> different fractions <strong>of</strong> cow urine. Indian J Nat Prod 2004;20:14-6. 

15. Jain NK, Gupta VB, Garg R, Silawat N. Efficacy <strong>of</strong> cow urine therapy 

on various cancer patients in Mandsaur District, India -A survey. Int 

J Green Pharm 2010;4:29-35. 

16. Kumar AA. Study on Various Biochemical Constituents in the Urine 

<strong>of</strong> Cow, Buffalo and Goat. Thesis Submitted to the C.S.A. Univ Agr 

Techn, Kanpur (U.P.); 2001. p. 13. 

17. Badadani M, SureshBabu SV, Shetty KT. Optimum conditions <strong>of</strong> 

autoclaving for hydrolysis <strong>of</strong> proteins and urinary peptides <strong>of</strong> prolyl 

and hydroxyprolyl residues and HPLC analysis. J Chromatogr B Analyt 

Technol Biomed Life Sci 2007;847:267-74. 

18. Upadhyay RK, Dwivedi P, Ahmad S. Antimicrobial activity <strong>of</strong> photoactivated 

cow urine against certain pathogenic bacterial strains. Afr 

J Biotechnol 2010;9:518-22. 

19. Naotoshi K, Osamu Y, Yoshihiko S, Fuminobu M, Masahiro Y, 

Yoshimitsu M. Clinico-pathological findings in peripartum dairy 

cows fed anion salts lowering the dietary cation-anion difference: 

Involvement <strong>of</strong> serum inorganic phosphorus, chloride and plasma 

estrogen concentrations in milk fever. Jpn J Vet Res 2007;55:3-12. 

20. Türi M, Türi E, Kõljalg S, Mikelsaar M. Influence <strong>of</strong> aqueous extracts 

<strong>of</strong> medicinal plants on surface hydrophobicity <strong>of</strong> Escherichia coli 

strains <strong>of</strong> different origin. APMIS 1997;105:956-62. 

21. Chauhan RS, Singhal L. Harmful effects <strong>of</strong> Pesticides and their control 

through cowpathy. Int J Cow Sci 2006;2:61-70. 

22. Singh UP, Maurya S, Singh A, Nath G, Singh M. Antimicrobial 

efficacy, disease inhibition and phenolic acid-inducing potential <strong>of</strong> 

chlor<strong>of</strong>orm fraction <strong>of</strong> cow urine. Arch Phytopathol Plant Protect 

2012;45:1546-57. 

23. Chauhan RS. Panchagavya therapy (cow pathy)- Current status and 

future directions. Indian Cow 2004;1:3-7. 

24. Singla S, Garg R. Cow urine: An elixir. Innov J Ayur Sci 2013;1:31-5. 

25. Kumar S. Analysis <strong>of</strong> cow’s urine for detection <strong>of</strong> lipase activity and 

anti-microbial properties. J Pharm Biol Sci 2013;7:1-8. 

26. Pescheck-Böhmer F, Schreiber G. Healing yourself using urine. Urine 

Therapy: Nature’s Elixir for Good Health. Rochester: Inner Traditions, 

Bear & Company; 1999. p. 152. 

27. Minocheherhomji FP, Vyas BM. Study <strong>of</strong> the antimicrobial activity 

<strong>of</strong> cow urine and medicinal plant extracts on pathogenic human 

microbial strains. Int J Adv Pharm Biol Chem 2014;3:836-40. 

28. Rana R, De S. In vitro antimicrobial screening <strong>of</strong> cow urine – A 

potential natural anti-microbial agent. Int J Bioassays 2013;2:436-9. 

29. Ahuja A, Kumar P, Verma A, Tanwar R. Antimicrobial activities <strong>of</strong> cow 

urine against various bacterial strains. Int J Recent Adv Pharm Res 

2012;2:84-7. 

30. Kekuda PT, Nishanth BC, Kumar PS, Kamal D, Sandeep M, 

Megharaj HK. Cow urine concentration: A potent agent with 

antimicrobial and anthelmintic activity. J Pharm Res 2010;3:1025-7. 

31. Tyagi PK, Tyagi S, Sarsar V, Pannu R. Cow urine: An antimicrobial 

activity against pathogens and their possible uses. Int J Pharm Res 

Sch 2013;2:427-33. 

32. Sarsar V, Selwal KK, Selwal MK, Pannu R, Tyagi PK. Evaluation <strong>of</strong> 

antibacterial activity <strong>of</strong> photoactivated cow urine against human 

pathogenic strains. Environ Exp Biol 2013;11:201-3. 

33. Shah CP, Patel DM, Dhami PD, Kakadia J, Bhavsar D, Vachhani UD, 

et al. In vitro screening <strong>of</strong> antibacterial activity <strong>of</strong> cow urine against 

pathogenic human bacterial strains. Int J Curr Pharm Res 2011;3:91-2. 

34. Sathasivam A, Muthuselvam M, Rajendran R. Antimicrobial activities 

<strong>of</strong> cow urine distillate against some clinical pathogens. Glob J 

Pharmacol 2010;4:41-4. 

35. Vats S, Kumar R, Negi S. Natural food that meet antibiotics resistance 

challenge: In vitro synergistic antimicrobial activity <strong>of</strong> Azadirachta 

indica, Terminalia chebula, Piper nigrum and photoactivated cow 

urine. Asian J Pharm Biol Res 2012;2:122-6. 

36. Yadav H, Yadav M, Jain S, Bhardwaj A, Singh V, Parkash O, et al. 

Antimicrobial property <strong>of</strong> a herbal preparation containing Dalbergia 

sissoo and Datura tramonium with cow urine against pathogenic 

bacteria. Int J Immunopathol Pharmacol 2008;21:1013-20. 

37. Singh S. Cow urine has anti-leishmania donovani effect in vitro. Int 

J Cow Sci 2005;1(2):72-3. 

38. Biddle S, Teale P, Robinson A, Bowman J, Houghton E. Gas 

chromatography-mass spectrometry/mass spectrometry analysis 

to determine natural and post-administration levels <strong>of</strong> oestrogens 

in bovine serum and urine. Anal Chim Acta 2007;586:115-21. 

39. Rajapandiyan K, Shanthi S, Murugan AM, Muthu GA, Singh AJ. 

Azadirachta indica - Cow urine extract, a novel controlling agent 

towards clinically significant multi drug resistant pathogens. J Appl 

Pharm Sci 2011;1:107-13. 

40. Kumar S. Analysis on the natural remedies to cure dandruff/skin 

disease-causing fungus - Malassezia furfur. Adv BioTech 2013;12:1-5. 

41. Vijayalakshmi R, Saranya VT. Effect <strong>of</strong> “Go-Mutra” on plant growth and 

its antifungal and antibacterial activity. J Herb Sci Technol 2010;6:6-11. 

42. Deshmukh SS, Rajgure SS, Ingole SP. Antifungal activity <strong>of</strong> cow urine. 

IOSR J Pharm 2012;2(5):27-30. 

43. Sanganal JS, Jayakumar K, Jayaramu GM, Tikare VP, Paniraj K, 

Swetha R. Effect <strong>of</strong> cow urine on wound healing property in wister 

albino rats. Vet World 2011;4:317-21. 

44. Maheshwari AK, Gupta AK, Das AK. Effect <strong>of</strong> cow urine on wounds. 

Indian Cow 2004;1:19-24. 

45. Kumar R, Kumar A, Kumar K, Gupta V, Shrivas T, Tripathi K. Synergistic 

anthelmintic activity <strong>of</strong> different compositions <strong>of</strong> panchagavya and 

Bauhinia variegate Linn. Int J Phytopharmacol 2014;5:120-2. 

46. Tambekar DH, Kerhalkar SA. Cow urine: A bioenhancer for antibiotic. 

Asian J Microbiol Biotechnol Environ Sci 2006;8:329-33. 

47. Khanuja SP, Kumar S, Shasany AK, Arya JS, Darokar MP. Use 

<strong>of</strong> bioactive fraction from cow urine distillate (‘Go-mutra’) as a 

bioenhancer <strong>of</strong> anti-infective, anti-cancer agents and nutrients. US 

Patent US 7235262; 2007. 

48. Khanuja SPS, Kumar S, Shasany AK, Arya JS, Darokar MP, Singh M, 

et al. Pharmaceutical composition containing cow urine distillate and 

an antibiotic. US Patent US 6410059 B1; 2002. 

49. Tatiraju DV, Bagade VB, Karambelkar PJ, Jadhav VM, Kadam V. Natural 

bioenhancers: An overview. J Pharmacogn Phytochem 2013;2:55-60. 

50. Chawla PC. Resorine a novel CSIR drug curtails TB treatment. CSIR 

News 2010;60:52-4. 

51. Poornima G, Abhipsa V, Rekha C, Manasa M, Kekuda PT. Antibacterial 

activity <strong>of</strong> combination <strong>of</strong> Polyalthia longifolia thw. extract, cow urine 

distillate and Streptomycin. Res J Pharm Tech 2012;5:927-30. 

52. Kumar A, Kumar P, Singh LK, Agrawal DK. Pathogenic effects <strong>of</strong> 

free radicals and their prevention through cowpathy. Indian Cow 

2004;6:27-34. 

53. Ambwani S. Molecular studies on apoptosis in avian lymphocytes 

induced by pesticides. PhD Thesis Submitted to Department <strong>of</strong> 

Biotechnology and Molecular Biology, College <strong>of</strong> Basic Sciences 

and Humanities, GBPAUT, Pantnagar, India; 2004. 

54. Raja W, Agrawal RC. Chemopreventive potential <strong>of</strong> cow urine against 

J Intercult Ethnopharmacol ● Apr-Jun 2015 ● Vol 4 ● Issue 2 185


7, 12 dimethyl benz(a) anthracene-induced skin papillomasgenesis 

in mice. Acad J Cancer Res 2010;3(1):7-10. 

55. Dutta D, Devi SS, Krishnamurthi K, Chakrabarti T. Anticlastogenic 

effect <strong>of</strong> redistilled cow’s urine distillate in human peripheral 

lymphocytes challenged with manganese dioxide and hexavalent 

chromium. Biomed Environ Sci 2006;19:487-94. 

56. Verma A, Kumar B, Manish KS, Kharya MD. Immunomodulatory 

potential <strong>of</strong> cow urine. Der Pharm Lettre 2011;3:507-13. 

© SAGEYA. This is an open access article licensed under the terms 

<strong>of</strong> the Creative Commons Attribution Non-Commercial License (http:// 

creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted, 

noncommercial use, distribution and reproduction in any medium, provided 

the work is properly cited. 

Source <strong>of</strong> Support: Nil, Conflict <strong>of</strong> Interest: None declared. 


Mol Biol Rep (2014) 41:1967–1976 

DOI 10.1007/s11033-014-3044-6 

Effect <strong>of</strong> treatment <strong>of</strong> cow’s urine ‘‘Gomutra’’ and antioxidants 

in alleviating the lindane-induced oxidative stress in kidney 

<strong>of</strong> Swiss mice (Mus musculus) 

Girima Nagda • Devendra Kumar Bhatt 

Received: 5 December 2012 / Accepted: 4 January 2014 / Published online: 16 January 2014 

Ó Springer Science+Business Media Dordrecht 2014 

Abstract The study aimed to evaluate the effect <strong>of</strong> cow 

urine and combination <strong>of</strong> antioxidants against lindane 

induced oxidative stress in Swiss mice. Male healthy mice, 

8–10 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 <strong>of</strong> vitamin C and E were significantly 

decreased compared to control. Administration <strong>of</strong> cow urine 

and antioxidants alleviated the levels <strong>of</strong> these biochemical 

parameters. 

Keywords Antioxidants Cow urine Kidney Lindane 

Oxidative stress 

G. Nagda (&) D. K. Bhatt 

Cancer Biology and Toxicology Research Laboratory, 

Department <strong>of</strong> Zoology, University College <strong>of</strong> Science, 

M L Sukhadia University, Udaipur 313 001, India 

e-mail: girima10@gmail.com 


Kidney is vital organ <strong>of</strong> the body responsible for segregating 

the metabolic waste products from the blood. 

Accumulation <strong>of</strong> metabolites <strong>of</strong> xenobiotics contributes 

significantly to its susceptibility to damage kidney [24]. 

Any nephrotoxic insult would result in accumulation <strong>of</strong> 

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 <strong>of</strong> exposures to halogenated 

hydrocarbons, such as lindane, carbon tetrachloride 

and trichloroethylene, and the heavy metals cadmium 

and lead [3, 35, 36, 48, 59]. Some <strong>of</strong> 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 <strong>of</strong> HCH possesses the property 

<strong>of</strong> persistence, bioaccumulation and long term toxicity 

[32] and fulfills the criteria <strong>of</strong> POPs i.e., persistent organochlorine 

pesticides. India has total installed capacity <strong>of</strong> lindane 

(technical) production <strong>of</strong> 1,300 tonnes per annum (tpa), 

with two companies producing: Kanoria Chemicals and 

Industries Ltd with a capacity <strong>of</strong> 1,000 tpa, and India Pesticides 

Limited with 300 tpa capacity. According to data 

available from Department <strong>of</strong> Chemicals and Petrochemicals, 

Ministry <strong>of</strong> Chemicals and Fertilizers, between 1995 

and 2005, India has produced 5,387 tonnes <strong>of</strong> technical grade 

lindane. In India, lindane formulations are registered for use 

in pharmaceutical products for control <strong>of</strong> head lice and 

scabies on people, to control fly, flea, cockroach, mosquito, 

bed bug, and beetle populations and for the control <strong>of</strong> 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 <strong>of</strong> the exposure and affect 

123

1968 Mol Biol Rep (2014) 41:1967–1976 

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 <strong>of</strong> free radicals and generation <strong>of</strong> reactive 

oxygen species (ROS) [67]. 

