Protective Activity of Camel ’ s Milk and Urine Mixture ( Camelus dromedarius ) against Ethanol-Induced Hepatotoxicity in Rats

This study was implemented at the University of Khartoum to investigate the protective effect of camel milk mixed with camel urine against alcohol-induced liver damage in Wistar albino rats. The reasons for combining camel milk and urine together are because that in Sudan this combination is used extensively in traditional medicine and they claimed that this combination cures a lot of diseases including liver disease and jaundice; 25 Wister Albino rats of both sexes were brought and divided into 5 groups. Group 1 (control group) received normal saline, group 2 received ethanol 10% at dose of (0.5 g/100g body weight) and group 3 rats received Silymarin (5 mg/100g body weight) plus ethanol 10% (0.5 g/100g). Group 4 received a mixture of camel milk and urine (1:1) at the rate of 2 ml/100g and group 5 rats were administered with the mixture of camel’s milk and urine as in group 4 plus ethanol 10% (0.5 g/100g). All treatments given by an oral intubation, experiment lasts for 28 days, rats were euthanized, serum samples and liver sections obtained. Oral administration of 10% ethanol (0.5 g/100g) induced liver damage which was clear in group 2 in a form of generalized necrosis, fatty change and congestion, beside high elevation of serum enzymes (AST, ALT, and ALP). Giving camel’s milk and urine mixture to group 5 with ethanol 10% (0.5 g/100g), resulted in significant reduction of levels of serum enzymes (AST, ALT, and ALP) and in stabilization of serum metabolites (total protein, albumin and bilirubin). The degree of reduction was lower when significantly compared to (Silymarin + ethanol) group, and all results were verified by the histopathological changes. Hepatoprotective effects of camel’s milk and urine mixture were recorded and this could be attributed to antioxidant activity or to its chelate effects on toxicants. Drinking raw camel milk and urine mixture, How to cite this paper: Elhag, A.E., Bernard, F. and El Badwi, S.M.A. (2017) Protective Activity of Camel’s Milk and Urine Mixture (Camelus dromedarius) against Ethanol-Induced Hepatotoxicity in Rats. Advances in Bioscience and Biotechnology, 8, 378-387. https://doi.org/10.4236/abb.2017.810027 Received: August 9, 2017 Accepted: October 13, 2017 Published: October 16, 2017 Copyright © 2017 by authors and Scientific Research Publishing Inc. This work is licensed under the Creative Commons Attribution International License (CC BY 4.0). http://creativecommons.org/licenses/by/4.0/ Open Access


Introduction
Liver is the first organ to metabolize all foreign compounds and hence it's susceptible to many different disorders [1].Among the compounds provoking such disorders, alcohol is one of the main causes of end-stage liver damage in a form of cellular necrosis and it is the second most common reason for liver transplantation in the United States [2].Both acute and chronic ethanol consumption leads to formation of cytokines, especially TNF-alpha by hepatic Kupffer cells, the increase in tissue lipid peroxidation and the depletion in the tissue glutathione (GSH) [3], all this elements contributing to significant liver injury [4].
Moreover, alcoholic liver damages are related to the increase of serum levels of many biochemical markers like aspartate transaminase (AST), alanine transaminase (ALT), serum alkaline phosphatase (ALP), triglycerides, cholesterol and bilirubin [5].
In a context of low efficiency of available medical treatments for the liver protection from damage or help to regenerate hepatic cells, there is urgent need, therefore, for effective drugs to replace and supplement those in current use [6] [7] [8].In spite of lack of clinical studies, camel milk and urine are regarded traditionally as a high quality drink and since ancient times people use them for curing a number of diseases [9] [10].It has been reported that, the camel's milk and urine has been used to cure diseases caused by chronic imbalance of the liver [11] [12].
In-vivo, designed series of experiments in which mixture of camel urine and milk was given to Albino mice showed no effects on the normal liver and stomach [13], this indicated the biological effect of the mixture is safe product.
Therefore, the aims of this study were to evaluate the protective effect of the mixture of camel's milk and urine on alcohol-induced hepatotoxicity in rats and to make more considerable pharmacological and clinical investigation on the therapeutic activity of this mixture.Medicine, University of Khartoum.After one week adaptation period, housed in cages, maintained in a light room under normal environmental conditions with temperature (28˚C ± 2˚C) plus relative humidity (61%), with free access to water and food, they were divided into 5 groups.All rats were apparently healthy and identified by color tail marks.The research was carried out according to the rules governing the use of laboratory animals as acceptable international.

