PHYSIOLOGICAL AND HISTOPATHOLOGCAL STUDY FOR THE EFFECT OF BARLEY (HORDEUN VULGARE) FLOUR ON INDUCED DIABETIC

The aim of this study is to evaluate the effect of barley consumption on sugar reduction in diabetic patients in male and female rats. The mice were divided into six groups with 10 mice per group. The mice injected the substance with 200 mg / kg body weight to induce diabetes. The study included 60 mice of both sexes, injected with aloxane and a dose of 200 mg / kg body weight to induce diabetes. The first group was represented by the control group as its members took standard food from the experimental animal food. The second group, which represents the induced control group as its members took the general measure of food, but injected the substance of the aloxane for comparison. While the third and fourth groups were fed an equal mixture of wheat flour and barley in the form of dry paste for both females and males for mice, respectively. The fifth and sixth groups were fed barley flour only for both sexes. Measure both body weight and blood sugar at the beginning and end of the two weeks of the experiment, where it was fed daily with various food treatments mentioned previously. Different blood parameters were measured, The physiological and histological changes of liver, kidney and brain were also studied. The results showed that there was a significant decrease in the percentage of blood clocates in groups with diabetes, and showed groups treated with barley decreased cholesterol and triglyceride with the emergence of changes in the tissue in the liver and kidneys, while the brain appeared in its natural form. Barley has a protective effect against diabetes.


INTRODUCTION
Barley plant is the fourth most produced cereal in the world after wheat, maize and rice, scientifically known as Hordeum vulgare L. and an ancient grain. "Barley is a very good source of molybdenum, manganese, dietary fiber, and selenium (13)". Also, (23) presented that barley supplies a good source of the copper, chromium, phosphorus, magnesium, niacin and vitamin A. Barley is supplying a high nutritional values of β-glucan as a soluble and insoluble fibers which act to reduce blood cholesterol, lowered postprandial blood glucose and insulin levels and risk factor of heart disease (18; 23). Barley content of ß-glucan was varing between 2 and 9% (10; 27). Instead of that barley is very important for supplying protein (7), vitamins (vit. E) and many essential minerals such as iron and zinc (4; 20). The presence of soluble fiber in the bowel leads to delayed gastrointestinal passage and is suppressed in the large intestine. Also, there is a sufficient time to produce short chain, low-molecular fatty acids that are thought to be beneficial and closely linked to lowering blood cholesterol (14). Now days, there is a renewed interest in supplementing barley for many bakery products and meals for human food. Barley products are highly content of fiber, low fat that help to promote satiety score (12; 22) and that can be useful tools for weight loss and weight maintenance (29). Also, many studies have been founded that barley β-glucans effectively lower blood glucose levels, which is important in the prevention and management of type 2 diabetes (11; 17) and also increase satiety, which supported weight management (8; 28). Furthermore, barley's nutritional advantages, barley also has an excelent taste. So, barley through its functional and sensory properties, is considered a promising future for the production of various healthy foods and pastries (25). Thus, the current study was aimed to determine the effect of consumption of barley flour in reducing blood sugar and other blood parameter in laboratory diabetes white mice after eating barley paste for two weeks. Also it was conducted to determine the effect of barly flour consumsson on histopathical changes in liver, kidney and brain tissues of the mice.

MATERIALS AND METHODS Preparation of barley flour
Five kilo grams of white barley seeds were taken and washed in tap water to remove the suspended soil, gravel, straw and exotic impurities, and then left for half an hour to get rid of excess water. Then, It was published in trays of stainless steel under the sun until dry, and then removed from anything strange, then grind well in a laboratory mill then, sieved with sieve at diameter 250µm to fine powder in sealed nylon bags and kept in dry placed till used. Preparation of dried barley and wheat flour paste: One hundred gms of barley flour and 100 gms. were weighed Of wheat flour and mixed well with lukewarm water until got a stiff dough then cut into small pieces and left to dry in sun for several days until it used to mice daily.

Preparation of dry barley paste alone
One killo gm of barley flour was weighed, mixed well with amount of lukewarm water until got a stiff dough, then cut into small pieces and left to dry for several days until it used to mice daily.