Oxidative stress is defined as a disruption <strong>of</strong> the prooxidant–antioxidant 

balance in favor <strong>of</strong> the former, leading 

to potential damage [37]. It is a result <strong>of</strong> one <strong>of</strong> three 

factors: (i) an increase in ROS, (ii) an impairment <strong>of</strong> 

antioxidant defence systems, or (iii) an incapacity to repair 

oxidative damage. 

A number <strong>of</strong> studies indicate the toxicity <strong>of</strong> lindane on 

kidney [22, 48]. It has been considered that ROS play an 

important role in the pathogenesis <strong>of</strong> renal injury. Since the 

entire range <strong>of</strong> 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 <strong>of</strong> a wide variety <strong>of</strong> 

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 <strong>of</strong> 

the cell [17]. The excessive free radicals or the suppression 

<strong>of</strong> antioxidant defense <strong>of</strong> 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 <strong>of</strong> the body fails. In such situations, 

the use <strong>of</strong> exogenous substances with antioxidative 

potential becomes important [64]. 

Antioxidants have gained immense importance in recent 

years. The potency <strong>of</strong> 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 <strong>of</strong> cow’s 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 <strong>of</strong> 

cow urine has shown that it contains nitrogen, sulphur, 

phosphate, sodium, manganese, carbolic acid, iron, silicon, 

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 <strong>of</strong> 

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 <strong>of</strong> information regarding the role <strong>of</strong> 

fresh cow urine and combination <strong>of</strong> 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 

<strong>of</strong> mice has been investigated. Moreover, the use <strong>of</strong> 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 <strong>of</strong> analytical grade. Cow urine: Urine 

<strong>of</strong> 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 8–10 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 

<strong>of</strong> 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 <strong>of</strong> animals was according to the guidelines <strong>of</strong> 

123

Mol Biol Rep (2014) 41:1967–1976 1969 

Committee for the Purpose <strong>of</strong> Control and Supervision <strong>of</strong> 

Experiments on Animals (CPCSEA), Ministry <strong>of</strong> Environment 

and Forests, Govt. <strong>of</strong> 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 <strong>of</strong> 

treatment was based on occupational exposure <strong>of</strong> workers 

i.e., for 2 months during active malaria vector control 

programme. Dose selected for lindane was 40 mg/kg body 

wt. and duration <strong>of</strong> 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 <strong>of</strong> 

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 <strong>of</strong> lindane, vitamin E and lipoic acid were prepared 

by dissolving in olive oil. Dose <strong>of</strong> vitamin C was 

prepared in distilled water. Cow urine was administered 

without any modification. 

Experimental protocol 

A sub chronic study was done for 60 days and oral route <strong>of</strong> 

dose administration was chosen for all treatments. Mice 

were divided into eight groups with minimum <strong>of</strong> 8–10 

animals in each group. 

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 <strong>of</strong> 

60 days. 

Mice were sacrificed by cervical dislocation at the 

end <strong>of</strong> the scheduled period <strong>of</strong> 60 days and 24 h after 

the last dose treatment. Both the kidneys were blotted 

free <strong>of</strong> 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 <strong>of</strong> 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 

<strong>of</strong> 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 

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 <strong>of</strong> P \ 0.05, 0.01 was 

considered as statistically significant. 

S. no. Group no. Group code Treatment Dose Duration 

1 I C Control: vehicle only Olive oil only 60 days 

2 II L Lindane 40 mg/kg b.w. 60 days 

3 III A Antioxidants alone Combined dose <strong>of</strong> 125 mg/kg b.w. 60 days 

4 IV A?L Antioxidants?lindane Antioxidant dose <strong>of</strong> 125 mg/kg b.w. followed 

60 days 

by lindane at 40 mg/kg b.w. 

5 V U Cow urine alone 0.25 ml/kg b.w. cow urine 60 days 

6 VI U?L Cow urine?lindane 0.25 ml/kg b.w. cow urine ?40 mg/kg b.w. lindane 60 days 

7 VII U?A Cow urine?antioxidants 0.25 ml/kg b.w. cow urine ?125 mg/kg b.w. antioxidants 60 days 

8 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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1970 Mol Biol Rep (2014) 41:1967–1976 

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) <strong>of</strong> 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 <strong>of</strong> 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 <strong>of</strong> CAT (lmol <strong>of</strong> H 2 O 2 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 <strong>of</strong> 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 <strong>of</strong> GPx (mg <strong>of</strong> 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 <strong>of</strong> SOD (50 % inhibition <strong>of</strong> NBT/min/ 

mg protein) in various groups (a 60 day assessment). a compared to 



Graph. 5 Mean ± SD values <strong>of</strong> 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 


123

Mol Biol Rep (2014) 41:1967–1976 1971 

control group. The pretreatment groups A?L, U?L and 

U?A?L showed a significant increase (P \ 0.01) <strong>of</strong> about 

57.74, 77.64, and 98.26 % respectively, as compared to 

lindane group. The increasing levels <strong>of</strong> 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 <strong>of</strong> 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) <strong>of</strong> 32.12 % in the CAT activity as 

compared to control. A significant increase (P \ 0.01) <strong>of</strong> 

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 <strong>of</strong> 9.82 % as 

compared to lindane but the decrease was not significant. 

The maximum % <strong>of</strong> increase was observed in the A?L 

group. The increasing order <strong>of</strong> 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 <strong>of</strong> endogenous vitamin E (mg/g tissue) 




As compared to control animals, the lindane intoxicated 

animals showed a significant decrease (P \ 0.01) <strong>of</strong> 

45.19 % in the GPx levels. All the pretreatment groups 

showed promising results and brought about a significant 

rise <strong>of</strong> 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 <strong>of</strong> 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 <strong>of</strong> 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) <strong>of</strong> 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 <strong>of</strong> 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) 

<strong>of</strong> 41.18, 31.87, and 23.84 % was seen in the respective 

groups A?L, U?L and U?A?L. The increasing order <strong>of</strong> 

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) <strong>of</strong> 15.01 % was observed 

in the total protein levels <strong>of</strong> 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) <strong>of</strong> 20.00, 

123

1972 Mol Biol Rep (2014) 41:1967–1976 

17.08, and 11.95 % respectively, in the levels <strong>of</strong> total 

protein as compared to lindane group. The best results were 

shown by the pretreatment <strong>of</strong> combination <strong>of</strong> 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 <strong>of</strong> 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) <strong>of</strong> 34.94 % was registered 

in the endogenous levels <strong>of</strong> 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 <strong>of</strong> 

vitamin E by 86.65, 47.30, and 92.32 %, respectively in 

comparison to lindane group. The increasing order <strong>of</strong> 

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) 

<strong>of</strong> 50.95 % was observed in the endogenous levels <strong>of</strong> 

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 <strong>of</strong> A?L, U?L and U?A?L group 

showed an increase <strong>of</strong> 140.95, 100.51, and 107.18 %, 

respectively. The increasing order <strong>of</strong> 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. 


The results <strong>of</strong> the present study clearly demonstrate that 

LPO significantly increased in kidney after in vivo treatment 

<strong>of</strong> lindane. Thus, results <strong>of</strong> 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], CCl 4 [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 

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 <strong>of</strong> 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 <strong>of</strong> combination 

<strong>of</strong> antioxidants and cow urine (U?A?L) was the most 

effective in lowering the LPO levels followed by combination 

<strong>of</strong> antioxidant (A?L) and cow urine pretreatment 

(U?L). 

Due to increase in LPO the amount <strong>of</strong> free radicals 

crosses the threshold level. Nevertheless, decrease in the 

activities <strong>of</strong> CAT, SOD, GPx and GSH further deteriorates 

the situation and enhance the formation <strong>of</strong> 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 <strong>of</strong> cyt P450 system resulting in 

the generation <strong>of</strong> superoxide radicals [21]. 

Similar to earlier reports, results <strong>of</strong> 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], CCl 4 [7], lead [55], cisplatin [4], alcohol [60] and 

gentamicin [1] toxicity. 

The decreased activity <strong>of</strong> SOD in kidney in lindane 

treated mice may be due to the enhanced lipid peroxidation 

or inactivation <strong>of</strong> the antioxidative enzymes or 

could result from inactivation by hydrogen peroxide or 

glycation <strong>of</strong> the enzyme [62]. This would cause an 

increased accumulation <strong>of</strong> superoxide radicals, which 

could further stimulate lipid peroxidation. A decrease in 

SOD activity favors the accumulation <strong>of</strong> superoxide 

radicals,whichinturn inhibit CAT [33] asalsoseenin 

the present study. 

All the pretreatment groups showed alleviation in the 

levels <strong>of</strong> SOD and CAT. Earlier reports have shown that 

vitamin C and E are capable <strong>of</strong> increasing the renal SOD 

and CAT levels [4, 7, 46]. Combination <strong>of</strong> 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 <strong>of</strong> lindane treated mice. Amongst 

the three pretreatments the maximum amelioration in the 

levels <strong>of</strong> SOD was observed in case <strong>of</strong> U?A?L group 

followed by U?L group and was least in A?L group. 

Moreover, in the present study the protection <strong>of</strong>fered 

by combination <strong>of</strong> antioxidants (A?L group) in ameliorating 

the CAT levels was the maximum followed by 

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Mol Biol Rep (2014) 41:1967–1976 1973 

combination <strong>of</strong> antioxidants and cow urine (U?A?L 

group) and minimum in the only cow urine (U?L) group. 

The reduction in the activity <strong>of</strong> SOD, CAT enzymes 

may result in a number <strong>of</strong> deleterious effects due to the 

accumulation <strong>of</strong> superoxide anion and hydrogen peroxide 

[58]. The elimination <strong>of</strong> H 2 O 2 is either effected by CAT or 

GPx [69]. 

The level <strong>of</strong> GPx in the exposed group were lowered 

which is in accordance with the earlier studies where lindane 

caused a similar decrease in the levels <strong>of</strong> 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 CCl 4 [7] toxicity. 

The decreased levels can be attributed to either increased 

H 2 O 2 generation or decreased GSH concentration because 

GSH is one <strong>of</strong> the substrates for GPx [57]. A decreased 

GSH concentration was observed in the present study. 

Decreased activity <strong>of</strong> GPx may also result from radical 

induced inactivation and glycation <strong>of</strong> the enzymes [26]. 

Because <strong>of</strong> the decreased GPx activity the accumulation 

<strong>of</strong> H 2 O 2 may cause inhibition <strong>of</strong> SOD activity [10]. During 

oxidative stress, inactivation <strong>of</strong> GPx may occur, and on the 

other hand superoxide anion (O 2 •- ) 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 <strong>of</strong> GPx. It has been shown in earlier 

reports that vitamin C and E are capable <strong>of</strong> increasing the 

renal GPx levels [4, 7, 46] and combination <strong>of</strong> 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 <strong>of</strong> 

U?A?L group followed by U?L group and was least in 

A?L group. 

In the present study, the levels <strong>of</strong> 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 <strong>of</strong> 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 <strong>of</strong> GSH for 

metabolism <strong>of</strong> lipid hydroperoxides by GPx or interaction 

<strong>of</strong> GSH with free radicals. Similar analogy is being also 

drawn in the earlier reports [5, 23]. 

The levels <strong>of</strong> GSH were significantly ameliorated after 

the pretreatment with combination <strong>of</strong> antioxidants namely, 

vitamin C, E and a- lipoic acid (A?L group), cow urine 

(U?L group) and combination <strong>of</strong> 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 

were ameliorated by combination <strong>of</strong> 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 H 2 O 2 accumulation. The increased H 2 O 2 in turn could 

cause SOD inhibition resulting in increased production <strong>of</strong> 

superoxide radicals. Thus, increased production <strong>of</strong> superoxide 

radicals would inhibit both CAT and GPx. Treatment 

with the adopted formulation reduced the level <strong>of</strong> lipid 

peroxides indicating the effective antioxidant property <strong>of</strong> 

the combination as well as cow urine alone in the moderation 

<strong>of</strong> tissue damage. 

Antioxidant defense system protects the aerobic organism 

from the deleterious effects <strong>of</strong> 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 <strong>of</strong> 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 <strong>of</strong> vitamin C and E, 

consequently, depleting their levels. The observed decrease 

in the level <strong>of</strong> kidney ascorbic acid and alpha tocopherol in 

lindane treated group could be as a result <strong>of</strong> increased 

utilization <strong>of</strong> 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 <strong>of</strong> the active forms <strong>of</strong> vitamin C and 

vitamin E by neutralizing the free radicals. When there is a 

reduction in the GSH the cellular levels <strong>of</strong> 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 <strong>of</strong> GSH, vitamin C and 

vitamin E on lindane administration were observed in our 

study. 

The pretreatment with combination <strong>of</strong> antioxidants in 

group A?L, with cow urine in group U?L and combination 

<strong>of</strong> antioxidants and cow urine both in group U?A?L, 

resulted in significant increase in the endogenous levels <strong>of</strong> 

vitamin C and E. The amelioration in the endogenous 

levels <strong>of</strong> 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 <strong>of</strong> 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 

<strong>of</strong> 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 Mol Biol Rep (2014) 41:1967–1976 

(U?L) group also showed significant elevation in the 

levels <strong>of</strong> the two vitamins. This could be attributed to the 

composition <strong>of</strong> cow urine which is said to contain vitamins. 

The analysis <strong>of</strong> total proteins is important for estimating 

the degree <strong>of</strong> damage in the body. The protein pr<strong>of</strong>ile <strong>of</strong> the 

cells is indicative <strong>of</strong> the physiological status <strong>of</strong> 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 <strong>of</strong> total proteins due to lindane have been 

reported [9, 42, 48]. It is also reported that renal toxicity 

due to CCl 4 [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 <strong>of</strong> the tissue either by inhibiting RNA 

synthesis or inhibiting <strong>of</strong> 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 <strong>of</strong> either vitamin C, E or in combination 

and combination <strong>of</strong> vitamin C, E and lipoic acid and 

resveratrol elevated the decreased protein levels [7, 9, 42]. 