Collection of Camel's Milk and Urine
Fresh milk was collected from female camels of different ages and lactation periods during early morning milking time, while fresh urine from young female camels (Bekra) (6 month up to 3 years old) was collected early morning during normal urination from Camel Research Centre, University of Khartoum, this was done daily for 28 days during all experiment period.
All treatments were continued for 28 days.

Serum Enzymes and Serum Metabolites
Blood samples were collected from the orbital plexus of rats on days zero, 15 and 29 of the experiment.The markers of liver damages as aspartate aminotransferase (AST), alanine amino transferase (ALT) and alkaline phosphatase (ALP) were determined [14].In addition, serum total protein, albumin and bilirubin concentrations were also determined [15].All samples were analyzed by using commercial kits (Randox Laboratories Ltd., U.K.).

Histopathological Methods
Clinical signs were recorded and the postmortem examination was carried after slaughtering of the rats at day 29.Then 25 livers specimens were collected and fixed in 10% buffered formal saline, and transported to laboratory for histopathological processing and investigations.Specimens embedded in paraffin wax

Statistical Analysis
Data were entered and analyzed using SAS statistical package.Numerical data were expressed as means and standard errors.Significance of difference between means were tested by one-way ANOVA, depending on the number of compared groups; with a p value of ≤0.05 considered statistically significant [16].

Clinical Signs
There were no abnormal clinical signs in all groups except Ethanol rats (group 2) where the rats presented depression and general unthriftiness from day 13 throughout.

Postmortem Findings
Regarding the pathological lesions of liver, the rats from Ethanol group showed fatty changes, slight congestion and adhesion in lobes, while in group 4 (CMCU group) and Ethanol plus CMCU group, no postmortem changes were observed.
A slight fatty change of liver with grey spots was observed in Ethanol plus Silymarin group.

Changes in Serum Enzymes
Table 1, demonstrated the changes in serum enzymes as; in intoxicated rats (group 2), the activities of enzymes AST, ALT and ALP were significantly increased when compared to the untreated control groups while the administration of camel's milk and urine CMCU to intoxicated rats (group 5) resulted in significant amelioration of the levels of the enzymes AST, ALT, and ALP, when compared to the group treated with ethanol (group 2).Moreover, this improvement is higher in Ethanol plus CMCU rats (group5) than in rats treated with Silymarin and ethanol (group 3).

Changes in Serum Metabolites Parameters
Table 2 showed the changes in serum metabolites in treated groups which revealed that, in rats treated with ethanol only (group 2) the concentration of total protein, albumin and bilirubin were significantly increased when compared to the untreated control group 1.Administration of camel milk and urine CMCU to intoxicated rats (group 4) resulted in significant fall in the concentration of total protein, albumin and bilirubin, when compared to the group treated with ethanol (group 2) even more than Ethanol plus Silymarin rats (group 3).

Histopathological Changes
As showed in Figures 1(a)-(d), no pathological changes were observed in con-Advances in Bioscience and Biotechnology  Group 1 (Control); Group 2 (10% Ethanol at 5 g/kg); Group 3 (Silymarin at 50 mg/kg + 10% Ethanol at 5 g/kg); Group 4 (1:1 mixture of camel milk and urine at "2 ml/100gm B. W."); Group 5 (1:1 mixture of camel milk and urine at "2 ml/100gm B. W." + 10% Ethanol at 5 g/kg).Values are means ± standard errors.Means in the same column with the same letters are not significantly different.(P < 0.05).Group 1 (Control); Group 2 (10% Ethanol at 5 g/kg); Group 3 (Silymarin at 50 mg/kg + 10% Ethanol at 5 g/kg); Group 4 (1:1 mixture of camel milk and urine at "2 ml/100gm B. W."); Group 5 (1:1 mixture of camel milk and urine at "2 ml/100gm B. W." + 10% Ethanol at 5 g/kg).Values are means ± standard errors.Means in the same column with the same letters are not significantly different.(P < 0.05).trol group while liver damage was seen in a form of centrilobular necrosis and lymphocytic infiltration in Ethanol groups.In CMCU rats there was almost normal architecture.In Ethanol plus CMCU group, there was slight fatty change and lymphocytic infiltration, while in Ethanol plus Silymarin group there was generalized fatty change.

