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Raad et al., IJPSR, 2013; Vol. 4(4): 1534-1539. ISSN: 0975-8232

International Journal of Pharmaceutical Sciences and Research 1534

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 of Biochemistry, Dr. Babasaheb Amberdkar Marathwada University 1, Aurangabad, Maharashtra,

India

MGM’s, Institute of Biosciences and Technology 2, Auranagabad, Maharashtra, India

ABSTRACT: Cow urine therapy and all traditional practices from

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

proved to be a boon in the areas of agriculture, science and technology,

industry, energy, medicine etc for the development of any nation, in

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

antibacterial potentials of cow urine were investigated. Total 14

pathogenic and non-pathogenic bacterial cultures were used as test

organism against 10 different cow urine samples. The highest zone of

inhibition was shown by sample G against P aeruginosa NCIM 2945

(1.8cm) while the smallest zone of inhibition was shown against E. coli

NCIM 2065(0.3 cm) by sample A. Based on cumulative effect against

the test organism, the urine sample G was found to be the most efficient

inhibiting all the 14 test cultures. The antibacterial activity reported by

sample G was comparable with standard antibiotics. A higher zone of

inhibition was observed by sample G against P aeruginosa NCIM 2945

as compared to that of Gentamicin, Oxacillin and vancomycin. Though

the urine sample G showed a strong antibacterial activity against all the

test organisms, but the activity was reported low against the entire Gram

positive bacteria compared to Gram negative bacteria. The presence of

proline which is considered as a major amino acid in antimicrobial

peptides was also observed in the urine sample G.

INTRODUCTION: It is widely accepted among

clinicians, medical researchers, microbiologists and

pharmacologists, that antibiotic resistance will, in the

very near future, leave healthcare professionals

without effective therapies for bacterial infections.

QUICK RESPONSE CODE

IJPSR: ICV (2011)- 5.07

Article can be accessed online on:

www.ijpsr.com

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

all Staphylococcus aureus strains found in many

medical institutions are resistant to antibiotics such

as methicillin 1.

Presently we face a global public health crisis, as

infectious diseases top the list for causes of death

worldwide.

While it is likely that antibiotic resistance contributes

significantly to this problem, data on consumption

and resistance to antibiotics are limited for most

countries 2

and the relationship of resistance to

morbidity and mortality is quantitatively unclear.

Keywords:

Cow urine, Antibacterial activity, Zone

of inhibition, Antimicrobial Peptides

Correspondence to Author:

Dr. Devendra V. Deshmukh

Assistant Professor, MGM’s, Institute

of Biosciences and Technology, N-6,

CIDCO, Aurangabad, India

E-mail: [email protected]



Cow, Bos indicus is a most valuable animal in all

community. The cow urine is useful in number of

disease particularly in gulma, filaria, cancer ets. It is

also used with herbs to cure diseases like fever,

epilepsy, anemia, abdominal pain, constipation, etc

by the traditional healers 3 4

. Immunomodulatory 5,

hypoglycemic 6 and cardio-respiratory effects

7.

Recently the cow urine has been granted U.S. Patents

(No. 6,896,907 and 6,410,059) for its medicinal

properties, particularly for its use along with

antibiotics for the control of bacterial infection and

fight against cancers. Medicinal usage of cow urine

are extensively searched and scientifically endorsed 8.

In the Present study the antibacterial potentials of the

cow urine have been investigated against 14 different

pathogenic and nonpathogenic bacteria.

MATERIALS AND METHODS:

Collection of the urine sample: 10 urine samples

were collected from different cows from the farm; all

the samples were collected from milking cows.

Random sampling was a method of choice for

collection of the samples. Samples were collected in

sterile containers, 20 ml of middle stream urine was

collected and brought to the laboratory and stored in

fridge until further use. The samples were designated

as sample A, B, C to Sample J.

Qualitative test for proteins: The qualitative test of

protein was performed as according to Martin and

Mittelman 9. The urine Samples were centrifuged at

3000 rpm for 10 mins for the removal of sediments.

After centrifugation the supernatant was collected

and heat test for proteins was performed to observe

the presence of protein.

Quantitative estimation of Protein: The Folin

Lowry method was a method of choice for estimation

of protein. Aliquots of protein standard solution were

pipetted out as into a series of tubes as 0.1, 0.2….1.0

ml and the total volume was made to 4 ml with

distilled water. To each tube 5.5 ml of alkaline mix

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

to stand for 15 min, at room temperature. 0.5 ml of

FC reagent was pipetted out into each tube, mixed

thoroughly and kept in dark for 30 min. The blue

color formed was measured at 650 nm against a

proper blank. The same was conducted for the

samples 10

.

Antimicrobial activity:

Test bacterial cultures: Fourteen bacterial cultures

from laboratory repository viz. Escherichia coli

NCIM 2345, Escherichia coli NCIM 2065,

Escherichia coli NCIM 2310, Bacillus subtilis NCIM

2113, Bacillus licheniformis NCIM 2015, Bacillus

megaterium NCIM 2083, Staphylococcus aureus

NCIM 2124, Staphylococcus aureus NCIM 2079,

Staphylococcus aureus NCIM 2125, Pseudomonas

aeruginosa NCIM 2945, Pseudomonas aeruginosa

NCIM 2053, Proteus vulgaris NCIM 2857, Kebshella

pneumonie NCIM 2957 and Salmonella typhimurium

NCIM 2501 were used in the study. Freshly grown

12 h old cultures in nutrient broth were used as the

inoculum in antibacterial assays.

Disc Preparation: Paper disc of filter paper

Whattman No. 1 were prepared. The discs were

sterilized by autoclave at 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 of urine

samples against the test organisms was done by disc

diffusion assay 11

. Petri plate containing 15 ml of

solidified nutrient agar was spread inoculated with

100 μl of 12 h old test bacterial cultures. Presterilized

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

with 50 µl of urine and dried to be used in assays.

The plates were kept at 4oC for 10 min before they

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

assessed by measuring the diameter of growth

inhibition zone around the discs. Sensitivity of test

organisms was also checked against commercial

discs (Hi Media, India) containing standard

antibiotics.

Paper chromatography: The urine sample showing

highest protein content and antibacterial activity was

analyzed for the presence of amino acids using paper

chromatography technique . A strip of wattman’s

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

from one end of the length a line was drawn with the

help of a pencil. At the centre of the line a tiny spot

of the sample was placed. The spot was allowed to

dry and then placed in the chamber containing the

saturated solvent system (Butanol : Acetic acid :

Water, 4 : 1 : 5). The chromatogram was allowed to

run upto ¾ th

the paper and then taken out and dried

in an oven and then spayed with locating reagent



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

was calculated.

RESULTS:

Qualitative test for proteins: All the urine samples

tested for the presence of protein gave a positive

result. All the test tubes contain the urine sample

showed cloudiness with granules which gave a

positive test for protein in the urine sample.

Protein estimation by Lowry method: The

qualitative estimation of protein gave mixed results.

Sample G gave the highest protein content 520

µgm/ml while sample J showed the lowest

concentration of protein (Table 1).

Antibacterial assay: Antibacterial activities of all

the urine samples were tested using disc diffusion

method. On the basis of cumulative antibacterial

effect against all cultures under test, sample G

appeared as most effective. A highest cumulative

inhibition against all the fourteen bacterial cultures

was 15 cm for the urine sample G while the lowest

effect was shown by sample A (Table 2).

TABLE 1: PROTEIN ESTIMATED FROM ALL THE 10

URINE SAMPLES USING FOLIN LOWRY METHOD

Sample Absorbance at

660 nm

Concentration of

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

cultures

Urine samples

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

NCIM 2083 R R

0.9

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

S aureus

NCIM 2124 R R R R R

0.5

(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 of 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

Bacterial cultures

Standard Antibiotics

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 of spot

and the Rf value of the spot reveled the amino acid

present in the urine sample. From the calculated Rf

value it was clear that amino acid proline was

prominent amino acid and was confirmed with the Rf

value of standard proline.

DISCUSSION: Commonly, antibiotics are widely as

conservative treatment in various microbial

infections and diseases 12

. Considering the enormous

quantity of antibiotics used, the situation should have

been that there would be no infectious diseases. But,

the fact is that the problems of infectious diseases are

increasing day‐by‐day. Some of the major hindrances

are that bacteria have genetic ability to transmit and

acquire resistance towards the drugs 13

and there are

also adverse effects of drugs on the host.14

Therefore

to combat such problems many natural products have

been explored. The nature is an almost infinite

resource for drug development and discovery. It has

endowed with a complete repository of remedies to

cure all ailments of mankind, as it has always been a

first rate drug store with enormous range of plants,

micro organisms and animals.15

The ancient literature of cow urine has always

focused on prevention of disease and maintaining the

health and treatment of diseases. Cow urine acts like

a magical potion for the treatment of the disease like

cancer, asthma, chronic renal failure, hepatitis ABC,

urological disorders, respiratory diseases and also

plays its part as antimicrobial against disease like

Eczema, Psoriasis, acne vulgaris, scabies and other

various kinds of allergies. Urine contains volatile

salts which are beneficial to the human body because

these salts destroy acidity and get rid of pain in

kidney, intestine, and womb; furthermore urine, a

natural tonic, eliminates giddiness, tension in nerves,

lazy feeling, hemicrama, paralysis, common cold,

diseases of brain, nerves and joints.

In the present study the antibacterial potential of 10

different urine samples from cows at the MGM’s

farm house was revealed. The variation in the color

of the urine samples may be due to the amount and

type of fodder consumed and the protein content in

them.

FIGURE 1: COLOR VARIATION IN THE 10 URINE

SAMPLES TAKEN FROM COWS.

According to Figure 3, 50% showed yellow color ,

weak yellow for 30% of the urine samples, while

20% of the urine samples showed deep yellow

coloration.



Cow urine contains different constituents; it is rich in

potassium, chloride, calcium, estrogen, phosphorous,

urinary proteins 16

. Various research have also found

different components like urea, uric acid, nitrogen,

sulfur, copper, iron, sodium, other salts, carbolic

acid, ammonia, sugar lactose, Vitamin-A,B,C,D,E,

gonadotropin, phenols and also some anticancer

substances.

All the cow urine samples showed the presence of

protein. Vats and Kanupriya 17

has reported that the

components of cow urine are responsible for

showing antimicrobial activity.

The presence of protein in all the samples was clear

evidence that all the samples do contain the presence

of bioactive compounds. Marshall and Arenas 18

pointed out the use of the importance of naturally

occurring peptides and their use as an alternative to

chemical antibiotics and their role as antimicrobials.

The antibacterial potentials of the cow urine was

tested against some pathogenic and non pathogenic

bacteria (Table 2). The highest zone of inhibition

was shown by sample G against P aeruginosa NCIM

2945 (1.8cm) while the smallest zone of inhibiton

was shown against E. coli NCIM 2065(0.3 cm) by

sample A.

FIGURE 2: SENSITIVITY PATTERN OF THE TEST

CULTURES AGAINST 10 URINE SAMPLES

All the samples showed the presence of antibacterial

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

maximum activity of all the 10 urine samples was

against Gram negative bacteria than gram positive

bacteria. Similar results were obtained by Edwin et

al 19

. Where they have reported the antibacterial

effect of cow urine against gram negative and gram

positive bacteria.

The gram negative bacteria were more efficiently

inhibited than gram positive bacteria. Sathasivam et

al 20

has also reported the antibacterial activity of the

cow urine distillate against 4 gram negative bacteria.

A synergistic effect of Azadirachta indica and cow

urine against some gram negative bacteria and yeast

was observed by Vats and Miglan 17

. Though all the

urine samples showed the antibacterial activity,

sample G was a promising candidate showing

antibacterial activity against all given test organisms.

FIGURE 3: ZONE OF INHIBITION (in cm) RECORDED

BY SAMPLE G AGAINST ALL THE 14 BACTERIAL

CULTURES

As according to figure 3, the sample G gave the

highest zone of inhibition against P aeruginosa

NCIM 2945(1.8 cm) followed by inhibition against

E.coli NCIM 2345 and S typhimurium NCIM 2501

(1.5). The antibacterial activity shown by the urine

sample G was comparable with the antibacterial

activity by standard antibiotics.

A higher zone of inhibition was observed by sample

G against P aeruginosa NCIM 2945 as compared to

that of Gentamicin, Oxacillin and vancomycin.

Though the urine sample G showed a strong

antibacterial activity against all the test organisms,

but the activity was reported low against all the S

aureus cultures, specifically S aureus NCIM 2079,

(0.5 cm).

The chromatography of the sample revealed the

presence of proline. The amino acid proline is

considered as a major amino acid in antimicrobial

peptides 21

.



CONCLUSION: The Antibacterial property of the

cow urine was reveiled using biological assay. Total

10 urine samples were tested against 14 different

strains of bacteria. The ability of the cow urine

sample was more to inhibit the gram negative

bacteria than that of gram positive bacteria. The

highest zone of inhibition that was observed was

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

which has inhibited the growth of all the test

organism and when compared with standard

antibiotic proved to be more promising. The presence

of amino acid proline in the sample G has proved its

potential similar to peptide antibiotics.

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Skinhoj P, Rosdahl VT. Clinical features of Staphylococcus

aureus endocarditis: a 10-year experience in Denmark.

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

antibiotic usage: report of Task Force 1. Rev Infect Dis

1987; 9: S232–243.

3. Pathak ML, Kumar A. Cow praising and importance of

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

Protective effect of diatillate and redistillate of cow,s urine in

human polymorphonuclear leukocytes challenged with

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susceptibility testing by a standardized single disc method.

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and antibiotic sensitivities of purulent dacryocystitis. Orbit;

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13. Cohen ML. Epidemiology of drug resistance: implications

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

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Indian medicinal plants for their antimicrobial properties. J

Ethnopharmacol 1998; 62: 183‐193.

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How to cite this article:

Raad S, Deshmukh DV, Harke SN and Kachole MS: Antibacterial activity of Cow urine against some Pathogenic and Non-

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

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Pharmaceutical Biology

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Antidiabetic Activity of Cow Urine and a HerbalPreparation 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) AntidiabeticActivity of Cow Urine and a Herbal Preparation Prepared Using Cow Urine, PharmaceuticalBiology, 46:10-11, 789-792

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Pharmaceutical Biology2008, Vol. 46, Nos. 10–11, pp. 789–792

Antidiabetic Activity of Cow Urine and a Herbal Preparation

Prepared Using Cow Urine

E. Edwin Jarald,1 S. Edwin,1 V. Tiwari,1 R. Garg, and E. Toppo1

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

Abstract

An herbal preparation prepared by the traditional heal-ers of Mandsaur using cow urine and Gymnema sylvestreR. Br. (Asclepiadaceae), Momordica charantia L. (Cu-curbitaceae), Eugenia jambolana Lam. (Myrtaceae), Ae-gle marmelos Correa (Rutaceae), Cinnamomum tamalaBuch.-Ham. (Lauraceae), Aloe barbadensis Linn. (Lili-aceae), and Trigonella foenum-graecum L. (Leguminosae)is being used in the treatment of diabetes. In order to sci-entifically appraise the claim, this preparation was stud-ied for antidiabetic activity and also compared with theherbal preparation prepared using water. Fresh cow urinewas also used in the study to identify the synergistic ef-fect. The preparations were tested for antidiabetic activityin alloxan-induced diabetic rats at two dose level, 200 and400 mg/kg, respectively. The study was done for a period of21 days. The activity was compared with reference standard,insulin (1 unit/kg, i.p.) and control. The herbal preparationssignificantly (P< 0.05, P < 0.01) lowered the blood sugarlevel of hyperglycemic rats in a dose-dependent manner.Comparatively, the cow urine preparation showed better ac-tivity than did the preparation prepared using water. Freshcow urine also exhibited significant antidiabetic effect. Thisstudy supports the claim of the local traditional healers.

Keywords: Alloxan monohydrate, antidiabetic, cow urine,herbal preparation.

Introduction

Diabetes mellitus is a group of metabolic diseases char-acterized by hyperglycemia, hypertriglyceridemia, and hy-percholesterolemia, resulting from defects in insulin se-

Accepted: April 2, 2008

Address correspondence to: E. Edwin Jarald, Assistant Professor, Herbal Drug Research Lab, B. R. Nahata College of Pharmacy &Contract Research Centre, Mhow Neemuch Road, Mandsaur 458001, Madhya Pradesh, India. E-mail: [email protected]

cretion or action or both (Nyholm et al., 2000). Diabetesmellitus is a metabolic disease as old as mankind, and itsincidence is considered to be high (4–5%) all over theworld. Oral hypoglycemic drugs, such as sulfonylureas andbiguanides, have been used in the treatment of diabetesmellitus (Okinea et al., 2005). In spite of the introduc-tion of hypoglycemic agents, diabetes and related compli-cations continue to be a major medical problem. Since timeimmemorial, patients with non–insulin-dependent diabeteshave been treated orally in folk medicine with a variety ofplant extracts. In India, a number of plants are mentioned inancient literature (Ayurveda) for the cure of diabetic condi-tions known as “madhumeha,” and some of them have beenexperimentally evaluated and the active principles isolated(Som et al., 2001).

Cow urine is used along with herbs to treat various dis-eases like fever, epilepsy, anemia, abdominal pain, consti-pation, and so forth, by traditional healers all over India(Pathak & Kumar, 2003a; Krishnamurthi et al., 2004). Thetraditional healers (“Gayathri Parivar”) in Mandsaur use anherbal preparation prepared using cow urine for the treat-ment of diabetes. The traditional healers prepare a decoctionusing cow urine instead of water that contains the follow-ing herbs: Gymnema sylvestre R. Br. (Asclepiadaceae), Mo-mordica charantia L. (Cucurbitaceae), Eugenia jambolanaLam. (Myrtaceae), Aegle marmelos Correa (Rutaceae),Cinnamomum tamala Buch.-Ham. (Lauraceae), Aloe bar-badensis Linn. (Liliaceae), and Trigonella foenum-graecumL. (Leguminosae). The aim of this work was to validate thefolk claim. In order to create a logic base behind this treat-ment, the preparation using cow urine was compared withthe preparation using water. Fresh cow urine was also usedin this antidiabetic study to investigate the synergistic effectif any.

DOI: 10.1080/13880200802315816 C© 2008 Informa UK Ltd.

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Materials and Methods

Procurement of materials

The urine of a 2-year-old virgin Gujarati Indian cow knownas “Geer cow” was used in the study. The study was per-formed after getting a certificate from the veterinary doctorstating that the cow was free from diseases. Fresh cow urinewas collected daily and used after filtration. The plant drugswere collected from the Gayathri Parivar (local traditionalhealers) in order to minimize the variation in the claimedtherapeutic effect. The collected plant materials were pos-itively identified by Dr. H.S. Chatree, Botanist, Govt. Artsand Science College, Mandsaur, and the voucher specimenswere retained in our department for future reference.

Preparation of extracts

The herbal preparations using cow urine and distilled wa-ter were made using the above-mentioned different plantspecies. Equal quantities of air-dried samples of each plantspecies were ground and mixed with 10-times the equiva-lent volume of cow urine and water separately and boiledfor 4 h. The extracts were filtered and evaporated in a distil-lation assembly to get the residue. The percentage yield ofextracts prepared using cow urine and distilled water was12.5% and 11.0%, w/w, respectively. Preliminary chemi-cal investigation was carried out in the extracts to identifythe nature of constituents present in the extracts (Brain &Turner, 1975; Khandelwal, 2005).