The most effective pretreatment was that <strong>of</strong> combination <strong>of</strong> 

antioxidants (A?L group); cow urine pretreatment group 

(U?L) was moderately effective and least effective was 

pretreatment with combination <strong>of</strong> 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 <strong>of</strong> distillate and redistillate <strong>of</strong> cow 

urine have been reported. Attenuation <strong>of</strong> CCl 4 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 <strong>of</strong> 

cow urine therapy has been evaluated on cancer patients 

[28]. The amelioration <strong>of</strong> 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 <strong>of</strong> 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 <strong>of</strong> antioxidants 

and cow urine act synergistically in reducing lindane 

induced dysfunction. The analysis <strong>of</strong> the results <strong>of</strong> the 

present study reveals the efficacy <strong>of</strong> cow urine against 

oxidative stress. It can be safely concluded that the suggested 

combination <strong>of</strong> vitamin C, vitamin E, alpha lipoic 

acid and cow urine can prove to be beneficial in a number <strong>of</strong> 

ailments. The highlight <strong>of</strong> the investigation is the efficiency 

<strong>of</strong> cow urine against the pesticide toxicity which can open 

new insights in the field <strong>of</strong> medicine. Cow urine can prove 

to be an effective co-remedy for oxidative stress. This study 

emphasizes the importance <strong>of</strong> antioxidants and cow urine 

which could be beneficial in the therapeutic world for the 

treatment <strong>of</strong> various disorders implicating oxidative stress. 

References 

1. Abdel-Raheem IT, Abdel-Ghany AA, Mohamed GA (2009) 

Protective effect <strong>of</strong> quercetin against gentamicin induced nephrotoxicity 

in rats. Biol Pharm Bull 32(1):61–67 

2. Achliya GS, Kotagale NR, Wadodkar SG, Dorle AK (2003) 

Hepatoprotective activity <strong>of</strong> Panchagavya Ghrita against carbon 

tetrachloride-induced hepatotoxicity in rats. Ind J Pharmacol 

35:308–311 

3. Adewole SO, Salako AA, Doherty OW, Naicker T (2007) Effect 

<strong>of</strong> melatonin on carbon tetrachloride-induced kidney injury in 

Wistar rats. Afr J Biomed Res 10:153–164 

4. Ajith TA, Usha S, Nivith AV (2007) Ascorbic acid and a- 

tocopherol protect anticancer drug cisplatin induced nephrotoxicity 

in mice: a comparative study. Clin Chim Acta 375:82–86 

5. Anbarasi K, Vani G, Balakrishna K, Shyamala CSD (2006) Effect 

<strong>of</strong> bacoside A on brain antioxidant status in cigarette smoke 

exposed rats. Life Sci 78:1378–1384 

6. Bae EH, Lee JU, Kwon Ma S, Kim IJ, Frokiaer J, Nielsen S, Kim 

SY, Kim SW (2009) a-Lipoic acid prevents cisplatin-induced acute 

kidney injury in rats. Nephrol Dial Transplant 24:2692–2700 

7. Balahoroglu R, Dulger H, Ozbek H, Bayram I, Sekeroglu MR 

(2008) Protective effects <strong>of</strong> antioxidants on the experimental liver 

and kidney toxicity in mice. Eur J Gen Med 5(3):157–164 

8. Bano M, Bhatt DK (2007) Neuroprotective role <strong>of</strong> a novel 

combination <strong>of</strong> certain antioxidants on lindane (c-HCH) induced 

toxicity in cerebrum <strong>of</strong> mice. Res J Agric Bio Sci 3:664–669 

9. Bano M, Bhatt DK (2010) Ameliorative effect <strong>of</strong> a combination 

<strong>of</strong> vitamin E, vitamin C, alpha-lipoic acid and stilbene resveratrol 

on lindane induced toxicity in mice olfactory lobe and cerebrum. 

Indian J Exp Biol 48:150–158 

10. Bast A, Haenen GRMM, Doelman CJA (1991) Oxidants and 

antioxidants: state <strong>of</strong> the art. Am J Med 91:25–135 

11. Bist R, Bhatt DK (2009) The evaluation <strong>of</strong> effect <strong>of</strong> alpha- lipoic 

acid and vitamin E on the lipid peroxidation, gamma-amino 

butyric acid and serotonin level in the brain <strong>of</strong> mice (Mus musculus) 

acutely intoxicated with lindane. J Neurol Sci 276:99–102 

12. Blum J, Fridovich I (1985) Inactivation <strong>of</strong> glutathione peroxidase 

by superoxide radical. Arch Biochem Biophys 240:500–508 

13. Brzoska MM, Moniuszko-Jakoniuk J, Pilat-Marcinkiewicz B, 

Sawicki B (2003) Liver and kidney function and histology in rats 

exposed to cadmium and ethanol. Alcohol Alcohol 38(1):2–10 

14. Chauhan RS, Singh BP, Singhal LK (2001) Immunomodulation 

with kamdhenu Ark in mice. J Immunol Immunopathol 71:89–92 

15. CIBRC (2005) Central Insecticide Board and Registration 

Committee, Dept. Of Plant Protection and Quarantine, Ministry 

<strong>of</strong> Agriculture, India. http://www.ipen.org/ipenweb/documents/ 

work%20documents/lindanes%20dirty%20secret.pdf 

123

Mol Biol Rep (2014) 41:1967–1976 1975 

16. Cohen G, Dembiec D, Marcus J (1970) Measurement <strong>of</strong> catalase 

activity in tissue extracts. Anal Biochem 34:30–38 

17. Davies K (1995) Oxidative stress: the paradox <strong>of</strong> aerobic life. 

Biochem Soc Symp 61:1–31 

18. Desai ID (1984) Vitamin E methods for animal tissues. Methods 

Enzymol 105:138–143 

19. Dutta D, Devi SS, Krishnamurthi K, Chakrabarti T (2006) Anticlastogenic 

effect <strong>of</strong> redistilled cow’s urine distillate in humanperipheral 

lynphocytes challenged with manganese dioxide 

and hexavalent chromium. Biomed and Environ Sci 19:487–494 

20. Elegbe RA, Oyebola DDO (1976) Cow’s urine poisoning in 

Nigeria: the cardiotoxic effects <strong>of</strong> cow’s urine in dogs. Trans R 

Soc Trop Med Hyg 71:127–132 

21. English D, Schell M, Siakotos A, Gabig TG (1986) Reversible 

activation <strong>of</strong> the neutrophil superoxide generating system by 

hexachlorocyclohexane: correlation with effects on a subcellular 

superoxide-generating fraction. J Immunol 137:283–290 

22. Fidan AF, Cigerci IH, Sozbilir NB, Kucukkurt I, Yuksel H, Keles 

H (2008) The effects <strong>of</strong> the dose-dependent hexachlorocyclohexane 

(Lindane) on blood and tissue antioxidant defense systems, 

lipid peroxidation and histopathological changes in rats. 

J Anim Vet Adv 7:1480–1488 

23. Flohe L (1982) Glutathione peroxidase brought into focus. In: 

Pryor WA (ed) Free radicals in biology. Academic Press, New 

York, p 233 

24. Goldstein RS, Schnellmann RG (1996) Toxic responses <strong>of</strong> the 

kidney. Cassrrett and Doull’s toxicology, 5th edn. McGraw Hill, 

New York 

25. Gururaja MP, Joshi AB, Joshi H, Sathyanarayana D, Subrahmanyam 

EV, Chandrashekhar KS (2009) Attenuation <strong>of</strong> carbon 

tetrachloride-induced hepatotoxicity by cow urine distillate in 

rats. Biomed Environ Sci 22(4):345–347 

26. Hodgson EK, Fridovich I (1975) The interaction <strong>of</strong> bovine 

erythrocyte superoxide dismutase with hydrogen peroxide and 

inactivation <strong>of</strong> the enzyme. Biochemistry 14:5294–5299 

27. Isik B, Bayrak R, Akcay A, Sogut S (2006) Erdosteine against 

acetaminophen induced renal toxicity. Mol Cell Biochem 287: 

185–191 

28. Jain NK, Gupta VB, Garg R, Silawat N (2010) Efficacy <strong>of</strong> cow 

urine therapy on various cancer patients in Mandsaur district, 

India—a survey. Int J Green Pharm 4(1):29–35 

29. Kadkhodaee M, Khastar H, Faghihi M, Ghaznavi R, Zahmatkesh M 

(2004) Effects <strong>of</strong> co-supplementation <strong>of</strong> vitamins E and C on gentamicin-induced 

nephrotoxicity in rat. Exp Physiol 90(4):571–576 

30. Kakkar P, Das B, Viswanathan PN (1984) A modified spectrophotometric 

assay <strong>of</strong> superoxide dismutase. Indian J Biochem 

Biophys 21:130–132 

31. Kelly JF (1997) The urine cure and other curious medical treatments. 

http://wfmu.org/LCD/_Articles/LCD_19/Urine.html 

32. Khanna RN, Das M, Anand M (2002) Influence <strong>of</strong> phenobarbitol 

and CCl 4 on the modulation <strong>of</strong> tissue retention pr<strong>of</strong>ile <strong>of</strong> hexachlorocyclohexane 

in rats. Biomed Environ Sci 15:119–129 

33. Kono Y, Fridovich I (1982) Superoxide radical inhibits catalase. 

J Biol Chem 257:5751–5754 

34. Krishnamurthi K, Dutta D, Devi SS, Chakrabarti T (2004) Protective 

effect <strong>of</strong> distillate and redistillate <strong>of</strong> cow’s urine in human 

polymorphonuclear leukocytes challenged with established 

genotoxic chemicals. Biomed Environ Sci 17:57–66 

35. Kumar MR, Reddy KS, Reddy AG, Anjaneyulu Y, Kalakumar B, 

Reddy GD (2009) Amelioration <strong>of</strong> lead induced nephrotoxicity by 

certain adaptogens in broilers. Indian J Vet Pathol 33(2):19–190 

36. Lash HL, Qian W, Putt DA, Hueni SE, Elfarra AA, Krause RJ, 

Parker JC (2001) Renal and hepatic toxicity <strong>of</strong> trichloroethylene 

and its glutathione derived in rats and mice: sex-, species-, and 

tissue dependent differences. J Pharmacol Exp Ther 297(1): 

155–164 

37. Lightboy JH, Stevenson LM, Jackson F, Donaldson K, Jones DG 

(2001) Comparative aspects <strong>of</strong> plasma antioxidant status in sheep 

and goats and the influence <strong>of</strong> experimental abomasal nematode 

infection. J Comp Pathol 124:192–199 

38. Lowry OH, Roesborough MJ, Farr AL, Randall RJ (1951) Protein 

measurement with Folin–Phenol reagent. J Biol Chem 193:265 

39. Lynch MJ, Raphael SS, Miller LD, Spare DD, Inwood MJH (1969) 

Medical laboratory technology and clinical pathology. W.B. Saunders 

Co., Igaku shoin Ltd, Philadelphia, Tokyo, pp 2–629 

40. Moron MA, Depierre JW, Mannervick B (1979) Levels <strong>of</strong> glutathione, 

glutathione redutase, glutathione S-transferase activities 

in rat lung and liver. Biochem Biophys Acta 582:67–78 

41. Muzakova V, Kandar R, Vojtisek P, Skalicky J, Vankova R, 

Cegan A, Cervinkova Z (2001) Antioxidant vitamin levels and 

glutathione peroxidase activity during ischemia/reperfusion in 

myocardial infarction. Physiol Res 50:389–396 

42. Nagda G, Bhatt DK (2011) Alleviation <strong>of</strong> lindane induced toxicity 

in testis <strong>of</strong> Swiss mice (Mus musculus) by combined treatment 

with vitamin C, vitamin E and a-Lipoic acid. Indian J Exp 

Biol 49:191–199 

43. Nouri M, Rahbani-Nobar M, Argani H, Rokhforooz F (1999) 

Superoxide dismutase and glutathione peroxidase in hemodialyzed 

patients and renal transplant recipients and their relationship 

to osmotic fragility. Med J Islam Acad Sci 12(2):33–38 

44. Ojewole JA, Olusi SO (1976) Effects <strong>of</strong> cow’s urine concoction 

on plasma glucose concentration in fasted rats. Trans R Soc Trop 

Med Hyg 71:241–245 

45. Omaye ST, Turbull TP, Sauberchich HC (1979) Selected methods 

for determination <strong>of</strong> ascorbic acid in cells, tissues and fluids. 

Methods Enzymol 6:3–11 

46. Oncu M, Gultekin F, Karaoz E, Altuntas I, Delibas N (2002) 

Nephrotoxicity in rats induced by chlorpyrifos-ethyl and ameliorating 

effects <strong>of</strong> antioxidants. Hum Exp Toxicol 21(4):223–230 

47. Osfor MMH, Ibrahim HS, Mohamed YA, Ahmed SM, Abd El 

Azeem AS, Hegazy AM (2010) Effect <strong>of</strong> alpha lipoic acid and 

vitamin E on heavy metals intoxication in male albino rats. J Am 

Sci 6(80):56–63 

48. Padma VV, Sowmya P, Felix TA, Baskaran R, Poornima P 

(2011) Protective effect <strong>of</strong> gallic acid against lindane induced 

toxicity in experimental rats. Food Chem Toxicol 49:991–998 

49. Pathak ML, Kumar A (2003) Gomutra a descriptive study. 

Sachitra Ayurveda 7:81–84 

50. Poirier B, L-Bournville M, Conti M (2000) Oxidative stress occur 

in absence <strong>of</strong> hyperglycemia and inflammation in the onset <strong>of</strong> 

kidney lesions in normotensive obese rats. Nephrol Dial Transplant 

15:467–476 

51. Prabhakaran S, Devi KS (1993) Impact <strong>of</strong> protein deficiency and 

exposure to HCH or malathione on lipid metabolism in pregnant 

rats. Indian J Biochem Biophys 30:234–238 

52. Raja W, Agarwal RC (2010) Chemoprotective potential <strong>of</strong> cow 

urine against 7, 12-dimethylbenz(a) antracene-induced skin papillomagenesis 

in mice. Acad J Can Res 3(1):7–10 

53. Ray G, Hussain SA (2002) Oxidants, antioxidants and carcinogenesis. 

Indian J Exp Biol 42:1213–1232 

54. Rotruck JT, Pope AL, Ganther HE, Swanson AB (1973) Selenium: 

biochemical roles as a component <strong>of</strong> glutathione peroxidise. 

