Therapeutic and Preventive Effects of a New Type Healthy and Viable Food Supplement on Fatty Liver and Blood Lipids in Animal Model


 Background: Fatty liver disease is one of the most common liver complications worldwide. Also, blood lipids are elevated cholesterol or triglyceride levels from the normal amount in the blood that causes fatty liver disease. The aim of this study was to investigate the therapeutic effects of a new healthy and viable food supplement from wheat on fatty liver disease and blood lipids in animal model.Methods: The new NBS healthy and viable food supplement was prepared by a green route. The NBS healthy and live food powder had various vitamins, macro and micro molecules, and ingredients such as B1, B2, B3, B5, B6, B9, C, K, A, E, D, phosphorus, potassium, sulfur, magnesium, calcium, iron, manganese, zinc, copper, omega-3 and etc. For therapeutic and preventive effects of the new healthy and viable dietary supplement on fatty liver and blood lipids, 25 Wistar rats weighing 180–220 g were used. The rats were divided into 5 groups and were treated with 25, 50 and 100 mg/kg of healthy and live medication.Results: Investigation of the interaction between the concentrations of the medication supplement showed that 100 mg/ kg has the most therapeutic effect against oasises in fatty liver disease. Also, it was found that the concentration of 1000 mg/ kg has the most reducing effect on the level of lipid profile.Conclusions: The new food supplement reduces the level of hepatic macrovesicles, microvesicles, and the steatosis symptoms without specific hepatic complications. Also, the healthy food causes reduction of lipid parameters.


Introduction
Fatty liver disease is a chronic inflammation of the human body that has led to a substantial increase in our population. The importance of this disease is due to the destruction of liver cells and in the absence of early diagnosis and proper treatment in which can lead to progressive and irreversible liver disease called cirrhosis. Hypertension, hyperlipidemia, obesity and diabetes that all of them are components of metabolic syndrome, have been observed with fatty liver disease. For this reason, some researchers may know fatty liver disease as insulin resistance, or metabolic syndrome. Insulin resistance has adverse effects on vital organs such as the heart and brain blood vessels, kidney, peripheral nerves and ultimately the liver. In the other word, fatty liver disease can be a sign of resistance to insulin and for this reason, early diagnosis and appropriate treatment will prevent of the liver damages and the cardiovascular complications that are the most important cause of death in patients with fatty liver [1]. Lipid-lowering drug gemfibrozil is that medicines improve symptoms in patients with fatty liver is laboratory [2]. Statins are other lipid-lowering drugs, particularly cholesterol, which also caused symptoms improvement in laboratory [3], however, the Ursodeoxycholic acid as a protective liver cells is not very useful in recent studies [4]. Based on the available information, the treatments of fatty liver are weight loss, elimination of drugs and toxins as well as diabetes and blood lipid controls [1]. However, lack of proper treatment and numerous side effects of existing chemical drugs including gastric bloating, stomach ache and heartburn, cutaneous rash and nausea or vomiting may continue to occur in the area of drug medications. Thus, the aim of this study was to evaluate the therapeutic activity of a new healthy and viable food supplement, without side effects, against fatty liver disease.
On the other hand, blood lipids are elevated cholesterol or triglyceride levels from the normal amount in the blood that causes fatty liver disease. The amount of these substances in the blood increases as a result of dietary intake is more than needed or when the metabolism of fat develops. High levels of fat or cholesterol can lead to complications such as atherosclerosis (stiff and vasodilated), high blood pressure, cardiovascular disease, increased risk of stroke, fatty liver, and so on.
Inappropriate diet therapy, the use of fatty and high-calorie foods, inappropriate culture of over consumption of fast foods, inappropriate culture of the consumption of red meat in society and machinery life has caused many people have a problem with high fat and blood cholesterol or obesity. Cardiovascular diseases are now the leading cause of death in industrialized countries. Reports suggest that the disease has caused death in 95,000 people in the United States in 1998, and in 2000, this country incurred about $ 118 billion in costs for this disease. Increased blood lipids, especially cholesterol as an important factor in exacerbating this disease. Now, there are over 100 million Americans adult with high blood cholesterol and about 50 million of these need treatment [5][6][7]. Over the past few decades, most countries have increased the use of alternative therapies, especially herbal therapies and dietary supplements, to improve a variety of diseases, including high levels of blood lipids.
One of the major problems of physicians and consumers of herbal medicines is insufficient information on drug health and its effect on disease. Fortunately, over the past 30 years, there has been a great deal of research on the effectiveness of medicinal herbs used in the traditional medicine that prove their efficient or inefficient. Recent research into the nutritional supplements and herbal drugs used in traditional medicine suggests that their compounds, including dietary fiber, vitamins, flavonoids, sterols, and other antioxidant compounds, can reduce LDL oxidation and free radical oxygen uptake and probably, with effect on the immune system and metabolic disorders improve this disease [8][9][10][11][12].
Considering the mentioned issues and the increasing in the number of diseases associated with lipid profile and the lack of definitive treatment of this disease, the present study was designed to investigate the reducing effect of live and healthy food powder on the level of lipid profile.

