The Possible Improving Effects of γ-Irradiated and / or Extruded Soy Flour on Hypercholesterolemic Rats

Hypercholesterolemia is serious conditions that can cause fatal complications without careful management. Among the dietary supplementation with functional food, soybeans possess variety of antioxidant compounds that may lower incidence of hypercholesterolemia and degenerative cardiovascular disease. Thus, the purpose of this study is to determine the effect of gammairradiated and/or extruded soy flour on hypercholesterolemic rats. Processing of soy flour by γ-irradiation and/or extrusion reduced the amount of antinutritional factors such as tannin and trypsin inhibitor and resulted in different changes in the total amino acids and fatty acid contents. The animals maintained on the HCD showed remarkable decrease in the level of HDL-C associated with significant increase in the values of serum total lipid, total cholesterol, triglyceride, LDL-C, vLDL-C and the risk ratio in addition to serum concentration of urea, creatinine and uric acid in comparison with those of the control group. However, dietary supplementation of raw and treated soy flour resulted in reduction in the bad changes induced by HCD in the above mentioned parameters. In conclusion, treated soy flour supplementation in diet of rats pointed out to its hypocholesterolemic effect and its ability to improve lipid profile and kidney function of hypercholesterolemic rats.


Introduction
Hypercholesterolemia is the presence of a high level of total cholesterol in the bloodstream (Barbana et al., 2010) and it is closely associated with obesity, diabetes mellitus, and several other metabolic syndromes (Aleisa et al., 2013).Also, it seems to be an important risk factor accounting for severe atherosclerotic diseases (Augusti et al., 2012).
Several studies indicated that soybean (Glycine max (L.) Merrill) is an effective dietary component and contains many bioactive components such as soy protein and soy isoflavones that can improve lipid profiles and reduce risk factors for chronic diseases like hypercholesterolemia and cardiovascular disease (Llaneza et al., 2011).
Moreover, soybean contains several antinutritional factors, which could limit their consumption such as trypsin inhibitors (TI) and tannin content (Gu et al., 2010) and removal of undesirable components is essential to improve the nutritional quality of soy.Various conventional and simple processing methods have been used such as gammairradiation (Dixit et al., 2011) and extrusion (Frias et al., 2011) in order to inactivate or reduce the antinutritional substances.
Therefore, the objective of the present study was oriented to the use of γ-irradiation and/or extrusion for in-activation or removal of certain antinutritional factors as well as to study the biochemical effect of γ-irradiated and/ or extruded soy flour on lipid profile in hypercholesterolemic rats.

Material
Freshly dried soybean seeds were obtained from the Agriculture Research Institute, Ministry of Agriculture and Land Reclamation, Giza, Egypt.Seeds were sorted by discarding damaged and immature ones.They were stored in air-tight containers at room temperature (25±1 °C) prior to further use.

Processing of soy bean seeds a-Gamma irradiation treatment
Soybean seeds were packed in polyethylene bags, and sealed by heat.Each bag contained about 500 g for gamma irradiation treatment.They were subjected at ambient temperature to gamma irradiation from 60Co source at the National Center for Radiation Research and Technology (NCRRT) -Nasr City, Cairo, Egypt.The facility used was the Indian Gamma Chamber 400 A, 60 Co facility.The doses applied were 5, 10 kGy, delivered at a dose rate

Animals
The experiments were conducted on male albino senile rats (130-150 g).The animals were housed under conditions of controlled temperature (30±2 ºC) with natural light.
The animals were randomly divided into 8 groups, each consisted of 10 rats.
• Group I: Fed on the basal diet for 10 weeks and considered as control group.
• Group II: Fed on hypercholesterolemic diet for 10 weeks and considered as a positive control group.
• Group III: Fed on hypercholesterolemic diet containing raw soybean flour for 10 weeks and considered as a treated control group.
• Group VI: Fed on hypercholesterolemic diet with extruded soybean flour for 10 weeks.
At the end of the experimental period, the rats in each group were fasted overnight, anaesthetized with diethyl ether and sacrificed.Blood samples were collected by heart puncture, allowed to coagulate and centrifuged to obtain serum for biochemical analysis.

