II. REVIEW OF LITERATURE

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1 II. REVIEW OF LITERATURE 2.1 Composition of urine Burger and Smith (1987) opined that composition and characteristics of cow urine were directly related to the diet of animals. Abigail et al. (2001) compared the urine composition of healthy Labrador Retrievers and Miniature Schnauzers. They found that differences in urine composition exist between breeds fed the same diet. Cow s urine was found to contain various inorganics, including silver, traces of gold, Na-K in ratio of 4:1 (36%: 9% in dried urine), apart from about 3% urea. Further important findings were, that fresh cow-urine contains mg oestrogens/100 ml; µg of cortico-steroids/100 ml and mg of 17- keto-steroids/100 ml (Apte, 2002). Kumar (2001) studied the various biochemical constituents in the urine of cow, buffalo and goat and concluded that there was a species variation in levels of biochemical constituents. Jane et al. (2006) assessed the impact of preservation, storage temperature and time since collection on nonpersistent contaminants in urine samples. In spiked urine samples stored in three types of urine vacationers (no preservative, boric acid and chlorhexidine), they measured five groups of contaminants to assess the levels of these analytes at five time points (0, 24, 48, and 72 h and 1 week) and at two temperatures (room temperature and 41 0 C). The target chemicals were bisphenol A (BPA), metabolites of organophosphate (OP), carbamate and pyrethroid insecticides, chlorinated phenols and phthalate monoesters and were measured using five different mass spectrometry-based methods. Three samples were analyzed at each time point, with the exception of BPA. Repeated measures

2 analysis of variance was used to evaluate effects of storage time, temperature and preservative. Stability was summarized with percent change in mean concentration from time 0. In general, most analytes were stable under all conditions with changes in mean concentration over time, temperature and preservative being generally less than 20%, with the exception of the OP metabolites in the presence of boric acid. The effect of storage temperature was less important than time since collection. The precision of the laboratory measurements was high allowing observation of small differences, which may not be important when categorizing individuals into broader exposure groups. Loomis and Kromhout (2004) were able to identify very small differences in urine concentration, generally much smaller than the between individual variation. Iona et al. (2004) estimated the total protein in human urine for quality assurance in the determination of human urinary proteins. The material was prepared by freeze-drying of a pooled urine sample obtained from healthy volunteers. Protein concentrations in both batches were within physiological values, close to their upper limit. Results obtained by the individual methods were significantly different due to various reaction mechanisms involved. It was therefore necessary to certify the mean values and their uncertainties individually for each method. Ketone body concentrations were determined in six body fluids of cows in late pregnancy and their fetuses (blood plasma and urine of cows, amniotic and allantoic fetal fluids and fetal blood serum and urine) using the head-space gas chromatographic method (Hradecky and Kudla, 2005). Ketone bodies were below 10% in cow blood plasma and urine, fetal serum and urine and allantoic fluid and below 20% in amniotic fluid. Kirchmann and Pettersson (1994) found that stored human urine had ph values of 8.9 and was composed of eight main ionic species (> 0.1 meq L 1 ), the

3 cations Na, K, NH 4, Ca and the anions, Cl, SO 4, PO 4 and HCO 3. Nitrogen was mainly (> 90%) present as ammoniacal N, with ammonium bicarbonate being the dominant compound. Urea and urate decomposed during storage. Heavy metal concentrations in urine samples were low compared with other organic fertilizers, but copper, mercury, nickel and zinc were times higher in urine than in precipitation and surface waters. Edin-Liljegren et al. (1992) evaluated how the time needed to increase urine ph varies between different urine samples and how it depends on urine composition when, a standardized amount of urease was added to different human urine samples. Rajanarayanan and Archunan (2004) indicated clearly that the occurrence of specific pheromonal compound (s) present in the urine and vaginal mucus during estrus significantly influence the flehmen behaviour in male buffaloes. Salwa et al. (2004) have done work on physical and biochemical contents of camel, cattle, goat and human urine. They used twenty-eight urine samples which were collected from healthy, male and female, young and adult camels (Camelus dromedarus), cattle, goat and human. Samples were physically, chemically, microscopically and biochemically examined for determination of ph, specific gravity, chloride, phosphate, sulphate, bicarbonate, uric acid, urea, creatinine, creatine, total protein, albumin, macro elements (Ca ++, Mg +, Na + and K + ) and micro elements (Zn ++, Co ++ ). 2.2 Uses Council for Scientific Industrial Research (CSIR), India had obtained US patent (No / ) for cow urine distillate, which claimed a novel pharmaceutical composition (Khanuja, 2002) and is effective as an antimicrobial and antifungal. Panchagavya is one such formulation mentioned in Ayurveda,

