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SHORT TERM EFFECT OF DOSE DEPENDENT CAMEL MILK IN ALLOXAN INDUCED DIABETES IN FEMALE ALBINO RATS.

Byline: M. Usman, M. Z. Ali, A. S. Qureshi, M. K. Ateeq and F. U. Nisa

ABSTRACT

Current project was designed to evaluate the antidiabetic effect of camel milk on hematology, serum profile and histology of selected organs. Forty female albino rats were divided into 4 groups (n=10): Group 1 as placebo. Diabetes was induced by AlloxanA(r) in remaining groups. Group 2 served as diabetic control while group 3 and 4 were offered camel milk orally @ 20 and 40ml/kg/day, respectively, for 60 days. Weekly blood glucose was measured. Animals were slaughtered on day 60 to collect: blood for hematology and tissue for histology. Microscopy was done for degenerative changes in uterus (epithelial height, gland area, thickness of endometrium and myometrium), liver and kidneys. Diabetes revealed significant (P<0.05) adverse effect on all parameters. Camel milk exhibited more significant (P<0.05) mitigating influence on diabetic altered hematology and histology of uterus, liver and kidney @ 40ml/kg compared to 20 ml/kg.

Camel milk @ 20 ml/kg significantly (P<0.05) reduced blood glucose in 3rd week and remained constant in trial while this reduction was significant (P<0.05) at 2nd week and became more significant (P250mg/dL were declared as diabetic and used for the present study.

Collection of camel milk: Fresh raw camel milk (CM) was used in this study. CM was collected in the sterile bottles on daily basis from a local camel market of Faisalabad. The bottles were kept in cold container and transported to laboratory.

Research Design: All experimental animals were randomly divided into 4 groups: each having ten rats. Group 1 was considered as placebo. Diabetes was induced in rest of the groups such as Group 2 kept as diabetic control while CM was administered orally to group 3 and 4 @ 20 and 40ml/kg/day respectively for a period of 60 days.

Collection of samples: Fasting blood glucose concentration was measured on weekly basis with the help of blood glucose monitoring system (On CallA(r) EZ II, AconA(r) Laboratories, Inc. USA). Collection of samples was done on day 60 after euthanizing the animals. Blood sample (2ml) was taken from heart before slaughter and collected in two vacutainers: one containing EDTA for hematology and second without EDTA for the estimation of liver and kidney function tests. Serum was separated by centrifugation at 1500 rpm for 15 minutes and stored at -20AdegC for its further analysis. Samples of uterus, liver and kidneys were collected immediately after the slaughtering. Uterus was fixed in Bouin's fluid while liver and kidneys were fixed in 10% neutral buffered formalin after washing with normal saline.

Microscopic analysis: Tissues were cut into thin slices and processed by paraffin preparation technique. Sections were cut at 5 um and subjected to Hematoxylin and Eosin (HandE) staining procedure of Bancroft et al. (2013). Microscopic slides were examined at 100X to measure the uterine height (um) of epithelium, endometrial glands area and thickness (um) of endometrium and myometrium using automated image analysis system image JA(r). Vacuolar and degenerative changes in tubules of kidneys and radial arrangement of hepatocytes around the central vein along with the necrosis of hepatocytes were also observed.

Statistical Analysis: Descriptive statistics of each parameter under study was calculated with the help of computer software Microsoft ExcelA(r). The means of parameters were compared with one-way analysis of variance (ANOVA) except fasting blood glucose for which repeated measurement ANOVA under Complete Randomize Design (CRD)was performed by SASA(r). The group means were compared with help of the Least Significance Difference (LSD) test. The level of significance was kept at 5 percent.

