[0001] Technical Field:
The present invention relates to novel scheme and method for the development of marker for the detection of urine of cow of Indian breed amongst the urines of other breed cow urine, goat and buffalo urine." obtained from the Indian breed cow and its differentiation from conmen adulterants such other breed cow urine, goat and buffalo urine in order to prevent the adulteration in the urine of cow of Indian breed. Urine is usually yellow or clear, depending upon the health and diet of cow. It usually has an ammonia-like odor due to the nitrogenous wastes that make up about 5% of the fluid (the remaining 95% is water).
Indian breed cow urine used as key therapeutic medicine in ayurvedic system of Medicine for the detoxification of bhasma. Purification of metal in ayurvedic system
is the of the important features and the quality of metallic ayurvedic medicines is very much depends on the urine of cow for purification and detoxification of these metals. The present invention is also relates to a process of collection of urine in hygienic conditions, for the purposes of detection of specific marker in order to differentiate between Indian breed cow urine and goat and buffalo urine which are the most conmen adulterant in the urine of Indian breed cow.
1 he focus of the invention is to highlight the value of the Indian breed cow urine during recent time and our invention is keen to provide the right way for adopting the pure Indian breed cow urine from suppliers of Indian breed cow urine by Ayurvedic Medicines Manufactures. Practicing Vadiyas and other common users of Indian breed cow urine by using my newly invented scheme and method expressed later in this invention which was never available earlier anv where in the universe.
6
A scheme and method of development of marker for identification .detection and differentiation of Indian breed cow urine with conmen adulterants is obtained via a process of stated in the "METHOD AND MATF.RIAL" section of specification.
Background & prior art
Urine is a slightly acidic fluid which carries waste from the kidneys to the outside world. fhe kidneys have millions of nephrons which filter toxins, waste, ingested water and mineral salts out of the bloodstream, fhe kidneys regulate blood acidity by excreting excessive alkaline salts when necessary. The chief constituent of the nitrogenous wastes in urine is urea. a product of protein decomposition. Urea is. among other things, a diuretic. .Average adult urine production is from one to two quarts a day. fhe bladder.

where urine is stored for discharge, holds on average about 16-20 ounces of fluid, though the average discharge is about half that amount. In addition to uric acid, ammonia, and creatine, urine consists of many other waste products in minute quantities.
Principle for therapeutic efficacy of cow urine:-Being a waste product does not mean that a substance is toxic or harmful. It means that the body cannot ab.sorb the substance al the present time. We might think of many of urine's constituents as if they were leftcners from a meal. We could throw the excess food away or we could eat it later after dilutuig it substantially with water and putting it in the blender. With urine. unfortunately. we cannot ingest waste products in the form they had when first ingested, if by chance some poisonous or harmful material enters cow's food, she absorbs it in her flesh. She does not let it go into cow urine, cow dung or milk or releases in very small quantity. These results have been compared with other animals by feeding them various items and then testing milk and urine. Therefore cow urine and dung is pure and removes toxins. Cow milk is certainly anti-toxin. Cow urine is included in "panchgavya". "Panchguvya" is said to be curer of all diseases. Panchagavya is made up of five cow products viz. milk. curd. ghee, urine and dung.
lor most people most of the lime, one's own urine is not likely to be harmful. However, it is not likely to be healthful or useful except for those rare occasions when one is buried beneaih a building or lost at sea for a week or two. In such situations drinking one's own urine might be the difference between life and death. ,A.s a daily tonic, there are much tastier ways to introduce healthful products into one's blood stream.
According to the Indian sagas the cow is the mother of all human beings and the specific use of cow''s urine can help to cure many diseases. .Medicinal use of cow urine
"Ja Gluir tulsi Aru gay, ta GItar vaitlya kabhu najaye" meaning A house having I uisi (sacred basil) plant and cow is never visited by a doctor.
In ayurved poisonous material are purified by cow urine only if cows live without slaughter for their whole life they remove poison and toxins from man\ sources of environment, "go nnitre tridinum sihupyuy visham leu vishiidhyuli" Meaning use of cow urine for three days cleanse poison.
" Yalvcigiislhi gutam papam dehe lishlhli inumkc
prci.snul pcinchgavyasya dahasagnirivendhnam"
Meaning: From skin to bones, what ever sins (disea.ses) are in my body, are destroyed bv panchagavya just as fire destroys fuelCow urine makes one disease free by pruhhav (natural effect). "Acinlya shakli Hi prcihhciv" meaning: Inconceivable power is called "Pnihhav". Prahhav is that power which cannot be conceived and described. Qualities of cow urine are stated in ayurveda.