Biochemical evaluation of serum of rats in two groups of control and high fat diet
In order to create steatosis, rats were fed with high fat diet. The fat emulsion according to the method described by Zou et al. [13], were contains 400 grams of corn oil, 150 grams of sucrose, 80 grams of whole milk powder, 100 grams of cholesterol, 10 grams of sodium deoxycholate, 36.4 grams of polysorbate 80, 31.1 grams of propylene glycol, 2.5 grams multivitamin, 10 grams of salt, 1.4 gram of mixed minerals and 300 ml of distilled water. The mice were fed a high fat emulsion via gavage, in the amount of 10 ml/kg, daily for 4 weeks.
At the same time the control group were given equal volumes of saline via gavage daily.
After 30 days of treatment, to evaluate the effect of healthy and viable dietary supplement on fatty liver induced by high fat diet in rats, blood sampling and evaluation of the parameters level was performed using capillary tube and biochemical kits, respectively. Also, for histopathological study of the studied animals, they killed using mechanical method (spinal cord dissection). From these samples, sections of 4-6 micron was prepared using standard methods of the tissue processing and histopathological sections, and stained with hematoxylin-eosin.

Blood biochemical parameters of rats receiving different concentrations of dietary supplement and healthy living
After 30 days of treatment, to evaluate the effect of different concentrations of healthy and live dietary supplement on fatty liver caused by high fat diet in rats, capillary blood sampling was performed. The levels of the parameters were measured using biochemical kits, the results of these experiments were recorded.

Liver pathology of mice treated with live and healthy dietary supplements
The rats in the treatment groups at concentrations of 25, 50 and 100 mg/ ml, healthy and viable dietary supplement, after 4 weeks of treatment, blood samples were collected using mechanical method (spinal cord dissection). All mice were sacrificed and tissue samples were fixed in 10% formalin. The above specimens were prepared using high-pressure silica gel and from three phylogenetic bases, 4-6 mm slices were prepared and stained with hematoxylineosin. The results of these experiments were recorded.

Blood lipids profiles
In fatty diet groups, high fat emulsion was used for fatty diet induction, according to the method presented by Zou et al. [13], as mentioned in fatty liver section. At the end, blood samples were taken from the mice and the level of blood lipid parameters was measured.
After a 30 day treatment, the effect of food powder (healthy and alive) on fatty profiles of high fat diet recipients was performed using a blood sampler tube from the rat's eye. The level of the parameters was measured using biochemical kits.

Statistical Analysis
In order to analysis the data in this study, we first make sure that the distribution of data is normal, using Kolmogorov-Smirnov test. Also, in this study, in order to evaluate the significance of the data, it is recommended to use ANOVA test. ANOVA test was used to investigate the differences between and within groups. P<0.05 indicates statistical significance. In order to clarify this issue, by Scheffe post hoc test, this significance was tested one by one between groups.

Biochemical evaluation of serum of rats in two groups of control and high fat diet
According to the results and comparing the results of two groups of mice (control and a high fat diet), can be stated that the levels of the serum alanine amino transferase (ALT), aspartate aminotransferase (AST), serum total bilirubin (TB) and alkaline phosphatase (ALP) compared with healthy control group, have a significant increase and total protein (TP) and albumin (Alb) have significant decrease. According to the biochemical tests results of the level of liver parameters and comparison of the results of healthy group with the patient group (high fat diet) ( Table 1(a-b)) ( Fig. 1(a-f)) and also according to histopathologic changes in the liver tissue of control rats ( Fig. 2(a-d)), it can be concluded that the diet used in this study caused fatty liver disease. Fig. 2(a-b) show the microscopic examination of the liver tissue of a rat in the control group in which hepatocytes and liver tissue structure are normal. In contrast, the microscopic examination of liver tissue with high-fat diet fed that prove the formation of the large fat macrovesicles (rounded bodies, white and hollow), are shown in Fig. 2(c-d).

Blood biochemical parameters of rats receiving different concentrations of dietary supplement and healthy living
According to the results of tests of biochemical studied mice, it can be concluded that the dietary supplement alive can return biochemical parameters to normal levels. Investigation of the interaction between the concentrations of healthy and live medication supplement showed that 100 mg / kg showed the most effective therapeutic effect and 25 mg / kg the least effect against steatosis (5% probability level).