RESULTS

Multiple mean comparison of fasting blood glucose concentration and live weight gain of all groups of rats is given in Table 1. AlloxanA(r) caused significant (P<0.05) rise in the blood glucose concentration. Following the oral therapy of CM @ 20 and 40ml/kg/day, the blood glucose concentration reduced significantly (P<0.05). The live weight gain was significantly (P<0.05) high in CM @ 40 group as compared to other groups. Table 2 described that diabetes caused significant (P0.05) except for hemoglobin. CM @ 40ml/kg revealed significant (P<0.05) increase in these parameters especially in platelets, hemoglobin, MCV and MCH which was near to the placebo.

The significant (P<0.05) reduction was seen in the uterine histometry including epithelial height, endometrium thickness and its glands area, inner circular and outer longitudinal smooth muscle thickness of myometrium in the diabetic rats. Oral administration of CM showed significant (P<0.05) recovery towards normal in these parameters especially @ 40 ml/kg (Table 3, Figure 1). Micrograph of hepatic tissue, in placebo rats, showed radial arrangement of hepatocytes around the central vein along with the portal triad with bile duct, portal vein and hepatic artery. Hyperglycemic rats showed the disrupted radial arrangements of hepatocytes around central vein along with the necrosis of hepatocytes. Oral administration of CM recovered the radial arrangement of hepatocytes and reduced the necrosis of hepatocytes with less hemorrhages but best results were displayed in group treatment with CM @ 40 ml/kg (Figure 2).

Histological examination of kidney from placebo rats showed normal glomerular structure along with renal tubules while diabetic kidneys showed glomerular damage, excessive tubular degeneration and hemorrhages. CM @ 20 ml/kg showed no improvement in the cellular structures of damaged glomerulus and renal tubules. Nephroprotective effect was observed in the rats treated with CM @ 40ml/kg with less hemorrhages and tubular degeneration and nearly normal glomerular structure (Figure 3). Diabetes caused marked (P<0.05) increase in the Aspartate transaminase (AST), Alanine transaminase (ALT), serum urea and creatinine level as compared to the placebo (Figure 4). It was observed that long term CM treatment significantly (P<0.05) decrease diabetic AST level at both doses while significant (P<0.05) reduction in ALT level was only observed in group treated with CM @ 40ml/kg.

CM therapy significantly (P0.05) on the creatinine level (Figure 5).

Table 1. Comparison of dose dependent effect of camel milk in different groups on fasting blood glucose and live weight gain.

Groups###Blood glucose (Mean+-SE) mg/dL

###W-1###W-2###W-3###W-4###W-5###W-6###W-7

Placebo###95.6+-21.22C###120+-11.84C###119.3+-1.74C###132.3+-21.92C###125.75+-21.34C###132.8+-13.84C###130.5+-11.84C

Diabetic###495.86+-21.74A###408.57+-7.75A###457.57+-20.8A###419.00+-21.30A###457.57+-20.79A###486.86+-22.45A###491.00+-20.4A

control

CM @20###503.67+-14.25A###399.00+-7.64A###406.00+-6.66AB###333.67+-20.90B###305.67+-18.70B###315.33+-21.40B###306.33+-18.9B

CM @40###482.25+-19.98A###407.38+-7.16A###342.63+-15.4B###291.88+-18.45B###251.00+-16.30B###241.67+-10.00C###226.00+-7.48C

###Live Weight Gain (Mean+-SE) grams

Placebo###95.00+-11.58A###120.00+-6.36A###119.00+-2.08A###123.30+-11.2A###125.74+-12.4A###132.8+-8.2A###130.5+-10.4A

Diabetic###164.00+-3.91A###155.57+-11.58A###168.14+-12.64A###158.14+-11.6B###156.57+-11.2B###151.14+-10.0B###147.57+-9.33B

control

CM @20###153.00+-1.53A###156.00+-2.08A###181.33+-6.36A###181.00+-4.51AB###179.33+-4.98AB###180.00+-3.61AB###176.67+-3.33AB

CM @40###160.75+-4.60A###169.13+-6.08A###187.13+-5.73A###192.38+-5.41A###197.17+-6.59A###198.83+-6.55A###211.50+-10.2A

Table 2. Dose dependent effect of camel milk on hematological parameters in Alloxan induced diabetic fe male albino rats.