Laboratory animals
Sixty fifty adult albino mice (male and female) age 8-10 weeks weighing between 20-25gm. used for experiment that obtained from animal house of Al-Razi center. All the experiments were done by the veterinary staff lab. of veterinary drug Research Center which had approved all the experimental protocols for ethical reason. The mices were given a normal standard food while providing the drink water from the tap for a whole week to get well adapted. Then after passing the week, glucose level was measured after pulling blood out of the tail vein. Mices were divided into six groups (10 mice) for each one (table 1). Animals were fasted for 12 hr., then five groups were induced for diabetes by intraperitoneal injection of a single dose of 200 mg /kg of alloxan (Alexis Biochemical, Lot-L24553) as (DC), while one group was assigned to be non-diabetic control (N-DC). After given an alloxan, the blood glucose level of all animals was analyzed every day from the tail vein during one week. The animals that showed stable hyperglycemia > 200 mg/dL were used for experiments as diabetics and the others were eliminated. Blood glucose level was tested by using the test strips for the Glucose (Accua chek, Germany). Then all groups except control group (N-DC) were feded barley flour only or combined flour of barly and wheat. All mice groups were individually housed in stainless steel cages in a room at 21±3Cº for 12hr dark cycle and 60-65% humidity. Each mice had free access to its respective diet and drinking water during the experimental period and weighing daily. At the end of the 14 th day of the experiment, mices were given a lethal overdose of isoflorane by inhalation. Then instantly the blood samples were taken from the heart by syringe and layed on in box full of ice, then blood of all samples were immediately centrifuged at 3000 g for 5 min at 4•C, after that serum was separated, and it was kept at −80•C until analyzed.

Comercial standard diet (CSD): Brown Rice, Oats and Wheat, Soy and Fish Meal. Chemical composition of barley flour
Moisture (44-16 A), crude ash (08-07) were determined by incineration in a muffle furnace at 580 0 C for 8hr.; fat was estimated by using Soxlet extraction (30-10) according to AACCapproved methods (1). The kjeldahl method was used for determination the crude protein level, its expressed by conventional factor Nx 6.25 where the multiplying total content of nitrogenous compound were corresponding to the total nitrogen content. Dietary fiber was estimated according to (Asp, et al. 1983) method; Dry matter evalution by drying in an electric oven with ventilation (JR AD/ Italy, www jradco.org) at 105 0 C until reaching constant weight (2); total carbohydrate was estimated by difference.= Determination of blood glucose level Blood glucose was estimated by drawing blood from the tail vein by using the sugar test strips (Accua chek, Germany) and the examination was done daily after the administration also recorded the changes in the mice weights treated and untreated daily.

Blood analysis & lipid profile
At the end of the experiment, animals were slaughtered and blood was withdrawn directly from the heart. Blood hematosis parameters (WBC, RBC,HGB, HCT, MCV, MCh, MCHC and PLT) were determined, also serum glucose, triglycerides (TGs) and total cholesterol were estimated by using enzymatic method at Al-Maghreb Lab, Baghdad Histopathological changes After slaughtering all mice, the important organs were taken to evaluate the histopathology changes in kidney, liver and brain of the mice that induced with alloxan. Kidney, liver and brain biopsies were taken on the 14 th day of the study.  (Table 3). Also, barley flour contain relatively high dietary fiber ratios that are important to human health. This finding were accordance with finding by (4). Also, (7) found almost approximately accordance percentages of chemical content.  (Table 4), there were an increase in females weight group, which were given barley and wheat flour paste, and the males group was not significant differences compared with control group. While, groups 5 & 6 there were no significant differences by consumption barly flour only Table 4.The role of barley in an increasing the weight may be due the nature of the functional ingredients contained which lead to obesity as presented by (9). The result was shown that the barley works as appetizer and incease the intake of food and its play an important role in the incidence of obesity in both sex and regulates the body weight through different mechanisms and reduced energy spending as pointed by (21). Also, it was presented thay barley has the ability to increase fat synthesis through stimulating cholesterol synthesis in liver, decreasing bile acid secretion and reducing the activity of the acyl-transferase enzyme to increase the absorption of fat droplet in GIT (18). In this study, it was notice in all mice groups that given barley were appeared tendency to stable condition without any signs of nervous or stress during the period of experiment that was considered the positive effects of barley and as actual effect in reducing disorders of the central nervous system (19).