Results of liver pathology of mice treated with live and healthy dietary supplements
Also, according to the observations of the blood biochemical tests of mice receiving healthy and live medication supplement (Table 2(a-c)), and also the results of liver pathology images ( Fig. 3(a-f), It can be concluded that a healthy and viable dietary supplement return the level of hepatic parameters to normal state and reduces the level of hepatic macrovesicles, microvesicles, and the steatosis symptoms without specific hepatic complications. In microscopic studies, no abnormal state in the mice liver of the control group were observed.
While, in mice fed with only high fat diet for 30 days, severe liver steatosis as macrovesicles, and sometimes microvesicles fat, accompanied by hepatocytes swelling, had been created ( Fig. 2). In the high fat diet group treated with healthy dietary supplements, the incidence of fatty change in hepatocytes significantly was prevented. Pathology of liver tissue of mice treated with a concentration of 25 show no changes on the large fat macrovesicles (rounded bodies, white and hollow) in patient groups ( Fig. 3(a-b)). In contrast, pathology of liver tissue of mice treated with a concentration of 50 and 100 show significant changes on the large fat macrovesicles (rounded bodies, white and hollow) in which fats are sporadic and mild. (Fig.   3(c-f)).
Investigation of the interaction between the concentrations of healthy and live medication supplement showed that 100 mg/ kg showed the most effective therapeutic effect and 25 mg/ kg the least effect against steatosis (5% probability level).
Meanwhile, in order to evaluate the significance of the data, it is recommended to use ANOVA test. ANOVA test was used to investigate the differences between and within groups. The results of this test showed that there was a significant difference between the groups with 5% probability level. In order to clarify this issue, by Scheffe post hoc test, this significance was tested one by one between groups. The results can be seen in Table 3.

Blood lipids profiles
In this research, comparing the results of two groups of control and high fat diet, it can be concluded that in rats fed with high fat diet, serum levels of glycoside, LDL increased significantly and with attention the results of biochemical tests on blood lipids can be found that the diet used in this study induced lipid-related disease in rats (Tables 4-8). According to the results of blood biochemical tests in the studied mice, it can be concluded that dietary powder (healthy and alive nutrition) causes reduction of blood lipid parameters in the studied mice. In the study of the interaction of the concentrations of food powder (healthy and alive nutrition), it was found that the concentration of 1000 mg/ kg had the most and the concentration of 250 mg/ kg had the least therapeutic effect on increasing the blood lipid profiles.
In order to analysis the data in this study, we first make sure that the distribution of data is normal, using Kolmogorov-Smirnov test. In this regard, by using the information test obtained by observing Sig, it can be stated that these data have a normal distribution.
Therefore, parametric tests should be used to analysis the differences between groups. In this study, in order to evaluate the significance of the data, it is recommended to use ANOVA test.
ANOVA test was used to investigate the differences between and within groups. The results of this test showed that there was a significant difference between the groups with 5% probability level. In order to clarify this issue, by Scheffe post hoc test, this significance was tested one by one between groups. The results can be seen in Table 9.

Discussion
The liver is one of the important members of the body that detoxify from drugs, disposal of waste products resulting from demolition and renovation as bile red blood cells, the production of blood clotting factors, glucose stored as glycogen and the regulation of sugar and fat metabolism are the most important roles of liver in the body. However, the role of liver should not be ignored in fat absorption and defense against microbes and toxins absorbed by fatty food. Fatty liver in medicine is a reversible condition of the accumulation of fat vacuoles in the liver cells, which is characterized by liver inflammation. This condition may occur in people who drink alcohol, but in Iran the disease has other causes and is called non-alcoholic fatty liver. The non-alcoholic form of the disease occurs in a number of clinical disorders such as diabetes, obesity and malnutrition. Fat existence in the liver is normal, but if this amount exceeds 2 to 5 percent of the total liver weight, the person develops fatty liver disease. It has no specific symptoms, but not observing and progression this disease, it was causes indigestion and eventually leads to death [14]. Background disorders should be treated to improve fatty liver. Treatment is currently focused on controlling the medical problems and conditions that underlie fatty liver. Various new drugs, such as metformin, have been introduced for the treatment of fatty liver, but their definitive effect has not been established.
Daily exercise and regular consumption of fruits and vegetables have beneficial effects on the disease. Gradual weight loss is necessary and beneficial in obese people, but sudden weight loss exacerbates the disorder. Vitamin E at a dose of 4 mg/ day reduces the damage to liver cells and improves liver enzymes, but because it is associated with an increased risk of heart disease, long-term use is not recommended.
Increase the activity of liver function biomarkers, includes AST, ALP and ALT in serum, is indicative of liver damage [15], since the change in the above markers during the liver steatosis previously have been reported [16][17]. So in this study, serum levels of these enzymes were studied. In this research, the levels increase of the serum ALT, AST and ALP in the serum of the high fat diet mice was observed that indicate damage to the liver cells and

Conclusions and implications
In this research, based on the results of the biochemical tests of blood of the studied mice, it can be concluded that the new NBS healthy and viable food supplement will restore the level of liver parameters to normal. Interaction effects of the concentrations of healthy and live dietary supplements showed that 100 mg/ kg showed the most effective therapeutic effect and 25 mg / kg showed the least effect against acaiosis. Based on the observations from liver pathology images, it can be concluded that a healthy and viable dietary supplement reduces the level of hepatic macrovesicles, microvesicles, and the steatosis symptoms without specific hepatic complications. Also, the results of this study showed that the use of the new NBS healthy and viable dietary supplement would normalize the fat-related factors in blood serum, so that can use this compound as a lipid lowering agent. Given the similar results, clinical studies are needed to extend the results to the community. Table 1. Test results of liver enzymes and protein levels in control and patient rats (a and b). Table 2. Test results of liver enzymes and protein levels of mice treated with the concentrations of (a) 25, (b) 50, and (c) 100 healthy and alive nutrients.        Protein and (f) ALP blood levels of healthy group and high fat diet.