Groups###Hematology(Mean+-SE)

###RBCs(1012/L)###PLT###WBC###MCV(fl)###Hb(g/dl)###Lymphocyte###MCH(pg)

###(109/L)###(109/L)###(%)

Placebo###8.94+-0.12 A###851+-15 A###17.20+-1.05 A###55.63+-5.41 A###15.03+-0.45 A###65.20+-2.03 A###16.93+-1.78A

Diabetic control###4.81+-0.38 C###398+-179 B###5.63+-1.18 C###51.65+-1.47 B###11.00+-0.53 C###45.87+-1.61 B###12.63+-0.85 B

CM @20###4.88+-0.32 C###455+-39 B###7.17+-0.58 C###51.87+-1.27 B###12.60+-0.42 B###46.23+-2.13 B###14.23+-0.41 B

CM @40###6.09+-0.42 B###751+-104 A###10.43+-2.51 B###56.52+-1.05 A###14.72+-0.49 A###60.32+-2.83 A###16.55+-0.78 A

Table 3. Effect of different doses of camel milk on histometry of uterus in Alloxan induced diabetic female albino rats.

Groups###Epithelial###Endometrium###Endometrium###Inner circular###outer###myometrium

###height(um)###thickness(um)###glands(um2)###smooth muscle###longitudinal###thickness(um)

###layer (um)###smooth muscle

###layer (um)

Placebo###103.95+-3.07AB###200.24+-18.44A###31.883+-9.3A###277.02+-07.22A###396.26+-26.03A###673.28+-23.75A

Diabetic###87.86+-5.18C###152.95+-13.05B###25.837+-4.5B###161.70+-09.01C###121.03+-05.06C###282.73+-10.99C

control

CM###95.79+-3.12BC###184.70+-13.72AB###30.114+-10.0C###140.61+-10.94C###168.25+-23.59C###304.69+-27.04C

@20

CM###113.09+-3.99A###229.96+-18.95A###31.617+-9.2C###227.05+-15.48B###240.06+-20.03B###467.11+-21.18B

@40

DISCUSSION

Diabetes mellitus (DM) has been recognized as the most common metabolic disorder that impairs blood glucose level which leads to high microvascular complications, angiopathy, nephropathy, retinopathy, hepatopathy and neurological defects (Sharma et al., 2010; Khan et al., 2013). The prevalence of this disorder has been increasing day by day and becoming a major threat to the global health. Health departments of all over the world are utilizing their sources to combat this threat (Marx, 2002; Baragob, 2015). Camel milk (CM) has many qualities to normalize blood glucose level and restore body functions. This action of camel milk may be due to the presence of insulin like protein (45-128 IU/L), high amount of zinc and vitamin C (Rahimi et al., 2011; Mullaicharam, 2014: Ali et al., 2017). DM can be induced by synthetic drugs like AlloxanA(r) and StreptozotocinA(r) in rabbits and rats (Alam et al., 2005; Hassan and Bayoumi, 2010).

AlloxanA(r) monohydrate solution can be prepared in normal saline having pH 7 and citrate buffer having pH 4.5 (Ajiboye and Ojo, 2014). In the present study, AlloxanA(r) (prepared in the normal saline) was administered intraperitonealy @ 150mg/kg b.w. to overnight fasted rats. DM was confirmed by measuring blood glucose level after 3 days. This result was according to Doss et al. (2009) and Ali et al. (2017). In the present study, AlloxanA(r) administration induced an elevated blood glucose level in rats as observed by Hassan and Emam (2012). Both the treatments of CM @ 20 and 40 ml/kg bw significantly (P<0.05) decreased this elevated blood glucose level. However, multiple comparison showed highly significant (P<0.01) in reduction in CM @ 40ml/kg bw. This anti-hyperglycemic effect may be due to the presence of high concentration of insulin like protein that can tolerate the stomach acidic pH (Agrawal et al., 2005; Mullaicharam, 2014).