Chemical description of cow urine as per modern concepts and cure of diseases accordingly.
Table - Chemical contents of cow urine and cure of diseases as per them.
S. No. Name of chemical Effect of chemical on diseases
1. Nitrogen N2 ,NH2 Removes blood abnormalities and toxins. Natural stimulant of
urinary track, activates kidneys and it is diuretic.
2. Sulphur S Supports motion in large intestines. Cleanses blood.
3. Ammonia NH3 Stabilise bile, mucous and air of body Stabilises blood
formation.
4. Copper Cu Controls built up of unwanted fats
5. Iron Fe Maintains balance and helps in production of red blood cells &
haemoglobin. Stabilises working power.
6. Urea CO(NH2)2 Affects urine formation and removal. Germicidal.
7. Uric Acid C5H4N403 Removes heart swelling or inflammation. It is diuretic therefore
destroys toxins.
8. Phosphate P Helps in removing stones from urinary track.
9 Sodium Na Purifies blood. Antacid
10. Potassium K Cures hereditary rheumatism. Increases appetite. Removes
muscular weakness and laziness.
11 Manganese Mn Germicidal, stops growth of germs, protects decay due to
gangrene.
12. Carbolic acid HCOOH (iermicidal. stops growth of germs and decay due to gangren
13. Calcium Ca Blood purifier, bone stregthener. germicidal. ?? Rakta skandak
14. Salt NaCl Sanyas vishamta ?? decreases acidic contents of blood.
germicidal
I 5. Vitamins A,B,C.D,f: Vitamin B is active ingredient for energetic life and saves from
nervousness and thirst, strengthens bones and reproductive ingredient for energetic life and saves from nervousness and thirst, strengthens bones and reproductive power.
16. Other Minerals Increase immunity
17. Lactose C6H12(36 Gi\es satisfaction. Strengthens Mouih ?? strengths heart.

removes thirst and nervousness.
18. Enzymes Make healthy digestive juices, increase immunity
19. Water (H2()) . It is hfe giver. Maintains fluidity of blood, maintains body
temperature
20. Hipuric acid Removes to.xins through urine
21. Creatinin Germicide
22. Aurum Hydroxide It is germicidal and increases immunity power. AuOH is highly
AuOH antibiotic and anti-toxic
23. Eight month pregnent cow's cow urine contain hormones v\hich
are healthy and nutrituous.
In the review of modern research Indian breed cow urine removes the blood abnormalities and toxins. Naturally stimulates the urinary track, activates kidneys .anti tumors and anti-intlammatory properties .Indian breed cow urine also maintains tluidily of blood, maintains body temperature.
[0031] Objectives:
1. .An objective of the present invention is to provide a scheme and method of for the
therapeutic use of use of Indian breed cow urine based on the clinical trials.
2. .Another objective of the present invention is to provide specific, accurate &
reproducible scheme and method in order to Identity. Detect & Differentiate the Indian
breed cow urine & adulterated urine by the specific markers present in of Indian breed
cow urine and absent in other breed cow, goat and buffalo urine.

Need of My Invention: -
Indian breeds of cows are resistant to several infectious diseases. This property seems to be gradually declining because of the uncontrolled cross breeding, The immunological characters of pure indigenous breeds were used for medical treatment in ancient times, fhere is a practice of administering the excreta of a just-born calf to infants & children for the purpose of achieving immunity against certain diseases. A patient suffering from tuberculosis in ancient days was advised to live amongst cattle for a period of a month or two. These practices are indicative of realization of special characters of immunity amongst Indian breeds. A methodical research in this area by immunologists can be beneficial.
The urine of Indian breed cow is considered the best urine for the Ayurvedic purposes. But due to the heavy demand and non availability of Indian bread cows the adulteration imitation, duplication of Indian breed cow urine is very common, fhe possible adulterants for the adulteration in Indian breed cow urine are other breed cow, goat and buffalo urine, fhe pries difference between pure Indian breed cow urine and fake adulterated other breed cow urine is high, fhe common source of adulteration as we identified is the urine of foreign breed cow urine, goat and buffalo urine, fhe adulterants are cheap and easily available. Till the date there is no method reported by any one in the vsorld for the differentiation between Indian bread cow urine and other animals urine used as therapeutic agent in Ayurvedic System of Medicine. The inventor is proposing the scheme and various analytical methods by which differentiation can be done between two types of urines. The proposed schemes and methods are reliable, robust and very swift. The differentiation is necessary for the desired therapeutic effects and for the safet\ of humans. We have invented the present scheme & method for present need, our method is very specific accurate & reproducible in order to detect & differentiate between Indian breed cow urine and other breed cow urine. We have analyzed the difference between the composition (especially in terms of therapeutic compounds) of secretion & excretion of pure indigenous breed & exotic and cross breeds of cows.

