MODULATING EFFECT OF CAMEL’S MILK ON ALLOXAN-INDUCED EXPERIMENTAL DIABETES IN LABORATORY ANIMAL MODEL

MODULATING EFFECT OF CAMEL’S MILK ON ALLOXAN-INDUCED EXPERIMENTAL DIABETES IN LABORATORY ANIMAL MODEL

RASHA G. SAYED^*; AHMED ABDEL-HAMEID AHMED^*; NASR-ELDIN M. AREF^** and MOHAMMED SAYED^*

^* Department of Food Hygiene, Faculty of Veterinary Medicine, Assiut University, Assiut 71526 – Egypt.

^** Department of Animal Medicine, Faculty of Veterinary Medicine, Assiut University, Assiut 71526 – Egypt.

   E-mail: nasreldeen.aref@vet.au.edu.eg

INTRODUCTION

Diabetes mellitus is a global major health problem (Macedo et al., 2002), characterized by metabolic disorder and hyperglycemia results from insufficient insulin hormone with or without abnormal resistance to insulin effect (Tirerney et al., 2002).It becomes the third killer of mankind after cancer and cerebrovascular & cardiovascular diseases (Li et al., 2004). It is also one of major endocrine dysfunctions in pet animals (Davison et al., 2005; Catchpole et al., 2008).

In modern medicine, no satisfactory effective therapy is available to cure diabetes mellitus, although it can be managed by insulin treatment. Pharmaceutical drugs used in diabetic therapy are effective; however they may have undesirable side effects or contraindications. Additionally, they are too expensiveand several patients might have needle phobia (Pari and Satheesh, 2004). All these factors force diabetic patients to adopt alternative therapies. Therefore, the research for more effective and safer hypoglycemic agent has continued to be an area of an active research (Lemhadri et al., 2004; Stanely–Prince et al., 2004).

Beside the nutritive value of camel’s milk which is extremely well adapted for human requirements, it appears to be an additional factor as medical or natural healing properties. Using camel’s milk for treatment is itself a point of contention. Historically camel’s milk has been used for a number of medical problems and according to FAO, camel's milk is the healthiest milk produced by animals (Yagil, 1982).Its consumption may help in reducing the nutritional deficiencies and morbidities in adult communities (Singh et al., 2009). It was found that one of camel's milk proteins has many characteristic similarities with insulin (Beg et al., 1986). Radioimmunoassay tests of camel's milk has revealed high concentration of insulin i.e. 52 micro unit/ml (Agrawal et al., 2003).

Against this background, the present study was carried out to investigate the therapeutic effect of camel’s milk on diabetes mellitus in diabetic-induced animal model and compare its effect with the cow's milk.

MATERIALS and METHODS

Animals

Thirty five female white albino rats of approximately same age group (8-9 weeks old) weighing between 120-150 g were included in this study. All rats were obtained from Laboratory Animals House, Faculty of Medicine, Assiut University. Standard operating procedures for handling of animals in compliance with the guidelines and recommendations for the Institutional Animal Care and Use Committee (IACUC) of Assiut University were adopted.Animals were kept on commercial rat chow and water ad libitium for one week before initiating the experiment to acclimatize the laboratory condition (temperature 22 ^oC, relative humidity 50%, 12 h light and 12 h dark) (Agrawal et al., 2005).

Milk samples

Camel's milk was obtained from she-camels (Camelous dromedaries) belonging to individuals, while cow's milk was obtained from Veterinary Teaching Hospital, Assiut University-Egypt.

Both types were used without any treatment or dilution. The clot on boiling and acidity of the milk samples were checked to monitor the freshness.

Experimental Design

1. Animal groups

a)   Optimizing group: 20 rats were used to determine the diabetic dose of alloxan monhydrate (EL-Gomhorya Co, Egypt) that develops stable diabetes without toxicity. Dose-response relationship was established at 100, 140, 180 and 200 mg/kg B.W. Each dose was administered into 5 rats.

b)   Experimental group: 15 rats were used to test the effect of camel’s and cow’s milk in diabetic rats.