Science 179:588–590 

55. Salawu EO, Adeleke AA, Oyewo OO, Ashamu EA, Ishola OO, 

Afolabi AO, Adesanya TA (2009) Prevention <strong>of</strong> renal toxicity 

from lead exposure by oral administration <strong>of</strong> Lycopersicon esculentum. 

J Toxicol Environ Health Sci 1(2):022–027 

56. Sauviat MP, Pages N (2002) Cardiotoxicity <strong>of</strong> lindane: a gamma 

isomer <strong>of</strong> hexachlorohexane. J Soc Biol 196:339–348 

57. Scibior D, Skrzycki M, Podsiad M, Czeczot H (2008) Glutathione 

level and glutathione-dependent enzyme activities in blood serum <strong>of</strong> 

patients with gastrointestinal tract tumors. Clin Biochem 41:852–858 

123

1976 Mol Biol Rep (2014) 41:1967–1976 

58. Searle AJ, Wilson RL (1980) Glutathione peroxidase: effect <strong>of</strong> 

superoxide, hydroxyl and bromine free radicals on enzyme 

activity. Int J Radiant Biol 37:213–217 

59. Shaikh ZA, Vu TT, Zaman K (1999) Oxidative stressasamechanism 

<strong>of</strong> chronic cadmium-induced hepatotoxicity and renal toxicity and 

protection by antioxidants. Toxicol Appl Pharmacol 154(3):256–263 

60. Shanmugam KR, Ramakrishna CH, Mallikarjuna K, Reddy KS 

(2010) Protective effect <strong>of</strong> ginger against alcohol-induced renal 

damage and antioxidant enzymes in male albino rats. Indian J 

Exp Biol 48:143–149 

61. Sivanesan D, Begum H (2007) Preventive role <strong>of</strong> Gyanandropsis 

gynandra L., against aflatoxin B 1 induced lipid peroxidation and 

defense mechanism in rat. Indian J Exp Biol 45(3):299–303 

62. Sozmen EY, Sozmen B, Delen Y, Onat T (2001) Catalase/superoxide 

dismutase (SOD) and catalase/paraoxonase (PON) ratios may 

implicate poor glycemic control. Arch Med Res 32:283–287 

63. Srivastava A, Shivanandappa T (2005) Hexachlorocyclohexane 

differentially alters the antioxidant status <strong>of</strong> the brain regions in 

rat. Toxicology 214:123–130 

64. Tasaduq SA, Singh K, Sethi S, Sharma SC, Bedi KL, Singh J, 

Jaggi BS, Johri RK (2003) Hepatocurative and antioxidant pr<strong>of</strong>ile 

<strong>of</strong> HP-1, a polyherbal phytomedicine. Hum Exp Toxicol 22(12): 

639–645 

65. Vela C, Cristol JP, Maggi MF (1999) Oxidative stress in renal 

transplant recipients with chronic rejection: rationale for antioxidant 

supplementation. Transplant Proc 31:1310–1311 

66. Verschurren RK, Engelina MD, Berkvens JB, Helleman PW, 

Rauws AG, Schuller PL, Vanesch GJ (1976) Toxicity <strong>of</strong> methyl 

mercuric chloride in rats (I) short term study. Toxicology 6(1): 

85–96 

67. Videla LA, Barros SBM, Junqueira VBC (1990) Lindane-induced 

liver oxidative stress. Free Rad Biol Med 9:169–179 

68. Wilber KM, Baerheim F, Shapiro OW (1949) The thiobarbituric 

acid reagent as a test for the oxidation <strong>of</strong> unsaturated fatty acid by 

various reagents. Arch Biochem Biophys 24:304–311 

69. Zini A, Schlegel PN (1996) Catalase mRNA expression in the 

male rat reproductive tract. J Androl 17:473–480 

123

ISSN NO 2320-5407 International Journal <strong>of</strong> Advanced Research (2013), Volume 1, Issue 5, 71-78 

Journal homepage: http://www.journalijar.com 

INTERNATIONAL JOURNAL 

OF ADVANCED RESEARCH 

RESEARCH ARTICLE 

Evaluation <strong>of</strong> Anticancer properties <strong>of</strong> Taxusbaccata and Badri cow urine in mice: Clinicohematological 

study 

Ankita Joshi, and R.S. Chauha 

1. Post Doctoral Fellow, Cell Culture Laboratory, Institute <strong>of</strong> Biotechnology, G.B. Pant University <strong>of</strong> Agriculture 

and Technology, Patwadangar-263128, Nainital, Uttarakhand, INDIA. 

2. Campus Director, Institute <strong>of</strong> Biotechnology, G.B. Pant University <strong>of</strong> Agriculture and Technology, Patwadangar- 

263128, Nainital, Uttarakhand, INDIA. 

Manuscript Info 

Manuscript History: 

Received: 12 June 2013 

Final Accepted: 23 June 2013 

Published Online: July 2013 

Key words: 

Badricow urine, 

Taxusbaccata, Mice, 

Body weight, hematology. 

Abstract 

In the present investigation, the anticancerous effect <strong>of</strong> Taxusbacaata and 

distilled Badri cow urine was studied in mice for clinicohematology and 

body weight for a period <strong>of</strong> six month at an interval <strong>of</strong> 15 days. The study 

revealed that the values <strong>of</strong> total leucocyte count (TLC), absolute lymphocyte 

count (ALC) and absolute neutrophil count (ANC) were significantly 

increased in the treated groups <strong>of</strong> 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 <strong>of</strong> body which in 

turn helps in further healing <strong>of</strong> cancer. 

Copy Right, IJAR, 2013,. All rights reserved. 


Cancer is a disease involving dynamic changes in 

genome and is characterized by the uncontrolled, 

uncoordinated and purposeless proliferation <strong>of</strong> 

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% <strong>of</strong> population obtains medical 

help from private practitioners and half <strong>of</strong> 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 <strong>of</strong> 

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 <strong>of</strong> free radicals are 

prevented through cow urine therapy. Use <strong>of</strong> cow 

urine one can get the charm <strong>of</strong> 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 <strong>of</strong> 

71


4 m probably only being reached in around 2,000- 

4,000 years. Thuner is the oldest plant at high altitude 

region <strong>of</strong> Uttarakhand. Most parts <strong>of</strong> 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 

<strong>of</strong> extracts <strong>of</strong> leaves and bark showed the presence <strong>of</strong> 

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 <strong>of</strong> taxol; 

the anti-cancer drug, but has not been widely 

exploited in this connection (Hartzell, 2003). 

Considering the severity <strong>of</strong> cancer as a 

disease <strong>of</strong> man and animals and complexity <strong>of</strong> 

therapeutic approaches and their harmful side effects, 

it was planned to study the effect <strong>of</strong> Taxusbaccata 

preparation along with cow urine distillate in mice as 

measured through clinical haematology 


1. Extract preparation 

Extracts <strong>of</strong> leaves and bark <strong>of</strong> 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 <strong>of</strong> 

Biotechnology, G.B. Pant University <strong>of</strong> Agriculture 

and Technology, Patwadangar, Nainital, Uttarakhand, 

India. A total <strong>of</strong> 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 <strong>of</strong> 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 <strong>of</strong> leaves <strong>of</strong> Taxusbaccata)(8 

mice), (Ethanolic extract <strong>of</strong> leaves <strong>of</strong> 

Taxusbaccata)(8 mice), G(Methanolic extract <strong>of</strong> bark 

<strong>of</strong> Taxusbaccata)(8 mice), H(Ether extract <strong>of</strong> Bark <strong>of</strong> 

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 <strong>of</strong> diethyl nitrosamine (DEN) @ 200 

μl/kg body weight was given to each mice <strong>of</strong> 

negative control group and tests groups. 500 ml <strong>of</strong> 

each extract were made by adding 20% <strong>of</strong> extract in 

500ml <strong>of</strong> distilled water (Kumar et al., 2004b). The 

mice <strong>of</strong> 9 test groups were given different extracts <strong>of</strong> 

taxusbaccata alone and in combination with CUD 

(2ml/day/mice), daily p.o., from day 1 for 6 months; 

however, the mice <strong>of</strong> negative control group were 

maintained with routine feed and water.Body weight 

<strong>of</strong> mice were taken regularly at an interval <strong>of</strong> 15 day 

till the end <strong>of</strong> the experiment.Total leucocyte count 

(TLC), absolute neutrophil count (ANC), absolute 

leucocyte count (ALC), hemoglobin, total erythrocyte 

count (TEC) and hemoglobin (Hb) content <strong>of</strong> all the 

experimental animals in different groups were 

determined regularly at an interval <strong>of</strong> 15 day till the 

end <strong>of</strong> 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 <strong>of</strong> mice were taken in gram at an 

interval <strong>of</strong> 15 days till the end <strong>of</strong> experiment. Data <strong>of</strong> 

body weight change during experiment were givens 

in table-1. Initially, the mean bodyweight <strong>of</strong> control 

was 21.47±1.21 gm and after 6 month the mean body 

weight <strong>of</strong> mice was 25.86±1.87 gm. In DEN 

(negative control) treated group the initial mean body 

weight at zero day <strong>of</strong> experiment was 22.73±1.33 gm, 

which decreased to 19.16±1.81 gm at the end <strong>of</strong> 

experiment. But in CUD treated group mean body 

weight at zero day was 22.43±1.36 gm and at the end 

<strong>of</strong> experiment it was 23.91±1.21 gm.CUD treated 

group in which the carcinogen has been given, the 

initial mean body weight at zero day was 21.42±1.56 

gm. After the end <strong>of</strong> experiment, the mean body 

weight was 21.06±1.91 gm. In test group A, the zero 

day mean body weight 23.13±1.54 gm, which 

marginally increased at the end <strong>of</strong> experiment to 

23.52±1.01gm.In the group CUD+A the mean in 

body weight at zero day was 21.36±1.47 gm which 

was 26.48±0.902 at the end <strong>of</strong> experiment. In the 

group CUD+B, the mean body weight was 

22.76±1.44 gm at zero day and was 24.80±1.09 gm at 

the end <strong>of</strong> experiment. In CUD+G and CUD+H 

groups, the mean body weight at zero day was 

22.97±1.37 gm and 22.81±1.21 gm which was 

increased to 24.67±0.941 gm and 24.60±1.01 gm at 

the end <strong>of</strong> the experiment. In group B, G and H, the 

mean body weight at zero day was found 22.81±1.26 

gm, 22.41±1.32 gm and 22.51±1.28 gm respectively 

and at the end <strong>of</strong> experiment the body weight reaches 

72


to 23.40±1.02, 23.33±1.91 and 23.28±1.89, respectively. 

Table 1: Body weight in gm <strong>of</strong> experimental mice at an interval <strong>of</strong> 15 days (Mean±SE). 

Groups/Days 0 Day 15 30 45 60 75 90 105 120 135 150 165 180 

Control 21.47±1.21* 22.52±1.32 22.92±1.47 23.17±1.07 23.19±1.36 24.31±1.41 24.56±1.53 25.07±1.43 25.19±0.947 25.37±1.12 25.69±1.49 25.72±0.989 25.86±1.87 

CUD (no 22.43±1.36* 22.48±1.29* 22.51±1.33 22.54±1.42* 22.61±1.62** 22.79±1.46 22.94±1.53 23.08±1.67* 23.48±1.82 23.54±1.29* 23.67±1.68 23.82±1.92 23.91±1.21** 

DEN) 

DEN 22.73±1.33** 23.12±1.42 23.49±1.36* 23.79±1.21* 24.01±1.36 24.67±1.17** 24.92±1.07* 25.16±0.941 24.01±1.21* 23.72±1.06 22.09±1.09* 21.87±1.31* 19.16±1.81 

CUD 21.42±1.56* 21.62±1.41* 21.71±1.52* 22.07±1.02 22.67±1.17** 22.89±1.19 23.12±1.23* 22.92±1.16 22.66±0.941 22.52±1.12 22.26±1.07 22.12±1.13* 21.06±1.91* 

A 23.13±1.54* 23.63±1.71 24.76±1.23 24.93±1.32** 25.11±1.16* 25.72±1.21 25.91±1.47 24.37±1.41** 24.87±1.31 23.85±1.03** 23.71±1.07 23.68±1.13 23.52±1.01* 

B 22.81±1.26 23.12±1.21** 23.71±1.28 23.91±1.17 24.11±1.24* 24.52±1.31** 25.03±1.28* 24.76±1.19* 24.10±1.07* 23.81±1.23 23.71±1.16* 23.67±1.09 23.40±1.02* 

G 22.41±1.32 22.91±1.61* 23.14±1.10** 23.57±1.17 23.87±1.36* 24.10±1.30 24.47±1.28 24.32±1.21* 24.10±1.07 23.73±1.22* 23.64±0.940 23.50±1.11 23.33±1.91* 

H 22.51±1.28* 22.68±1.31 22.91±1.12* 23.41±1.21** 23.62±1.31 23.82±1.40* 23.98±1.33 23.90±1.21 23.81±1.17 23.63±1.07 23.48±1.21 23.39±1.07** 23.28±1.89 

A+CUD 21.36±1.47** 21.73±1.32* 22.07±1.12** 22.39±1.39 22.87±1.42* 23.01±1.25 23.93±1.61** 24.03±1.71* 24.18±0.981** 25.81±1.42* 25.91±1.07* 26.36±1.03 26.48±0.902** 

B+CUD 22.76±1.44* 22.91±1.51* 23.57±1.07 23.91±1.40** 23.96±1.31 24.03±1.61 24.18±1.42* 24.43±1.27 24.89±1.19 24.97±1.36* 24.91±1.17 24.88±1.13 24.80±1.09 

G+CUD 22.97±1.37 23.07±1.27 23.46±1.21* 23.71±1.41** 23.96±1.31* 24.10±1.31* 24.57±1.19 24.81±1.17** 24.95±1.16* 24.89±1.27* 24.84±1.11 24.72±1.08* 24.67±0.941 

H+CUD 22.81±1.21* 22.90±1.20* 22.96±1.17 23.07±1.31 23.48±1.32** 23.69±1.20 23.84±1.23 23.96±1.19 24.97±1.23 24.90±1.21 24.78±1.31** 24.67±1.08 24.60±1.01* 

Significant difference in comparison to control (*p≤0.5 and **p≤0.01) 

Table 2: Total erythrocyte count (TEC) (x 10 6 /cumm) <strong>of</strong> experimental mice at an interval <strong>of</strong> 15 days (Mean±SE). 