DM alters the uterine histology and causes atrophy of endometrium and myometrium which hampers its normal function. DM lowers the number of myofibrils which results in thinning of myometrium (McMurtrie et al., 1985). In present study, DM caused a significant (P<0.05) decrease in endometrium (epithelial height, endometrium thickness and glands area). Similarly, a significant (P<0.05) decrease was found in myometrium thickness (inner circular and outer longitudinal muscles). These results were in accordance to the Tatewaki et al. (1989) and Favaro et al. (2010). The histological changes in the myometrium smooth muscle layer depend upon the duration of diabetes. Short duration (7-8 weeks) of hyperglycemia had not developed significant abnormal alterations in myometrium (Favaro et al., 2010). CM @ 20ml/kg bw significantly (P0.05) on diabetic myometrium.

CM @ 40ml/kg bw significantly (P< 0.05) improved both the endometrium and myometrium thickness thus restored the histology of uterus towards normal. In the present study, DM significantly (P<0.05) decreased the hematological parameters (RBCs, Hb, MCH, MCV). This decrease could be attributed to high level of non-enzymatic glycosylation of the cell membranes of RBCs which caused lysis of RBCs and ultimately resulted in anemia (Oyedemi et al., 2011a; Oyedemi et al., 2011b; Basker et al., 2006). CM showed significant (P0.05) @ 20ml/kg bw. This antihyperglycemic effect of CM may be due to presence of high level of antioxidant and some compounds that favor the erythropoietin production. Blood clotting mechanism affected in DM is mainly due to poor glycemic control and insulin deficiency.

DM caused a decrease in platelets count which consequents in internal and external blood loss and leads to death (Jarald et al., 2008; Oyedemi et al., 2010). CM treatment caused a significant (P0.05) @ 20ml/kg bw This antidiabetic therapy of CM which showed an increase in the platelets count may be due to high level of insulin present in it. AlloxanA(r) suppresses the immunity of animals by damaging and/or decreasing the WBCs count (Erukainure et al., 2013). This suppression may be due to poor leukocytosis in the bone marrow (Torell et al., 1986; Oydemi et al., 2011). In this trial, diabetes significantly (P<0.05) decreased WBCs count but CM treatment caused a significant (P0.05) by CM @ 20 ml/kg bw. The restoration of histological structures toward normal may be due to the presence of higher levels of antioxidant and insulin (Mullaicharam, 2014). A significance (P<0.05) increase was observed in the liver enzymes (AST and ALT) in the diabetic rats at the end of trial. These findings are similar to that of khan et al. (2013).

The elevated level of liver enzymes reflects the necrosis and damaging of hepatocytes which results in the leakage of AST and ALT from hepatocytes (Manal and Moussa, 2014). In present study, camel milk significantly (P<0.05) decreased these diabetogenic values of liver enzymes and these results were supported by the findings of Khan et al. (2013) and Manal and Moussa (2014). Creatinine and serum urea are the two kidney function markers. Data analysis of present study showed that diabetes caused significant (P<0.05) increased in these markers as compared to placebo. The elevated level of these parameters could be due to the diabetic alterations in the structure of the kidney tubules. Sunil et al. (2011) observed the same trend in kidney function markers. Therapy of camel milk significantly (P<0.05) recovered the serum urea level of diabetic rats. Therapeutic activity of fresh raw camel milk was found significant in AlloxanA(r) induced female diabetic rats.

Based on this study, results showed that camel milk has potential to improve diabetes related reproductive and hematological complications especially @ 40 ml/kg bw. This result may have implications in the clinical management of diabetes mellitus in human.

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Publication:Journal of Animal and Plant Sciences
Date:Oct 31, 2018
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