Abstract: Background and Objective: The increasing of environmental pollution and changing of the dietary habits and other lifestyle are associated with increasing the risk factors and oxidative stress. Dairy manipulation and traditional plant therapies are commonly prescribed in various countries. Therefore, the aim of the present study was to investigate the antidiabetic and immunoprophylactic effect of a combination of camel milk filtrate with bitter gourd (Momordica charantia). Materials and Methods: Fresh camel milk was subjected to membrane filtration system and milk filtrate was used. Bitter gourd was cut into small size cubes and put in a high speed electric blender to make juice. The biological evaluation of camel milk filtrate and bitter gourd juice against alloxan-induced diabetic rats were studied. The immunological analyses and some blood biochemical markers were carried out using official methods. Results: Administration of camel milk filtrate and bitter gourd juice showed a hypoglycemic and immunoprophylactic effect. Blood glucose levels were decreased in alloxan-induced diabetic rats fed on camel milk filtrate and bitter gourd juice groups compared with alloxan-induced diabetic group fed on basal diet (positive control). In addition, administration of camel milk filtrate and bitter gourd juice was associated with a reduction in the serum levels of Alanine aminotransaminase (ALT) and Aspartate aminotransaminase (AST), when compared with positive control. Conclusion: Administration of camel milk filtrate and bitter gourd juice could have hypoglycemic effect in alloxan-induced diabetic rats. This immunoprophylactic and protective effect probably due to micronutrients found in the combination and synergistic effect of camel milk filtrate and bitter gourd against oxidative stress and diabetes.
INTRODUCTION
The development of industrial technology and excessive use of the agricultural techniques, food additives, pesticides, pharmaceutical chemicals and heavy metals pollution increased the risk factors for oxidative stress. Diabetes mellitus (DM) is a major metabolic disorder characterized by chronic hyperglycemia as a result of a relative or absolute lack of insulin or its action1.
Oxidative stress plays an important role in chronic complications of diabetes and is associated with increased lipid peroxidation2. An increasing in oxidative stress indices (lipid peroxidation and protein oxidation) was observed in the plasma of diabetic patients3. Besides, glucose autoxidation was found to be the cause of protein damage in the experimental model of DM and aging4.
Camel milk is an important source of proteins for people who are usually living in the desert of the world. It was claimed to have protective health effects and therapeutic values. Moreover, camel milk is well known by its medical properties that have been widely exploited for human health5 in a lot of developing countries6. The beneficial effects of camel milk, as can be depicted from various studies, can be attributed to many factors. Camel milk is considered to have anti-cancer7, hypo-allergic8 and anti-diabetic properties9,10. High contents of unsaturated fatty acids in camel milk contribute to its overall dietary quality11,12. Other components such as protective milk proteins (i.e., lactoferrin, immunoglobulins and lysozyme) were reported to play a central role in the determination of this properties13,14.
The membrane filtration systems used worldwide in dairy industry since the 1970’s. Ultrafiltration (UF) and reverse osmosis membrane systems implemented in the 1980’s. Microfiltration (MF) is a low pressure-driven membrane filtration process, which is based on a membrane with an open structure allowing dissolved components to pass while most non-dissolved components are rejected by the membrane. In the dairy industry, microfiltration is widely used for bacteria reduction and fat removal in milk and whey as well as for protein and casein standardization. Ultra-filtration is characterized as having a molecular weight cut-off range from about 3000-75,000. In dairy factories most common cut-off is the dairy standard of 10,000 MW that is commonly used for fractionating whey proteins from lactose. In the dairy industry, microfiltration and ultrafiltration is used for a wide range of applications such as protein standardization of cheese milk, processing of milk powders, production of fresh cheese, concentration of milk proteins and decalcification of permeates as well as reduction of lactose in milk15.