Groups/Days 0 15 30 45 60 75 90 105 120 135 150 165 180 

Control 6.82±0.13 6.86±0.17 6.92±0.27 6.97±0.16 7.06±0.31 7.14±0.24 7.16±0.41 7.18±0.37 7.19±0.34 7.20±0.17 7.21±0.19 7.23±0.23 7.25±0.27 

CUD (NO 

DEN) 

6.29±0.52* 6.36±0.41* 6.52±0.63 6.70±0.13* 6.83±0.46 6.94±0.32 7.03±0.21** 7.19±0.61* 7.22±0.73* 7.25±0.49* 7.29±0.36 7.34±0.51** 7.40±0.42 

DEN 6.87±0.51 6.89±0.62** 6.96±0.71* 6.98±0.18* 7.08±0.42* 7.11±0.52* 7.01±0.63 6.94±0.87* 6.66±0.33* 6.06±0.61 5.04±0.12** 4.71±0.43 3.61±0.46* 

CUD 6.42±0.83 6.48±0.17 6.63±0.21* 6.69±0.47* 6.78±0.82* 6.83±0.27* 6.91±0.16* 7.03±0.51* 7.14±0.21 7.01±0.32** 6.81±0.27 6.71±0.61 6.68±0.53 

A 6.31±0.37** 6.38±0.41 6.48±0.28* 6.63±0.46* 6.89±0.72* 6.96±0.69 7.09±0.33* 7.47±0.38 7.34±0.28 7.12±0.48 7.19±0.59* 7.23±0.72 7.32±0.58 

B 6.33±0.61 6.45±0.42* 6.61±0.47 6.75±0.51 6.95±0.80 7.08±0.72** 7.22±0.61 7.45±0.56 7.29±0.51* 7.09±0.62 7.15±0.68* 7.21±0.52* 7.29±0.57** 

G 6.39±0.41 6.41±0.39 6.54±0.41* 6.71±0.43 6.90±0.53 7.05±0.62** 7.17±0.47 7.39±0.54** 7.06±0.57 6.89±0.62 7.05±0.41* 7.15±0.46 7.20±0.37 

H 6.37±0.52* 6.39±0.59* 6.53±0.47 6.68±0.45 6.70±0.52** 6.72±0.58 6.96±0.43 7.15±0.48 7.41±0.51** 7.69±0.48 7.35±0.61 7.23±0.42* 7.18±0.41** 

A+CUD 6.31±0.97** 6.46±0.818 6.79±0.17* 6.98±0.42** 7.09±0.78 7.35±0.18* 7.49±0.19** 7.83±0.63* 7.98±0.76 8.38±0.84 8.73±0.39** 8.83±0.36* 8.90±0.47 

B+CUD 6.37±0.53 6.40±0.61** 6.66±0.59 6.77±0.41* 6.81±0.47* 6.96±0.53 7.09±0.12 7.18±0.35 7.21±0.61* 7.40±0.69** 7.77±0.63 8.15±0.71 8.59±0.62* 

G+CUD 6.42±0.59* 6.44±0.53 6.79±0.49 6.83±0.50 6.93±0.60 7.07±0.59* 7.18±0.57 7.310.51 7.77±0.53 7.86±0.59 7.72±0.47* 8.19±0.44 8.51±0.42 

H+CUD 6.35±0.61 6.40±0.57 6.55±0.64** 6.72±0.51* 7.13±0.57 7.45±0.63 7.68±0.53* 7.88±0.56* 8.04±0.47** 8.13±0.52 8.29±0.56 8.44±0.48** 8.48±0.43* 


Haematological parameters 

Data <strong>of</strong> TEC is expressed in number <strong>of</strong> 

cellsx10 6 /cumm and is mentioned in Table 2. Initially 

the TEC <strong>of</strong> control was 6.82±0.13 x 10 6 /cumm and 

after 6 month, TEC <strong>of</strong> experimental mice was 

7.25±0.27 x 10 6 /cumm. In DEN treated (negative 

control) group the initial TEC at zero day <strong>of</strong> 

experiments was 6.87±0.51 x 10 6 /cumm which 

decreased to 3.61±0.46 x 10 6 /cumm significantly at 

the end <strong>of</strong> experiment. But in CUD treated group, the 

TEC at zero day was 6.29±0.52 x 10 6 /cumm and 

7.40±0.42 x 10 6 /cumm at the end <strong>of</strong> experiment. 

CUD treated group in which the carcinogen had also 

been given, the initial TEC at zero day was 6.42±0.83 

x 10 6 /cumm. After the end <strong>of</strong> experiment, it was 

6.68±0.53x 10 6 /cumm. In test group A, the zero day 

73


TEC was 6.31±0.37 x 10 6 /cumm. The TEC decreased 

to 7.32±0.58 x 10 6 /cumm at 180 day <strong>of</strong> experiment. 

In group CUD+A the total erythrocyte count, at zero 

day was 6.31±0.97 x 10 6 /cumm which was increased 

to 8.90±0.47 x 10 6 /cumm at the end <strong>of</strong> experiment. 

Group CUD+B had 6.37±0.53 x 10 6 /cumm TEC at 

zero day and 8.59±0.62 x 10 6 /cumm at the end <strong>of</strong> 

experiment, respectively. In CUD+G and CUD+H 

groups the TEC at zero day were 6.42±0.59 x 

10 6 /cumm and 6.35±0.61 x 10 6 /cumm, respectively 

and were 8.51±0.42 x 10 6 /cumm and 8.48±0.43 x 

10 6 /cumm at the end <strong>of</strong> the experiment. In group B, G 

and H, the TEC at zero day was found 6.33±0.61 x 

10 6 /cumm, 6.39±0.41 x 10 6 /cumm and 6.37±0.52 x 

10 6 /cumm, respectively and at the end <strong>of</strong> experiment 

increased to 7.29±0.57 x 10 6 /cumm, 7.20±0.37 x 

10 6 /cumm and 7.18±0.41 x 10 6 /cumm. 

Data <strong>of</strong> TLC is expressed in no. <strong>of</strong> cells x 10 3 /cumm 

and is presented in Table-3.Initially the TLC count <strong>of</strong> 

control group was 8.91±0.30 x 10 3 /cumm and after 6 

month the TLC was 12.11±0.04 x 10 3 /cumm. In DEN 

(negative control) group the initial TLC at zero day 

<strong>of</strong> experiments was 9.12±3.20 x 10 3 /cumm which 

was decreased to 3.02±1.45 x 10 3 /cumm. But in CUD 

treated group, the TLC at zero day was 8.65±4.31 x 

10 3 /cumm and 9.53±4.67 x 10 3 /cumm at the end <strong>of</strong> 

experiment. In CUD treated group, the initial 

TLC at zero day was 9.02±5.31 x 10 3 /cumm which 

decreased to 7.81±3.11 x 10 3 /cumm. In test group 

like A, the zero day TLC was 9.08±5.68 x 10 3 /cumm 

which was decreased to 8.64±3.08 x 

10 3 /cumm.CUD+A had the zero day mean TLC 

count as 8.97±4.02 x 10 3 /cumm which was increased 

to 12.61±2.17 x 10 3 /cumm, at the end <strong>of</strong> experiment. 

Group CUD+B has 8.89±6.31 x 10 3 /cumm at zero 

day and was 10.55±3.18 x 10 3 /cumm at the end <strong>of</strong> 

experiment.In group B, G and H, the TLC at zero day 

was observed as 9.01±6.81 x 10 3 /cumm, 9.10±6.07 x 

10 3 /cumm and 9.07±7.03 x 10 3 /cumm, respectively 

and at the end <strong>of</strong> experiment 8.59±3.19 x 10 3 /cumm, 

8.33±3.61 x 10 3 /cumm and 8.30±4.1 x 10 3 /cumm 

respectively. In groups CUD+G and CUD+H the 

TLC at zero day was 9.11±5.98 x 10 3 /cumm, 

9.03±6.19 x 10 3 /cumm and was 10.21±3.81 x 

10 3 /cumm and 10.05±3.14 x 10 3 /cumm at the end <strong>of</strong> 

the experiment, respectively. 

Data <strong>of</strong> ALC is expressed in no. <strong>of</strong> cells x 

10 3 /cumm and is presented in Table-4.Initially the 

ALC count <strong>of</strong> control group was 4.30±0.69 x 

10 3 /cumm and after 6 month the ALC was 5.92±0.98 

x 10 3 /cumm. In DEN (negative control) group the 

initial ALC at zero day <strong>of</strong> experiments was 4.41±0.81 

x 10 3 /cumm which was decreased to 1.36±0.47 x 

10 3 /cumm. But in CUD treated group, the ALC at 

zero day was 4.13±0.70 x 10 3 /cumm and 4.63±0.43 x 

10 3 /cumm at the end <strong>of</strong> experiment.In CUD treated 

group, the initial ALC at zero day was 4.43±0.84 x 

10 3 /cumm which decreased to 3.76±0.53 x 

10 3 /cumm. In test group like A, the zero day ALC 

was 4.42±0.91 x 10 3 /cumm which was decreased to 

4.19±0.27 x 10 3 /cumm.CUD+A had the zero day 

mean ALC count as 4.39±0.87 x 10 3 /cumm which 

was increased to 6.16±0.42 x 10 3 /cumm, at the end <strong>of</strong> 

experiment. Group CUD+B has 4.36±0.89 x 

10 3 /cumm at zero day and was 5.16±0.80 x 

10 3 /cumm at the end <strong>of</strong> experiment.In group B, G and 

H, the ALC at zero day was observed as 4.40±0.94 x 



4.18±0.94 x 10 3 /cumm, 4.01±0.37 x 10 3 /cumm and 

3.98±0.77 x 10 3 /cumm respectively. In groups 

CUD+G and CUD+H the ALC at zero day was 


was 4.90±0.28 x 10 3 /cumm and 4.89±0.11 x 

10 3 /cumm at the end <strong>of</strong> the experiment, respectively. 

Data <strong>of</strong> ANC is expressed in no. <strong>of</strong> cells x 

10 3 /cumm and is presented in Table-5.Initially the 

ANC count <strong>of</strong> control group was 4.57±0.72 x 

10 3 /cumm and after 6 month the ANC was 6.00±0.99 

x 10 3 /cumm. In DEN (negative control) group the 

initial ANC at zero day <strong>of</strong> experiments was 

4.69±0.83 x 10 3 /cumm which was decreased to 

1.49±0.52 x 10 3 /cumm. But in CUD treated group, 

the ANC at zero day was 4.38±0.78 x 10 3 /cumm and 

4.71±0.47 x 10 3 /cumm at the end <strong>of</strong> experiment. 

In CUD treated group, the initial ANC at 

zero day was 4.51±0.97 x 10 3 /cumm which decreased 

to 3.88±0.56 x 10 3 /cumm. In test group like A, the 

zero day ANC was 4.60±0.97 x 10 3 /cumm which was 

decreased to 4.30±0.31 x 10 3 /cumm.CUD+A had the 

zero day mean ANC count as 4.46±0.89 x 10 3 /cumm 

which was increased to 6.25±0.45 x 10 3 /cumm, at the 

end <strong>of</strong> experiment. Group CUD+B has 4.41±0.92 x 

10 3 /cumm at zero day and was 5.24±0.82 x 

10 3 /cumm at the end <strong>of</strong> experiment.In group B, G and 

H, the ANC at zero day was observed as 4.58±0.99 x 




4.09±0.79 x 10 3 /cumm respectively. In groups 

CUD+G and CUD+H the ANC at zero day was 


was 5.01±0.29 x 10 3 /cumm and 4.96±0.12 x 

10 3 /cumm at the end <strong>of</strong> the experiment, respectively. 

74


Table 3: Total leucocyte count (TLC) (x 10 3 /cumm) count <strong>of</strong> experimental mice at an interval <strong>of</strong> 15 days (Mean±SE). 