Animals and treatment

After getting approval from the institutional animal ethicalcommittee (reg. no. – 918/ac/05/CPCSEA), male Wistarstrain rats (weighing between 150 and 200 g) procuredfrom the animal house of B. R. Nahata College of Phar-macy, Mandsaur, were used for the investigation. The ani-mals were housed in standard environmental conditions oftemperature (21 ± 2◦C), humidity (55 ± 10%), and a 12-hlight-dark cycle. Rats were supplied with standard pelletdiet and water ad libitum.

Acute toxicity studies

The acute toxicity test of the preparations and cow urine wasdetermined according to the OECD guidelines (No. 420,Organization for Economic Cooperation and Develop-ment). Female albino mice (20–25 g) were used for thisstudy. Dosing amounts for sample in liquid form were cal-culated with the help of density or specific gravity. Afterthe sighting study, a starting dose of 2000 mg/kg (p.o.) ofthe test samples was given to various groups of five animalseach. The treated animals were monitored for 14 days formortality and general behavior. No deaths were observedthrough the end of the study. The test samples were found

to be safe up to the dose of 2000 mg/kg, and doses of 200and 400 mg/kg were chosen for further experimentation.

Antihyperglycemic activity

Diabetes was induced in rats by injecting 150 mg/kgof alloxan monohydrate intraperitoneally in 0.9% w/vNaCl (Ainapure et al., 1985; Porchezian et al., 2000).Seventy-two hours after injection, blood glucose level wasmeasured, and the diabetic rats were divided into eightgroups of six animals each. Insulin [1 unit/kg (i.p.)] wasused as standard drug (Mukherjee, 2002). The first groupwas kept as vehicle control, the second was treated withinsulin, and the third to eighth groups were treated withherbal preparations prepared using cow urine, distilledwater, and pure cow urine at two dose levels, 200 and 400mg/kg (p.o), respectively. One more group was includedin the study to determine the effects of fresh cow urine inthe blood glucose level of normal rats. Fresh cow urine ata dose of 400 mg/kg was given to the rats in this group for21 days. The treatment was given once daily for 21 days.Blood samples were collected at regular intervals afterfasting overnight, before treatment, from rat-tail vein undermild anesthesia and monitored. The blood sugar level wasmonitored using Accu-chek Active Test strips in Accu-chekActive Test meter (Roche Diagnostics, Germany).

Statistical analysis

Data were expressed as mean ± SEM, and the obtained datawere subjected to one-way ANOVA followed by Dunnet’stest. The p values less than 0.05 were considered as signif-icant.

Results

The phytochemical investigations performed in the extractsrevealed the presence of alkaloids, tannins, flavonoids, car-bohydrates, and saponins in both the extracts. The resultsof antidiabetic activity of cow urine and herbal preparationsprepared using cow urine and water are presented in Table 1.

The basal blood glucose levels of all the groups werestatistically not different from each other. Three days afteralloxan administration, blood glucose values were 5-foldhigher in all the groups and were not statistically differentfrom each other. After 21 days, values of blood glucose weredecreased in all the treatment groups (P < 0.05, P < 0.01).The value in diabetic control group remained stable. Thepreparations exhibited activity in a dose-dependent manner.The activities of the preparations were found significantfrom the 7th day onwards, whereas the activity of cow urinewas found significant only after 21 days of treatment. Nor-mal rats treated with cow urine for 21 days did not show anyelevation in their blood glucose levels. Comparatively, thepreparations containing cow urine were found to be betterthan the herbal preparation prepared using distilled water.

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Antidiabetic activity of cow urine and cow urine preparation 791

Table 1. Effect of 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 ± 15.20CUP 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 of all.” A composition containingcows excretions—urine, dung, milk, curd, and ghee—fiveingredients together known as “panchagawya,” is given towomen after delivering a baby. Panchagawya is the main in-gredient of many Ayurvedic preparations (Pathak & Kumar,2003b). Cow urine, one of the ingredients in panchagawya,is believed to have many therapeutic values. In India, cowurine is used by the majority of the rural population as afolklore remedy in almost all the states. Agencies in Gujarathave been marketing cow urine preparations from multipleoutlets, advertising that they are sterilized and completelyfresh, with prices ranging from Rs. 20 to Rs. 30 per bot-tle. Keeping in view the enormous role of cow’s urine inmedicinal and veterinary medicine, a scientific experimentwas performed in rats to elucidate the effect of cow urineand cow urine containing preparation as an antidiabetic.

Alloxan produces hyperglycemia by a selective cytotoxiceffect on pancreatic beta cells. One of the intracellularphenomena for its cytotoxicity is through generation offree radicals demonstrated both in vivo and in vitro (Yadavet al., 2002). Our investigations indicate the efficiency ofthe herbal preparations in maintaining blood glucose lev-els in alloxan-induced diabetic rats. The glucose-loweringactivity observed in diabetic animals may be due to stim-ulation of beta cells of pancreatic islets or stimulation ofglycogenesis (Miura et al., 2001). This may be due to thepresence of some hypoglycemic principles in the plantsused in these preparations because all these plants are wellknown for their antidiabetic action (Grover et al., 2002;Kar et al., 2003; Mohamed et al., 2006; Pulok et al., 2006),and these plants have different types of mechanisms inreducing blood glucose levels. Comparatively, the prepara-tion using cow urine was found to exhibit better activitythan did the one prepared using distilled water. This could

not be correlated with the nature of the phytoconstituentspresent in the extracts because both extracts contains thesame nature of constituents. The interesting observation inour study was the antidiabetic activity of pure cow urine.The hypoglycemic effect was not observed in the normalrats treated with fresh cow urine, and this indicates that thepossible mechanism behind the antidiabetic effect of freshcow urine may be due to its stimulation in peripheral use ofglucose. According to literature, cow urine was found to ex-hibit an antioxidant effect (Krishnamurthi et al., 2004). Freeradicals are implicated in wide range of diseases includingdiabetes; the antioxidant activity of cow urine also may beone of the reasons for its observed antidiabetic effect.

Chemoprofiling of cow urine in our laboratory confirmedthe presence of protein, urea, uric acid, creatinine, phenol,aromatic acids, enzymes such as acid phosphatase, alka-line phosphatase, amylase, and vitamins (Gowenlock &McMurray, 1988). Along with these, there may be someother constituents that may be responsible for the observedactivity. From these observations, it was clear that the betteractivity of herbal preparation prepared using cow urine maybe due to its synergistic effect with cow urine or, accordingto ancient literature, cow urine is a wonderful solvent forextraction, and so it is the ability of cow urine to extract outmore active constituents from the herbal drugs and therebyincrease antidiabetic activity.

Further pharmacological investigations are needed toelucidate the mechanism of the observed antihyperglycemiceffect. This study supports the claim of the traditional heal-ers of Mandsaur.

Acknowledgement

The authors are thankful to Gayathri Parivar (local tradi-tional healers) for providing the necessary information tocarry out this research work.

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792 E.E. Jarald et al.

References

Ainapure SS, Arjaria PD, Sawant VR, Baid PS, Maste SS, VardaAB (1985): Hypoglycemic activity of an indigenous prepa-ration. Indian J Pharmacol 17: 238–239.

Brain KR, Turner TD (1975): The Practical Evaluation ofPhytopharmaceuticals. Bristol, Wright-Scientechnica,pp. 10–30.

Gowenlock AH, McMurray RJ (1988): Varley’s Practical ClinicalBiochemistry. New Dehli, CBS Publishers and Distributors,pp. 149–153.

Grover JK, Yadav S, Vats V (2002): Medicinal plants of India withanti-diabetic potential. J Ethnopharmacol 81: 81–100.

Kar A, Choudhary BK, Bandyopadhyay NG (2003): Comparativeevaluation of hypoglycaemic activity of some Indian medic-inal plants in alloxan diabetic rats. J Ethnopharmacol 84:105–108.

Khandelwal KR (2005): Practical Pharmacognosy. Pune, NiraliPrakashan, pp. 152–154.

Krishnamurthi K, Dutta D, Devi SS, Chakrabarti T (2004): Protec-tive effect of distillate and redistillate of cow’s urine in humanpolymorphonuclear leukocytes challenged with establishedgenotoxic chemicals. Biomed Environ Sci 17: 57–66.

Miura T, Itoh C, Iwamoto N, Aato M, Kawai M, Park SR, Suziki I(2001): Hypoglycemic activity of the fruit of the Momordicacharantia 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 an-tidiabetic activity—A review of ten years of herbal medicineresearch (1990–2000): Int J Diabetes Metab 14: 1–25.

Mukherjee KP (2002): Quality Control of Herbal Drugs.New Delhi, New Business Horrizons, p. 525.

Nyholm B, Porksen N, Juhl CB, Gravholt CH, Butler PC, WeekeJ, Veldhuis JD, Pincus S, Schmitz O (2000): Assessmentof insulin secretion in relatives of patients with type 2 (non-insulin-dependent) diabetes mellitus: evidence of early β-celldysfunction. Metabolism 49: 896–905.

Okinea LKN, Nyarkob AK, Osei-Kwabenaa N, Oppongc IV,Barnesa F, Ofosuheneb M (2005): The antidiabetic activityof the herbal preparation ADD-199 in mice: A comparativestudy with two oral hypoglycaemic drugs. J Ethnopharmacol97: 31–38.

Pathak ML, Kumar A (2003a): Cow praising and importanceof Panchyagawya as medicine. Sachitra Ayurveda 5: 56–59.

Pathak ML, Kumar A (2003b): Gomutra a descriptive study. Sa-chitra Ayurveda 7: 81–84.

Porchezian E, Ansari SH, Shreedharan NKK (2000): Antihyper-glycemic activity of Euphrasia officinale leaves. Fitoterapia71: 522–526.

Pulok KM, Kuntal M, Kakali M, Peter JH (2006): Leadsfrom 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 of an antidia-betic extract of Catharanthus roseus on enzymic activities instreptozotocin induced diabetic rats. J Ethnopharmacol 76:269–277.

Yadav S, Vats V, Dhunnoo Y, Grover JK (2002): Hypoglycemicand antihyperglycemic activity of Murraya koenigii leaves indiabetic rats. J Ethnopharmacol 82: 111–116.

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Global Journal of Pharmacology 4 (1): 41-44, 2010ISSN 1992-0075© IDOSI Publications, 2010

Corresponding Author: Arunkumar Sathasivam, Muthaiyah Research Foundation, Thanjavur, Tamilnadu, India-613 005.Mob: 09486131235, Email: [email protected]

41

Antimicrobial Activities of Cow Urine Distillate Against Some Clinical Pathogens

Arunkumar Sathasivam, M. Muthuselvam and Rajasekran Rajendran1 2 1

Muthaiyah Research Foundation, Thanjavur, Tamilnadu, India - 613 0051

Department of Microbiology, Marudupandiyar College, Thanjavur, Tamilnadu, India2

Abstract: From the ancient period cow’s urine has been used as a medicine. In India, drinking of cow urine hasbeen practiced for thousands of years. Panchagavya is a term used in Ayurveda to describe five importantsubstances obtained from cow namely Urine, Dung, Milk, Ghee and Curd. The present study analyzes theantibacterial and antifungal activity of Cow Urine Distillate against the clinical pathogenic microorganisms.Antibacterial activity of Cow Urine Distillate (5, 10 and 15µl) was analyzed against the Bacillus subtilis,Pseudomonas aeruginosa, Klebsiella pneumoniae and Salmonella typhi. Maximum antibacterial activity wasobserved in Pseudomonas aeruginosa (7.06±0.05, 8.08±0.18 and 10.4±1.23, mm in diameter, respectively) andSalmonella typhi (6.3±1.23, 8.06±0.17 and 10.4±1.2, mm in diameter, respectively). Antifungal activity of cowurine distillate was analysed against Aspergillus niger and Aspergillus flavus. When the two fungal organismswere 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 inhibitoryactivity can be used in the control of bacteria and fungi of various origins.

Key words: Cow Urine Distillate Antibacterial and Antifungal Activity

INTRODUCTION treatment of falling body parts, discharging lymphs and

In Veda, cow’s urine was compared to the nectar. In some other ingredients) has been recommended for bath,substrata, several medicinal properties of cow’s urine anointing and intake [2]. The cow urine distillate has beenhave been mentioned and are known to cause weight loss, patented as activity enhancer and availability facilitatorreversal of certain cardiac and kidney problems, for bioactive molecules including anti- infective andindigestion, 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 of cow urine in herbal insecticides [4]. Feeding ofmost effective substance secretion of animal origin with cow urine increased the feed intake in white legborninnumerable therapeutic values. It has been recognized as layers [5]. The present study was carried out to preparewater of life or “Amrita” (Beverages of immortality), the cow urine distillate and to determine the antibacterial andnectar of the God. In India, drinking of cow urine has been antifungal activities.practiced for thousands of years. Panchagavya is a termused in Ayurveda to describe five important substances MATERIALS AND METHODSobtained from cow namely Urine, Dung, Milk, Ghee andCurd. A number of formulations mentioned in Ayurveda Collection of Sample: Cow urine sample was collecteddescribe the use of Panchagavya components either alone from cow farm (at Avanam, Thanjavur Dt) using sterileor in combination with drugs of herbal, animal or mineral container and stored for further uses.origin [1].

An exhaustive reference of cow’s urine having CUD Preparation: Cow urine was distilled at 100°C usingcurative properties in skin diseases, especially leprosy, distillation apparatus [6]. The single distilled cow urineis referred to in Caraka samhita. Furthermore, in the was acidified by lowering the pH below 2.0 with the

organism infested organs, use of cow’s urine (along with

Global J. Pharmacol., 4 (1): 41-44, 2010

42

addition of 85% orthophosphoric acid. The cow urine was plates with equal distance positive control disc wasagain distilled at 100°C using a distillation apparatus to also maintain. All the bacterial plates were incubated atremove 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 of the minimum zone of

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 werepneumoniae (MTCC 7407), Salmonella typhi, Aspergillus calculated to facilitate the comparison of the data [8].niger and Aspergillus flavus were collected form microbialtype culture collection centre (MTCC) at Chandigarh. RESULTS AND DISCUSSION

Disc Preparation: 5 mm (diameter) discs were prepared Antibacterial Activity: Antibacterial activity of cow urinefrom 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 andthe moisture discs were dried on hot air oven at 50°C. Salmonella typhi (Table 1, Fig. 1 and Plate 1). 5, 10 andThe sterile discs were rinsed in cow urine distillate at 15µl concentrations of cow urine distillate discs weredifferent concentration (5, 10, 15µl). taken for the study. Among the three concentrations

Antibacterial and Antifungal Activity of Cow Urine concentration when compared with 5 and 10µl. MaximumDistillate: The antimicrobial and antifungal activity antibacterial activity was observed in Pseudomonasstudies were carried out by disc diffusion technique [7]. aeruginosa (12.6±0.05, 13.8±0.18 and 15.4±1.23, mm inThe 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 theAspergillus niger and Aspergillus flavus were standard antibiotic (Ampicillin). US patent was obtainedspreaded over the Mueller Hinton ager plates by by CSIR (Counsil for Scientific Industrial Research) Indiausing separate sterile cotton swabs. After the which claimed a novel pharmaceutical compositionspreading the different concentrated cow urine distillate present in cow urine distillate and is effective as andiscs were placed separately on the organism inoculated antifungal and antibacterial [6].

inhibition was measured in mm. For each test, three

highest antibacterial activity was noted in 15µl

Table 1: Antibacterial activity of cow urine distillateZone of inhibition (mm in diameter) (M±SD) (n=3)-----------------------------------------------------------------------------------------------------------------------------------------Concentration of 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±0.012. Pseudomonas aeruginosa 12.6±0.04 13.6±0.17 15.4±1.23 11.2±0.013. Klebsiella pneumoniae 7.3±0.25 7.3±0.25 11±0.14 9.5±0.054. Salmonella typhi 12±1.23 13.6±0.17 15.4±1.23 9.6±0.02Values are triplicate mean ± Standard deviationAmp* - Standard antibiotic disc Ampicillin (30mg/disc)

Table 2: Antifungal activity of cow urine distillateZone of inhibition (mm in diameter) (M±SD) (n=3)-------------------------------------------------------------------------------------------------------------------------------------------Concentration of CUD (µl)-------------------------------------------------------------------------------------------------------------------------------------------

S.NO. Fungi 5 10 151. Aspergillus niger 8±0.14 11.3±1.2 12.6±0.042. Aspergillus flavus 7.3±0.25 10±0.25 11±1.2Values are triplicate mean ± Standard deviation

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Fig. 1: Antibacterial activity of cow urine distillate

Fig. 2: Antifungal activity of cow urine distillate

Plate 1: Antibacterial activity of CUD against Pathogenic bacteria

Plate 2= Antifungal activity of CUD against Pathogenic fungi


44

Antifungal Activity: Antifungal activity of cow urine REFERENCESdistillate was analysed against Aspergillus niger andAspergillus flavus. The investigated results were 1. Shah, E., 1997. Herbal composition in cow urinepresented 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 of medicine in ancientsuppression 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 Leshmaniaresult 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 Bulletinwas taken into consideration and antifungal effect was 5. Asian agri-History Foundation, Secunderabad-evaluated. 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 of indigenous cow urine on nutrient utilization

It was concluded that the cow urine distillate has of white leghorn layers. International J. Cow Sci.,antibacterial and antifungal activities the inhibitory 1: 36-38.activity can be used in the control of bacteria and fungi 6. Khanuja, S.P.S., 2002. Pharmaceutical compositionof 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 andsectors. Further studies analyze which components are M. Turck, 1966. Antibiotic susceptibility testing byresponsible for antimicrobial activity and animal model standard single disc diffusion method. Americancould reveal antibacterial and antifungal activity of cow J. Clinical Pathol., 45: 493-496.urine distillate in vivo. 8. Salil Bose, 1982. Biostatistics in Elementary

ACKNOWLEDGMENTS 9. Prashith Kekuda, T.R., R. Kavya, R.M. Shrungashree

The authors are thankful to Muthaiyah Research urine distillate. (http://www.microbiocare.com/).Foundation, Thanjavur for offering facilities to carry outthis study.

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

and S.V. Suchitra, 2007. Antifungal activity of cow

Journal of Intercultural Ethnopharmacology

DOI: 10.5455/jice.2015022210032www.jicep.com

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

INTRODUCTION

Infectious diseases remain a major threat to the public health despite tremendous progress in human medicine. Emergence of widespread drug resistance to the currently available antimicrobials is a matter of deep concern. A high percentage of nosocomial infections are caused by highly resistant bacteria such as methicillin-resistant Staphylococcus aureus or multidrug-resistant (MDR) Gram-negative bacteria. Each year in the United States, about 2 million people become infected with antibiotic resistant bacteria and at least 23,000 people die every year as a consequence of these infections. Many more people die from other conditions that are complicated by an antibiotic-resistant infection [1]. In 2012, there were about 450000 new cases of MDR tuberculosis. Extensively drug-resistant tuberculosis has been identified in 92 countries. Development of resistance to oral drug of choice fluoroquinolones, for urinary tract infections caused by Escherichia coli is very widespread, often sensitivity remains only for injectables [2]. Infections caused by resistant microorganisms often fail to respond to the standard treatment, resulting in prolonged illness, higher health care expenditures, and a greater risk of death. There is a dire need for the development of new antimicrobial agents with sensitivity intact against microorganisms [3,4]. The rational designing of novel drugs from traditional medicines to treat

these difficult to treat infections offers a new prospect for the modern health-care system.