Momordica charantia commonly known as bitter gourd or bitter melon is a tropical and a subtropical climber of the family Cucurbitaceae. It is a green fruit, although bitter, that has been used to fight cancer, diabetes and many infectious diseases. It is one of the most favorite vegetables in China, Malaysia, India and tropical Africa. All parts of the plant, including the fruit taste is very bitter16. It has been reported that oral administration of M. charantia could lead to the secretion of insulin from endocrine pancreatic β cells17. In Ayurvedic medicine, various parts of Momordica charantia are recommended for many of certain diseases like anemia, blood diseases, cholera, ulcer, bronchitis, diarrhea, dysentery, sexual tonic and as a cure for gonorrhea18.
The aim of the present study was to investigate the antidiabetic and immunoprophylactic effect of a combination of camel milk filtrate with bitter gourd (Momordica charantia) against alloxan-induced oxidative stress and diabetes in rats.
MATERIALS AND METHODS
Camel milk samples were obtained from healthy lactating animals in Al-Qassim area and then stored at -20°C until analysis. Bitter gourd (Momordica charantia) was purchased from Indian fresh fruits and vegetables market, Saudi Arabia. Chemicals and pure reagents were purchased from Sigma (Sigma-Aldrich, St. Louis, MO, USA) and Roche Diagnostics (Roche Professional Diagnostics , Rotkreuz, Switzerland.
Production of camel milk filtrate: Fresh camel milk was subjected to membrane filtration system using a membrane with a molecular weight cut off allowed to pass some of protein and peptides in final milk filtrate. The QuixStand™ benchtop membrane filtration system was used in the present experiment to produce camel milk filtrate (Fig. 1). The configuration of this system is designed to accommodate a full range of hollow fiber ultrafiltration (UF) and microfiltration (MF) cartridges. The pore size of the UF cartridge is ranging from 1,000-750,000 nominal molecular weight cutoff (NMWC) and the pore size for MF cartridge is ranging from 0.1-0.65 micron. In the present study, MF cartridge was used at a pore size of 0.1 micron to produce camel milk filtrate. The obtained camel milk filtrate was collected, then pasteurized and stored in a sterile dark bottles at -4°C until use in the experiment.
| Fig. 1: | QuixStand benchtop membrane filtration system was used in the experiment to form camel milk filtrate rich in protein and peptides |
Preparation of aqueous extracts of bitter gourd (Momordica charantia) juice: Firstly, the outer green flesh of the bitter gourd was cut into small to medium-size cubes with a sharp knife. The seeds and white flesh were removed from the bitter gourd with a spoon to leave the green outer section, then put in a high speed electric blender to make juice. The final concentration of juice was 20% by adding distilled water. The extract juice was filtered twice through cheese-cloth (50% cotton/50%polyester) and was preserved in sterile dark bottles (500 mL) at -4°C for using in experiment.
Chemical composition of camel milk filtrate and bitter gourd juice: Chemical composition of camel milk filtrate and bitter gourd juice were carried out according the official methods of analysis19.
Experimental animals: Forty male Swiss albino rats, weighing about 150±40 g were obtained from the Experimental Animal Unit, College of Pharmacy, King Saud University, Riyadh, Saudi Arabia. Animals were housed in cages and assigned to be given a basal diet pellets (Protein 20.0%, fat 4.0%, fiber 4%, vitamin mixtures 1% and salt mixtures 4%) for 2 weeks for adaptation. Clean drinking water was allowed according to AIN-93 guidelines20. Animal procedures were performed in accordance with the ethics committee of Qassim University and according to the Guide for the Care and Use of Laboratory Animals of the National Institute of Health. Animals were kept under standard conditions of temperature and humidity along the experimental period (42 days). Rats were randomly divided into five-test groups (n = 8 rats/group).
