STUDY OF THE EFFECT OF CHITOSAN EXTRACTED FROM THE MUSHROOM ON THE EXPERIMENTALLY INDUCED HYPERLIPIDEMIA IN MALE RABBITS

The present study aimed to identify the therapeutic evaluation of chitosan extracted from the fungus cushroom and pure chitosan on glucose and lipid profile in the blood of 35 male rabbits with hyperlipidemia induced experimentally by cholesterol. The tests included estimation of glucose levels, total cholesterol, triglycerides, high-density lipoproteins, low-density lipoproteins, and very low-density lipoproteins. hyperlipidemia was induced in the male rabbits used in the study which was administered orally with cholesterol 150mg/kg body weight for a week. rabbits were divided into seven groups: control, cholesterol, pure chitosan, mushroom chitosan, cholesterol and pure chitosan, cholesterol and mushroom chitosan and cholesterol and simvastatin. The results of the study showed, the hyperlipidemia induced experimentally resulted a significant increase (P<0.05) in TC, TG, LDL, and VLDL, while no significant difference in HDL compared with control group, on the otherwise the glucose level significantly increase than control. Also, groups of animals treatment with pure chitosan and mushroom chitosan showed a significant decrease (P<0.05) in glucose, TC, TG, LDL, and VLDL, and no significant difference in HDL compared with control group. While, the groups showed treatment with cholesterol and pure chitosan, cholesterol and mushroom chitosan, cholesterol and simvastatin a significant decrease (P<0.05) in glucose, TC, TG, LDL, and VLDL, and a significant increase (P<0.05) in HDL compared with the cholesterol group. The research study revealed that chitosan extracted from mushroom can control the levels of fat concentrations and their complications, in addition to its important role in biochemical variables, and treatment of most disease cases, especially cardiovascular disease.


INTRODUCTION
Nutritional fungi, including mushroom, are rich in carbohydrates that include dietary fibers and polysaccharides such as gluconate, glycogen, monosaccharides and disaccharides, and it are rich in protein content because it contains most of amino acids, and it also contain low levels of fats, and they are in the form of sterols. And unsaturated fatty acids, as well as containing low levels of sodium and high levels of potassium, iron and selenium, in addition to containing vitamins and antioxidants In view of the importance of chitosan from a medical and preventive point of view, this study aimed at the therapeutic evaluation of chitosan extracted from Mushroom against hyperlipidemia induced in rabbitsand compare its effect with simvastatin.

MATERIALS AND METHODS Collection and preparation of Sample
Samples of mushrooms were collected from Al-Wadq Farm, Baghdad, Iraq. The samples were washed well with running tap water several times to get rid of soluble organic matter, adherent proteins and other impurities. Then they were dried for 4 consecutive days by exposing them to the heat of the sun, then grinded to obtain a homogeneous fine powder. Keep it in closed plastic containers at room temperature until use.

Extraction of Chitosan
Chitosan was extract according to the method used by (Kamil et al., 2002) with some modifications made to it.

Diagnosis of chitosan
The chitosan was diagnosed using a Fourier Transform InfraRed Spectrophotometer (FT-IR) Shimadzu company/ Japan affiliated to the laboratories of the Department of Chemistry, College of Science, University of Tikrit (Vaingankar & Juvekar 2014). And a person was also diagnosed by dissolving it in 5% acetic acid, because the dissolution process is in itself one of the evidence for the formation of chitosan.

Initialization of laboratory animals
The experiment was using 35 rabbits male were getting from the local market with weights ranging 1.5-2.0 kg and ages 5-8 months, the animals were divided and distributed uniformly of weight and placed in tight wooden cages with dimensions of 50 × 60 × 150cm. The animals were subjected to laboratory conditions with a light cycle divided into 12 hours of light and 12 hr of darkness.