Ayurvedic texts (Sushruta Samhita, Ashtanga Sangrah and Bhav Prakash Nighantu) describe cow urine (CU) (gomutra) as an effective medicinal substance/secretion of animal origin with innumerable therapeutic uses. Cow (Kamadhenu) has been considered as a sacred animal in India. In Rigveda (10/15), CU is compared to nectar. In Susruta (45/221) and in Charak (sloka-100) several medicinal properties of CU have been mentioned such as weight loss, reversal of certain cardiac and renal diseases, indigestion, stomach ache, diarrhea, edema, jaundice, anemia, hemorrhoids and skin diseases including vitiligo. Gomutra is capable of removing all the imbalances in the body, thus maintaining the general health [5]. CU contains 95% water, 2.5% urea, minerals, 24 types of salts, hormones, and 2.5% enzymes. It also contains iron, calcium, phosphorus, carbonic acid, potash, nitrogen, ammonia, manganese, iron, sulfur, phosphates, potassium, urea, uric acid, amino acids, enzymes, cytokine and lactose [6].

CU is an effective antibacterial agent against a broad spectrum of Gram-negative and Gram-positive bacteria and also against some drug-resistant bacteria. It acts as a bio-enhancer of some antimicrobial drugs. It has antifungal, anthelmintic, antineoplastic action, is useful in hypersensitivity reactions and

Chemotherapeutic potential of cow urine: A reviewGurpreet Kaur Randhawa1, Rajiv Sharma2

1Department of Pharmacology, Government Medical College, Amritsar, Punjab, India, 2Department of Medicine, Guru Nanak Dev Hospital, attached to Government Medical College, Amritsar, Punjab, India

Address for correspondence:Address for correspondence:Gurpreet Kaur Randhawa, Department of Pharmacology, Government Medical College, Amritsar, Punjab, India. E-mail: [email protected]

Received: Received: January 16, 2015

Accepted: Accepted: February 22, 2015

Published: Published: March 07, 2015

ABSTRACTIn the grim scenario where presently about 70% of pathogenic bacteria are resistant to at least one of the drugs for the treatment, cue is to be taken from traditional/indigenous medicine to tackle it urgently. The Indian traditional knowledge emanates from ayurveda, where Bos indicus is placed at a high pedestal for numerous uses of its various products. Urine is one of the products of a cow with many benefits and without toxicity.Various studies have found good antimicrobial activity of cow’s urine (CU) comparable with standard drugs such as ofloxacin, cefpodoxime, and gentamycin, against a vast number of pathogenic bacteria, more so against Gram-positive than negative bacteria. Interestingly antimicrobial activity has also been found against some resistant strains such as multidrug-resistant (MDR) Escherichia coli and Klebsiella pneumoniae. Antimicrobial action is enhanced still further by it being an immune-enhancer and bioenhancer of some antibiotic drugs. Antifungal activity was comparable to amphotericin B. CU also has anthelmintic and antineoplastic action. CU has, in addition, antioxidant properties, and it can prevent the damage to DNA caused by the environmental stress. In the management of infectious diseases, CU can be used alone or as an adjunctive to prevent the development of resistance and enhance the effect of standard antibiotics.

KEY WORDS:Antibiotic, antifungal, antineoplastic, bioenhancer, Bos indicus, immune-enhancer

Review Article

Randhawa and Sharma: Chemotherapeutic potential of cow urine

J Intercult Ethnopharmacol ● Apr-Jun 2015 ● Vol 4 ● Issue 2 181

in numerous other diseases including increasing the life-span of a person. Recent researches have shown that CU is an immune-enhancer also [7-9]. Therapeutic properties of CU have been validated by modern science also.

MECHANISM OF ACTION OF CU

Different fractions of CU possess antimicrobial activity due to the presence of certain components like volatile and nonvolatile ones [10-13]. Presence of urea, creatinine, swarn kshar (aurum hydroxide), carbolic acid, phenols, calcium, and manganese has strongly explained the antimicrobial and germicidal properties of CU [14-16]. Presence of amino acids and urinary peptides may enhance the bactericidal effect [17] by increasing the bacterial cell surface hydrophobicity. CU enhances the phagocytic activity of macrophages. Higher amounts of phenols in fresh CU than CU distillate (CUD) makes it more effective against microbes.

After photo-activation, few biogenic volatile inorganic and organic compounds such as CO2, NH3, CH4, methanol, propanol and acetone, and some metabolic secondary nitrogenous products are also formed [18]. Photo-activated CU (PhCU) becomes highly acidic in comparison to fresh CU. An increase in bactericidal action may be due to a significant decrease in pH [12], presence of inorganic phosphorus, chloride and dimethylamine may also play an important role [19], along with increased formation of some reactive compounds like formaldehyde, sulfinol, ketones and some amines during photo-activation and long term storage [20]. CU prevents the development of antibacterial resistance by blocking the R-factor, a part of plasmid genome of bacteria [21].

CU contains phenolic acids (gallic, caffeic, ferulic, o-coumaric, cinnamic, and salicylic acids) which have antifungal characteristics [22].

Antioxidant property of uric acid and allantoin present in CU correlates with its anticancer effect. CU reduces apoptosis in lymphocytes and helps them to survive better [5]. This action may be due to the free radical scavenging activity of the urine components, and these components may prevent the process of aging [10]. It efficiently repairs the damaged DNA [5].

Daily consumption of CU improves immunity due to the presence of swarn kshar and fastens the wound healing process, which is due to allantoin [8]. CU enhances the immunocompetence by facilitating the synthesis of interleukin-1 and -2 [23,24], augments B - and T- lymphocyte blastogenesis, and IgA, IgM and IgG antibody titers [25].

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 of 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 aureofaciens, Lactobacillus acidophilus and Bacillus subtilis, and Leishmania donovani has been observed in various studies. In these studies the antimicrobial activity of CU was found to be comparable with ofloxacin, ciprofloxacin, ampicillin, chloramphenicol, nalidixic acid, rifampicin, tetracycline, streptomycin, cefpodoxime and gentamycin in different studies [27-36].

Studies with Indigenous Bos indicus Breeds of Cow

Fresh CU (FCU), Sterile, PhCU and CUD from a healthy Geer cow, was used to assess the antibacterial effect against different strains of bacteria. Against E. coli, FCU had the bigger mean of inhibition zone (15 mm) than Sterile, PhCU, and CUD (~10 mm). FCU had good activity of 15, 16 and 20 mm of inhibition against E. coli, B. subtilis, and S. typhi, respectively. Other forms of CU showed activity against E. coli, S. typhi, P. vulgaris, S. aureus and B. subtilis [27].

Rana and De [28] observed a greater activity against Gram-positive than Gram-negative bacteria with CU obtained from Geer cow. The minimum inhibitory concentration (MIC) in all the four tested Gram-positive bacteria was 134 mg/ml. Among Gram-negative organisms, P. aeruginosa was more sensitive (MIC 134 mg/ml) than P. vulgaris (MIC 268 mg/ml). Mean zone of inhibition (mm)± standard error of the mean against B. subtilis was found to be 18.67±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 of cefpodoxime (19) but less than with gentamycin (26). Activity (16) against S. aureus and S. epidermidis was <25 for Gentamycin and ~23 with Cefpodoxime. No inhibition against P. aeruginosa was observed with Cefpodoxime while CU had an inhibition of 19.33 ± 1.15 mm and Gentamycin 35 mm. Against P. vulgaris inhibition was comparable between CU (16 ± 1.73), gentamycin (21) and cefpodoxime (20). There was comparable inhibition of P. vulagris by CU (16 ± 1.73), gentamycin (21) and cefpodoxime (20). Against K. pneumoniae, inhibition observed with CU (15.67 ± 0.57) was less than gentamycin (34) and cefpodoxime (20).

Comparatively FCU obtained from Gujarati Geer cow was found to have more antimicrobial activity than its distillate (Table 1).

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

The antibacterial efficacy (as mean zone of inhibition in mm) of CU Concentrate (CUC) obtained from Karnataka breed, Amrit



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 of PhCU of Hariana breed was found to be comparable in efficacy to Tetracycline (30 μg mL). Antimicrobial activity (mean zone of inhibition in mm) of PhCU and Tetracycline, respectively against B. cereus was 17 and 22, S. aureus was 18 and 21, S. typhimurium was 21 and 22, A. hydrophila was 22 and 24, E. aerugenes was 13 and 18 and M. luteus was 15 and 17 [31]. Similar results were found in another study with PhCU of Hariana breed against these bacteria [32].

Studies where breed of cow is not mentioned

In an in-vitro test, activity of FCU was comparable to Streptomycin. Similar mean zone of inhibition (mm) was seen against gram positive organisms E. coli (16:16:13), K. pneumonia (15:17:12) and P. aeruginosa (17:19:15) with FCU and Streptomycin and lesser with PhCU (by keeping urine in sunlight in sealed glass bottles for 72 h), respectively. Comparatively lesser antibacterial activity against gram negative organisms S. aureus (18:26:17), coagulase negative Staphylococci (18:29:15), B. subtilis (20:29:15), and S. pyogenes (20:26:14) was seen for FCU than streptomycin, and still lesser than with PhCU [33]. No antibacterial activity was seen for CUD, which is contradictory to some previous reports [34].

Vats et al. [35] studied the synergistic antimicrobial effect of PhCU and herbs against bacterial and fungal strains. PhCU and Azadirachta indica combination showed a remarkable synergistic antimicrobial activity against Candida tropicalis, Candida glabrata, P. aeruginosa, and S. aureofaciens. PhCU and Terminalia chebula showed maximum activity against S. auereofaciens (45 mm), and P. aeruginosa (zone of inhibition of 40 mm). Piper nigrum, T. chebula and PhCU in combination were most effective against C. glabrata (35 mm) and C. tropicalis (45) mm.

Upadhyay et al. [18] found in in-vitro tests that PhCU has better bactericidal activity against S. aureus, B. cereus, L. acidophilus, M. luteus, K. pneumonia, S. pneumonia and E. coli, when compared with Tetracycline, Ampicillin and Ciprofloxacin. PhCU showed MIC value of 0.25 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 of 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 of CUD (5, 10 and 15 μl) against the B. subtilis, P. aeruginosa, K. pneumoniae and S. typhi. Antibacterial activity (mean zone of inhibition in mm) was observed against B. subtilis (7.6 ± 0.04, 8.6 ± 0.17, 8.8 ± 0.17, respectively) P. aeruginosa (12.6 ± 0.04, 13.6 ± 0.17, 15.4 ± 1.23, respectively) and S. typhi (12 ± 1.23, 13.6 ± 0.17, 15.4 ± 1.23, respectively). This antibacterial activity was more than the positive control of ampicillin (30 mg/disc), which was 7.1 ± 0.01 mm against B. subtilis, 11.2 ± 0.01 mm against P. aeruginosa and 9.6 ± 0.02 mm of inhibition zone against S. typhi. Antibacterial activity against K. pneumonia was 11 ± 0.14 mm with 15 μl of CUD, which was more than the activity (9.5 ± 0.05 mm) with Ampicillin [34].

Yadav et al. reported the antimicrobial property of a herbal formulation containing CU, Dalbergia sissoo, and Datura stramonium. The antimicrobial activity of CU alone was also found to be significant (P > 0.001). It was found that CU extract showed the highest inhibition in gram-positive S. aureus (CI, 213%) and comparable activity in S. pneumoiae (95%) compared to chloramphenicol (30 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 of existing antibiotics in human and veterinary medicine, and also in agriculture, threat from antimicrobial resistance is increasing. Resistant strains like Penicillin- and Methicillin- resistant S. aureus, vancomycin resistant Enterococcus, and ciprofloxacin resistance P. aeruginosa are an ever increasing global threat. After photoactivation and purification, CU has been found to be effective against certain drug resistant bacterial strains [38]. CU extract of A. indica showed better MIC values than the organic fractions for MDR E. coli (12.68 mm) and K. pneumonia (9 mm). CU extracts of A. indica showed >8.66 mm zone of inhibition for MDR S. aureus, P. aeruginosa and P. vulgaris [39].

Table 1: Antimicrobial activity of 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 24CUD 20 20 18 20 20 21Ofloxacin 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



FUNGICIDE AND BIOFUNGICIDE

Fungicidal effect against Aspergillus fumigatus, Aspergillus flavus, Aspergillus niger, Aspergillus, Malassezia, C. tropicalis and C. glabrata has been observed in various studies. CU was highly stable and capable in inhibiting the growth of Malassezia fungi (90-95%) responsible for causing dandruff for a longer time (4-5 days), than rice water (due to B. cereus growth in rice water) which was stably capable of inhibiting 85-90% of the growth for 3-4 days. Neem leaves extract and Lemon Juice extract were comparatively less effective in this study [40].

15% CU was most active against Aspergillus, Rhizophus and the percentage of inhibition obtained with it was 85% [41]. 5% CUC showed maximum antifungal activity against A. niger (93%), followed by A. oryzae (92.67%) and A. flavus (83%)[30]. CUD showed better antifungal activity against A. fumigatus and C. albicans with mean zone of inhibition of 13 and 11 mm than PhCU [27]. More fungal growth suppression (as mm in diameter) was observed with CUD in A. niger (8 ± 0.14, 11.3 ± 1.2 and 12.6 ± 0.04, respectively) than A. flavus (7.3 ± 0.25, 10 ± 0.26 and 11 ± 1.2, respectively) with the use of 5, 10 and 15 μl of CUD [34].

In vitro antifungal activity (in mm) of Geer CU against A. flavus (17.33 ± 0.57) was in between 50 μg of amphotericin B (15) and 10 μg of clotrimazole (24) and against C. albicans, activity was similar with CU (18.67 ± 1.15) and amphotericin B (19), but less than clotrimazole (30) [28]. Antifungal activity of Geer CU is better than the others where source of CU is not mentioned.

In an in vitro study, it was found that the urine samples of outdoor feeding cow (OCU) was more effective and inhibited growth of fungi more strongly as compared to indoor feeding CU (ICU). This inhibition was concentration dependent. No growth of Penicillium notatum, Trichoderma viridae, and Alternaria solanii was observed with 10% OCU and with 20% ICU and that of Claviceps purpurea, Rhizopus oligosporius, C. albicans and A. candidus, no growth was observed with 20% of OCU only [42].

ANTISEPTIC

Sanganal et al. observed the enhanced wound healing activity of CU in Wistar albino rats [43]. On 4th day, the external application of CU showed significant and progressive increase in wound healing in rats compared to different concentrations of CU and 1% w/w nitrofurazone ointment locally. Similar findings were also observed by Maheshwary et al. [44].

ANTHELMINTIC ACTIVITY

CUC was found to be more effective than piperazine citrate as anthelmintic agent at both 1% and 5% concentrations. For anthelmintic activity, adult Indian earthworm Pheretima posthuma was studied due to its anatomical and physiological resemblance with the intestinal roundworm parasite of human

beings. Paralysis of earthworm occurred in 53 and 48 min with 1% piperazine and CUC, respectively and 16 and 13 min with 5% piperazine and CUC, respectively. Time taken for the death of earthworms decreased from 72 min with 1% piperazine to 60 min with 1% CUC, respectively. It further decreased from 28 min with 5% piperazine to 18 min with 5% CUC, respectively [30].

Different compositions of Panchgavya (five products of cow namely milk, curd, ghee, urine and dung) alone and combination of Panchgavya and ethanolic extract of Bauhinia variegata Linn (10%, 50%, 75% in Panchgavya) were found to have excellent anthelmintic activity against adult Indian earthworm (P. posthuma) when we compared to control Piperazine (50 and 100 mg/ml). In combination, the anthelmintic activity was synergistic and with increasing doses, time (in minutes) of onset of paralysis and death in earthworm decreased [45].

BIOENHANCER

A ‘bioenhancer’/‘biopotentiator’ is an agent capable of enhancing the bioavailability and efficacy of a drug with which it is co-administered, without any pharmacological activity of its own at the therapeutic dose used. In ayurveda, this concept is known as ‘yogvahi’ and is used to increase the effect of medicines by increasing the oral bioavailability (especially of medicines with poor oral bioavailability), decreasing their dose and adverse effects, and were used to circumvent the parentral routes of drug administration. We can develop more such useful and economically viable drug combinations, by integrating the knowledge of time tested ayurveda with modern methods of research [8]. CU is the only agent of animal origin which acts as bioenhancer of antimicrobial, antifungal, and anticancer agents [30]. The indigenous CU contains ‘Rasayana’ tatva, which is responsible for modulation of the immune system and also act as a bioenhancer [21].

CUD is more effective bioenhancer than CU [30,46]. CUD enhances the transport of antibiotics like rifampicin, tetracycline and ampicillin across the gut wall by 2-7 folds [47]. It also enhances the potency of taxol against MCF-7 cell lines [48]. It enhances the bioavailability of rifampicin by 80 fold in 0.05 microgm/ml concentrations, ampicillin by 11.6 fold in 0.05 μ g/ml concentrations and clotrimazole by 5 fold in 0.88 μ g/ml concentration [49]. The activity of rifampicin increases by about 5-7 folds against E. coli and 3-11 folds against Gram-positive bacteria, when used along with CU [50]. Potency of paclitaxel has been observed to increase against MCF-7, a human breast cancer cell line in in-vitro assays [49]. The bioenhancing ability is by facilitating the absorption of drugs across the cell membrane. The CU has been granted US Patents for its medicinal properties, particularly as a bioenhancer along with antibiotics, antifungal and anticancer drugs (6896907, 6410059).

CUD alone caused more inhibition of Gram-positive bacteria. Inhibition caused by streptomycin (1 mg/ml) alone was higher (31-34 mm) than that of CUD alone (19-22 mm). With the



combination of Pinguicula longifolia, CUD and streptomycin together, S. aureus was inhibited to a more extent (38 mm) followed by P. aeruginosa (37 mm) and an equal inhibition was exhibited by B. subtilis and E. coli (36 mm) [51]. S. aureus and P. aeuroginosa have been recognized as most common bacteria, which have developed resistance against several antibiotics and is a major hospital borne pathogen, which is particularly dangerous to immunocompromised patients. This study is of importance in this scenario.

This bioenhancing activity of CU has been aptly and widely used in various ayurvedic formulations. It is one of the constituents of Hingwadh ghrita, Lashunadh ghrita, Sidhartak ghrita for psychiatric illness used in abdominal tumors and in other formulations like Mandurvatak, Darvi ghrita, and Punnarvamandur. CU is used as yogvahi along with Hareetakyadi yog, Swarnkshiryad yog, Swarnmakshik bhasma and Gvakshyadi churana. These preparations are commercially available in the Indian market. Ghritas are available as semisolid preparations while bhasms, yogs, and churans are in the powder form.

ANTICANCER PROPERTIES

CU has antioxidant properties and is a free radical scavenger, and thus it neutralizes the oxidative stress. Scientists proved that the pesticides even at very low doses cause apoptosis of lymphocytes and tissues through fragmentation of DNA while CU helps the lymphocytes to survive by inhibiting their apoptosis and by repairing the damaged DNA and is, therefore, effective as anti-cancer therapy [52,53].