Determination the blood glucose level
The results in Table 5 shows that it was found after 14 days of barley flour feeding along period of the experiment, a significant reduction in FBS in group 5 (DMB) comparing with control and diebete control group. (p <0 .05), as well as in group 6 (FB) (p = .03) was seen in comparison with group 1 (N-DC) & 1a (DC). Also, there were a significant differences in group 4 (FB). Thus, it was concluded the directly effect of barley on blood glucose level. Wheras, a slight significant decrease in the groups that given the barley and flour compared with control group. This confirms the ability of barley flour to decrease blood glucose when taken appropriately (9; 16). The results were proved the ability of barley to decrease the painful symptom of diabetes in fat mice, and may act as a therapeutic supplement to regulate blood glucose levels due to fiber content in barley and also help to prevent blood sugar levels from rising too high in people with diabetes (15). "In addition, the barley and other whole grains are rich sources of magnesium, a mineral that acts as a co-factor for more than 300 enzymes, including enzymes involved in the body's use of glucose and insulin secretion (16; 21)".   Table 6, was noted that the barley has an effect on red blood cells and hemoglobin in groups given barley or (barley & wheat flour) paste, due to the presence of proteins in sufficient amounts of barley that helps the blood system to produce important components (9). Also the results were shown decreasing in a number of platelets after given the groups both paste (barley only or barley-wheat) paste, that may be due to the detect in bone marrow or deficiency resulting from break in the platelets after leaving the bone marrow and pass through the blood vessels to spleen and sticking together with active molecules of barley thus make it a strange body for the immune system that led to attack and the hemolysis (21). Barley also rich in beta-glucan, which plays a role in lowering cholesterol, especially LDL cholesterol and maintaining the health of the blood system. Result of this study showed a high significant differences in cholesterol and triglyceride content in all mices that fed barley flour only, also there were a significant differences in mices groups that fed a mixed equally amount of barley and wheat flour of both sex of mices. This finding was comparable with others who stated that barley fiber reduces glucose, cholesterol and triglycerides through the increase the activity of GIT (18). The low cholesterol level have been founded may be due to the ability of barley to effect on protein receptors that in turn effect on the liver and led to reduce the production of triglycerides (5; 6; 24). "In addition, the soluble barley's dietary fiber available in barley is an appropriate environment for friendly bacteria in the large intestines. When these helpful bacteria ferment soluble barley's fiber, they produce a shortchain fatty acid as butyric acid, which serves as the primary fuel for the cells of the large intestine and helps maintain a healthy colon. Also, these helpful bacteria create two other short-chain fatty acids, propionic and acetic acid, which are used as fuel by the cells of the liver and muscles (26). As well as barley's dietary fiber contain a high beta glucan, which assisted to lower the cholesterol by binding with bile acids and removing them from the body through feces. Bile acids have been shown to be useful for cholesterol-producing liver enzymes, so when bile acids are secreted into the gut, they mix with the dietary fiber. The liver builds up these new bile acids using a higher amount of cholesterol to manufacture these acids and thus reduces any cholesterol in the blood (13)".

Histopathological changes in liver
The liver section as shows in Fig. 1, section A, the control grou showed the section reveal radial arrangement of hepatocyte, sinusoid originates at lobules margin toward the hepatic vein (c.v. ) (H&E stain X). While, section B, diabetic mice that were fedding standard food had congestion and necrosis due to the high level of glucose which effect badly the liver of mice. section C group 3 &4, that mice were fedding barley and wheat flour also showed heavy congestion with infiltration of inflammatory cell, dilation of number of hepatocyte cells with apoptosis (H&E stain X40). In section D, group 5 &6, mice were fedding barle flour only, were also shown still a congestion in liver vessels with infiltration of inflammatory cells and enlargement of hepatic cells in the liver with the emergence of kupffer cells. This may be need more time, until barley flour effects positively to heal the kidney tissues.  Fig. 2, section. A, the control group showed the appearance of renal tissue which consist glomeruli and renal tubules ( Proximal and distal convoluted tubules ). While, section B, diabetic mice that were fedding standard food kidney was showed hemorrhage in its tissue. dialation in glumeruli and convoluted tubules, presence of vacuoles with degenerative changes ( H&E X40). Section C group 3 &4, that mice were fedding barley and wheat flour also were showed hemorrhage in kidney tissue, dialation in glumeruli and convoluted tubules, presence of vacuoles with diffusion of large number of inflammatory cells ( H&E X40). In section D, group 5 &6, mice were fedding barle flour only, were also shown still a showing hemorrhage in kidney tissue, dialation in glumeruli and convoluted tubules, presence of vacuoles with diffusion of large number of inflammatory cells. This may be need more time, until barley flour effects positively to heal the kidney tissues ( H&E X40). Histopathological changes in brain It was found that barley concumption had not any effection on brain tissues as shown normal glial cells and other cells section 1 in Fig. 3, compared with normal group as clear in Fig. 3. without exception, as well the histological change in liver and kidney may probably due to giving a paste in continuous period, while there were no strange change in brain tissues of any. So it is necessary to urge to provide barley flour and to educate the owners of factories for the manufacture of cereals for the production barly , s pastas, noodles, bread, roller-milled barley bran and flour, panacakes, muffins, doughnuts, biscuits, waffles…etc. of such products because of the lack of Iraqi markets for such products and their importance to consumer health, which is looking for the provision of these products because there is currently awareness and nutrition awareness of barley and its importance For public health and for diabetics, especially through private satellite channels, health and food.=