| • | Group 1 was kept as untreated, control group (negative control) fed on the basal diet only and normal saline |
| • | Group 2-5 were treated with alloxan as inducer of diabetes |
| • | Group 2 was fed on the basal diet only and used as a positive control |
| • | Group 3 was fed on basal diet+camel milk filtrate replacing drinking water |
| • | Group 4 was fed on basal diet+bitter gourd juice replacing drinking water |
| • | Group 5 was fed on basal diet+camel milk filtrate+bitter gourd juice (1:1) replacing drinking water |
The feeding experiment have been continued for 6 weeks. At the end of the experimental period and after an overnight fast, rats were anesthetized by exposure to an atmosphere of a ratio 1:2:3 of ethanol: chloroform: diethyl ether, respectively21. After anesthetizing rats were sacrificed and a blood sample from each rat was collected using blood capillary tubes. Blood samples were then centrifuged at 3500 rpm for 15 min. Sera were harvested in labeled tubes and deep frozen (-20°C) for biochemical analysis. This experiment have been conducted during 2014-2015.
Agarose gel electrophoresis of serum proteins: Changes in serum proteins of rats fed on camel milk filtrate and bitter gourd juice were monitored using agarose gel electrophoresis method reported by Werner and Reavill22 using a kit and an automated SAS electrophoresis unit (Helena, UK). Electrophoresis was conducted at a voltage of 100 V for 12 min on a high resolution agarose gel. After electrophoresis, gels were transferred to SAS 4 unit (Helena, UK) for staining, de-staining and drying. Gels were then photo scanned and interpretation of the revealed peaks was carried out using a densitometer software (Platinum, Helena, UK).
Biochemical analysis: Blood glucose concentration were determined by the Haemo-Glukotest 20-800-R and measurements read with a Reflolux test (Boehringer-Mannheim)23. The serum levels of alanine aminotransaminase (ALT) and aspartate aminotransaminase (AST) were determined according to the method described by Reitman and Frankel24. Urea was determined according to the method of Tietz25. Creatinine was determined according to the method of Bousnes and Taussky26. Triglycerides were determined according to the method of Stein and Myers27.
Statistical analysis: Mean, standard deviation and coefficient of variation of the obtained data were calculated and conducted according the method described by Miller and Miller28.
RESULTS
The chemical composition of camel milk, bitter gourd juice and camel milk filtrate is shown in Table 1. Table 2 shows the glucose level in serum of alloxan-induced diabetes rats fed on camel milk filtrate and bitter gourd juice.
The obtained results showed that the glucose levels were decreased in serum of alloxan-induced diabetic rats fed on camel milk filtrate and bitter gourd juice groups compared with alloxan-induced diabetic group fed on basal diet (positive control). The mean values of glucose level (mg dL–1) were 116.5±2.64, 121.25±5.188 and 114.5±8.88 in serum group fed on camel milk filtrate, bitter gourd juice and their mixture, respectively, compared with negative (114.75±3.77) and positive (399.25±68.67) control groups.
Table 3 and 4 show alanine aminotransaminase (ALT) and aspartate aminotransaminase (AST) levels in serum of alloxan-induced diabetes rats fed on camel milk filtrate, bitter gourd juice and their combination.
The mean values of ALT levels (U L–1) in serum were 32±4.53, 30±3.57 and 31±4.83 when rats were fed on camel milk filtrate, bitter gourd juice and their mixture, respectively, compared with positive control (56±6.97). Also, the AST levels (U L–1) in serum were141±12.47, 130±9.67 and 136±9.73 when rats were fed on camel milk filtrate, bitter gourd juice and their mixture, respectively, compared with positive control (192±11.43).
Table 5 shows the triglycerides levels (mg dL–1) in serum of alloxan-induced diabetes rats fed on camel milk filtrate and bitter gourd juice. It can be observed that no significant effect of all treatments on the serum levels of triglycerides when compared with control.