4 which is prepared with five components derived from cow viz., milk, curd, ghee, urine and dung. These formulations were claimed to be useful against liver disorders, fever and inflammations and has hepatoprotective effect in toxicity induced in rats (Achliya et al., 2003) and immunostimulant activity in rats (Fulzele et al., 2003). During the past few years, cow urine therapy has provided promising and authentic results for the treatment of cancer, a deadly malady which is being faced by the mankind and the incidences of which are ever increasing in the current scenario of changed lifestyle and food habits along with exposure to predisposing factors of carcinogens such as tobacco chewing, smoking, alcohol intake, environmental pollutants, occupational health hazards etc. Anti-cancer potential of cow urine therapy has been reflected by several case reports, success stories and practical feed back of patients for the treatment of cancer. Cow urine enhances the immunocompetence and improves general health of an individual; prevent the free radicals formation and acts as anti-aging factor; reduces apoptosis in lymphocytes and helps them to survive; and efficiently repairs the damaged DNA, thus is effective for the cancer therapy (Dhama et al., 2005). The redistillate of cow's urine was found to possess total antioxidant status of around 2.6 µmol, contributed mainly by volatile fatty acids (1500 mg/l) as revealed by the GC-MS studies. These fatty acids and other antioxidants might cause the observed protective effects (Krishnamurthi et al., 2004). Garg et al. (2005) evaluated the effect of distilled cow urine on the nutrient utilization by the white leghorn layers. The results showed that there was increase in feed intake, decreased feed conversion ratio and feed efficiency ratio. Digestibility of dry matter, crude protein, ether extract, crude fiber and organic matter increased significantly in the cow urine treated group.

5 Mathivanan et al. (2006) conducted an experiment on study the effect of panchagavya and Andrographis paniculata as alternatives to antibiotic growth promoter on broiler production and carcass characteristics. The result revealed that the mean body weight and weight gain of panchagavya and Andrographis paniculata fed groups were significantly (P<0.05) higher. Achliya et al. (2003) studied hepatoprotective effect of panchgavya and found that treatment with panchgavya in doses ( mg/kg) significantly prevented the rise in the levels of serum marker enzymes viz., GPT, GOT, ACP and ALP and the results obtained were comparable with those of silymarin treated group. Dutta et al. (2006) studied the anticlastogenic effect of redistilled cow's urine distillate (RCUD) in human peripheral lymphocytes (HLC) challenged with manganese dioxide and hexavalent chromium. The redistilled cow's urine distillate posseses strong antigenotoxic and anticlastogenic properties against HPNLs and HLC treated with Cr+6 and MnO 2. This property is mainly due to the antioxidants present in RCUD. Oyekan and Laniyonu (1984) studied the effects of cow's urine concoction (CUC) with cow's urine as the diluting medium and of the same concoction with redistillate water as the diluting medium (CUPRw), together with each of the constituents in both media were assessed on such parameters as reaction to spinal cord reflexes e.g. startle, nociception and righting, limb abduction, locomotor anomaly and limb paralysis. The results showed that the CUPR affected all the parameters positively while the CUPRw also had positive effects on the parameters with the exception of response to startle reflex. Cow's urine alone gave no appreciable results, neither did the administration of some other constituents of the concoction in distilled water or cow's urine affected the parameters appreciably.

6 Oyebola and Ariwodola (1984) studied the effect of ganglionic blockade and adrenalectomy on the cardio-respiratory response to cow's urine concoction in rats. It was established that in CUC-induced death, respiratory failure precedes circulatory failure. However, failures of both respiration and circulation were the causes of death from CUC. Oyebola and Ariwodola (1985) studied the influence of alpha and beta adrenoceptor blockers on the cardio-respiratory effects of cow's urine concoction (CUC) in rats. The study showed that phenoxybenzamine abolished the pressor response observed in the untreated animal while propranolol had no effect on blood pressure response. The alpha adrenoceptor blocked animals also developed significant post-injection hypotension. CUC caused an initial depression of respiration with short apnoeic phases and an increase in pulmonary ventilation. The latter was significantly decreased by alpha and beta adrenoceptor blockers. Achliya et al. (2004) studied neuropharmacological actions of panchagavya formulation containing Emblica officinalis Gaerth and Glycyrrhiza glabra Linn. in mice. The results suggested that the panchagavya formulation is sedative in nature. Khan and Srivastava (2005) illustrated the antitoxic and bioenhancing role of Kamdhenu Ark (cow's urine distillate) in fertility rate of male mice affected by cadmium chloride toxicity. They found that, animals exposed with CdCl 2 only showed 0% fertility rate. However, the animals exposed with CdCl 2 +Kamdhenu Ark+ZnSO 4 showed 90% fertility rate with 100% viability and lactation indices. Besides this, the fertility index was also found to be 88% in group treated with CdCl 2 +Kamdhenu Ark. Thus, these results indicated that Kamdhenu Ark work as an antitoxic against the cadmium chloride toxicity and it can be used as a bio enhancer of Zn.