Groups/Days 0 15 30 45 60 75 90 105 120 135 150 165 180 


CUD (NO 8.65±4.31 8.71±4.36** 8.86±4.51* 8.93±4.70 9.08±5.01* 9.13±5.21 9.18±3.24* 9.20±4.22 9.27±4.56** 9.32±4.61 9.38±4.28 9.46±4.39* 9.53±4.67 

DEN) 

DEN 9.12±3.20** 9.21±3.52* 9.43±4.07** 9.89±3.81 8.07±3.61 7.42±3.82 6.41±2.01 6.12±3.10* 5.87±2.12* 4.31±3.19 4.16±2.16 4.08±1.87* 3.02±1.45* 

CUD 9.02±5.31* 9.13±5.27 9.31±5.17 9.67±4.81** 9.89±4.72 10.07±4.55 9.91±4.31** 9.73±4.24 9.41±4.27* 9.01±3.69 8.23±3.16** 8.11±3.08* 7.81±3.11 

A 9.08±5.68* 9.15±5.51* 9.28±5.42 9.57±5.23 9.79±5.05** 9.98±4.93 9.78±4.61* 9.61±4.34 9.32±4.21* 8.91±4.07 8.83±3.21 8.79±3.12 8.64±3.08 

B 9.01±6.81* 9.11±6.72 9.19±6.61** 9.41±6.52 9.61±6.37* 9.77±5.83* 9.58±5.61 8.91±5.37 8.87±5.21* 8.81±5.08 8.79±4.61* 8.68±4.52 8.59±3.19** 

G 9.10±6.07 9.16±5.91* 9.24±5.82 9.33±5.74** 9.51±5.41* 9.67±5.34* 9.48±5.28 8.87±5.05 8.76±4.81 8.69±4.23* 8.59±4.07 8.41±3.82 8.33±3.61 

H 9.07±7.03 9.11±6.87 9.15±6.67** 9.21±6.41 9.38±5.91 9.47±5.80 9.31±5.47* 8.83±5.32** 8.73±5.16 8.62±4.77* 8.57±4.41* 8.46±4.21* 8.30±4.10 

A+CUD 8.97±4.02** 9.21±4.47 9.49±3.32 9.89±3.77* 10.08±2.01* 10.33±2.61* 10.69±3.16* 10.93±4.03 11.31±2.12** 11.67±3.41* 11.91±4.91* 12.25±3.53* 12.61±2.17** 

B+CUD 8.89±6.31* 9.12±6.27* 9.25±6.08 9.49±5.61* 9.68±5.32* 9.87±5.17* 9.67±5.03 9.47±4.71 9.73±4.57 9.84±4.31 10.08±4.17 10.67±4.08* 10.55±3.18 

G+CUD 9.11±5.98 9.18±5.81* 9.27±5.61 9.39±5.47* 9.59±5.27 9.71±5.17 9.51±5.09** 9.30±4.81 9.26±4.67 9.42±4.51 10.02±4.41** 10.17±4.32* 10.21±3.81* 

H+CUD 9.03±6.19 9.13±6.01 9.19±5.81** 9.27±5.72 9.44±5.51 9.59±5.42* 9.40±5.31 9.31±5.06* 9.12±4.87* 9.09±4.62* 9.51±4.41 9.88±4.17 10.05±3.14 

Significant difference in comparison to control(*p≤0.5 and **p≤0.01) 

Table 4: Absolute lymphocyte count (ALC) (x 10 6 /cumm) <strong>of</strong> experimental mice at an interval <strong>of</strong> 15 days (Mean±SE). 

Groups/Days 0 15 30 45 60 75 90 105 120 135 150 165 180 


CUD (NO 4.13±0.70* 4.24±0.72 4.31±0.69* 4.37±0.58* 4.43±0.42 4.45±0.39* 4.48±0.69** 4.49±0.84 4.52±0.92* 4.54±0.14 4.55±0.28* 4.61±0.63* 4.63±0.43 

DEN) 

DEN 4.41±0.81* 4.43±0.61 4.56±0.71 4.86±0.24* 3.92±0.92** 3.59±0.43* 3.05±0.64 2.94±0.59 2.81±0.36 2.08±0.31* 1.92±0.27* 1.90±0.17 1.36±0.47* 

CUD 4.43±0.84* 4.41±0.64 4.54±0.84 4.74±0.18 4.83±0.29 4.85±0.36 4.85±0.47* 4.72±0.53* 4.59±0.61 4.35±0.74* 3.96±0.89 3.89±0.92 3.76±0.53 

A 4.42±0.91 4.44±0.738 4.51±0.42** 4.67±0.75 4.78±0.94* 4.86±0.86 4.78±0.73 4.66±0.21** 4.53±0.28* 4.36±0.69 4.28±0.74** 4.25±0.18** 4.19±0.27* 

B 4.40±0.94 4.42±0.57 4.44±0.55 4.60±0.85 4.69±0.69 4.77±0.54** 4.65±0.22* 4.33±0.51 4.34±0.49* 4.26±0.16 4.24±0.21* 4.22±0.86 4.18±0.94* 

G 4.48±0.73* 4.41±0.82** 4.51±0.98* 4.56±0.37 4.64±0.76 4.76±0.41 4.62±0.47* 4.32±0.53 4.27±0.84 4.23±0.61 4.15±0.65 4.10±0.21* 4.01±0.37* 

H 4.44±0.84 4.40±0.34* 4.48±0.76 4.50±0.26** 4.54±0.86* 4.63±0.16 4.56±0.36 4.30±0.61* 4.24±0.42* 4.15±0.69** 4.16±0.48 4.09±0.59* 3.98±0.77 

A+CUD 4.39±0.87 4.49±0.29 4.62±0.59 4.84±0.43* 4.90±0.33 5.02±0.77 5.21±0.61 5.33±0.98 4.55±0.71 5.71±0.14 5.83±0.21* 5.98±0.63 6.16±0.42** 

B+CUD 4.36±0.89** 4.47±0.19* 4.51±0.41** 4.62±0.82* 4.74±0.27 4.83±0.87* 4.72±0.91** 4.61±0.84** 4.73±0.16** 4.76±0.72 4.91±0.18 5.21±0.68* 5.16±0.80 

G+CUD 4.41±0.80* 4.48±0.47 4.53±0.63 4.58±0.92 4.65±0.46** 4.74±0.18* 4.66±0.78 4.56±0.20 4.52±0.91 4.57±0.39* 4.87±0.65* 4.91±0.21 4.90±0.28 

H+CUD 4.42±0.79 4.45±0.77 4.48±0.27* 4.52±0.39 4.61±0.65 4.69±0.23 4.59±0.88 4.54±0.49 4.46±0.64* 4.43±0.72* 4.63±0.47 4.86±0.30* 4.89±0.11* 


Table 5: Absolute Neutrophil count (ANC) (x 10 6 /cumm) <strong>of</strong> experimental mice at an interval <strong>of</strong> 15 days (Mean±SE). 

Groups/Days 0 15 30 45 60 75 90 105 120 135 150 165 180 


CUD (NO 4.38±0.78** 4.33±0.74** 4.40±0.72 4.44±0.60 4.52±0.45* 4.53±0.43 4.56±0.72 4.57±0.86* 4.60±0.97 4.62±0.19 4.67±0.34 4.70±0.65 4.71±0.47* 

DEN) 

DEN 4.69±0.83 4.56±0.65** 4.68±0.75 4.92±0.26 4.01±0.96 3.68±0.46 3.14±0.67** 3.00±0.61* 2.90±0.41 2.13±0.34 2.00±0.31 1.99±0.23* 1.49±0.52 

CUD 4.51±0.87 4.50±0.69* 4.60±0.89* 4.81±0.21 4.92±0.31 4.99±0.37 4.93±0.49 4.83±0.57 4.67±0.66** 4.47±0.77 4.07±0.92** 3.99±0.95* 3.88±0.56** 

A 4.60±0.97* 4.53±0.79 4.61±0.46 4.75±0.77 4.87±0.98 4.96±0.89* 4.86±0.74** 4.78±0.26 4.61±0.32 4.42±0.75* 4.39±0.77 4.36±0.23 4.30±0.31 

B 4.58±0.99* 4.51±0.58** 4.56±0.57** 4.68±0.87* 4.78±0.69* 4.85±0.56 4.77±0.26 4.40±0.55 4.40±0.51 4.38±0.19* 4.35±0.26 4.31±0.89 4.26±0.96 

G 4.56±0.75 4.54±0.86 4.62±0.99 4.64±0.39* 4.73±0.79* 4.80±0.43** 4.71±0.49 4.41±0.54** 4.35±0.85* 4.31±0.66* 4.26±0.66 4.18±0.24 4.11±0.38 

H 4.51±0.87** 4.52±0.37 4.53±0.80* 4.58±0.29 4.66±0.88 4.71±0.17* 4.61±0.38 4.39±0.63 4.32±0.46 4.27±0.74 4.25±0.53* 4.20±0.61 4.09±0.79 

A+CUD 4.46±0.89* 4.58±0.31* 4.70±0.61** 4.92±0.45* 4.99±0.35** 5.11±0.78* 5.32±0.64* 5.41±0.99* 5.62±0.74* 5.80±0.15** 5.91±0.23 6.10±0.65** 6.25±0.45* 

B+CUD 4.41±0.92 4.51±0.23 4.63±0.46 4.73±0.86 4.80±0.29* 4.91±0.89 4.80±0.93 4.70±0.86 4.81±0.18 4.89±0.74* 5.00±0.20* 5.30±0.69 5.24±0.82 

75


G+CUD 4.52±0.84 4.56±0.49 4.61±0.66* 4.67±0.95* 4.77±0.48 4.80±0.20 4.73±0.79** 4.62±0.23 4.60±0.95* 4.68±0.44 4.98±0.67 5.00±0.24* 5.01±0.29* 

H+CUD 4.50±0.85* 4.54±0.81* 4.56±0.29 4.61±0.40 4.70±0.66* 4.75±0.25 4.67±0.90 4.61±0.50* 4.53±0.65 4.51±0.73 4.72±0.49** 4.95±0.32 4.96±0.12 

Table 6: Haemoglobin content (Hb) <strong>of</strong> experimental mice at regular interval <strong>of</strong> 15 days (gm%, mean±SE). 

Groups/Days 0 15 30 45 60 75 90 105 120 135 150 165 180 


CUD(NO 

DEN) 

12.19±1.31* 12.21±1.42 12.27±1.51* 12.49±1.36 12.62±1.47* 12.85±1.51 12.99±1.62* 13.41±1.73* 13.53±1.81* 13.66±1.74* 13.70±1.32 13.76±1.21* 13.78±1.19 

DEN 12.40±1.36* 12.42±1.38 12.43±1.29 12.45±1.31** 12.46±1.33 12.25±1.36* 12.08±1.39 11.82±1.36* 11.65±1.30 10.52±1.32* 10.09±1.21 9.36±1.19* 8.02±1.07 

CUD 12.32±1.31 12.45±1.28** 12.86±1.33 12.90±1.37 13.06±1.29* 13.40±1.42 13.87±1.47 13.67±1.40* 13.46±1.33** 13.22±1.28* 12.79±1.21* 

12.09±1.19** 

12.34±1.28 

A 12.27±1.17** 12.43±1.27 12.84±1.29 12.87±1.32* 13.07±1.33 13.38±1.38* 13.85±1.41 14.04±1.43 13.95±1.47 13.80±1.27 13.76±1.23* 13.71±1.19 13.63±1.13 

B 12.31±1.40 12.39±1.35* 12.79±1.25 12.83±1.38** 13.03±1.27 13.30±1.30 13.79±1.37** 14.02±1.39* 13.95±1.41* 13.75±1.26 13.71±1.19** 13.67±1.21** 13.56±1.18 

G 12.34±1.28* 12.37±1.21 12.72±1.24** 12.79±1.41 13.04±1.32** 13.25±1.19** 13.72±1.29 13.96±1.26 13.91±1.25* 13.70±1.32* 13.65±1.27 13.61±1.21 13.49±1.29 

H 12.34±1.42 12.36±1.40 12.68±1.23** 12.74±1.21 12.95±1.31* 13.10±1.22 13.67±1.28 13.87±1.27 13.82±1.30 13.65±1.33* 13.59±1.24** 13.55±1.32* 13.40±1.25 

A+CUD 12.41±1.48* 12.48±1.47 12.89±1.36** 12.94±1.39 13.08±1.33* 13.42±1.41* 13.89±1.52* 14.06±1.47** 14.64±1.37 14.82±1.42 15.07±1.37 15.21±1.27 15.38±1.31* 

B+CUD 12.29±1.42* 12.40±1.37 12.81±1.27 12.85±1.36** 13.05±1.31 13.35±1.32 13.81±1.39 14.01±1.41 14.22±1.42 14.48±1.25 14.74±1.21* 14.89±1.22* 14.90±1.17* 

G+CUD 12.27±1.41 12.36±1.39** 12.76±1.21 12.80±1.29* 13.01±1.25 13.27±1.28 13.76±1.31* 13.98±1.30* 14.03±1.27* 14.33±1.21** 14.69±1.23 14.74±1.17 14.86±1.14 

H+CUD 12.30±1.39* 12.35±1.35 12.70±1.27* 12.76±1.23 12.98±1.33** 13.12±1.29* 13.70±1.20 13.90±1.24 13.97±1.26 14.08±1.31 14.62±1.28* 14.70±1.22 14.78±1.11** 


Data <strong>of</strong> Hemoglobin is expressed in gm% and is 

presented in Table-6. Initially the Hb content <strong>of</strong> 

control was 12.45±1.42gm% and after 6 month the 

Hb content <strong>of</strong> mice was 12.68±1.36gm%. In DEN 

(negative control) group the initial Hb content at zero 

day <strong>of</strong> experiment was 12.40±1.36gm%, which 

decreased to 8.02±1.07gm%, at the end <strong>of</strong> 

experiment. But in CUD treated group, their Hb 

content at zero day was 12.19±1.31gm% and 

13.78±1.19gm% at the end <strong>of</strong> experiment. In CUD 

treated group in which the carcinogen has been given, 

the initial Hb content at zero day was 

12.32±1.31gm%, after the end <strong>of</strong> experiment the Hb 

content was 12.09±1.19gm%. In test group A, the 

zero day Hb content was 12.27±1.17gm%. The Hb 

content increased to 13.63±1.13gm% at the end <strong>of</strong> 

experiment. In group B, G and H, the Hb content at 

zero day was found 12.31±1.40gm%, 

12.34±1.28gm% and 12.34±1.42gm%, respectively 

and at the end <strong>of</strong> experiment the Hb content reached 

to 13.56±1.18gm%, 13.49±1.29gm% and 

13.40±1.25gm%, respectivelyCUD+A had Hb 

content at zero day 12.41±1.48gm% which was 

increased to 15.38±1.31gm%, at the end <strong>of</strong> 

experiment. Group CUD+B had 12.29±1.42gm% Hb 

content on zero day which was increased to 

14.90±1.17gm% at the end <strong>of</strong> experiment. In 

CUD+G and CUD+H, the Hb content at zero day was 

12.27±1.41gm%, 12.30±1.39gm% and was 

14.86±1.14gm% and 14.78±1.11gm% at the end <strong>of</strong> 

the experiment, respectively. 