Table 6 and 7 show the urea and creatinine levels in serum of alloxan-induced diabetes rats fed on camel milk filtrate and bitter gourd juice.
| Table 1: | Chemical composition of camel milk, bitter gourd juice and camel milk filtrate |
| Table 2: | Levels of blood glucose between alloxan-induced diabetes rats groups fed on camel milk filtrate and bitter gourd juice |
| Min: Minimum values, Max: Maximum values SD: Standard deviation, CV: Coefficient of variation | |
| Table 3: | Levels of alanine aminotransaminase (ALT) activity between alloxan-induced diabetes rats groups fed on camel milk filtrate and bitter gourd juice (U L–1) |
| Min: Minimum values, Max: Maximum values, SD: Standard deviation, CV: Coefficient of variation | |
| Table 4: | Levels of aspartate aminotransaminase (AST) activity between alloxan-induced diabetes rats groups fed on camel milk filtrate and bitter gourd juice (U L–1) |
Min: Minimum values, Max: Maximum values SD: Standard deviation, CV: Coefficient of variation | |
| Table 5: | Levels of triglycerides between alloxan-induced diabetes rats groups fed on camel milk filtrate and bitter gourd juice (mg dL–1) |
Min: Minimum values, Max: Maximum values, SD: Standard deviation, CV: Coefficient of variation | |
| Table 6: | Levels of urea between alloxan-induced diabetes rats groups fed on camel milk filtrate and bitter gourd juice (mg dL–1) |
Min: Minimum values, Max: Maximum values, SD: Standard deviation, CV: Coefficient of variation |
|
| Table 7: | Levels of creatinine between alloxan-induced diabetes rats groups fed on camel milk filtrate and bitter gourd juice (mg dL–1) |
Min: Minimum values, Max: Maximum values, SD: Standard deviation, CV: Coefficient of variation | |
The urea levels (mg dL–1) in serum were 30±3.11, 26±1.50 and 27±2.84 in rats’ groups fed on camel milk filtrate, bitter gourd juice and their mixture, respectively, compared with positive control (19±3.11). The highest levels of urea were observed when rats were given camel milk filtrate.
| Table 8: | Changes in albumin levels (%) between alloxan-induced diabetes rats groups fed on camel milk filtrate and bitter gourd juice |
Min: Minimum values, Max: Maximum values, SD: Standard deviation, CV: Coefficient of variation, ↓: Decrease ↑: Increase |
|
| Table 9: | Changes in α1-globulin (alpha1-globulin) levels (%) between alloxan-induced diabetes rats groups fed on camel milk filtrate and bitter gourd juice |
Min: Minimum values, Max: Maximum values, SD: Standard deviation, CV: Coefficient of variation, ↓: Decrease, ↑: Increase |
|
| Table 10: | Changes in α2-globulin (alpha2-globulin) levels (%) between alloxan-induced diabetes rats groups fed on camel milk filtrate and bitter gourd juice |
Min: Minimum values, Max: Maximum values, SD: Standard deviation, CV: Coefficient of variation, ↓: Decrease, ↑: Increase | |
| Table 11: | Changes in β-globulin (beta-globulin) levels (%) between alloxan-induced diabetes rats groups fed on camel milk filtrate and bitter gourd juice |
Min: Minimum values, Max: Maximum values, SD: Standard deviation, CV: Coefficient of variation, ↓: Decrease, ↑: Increase |
|
| Table 12: | Changes in γ-globulin (gamma-globulin) levels (%) between alloxan-induced diabetes rats groups fed on camel milk filtrate and bitter gourd juice |
Min: Minimum values, Max: Maximum values, SD: Standard deviation, CV: Coefficient of variation, ↓: Decrease, ↑: Increase |
|
The creatinine levels (mg dL–1) in serum were 0.48±0.013, 0.38±0.015 and 0.47±0.015 in the rats’ groups that were fed on camel milk filtrate, bitter gourd juice and their mixture, respectively, compared with both negative (0.42±0.074) and positive control groups (0.45±0.092).
Table 8-12 show change in serum proteins (%) of alloxan-induced diabetic rats that were fed on camel milk filtrate, bitter gourd juice and their mixture. The obtained results showed that the levels of albumin were 45.12±0.70, 45.93±2.32 and 44.6±0.95 when rats fed on camel milk filtrate, bitter gourd juice and their mixture, respectively, compared with both negative (41.96±0.91) and positive (40.52±0.70) control groups (Table 8).