Experience Design
The experimental animals were divided and distributed uniformly in terms of weight into Seven groups, 5 animals for each group. The animals were treated once daily for 21 days. Hyperlipidemia was induced in 4 groups of experimental animals using cholesterol by oral injection 150 mg/ kg dissolved in soybean oil a period of two weeks, and this was confirmed by conducting lipid tests for the animal group, then the animals were left for 48 hour, the treatment stage began during the period of 21 days, according to the following groups: (1): (Control) was given the standard feed and water.

Collection of blood samples
After the end of experiment, the animals were anesthetized with chloroform to collect blood samples from the jugular vein and placed in gel tube, were centrifuged (4000 rotation/min) for 15 min, to obtain the serum was stored in plan tube a temperature of -20°C until the analysis was conducted.

Biochemical of blood tests
The device was chemical analyzer USA(Smart-150) to estimate all the tests glucose, total cholesterol (TC), triglycerides (TG), high-density lipoproteins (HDL), low-density lipoproteins (LDL), and very low-density lipoproteins (VLDL). According to the manufacturer of the solutions.

Statistical Analysis
The data of results in the present study were analyzed by using the ANOVA analysis, utilized the general linear model of the Statistically Analysis System. Also, significant differences were evaluated by using Duncan's multiple-range test (Duncan, 1955), and significance level is based on level of probability (P<0.05).

RESULTS AND DISCUSSION
Chitosan was obtained from mushroom as in (Figure 2).

FTIR spectroscopy
The FTIR spectrum of the chitosan model extracted from mushroom, (Figure 3) shows the amino group (NH 2 ) is the most important active group, that absorption peak appeared at the frequency 1490 cm -1 , where the appearance of this group on the carbon 2 site of glucose amine is an evidence of the presence of chitosan, and these results are close to what he found (Kumirska et al., 2010).While the active group, which represents the bundle of N -H groups, its absorption peak appeared at frequencies 3265 cm -1 . While the active group, which represents the hydroxyl stretching band, its absorption peak appeared at frequencies 3366 cm -1 , as this group appears in chitosan and chitin because it is not affected by the removal process Acetyl groups or the hydrolysis process is therefore a reference to ensure the presence of chitin and chitosan (Ebrahimzadeh et al., 2013).The beams at frequencies, 1628 cm -1 refer to the group C = O in the primary group (Amide Ι), and their intensity depends on the degree of removal of the acetyl group (Arantes et al., 2014). The absorption bands that appeared at the frequencies, 2879 cm -1 which belong to the stretches of the C -H group, this result agreed with study (Al-Abbasy et al., 2018). The glycosidic bond of the β-anomer of chitosan, its absorption peak appeared at frequencies 895 cm -1 , this result is in agreement with what was mentioned (Wu et al., 2019).