7 The effect of camel urine on the cytological and biochemical changes induced by cyclophosphamide in mice was also studied (Harbi et al., 1996). 2.3 Toxicity Oyebola (1983) presented a review of current information on the composition, pharmacological actions and mode of death from cow's urine concoction toxicity. The chemical composition and pharmacological actions of the individual components of CUC are also reviewed. Observations of CUC poisoning in man and experimental animals showed that the main effects of CUC are severe depression of respiration, cardiovascular system, the central nervous system and hypoglycemia. These toxic effects acting singly or in combination are believed to be the cause(s) of death from CUC. Research on the effects of variation in the quantities of the constituents of cow's urine concoction on its acute toxicity in mice carried out by Oyebola and Adetuyibi (1979). Oyebola and Elegbe (1975) carried out experimental investigations on toxicity in mice by cow urine concoction and the effect of the concoction was compared with that of pure nicotine. The effect of each component used in preparing the concoction was also tested. Results of the experiments shown that both "cow's urine" and nicotine cause excitement in low doses and cause convulsion and/or death in higher doses. In addition depress respiration. Elegbe and Oyebola (1977) reported cardio respiratory adverse effects of cow's urine in dogs. Ayorinde et al. (1982) noted severe poisoning when cow s urine concoction was administered to Yoruba-speaking people of Nigeria where cow s urine concoction commonly used in convulsing patients.

8 Jimoh et al. (2000) studied the effect of chronic consumption of cow's urine concoction (CUC) and the role of tobacco leaves on the gastric mucosa of albino rats. The weight of the rats were monitored over the four weeks period. The histology of the mucosa was analyzed after staining with haematoxylin and eosin dyes. The results showed changes in the staining characteristics and growth retardation in all the three groups exposed to the formulations. Elegbe et al. (1976) found the cardio toxic effects of cow's urine concoction on anaesthetized cats in Nigeria. Bhalla and Thami (2005) observed the skin lesions a few hours after the second dose of gomutra gargle followed by ingestion. There was no past or family history of urticaria, angioedema and food or drug allergy. Although the patient had used cow's milk and milk products in the past without any allergy, he apparently had had no exposure to cow's urine. 2.4 Patents Cow urine has been granted U.S. Patents (No and ) for its medicinal properties, particularly for its use along with antibiotics for the control of bacterial infection and fight against cancers. U.S. Patent, titled "Pharmaceutical compositions containing cow urine distillate and an antibiotic" issued to S.P.S. Kanuja and 13 others and assigned to the Council of Scientific and Industrial Research (CSIR), attracted global attention. This invention related to new use for a known and abundantly available cow urine distillate as an enhancer of antibiotic action on the target. The molecule of invention helps in the absorption of antibiotics across the cell membrane in animal cells, gram positive and gram negative bacteria. Similar activities can also be obtained by using the distillate of the urine of cow at C and from the concentrate, which is lyophilized and dissolved for further use. Further the urine distillate from buffalo, camel and deer provides similar activity on bioavailability.

9 A pharmaceutical composition comprising of an antibiotic and cow urine distillate in an amount effective to enhance antimicrobial effect of the antibiotic is disclosed. The antibiotic can be an antifungal agent. The antimicrobials can be a quinolone or a fluoroquinolone. The antifungal agent can be azoles, clotrimazole, mystatin or amphotericin. When the antibiotic is an anti-tuberculosis agent, selected from the group consisting of isoniazid, pyrazinamide and ethambutal, anti-tuberculosis effect of said agent is enhanced 2 to 80 folds. United States Patent No was given for use of animal urine for efficient and quality vermicomposting and recycling slow degrading and unconventional substrates and the process for the same. This patented invention relates to an efficient process of vermicomposting and production of high-quality vermicompost from agro-waste (including distillation waste) using animal urine such as cattle urine. 2.5 Immunological effect Olusi and Ojewole (1978) found that oral administration of cow's urine concoction in rats causes the activation of the third complement component in the serum. The product of this activation has some histamine-releasing effects and causes a characteristic acute fall in neutrophil and monocyte counts in the peripheral blood which is reversed within four hours. The importance of these observations to the areas of the world where cow's urine concoction is used for the treatment of childhood convulsion is discussed. Kumar et al. (2004) investigated 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 in chicks. The increase in lymphocyte proliferation activity was maximum during first two weeks of development. During the experimental period cow urine enhanced the T- and B-