Chemopreventive potential of CU was observed in a study, which was conducted on 70 Swiss albino mice for 16 weeks. Papillomas were induced by 7, 12 dimethyl benzanthracene and later promoted by repeated application of croton oil. In mice treated with CU, the incidence of tumor (papillomas), tumor yield, and its burden was statistically less than the untreated group [54].

Jain et al. studied the effect of CU therapy on various types of cancers in Mandsaur area. The severity of symptoms (pain, inflammation, burning sensation, difficulty in swallowing, and irritation) decreased from day 1 to day 8 with CU therapy. Percent of patients with severe symptoms decreased from 82.16 to 7.9 on day 8, patients with moderate symptoms increased from 15.8 to 55.3 and with mild symptoms, patients increased from 1.58 to 36.34. The severity of symptoms decreased further with continued CU therapy [15].

Dutta et al. reported the anti-clastogenic and anti-genotoxic effect of redistilled CUD (RCUD) in human peripheral lymphocytes, which have been challenged with manganese dioxide (MnO2) and hexavalent chromium (Cr+6). Protection in number of chromosomal aberrations and frequency of micronuclei were more prominent when these cells were pretreated with CU than simultaneous treatment with CU [55].

IMMUNOSTIMULANT

The use of herbs and minerals (like chavanprash and panchgavya) for improving the overall resistance of the body against common infections and pathogens has been a guiding principal of Ayurveda. Ancient books on Ayurveda state that consuming CU daily increases the resistance to diseases by up to 104%. CU enhances the humoral, and cell-mediated immune response in mice [5]. CUD was found to augment B- and T-lymphocyte blastogenesis; IgG, IgA and IgM antibody titers in mice. It has been observed that CU also increases the phagocytic activity of macrophages and is thus helpful in the prevention and control of bacterial infections. The level of both interleukins -1 and - 2 in mice was increased by 30.9% and 11.0%, respectively, and in rats these levels were increased significantly by 14.75% and 33.6%, respectively [52]. CUD has been reported to be a potent and safe immunomodulator, which increases both humoral, and cell-mediated immunity in mice.

Cell-mediated immune response was evaluated on various parameters in a study by Verma et al. using CU for 30 days. There was a 55% increase in phagocytic index, and a significant increase (16%) in neutrophil adhesion on regular use of whole freeze dried CU. CU has the potential to boost the immune functions by increasing the white blood cells counts and subsequently reducing the red blood cells count to a certain extent [56].

Traditional uses of CU

Some of the traditional uses of CU are in fever, where CU along with pepper, curd and ghee is used; in leprosy, CU is used along with dhruhardi and in deformities associated with leprosy, it is used with Nimbuchal, whereas in chronic leprosy, CU is used along with leaves of Vasaka and kanar, and bark of kuraila and neem [11]. Local traditional healers in Mandsaur prescribe CU for worm infestations, to develop immunity and to avoid aging. They suggest 10-25 ml of CU to be taken on an empty stomach for the same [15].

CONCLUSIONS

On analyzing the effect of different preparations of CU, FCU had better activity than CUD [27-32]. Activity of FCU and CUD from indigenous cows was similar to streptomycin and tetracycline. Ayurveda also mentions that FCU of indigenous cows’ is the best.

More well-planned studies in human subjects are required to fully assess its potential as an effective antimicrobial agent as most of the studies quoted are in vitro studies. Comparative studies between CU obtained from indigenous breeds and of inbred strains may be undertaken, as ayurveda was written when inbred strains of cows were not present. Future development of newer drugs can involve CU in its repository.



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© SAGEYA. This is an open access article licensed under the terms of the Creative Commons Attribution Non-Commercial License (http://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 of Support: Nil, Confl ict of Interest: None declared.

Effect of treatment of cow’s urine ‘‘Gomutra’’ and antioxidantsin alleviating the lindane-induced oxidative stress in kidneyof Swiss mice (Mus musculus)

Girima Nagda • Devendra Kumar Bhatt

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

� Springer Science+Business Media Dordrecht 2014

Abstract The study aimed to evaluate the effect of cow

urine and combination of antioxidants against lindane

induced oxidative stress in Swiss mice. Male healthy mice,

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 adminis-

tered 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 con-

tinuously for 60 days. Lindane treated group showed

increased lipid peroxidation, whereas glutathione, glutathi-

one peroxidase, superoxide dismutase, catalase, protein and

endogenous levels of vitamin C and E were significantly

decreased compared to control. Administration of cow urine

and antioxidants alleviated the levels of these biochemical

parameters.

Keywords Antioxidants � Cow urine � Kidney � Lindane �Oxidative stress

Introduction

Kidney is vital organ of the body responsible for segre-

gating the metabolic waste products from the blood.

Accumulation of metabolites of xenobiotics contributes

significantly to its susceptibility to damage kidney [24].

Any nephrotoxic insult would result in accumulation of

waste materials in the blood which in turn may lead to

other toxic manifestations in the body. Toxic injury to the

kidney is known to occur as a result of exposures to hal-

ogenated hydrocarbons, such as lindane, carbon tetrachlo-

ride and trichloroethylene, and the heavy metals cadmium

and lead [3, 35, 36, 48, 59]. Some of these toxicants cause

acute injury to the kidney, while others produce chronic

changes that can lead to end-stage renal failure or cancer.

Lindane, the gamma isomer of HCH possesses the prop-

erty of persistence, bioaccumulation and long term toxicity

[32] and fulfills the criteria of POPs i.e., persistent organo-

chlorine pesticides. India has total installed capacity of lin-

dane (technical) production of 1,300 tonnes per annum (tpa),

with two companies producing: Kanoria Chemicals and

Industries Ltd with a capacity of 1,000 tpa, and India Pes-

ticides Limited with 300 tpa capacity. According to data

available from Department of Chemicals and Petrochemi-

cals, Ministry of Chemicals and Fertilizers, between 1995

and 2005, India has produced 5,387 tonnes of technical grade

lindane. In India, lindane formulations are registered for use

in pharmaceutical products for control of head lice and

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

bed bug, and beetle populations and for the control of pests in

cotton, sugarcane, pumpkin, cabbage, onion, apple, walnut,

maize, okhra, potato, tomato, cauliflower, radish, cucumber

and beans [15]. Lindane is highly lipophilic and absorbed by

the respiratory, digestive or cutaneous pathways. Its accu-

mulation depends on the duration of the exposure and affect

G. Nagda (&) � D. K. Bhatt

Cancer Biology and Toxicology Research Laboratory,

Department of Zoology, University College of Science,

M L Sukhadia University, Udaipur 313 001, India

e-mail: [email protected]

123

Mol Biol Rep (2014) 41:1967–1976

DOI 10.1007/s11033-014-3044-6

tissues in the following order: fat [ brain [ kidney [muscle[ lung[heart[ spleen[ liver[blood[56].Toxicity

induced by lindane is attributed to oxidative stress as it

induces the release of free radicals and generation of reactive

oxygen species (ROS) [67].

Oxidative stress is defined as a disruption of the pro-

oxidant–antioxidant balance in favor of the former, leading

to potential damage [37]. It is a result of one of three

factors: (i) an increase in ROS, (ii) an impairment of

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

oxidative damage.

A number of studies indicate the toxicity of lindane on

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

important role in the pathogenesis of renal injury. Since the

entire range of toxic metabolites in the body is excreted

mainly from the kidney, this organ is endowed with sig-

nificant antioxidant defense system next only to liver. This

is understandable because ROS play a key intermediary

role in the pathophysiologic processes of a wide variety of

clinical and experimental renal diseases [50, 65].

The human body has a strong inherent synergistic and

multilevel defense mechanism, to combat and counteract

the damage caused by free radicals [41]. This defense is

mediated by endogenous antioxidant system which either

prevent these reactive species from being formed, or cause

their removal before they can damage vital components of

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

of antioxidant defense of body results into toxicity. In such

conditions, though the body tissue is endowed with enzy-

matic and non-enzymatic protective systems, but it seems

that the homeostasis of the body fails. In such situations,

the use of exogenous substances with antioxidative

potential becomes important [64].

Antioxidants have gained immense importance in recent

years. The potency of various antioxidants on different

organ systems has been investigated against lindane tox-

icity [8, 9, 11, 42, 63].

Cow urine or Gomutra is considered sacred in Hindu

mythology and from ancient times it has been used as a

medicine in India. The medicinal properties of 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 of

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 con-

tains those substances, which are present in the human

body and thus its consumption maintains the balance of

these substances and cures incurable diseases [49]. Cow

urine is also used along with herbs to treat various diseases

like fever, epilepsy, anemia, abdominal pain, constipation

etc. by the traditional healers [34].

There is a paucity of information regarding the role of

fresh cow urine and combination of vitamin E, vitamin C

and alpha lipoic acid against lindane toxicity in kidney.

Therefore, in the present study their role in alleviating the

oxidative stress induced by lindane intoxication in kidney

of mice has been investigated. Moreover, the use of cow

urine from ancient times has shown to be quite effective

but its efficiency against lindane induced toxicity and its

combined effect along with the various antioxidants is

hitherto unreported. Hence, present study was undertaken

to fill the lacuna in this regard.

Materials and methods

Chemicals

Lindane (c-HCH) was obtained from Sigma chemicals St.

Louis, Mo, USA (CAS No. 58-89-9 and purity 97 %). vita-

min E, vitamin C, a-lipoic acid, sodium azide, thiobarbituric

acid, dinitrophenylhydrazine (DNPH), 2, 2 dipyridyl, and

phenazine methosulphate were obtained from Himedia,

India. Dithiobisnitro benzene (DTNB), reduced glutathione

and bovine serum albumin were purchased from Sisco

Research Laboratories, Mumbai, India. All other chemicals

and solvents used were of analytical grade. Cow urine: Urine

of young cow was collected from local cowshed and stored in

an air tight bottle for further use.

Animals and treatment

Male Swiss mice, weighing 30 ± 5 g and 8–10 weeks old

were procured from Cadila Health Care Institute, Ahme-

dabad. Animals were maintained on sterilized rice husk

bedding in polypropylene cages and kept at a temperature

of about 23 ± 3 �C with 12 ± 1 h L:D cycle. Animals

were fed on standard pelletal diet (Pranav Agro, Baroda).

Food and water were ad libitum. Experimental protocol

was approved by the Institutional Animal Ethics Commit-

tee. Handling of animals was according to the guidelines of

1968 Mol Biol Rep (2014) 41:1967–1976

123

Committee for the Purpose of Control and Supervision of

Experiments on Animals (CPCSEA), Ministry of Envi-

ronment and Forests, Govt. of India.

Dose selection

Dose for lindane was selected after conducting pilot

experiments in our laboratory. LD50 for lindane was found

to be at 60 mg/kg body wt. considering this aspect, a dose

level which may show adverse effect on kidney was

selected as the dose for the present study. Duration of

treatment was based on occupational exposure of workers

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

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

wt. and duration of treatment was for 2 months i.e.,

60 days. There was no mortality in exposure group during

the study.

Doses for antioxidants were calculated keeping the

doses prescribed for humans and also in accordance with

the previous reports [8, 9, 42]. The combined dose of

antioxidants selected was 125 mg/kg body wt. which

included vitamin C 50 mg/kg body wt., vitamin E 50 mg/kg

body wt. and a-lipoic acid 25 mg/kg body wt. Cow urine

was administered at a dose equivalent to the corresponding

dose for human in ml/kg b.w. i.e., 0.25 ml/kg b.w.

Doses of lindane, vitamin E and lipoic acid were pre-

pared by dissolving in olive oil. Dose of 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 of

dose administration was chosen for all treatments. Mice

were divided into eight groups with minimum of 8–10

animals in each group.

In the group IV and VIII the antioxidants and cow

urine were administered 1 h prior to lindane administra-

tion. In group VI and VII cow urine was administered

10 min before the antioxidants administration. All the

treatments were given continuously for a period of

60 days.

Mice were sacrificed by cervical dislocation at the

end of the scheduled period of 60 days and 24 h after

the last dose treatment. Both the kidneys were blotted

free of blood, weighed and to maintain uniformity in

all groups the right kidney was used for biochemical

analysis. The right kidney (just to maintain uniformity

amongst animals of all groups) was washed with ice

cold physiological saline and a 10 % w/v homogenate

was prepared in 0.1 M phosphate buffer (pH 7.4). The

homogenate was centrifuged at 6,0009g for 10 min

to obtain the supernatant. Supernatant was diluted

five times and used for estimating the biochemical

parameters.

Biochemical analysis

The kidney tissue homogenate was used for the estimation

of lipid peroxidation (LPO) [68], superoxide dismutase

(SOD) [30], catalase (CAT) [16], glutathione peroxidase

(GPx) [54], glutathione (GSH) [40], protein [38], vitamin E

(a-tocopherol) [18] and vitamin C (ascorbic acid) [45].

Statistical evaluation

Values are mean ± SD and the results obtained were

analyzed using one way ANOVA. Inter group comparisons

were performed by using the least significance difference

(LSD) test. A probability value of P \ 0.05, 0.01 was

considered as statistically significant.

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 of 125 mg/kg b.w. 60 days

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

by lindane at 40 mg/kg b.w.

60 days

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

Mol Biol Rep (2014) 41:1967–1976 1969

123

Results

The changes in various biochemical parameters in different

groups have been presented in Graphs 1, 2, 3, 4, 5, 6, 7, 8.

Effect on LPO (Graph 1)

A significant increase (P \ 0.01) of 32.21 % was observed in

the LPO levels after lindane intoxication as compared to con-

trol. All the pretreatment groups showed a significant decline

(P\ 0.01) in the LPO levels. 29.21, 27.66, and 29.47 %

decline was observed in the A?L, U?L, and U?A?L groups

respectively as compared to lindane group. The increasing

order of the LPO levels in various groups was as follow:

L [C [ U?L [ A?L [ U?A?L [ U?A [ U [ A.

Graph. 1 Mean ± SD values of LPO (nmoL TBARS/g tissue) in

various groups (a 60 day assessment). a when compared to control, b

when compared to lindane, NS non significant, * significant

(P \ 0.05), ** highly significant (P \ 0.01)

Graph. 2 Mean ± SD values of SOD (50 % inhibition of NBT/min/

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

control, b when compared to lindane, NS non significant, * significant


Graph. 3 Mean ± SD values of CAT (lmol of H2O2 decomposed/

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

to control, b when compared to lindane, NS non significant,

* significant (P \ 0.05), ** highly significant (P \ 0.01)

Graph. 4 Mean ± SD values of GPx (mg of GSH consumed/min/

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



Graph. 5 Mean ± SD values of glutathione (GSH) (mg/gm tissue)

in various groups (a 60 day assessment). a compared to control,

b when compared to lindane, NS non significant, * significant


1970 Mol Biol Rep (2014) 41:1967–1976

123

Effect on SOD (Graph 2)

The SOD levels declined significantly (P \ 0.01) up to

45.96 % in lindane intoxicated mice as compared to

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

U?A?L showed a significant increase (P \ 0.01) of about

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

lindane group. The increasing levels of SOD in various

groups were as follow: L \ A?L \ U?L \ C \ U?A?

L \ A \ U?A \ U.

Effect on CAT (Graph 3)

Lindane induced kidney toxicity showed a significant

decline (P \ 0.01) of 32.12 % in the CAT activity as

compared to control. A significant increase (P \ 0.01) of

58.91 % in A?L, 32.85 % in U?L and 51.97 % in

U?A?L was observed when compared to lindane. The

cow urine alone group also showed a decrease of 9.82 % as

compared to lindane but the decrease was not significant.

The maximum % of increase was observed in the A?L

group. The increasing order of the enzyme level in all the

eight groups was as follow: L \ U?L \ C \ U?A?L \A?L \ U \ U?A \ A.

Effect on GPx (Graph 4)

As compared to control animals, the lindane intoxicated

animals showed a significant decrease (P \ 0.01) of

45.19 % in the GPx levels. All the pretreatment groups

showed promising results and brought about a significant

rise of 101.89, 146.37, and 215.30 % in the GPx levels in

the groups A?L, U?L and U?A?L, respectively. The

increasing order of GPx levels in various groups was as

followed: L \ C \ A?L \ U?L \ U?A?L \ A \ U \U?A.

Effect on GSH (Graph 5)

A significant decline (P \ 0.01) of 29.24 % was observed

in the GSH levels after lindane intoxication as compared to

control animals. The pretreatment groups showed a non-

significant decrease in the levels of GSH as compared to

control which accounted to 0.10 % in the A?L group,

6.69 % in U?L group and 12.37 % in U?A?L group. But

when compared to lindane a significant increase (P \ 0.01)

of 41.18, 31.87, and 23.84 % was seen in the respective

groups A?L, U?L and U?A?L. The increasing order of

GSH levels in different groups was as follow: L \ U?

A?L \ U?L \ U?A \ A?L \ C \ U \ A.

Effect on protein (Graph 6)

A significant decrease (P \ 0.01) of 15.01 % was observed

in the total protein levels of lindane intoxicated animals as

compared to control. The pretreatment groups A?L, U?L

and U?A?L showed a significant rise (P \ 0.01) of 20.00,

Graph. 6 Mean ± SD values of total protein (mg/100 mg tissue

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



Graph. 7 Mean ± SD values of endogenous vitamin E (mg/g tissue)




Graph. 8 Mean ± SD values of endogenous vitamin C (mg/g tissue)




Mol Biol Rep (2014) 41:1967–1976 1971

123

17.08, and 11.95 % respectively, in the levels of total

protein as compared to lindane group. The best results were

shown by the pretreatment of combination of antioxidants

which resulted in about 20.00 % rise in the total protein

levels which had decreased to 15.01 % after lindane

intoxication. The increasing order of protein levels in

various groups is as follows: L \ U?A?L \ U?A \ U?

L \ C \ A?L \ U \ A.

Effect on endogenous vitamin E (Graph 7)

A significant decrease (P \ 0.01) of 34.94 % was regis-

tered in the endogenous levels of vitamin E in lindane

group as compared to control. A?L, U?L and U?A?L

groups significantly (P \ 0.01) alleviated the levels of

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

comparison to lindane group. The increasing order of

vitamin E levels in all the groups was as follow:

L \ U?L \ C \ U \ A?L \ U?A?L \ A \ U?A.

Effect on endogenous vitamin C (Graph 8)

As compared to control, a significant decrease (P \ 0.01)

of 50.95 % was observed in the endogenous levels of

vitamin C in animals toxicated with lindane. This decrease

was alleviated significantly (P \ 0.01) when the different

pretreatments were given. In comparison to lindane treated

animals, the animals of A?L, U?L and U?A?L group

showed an increase of 140.95, 100.51, and 107.18 %,

respectively. The increasing order of the vitamin C levels

in all the eight groups was as follow: L \ U?L \ C \U?A?L\ A?L \ U \ U?A \ A.

Discussion

The results of the present study clearly demonstrate that

LPO significantly increased in kidney after in vivo treat-

ment of lindane. Thus, results of the present study are in

agreement with previous reports where increase in LPO

was also observed due to lindane in different tissues [11,

22, 42, 48, 67]. Moreover, renal LPO levels also increase

due to aflatoxin [61], CCl4 [7], chlorpryfos-ethyl [46], lead

[55], cisplatin [4] and gentamicin [1] toxicity. The increase

in LPO results owing to increase in ROS or alternatively

lindane might inhibit antioxidant molecules and antioxi-

dant enzymes. The support for such an assumption comes

from the findings that lindane reduces antioxidant mole-

cules 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 sup-

plementation with vitamin C and E attenuated the LPO

levels decreased due to cisplatin [4] and chlorpyrifos [46].