Total Leucocyte count (TLC) in experimental mice 

76


Absolute lymphocyte count (AlC) <strong>of</strong> experiment <strong>of</strong> mice. 

Haemoglobin content (Hb) in experimental mice 

Absolute neutrophil count (AlC) <strong>of</strong> experiment <strong>of</strong> mice. 

Total erythrocyte count (TEC) in experimental mice 


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 <strong>of</strong> 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 <strong>of</strong> mice was decreased substantially in 

DEN treated mice indicating in the development <strong>of</strong> 

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 <strong>of</strong> 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 <strong>of</strong> DEN; 

however, herbal formulations <strong>of</strong> extracts and CUD 

were found to be a preventive agent against the 

carcinogenic effects <strong>of</strong> DEN. DEN is already known 

chemical carcinogen.Increased immunocompetence 

<strong>of</strong> an individual is a very essential parameter to 

prevent the development <strong>of</strong> cancers by several 

mechanisms, <strong>of</strong> which the upregulation <strong>of</strong> 

lymphocyte proliferation and stimulation activity, 

increased macrophage activity, higher antibody 

production and increased synthesis and secretion <strong>of</strong> 

cytokines (IL-1, Il-2) plays significant role by 

enhancing the recognition <strong>of</strong> tumor cells by the 

immune cells <strong>of</strong> the body and cytotoxic activities <strong>of</strong> 

the tumor killing cells, the lymphocytes. Using herbs 

for cancer treatment can help the body to support its 

77


healing power. In the present investigation, both 

doses <strong>of</strong> QC (5 and 25 mg/kg) led to a significant 

decrease in the number as well as the mean area <strong>of</strong> 

GST-P positive foci, TUNEL positive apoptotic cells, 

p53 positive hepatocytes, and restoration <strong>of</strong> 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 <strong>of</strong> the cancer caused by 

DEN. 

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 <strong>of</strong> the highly 

proliferating cells through their destruction by the 

white blood cells. Erythrocytosis and increased 

hemoglobin content are the indication <strong>of</strong> good health 

and recovery and neutralization <strong>of</strong> the effect <strong>of</strong> DEN 

by T. baccata and CUD. Joshi et al., 2013 

investigated that the immunomodulatory effect <strong>of</strong> 

distilled Gir cow urine in rabbits 

throughhaematological parameters. The study 

revealed that the values <strong>of</strong> 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 <strong>of</strong> enhanced 

vitality <strong>of</strong> mice. Similarly leukocytosis, 

lymphocytosis and neutrophilia are the immune cell 

showing immunopoturtration, which is considered 

protective against cancer and an indication <strong>of</strong> a good 

prognosis (chauhan, 2005). It further needs a detailed 

study for further confirmation. 

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): 69–90. 

Joshi A.,Bankoti K.,Bisht T. and Chauhan R.S. 

Immunomodulatory effect <strong>of</strong> Gir cow urine distillate 

in Rabbits. Journal <strong>of</strong> Immunology and 

Immunopathology, 2012, 14(1): 57-61. 

Kumar A.,Kumar P.,Singh L.K and Agrawal D.K. 

Pathogenic effect <strong>of</strong> 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). 

Udupa A.L.,Kulkarni D.R. and Udupa S.L. Effect <strong>of</strong> 

Tridaxprocumbens extract on wound healing, Int. J. 

Pharmacognosy, 1995, 1: 37-40. 

Ramji C and You W. Differential sensitivity <strong>of</strong> 

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 <strong>of</strong> adult self-renewing 

haematopoietic stem cells". Nature. 2003, 423 

(6937): 302–5. 

Kinzler K.W and Vogelstein B.The genetic basis <strong>of</strong> 

human cancer.Medical Pub. Division, 2002, 2:1-5. 

IARC.Cancer an entropic disease, 2003, 143: 112- 

131. 

Bedi P.S, Singh H.,Johri H.S and Agarwal R.N. 

Effect <strong>of</strong> 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 <strong>of</strong> Ancient 

Science. Employment News, 2005, 30(15):1-2. 

Pal S and Mittal K. Use <strong>of</strong> alternative cancer 

medicine in India. Lancetoncol.2004, 3: 394-395. 

Govindachari T.R., Suresh G. and Masailmani S. 

Antifungal activity <strong>of</strong> Azadirachtaindica leaf hexane 

extract fitoterpia, 1999, 70: 427-420. 

References 

Hartzell M. Carcinogenic effect <strong>of</strong> Taxusbacata in 

diabetic condition., 2003, 45:147-168. 

*********** 

78

Original Research Paper 

Lipid-lowering activity <strong>of</strong> Cow urine ark in guinea pigs fed with a high 

cholesterol diet 

Chawda Hiren Manubhai 1 , Mandavia Divyesh Rasiklal 1 , Baxi Seema Natvarlal 2 , Vadgama 

Vishalkumar Kishorbhai 1 , Tripathi ‎Chandrabhanu Rajkishor 1* ‎ 

1 Department <strong>of</strong> Pharmacology, Government Medical College, Bhavnagar-364001, Gujarat, India 

2 Department <strong>of</strong> 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 <strong>of</strong> CUA and its possible 

implication in metabolic syndrome.‎ 

Materials and Methods: Thirty guinea pigs <strong>of</strong> 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 <strong>of</strong> study period, respectively. Serum lipid pr<strong>of</strong>ile 

(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 <strong>of</strong> the study. Histological study <strong>of</strong> 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) <strong>of</strong> ‎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 <strong>of</strong> 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 <strong>of</strong> Cow urine ark 

in guinea pigs fed with a high cholesterol Diet. Avicenna J Phytomed, 2014; 4 (5): 354-363. 


Cardiovascular diseases (CVD) are 

emerging problem in developing countries 

‎(Freedman, 2003; Badimon et al., 2010). 

CVD may become the reason for the loss 

<strong>of</strong> 17.9 ‎million Potentially Productive 

Years <strong>of</strong> Life Lost (PPYLL) in India, by 

2030 (Anchala et al., 2012). ‎Dyslipidaemia 

is well recognized risk factor for the 

development <strong>of</strong> cardiovascular diseases 

AJP, Vol. 4, No. 5, Sep-Oct 2014 354

Lipid-lowering activity <strong>of</strong> Cow urine ark ‎ 

‎(Freedman, 2003; Badimon et al., 2010). 

Reactive oxygen species induce the 

oxidative stress, ‎which play significant 

role in the development <strong>of</strong> 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 <strong>of</strong> 

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 <strong>of</strong> 

dyslipidaemia (Suanarunsawat et al., 

2011). ‎ 

Cow urine, known‎as‎“Amrita”‎or‎water‎ 

<strong>of</strong> life as mentioned in Ayurveda ‎(Dhama 

et al., 2005), is one <strong>of</strong> the ingredients <strong>of</strong> 

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 <strong>of</strong> 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 <strong>research</strong> center, Ahmedabad. 

(Batch no: ARD2110109)‎ 

Animal preparation 

Thirty guinea pigs weighing 520 – 860 

g <strong>of</strong> either sex were procured from the 

Central ‎Animal House <strong>of</strong> Government 

Medical College, Bhavnagar, which is 

registered in the ‎Committee for the 

Purpose <strong>of</strong> Control and Supervision <strong>of</strong> the 

Experiments on Animals ‎(CPCSEA), New 

Delhi, India. CPCSEA guidelines were 

followed during animal ‎experiments <strong>of</strong> our 

study. The guinea pigs were housed in 

stainless steel cages under 12 ‎hour lightdark 

cycle room with controlled 

temperature at 23±2°C, being fed with 

standard ‎laboratory food and water ad 

libitum. After the proper acclimatization 

for 15 days, they were ‎divided into five 

groups <strong>of</strong> 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 

<strong>of</strong> CUA (0.8 ‎ml/kg); Group IV: High fat 

diet plus a higher dose <strong>of</strong> CUA (1.6 

ml/kg); Group V: High fat ‎diet plus 

rosuvastatin (1.5 mg/kg)‎ 

Diet composition ‎ 

Normal diet: in the morning, mixtures 

<strong>of</strong> 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 <strong>of</strong> wheat and 

‎Bengal gram flour, followed by 40 g <strong>of</strong> the 

mixtures mentioned above, in the normal 

diet/animal. ‎In the evening: green leafy 

vegetables, 30 g/animal.‎ 

AJP, Vol. 4, No. 5, Sep-Oct 2014 355

Chawda Hiren et al.‎ 

Methodology 

The study was conducted in Animal 

room, Department <strong>of</strong> Pharmacology, 

Government ‎Medical College, Bhavnagar, 

Gujarat, after approval from the 

Institutional Animal Ethics ‎Committee <strong>of</strong> 

the same institute. The baseline blood 

sample was collected from a lateral 

‎saphenous vein <strong>of</strong> the hind paw <strong>of</strong> each 

guinea pig in overnight fasting state. 

Blood samples were‎ sent to Clinical 

Biochemistry Laboratory <strong>of</strong> our institute 

which is accredited by National 

‎Accreditation Board for Testing and 

Calibration Laboratories (NABL), for the 

serum lipid ‎pr<strong>of</strong>ile, 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 <strong>of</strong> the 

experiment, animals <strong>of</strong> group I and II ‎were 

given distilled water daily, animals <strong>of</strong> 

group III, IV and IV were given a lower 

dose ‎<strong>of</strong> CUA, higher dose <strong>of</strong> 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 <strong>of</strong> all groups ‎were 

sacrificed after blood <strong>collection</strong> from the 

lateral saphenous vein in the overnight 

fasting ‎stat at the end <strong>of</strong> 60 days. 

Bloodsmples were sent to labratory for the 

analysis <strong>of</strong> the serum lipid pr<strong>of</strong>ile, liver 

and ‎cardiac enzymes. We obtained the 

liver and kidney from each animal <strong>of</strong> the 

five groups ‎for histopathological analysis, 

which was done by senior faculty from the 

Pathology ‎department <strong>of</strong> our institute.‎ 

Serum lipid pr<strong>of</strong>ile 

The serum levels <strong>of</strong> 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 <strong>of</strong> the total 

cholesterol and HDL-C.‎ 

Evaluation <strong>of</strong> 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 

<strong>of</strong> 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 <strong>of</strong> CUA on the weight <strong>of</strong> the 

animals 

Weighing <strong>of</strong> each animal in all groups 

was done before the start <strong>of</strong> the study and 

also at the ‎end <strong>of</strong> 60 days to rule out any 

effect <strong>of</strong> CUA on the weight <strong>of</strong> 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 <strong>of</strong> the groups. Scalinggrades 0, 

1, 2, 3 and 4 were given for no change, 

slight, mild, ‎moderate and severe changes, 

respectively regarding theseverity 

assessment <strong>of</strong> histopathological‎ results.‎ 

Statistical analysis:‎ 

All parameters were expressed as 

Mean±standard error <strong>of</strong> mean (S.E.M.). 

One-way ‎Analysis <strong>of</strong> Variance (ANOVA) 

followed by Tukey-Kramer Multiple 

comparison test was ‎used to compare the 

AJP, Vol. 4, No. 5, Sep-Oct 2014 356


inter group differences <strong>of</strong> lipid pr<strong>of</strong>ile, 

liver enzymes, cardiac enzymes and ‎extent 

<strong>of</strong> body weight gain at the end <strong>of</strong> 60 days. 

