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Pharmaceutical Biology 2008, Vol. 46, Nos. 10–11, pp. 789–792 Antidiabetic Activity <strong>of</strong> Cow Urine and a Herbal Preparation Prepared Using Cow Urine E. Edwin Jarald, 1 S. Edwin, 1 V. Tiwari, 1 R. Garg, and E. Toppo 1 1 Herbal Drug Research Lab, B. R. Nahata College <strong>of</strong> Pharmacy and Research Centre, Mandsaur, Madhya Pradesh, India Downloaded by [University <strong>of</strong> Gothenburg] at 21:45 17 September 2015 Abstract An herbal preparation prepared by the traditional healers <strong>of</strong> Mandsaur using cow urine and Gymnema sylvestre R. Br. (Asclepiadaceae), Momordica charantia L. (Cucurbitaceae), Eugenia jambolana Lam. (Myrtaceae), Aegle marmelos Correa (Rutaceae), Cinnamomum tamala Buch.-Ham. (Lauraceae), Aloe barbadensis Linn. (Liliaceae), and Trigonella foenum-graecum L. (Leguminosae) is being used in the treatment <strong>of</strong> diabetes. In order to scientifically appraise the claim, this preparation was studied for antidiabetic activity and also compared with the herbal preparation prepared using water. Fresh cow urine was also used in the study to identify the synergistic effect. The preparations were tested for antidiabetic activity in alloxan-induced diabetic rats at two dose level, 200 and 400 mg/kg, respectively. The study was done for a period <strong>of</strong> 21 days. The activity was compared with reference standard, insulin (1 unit/kg, i.p.) and control. The herbal preparations significantly (P< 0.05, P < 0.01) lowered the blood sugar level <strong>of</strong> hyperglycemic rats in a dose-dependent manner. Comparatively, the cow urine preparation showed better activity than did the preparation prepared using water. Fresh cow urine also exhibited significant antidiabetic effect. This study supports the claim <strong>of</strong> the local traditional healers. Keywords: Alloxan monohydrate, antidiabetic, cow urine, herbal preparation. Introduction Diabetes mellitus is a group <strong>of</strong> metabolic diseases characterized by hyperglycemia, hypertriglyceridemia, and hypercholesterolemia, resulting from defects in insulin se- cretion or action or both (Nyholm et al., 2000). Diabetes mellitus is a metabolic disease as old as mankind, and its incidence is considered to be high (4–5%) all over the world. Oral hypoglycemic drugs, such as sulfonylureas and biguanides, have been used in the treatment <strong>of</strong> diabetes mellitus (Okinea et al., 2005). In spite <strong>of</strong> the introduction <strong>of</strong> hypoglycemic agents, diabetes and related complications continue to be a major medical problem. Since time immemorial, patients with non–insulin-dependent diabetes have been treated orally in folk medicine with a variety <strong>of</strong> plant extracts. In India, a number <strong>of</strong> plants are mentioned in ancient literature (Ayurveda) for the cure <strong>of</strong> diabetic conditions known as “madhumeha,” and some <strong>of</strong> them have been experimentally evaluated and the active principles isolated (Som et al., 2001). Cow urine is used along with herbs to treat various diseases like fever, epilepsy, anemia, abdominal pain, constipation, and so forth, by traditional healers all over India (Pathak & Kumar, 2003a; Krishnamurthi et al., 2004). The traditional healers (“Gayathri Parivar”) in Mandsaur use an herbal preparation prepared using cow urine for the treatment <strong>of</strong> diabetes. The traditional healers prepare a decoction using cow urine instead <strong>of</strong> water that contains the following herbs: Gymnema sylvestre R. Br. (Asclepiadaceae), Momordica charantia L. (Cucurbitaceae), Eugenia jambolana Lam. (Myrtaceae), Aegle marmelos Correa (Rutaceae), Cinnamomum tamala Buch.-Ham. (Lauraceae), Aloe barbadensis Linn. (Liliaceae), and Trigonella foenum-graecum L. (Leguminosae). The aim <strong>of</strong> this work was to validate the folk claim. In order to create a logic base behind this treatment, the preparation using cow urine was compared with the preparation using water. Fresh cow urine was also used in this antidiabetic study to investigate the synergistic effect if any. Accepted: April 2, 2008 Address correspondence to: E. Edwin Jarald, Assistant Pr<strong>of</strong>essor, Herbal Drug Research Lab, B. R. Nahata College <strong>of</strong> Pharmacy & Contract Research Centre, Mhow Neemuch Road, Mandsaur 458001, Madhya Pradesh, India. E-mail: ejeru@rediffmail.com DOI: 10.1080/13880200802315816 C○ 2008 Informa UK Ltd.