Lipoic acid also ameliorates renal oxidative stress [6].

Moreover, combination of lipoic acid and vitamin E [11],

vitamin C, E, lipoic acid and resveratrol [8] and vitamin C,

E and lipoic acid [42] ameliorated the lindane induced

increased LPO levels.

The results reveal that the pretreatment of combination

of antioxidants and cow urine (U?A?L) was the most

effective in lowering the LPO levels followed by combi-

nation of antioxidant (A?L) and cow urine pretreatment

(U?L).

Due to increase in LPO the amount of free radicals

crosses the threshold level. Nevertheless, decrease in the

activities of CAT, SOD, GPx and GSH further deteriorates

the situation and enhance the formation of lipid peroxides.

It could be assumed that lindane might have caused LPO

by inhibiting the antioxidant enzymes, molecule, vitamin E

and C. The mechanism by which lindane induces oxidative

stress involves the activity of cyt P450 system resulting in

the generation of superoxide radicals [21].

Similar to earlier reports, results of the present study

also show a decrease in SOD and CAT levels in the lindane

treated group and in all such cases the main culprit being

superoxide radicals [9, 42, 48]. Renal SOD and CAT levels

were also lowered due to chlorpryfos-ethyl [46], aflatoxin

[61], CCl4 [7], lead [55], cisplatin [4], alcohol [60] and

gentamicin [1] toxicity.

The decreased activity of SOD in kidney in lindane

treated mice may be due to the enhanced lipid peroxi-

dation or inactivation of the antioxidative enzymes or

could result from inactivation by hydrogen peroxide or

glycation of the enzyme [62]. This would cause an

increased accumulation of superoxide radicals, which

could further stimulate lipid peroxidation. A decrease in

SOD activity favors the accumulation of superoxide

radicals, which in turn inhibit CAT [33] as also seen in

the present study.

All the pretreatment groups showed alleviation in the

levels of SOD and CAT. Earlier reports have shown that

vitamin C and E are capable of increasing the renal SOD

and CAT levels [4, 7, 46]. Combination of lipoic acid,

vitamin C, E and resveratrol [9] and lipoic acid and vitamin

C and E [42] attenuated the SOD and CAT levels in brain

and testis, respectively of lindane treated mice. Amongst

the three pretreatments the maximum amelioration in the

levels of SOD was observed in case of U?A?L group

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

Moreover, in the present study the protection offered

by combination of antioxidants (A?L group) in amelio-

rating the CAT levels was the maximum followed by

1972 Mol Biol Rep (2014) 41:1967–1976

123

combination of antioxidants and cow urine (U?A?L

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

The reduction in the activity of SOD, CAT enzymes

may result in a number of deleterious effects due to the

accumulation of superoxide anion and hydrogen peroxide

[58]. The elimination of H2O2 is either effected by CAT or

GPx [69].

The level of GPx in the exposed group were lowered

which is in accordance with the earlier studies where lin-

dane caused a similar decrease in the levels of GPx in

various tissues [42, 48]. Renal GPx levels were found to be

decreased due to chlorpryfos-ethyl [46], aflatoxin [61],

acetaminophen [27], cisplatin [4] and CCl4 [7] toxicity.

The decreased levels can be attributed to either increased

H2O2 generation or decreased GSH concentration because

GSH is one of the substrates for GPx [57]. A decreased

GSH concentration was observed in the present study.

Decreased activity of GPx may also result from radical

induced inactivation and glycation of the enzymes [26].

Because of the decreased GPx activity the accumulation

of H2O2 may cause inhibition of SOD activity [10]. During

oxidative stress, inactivation of GPx may occur, and on the

other hand superoxide anion (O2•-) itself can inhibit per-

oxidase function [12]. So GPx must be considered to be

complementary to SOD [43].

The pretreatment with vitamin C, E, lipoic acid and cow

urine in groups A?L, U?L and U?A?L significantly

improved the levels of GPx. It has been shown in earlier

reports that vitamin C and E are capable of increasing the

renal GPx levels [4, 7, 46] and combination of vitamin C, E

and lipoic acid ameliorated the testis GPx levels lowered

due to lindane [42]. It is also concluded that the significant

increase in the GPx levels was the maximum in case of

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

A?L group.

In the present study, the levels of GSH were decreased

significantly in lindane treated group indicating the oxidative

stress caused by lindane as also reported earlier [8, 22, 42, 48,

67]. Decrease in renal GSH levels has also been documented

in a case of alcohol [60], aflatoxin [61], gentamicin [1, 29],

acetaminophen [27] and cisplatin [4] toxicity.

The decrease in the GSH level as observed in the present

study can be due to increased utilization of GSH for

metabolism of lipid hydroperoxides by GPx or interaction

of GSH with free radicals. Similar analogy is being also

drawn in the earlier reports [5, 23].

The levels of GSH were significantly ameliorated after

the pretreatment with combination of antioxidants namely,

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

(U?L group) and combination of antioxidants and cow

urine (U?A?L group). Earlier studies have shown that

vitamin C, E and lipoic acid attenuates the decreased renal

GSH levels [4, 29]. Lindane induced decreased GSH levels

were ameliorated by combination of lipoic acid, vitamin C,

E and resveratrol [8] and lipoic acid, vitamin C and E [42]

in brain and testis, respectively. The significant increase in

GSH levels in the pretreatment groups was least in

U?A?L group; intermediate in U?L group and maximum

in A?L group.

The present study reveals that lindane inhibits GPx and

CAT due to its capacity to generate ROS, which will result

in H2O2 accumulation. The increased H2O2 in turn could

cause SOD inhibition resulting in increased production of

superoxide radicals. Thus, increased production of super-

oxide radicals would inhibit both CAT and GPx. Treatment

with the adopted formulation reduced the level of lipid

peroxides indicating the effective antioxidant property of

the combination as well as cow urine alone in the moder-

ation of tissue damage.

Antioxidant defense system protects the aerobic organ-

ism from the deleterious effects of reactive oxygen

metabolites. Vitamin E, a major lipophilic antioxidant and

vitamin C, play a vital role in the defense against oxidative

stress [53]. In our study, the levels of vitamin E and C were

decreased significantly during lindane intoxication. This is

in agreement with the previous reports that oxidative stress

results in deficiency in vitamin C and E [60, 61]. The

increased oxidative stress due to lindane intoxication might

have resulted in excess utilization of vitamin C and E,

consequently, depleting their levels. The observed decrease

in the level of kidney ascorbic acid and alpha tocopherol in

lindane treated group could be as a result of increased

utilization of these antioxidants in scavenging the free

radicals generated due to lindane.

Moreover, it is well established that GSH in blood keeps

up the cellular levels of the active forms of vitamin C and

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

reduction in the GSH the cellular levels of vitamin C is also

lowered, indicating that GSH, vitamin C, and vitamin E are

closely interlinked to each other [61]. In agreement with

these reports, the decreased levels of GSH, vitamin C and

vitamin E on lindane administration were observed in our

study.

The pretreatment with combination of antioxidants in

group A?L, with cow urine in group U?L and combina-

tion of antioxidants and cow urine both in group U?A?L,

resulted in significant increase in the endogenous levels of

vitamin C and E. The amelioration in the endogenous

levels of vitamin C was the maximum in A?L group,

followed by U?A?L group and minimum in U?L group.

In contrast, the significant increase in the levels of vitamin

E was the maximum in U?A?L group; intermediary in

A?L group and least in U?L group. The increase in levels

of vitamin C and E is obvious in the pretreatment groups

A?L and U?A?L as these were exogenously supplied

with both vitamin C and E, however, the cow urine alone

Mol Biol Rep (2014) 41:1967–1976 1973

123

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

levels of the two vitamins. This could be attributed to the

composition of cow urine which is said to contain vitamins.

The analysis of total proteins is important for estimating

the degree of damage in the body. The protein profile of the

cells is indicative of the physiological status of animal and

there exists dynamic equilibrium between the synthetic and

degenerative pathways with these biomolecules. In the

present study a decline in the total proteins was observed

after lindane intoxication which can be due to decreased

protein synthesis or increased protein loss. Similar reduc-

tion in the levels of total proteins due to lindane have been

reported [9, 42, 48]. It is also reported that renal toxicity

due to CCl4 [7] and gentamicin [1] causes a similar

decrease in protein levels. Decreased protein levels could

be attributed to decreased feed consumption, maldigestion

or malabsorption, hepatic dysfunction [13, 47]. Reduced

protein levels can also be ascribed to increased urinary

excretion owing to kidney damage [66] and glomerular

apparatus or reduced protein synthesis [39]. Prabhakaran

and Devi [51] have proposed that a toxicant can affect the

protein content of the tissue either by inhibiting RNA

synthesis or inhibiting of amino acids into the polypeptide

chain.

The pretreatment groups showed an elevation in the

protein levels which is in accordance with earlier reports

where administration of either vitamin C, E or in combi-

nation and combination of vitamin C, E and lipoic acid and

resveratrol elevated the decreased protein levels [7, 9, 42].

The most effective pretreatment was that of combination of

antioxidants (A?L group); cow urine pretreatment group

(U?L) was moderately effective and least effective was

pretreatment with combination of antioxidants and cow

urine both (U?A?L group).

Hypoglycemic [44], cardio-respiratory effect [20],

immunomodulatory [14], antigenotoxic and antioxidant

properties in vitro [34], anticlastogenic [19] and chemo-

protective [52] effects of distillate and redistillate of cow

urine have been reported. Attenuation of CCl4 induced

hepatotoxicity by Panchagavya ghrita (prepared by cow

milk, cow urine, cow dung, ghee and curd) [2] and cow

urine distillate [25] have also been reported. Efficacy of

cow urine therapy has been evaluated on cancer patients

[28]. The amelioration of oxidative stress by fresh cow

urine has not been reported so far and this work seems to be

the first report.

Thus it is inferred that the combination of antioxidants

taken in the present study are quiet helpful in mitigating and

modulating the oxidative stress in the kidney caused due to

lindane. Moreover, cow urine treatment also modulated the

oxidative stress parameters caused by lindane. From the

results it is apparent that the given combination of antiox-

idants and cow urine act synergistically in reducing lindane

induced dysfunction. The analysis of the results of the

present study reveals the efficacy of cow urine against

oxidative stress. It can be safely concluded that the sug-

gested combination of vitamin C, vitamin E, alpha lipoic

acid and cow urine can prove to be beneficial in a number of

ailments. The highlight of the investigation is the efficiency

of cow urine against the pesticide toxicity which can open

new insights in the field of medicine. Cow urine can prove

to be an effective co-remedy for oxidative stress. This study

emphasizes the importance of antioxidants and cow urine

which could be beneficial in the therapeutic world for the

treatment of various disorders implicating oxidative stress.

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ISSN NO 2320-5407 International Journal of Advanced Research (2013), Volume 1, Issue 5, 71-78

71

Journal homepage: http://www.journalijar.com INTERNATIONAL JOURNAL

OF ADVANCED RESEARCH

RESEARCH ARTICLE

Evaluation of Anticancer properties of Taxusbaccata and Badri cow urine in mice: Clinicohematological

study

Ankita Joshi, and R.S. Chauha

1. Post Doctoral Fellow, Cell Culture Laboratory, Institute of Biotechnology, G.B. Pant University of Agriculture

and Technology, Patwadangar-263128, Nainital, Uttarakhand, INDIA.

2. Campus Director, Institute of Biotechnology, G.B. Pant University of Agriculture and Technology, Patwadangar-

263128, Nainital, Uttarakhand, INDIA.

Manuscript Info Abstract

Manuscript History:

Received: 12 June 2013 Final Accepted: 23 June 2013

Published Online: July 2013

Key words: Badricow urine, Taxusbaccata, Mice,

Body weight, hematology.

In the present investigation, the anticancerous effect of Taxusbacaata and

distilled Badri cow urine was studied in mice for clinicohematology and

body weight for a period of six month at an interval of 15 days. The study

revealed that the values of total leucocyte count (TLC), absolute lymphocyte

count (ALC) and absolute neutrophil count (ANC) were significantly

increased in the treated groups of mice either by CUD alone and in

combination withTaxusbaccata extracts. At180th day, it was found that there

was an increase in body weight, Hemoglobin content (Hb), total erythrocyte

count (TEC), total leucocyte count (TLC), absolute lymphocyte count (ALC)

and absolute neutrophil count (ANC) levels in CUD +A treated group as

23%, 23.99%, 41%, 40%, 40.31%, and 40.13%, respectively. This clearly

indicate the, increase in vitality and defence mechanism of body which in

turn helps in further healing of cancer.

Copy Right, IJAR, 2013,. All rights reserved.

Introduction

Cancer is a disease involving dynamic changes in

genome and is characterized by the uncontrolled,

uncoordinated and purposeless proliferation of

malignant cells and their ability to spread, either by

growth in the adjacent tissue through invasion or by

implantation at distant sites through metastasis.

World cancer report issued by International Agency

for Research on Cancer (IARC) reported in 2003 that

cancer rate is set to increase at an alarming rate

globally. The 5-year relative survival rate for all

cancers diagnosed between 1999-2005 is 68%, up

from 50% in 1975-1977. (Kinzler and Vogelstein,

2002)(Jemal et al., 2011).

In India about 70% of population obtains medical

help from private practitioners and half of those who

seek medicinal help obtain it from alternative and

traditional medicine (Kumar et al., 2004). Poverty

and socioeconomic status are other hurdles in

treatment (Pal and Mittal, 2004). American cancer

society defines complementary and alternative

medicines (CAM) simply as anything which is not

conventional (Zollman and Vickers, 1999; Park et al.,

2003). There are various CAM used for cancer

patient worldwide viz. Herbal medicine, acupuncture,

Ayurveda, biological agents, traditional Chinese

medicines, meditation and yoga etc. However, use of

herbs for cancer treatment is very popular throughout

the world.

Distilled cow urine protects DNA and

repairs it rapidly as observed after damage due to

pesticides. It protects chromosomal aberrations by

mitocycin in human leukocyte. Cow urine helps the

lymphocytes to survive and not to commit suicide

(apoptosis). Pathogenic effect of free radicals are

prevented through cow urine therapy. Use of cow

urine one can get the charm of a youth as it prevents

the free radicals formation. Taxus baccata,

commonly known as THUNER, which is mainly

found in high altitude area like, Patwadangar,

Nainital India also had anticancer and antiviral

properties. It is a small to medium-sized evergreen

tree, growing 10-20 m tall, exceptionally up to 28 m.

It is relatively slow growing, but can be very long-

lived, with the maximum recorded trunk diameter of

http://www.journalijar.com/


72

4 m probably only being reached in around 2,000-

4,000 years. Thuner is the oldest plant at high altitude

region of Uttarakhand. Most parts of the tree are

toxic, except the bright aril surrounding the seed,

enabling ingestion and dispersal by birds. The major

toxin is the alkaloid taxane. Phytochemical analysis

of extracts of leaves and bark showed the presence of

lignans, flavonoid, glycosides, sterols, sugar, amino

acid, and triterpenoid, alkaloids, steroids, tannins,

mucilage, fixed oil, phenolic compounds and

protein.The leaves are the principal source of taxol;

the anti-cancer drug, but has not been widely

exploited in this connection (Hartzell, 2003).

Considering the severity of cancer as a

disease of man and animals and complexity of

therapeutic approaches and their harmful side effects,

it was planned to study the effect of Taxusbaccata

preparation along with cow urine distillate in mice as

measured through clinical haematology

Materials and Methods 1. Extract preparation

Extracts of leaves and bark of T. baccata were

prepared by applying the standard methods with

different solvents like; Aqueous, ethanol, methanol

and ether as described by Govindachariet al., (1999)

and Udupaet al., (1995).

In-vivo study

2. Experimental design

Present study was performed in mice maintained in

the experimental animal house in Institute of

Biotechnology, G.B. Pant University of Agriculture

and Technology, Patwadangar, Nainital, Uttarakhand,

India. A total of 97 animals were equally divided into

11 groups. The mice were housed in clean

polypropylene cages and fed adlibitum with

commercially available feed and water. The

experiment was carried out in accordance with the

Institutional Animal Ethical Committee (IAEC), G.B.

Pant University of Agriculture and Technology,

Pantnagar, Uttarakhand, India. The 11 groups

were:Control group(9 mice), Negativecontrol(DEN

treated mice)(8 mice), CUD(without DEN)(8 mice),

A (Aqueous extract of leaves of Taxusbaccata)(8

mice), (Ethanolic extract of leaves of

Taxusbaccata)(8 mice), G(Methanolic extract of bark

of Taxusbaccata)(8 mice), H(Ether extract of Bark of

Taxusbaccata)(8 mice), CUD(Cow Urine

Distillate)(with DEN)(8 mice), CUD+A(8 mice),

CUD+B(8 mice), CUD + G(8 mice), CUD+H (8

mice).

Single dose of diethyl nitrosamine (DEN) @ 200

μl/kg body weight was given to each mice of

negative control group and tests groups. 500 ml of

each extract were made by adding 20% of extract in

500ml of distilled water (Kumar et al., 2004b). The

mice of 9 test groups were given different extracts of

taxusbaccata alone and in combination with CUD

(2ml/day/mice), daily p.o., from day 1 for 6 months;

however, the mice of negative control group were

maintained with routine feed and water.Body weight

of mice were taken regularly at an interval of 15 day

till the end of the experiment.Total leucocyte count

(TLC), absolute neutrophil count (ANC), absolute

leucocyte count (ALC), hemoglobin, total erythrocyte

count (TEC) and hemoglobin (Hb) content of all the

experimental animals in different groups were

determined regularly at an interval of 15 day till the

end of the experiment as per in standard procedures

(Chauhan, 2005).

Results In-vivo study was carried out in mice using DEN as

carcinogen and plant extracts alone and/or

combination with CUD as test material for a period

six month.

Body Weight

Body weight of mice were taken in gram at an

interval of 15 days till the end of experiment. Data of

body weight change during experiment were givens

in table-1. Initially, the mean bodyweight of control

was 21.47±1.21 gm and after 6 month the mean body

weight of mice was 25.86±1.87 gm. In DEN

(negative control) treated group the initial mean body

weight at zero day of experiment was 22.73±1.33 gm,

which decreased to 19.16±1.81 gm at the end of

experiment. But in CUD treated group mean body

weight at zero day was 22.43±1.36 gm and at the end

of 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 of 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 of 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 of 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 of 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 of 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 of experiment the body weight reaches


73

to 23.40±1.02, 23.33±1.91 and 23.28±1.89, respectively.

Table 1: Body weight in gm of experimental mice at an interval of 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

DEN)

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 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 106/cumm) of experimental mice at an interval of 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 of TEC is expressed in number of

cellsx106/cumm and is mentioned in Table 2. Initially

the TEC of control was 6.82±0.13 x 106/cumm and

after 6 month, TEC of experimental mice was

7.25±0.27 x 106/cumm. In DEN treated (negative

control) group the initial TEC at zero day of

experiments was 6.87±0.51 x 106/cumm which

decreased to 3.61±0.46 x 106/cumm significantly at

the end of experiment. But in CUD treated group, the

TEC at zero day was 6.29±0.52 x 106/cumm and

7.40±0.42 x 106/cumm at the end of experiment.