Biochemical tests of blood
The results of the current study showed in (Table 1)   It also showed a significant decrease (P<0.05) in the group treated with (MushroomChitosan), and there was no significant difference in the group treated with (Pure Chitosan) compared with the control group. While there was a significant decrease (P<0.05) in the groups treated animals with (cholesterol and pure chitosan, cholesterol and MushroomChitosan, cholesterol andsimvastatin) compared with the cholesterol group.
There is a relationship between high glucose and high blood lipid levels, and this is reflected in a group of mechanisms, including weak insulin signal in muscles with other tissues, according to the increase and accumulation of extracellular fats (Kopelman 2000).
The reason for the high blood sugar concentration may be attributed to the increase in the generation of free radicals that destroy beta cells in the pancreas and work to stop their work and destroy them, as free radicals stimulate the process of lipid peroxidation and breakdown of the RNA and inhibit the synthesis of the primary insulin, the blood sugar concentration increases, thus halting the degradation of blood sugar and stimulating the processes of blood sugar formation and glycogenolysis (Ayalaet al, 2014). After eating a meal, the blood sugar level will increase, and is preceded by inflammation and endoplasmic reticulum stress, which leads to an increase in insulin resistance and weakening of insulin secretion (Bender et al., 2014). Polyunsaturated fatty acids suppress liver fat synthesis (Albert  et al., 2014). The fatty acids reduce the reproduction of genes encoded for the breakdown of liver fats or glycolytic enzymes.
The presence of a significant decrease in the glucose concentration for groups of animals treated with Pure Chitosan and MushroomChitosan may be due to the fact that chitosan stimulates the secretion of insulin from the pancreatic islets directly, leading to facilitating the entry of calcium ions into the pancreatic beta cells, which is the key to the metabolic pathway in regulating the glucose response (Zhang & Lin 2004).
The results of the present study showed in (Table 1)  Also, the groups of animals treated with (pure Chitosan, MushroomChitosan) showed a significant decrease (P<0.05) in the concentrations of levels of TC, TG, LDL and VLDL, and no significant difference in HDL concentration compared with the control group.
While the groups treated with (cholesterol and pure chitosan, cholesterol and mushroomchitosan, cholesterol and simvastatin) showed a significant decrease (P<0.05) in the concentrations of total cholesterol, triglycerides, LDL and VLDL levels, and a significant increase in the HDL concentration compared with the cholesterol group.
The significant increase in the concentration levels of (TC), (TG), low-density lipoproteins (LDL), and very low-density lipoproteins (VLDL), in the blood serum of male rabbits infected with new hypercholesterolemia is a natural result as a result of feeding animals with cholesterol, which may be due to the increase in the resulting cholesterol esters This confirms the ability of cholesterol to raise the concentration of triglycerides, or it may be due to changes in the absorption process and excretion of steroids or a decrease in the level of bile salts, or the increase in cholesterol may be due to the presence of a disease affecting the liver, which leads to its inability to Benefit from cholesterol for conversion into HDL and LDL (Abdelhalim & Al-Ayed 2008).
HDL-C plays a cholesterol-lowering role as it picks up excess cholesterol from the blood and peripheral tissues to the liver, where it is converted into bile acids, and plays an important role in inhibiting the formation of atherosclerotic plaques in the arteries and for this reason it is known as good cholesterol (Kim et al., 2008), and it is possible to increase HDL-C, with treatment, is due to the activation and increase of the activity of the enzyme Lecithin-Cholesteryl acyl transferase (LCAT), which is the enzyme responsible for combining cholesterol with HDL, and inhibiting the enzyme Hepatic triglycerides lipase (HTL), which leads to rapid lipid catabolism through the suprahepatic tissue (Anila & Vijayalakshmi 2002).
LDL-C is responsible for transporting cholesterol to the cells of the body, it transports about 60-70% of total cholesterol, so the increase in TC levels is followed by an increase in LDL-C that cannot be eliminated in the fat metabolism process, and it is more likely that it will enter into the distance.Subepithelialis as a prelude to oxidation, and LDL-C carries oxidation by inhibitors of macrophages, and large fat-laden macrophages will leave pulp rich or full of fat and cholesterol after storage and then atherosclerosis begins (Beckman et al., 2002).
The significant decrease in the levels of cholesterol, triglycerides, LDL-C and VLDL-C levels, and an increase in HDL-C levels in the groups treated with chitosan may be due to the effect of chitosan that reduces the absorption of fat and cholesterol, and that chitosan is degraded into multiple sugars in the intestine, which causes an increase in Viscosity and reduced absorption of fat and cholesterol (Panith et al., 2016). In addition, the decomposition of chitosan into glucose amine reduces the formation of triglycerides in the liver (Kobayashi et  al., 2006). Chitosan is also considered a dietary fiber, and its action is to surround fat drops and prevent the digestive enzymes from reaching the fat, which leads to its excretion outside the body (Biskup et al., 2007).
Studies indicate that the positive charge carried by the chitosan molecule (amino groups) makes it associated with negatively charged substances such as fats, cholesterol and lipoproteins. Chitosan mixes with the fat in the food in the stomach and then interferes with the fat droplets to form a lipid-chitosan complex. In the small intestine, thus preventing lipolysis and thus the secretion of undigested fats, including cholesterol (Santas et al., 2012). The results of the current study are consistent with what was reported by (Bahijri et al., 2017)