10 cell blastogenesis by 1.81% and 2.21% respectively. However, Chauhan et al. (2001) reported that cow urine significantly enhances T- and B-cell proliferative activity in mice. Ylonen et al. (1992) reported that a total of two main allergens were found in cow dander (20 and 22 kd) and one in cow urine (20 kd). The 20 kd component was shown to be the most important allergen in cow antigen extracts. 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 diseases (Chauhan et al., 2004). The specificity of immune system depends upon the number and activity of lymphocytes. Chauhan et al. (2001) 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 (2001) and Chauhan et al. (2004) reported increase in both cellular and humoral immune responses due to cow urine. 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. They showed cow urine marginally upregulates lypmphoblastogenesis in developing stages of chicks. This means immune system develops at an early stage and neonatal mortality can be reduced with the use of cow urine. Hayakawa and Takenaka (1999) examined the potential for preparing template DNA in polymerase chain reactions (PCR) from urine in Japanese macaques (Macaca fuscata). Microsatellite band patterns from urine samples showed close agreement with those of blood and fecal samples, and only a few hundred micro litre of urine yielded a template DNA for PCR.

11 2.6 Agriculture uses Urine has applications in gardening and agriculture as a fertilizer. Gardeners often recommend a dilution of parts water to one part of urine for application to pot plants and flower beds during the growing season; pure urine can chemically burn the roots of some species. Urine typically contains more than 50% of the nitrogen and phosphorus and potassium content of whole sewage, and is widely considered as good as or better than commercially-available chemical fertilizers or stabilized sludge from sewage plants. Urine is also used in composting to increase the nitrogen content of the mulch, accelerating the composting process and increasing its final nutrient values. Urine is also being actively considered as a fertilizer for use in food-crop agriculture in developed countries. Studies into its feasibility and safety usually indicate that it is an acceptable alternative to chemical fertilizers and stabilized sludge. However, the technology to implement its use on a large scale has not been developed, and is considered too expensive. There are also concerns over its safety regarding the potential for transmitting infectious disease and refluxing xenobiotic compounds (associated with toilet-cleaning products and prescribed drugs expelled in urine) in the human food chain. Proponents of adopting urine for this use usually claim the risks to be negligible or acceptable, and point out that sewage causes more environmental problems when it is treated and disposed of compared with when it is used as a resource. Critics generally agree that more research is needed into how the resource is to be collected, processed and handled. A few people use urine as a crop fertilizer. These include organic farming cooperatives and eco-villages where special urine-diverting toilets with collecting tanks are installed. Many of these also employ concepts such as grey water irrigation and the composting of fecal matter. Many are the subject on ongoing feasibility studies sanctioned by governments and private organizations. These

12 people generally reject safety concerns over its use on food crops provided that it is used with common sense. For example, application to fruit trees is considered safer than to bushes and especially root crops. It is also considered sensible to cease application at a safe interval before harvesting. However, the use of urine for this purpose is even rarer than its use on ornamental gardens. In developing countries, the application of pure urine to crops is also rare. However, whole, untreated sewage, termed night soil, is often applied to crops and is considered essential. It is worth noting that this practice is not new and has been applied, along with crop rotation schemes, for thousands of years. Ruchi et al. (2005) studied the Molluscicidal activity of cow urine alone and with different additives against Lymnaea acuminata. The toxicity of cow urine kept for 15 days in sunlight (8 h/day) or ambient laboratory conditions against the snail was time - and concentration-dependent. The ph of fresh cow urine is 7.4 which changes to 10.9 when kept for 15 days in laboratory; exposure to sunlight for the same period alter the ph to Binary combinations (1:1) of freezedried cow urine kept for 15 days separately with each of Allium sativum (Liliaceae) bulb powder, Azadirachta indica (Meliaceae) oil, Annona squamosa (Annonaceae) seed powder, Ferula asafoetida (Apiaceae) root latex and tea leaves, and Camellia sinensis (Theaceae) were more toxic to the snail than treatment with urine alone. Additives to cow urine in sunlight were more effective than those under laboratory conditions. The effects of dairy cow urine and defoliation severity on biological nitrogen fixation and pasture production of a mixed ryegrass-white clover sward were investigated (John et al., 2003). This study showed that urine has a prolonged effect on reducing N 2 fixation in pasture. In addition, defoliation severity is a potential pasture management tool for strategically enhancing N 2 fixation.

13 Saadullah et al. (1981) determined the effect of treating straw with animal urine. Due to treatment with urine, the crude protein content of the straw was improved from 3.3% to 5.6%. The nitrogen balance for 24 hours was improved from 2.94 g to 1.15 g. Dry matter, organic matter and crude fibre digestibilities went up. The intake of digestible dry matter went up by more than 70%. It is concluded that animal urine can be as efficient as urea as source of urea for treatment.