The serum levels of α1-globulin were 14.52±0.98, 13.57±1.65 and 13.49±0.86 in rats’ groups fed on camel milk filtrate, bitter gourd juice and their mixture, respectively, compared with negative (15.12±1.30) and positive (16.02±1.96) control groups (Table 9). The serum levels of α2-globulin were 6.12±0.28, 7.56±0.89 and 6.63±0.55 in rats’ groups fed on camel milk filtrate, bitter gourd juice and their mixture, respectively, compared with negative (7.25±1.05) and positive (8.44±0.44) control groups (Table 10). The serum levels of β-globulin were 19.02±0.46, 18.95±2.01 and 19.39±1.03 in rats’ groups fed on camel milk filtrate, bitter gourd juice and their mixture, respectively, compared with negative (20.32±0.85) and positive (19.70±0.37) control groups (Table 11). The serum levels of γ-globulin were 18.92±3.09, 13.58±0.96 and 17.53±1.23 in rats’ groups fed on camel milk filtrate, bitter gourd juice and their mixture, respectively, compared with negative (11.72±2.34) and positive (12.98±2.34) control groups (Table 12).
DISCUSSION
In arid and semi-arid regions, camel milk represents an important dietary component with high nutritional values for humans29. Ultrafiltration process of milk by using 100,000 molecular weight cut off membrane produces a milk rich in α-lactalbumin, β-lactoglobulin and peptides. Milk and whey contains many proteins with excellent properties which are also of a high nutritional value. Now-a-days, a large amount of whey protein concentrate with high nutritional value are available due to popularity of membrane filtration technology in dairy industries. Therefore, whey protein concentrate has become increasingly important in the field of functional food, dietetic and pharmaceutical industries30. Surprisingly, It has been found that the ratio of α-lactalbumin to β-lactoglobulin in the filtrate is substantially greater than that in the starting material when a 10,000 molecular weight cut off membrane were used. In addition, the ratio of α-lactalbumin/β-lactoglobulin in the filtrate using a 100,000 molecular weight cut off membrane was not significantly lower than that obtained from a 50,000 molecular weight cut off membrane. It was expected that the rate of permeation of α-lactalbumin through the 100,000 molecular weight cut off membrane is significantly higher than that through a 50,000 molecular weight cut off membrane30.
The obtained results indicated that feeding alloxan-induced diabetic rats on camel milk filtrate and bitter gourd juice might showing hypoglycemic, immunoprophylactic and decreased blood glucose level. This positive effect probably due to many potentially bioactive compounds found in both camel milk filtrate and bitter ground juice. There are elevation in blood glucose levels in positive control group. Administration of camel milk filtrate and bitter gourd juice in alloxan-induced diabetic rats caused a high reduction in the blood glucose levels compared to the diabetic control rats.
The increased blood urea in male rats fed on camel milk filtrate compared with control groups could be due to higher micronutrients and protein content in camel milk filtrate as resulted from membrane filtration system. Also, elevated blood urea is known to be a function of or related to increased protein catabolism in mammals and/or the conversion of ammonia to urea as a result of increased synthesis of arginase enzyme involved in urea production. Serum urea levels are known to be elevated by high-protein diets. Although daily production of creatinine is relatively constant and serum levels are unaffected by diet, but its levels are generally used as an indicator of kidney function31.
Table 8-12 show change in serum proteins (%) of rats fed on camel milk filtrate and bitter gourd juice and treated with alloxan for inducing diabetes. Induction of diabetes in rats by alloxan resulted in a decrease in the concentration of albumin, beta and gamma-globulins and slight increase in alpha globulin as can be noticed in positive control group. This reduction in albumin and gamma-globulins levels were shifted after giving rats camel milk filtrate and bitter gourd. In addition, the levels of alpha and beta globulins in the same group were similar with that found in the negative control group.
The data revealed that administration of camel milk filtrate and bitter gourd juice augmented the immune system by elevating albumin and gamma-globulins which might overcame negative effects of alloxan. Previously, patients with certain autoimmune and allergic diseases, such as systemic lupus, multiple sclerosis, autoimmune thyroiditis or atopic eczema, often show an increased lymphocyte stimulation by oxidative stress agents in vitro32. A hallmark of such autoimmune induction is the accompaniment of an immune shift, in which there is usually an initial skewing toward a Th2-like immune environment33.