1)   Subgroup 1: Five diabetic rats received no milk (positive control).

2)   Subgroup 2: Five diabetic rats received camel’s milk

3)   Subgroup 3: Five diabetic rats received cow’s milk

Milk was given to them daily by oral cannula (20 ml) in additions to free milk ad libitium.

2. Experimental induction of diabetes and studying the antidiabetic effect of camel’s and cow’s milk.

2.1. Determination of the diabetic dose of alloxan monohydrates in dose response relationship

Alloxan monohydrate has selective destructive cytotoxic effect on the cells of pancreas (Bolaffi et al., 1986). To induce stable diabetes, briefly four different doses (100, 140, 180 or 200 mg/kg B.W.) of alloxan monohydrate dissolved in sterile normal saline were freshly prepared and each dose was injected intraprotenially (Fig. 1) into overnight (16-18 h) fasted rats (n = 5) at 0.5 ml/rat. Animals were kept on glucose 5% for the next 24 h to avoid hypoglycemia that might be occurred (Tatiya et al., 2010).

2.2. Studying the antidiabetic effect of camel’s and cow’s milk

Fifteen rats were injected with the optimal dose of alloxan (140 mg/kg B.W.). These animals were divided into 3 subgroups (5 rats each): 1^st subgroup received no milk (positive control), 2^nd group received camel’s milk and 3^rd one received cow’s milk (Fig. 2). All animals were weekly monitored for their BGLs until the end of the study (5^th weeks). Succumbed rats before 5 weeks were recorded as unrecovered diabetic rat.

3. Blood samples

Blood samples were collected (0.5 ml/collection) weekly from the retro-orbital plexus of all animals using heparinized micro-capillary glass tubes into a clean dry plain Epindorf's tubes (Fig. 3). Samples were left to clot at room temperature for 30 min and centrifuged for 10 min at 4500 rpm for separation of the sera. Sera were aspirated by a micropipette into stopper Epindorf's tubes and BGL were determined immediately using glucose test kits.

4. Determination of blood glucose level (BGL)

Glucose oxidase method was adopted to determine BGL spectro-photometerically at 505 nm using a commercially available test kit (Biotechnology, Egypt).

5. Statistical analysis: Generated data of BGL from various groups were subjected to statistical analysis using SPSS (1999) program for windows version 10.0.1 (SPSS Inc., Chicago, IL) for calculation of mean and standard error.

RESULTS

Results of the effect of different doses of alloxan monohydrate on BGL were presented in Table 1 and depicted in Fig. 4 & 5 while the results of the effect of cow’s milk on diabetic group was presented in Table 2 and Fig. 6 compared to the modulating effect of camel’s milk (Table 2 and Fig. 7 & 8).

Table 1: The Effect of Different Doses of Alloxan Monohydrate on Blood Glucose Level

* Morbidity rate is the number of animals showed high BGL/total number of animals

N.B. Rats gave fasting blood glucose level > 250 mg/dl and remained alive after 2 weeks were considered positive stable BGL and included in the presented study.