CUD treated group in which the carcinogen had also

been given, the initial TEC at zero day was 6.42±0.83

x 106/cumm. After the end of experiment, it was

6.68±0.53x 106/cumm. In test group A, the zero day


74

TEC was 6.31±0.37 x 106/cumm. The TEC decreased

to 7.32±0.58 x 106/cumm at 180 day of experiment.

In group CUD+A the total erythrocyte count, at zero

day was 6.31±0.97 x 106/cumm which was increased

to 8.90±0.47 x 106/cumm at the end of experiment.

Group CUD+B had 6.37±0.53 x 106/cumm TEC at

zero day and 8.59±0.62 x 106/cumm at the end of

experiment, respectively. In CUD+G and CUD+H

groups the TEC at zero day were 6.42±0.59 x

106/cumm and 6.35±0.61 x 10

6/cumm, respectively

and were 8.51±0.42 x 106/cumm and 8.48±0.43 x

106/cumm at the end of the experiment. In group B, G

and H, the TEC at zero day was found 6.33±0.61 x

106/cumm, 6.39±0.41 x 10

6/cumm and 6.37±0.52 x

106/cumm, respectively and at the end of experiment

increased to 7.29±0.57 x 106/cumm, 7.20±0.37 x

106/cumm and 7.18±0.41 x 10

6/cumm.

Data of TLC is expressed in no. of cells x 103/cumm

and is presented in Table-3.Initially the TLC count of

control group was 8.91±0.30 x 103/cumm and after 6

month the TLC was 12.11±0.04 x 103/cumm. In DEN

(negative control) group the initial TLC at zero day

of experiments was 9.12±3.20 x 103/cumm which

was decreased to 3.02±1.45 x 103/cumm. But in CUD

treated group, the TLC at zero day was 8.65±4.31 x

103/cumm and 9.53±4.67 x 10

3/cumm at the end of

experiment. In CUD treated group, the initial

TLC at zero day was 9.02±5.31 x 103/cumm which

decreased to 7.81±3.11 x 103/cumm. In test group

like A, the zero day TLC was 9.08±5.68 x 103/cumm

which was decreased to 8.64±3.08 x

103/cumm.CUD+A had the zero day mean TLC

count as 8.97±4.02 x 103/cumm which was increased

to 12.61±2.17 x 103/cumm, at the end of experiment.

Group CUD+B has 8.89±6.31 x 103/cumm at zero

day and was 10.55±3.18 x 103/cumm at the end of

experiment.In group B, G and H, the TLC at zero day

was observed as 9.01±6.81 x 103/cumm, 9.10±6.07 x

103/cumm and 9.07±7.03 x 10

3/cumm, respectively

and at the end of experiment 8.59±3.19 x 103/cumm,

8.33±3.61 x 103/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 103/cumm,

9.03±6.19 x 103/cumm and was 10.21±3.81 x

103/cumm and 10.05±3.14 x 10

3/cumm at the end of

the experiment, respectively.

Data of ALC is expressed in no. of cells x

103/cumm and is presented in Table-4.Initially the

ALC count of control group was 4.30±0.69 x

103/cumm and after 6 month the ALC was 5.92±0.98

x 103/cumm. In DEN (negative control) group the

initial ALC at zero day of experiments was 4.41±0.81

x 103/cumm which was decreased to 1.36±0.47 x

103/cumm. But in CUD treated group, the ALC at

zero day was 4.13±0.70 x 103/cumm and 4.63±0.43 x

103/cumm at the end of experiment.In CUD treated

group, the initial ALC at zero day was 4.43±0.84 x

103/cumm which decreased to 3.76±0.53 x

103/cumm. In test group like A, the zero day ALC

was 4.42±0.91 x 103/cumm which was decreased to

4.19±0.27 x 103/cumm.CUD+A had the zero day

mean ALC count as 4.39±0.87 x 103/cumm which

was increased to 6.16±0.42 x 103/cumm, at the end of

experiment. Group CUD+B has 4.36±0.89 x

103/cumm at zero day and was 5.16±0.80 x

103/cumm at the end of experiment.In group B, G and

H, the ALC at zero day was observed as 4.40±0.94 x

103/cumm, 4.48±0.73 x 10

3/cumm and 4.44±0.84 x


4.18±0.94 x 103/cumm, 4.01±0.37 x 10

3/cumm and

3.98±0.77 x 103/cumm respectively. In groups

CUD+G and CUD+H the ALC at zero day was

4.41±0.80 x 103/cumm, 4.42±0.79 x 10

3/cumm and

was 4.90±0.28 x 103/cumm and 4.89±0.11 x

103/cumm at the end of the experiment, respectively.

Data of ANC is expressed in no. of cells x

103/cumm and is presented in Table-5.Initially the

ANC count of control group was 4.57±0.72 x

103/cumm and after 6 month the ANC was 6.00±0.99

x 103/cumm. In DEN (negative control) group the

initial ANC at zero day of experiments was

4.69±0.83 x 103/cumm which was decreased to

1.49±0.52 x 103/cumm. But in CUD treated group,

the ANC at zero day was 4.38±0.78 x 103/cumm and

4.71±0.47 x 103/cumm at the end of experiment.

In CUD treated group, the initial ANC at

zero day was 4.51±0.97 x 103/cumm which decreased

to 3.88±0.56 x 103/cumm. In test group like A, the

zero day ANC was 4.60±0.97 x 103/cumm which was

decreased to 4.30±0.31 x 103/cumm.CUD+A had the

zero day mean ANC count as 4.46±0.89 x 103/cumm

which was increased to 6.25±0.45 x 103/cumm, at the

end of experiment. Group CUD+B has 4.41±0.92 x

103/cumm at zero day and was 5.24±0.82 x

103/cumm at the end of experiment.In group B, G and

H, the ANC at zero day was observed as 4.58±0.99 x

103/cumm, 4.56±0.75 x 10

3/cumm and 4.51±0.87 x


4.26±0.96 x 103/cumm, 4.11±0.38 x 10

3/cumm and

4.09±0.79 x 103/cumm respectively. In groups

CUD+G and CUD+H the ANC at zero day was

4.52±0.84 x 103/cumm, 4.50±0.85 x 10

3/cumm and

was 5.01±0.29 x 103/cumm and 4.96±0.12 x

103/cumm at the end of the experiment, respectively.


75

Table 3: Total leucocyte count (TLC) (x 103/cumm) count of experimental mice at an interval of 15 days (Mean±SE). Groups/Days 0 15 30 45 60 75 90 105 120 135 150 165 180

Control 8.91±0.30 9.06±0.17 9.38±0.32 9.91±0.37 10.08±0.51 10.21±0.62 10.53±0.02 10.73±0.12 11.05±0.81 11.32±0.91 11.51±0.07 11.87±0.47 12.11±0.04

CUD (NO

DEN)

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 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 106/cumm) of experimental mice at an interval of 15 days (Mean±SE). Groups/Days 0 15 30 45 60 75 90 105 120 135 150 165 180

Control 4.30±0.69 4.41±0.42 4.59±0.24 4.81±0.49 4.92±0.19 4.98±0.33 5.17±0.54 5.23±0.68 5.41±0.57 5.53±0.28 5.62±0.31 5.81±0.73 5.92±0.98

CUD (NO DEN)

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 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 106/cumm) of experimental mice at an interval of 15 days (Mean±SE). Groups/Days 0 15 30 45 60 75 90 105 120 135 150 165 180

Control 4.57±0.72 4.50±0.43 4.67±0.29 4.90±0.51 5.00±0.23 5.07±0.38 5.22±0.55 5.32±0.69 5.50±0.59 5.61±0.37 5.71±0.37 5.90±0.77 6.00±0.99

CUD (NO DEN)

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 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


76

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) of experimental mice at regular interval of 15 days (gm%, mean±SE). Groups/Days 0 15 30 45 60 75 90 105 120 135 150 165 180

Control 12.45±1.42 12.47±1.51 12.53±1.52 12.51±1.42 12.55±1.47 12.50±1.44 12.52±1.41 12.55±1.37 12.56±1.35 12.58±1.39 12.62±1.34 12.66±1.21 12.68±1.36

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* 12.34±1.28

12.09±1.19**

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 of Hemoglobin is expressed in gm% and is

presented in Table-6. Initially the Hb content of

control was 12.45±1.42gm% and after 6 month the

Hb content of mice was 12.68±1.36gm%. In DEN

(negative control) group the initial Hb content at zero

day of experiment was 12.40±1.36gm%, which

decreased to 8.02±1.07gm%, at the end of

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 of 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 of 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 of

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 of 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 of

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 of 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 of

the experiment, respectively.

Total Leucocyte count (TLC) in experimental mice


77

Absolute lymphocyte count (AlC) of experiment of mice.

Absolute neutrophil count (AlC) of experiment of mice.

Total erythrocyte count (TEC) in experimental mice

Haemoglobin content (Hb) in experimental mice

Discussion In-vivo study with T. baccata leaves and bark extracts

alone and in combination with CUD were carried out

in experimental mice for a period of six months. With

an observation at 15 days interval in mice, an attempt

was made to produce cancer using DEN and various

clinicohematological parameters were observed. The

body weight of mice was decreased substantially in

DEN treated mice indicating in the development of

cancer, due to DEN.Ramji and You, (1992) reported

that aflatoxin has been directly related to under

weight status in children in Benin and Togo. Bedi et

al.,(1996) reported decreased in body weight in

Guinea fowl fed on aflatoxin B1. In present study

body weight in DEN treated mice was decreased at

the end of experiment. However, there was increase

in body weight in other test groups. This study

showed that the weight loss in DEN treated group

may be due to the carcinogenic effect of DEN;

however, herbal formulations of extracts and CUD

were found to be a preventive agent against the

carcinogenic effects of DEN. DEN is already known

chemical carcinogen.Increased immunocompetence

of an individual is a very essential parameter to

prevent the development of cancers by several

mechanisms, of which the upregulation of

lymphocyte proliferation and stimulation activity,

increased macrophage activity, higher antibody

production and increased synthesis and secretion of

cytokines (IL-1, Il-2) plays significant role by

enhancing the recognition of tumor cells by the

immune cells of the body and cytotoxic activities of

the tumor killing cells, the lymphocytes. Using herbs

for cancer treatment can help the body to support its


78

healing power. In the present investigation, both

doses of QC (5 and 25 mg/kg) led to a significant

decrease in the number as well as the mean area of

GST-P positive foci, TUNEL positive apoptotic cells,

p53 positive hepatocytes, and restoration of cellular

morphology. These results clearly indicate that

quercetin inhibits diethylnitrosamine-induced hepatic

preneoplastic lesions in medium-term rat liver

bioassay. In the mice given T. baccataalone and

along with CUD. The body weight either remain

constant or enhanced substantially. These

preparations as shown in the in-vitro study were

having anti-carcinogenic effect, which might be

altering the clinoco effects of the cancer caused by

DEN.

Various hematological parameters indicated the

leukocytosis, erythrocytosis higher hemoglobin

content in treated mice with T. baccata products

along with indigenous cow urine. While, in DEN

treated mice there was leucopenia, erythropenia and

decreased heamoglobin content. These findings are

further supported by the fact that CUD had the

immunomodulatory property which caused

leukocytosis leading to the control of the highly

proliferating cells through their destruction by the

white blood cells. Erythrocytosis and increased

hemoglobin content are the indication of good health

and recovery and neutralization of the effect of DEN

by T. baccata and CUD. Joshi et al., 2013

investigated that the immunomodulatory effect of

distilled Gir cow urine in rabbits

throughhaematological parameters. The study

revealed that the values of total leucocyte count

(TLC), absolute lymphocyte count (ALC) and

absolute neutrophil count (ANC) were significantly

increased in Group II, in which the rabbits were

given Gir cow urine distillate alone and Gir cow

urine distillate with citric acid, respectively. In the

present study the ALC and ANC increased 40.31%

and 40.13% inn extracts and/or CUD treated mice in

comparison to control or DEN treated mice. Increase

in TEC and Hb content is an indication of enhanced

vitality of mice. Similarly leukocytosis,

lymphocytosis and neutrophilia are the immune cell

showing immunopoturtration, which is considered

protective against cancer and an indication of a good

prognosis (chauhan, 2005). It further needs a detailed

study for further confirmation.

References

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 of Gir cow urine distillate

in Rabbits. Journal of Immunology and

Immunopathology, 2012, 14(1): 57-61.

Kumar A.,Kumar P.,Singh L.K and Agrawal D.K.

Pathogenic effect of free radicals and their prevention

throughCowpathy. The Indian Cow, 2004, 2:27-34.

Zollman A. and Vickers G. Dervan.Understanding

Cancer. Jefferson, NC: McFarland (1999).

Udupa A.L.,Kulkarni D.R. and Udupa S.L. Effect of

Tridaxprocumbens extract on wound healing, Int. J.

Pharmacognosy, 1995, 1: 37-40.

Ramji C and You W. Differential sensitivity of

human mammary epithelial and breast carcinoma cell

lines to curcumin .Br. Canc. Res. Treat., 1999,

54(1):269-79.

ParkI.K.,Qian D., KielM.,Becker M.W, Pihalja M.,

Weissman I.L., Morrison S.J and Clarke M.F. "Bmi-1

is required for maintenance of adult self-renewing

haematopoietic stem cells". Nature. 2003, 423

(6937): 302–5.

Kinzler K.W and Vogelstein B.The genetic basis of

human cancer.Medical Pub. Division, 2002, 2:1-5.

IARC.Cancer an entropic disease, 2003, 143: 112-

131.

Bedi P.S, Singh H.,Johri H.S and Agarwal R.N.

Effect of aflatoxin on growth and feed consumption

in Guinea fowl. XX World Poultry Congress, 1996,

4(3):665-667.

Chauhan R.S. Cowpathy: A new version of Ancient

Science. Employment News, 2005, 30(15):1-2.

Pal S and Mittal K. Use of alternative cancer

medicine in India. Lancetoncol.2004, 3: 394-395.

Govindachari T.R., Suresh G. and Masailmani S.

Antifungal activity of Azadirachtaindica leaf hexane

extract fitoterpia, 1999, 70: 427-420.

Hartzell M. Carcinogenic effect of Taxusbacata in

diabetic condition., 2003, 45:147-168.

***********

AJP, Vol. 4, No. 5, Sep-Oct 2014 354

Original Research Paper

Lipid-lowering activity of Cow urine ark in guinea pigs fed with a high

cholesterol diet

Chawda Hiren Manubhai1, Mandavia Divyesh Rasiklal

1, Baxi Seema Natvarlal

2, Vadgama

Vishalkumar Kishorbhai1, Tripathi ‎Chandrabhanu Rajkishor

1*‎

1Department of Pharmacology, Government Medical College, Bhavnagar-364001, Gujarat, India

2Department of Pathology, Government Medical College, Bhavnagar-364001, Gujarat, India

Article history: Received: Sep 28, 2013

Received in revised form:

Apr 5, 2014

Accepted: May 14, 2014

Vol. 4, No. 5, Sep-Oct 2014,

354-363.

* Corresponding Author: Tel: ‎+919825951678‎

Fax: ‎+9102782422011‎

[email protected]

Keywords:

Antioxidant activity

Cow urine ark

Dyslipidemia

Hypolipidemia

Guinea pig

Statin

Abstract Objectives: Cow urine ark (CUA), known‎ as‎ “Amrita”‎ as‎

mentioned in Ayurveda, contains‎ anti-hyperglycemic and

antioxidant effects. Therefore, we designed the present study to

evaluate the lipid ‎lowering activity of CUA and its possible

implication in metabolic syndrome.‎ Materials and Methods: Thirty guinea pigs of either sex were

divided into five groups: Group 1 and 2 serving as a vehicle ‎and

sham control, received normal and high fat diet for 60 days

respectively; Group 3, 4 and 5 ‎received high fat diet for 60 days

with CUA 0.8 ml/kg, 1.6 ml/kg and rosuvastatin (1.5 mg/kg) on

the‎last 30 days of study period, respectively. Serum lipid profile

(total cholesterol, triglycerides, LDL-‎C, VLDL-C, HDL-C, total

Cholesterol/HDL-C) and serum enzymes (ALT, AST, ALP, LDH

and CK-MB) ‎were performed in each group at the beginning and

end of the study. Histological study of liver and ‎kidney was done

in each group.

Results: CUA (0.8 ml/kg) significantly decreased the serum

triglycerides and VLDL-C, but CUA (1.6 ml/kg) ‎decreased the

total serum Cholesterol, triglycerides and VLDL-C (p < 0.05).

Higher dose (1.6 ml/kg) of ‎CUA also increased HDL-C level,

significantly (p < 0.05). CUA reduced serum AST, ALP and LDH

‎level, which was statistically significant as well, while it also

decreased the accumulation of lipid in hepatocytes as ‎compared to

sham control.‎

Conclusions: CUA reduced triglycerides, increased HDL-C and

found to be hepatoprotective in ‎animals that are on a high fat diet.

Please cite this paper as:

Chawda Hiren M, Mandavia DR, Baxi SN, Vadgama VK, ‎Tripathi ‎CR‎.Lipid-lowering activity of Cow urine ark

in guinea pigs fed with a high cholesterol Diet. Avicenna J Phytomed, 2014; 4 (5): 354-363.

Introduction

Cardiovascular diseases (CVD) are

emerging problem in developing countries

‎‎(Freedman, 2003; Badimon et al., 2010).

CVD may become the reason for the loss

of 17.9 ‎million Potentially Productive

Years of Life Lost (PPYLL) in India, by

2030 (Anchala et al., 2012). ‎Dyslipidaemia

is well recognized risk factor for the

development of cardiovascular diseases

Lipid-lowering activity of Cow urine ark ‎

AJP, Vol. 4, No. 5, Sep-Oct 2014 355

‎‎(Freedman, 2003; Badimon et al., 2010).

Reactive oxygen species induce the

oxidative stress, ‎which play significant

role in the development of CVD and

atherosclerosis. Liver, kidney ‎and heart are

also under oxidative stress due to

hyperlipidemia in CVD (Kwiterovich,

‎‎1997; Vijayakumar et al., 2004). Other

crucial factors regarding CVD, associated

morbidity and mortality, are the insulin

resistance, diabetes, ‎elevated blood

pressure, obesity and dyslipidaemia that

are included in metabolic syndrome (Li et

al., 2013). Modern life style like smoking,

alcohol ‎consumption and junk food diet

are major obstacles in the management of

dyslipidaemia. ‎Elevated liver enzymes are

the major risk factors associated with the

current allopathic treatment ‎for

dyslipidaemia and thus, the alternative

medicines from Ayurveda, have been

getting attention ‎in the management of

dyslipidaemia (Suanarunsawat et al.,

2011). ‎

Cow urine, known‎as‎“Amrita”‎or‎water‎

of life as mentioned in Ayurveda ‎‎(Dhama

et al., 2005), is one of the ingredients of

Panchagavya Ghrita. Panchagavyapathy

was proved to be effective in life

threatening diseases such as diabetes,

cancer and AIDS ‎‎(Dhama et al., 2005;

Achliya et al., 2003; Jarald et al., 2008).