Discussion
In the current study in which we used the ethanol as a model for chronic hepatotoxicity in rats, a high increase in serum activities of liver enzymes AST, ALT, ALP and metabolites as total protein, albumin and bilirubin in rats treated with ethanol 10%, was recorded indicating that the dose of 0.5 g/100g can produce chronic liver toxicity, this finding was confirmed by histopatholohical finding as centrilobular necrosis and lymphocytic infiltration (Figure 1 were similarly consistent with the findings of many authors who reported that, alcohol (10% ethanol) intoxication significantly increased AST and ALT besides arising of the histopathology of hepatic injury [17].Also, they indicated that exposure of hepatocytes to ethanol alters the membrane structure and functions by increasing the leakage of enzymes into the circulation [18].On the other hand, some of them reported that, excess alcohol consumption has been linked with altered liver metabolism and liver damage [19].At reverse, the results obtained by others found a decrease in serum total proteins and albumin in ethanol administered rats [20].They suggested that this decrease was due to a reduction in the functional ability of liver by ethanol.
In our study treated rats showed, a decrease in serum activities of liver enzymes AST, ALT, ALP besides fall in levels of serum total protein, albumin and bilirubin, maintained near the normal range, especially in rats treated with camel's milk and urine plus alcohol.The degree of reduction was much lower than Ethanol plus Silymarin group.This could indicate improvement of liver function and protection from alcohol toxicity.The same results were reached by using camel's milk only as protection against CCL4 liver damage protocol instead of using alcohol [21].Our findings were confirmed by detection of necrosis, apoptosis, fatty accumulation and inflammatory cells infiltration on histological findings in ethanol treated group, and clearly alleviated in protected rats by camel's milk and urine CMCU (groups 4) while there were no pathological changes in group 5.These results were in agreement with those who suggested that camel's milk intake may play an important role in ameliorating alcoholic liver injury after using ethanol 56% at a dose of 6 g/kg/day [22], and also with the others who investigated the effects of camel's milk on improving the hepatic biochemical and cellular alterations induced by a high-fat, cholesterol-rich diet [23].The same findings were obtained from our previous study with camel's urine alone against alcohol induced liver damage in rats [24].Protection of the liver from chemical injuries was produced by different agents such as herbs as it was reported by many authors who found that if the rats pre-treated with some herbal extracts against CCL4 or paracetamol instead of camel's milk and urine against ethanol 10%, that will significantly controlled the changes in the biochemical parameters, and exhibited a sharp decrease of the levels in the serum enzymes indicators of suffering liver [25]- [30].Also similar results obtained by using montelukast sodium (Cysteinyl-leukotriene type 1 selective receptor antagonist) as hepatoprotective instead of camel's milk and urine [31].
The hepatoprotective effects of camel's milk and urine could be attributed to its possible chelating effects on toxicants specially alcohol [32], or to the high levels of Vitamins C, B2, E, magnesium and other trace elements on camel's milk and urine which may play a major role as antioxidants [33] and found to be useful in preventing the tissues injury caused by toxic agent [34].The biological function of camel urine was supported in literature by many researchers [35] [36] [37] [38].
A. E.

Conclusion
The oral administration of ethanol by a dose of 0.5 g/100g induced chronic damage of liver cells which was manifested biochemically in rats.Camel's milk and urine CMCU induced significant decreases in serum enzymes, indicators of liver suffering, testifying of a certain hepato-protective effect against chronic toxicity induced by ethanol even higher than using the reference drug (Silymarin).However, further studies are necessary, especially clinical investigations in order to confirm the potential effect of camel's milk mixed with urine as beneficial drink for the prevention of chronic liver toxicity.We recommend for future work purifying the macromolecules involved in the protective activity of camel milk and urine mixture, and test the protective effect for the purified portion.

25
Wistar albino rats of both sexes weighing 85 -105 g were used.They were obtained from the Animal House of the Veterinary Research Institute, Animal Resources Research Corporation, Soba, Khartoum.Rats kept within the premises of the Department of Pharmacology and Toxicology, Faculty of Veterinary Advances in Bioscience and Biotechnology

Figure 1 .
Figure 1.Histopathological changes in livers of rats treated with ethanol, camel milk and urine plus Silymarin (Arrowa indicate the followings): (a) = Section of liver cells of Ethanol rats (Group 2) (10% ethanol at 5 g/kg), showing centrilobular necrosis and lymphocytic infiltration; (b) = Section of liver cells of CMCU rats (Group 4) treated with mixture of camel milk and urine at "2 ml/100gm B. W.", showing almost normal architecture; (c) = Section of liver cells of Ethanol/CMCU rats (Group 5) which treated with mixture of camel milk & urine at "2 ml/100gm".+ 10% ethanol 5 g/kg, showing slight fatty change & lymphocytic infiltration; (d) = Section of liver cells of Ethanol/Silymarin rats (Group 3) treated with Silymarin at 50 mg/kg + 10% ethanol at 5 g/kg, showing generalized fatty change.

Table 1 .
Changes in serum enzymes in rats treated with mixture of camel milk and urine against ethanol induced-liver damage.

Table 2 .
Changes in serum metabolites concentration of rats treated with mixture of camel milk and urine against ethanol induced-liver damage.