** Mortality rate is the number of dead animals/total number of animals

Table 2: Weekly Mean ± SE of Blood Glucose Level (mg/dl) in Different Groups

DISCUSSION

Test 1: Optimizing the dose of alloxan monohydrate

Before initiating the experiment, the baseline for BGL was estimated in healthy animals to be 79.58 ± 2 mg/dl. Alloxan monohydrate at dose of 100, 140,180 and 200 mg/kg B.W. were injected intraperitoneally to the experimental rats. The data presented in Table 1 demonstrated that BGL at 100 mg/kg B.W. of alloxan monohydrate was 90-120 mg/dl and considered as suboptimal to induce diabetes BGL at 100 mg/kg BW was lower than what was previously recorded by Sushruta et al. (2006) and Dhasarathan and Theriappan (2010). The other 3 doses of alloxan monohydrate showed varying degree of severity of hyperglycemia. In the present study, alloxan monohydrate at dose 200 mg/kg B.W. was lethal and led to death of all rats within 3 days from injection. These animals suffered from severe loss of body weight, polyurea, diabetic coma and death. On contrary, Tripathi and Chandra (2009) found that 140–200 mg/kg B.W. alloxan doses were reported as non lethal. Our results also revealed that animals injected with alloxan at dose 180 mg /kg B.W. exhibited high fasting BLG with high mortality, while at dose 140 mg/kg B.W. resulted in a diabetic state (80 % morbidity) and no mortality. Alloxan monohydrate at dose 140 mg/kg B.W. was selected as optimal dose to induce stable diabetes in rats.

Test 2: Effect of camel's milk in comparison with cow's milk on alloxan-induced diabetic rats

This study was performed to observe the role of camel's milk in achieving glycemic control in alloxan-induced diabetic rats. The results of this experiment were presented in Table 2. During the experimental period, the initial mean BGL in diabetic control group was 399.5 ± 18.3 mg/dl and increased by the 5^th week to 488.2 ± 20.8 mg/dl. In camel's milk receiving group, diabetic rats had marked reduction in weekly BGL and one rat showed complete improvement after 8 weeks of treatment (BGL). The mean BGL came down from 429.24 ± 57 to 308.28 ± 52.2 mg/dl by the end of 5^th week. On contrary, diabetic rats received cow’s milk, showed increase in their mean BGL and 2 of them developed severe diabetes and succumbed. The mean BGL was increased from 404.6 ± 53.6 to 519.8 ± 131.5 mg/dl. The effect of cow's milk in our results is similar to the result recorded by Agrawal et al. (2005). This improvement may be due to presence of high concentration of insulin-like protein in camel's milk (Beg et al., 1986 and Yagil et al., 1994), According to Wangoh (1993) camel's milk does not form coagulum in acidic environment of the stomach. This lack of coagulum formation allows the camel's milk to pass through the stomach together with the specific insulin-like protein and remain available for absorption in the intestine. Also camel's milk is rich in mineral content and antioxidant (Farah, 1993). High concentrations of antioxidants may also make the insulin receptors to respond better to available insulin (Agrawal et al., 2004). Moreover, El-Said et al. (2010) reported that the pancreas of alloxan-induced rabbits receiving camel's milk showed high restoration number of islets of Langerhans among the pancreatic acini. Time-course evaluation of antibiabetic effect of camel’s milk on 5 rats is depicted in Fig. 6.

In the present study, we kept continue measuring the BGL after the testing period found that the diabetic group receiving camel's milk has long survival period. All diabetic rats in positive diabetic groups succumbed after the 5^th week while in diabetic group treated with cow’s milk died  before 5^th week (2 rats developed severe diabetes and died at the 3^rd weeks). The data in Fig. 7revealed that cow's milk had adverse effect on diabetic rats. This diabetogenicity of cow’s milk may be due to low concentration of insulin-like protein in cow’s milk (16.32 ± 5.98 micro unit/ml) (Shehadeh et al., 2001) or it might be due to milk clot in acid environment of the stomach(Breitling, 2002). The retention of milk clots in the stomach helps to accelerate the degradation of insulin. Vaarala et al. (1999) reported that bovine insulin acts as an immunogen and the IgG anti-bovine insulin may react with human insulin and damage β cells(Breitling, 2002).

From the result presented in our study, it could be concluded that raw camel's milk reduced BGL in diabetic rats. Thus, the therapeutic efficacy of camel's milk on alloxan-induced diabetic rats may have an importance implication for the clinical management in treatment of diabetes in target population.

Authors' contribution

Authors have formulated the research plan and conducted the study equally. Authors discussed the results, read and approved the final manuscript.

Acknowledgements

The authors are grateful to all staff at Laboratory Animals House, Assiut University-Egypt for their kind support during conducting this study.

Competing interests

None of the authors have any conflict of interest to declare.

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