790 E.E. Jarald et al. Downloaded by [University <strong>of</strong> Gothenburg] at 21:45 17 September 2015 Materials and Methods Procurement <strong>of</strong> materials The urine <strong>of</strong> a 2-year-old virgin Gujarati Indian cow known as “Geer cow” was used in the study. The study was performed after getting a certificate from the veterinary doctor stating that the cow was free from diseases. Fresh cow urine was collected daily and used after filtration. The plant drugs were collected from the Gayathri Parivar (local traditional healers) in order to minimize the variation in the claimed therapeutic effect. The collected plant materials were positively identified by Dr. H.S. Chatree, Botanist, Govt. Arts and Science College, Mandsaur, and the voucher specimens were retained in our department for future reference. Preparation <strong>of</strong> extracts The herbal preparations using cow urine and distilled water were made using the above-mentioned different plant species. Equal quantities <strong>of</strong> air-dried samples <strong>of</strong> each plant species were ground and mixed with 10-times the equivalent volume <strong>of</strong> cow urine and water separately and boiled for 4 h. The extracts were filtered and evaporated in a distillation assembly to get the residue. The percentage yield <strong>of</strong> extracts prepared using cow urine and distilled water was 12.5% and 11.0%, w/w, respectively. Preliminary chemical investigation was carried out in the extracts to identify the nature <strong>of</strong> constituents present in the extracts (Brain & Turner, 1975; Khandelwal, 2005). Animals and treatment After getting approval from the institutional animal ethical committee (reg. no. – 918/ac/05/CPCSEA), male Wistar strain rats (weighing between 150 and 200 g) procured from the animal house <strong>of</strong> B. R. Nahata College <strong>of</strong> Pharmacy, Mandsaur, were used for the investigation. The animals were housed in standard environmental conditions <strong>of</strong> temperature (21 ± 2 ◦ C), humidity (55 ± 10%), and a 12-h light-dark cycle. Rats were supplied with standard pellet diet and water ad libitum. Acute toxicity studies The acute toxicity test <strong>of</strong> the preparations and cow urine was determined according to the OECD guidelines (No. 420, Organization for Economic Cooperation and Development). Female albino mice (20–25 g) were used for this study. Dosing amounts for sample in liquid form were calculated with the help <strong>of</strong> density or specific gravity. After the sighting study, a starting dose <strong>of</strong> 2000 mg/kg (p.o.) <strong>of</strong> the test samples was given to various groups <strong>of</strong> five animals each. The treated animals were monitored for 14 days for mortality and general behavior. No deaths were observed through the end <strong>of</strong> the study. The test samples were found to be safe up to the dose <strong>of</strong> 2000 mg/kg, and doses <strong>of</strong> 200 and 400 mg/kg were chosen for further experimentation. Antihyperglycemic activity Diabetes was induced in rats by injecting 150 mg/kg <strong>of</strong> alloxan monohydrate intraperitoneally in 0.9% w/v NaCl (Ainapure et al., 1985; Porchezian et al., 2000). Seventy-two hours after injection, blood glucose level was measured, and the diabetic rats were divided into eight groups <strong>of</strong> six animals each. Insulin [1 unit/kg (i.p.)] was used as standard drug (Mukherjee, 2002). The first group was kept as vehicle control, the second was treated with insulin, and the third to eighth groups were treated with herbal preparations prepared using cow urine, distilled water, and pure cow urine at two dose levels, 200 and 400 mg/kg (p.o), respectively. One more group was included in the study to determine the effects <strong>of</strong> fresh cow urine in the blood glucose level <strong>of</strong> normal rats. Fresh cow urine at a dose <strong>of</strong> 400 mg/kg was given to the rats in this group for 21 days. The treatment was given once daily for 21 days. Blood samples were collected at regular intervals after fasting overnight, before treatment, from rat-tail vein under mild anesthesia and monitored. The blood sugar level was monitored using Accu-chek Active Test strips in Accu-chek Active Test meter (Roche Diagnostics, Germany). Statistical analysis Data were expressed as mean ± SEM, and the obtained data were subjected to one-way ANOVA followed by Dunnet’s test. The p values less than 0.05 were considered as significant. Results The phytochemical investigations performed in the extracts revealed the presence <strong>of</strong> alkaloids, tannins, flavonoids, carbohydrates, and saponins in both the extracts. The results <strong>of</strong> antidiabetic activity <strong>of</strong> cow urine and herbal preparations prepared using cow urine and water are presented in Table 1. The basal blood glucose levels <strong>of</strong> all the groups were statistically not different from each other. Three days after alloxan administration, blood glucose values were 5-fold higher in all the groups and were not statistically different from each other. After 21 days, values <strong>of</strong> blood glucose were decreased in all the treatment groups (P < 0.05, P < 0.01). The value in diabetic control group remained stable. The preparations exhibited activity in a dose-dependent manner. The activities <strong>of</strong> the preparations were found significant from the 7th day onwards, whereas the activity <strong>of</strong> cow urine was found significant only after 21 days <strong>of</strong> treatment. Normal rats treated with cow urine for 21 days did not show any elevation in their blood glucose levels. Comparatively, the preparations containing cow urine were found to be better than the herbal preparation prepared using distilled water.
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