Research studies on camel milk and its relation with diabetes indicated that drinking camel milk daily decreases the blood glucose level and reduces insulin requirement by 30%34. These studies revealed that camel milk may provide an insulin-like protein in a different form than in other mammals and/or delivers some other therapeutic compounds that boosts the health of diabetic patients. However, the mechanism is not yet fully understood34.
Clinical studies reported that the administration of insulin is incapable of overcoming mucosal barriers and is degraded by digestive enzymes before it enters the bloodstream35. On other hand, the insulin-like protein of camel milk might resist degradation in the stomach by nanoparticles and might be absorbed efficiently into blood stream to reach the target36. This might be due to the fact that camel milk does not coagulate in an acidic environment and it has a higher buffering capacity than the milk of other ruminants9. Also, it was reported that no differences were noted in the sequence of camel milk insulin-like protein and its digestion pattern in comparison with the other sources of milk36. Other studies found that amino acid sequence of some camel milk protein is rich in half cysteine which has a superficial similarity with the insulin family of peptides37. Hamers-Casterman et al.38 and Agrawal et al.34 reported that the small size and weight of camel milk immunoglobulin may offer enormous potential through interaction with the host cell protein and cause an induction of regulatory cells and finally result in a downward regulation of the immune system and β-cell salvage. Bottomley30 reported that some of whey proteins (α-lactalbumin and β-lactoglobulin) and milk peptides were passed into the permeate using ultrafiltration membrane system with a 30,000-50,000 molecular weight cut-off. In addition, the concentration of these proteins and peptides were increased when a 100,000 molecular weight cut off membrane was used.
Momordica charantia (bitter gourd or bitter melon) is a popular plant and used for the treating diabetes related condition in Asia, South America, the Caribbean, India and east Africa. Ayurvedic studies showed that fruits part of Momordica charantia contains an array of biologically active plant chemicals including proteins, flavonoids, triterpens, saponins, steroids, balkaloids and organic acids. This could explain the anti-bacterial, anti-fungal, anti-parasitic, anti-viral, anti-fertility, anti-tumorous, hypoglycemic and anti-carcinogenic properties of this plant39-40. Also, it is a potent hypoglycemic agent due to alkaloids, insulin like peptides and a mixture of steroidal sapogenins known as charantin10.
CONCLUSION
It is concluded that used camel milk filtrate produced from the microfiltration cartridges with pore sizes 0.1 micron and was a rich in low molecular weight protein, peptides and micronutrients. Administration of a combination of camel milk filtrate produced by membrane technology and bitter gourd juice decreased blood glucose level in rats and showed a hypoglycemic and immunoprophylactic characteristics. This effect may be due to the bioactive components found in camel milk filtrate and bitter gourd juice. Further studies are needed to clarify and identify the mode of action of each of these bioactive components.
SIGNIFICANCE STATEMENT
This study discovers the antidiabetic and immunoprophylactic effect of a combination of camel milk filtrate with bitter gourd (Momordica charantia) against alloxan-induced oxidative stress and diabetes in rats. The combination of camel milk filtrate with bitter gourd decreased the blood glucose levels and improves the immunoprophylactic and protective properties against oxidative stress and diabetes.
ACKNOWLEDGMENTS
The authors would like to thank Professor El-Agamy E. I professor of Dairy and immunology in Applied Medical Sciences Department, Community College, Qassim University, Saudi Arabia and Professor Abd El-Salam M. H. Professor of Dairy Science, National Research Centre, Egypt and Editors of Journal of Egyptian Dairy Science for their ongoing cooperation and suggestions. Also, authors would like to thank Dr. Hamad, E. M. for revising and editing language of this manuscript. This study did not receive any specific grant from funding agencies in the public, commercial or not-for-profit sectors.