Abstract
An herbal preparation prepared by the traditional healers of 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 of 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 of 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 of 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 of the local traditional healers.
Introduction
Diabetes mellitus is a group of metabolic diseases characterized by hyperglycemia, hypertriglyceridemia, and hypercholesterolemia, resulting from defects in insulin secretion or action or both (CitationNyholm 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 of diabetes mellitus (CitationOkinea et al., 2005). In spite of the introduction of 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 of plant extracts. In India, a number of plants are mentioned in ancient literature (Ayurveda) for the cure of diabetic conditions known as “madhumeha,” and some of them have been experimentally evaluated and the active principles isolated (CitationSom 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 (CitationPathak & Kumar, 2003a; CitationKrishnamurthi et al., 2004). The traditional healers (“Gayathri Parivar”) in Mandsaur use an herbal preparation prepared using cow urine for the treatment of diabetes. The traditional healers prepare a decoction using cow urine instead of 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 of 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.
Materials and Methods
Procurement of materials
The urine of 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 of extracts
The herbal preparations using cow urine and distilled water were made using the above-mentioned different plant species. Equal quantities of air-dried samples of each plant species were ground and mixed with 10-times the equivalent volume of 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 of 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 of constituents present in the extracts (CitationBrain & Turner, 1975; CitationKhandelwal, 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 of B. R. Nahata College of Pharmacy, Mandsaur, were used for the investigation. The animals were housed in standard environmental conditions of 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 of 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 of density or specific gravity. After the sighting study, a starting dose of 2000 mg/kg (p.o.) of the test samples was given to various groups of five animals each. The treated animals were monitored for 14 days for mortality and general behavior. No deaths were observed through the end of the study. The test samples were found to be safe up to the dose of 2000 mg/kg, and doses of 200 and 400 mg/kg were chosen for further experimentation.
Antihyperglycemic activity
Diabetes was induced in rats by injecting 150 mg/kg of alloxan monohydrate intraperitoneally in 0.9% w/v NaCl (CitationAinapure et al., 1985; CitationPorchezian et al., 2000). Seventy-two hours after injection, blood glucose level was measured, and the diabetic rats were divided into eight groups of six animals each. Insulin [1 unit/kg (i.p.)] was used as standard drug (CitationMukherjee, 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 of fresh cow urine in the blood glucose level of normal rats. Fresh cow urine at a dose of 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 of alkaloids, tannins, flavonoids, carbohydrates, and saponins in both the extracts. The results of antidiabetic activity of cow urine and herbal preparations prepared using cow urine and water are presented in .
Table 1. Effect of various preparations in alloxan-induced diabetic rats.
The basal blood glucose levels of 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 of 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 of the preparations were found significant from the 7th day onwards, whereas the activity of cow urine was found significant only after 21 days of 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.
Discussion
Cow, Bos indicus is a most valuable animal in all Veda; it is called “the Mother of all.” A composition containing cows excretions—urine, dung, milk, curd, and ghee—five ingredients together known as “panchagawya,” is given to women after delivering a baby. Panchagawya is the main ingredient of many Ayurvedic preparations (CitationPathak & Kumar, 2003b). Cow urine, one of the ingredients in panchagawya, is believed to have many therapeutic values. In India, cow urine is used by the majority of the rural population as a folklore remedy in almost all the states. Agencies in Gujarat have been marketing cow urine preparations from multiple outlets, advertising that they are sterilized and completely fresh, with prices ranging from Rs. 20 to Rs. 30 per bottle. Keeping in view the enormous role of cow's urine in medicinal and veterinary medicine, a scientific experiment was performed in rats to elucidate the effect of cow urine and cow urine containing preparation as an antidiabetic.
Alloxan produces hyperglycemia by a selective cytotoxic effect on pancreatic beta cells. One of the intracellular phenomena for its cytotoxicity is through generation of free radicals demonstrated both in vivo and in vitro (CitationYadav et al., 2002). Our investigations indicate the efficiency of the herbal preparations in maintaining blood glucose levels in alloxan-induced diabetic rats. The glucose-lowering activity observed in diabetic animals may be due to stimulation of beta cells of pancreatic islets or stimulation of glycogenesis (CitationMiura et al., 2001). This may be due to the presence of some hypoglycemic principles in the plants used in these preparations because all these plants are well known for their antidiabetic action (CitationGrover et al., 2002; CitationKar et al., 2003; CitationMohamed et al., 2006; CitationPulok et al., 2006), and these plants have different types of mechanisms in reducing blood glucose levels. Comparatively, the preparation using cow urine was found to exhibit better activity than did the one prepared using distilled water. This could not be correlated with the nature of the phytoconstituents present in the extracts because both extracts contains the same nature of constituents. The interesting observation in our study was the antidiabetic activity of pure cow urine. The hypoglycemic effect was not observed in the normal rats treated with fresh cow urine, and this indicates that the possible mechanism behind the antidiabetic effect of fresh cow urine may be due to its stimulation in peripheral use of glucose. According to literature, cow urine was found to exhibit an antioxidant effect (CitationKrishnamurthi et al., 2004). Free radicals are implicated in wide range of diseases including diabetes; the antioxidant activity of cow urine also may be one of the reasons for its observed antidiabetic effect.
Chemoprofiling of cow urine in our laboratory confirmed the presence of protein, urea, uric acid, creatinine, phenol, aromatic acids, enzymes such as acid phosphatase, alkaline phosphatase, amylase, and vitamins (CitationGowenlock & McMurray, 1988). Along with these, there may be some other constituents that may be responsible for the observed activity. From these observations, it was clear that the better activity of herbal preparation prepared using cow urine may be due to its synergistic effect with cow urine or, according to ancient literature, cow urine is a wonderful solvent for extraction, and so it is the ability of cow urine to extract out more active constituents from the herbal drugs and thereby increase antidiabetic activity.
Further pharmacological investigations are needed to elucidate the mechanism of the observed antihyperglycemic effect. This study supports the claim of the traditional healers of Mandsaur.
Acknowledgement
The authors are thankful to Gayathri Parivar (local traditional healers) for providing the necessary information to carry out this research work.
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