Paired t-test was used to compare intra 

‎group differences <strong>of</strong> lipid pr<strong>of</strong>ile, liver 

enzymes, cardiac enzymes and extent <strong>of</strong> 

body weight ‎gain. Value <strong>of</strong> p


Table 2. The effects <strong>of</strong> each treatment strategy on serum lipid pr<strong>of</strong>ile (% increment) on guinea pigsat the end <strong>of</strong> 

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) 

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) 

60 

Days 

60 

Days 

60 

Days 

60 

Days 

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 

92 ± 22.8 * 29.8 ± 7.5 * 63.2 ± 45.4 161.93 ± 45.5 * 29.8 ± 7.5 * 54.2 ± 30.7 

42.9 ± 25.3 - 26. 5 ± 12.7 # 70.8 ± 36 ## 78.9 ± 34.9 - 26.5 ± 12.7 # 5.9 ± 30 

27.2 ± 8.5 - 37.1 ± 8.2 # 50.5 ± 16 ## 72 ± 21.7 - 37.1 ± 8.2 # - 8.85 ± 13.2 

4.5 ± 4.9 ** - 8.82 ± 2.3 ** 234 ± 31 ** - 18.9 ± 3.9 ** - 8.8 ± 2.3 ** - 67.9 ± 2.1 ** 

Values are expressed as Mean ± standard error <strong>of</strong> 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 <strong>of</strong> 

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


Treatment Groups 

(n = 6) 

Time Period 

ALT 

(U/L) 

AST 

(U/L) 

ALP 

(U/L) 

LDH 

(U/L) 

CKMB 

(U/L) 

Vehicle control 

(Group 1) 

Sham control 

(Group 2) 


CUA (0.8 ml/kg) 

(Group 3) 


CUA (1.6 ml/kg) 

(Group 4) 


rosuvastatin 

(1.5 mg/kg) 

(Group 5) 

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 ** 

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 

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 

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 ± 31.12 392.19 ± 28.3 

Values are expressed as Mean ± standard error <strong>of</strong> mean; ALT: Alanine aminotransferase, AST: Aspartate 

aminotransferase,AP: Akaline phosphatase , LDH: Lactate dehyrogenase, CK-MB: Creatine kinase-MB, CUA: 

Cow urine ark; *p


Table 4. Effect <strong>of</strong> each treatment strategy on weight <strong>of</strong> guinea pigs 

Weight <strong>of</strong> 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 <strong>of</strong> mean; CUA: Cow urine ark; *p< 0.05 as compared to baseline 

value, paired t-test. 


In the present study, we selected guinea 

pig as the experimental animal for the 

‎evaluation <strong>of</strong> lipid lowering activity <strong>of</strong> 

Cow urine ark (CUA). Lipoprotein 

metabolism <strong>of</strong> ‎guinea pigs is closest to 

human and several lines <strong>of</strong> evidence 

proved that guinea pigs are ‎admirable 

models to assess hypolipidemic activity 

and lipoprotein metabolism <strong>of</strong> drugs 

‎(Fernandez and Volek, 2006).‎ 

The present study showed that 60 days 

<strong>of</strong> high cholesterol diet feeding, ‎raised the 

serum lipid pr<strong>of</strong>ile (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 <strong>of</strong>hepatic 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


Oxidative stress and free radical 

induced injuries play a major role in the 

‎pathogenesis <strong>of</strong> a number <strong>of</strong> diseases. 

Hyperlipidemia produces lots <strong>of</strong> 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 <strong>of</strong> ‎serum levels <strong>of</strong> AST, 

ALT, AP, LDH, and CK-MB suggested 

suppressed cardiac and ‎hepatic functions, 

due to the retention <strong>of</strong> lipid in liver and 

heart in sham control (Table 3 and ‎Figure 

1 B). CUA treatment decreased serum 

level <strong>of</strong> 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 <strong>of</strong> 

thiobarbituric acid, ascorbic acid, DPPH 

radical scavenging activity and ‎ABTS 

assay (Sachdev et al., 2012). Hence, the 

antioxidant activity <strong>of</strong> 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 

<strong>of</strong> 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 <strong>of</strong> lipid abnormality, 

including high levels <strong>of</strong> triglycerides, low 

‎levels <strong>of</strong> 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 <strong>of</strong> ‎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 <strong>of</strong> 

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 <strong>research</strong> 

center, Ahmedabad for providing 

rosuvastatin calcium powder as a free gift 

sample. ‎ 

Conflict <strong>of</strong> interest 

There is not any clash <strong>of</strong> attentiveness 

in this study. 

AJP, Vol. 4, No. 5, Sep-Oct 2014 361


References‎ 

Achliya GS, Kotagale NR, Wadodkar SG, 

Dorle AK. 2003. Hepatoprotective activity 

<strong>of</strong> ‎panchagavyaghrita against carbon 

tetrachloride induced hepatotoxicity in rats. 

Indian J ‎Pharmacol, 35: 308-311.‎ 

Ahmad-Raus RR, Abdul-Latif ES, 

Mohammad JJ. 2001. Lowering <strong>of</strong> lipid 

composition ‎in aorta <strong>of</strong> guinea pigs by 

Curcuma domestica. BMC Complement 

Altern Med, 1: 6-11.‎ 

Anchala R, Pant H, Prabhakaran D, Franco 

OH.2012.‎ ‘Decision‎ support‎ system‎ (DSS)‎ 

‎for prevention <strong>of</strong> cardiovascular disease 

(CVD) among hypertensive (HTN) patients 

‎ in‎ Andhra‎ Pradesh,‎ India’--a cluster 

randomised community intervention trial. 

BMC Public ‎Health, 12: 393-399. ‎ 

Badimon L, Vilahur G, Padro T.2010. 

“Nutraceuticals‎and‎atherosclerosis:‎human‎ 

trials.”‎‎CardiovasTher, 28: 202-215.‎ 

Bo S, Durazzo M, Gambino R, Berutti C, 

Milanesio N, Caropreso A, Gentile L, 

Cassader‎M, Cavallo-Perin P, Pagano 

G.2008. Associations <strong>of</strong> dietaryand serum 

‎copper with inflammation, oxidativestress, 

and metabolic variables in adults. J Nutr, 

138: ‎305-310.‎ 

Botham KM, Moore EH, De Pascale C, Bejta 

F. 2007.The induction <strong>of</strong> macrophage foam 

‎cell formation by chylomicron 

remnants.BiochemSoc Trans, 35: 454-458.‎ 

Cannon CP, Braunwald E, McCabe CH, et al. 

Intensive versus moderate lipid lowering 

with ‎statins after acute coronary 

syndromes. N Engl J Med. 2004; 350: 

1495-1504.‎ 

Demacker PN, Reijnen IG, Katan MB, Stuyt 

PM, Stalenhoef AF.1991. Increased 

removal ‎<strong>of</strong> remnants <strong>of</strong> triglyceride-rich 

lipoproteins on a diet rich in 

polyunsaturated fatty acids. Eur‎J Clin 

Invest, 21: 197-203.‎ 

Dhama K, Chauhan RS, Singhal L.2005. Anti- 

Cancer activity <strong>of</strong> cow urine: Current status 

‎and future directions. Int J Cow Sci, 1: 1- 

25.‎ 

Fernandez ML, Volek JS.2006. Guinea pigs: a 

suitable animal model to study lipoprotein 

‎metabolism, atherosclerosis and 

inflammation. NutrMetab (Lond), 3: 17-23.‎ 

Freedman‎ JE.2003.“High-fat diets and 

cardiovascular‎ disease.”Jacc,‎ 41:‎ 1750- 

1752.‎ 

Friedewald WT, Levy RI, Fredrickson 

DS.1972.Estimation <strong>of</strong> the concentration <strong>of</strong> 

low-‎density lipoprotein cholesterol in 

plasma, without use <strong>of</strong> the preparative 

ultracentrifuge.Clin‎Chem, 18: 499-502.‎ 

Fruchart JC.2013. Selective peroxisome 

proliferator-activated‎receptor‎α‎modulators‎ 

‎(SPPARMα):‎ the‎ next‎ generation‎ <strong>of</strong>‎ 

peroxisome proliferator-activated receptor 

α-agonists. ‎CardiovascDiabetol, 12: 1-8.‎ 

Gianturco SH, Ramprasad MP, Song R, Li R, 

Brown ML, Bradley WA.1998. 

‎Apolipoprotein B-48 or its apolipoprotein 

B-100 equivalent mediates the binding <strong>of</strong> 

‎triglyceride rich lipoproteins to their unique 

human monocytemacrophage receptor. 

‎ArteriosclerThrombVascBiol, 18: 968-976. ‎ 

Goldstein JL, Kita T, Brown MS.1983. 

Defective lipoprotein receptors and 

atherosclerosis.‎Lessons from an animal 

counterpart <strong>of</strong> familial 

hypercholesterolemia. N Engl J Med, 309: 

‎288-296.‎ 

Gururaja M P, Joshi A B, Joshi H, 

Sathyanarayana D, Subrahmanyam EVS, 

Chandrashekhar‎KS.2011. Antidiabetic 

potential <strong>of</strong> cow urine in streptozotocin 

induced diabetic rats. Asian J ‎Tradit Med, 

6: 8-13.‎ 

Jain NK, Gupta VB, Garg R, Silawat N.2010. 

Efficacy <strong>of</strong> cow urine therapy on various 

‎cancer patients in Mandsaur district, India: 

A survey. Int J Green Pharm, 4: 29-35.‎ 

Jarald EE, Edwin S, Tiwari V, Garg R, 

Toppo E.2008. Antidiabetic activity <strong>of</strong> cow 

urine ‎and a herbal preparation prepared 

using cow urine. Pharm Biol, 46: 789-792.‎ 

Kawakami A, Aikawa M, Alcaide P, 

Luscinskas FW, Libby P, Sacks FM.2006. 

‎Apolipoprotein CIII induces expression <strong>of</strong> 

vascular cell adhesion molecule-1 in 

vascular ‎endothelial cells and increases 

adhesion <strong>of</strong> monocytic cells. Circulation, 

114: 681-687.‎ 

Krishnamurthi K, Dutta D, Sivanesan SD, 

Chakrabarti T.2004. Protective effect <strong>of</strong> 

distillate ‎and redistillate <strong>of</strong> Cow's urine in 

human polymorphonuclear leukocytes 

challenged with ‎established genotoxic 

Chemicals. Biomed Environ Sci, 17: 247- 

256.‎ 

Kwiterovich‎ Jr.‎ PO.1997.‎ “The‎ effect‎ <strong>of</strong> 

dietary fat, antioxidants, and pro-oxidants 

on ‎blood lipids, lipoproteins, and 

AJP, Vol. 4, No. 5, Sep-Oct 2014 362


atherosclerosis.”‎ Journal‎ Am‎ Diabet‎ 

Association, 97: 31-41.‎ 

Lamb DJ, Avades TY, Ferns GA.2001. 

Biphasic modulation <strong>of</strong> atherosclerosis 

induced ‎by graded dietary copper 

supplementation in the cholesterolfedrabbit. 

Int J ExpPathol, 82: ‎287-294.‎ 

LaRosa JC, Grundy SM, Waters DD, Rader 

DJ, Rouleau JL, Belder R, Joyal SV, Hill 

‎KA, Pfeffer MA, Skene AM.2005. 

Intensive lipid lowering with atorvastatin in 

patients‎with stable coronary disease. N 

Engl J Med, 352: 1425-1435.‎ 

Li M, Li Y, Liu J.2013. Metabolic syndrome 

with hyperglycemia and the risk <strong>of</strong> 

‎ischemic stroke.Yonsei Med J, 54: 283- 

287.‎ 

Palmer AM, Murphy N, Graham A.2004. 

Triglyceride-rich lipoproteins inhibit 

cholesterol ‎efflux to apolipoprotein (apo) 

A1 from human macrophage foam cells. 

Atherosclerosis, 173: ‎27-38. ‎ 

Patel S, Puranik R, Nakhla S, Lundman P, 

Stocker R, Wang XS, Lambert G, Rye ‎KA, 

Barter PJ, Nicholls SJ, CelermajerDS.2009. 

Acute hypertriglyceridaemia in humans 

‎increases the triglyceride content and 

decreases the anti-inflammatory capacity <strong>of</strong> 

high ‎density lipoproteins. Atherosclerosis, 

204: 424-428.‎ 

Patel Y, Vadgama V, Baxi S, Chandrabhanu, 

Tripathi B. 2011. Evaluation <strong>of</strong> 

hypolipidemic‎activity <strong>of</strong> leaf juice <strong>of</strong> 

Catharanthusroseus (Linn.) G. Donn. in 

guinea pigs. Acta Pol ‎Pharm, 68: 927-935.‎ 

Pedersen TR, Faergeman O, Kastelein JJ, 

Olsson AG, Tikkanen MJ, Holme I, Larsen 

‎ML, Bendiksen FS, Lindahl C, Szarek M, 

Tsai J.2005. High-dose atorvastatin vs 

usual-dose ‎simvastatin for secondary 

prevention after myocardial infarction: the 

IDEAL study: a ‎randomized controlled 

trial. JAMA, 294: 2437-2445.‎ 

Randhawa GK.2010. Cow urine distillate as 

bioenhancer. J Ayurveda Integr Med, 1: 

240-‎241.‎ 

Sachdev D O, Gosavi D D, Salwe K J.2012. 

Evaluation <strong>of</strong> antidiabetic, antioxidant 

effect ‎and safety pr<strong>of</strong>ile <strong>of</strong> gomutra ark in 

Wistar albino rats.AncSci Life, 31: 84–89.‎ 

Sampson UK, Fazio S, Linton MF.2012. 

Residual cardiovascular risk despite 

optimal LDL ‎cholesterol reduction with 

statins: the evidence, etiology, 

andtherapeuticchallenges. Curr‎Atheroscler 

Rep, 14: 1-10.‎ 

Suanarunsawat T, Ayutthaya WD, Songsak T, 

Thirawarapan S, Poungshompoo 

S.2011.‎Lipid lowering and antioxidative 

activities <strong>of</strong> aqueous extracts <strong>of</strong> Ocimum 

sanctum L. ‎ leaves in rats fed with a highcholesterol 

diet. Oxid Med Cell Longev, 

2011:962025. ‎doi:10.1155/2011/962025. 

Epub 2011 Sep 21. ‎ 

Talayero B G, Sacks FM.2011. The Role <strong>of</strong> 

Triglycerides 

in 

Atherosclerosis.CurrCardiol‎Rep, 13: 544– 

552.‎ 

Vijayakumar RS, Surya D, Nalini N. 

2004.“Antioxidant‎efficacy‎<strong>of</strong>‎black‎pepper‎ 

(Piper ‎nigrum L.) andpiperine in rats with 

high fat diet induced oxidative stress.”‎ 

Redox Report, ‎9: 105-110, 2004. 

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