Examples:
brinc
\ rine was collected from the sets of Indian breed cow urine are other breed cow, goat and buffalo urine. Urine samples were frozen immediately after collection and stored at -80°C until processing.
Example 2:
1 Aperimental Materials
1. 17 hydroxy carticosteroids (Control sample )
2. Organic solvents
3. Solvent extraction apparatus
4. Glass wares. Plastic wares and DM water.
I he glasswares, plasticwares used in the present study were procured from M/s Borosil (Mumbai) and M/s Axygen. The chemicals were procured from Merck (Mumbai). Qualigens (Mumbai) and Sigma (USA).The materials/reagents used in various methods are mentioned in the relevant section of methodology.
EX T RACTION PROCESS FOR MARKER COMPOUND
Solvent system used for extraction - lithyl acetate
• Carti-costeroids - isolated by a modified method Cartland & Kuizenga"s method
Extracted with ether
↓
Evaporate the ether layer & find out the residue
↓
Dissolve the residue with ethylene chloride

↓
Filtrate the solution& evaporate up to dryness
↓
Residue is dissolve in 70. 50, 30% Methanol successively
↓
Petroleum ether soluble substance eliminated from each
↓
methanol solution
↓
30% methanol solution evaporated in reduced pressure
↓
Residue is dissolve in ethyl acetate wash with 1 % Na2CO3
↓
solution then by N/2 HCl
↓
After evaporating the solvent from the solution a dark gummy substance was obtained
↓
Dissolved in small quantity of chloroform
↓
Now analyze with chromatography
↓
Steroid is detected by UV light 253.7 nm Solvent system
Xylene: absolute methanol (3:1) Rf 0.13
Toluene: Ethyl Acetate: absolute methanol; H2O (9:1:5:5) Rf 0.30
Example 3:

HPTLC analysis of extracted urine exhibits the presence of 17 hydroxy carticosteroids was only detected in the Indian breed cow urine whereas other breed cow, goat and buffalo urine was not exhibited the 17 hydroxy carticosteroids in HPTLC analysis.
Example 4:
Novel method & scheme for loading of sample We have attempted the different method and mechanism for stationery phase systems for separation of specific marker compound in Indian breed cow urine with other breed cow, goat and buffalo urine
We used the cellulose, silica, silica 50.()()() and amino acid bond silica layers i'or the separation. Cellulose, silica, silica 50.000 and amino acid bond silica layers were not give the clear separation of samples. Finally we have developed a novel mechanism for stationery phase systems to eliminate the possibility of reducing the layer activity due to polar solvent and viscosity of the sample. We have taken commercially available plates with uniform thickness coating of silica gel-Ci or silica gel GF254 of uniform particle sizes on aluminum supports. We put the plates in vapors of iodine for few minutes before loading the sample. The pretreatment of the stationery phase give the clear separation due the partition, adsoroption and new anion exchange phenomena.
The treatment with iodine fumes formed the layer between the sample and adsorption media by which we eliminated the possibility of reducing of the layer activity and the sample binding onto the stationery phase due to the viscosity of the sample can be reduced and clear and be which allows the sample to run smoothly in solvent system and the clear and better resolution has been achieved. However use of non treated plates is not recommended because our treatment forms the layer between the sample and stationer} phase by which the binding of the sample can be reduced and clear and better separation can be achieved.

Example -5
The estimation of volatile fatty acids was carried out according to the protocol given in the 20th edition of the standard method APHA. AWWA and WPCF (1998). Ihe Indian cow's urine and other breed cow, goat and buffalo urine was extracted and esterified for the identification of volatile fatty acids using gas chromatography and mass spectrometer..
Ihe m-Methoxybezoic acid at Rt 5.3 was the marker compound in Indian bread cow urine.
We are the first user of scheme for extraction and separation of specific marker for the differentiation between Indian breed cow urine with other breed cow, goat and buffalo urine.

We Claim:
1. Our Claim in general is a novel scheme and method for the detection identification & differentiation between Indian breed cow urine and other breed cow urine, goat and buffalo urine which comprise complete scheme and method for sample preparation for detection and differentiation of Indian breed cow urine and other breed cow urine, goat and buffalo urine, marker isolation, marker development, characterization and identification of detoxifying compound present in Indian bred cow urine.
2. A novel scheme and method for the detection identification & differentiation of Indian breed cow urine with other breed cow, goat and buffalo urine
comprising:
a. Extraction procedure of Indian breed cow urine with other breed cow,
goat and buffalo urine
b. Pre treatment onto stationery phase before the application of Indian breed
cow urine with other breed cow, goat and buffalo urine
c. Development of inno\ati\e proportion of loading solution for sampling of
satwa from Giloy Potato and Maize onto stationery phase .
d. Development of newly designed proportion of solvent system for the
separation of satwa from Giloy Potato and Maize onto stationery phase;
c. Detection of the specific marker for the differentiation Indian breed cow
urine with other breed cow, goat and buffalo urine .
f. Identification of specific marker present in Indian breed cow urine with
other breed cow, goat and buffalo urine.

g. Identification of specific marker presents Indian breed cow urine and
absent in the other breed cow, goat and buffalo urine.
h. Densilometric scanning of separated peaks for the confirmation of pure &
fake samples cow urine.
2. The process as claimed in Claim 1. wherein the stationery phase systems for separation of Indian breed cow urine with other breed cow, goat and buffalo urine, m the vapors of iodine for five minutes before loading the sample.
3. The process as claimed in Claim 1. wherein the mobile phase for solvent system is selected from 1 oluene; Ethyl Acetate: absolute methanol.
4. 1 he process as claimed in Claim 1-H. wherein the Spectrum of each component is considered as individualization character of compound which differentiates between pure & fake cow urine sample.