Cow urine has medicinal ‎properties like

antimicrobial, antifungal and anticancer

which granted US Patents (No. ‎‎6,896,907

and 6,410,059) (Randhawa, 2010). Several

studies revealed that cow urine has

‎antidiabetic and antioxidant activity

(Sachdev et al., 2012; Krishnamurthi et al.,

2004). ‎Therefore, the present study was

planned to evaluate the lipid lowering

effect of Cow urine ark ‎‎(CUA) and also to

know its possible implication in metabolic

syndrome.‎

Materials and Methods Drugs and Chemicals

Cholesterol powder (analytical grade): was

purchased from High Purity Laboratory

Chemicals ‎Pvt. Ltd., Mumbai, India. Cow

urine ark (CUA) was obtained from Go

Vigyan‎Anushandhan Kendra, Sevadhanm,

Devalpur, Nagpur, Maharashtra, India and

(US Patent ‎No 6410 059/2002).

Rosuvastatin Calcium powder: (Gift

sample from Torrent ‎Pharmaceuticals Ltd.,

Torrent research center, Ahmedabad.

(Batch no: ARD2110109)‎

Animal preparation

Thirty guinea pigs weighing 520 – 860

g of either sex were procured from the

Central ‎Animal House of Government

Medical College, Bhavnagar, which is

registered in the ‎Committee for the

Purpose of Control and Supervision of the

Experiments on Animals ‎‎(CPCSEA), New

Delhi, India. CPCSEA guidelines were

followed during animal ‎experiments of our

study. The guinea pigs were housed in

stainless steel cages under 12 ‎hour light-

dark 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 of six guinea pigs.‎

Group I: Normal diet plus distilled

water (Vehicle control); Group II: High fat

diet plus ‎distilled water (Sham control);

Group III: High fat diet plus a lower dose

of CUA (0.8 ‎ml/kg); Group IV: High fat

diet plus a higher dose of CUA (1.6

ml/kg); Group V: High fat ‎diet plus

rosuvastatin (1.5 mg/kg)‎

Diet composition ‎

Normal diet: in the morning, mixtures

of cereals and pulses (60% wheat plus

35% Bengal ‎gram plus 15% peanuts), total

50 g/animal. In the evening: green leafy

vegetables, 30 g/ ‎animal.‎ High fat diet: in

the morning, cholesterol powder (500

mg/kg) mixed with 10 g of wheat and

‎Bengal gram flour, followed by 40 g of the

mixtures mentioned above, in the normal

diet/animal. ‎In the evening: green leafy

vegetables, 30 g/animal.‎

Chawda Hiren et al.‎

AJP, Vol. 4, No. 5, Sep-Oct 2014 356

Methodology

The study was conducted in Animal

room, Department of Pharmacology,

Government ‎Medical College, Bhavnagar,

Gujarat, after approval from the

Institutional Animal Ethics ‎Committee of

the same institute. The baseline blood

sample was collected from a lateral

‎saphenous vein of the hind paw of each

guinea pig in overnight fasting state.

Blood samples were‎ sent to Clinical

Biochemistry Laboratory of our institute

which is accredited by National

‎Accreditation Board for Testing and

Calibration Laboratories (NABL), for the

serum lipid ‎profile, liver and cardiac

enzymes. Animals were separated into

groups as mentioned above. Throughout

the study period in each group, diet ‎was

given according to the respective group

diet plan. During the last 30 days of the

experiment, animals of group I and II ‎were

given distilled water daily, animals of

group III, IV and IV were given a lower

dose ‎of CUA, higher dose of CUA and

rosuvastatin (1.5 mg/kg), respectively.

‎Distilled water, CUA and rosuvastatin

calcium were given orally by gavages

feeding tube ‎in a daily basis on mornings

in fasting state to ensure maximum

absorption. Animals of all groups ‎were

sacrificed after blood collection from the

lateral saphenous vein in the overnight

fasting ‎stat at the end of 60 days.

Bloodsmples were sent to labratory for the

analysis of the serum lipid profile, liver

and ‎cardiac enzymes. We obtained the

liver and kidney from each animal of the

five groups ‎for histopathological analysis,

which was done by senior faculty from the

Pathology ‎department of our institute.‎

Serum lipid profile

The serum levels of triglycerides, total

cholesterol and high density lipoprotein

cholesterol ‎‎(HDL-C) and Low density

lipoprotein cholesterol (LDL-C) were

analyzed by GPO PAP ‎METHOD, CHOD

PAP METHOD, IMMUNOINHIBITION

and ENZYME SELECTIVE ‎methods,

respectively. Very low density lipoprotein

cholesterol (VLDL-C) was calculated ‎by

the Friedwald method (Friedewald et al.,

1972) as well as the ratio of the total

cholesterol and HDL-C.‎

Evaluation of liver and cardiac enzymes

Liver function was evaluated by serum

alanine aminotransferase (ALT), aspartate

‎aminotransferase (AST), and alkaline

phosphatase (AP) levels. Cardiac injury

was assessed ‎by measuring the serum level

of creatine kinase MB subunit (CK-MB)

and lactate ‎dehydrogenase (LDH). ALT

and AST were examined by UV KINETIC

method and serum ‎alkaline phosphatase

was determinedby PNP AMP KINETIC

method. LDH and CK-MB ‎were analyzed

by IMMUNO-INHIBITION and UV

KINETIC, respectively.‎

Effect of CUA on the weight of the

animals Weighing of each animal in all groups

was done before the start of the study and

also at the ‎end of 60 days to rule out any

effect of CUA on the weight of the guinea

pig.‎

Histological analysis

The liver and kidney were isolated,

cleaned, dried, and fixed at 10% neutral

buffer formalin ‎followed by paraffin

embedding and stained with haematoxylin

and eosin (H&E) dye. All

‎histopathological slides were coded and

evaluated by a pathologist blindly without

‎knowledge of the groups. Scalinggrades 0,

1, 2, 3 and 4 were given for no change,

slight, mild, ‎moderate and severe changes,

respectively regarding theseverity

assessment of histopathological‎ results.‎

Statistical analysis:‎

All parameters were expressed as

Mean±standard error of mean (S.E.M.).

One-way ‎Analysis of Variance (ANOVA)

followed by Tukey-Kramer Multiple

comparison test was ‎used to compare the


AJP, Vol. 4, No. 5, Sep-Oct 2014 357

inter group differences of lipid profile,

liver enzymes, cardiac enzymes and ‎extent

of body weight gain at the end of 60 days.

Paired t-test was used to compare intra

‎group differences of lipid profile, liver

enzymes, cardiac enzymes and extent of

body weight ‎gain. Value of p<0.05 was

considered significant. The statistical

calculations were done ‎using

GraphPadInStat, Demo version 3.06.‎

Result Serum lipid profile

The baseline serum level of total

cholesterol, triglyceride, HDL-C, LDL-C

‎and VLDL-C are 46.33 ± 4.04, 92 ± 5.24,

4.5 ± 0.9, 23.56 ± 2.6 and 19.2 ± 1.16,

‎respectively in vehicle control (Table 1).

There are not statistically significant

differences in the ‎baseline values of each

variable in different treatment groups. The

serum level of the total ‎cholesterol,

triglyceride, HDL-C, LDL-C and VLDL-C

were 82.6 ± 4.65, 109.2 ± 7.63, 3.9 ‎‎± 0.8,

54.38 ± 7.1 and 22.5 ± 3.5, respectively at

the end of 60 days, which were statistically

‎significant as compared to the baseline

lipid profile, in Sham control [p<0.05;

Table 1]. The percentages of increment in

serum level of the total cholesterol,

triglyceride, HDL-C, ‎LDL-C and VLDL-C

were 92 ± 22.8 (%), 29.8 ± 7.5 (%), 63.2 ±

45.4 (%), 161.93 ± 45.5 (%) ‎and 29.8 ± 7.5

(%) in sham control, as showed in Table ‎‎2.‎

A significant reduction in serum

triglyceride and VLDL-C was observed in

high fat diet treated group with the lower

dose of cow urine ark (CUA), in

comparison to sham control. The serum

total cholesterol, serum triglyceride and

VLDL-C were ‎significantly decreased in

the higher dose of CUA as compared to

sham control [p<0.05; ‎‎(Table 1)].

However, the HDL-C level was

significantly increased with higher dose of

CUA (p < 0.05) but, ‎percentage of

increment in HDL-C with rosuvastatin

treatment was significantly greater than ‎the

lower and higher dose of CUA treatment.

The percentage of increment in

‎triglyceride and VLDL-C with

rosuvastatin treatment were- 8.82 ± 2.3

and - 8.8 ± 2.3, ‎respectively at 60 days,

while with lower ‎dose of CUA and higher

dose of CUA, the percentage results were -

26.5 ± 12.7 and - 37.1 ± 8.2, (Table 2).‎

Table 1. Effect of each treatment strategy on serum lipid profile in guinea pigs

Values are expressed as Mean ± standard error of mean; LDL: low density lipoprotein, VLDL: very low density

lipoprotein, HDL: high density lipoprotein; CUA: Cow urine ark; * p< 0.05 as compared to sham control,

ANOVA followed by Tukey-Kramer Multiple comparison test; **

p< 0.05 as compared to baseline level, paired

t-test.

Treatment Groups

(n = 6)

Time

Period

Total

Cholesterol

(mg/dl)

Triglycerides

(mg/dl)

HDL

Cholesterol

(mg/dl)

LDL

Cholesterol

(mg/dl)

VLDL

Cholesterol

(mg/dl)

Total

Cholesterol/

HDL-C

Vehicle control

(Group 1)

Base line 46.33 ± 4.04 92 ± 5.24 4.5 ± 0.9 23.56 ± 2.6 19.2 ± 1.16 11.4 ± 0.8

60 Days 46.53 ± 5.02 88.7 ± 6.65 4.4 ± 0.5 24.17 ± 2.13 18.7 ± 1.22 12.02 ± 0.6

Sham control

(Group 2)

Base line 46.2 ± 7.03 87.38 ± 6.2 4.1 ± 0.8 23.26 ± 3.47 17.3 ± 1.24 9.78 ± 1.5

60 Days 82.6 ± 4.65** 109.2 ± 7.63** 3.9 ± 0.8 54.38 ± 7.1** 22.5 ± 3.5** 15.39 ± 7.48

High fat diet plus

CUA (0.8 ml/kg)

(Group 3)

Base line 47.8 ± 3.43 75 ± 9.25 4.5 ± 0.6 28.33 ± 2.7 15 ± 1.85 12.53 ± 3.19

60 Days 64.9 ± 7.92 49.5 ± 2.65* 7.16 ± 1.6 47.6 ± 6.4 9.9 ± 0.5* 10.44 ± 1.86

High fat diet plus

CUA (1.6 ml/kg)

(Group 4)

Base line 46.23 ± 2.24 75.3 ± 6.01 4.3 ± 0.42 26.83 ± 4.26 15.06 ± 1.2 11.2 ± 1.45

60 Days 57.53 ± 2.9* 45.3 ± 3.21* 6.3 ± 0.6** 42.1 ± 1.9 9.06 ± 0.64* 9.29 ± 0.52

High fat diet plus

rosuvastatin

(1.5 mg/kg)

( (Group 5)

Base line 42.09 ± 8.64 86.83 ± 2.14 5.8 ± 0.7 26 ± 2.57 15.57 ± 2.3 8.8 ± 0.6

60 Days 46.32 ± 5.21* 76.42 ± 4.6* 9.6 ± 0.9* 28 ± 5.15* 15.08 ± 2.3* 4.55 ± 0.65*


AJP, Vol. 4, No. 5, Sep-Oct 2014 358

Table 2. The effects of each treatment strategy on serum lipid profile (% increment) on guinea pigsat the end of

60 days treatment

Values are expressed as Mean ± standard error of mean; LDL: low density lipoprotein, VLDL: very low density

lipoprotein, HDL: high density lipoprotein; CUA: Cow urine ark; * p< 0.05 as compared to vehicle control,

ANOVA followed by Tukey-Kramer Multiple comparison test; ** p< 0.05 as compared to sham control,

ANOVA followed by Tukey-Kramer Multiple comparison test;# p< 0.001 as compared to sham control,

ANOVA followed by Tukey-Kramer Multiple comparison test; ## p< 0.05 as compared to rosuvastatin

treatment group, ANOVA followed by Tukey-Kramer Multiple comparison test.

The baseline and 60 days values of

serum enzymes in each diet treatment

group were shown (Table3).In sham

control, there is a significant increase in

ALT, AST, ALP, LDH and CK-MB level

(p<0.05) at the end of 60 days, compared

to the baseline.ALT, AST and LDH levels

were significantly decreased in the high fat

diet group treated with low dose of CUA.

However, LDH level was significantly

decreased with the higher dose of CUA

compared to sham control [p<0.05;(Table

3)].High fat diet treated group with

rosuvastatin (1.5 mg/kg) showed

significant reduction in the serum total

cholesterol, LDL-C and ratio of total

cholesterol: HDL-C level and an increase

in HDL-C level as compared to sham

control [p<0.05; (Table 1)]. Compared to

the baseline level, there was an increase in

AST and ALP level in the rosuvastatin

treated group at the end of 60 days

period[p<0.05; (Table 3)].

The normal histological structure of

liver and kidney was shown in Figure 1 A

and 2 A, respectively. In sham control,

histological study of the liver showed

diffuse areas ballooning degeneration of

hepatocytes (grade 3+, 4+), Steatosis (up

to grade 2.5), fatty changes (midzone and

periportal) and congestion of central vein

and hepatic sinusoids (Figure 1 B) in all

animals, while the kidneys showed a

normal structure (Figure 2 B). In the group

treated with CUA and high fat diet, liver

histology showed no fatty changes, with

only ballooning degeneration (Grade 1+)

with repaired and regeneration process

(Figure 1 C) and the kidney examination

of this group were normal (Figure 2 C). In

rosuvastatin treated group, no

morphological changes in the liver and

kidney was detected (Figure 1 D and

Figure: 2 D).

Increase in mean body weight of all

groups at the end of 60 days was shown ‎in

Table 4 and the extent of weight gain was

not statistically significant between the

groups ‎‎(p<0.05).

Table 3.Effect of each treatment strategy on serum enzymes in guinea pigs

Treatment Groups(n = 6)

Time

Period

Total

Cholesterol

(mg/dl)

Triglycerides

(mg/dl)

HDL

Cholesterol

(mg/dl)

LDL

Cholesterol

(mg/dl)

VLDL

Cholesterol

(mg/dl)

Total

Cholesterol/

HDL-C

Vehicle control (Group 1) 60

Days - 2.4 ± 1.8 - 2.4 ± 1.8 - 2.4 ± 2.4 - 2.8 ± 3.8 - 2.3 ± 1.8 0.3 ± 3.4

Sham control (Group 2) 60

Days 92 ± 22.8* 29.8 ± 7.5* 63.2 ± 45.4 161.93 ± 45.5* 29.8 ± 7.5* 54.2 ± 30.7

High fat diet plus CUA

(0.8 ml/kg) (Group 3)

60

Days 42.9 ± 25.3 - 26. 5 ± 12.7# 70.8 ± 36## 78.9 ± 34.9 - 26.5 ± 12.7# 5.9 ± 30

High fat diet plus CUA

(1.6 ml/kg) (Group 4)

60

Days 27.2 ± 8.5 - 37.1 ± 8.2# 50.5 ± 16## 72 ± 21.7 - 37.1 ± 8.2# - 8.85 ± 13.2

High fat diet plus

Rosuvastatin

(1.5 mg/kg) (Group 5)

60

Days 4.5 ± 4.9** - 8.82 ± 2.3** 234 ± 31** - 18.9 ± 3.9** - 8.8 ± 2.3** - 67.9 ± 2.1**


AJP, Vol. 4, No. 5, Sep-Oct 2014 359

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)

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

Sham control

(Group 2)

Base line 61.17 ± 6.21 50.33 ± 5.3 85.83 ± 11.7 311.5 ± 39.05 285 ± 34.74

60 Days 104 ± 12.72** 162.4 ± 25.4** 145.6 ± 9.6** 456.5 ± 56.8** 360.23 ± 27.1**

High fat diet plus

CUA (0.8 ml/kg)

(Group 3)

Base line 57.14 ± 4.3 63.76 ± 14.9 126.5 ± 18.8 345.3 ± 22.6 433.56 ± 22.1

60 Days 50.83 ± 9.9* 72.66 ± 22.1* 139.9 ± 9.07 194.16 ± 28.12* 353.8 ± 36.5

High fat diet plus

CUA (1.6 ml/kg)

(Group 4)

Base line 51 ± 1.3 73 ± 14.7 86.6 ± 15 283.6 ± 27.2 317.33 ± 22.5

60 Days 63.8 ± 6.5 96.8 ± 14.6 124.3 ± 12.73 243.5 ± 37.4* 297.6 ± 17.5

High fat diet plus

rosuvastatin

(1.5 mg/kg)

(Group 5)

Base line 63.26 ± 13.2 62.26 ± 9.65 103.6 ± 12.3 241 ± 46.64 374.5 ± 42.25

60 Days 64.34 ± 5.5 169 ± 12.68** 161.5 ± 10.91** 301.8 ± 31.12 392.19 ± 28.3

Values are expressed as Mean ± standard error of mean; ALT: Alanine aminotransferase, AST: Aspartate

aminotransferase,AP: Akaline phosphatase , LDH: Lactate dehyrogenase, CK-MB: Creatine kinase-MB, CUA:

Cow urine ark; *p<0.05 as compared to sham control, ANOVA followed by Tukey-Kramer Multiple

comparison test;**

p< 0.05 as compared to baseline level, paired t-test.

Figure 1.Histological photograph of liver of guinea

pig (H&E 40x). (A) C, H and S indicates

normal structure of central vein, hepatocytes with

round nucleus and sinusoid, respectively; (B) Fatty

changes (Grade 4+) (arrow) and diffuse areas of

ballooning degeneration were shown in high fat

diet fed guinea pig; (C) Encircled area shown no

fatty changes, only ballooning degeneration (Grade

1+) with repaired and regeneration process in CUA

(1.6 ml/kg) treated group; (D) Normal

morphological changes seen in rosuvastatin (1.5

mg/kg) treated group.

Figure 2.Histological photograph of kidney of

guinea pig (H&E 40x). (A) G, T and I indicate

normal structure of glomeruli, tubule and

interstitum, respectively; (B) No fatty changes were

seen in high fat diet fed guinea pig; (C) and (D)

indicate normal histological appearance of kidney

in CUA (1.6 ml/kg) and rosuvastatin (1.5 mg/kg)

treated groups, respectively.

‎

A

B

C


AJP, Vol. 4, No. 5, Sep-Oct 2014 360

Table 4. Effect of each treatment strategy on weight of guinea pigs

Treatment group Weight of animal in grams

Baseline 60 days

Vehicle control 540.66 ± 6.14 554.16 ± 15.97

Sham control 632.33 ± 44.3 667.82 ± 45.32*

High fat diet plus CUA (0.8 ml/kg) 602.3 ± 29.3 637.6 ± 17.8*

High fat diet plus CUA (1.6 ml/kg) 604.3 ± 9.2 630.1 ±4.6*

High fat diet plus rosuvastatin(1.5mg/kg) 658 ± 36.8 682.5 ± 22.02*

Values are expressed as Mean ± standard error of mean; CUA: Cow urine ark; *p< 0.05 as compared to baseline

value, paired t-test.

Discussion

In the present study, we selected guinea

pig as the experimental animal for the

‎evaluation of lipid lowering activity of

Cow urine ark (CUA). Lipoprotein

metabolism of ‎guinea pigs is closest to

human and several lines of evidence

proved that guinea pigs are ‎admirable

models to assess hypolipidemic activity

and lipoprotein metabolism of drugs

‎‎(Fernandez and Volek, 2006).‎

The present study showed that 60 days

of high cholesterol diet feeding, ‎raised the

serum lipid profile (total cholesterol,

triglyceride, LDL-C and VLDL-C) and

‎induced the histopathological changes in

liver (Table 1, Figure 1 B). The liver plays

a major role in ‎equilibrium cholesterol

homeostasis (Suanarunsawat et al., 2011).

High cholesterol diet ‎increases the hepatic

cholesterol content and is responsible for

the elevated triglyceride synthesis and

‎cholesteryl ester-rich VLDL-C production

(Patel Y et al., 2011; Goldstein et al.,

1983; ‎Demacker et al., 1991). The

reduction in the amount ofhepatic LDL-‎C

receptors is caused by high fat diet which

diminishes cholesterol removal rate from it

(Patel et al., 2011; Goldstein et al., ‎‎1983;

Demacker et al., 1991). We opted a study

period, 60 days, which is sufficient to

‎produce fatty changes in guinea pigs, also

supported by previous studies (Patel et al.,

2011; ‎Ahmad-Raus et al., 2001). ‎

In the present study, CUA therapy for 30

days was found to be highly ‎effective to

reduce the total serum cholesterol,

triglycerides, VLDL-C [p<0.05; (Table 1

and ‎Figure 1 C)]. Elevated triglyceride

level is an independent risk factor for

atherosclerosis ‎‎(Beatriz et al., 2011).

Triglyceride rich lipoproteins (TRLs) have

apo C-III that stimulates ‎activation of pro-

inflammatory transcription factors nuclear

factor-κB,‎ processes‎ ultimately‎ ‎leads to

atherosclerosis (Kawakami et al., 2006).

Remnant species of TRLs can easily

accumulate ‎in endothelial cells and taken

up by macrophage to form foam cells

similar to oxidized LDL-‎C (Gianturco et

al., 1998; Botham et al., 2007). Foam cell

promotes the formation of fatty streak in

‎blood vessels that is an early marker of

atherosclerosis (Beatriz et al., 2011). TRLs

block ‎sterol efflux from monocytes and

macrophages which may diminish the

protective effect of ‎HDL-C (Palmer et al.,

2004; Patel et al., 2009).‎

Biochemical analysis of Cow urine

proved to have many constituents; ‎copper,

kallikrein, urokinase, nitrogen, uric acid,

hippuric acid, phosphate and others (Jain

et ‎al., 2010). Both, dietary and serum

copper were inversely associated with

fasting glucose, ‎total cholesterol, and

LDL-C according to Bo S et al (Bo et al.,

2008). Previous study also ‎indicated that

the supplementation of moderate dietary

copper inhibits atherogenesis in the

‎cholesterol-fed rabbit (Lamb et al., 2001),

thus the copper in CUA may be

responsible for its ‎lipid lowering activity.‎


AJP, Vol. 4, No. 5, Sep-Oct 2014 361

Oxidative stress and free radical

induced injuries play a major role in the

‎pathogenesis of a number of diseases.

Hyperlipidemia produces lots of free

radicals and ‎oxidative stress in blood

vessels along with atherosclerosis

progression, and it endangers ‎vital organs

such as liver, kidney, heart and brain

(Suanarunsawat et al., 2011).

Augmentation of ‎serum levels of AST,

ALT, AP, LDH, and CK-MB suggested

suppressed cardiac and ‎hepatic functions,

due to the retention of lipid in liver and

heart in sham control (Table 3 and ‎Figure

1 B). CUA treatment decreased serum

level of ALT, AST and LDH (p < 0.05),

and improvement in hepatocytes

histopathologically also seen (Table 3 and

Figure 1 C). CUA has ‎many volatile fatty

acids; acetic acid 2 propenyl ester, acetic

acid methyl ester, 2, 2, 3 ‎trichloro

propionic acid, butanoic acid-3methyl,

propyl ester, 1H indol-3-acetate, acetic

acid ‎phenyl ester and quinoline. They are

responsible for its antioxidant action

which is confirmed ‎by the estimation of

thiobarbituric acid, ascorbic acid, DPPH

radical scavenging activity and ‎ABTS

assay (Sachdev et al., 2012). Hence, the

antioxidant activity of CUA might be

‎responsible for the cytoprotective action

that was found in our study. ‎

Several studies reported that residual

cardiovascular risk is still apparent in ‎spite

of intensive statin therapy. Residual

cardiovascular risk with 80 mg

atorvastatin was ‎‎22.4%, 12% and 8.7%,

reported in the PROVE IT-TIMI study, the

IDEAL study and the ‎TNT study,

respectively (Cannon et al., 2004; LaRosa

et al., 2005; Pedersen et al., 2005).

‎Residual cardiovascular risk in patients

that were treated with statins is attributed

to the triglycerides and ‎low HDL-C which

is predominantly higher among diabetics

than non diabetics (Sampson et al., ‎‎2012).

Diabetes has a triad of lipid abnormality,

including high levels of triglycerides, low

‎levels of HDL-C and small, dense LDL-C

(Fruchart, 2013). CUA increases HDL-C

as well ‎as reducing triglycerides (Table 1).

These findings are in accordance with the

previous studies of ‎cow urine distillate in

diabetic wistar albino rats (Gururaja et al.,

2011). Thus, CUA may be ‎useful as an add

on therapy to reduce statin related residual

CV risk in diabetic patients.‎

Metabolic syndrome is a group of

interrelated metabolic abnormalities that

includes ‎insulin resistance, diabetes,

elevated blood pressure, obesity and

dyslipidaemia (Li et al., ‎‎2013). CUA has

lipid lowering, antidiabetic and antioxidant

activities (Sachdev et al., ‎‎2012). Cow

urine contains copper (Jain et al., 2010);

while previous studies revealed an inverse

‎association between diastolic blood

pressure and dietary copper intake (Bo et

al., 2008). ‎Cow urine has a diuretic action

which might be due to nitrogen, uric acid,

phosphates and hippuric‎acid (Jain et al.,

2010), Therefore cow urine may be

effective as an antihypertensive that has

the potential to‎ decrease all the metabolic

abnormalities and also a possible

‎implication in metabolic syndrome.‎

Hence, the present study revealed that

CUA has lipid lowering and ‎cytoprotective

effects that may be implicated in metabolic

syndrome. It is vital to mention that this

study has ‎several limitations since we did

not evaluate molecular mechanism,

objective evidence for ‎atherosclerosis and

metabolic syndrome.‎

From the current study, we concluded

that CUA reduces triglycerides, ‎improves

HDL-C and hepatoprotective in animals

with high fat diet; therefore it may be

useful in ‎diabetic dyslipidemic patients.‎

Acknowledgement‎

We would like to thank Torrent

Pharmaceuticals Ltd., Torrent research

center, Ahmedabad for providing

rosuvastatin calcium powder as a free gift

sample. ‎

Conflict of interest

There is not any clash of attentiveness

in this study.


AJP, Vol. 4, No. 5, Sep-Oct 2014 362

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Research Article

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

M. Vijaya Bhaskara Reddy1*, A. Karthik2, P. Sasikala1 1Department of LPM, College of Veterinary University, Sri Venkateswara Veterinary University, Tirupati, India

2Department of Microbiology, College of Veterinary University, Sri Venkateswara Veterinary University, Tirupati, India *Corresponding Author Email: [email protected]

Article Received on: 16/06/13 Revised on: 08/07/13 Approved for publication: 11/08/13

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

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


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and the aging process. Diabetes mellitus is one such disease, mostly spread over worldwide and the diabetic patients will continue to increase worldwide in the future. It has been postulated that the etiology of the complications of diabetes involves oxidative stress perhaps as a result of hyperglycemia. The elevated level of blood glucose in diabetes produces oxygen free radicals which cause membrane damage due to peroxidation of membrane lipids and protein glycation. It has been suggested that free radical activity is increased in diabetes. Under physiological conditions, glucose produces oxidants that exhibit reactivity similar to that of hydroxyl free radicals5. In recent years, there has been a renewed interest in variety of natural products with antioxidant potential which can play a major role in protecting against the molecular damage induced by reactive oxygen species. The present study was aimed to study the protective role of cow urine as an anti-diabetic and potentiality. Punganur cow the sacred Indian cow, Bos indicus, is believed to be a “mobile hospital” for the treatment of many diseases. A number of diseases can be cured by the use of medicines derived from the cow. Cow urine is described in detail in ancient Ayurvedic scriptures, such as Charaka samhita, Shushruta samhita and Brahad-Wagbhatt, as being bitter, pungent, spicy and warm. Now a day’s Cow urine is used as an insecticide and as a regulator for various disorders like intestinal gas, acidity and cough and is claimed to make humans wiser and can be used as a universal easily digestible medicine6. In classical texts Vedas like Ayurveda, like Charaka samhita and Shushruta samhita, several medicinal properties of cow urine are described very much with examples. Cow urine is known to cause weight loss, and reverse certain cardiac and kidney problems, as well as indigestion, stomach ache and edema. Cow urine is considered useful in treating renal colic, jaundice, anemia, diarrhea, gastric infection, piles and skin diseases including vitilago and considered as an appetizer and is known to reverse inflammation, and acts as a diuretic as well as a nephro-protective agent. However the anti-diabetic properties of cow urine have not been described in the literature. Further, although Indian Ayurvedic literature cites various medicinal properties of cow urine, there is very little scientific evidence to support7. Hence, the present study was undertaken. MATERIALS AND METHODS Cow Urine Distillate Preparation The first early morning voided urine of punganur cow (Bos indicus) was collected from the cow sheds Livestock Research Station, Palamaner, under Sri Venkateswara Veterinary University, immediately distilled by using double distillation unit (at 10000C using a temperature- controlled distillation apparatus and then stored below 1000C) used for further studies. Chemicals All chemicals and reagents used were of analytical grade and obtained from Sigma Chemical Company (St. Louis, MO, USA). The kits for the estimation of blood glucose levels and serum lipid profiles were obtained from Ranbaxy Diagnostics and Reckon Diagnostics Pvt. Ltd., India. The standard drug glibenclamide was purchased from a local pharmacy named Hetiro Pharamcy, Tirupati. Chittoor Dist, India.

Dose Selection Evaluations of the anti-diabetic activity of the cow urine distillate, three dose levels were selected. The rat dose was calculated from the human dose (60 ml per day), multiplied by a factor of 0.018 × 5 which is equal to 5.4 ml / kg body weight (first dose)8. The second dose selected was twice that of the first dose, i.e. 10.8 ml / kg body weight and the third dose were selected as 50 % of the first dose i.e. 2.7 ml / kg body weight. Animal Treatment Healthy Wistar albino rats (150 to 180 g) Animals were housed in a room with temperature maintained at 22 ± 20C and humidity 55 ± 4 %. They were fed with standard laboratory diet (SKS Feed, India). Rats were divided into 6 groups each containing 8 animals and allowed food and water ad libitum throughout the investigation. All the procedures are approved by the institutional animal ethics committee. Preparation of Streptozotocin Solution Preparation of 0.1 M citrate buffer solution of pH = 4.5: An accurately weighed quantity of Trisodium citrate (14.9 g) was dissolved in sufficient distilled water to produce 1000 ml and the pH was adjusted to 4.5 using conc. HCl. The solution of streptozotocin was prepared by dissolving the weighed quantity of streptozotocin in 0.1 M freshly prepared ice -cold citrate buffer (pH 4.5). Experimental Induction of Diabetes Diabetes was induced in rats by streptozotocin intraperitoneally injection at a dose level of 50 mg / kg b.wt. It is dissolved in citrate buffer (0.1M, pH 4.5) in the volume of 1 ml / kg. In order to prevent hypoglycemia during the first day after the streptozotocin administration, the diabetic rats were given 5 % w/v glucose solution orally. Three days after the injection, the blood glucose levels were measured and the animals with blood glucose levels above 300 mg/dl were considered to be diabetic and were used in the subsequent experiments. In all the experiments, rats were fasted for 16 h prior to streptozotocin injection. Animals were divided into six groups of 8 rats per group. The test samples were administered orally for 2 weeks9. Group I: Normal control group-Animals received only vehicle Group II: Diabetic control group (streptozotocin treated) - Animals received only vehicle. Group III: Standard drug group-Diabetic animals received daily a single oral dose of the reference drug glibenclamide (0.25 mg / kg) from day 1 to14. Group IV: Diabetic animals received daily a single oral dose of Cow urine distillate 2.7 ml / kg body weight from day 1 to14. Group V: Diabetic animals received daily a single oral dose of Cow urine distillate 5.4 ml / kg body weight from day 1 to 14. Group VI: Diabetic animals received daily a single oral dose of Cow urine distillate 10.8 ml / kg body weight from day 1 to 14. The effects of administration of cow urine distillate to diabetic rats were determined by measuring the fasting blood glucose levels, serum lipid profiles, liver glycogen levels and initial and final changes in body weight. Day 3 of induction was designated as day 1 for administration of the test sample


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

separated by centrifuging at 2000 rpm for 2 minutes. Blood glucose levels were measured using glucose kit. Statistical Analysis The data were expressed as Mean ± SEM and analyzed using one way analysis of variance (Anova), followed by a post hoc Sheffe’s multiple comparison tests using SPSS computer software version 10. The values were considered significant when P < 0.05.

Table 1: Effect of Cow Urine Distillate on Blood Glucose Levels in Streptozotocin-treated Diabetic Rats

Group Dose Glucose levels in blood (mg / dl)

Day 1 Day 5 Day 10 Day 14 Group I -- 85.89 ± 0.96bc 84.88 ± 1.44bc 84.89 ± 0.89bc 85.66 ± 0.73bc

Group II Diabetic control -- 336.10 ± 4.88a 359.54 ± 2.78ac 367.78 ± 1.65ac 374.84 ± 8.11ac

III- Standard glibenclamide 0.25 340.52 ± 0.99a 261.47 ± 1.01ab 194.88 ± 1.00ab 141.19 ± 2.87ab

Group IV 2.70 336.011 ± 2.89a 250.89 ± 1.77ab 231.11 ± 1.76ab 200.15 ± 1.70ab

Group V 5.40 341.44 ± 1.88a 250.11 ± 0.89ab 209.78 ± 2.88ab 173.96 ± 1.83ab

Group VI 10.80 339.91 ± 2.81a 230.96 ± 2.11ab 199.16 ± 1.79ab 145.59 ± 1.78ab

All the values are expressed as mean ± SEM (n = 8), values are statistically significant at P < 0.05 a P<0.05 when compared with the normal control group b P < 0.05 when compared with the diabetic control group c P < 0.05 when compared with the standard group

Table 2: Effect of Cow Urine Distillate on the Glycogen Content, Body Weight and Lipid Profiles in Streptozotocin-treated Diabetic Rats

Group Glycogen content mg / g Body weight % change Cholesterol mg / dl Triglycerides mg / dl HDL mg / dl Group I 35.26 ± 88bc +8.99 ± 1.89bc 62.019 ± 2.44bc 79.09 ± 1.16bc 59.16 ± 0.67bc

Group II Diabetic control 16.57 ± 1.01ac -13.87 ± 1.08ac 126.74 ± 0.71ac 189.24 ± 0.69ac 36.09 ± 1.11ac

III- Standard glibenclamide 30.89 ± 2.09ab -3.88 ± 0.79ab 68.45 ± 1.21ab 89.99 ± 1.10ab 49.71 ± 1.19ab

Group IV 23.88 ± 2.12ab -7.01 ± 2.03ab 89.03 ± 1.08ab 125.81 ± 1.006ab 38.21 ± 1.10ab

Group V 24.11 ± 0.99ab -7.10 ± 0.28ab 87.88 ± 0.10ab 120.19 ± 2.01ab 40.65 ± 1.11ab

Group VI 25.72 ± 2.71ab -5.12 ± 0.19ab 82.81 ± 1.11ab 134.11 ± 1.91ab 44.32 ± 1.011ab

All the values are expressed as mean ± SEM (n = 8), values are statistically significant at P < 0.05 a P < 0.05 when compared with the normal control group b P < 0.05 when compared with the diabetic control group c P < 0.05 when compared with the standard group

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

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


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diabetes12. Diabetic rats treated with the cow urine showed an improvement in weight gain compared with the diabetic control. The marked increase observed in serum triglycerides and cholesterol and the decrease in HDL in untreated diabetic rats is in agreement with the findings of Nikkila and Kekki13. Diabetic rats treated with the cow urine exhibited a significant decrease in cholesterol and triglycerides and an increase in HDL compared with the diabetic control. Glycogen syntheses in the rat liver and skeletal muscles are impaired during diabetes14. The decreased glycogen levels may probably be due to the lack of insulin in the diabetic state, which results in the inactivation of the glycogen synthase systems. In the present investigation, a significant increase in glycogen levels was observed in the treated groups, which might be due to the reactivation of the glycogen synthase system. Oxidative stress has been shown to play an important role in the etiology of diabetes15. Streptozotocin produces oxygen radicals in the body, which causes pancreatic injury and could be responsible for the increased blood glucose16. The compounds responsible for the anti-diabetic activity of cow urine are at presently not known. Studies have been carried out to examine the anti-oxidant potential of cow urine. For example17 described the anti oxidant properties of cow urine using two in vitro models, DPPH radical scavenging activity and superoxide scavenging activity using ascorbic acid as a reference standard.7 have described the antioxidant action of cow urine using an ABTS assay model and the antioxidant effect of cow urine was b due to the presence of volatile fatty acids. Further research in this field can be carried out by assessing the anti diabetic protective role of Punganur cow urine. Presence of antioxidants, free radical scavengers in cow urine could be responsible for its antidiabetic action. There are estimation that there are over 5.8 crore people who are diabetic in India. Diabetes is more common almost everywhere in the world. It is considered to be the root cause of many chronic diseases. Urine Therapy is the safest and easiest method of treatment to prevent, Control and cure Diabetes. It can safe guard all other complications arising from diabetes include heart disease, hypertension, and diabetic retinopathy. ACKNOWLEDGEMENT It is with most profound feelings of respect, sincerity and high regards that I express my indebtedness and deep sense of gratitude to my venerable brother, Pioneer and teacher M. Chandrasekhara Reddy and family K. Bharani, Master M. Druvin Reddy, SWE, Ohio, USA, for his meticulous guidance, Stimulating discussion, Awe inspiring Encouragement and suggestions to complete this work with confidence and for providing necessary financial asset facilities to carry out this study. I will ever remain grateful to him for his inspiring guidance, wise counsel unfailing attention. He has been a source of inspiration and confidence in my research and in my whole life. The authors are thankful to Dept. of LPM, SVVU for providing the facilities to carry out this study.

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Cite this article as: M. Vijaya Bhaskara Reddy, A. Karthik, P. Sasikala. Protective role of Punganur cow urine on streptozotocin induced diabetes in rats. Int. Res. J. Pharm. 2013; 4(8):164-167 http://dx.doi.org/10.7897/2230-8407.04832

Source of support: Nil, Conflict of interest: None Declared

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