Statistical analysis
Statistical analyses were performed using computer program Statistical Packages for Social Science (SPSS, 1998), and values compared with each other using suitable tests. of 4.75 kGy/h as calibrated using small pieces of the radio chromic film (McLaughlin et al., 1985), at the time of experimentation.

b-Heat processing (Extrusion)
The whole soybean seeds were cleaned, dehulled and then extruded by single screw extruder with three zones (At the soy processing unit, food Technology Research Institute, Agriculture Research Center, Giza, Egypt).The heat treatment rise gradually through extruder zones for short time (Wijeratne and Nelson, 1993).

c-Combination of γ-irradiation and extrusion
Soybean seeds were subjected to gamma irradiation at dose levels of (5 and 10 kGy) then followed by heat processing.

Biological study Diet
The raw and processed soy flour mixed with the other feed ingredients.Vitamin-mineral mixture was prepared by mixing analytically pure vitamin compounds with mineral salts and kept at 4 °C until used.The dietary mixture was freshly prepared every week.Control group received a basic diet (20% casein, 10% soybean oil, 5% cellulose, 4% mineral mix, 2% vitamin mix, 6% sucrose, and 53% cornstarch), hypercholestrolemic group received the same diets supplemented with 1% cholesterol, 15% sheep tail fat and 0.25 % cholic acid (Rossi, 2000), while other groups received hypercholesterolemic diet containing raw and treated soy flour obtained in Tab. 1.

Results and discussion
A significant reduction was noticed in the values of tannin content and trypsin inhibitor (TI) by processing of raw flour and the highest reduction was observed in irradiated (10 KGy) +extruded soy flour (Fig. 1).The animals maintained on the HCD showed significant high values of some lipid contents associated with remarkable decrease in the level of HDL-C in comparison with those of the control group.However, dietary supplementation of raw and treated soy flour resulted in reduction in the bad changes induced by HCD in the above mentioned parameters.
Rats received daily diet rich in cholesterol had an obvious increase in the serum concentration of urea, creatinine and uric acid compared to the control group.On the other hand, supplementation of HCD with raw and processed soy flour enhanced a significant reduction in the serum level of these constituents.
High dietary cholesterol intake is known risk factor for the development of steatohepatitis (non-alcoholic fatty liver disease), atherosclerosis in humans and animals (Townsend et al., 2008).Soybeans (Glycine max) is a species of legumes that becoming an important economic crop as a major source of protein, energy, polyunsaturated fats, fiber, vitamins, minerals, and other nutrients, for both humans and animals (Rettberg et al., 2011).
In this work, γ-irradiation treatment and/or extrusion reduced the trypsin inhibitor activity (TIA) of soy bean and the maximum reduction was observed in case of irradiation (10 kGy) + extrusion.Inactivation of trypsin inhibitor in irradiated samples could be attributed to the destruction of disulphide (-S-S-) groups (Tresina and Mohan, 2012).Abu-Tarboush (1998) found reduction of 34.9% on the trypsin inhibitory activity in soybean flour radiated with 10 kGy.The author attributed this reduction to the breakage of the trypsin Inhibitory structure with the radiation treatment.Thermal treatment had been reported to be a valuable process for the inactivation of TIA.The reduction in TIA following extrusion by up to 90% was reported in the literature for other foods particularly mungbean, cowpea and blends with other crops (Alonso et al., 2000a(Alonso et al., , 2000b)).
Also, the results revealed a significant reduction in tannins as a result of gamma irradiation.Similar results were reported by Musa et al. (2010).The reduction in tannin content is very favorable since this anti-nutritional factor has the capacity for decreasing protein digestibility.The reduction is probably due to chemical degradation by the action of the hydroxyl and superoxide anion radicals generated by the irradiation (Musa et al., 2010).The extrusion processing in this study resulted in decrease in tannin content.Alonso et al. (2000b) studied the effects of extrusion and conventional processing methods on protein and antinutritional factors in peas and they found varietal changes in their tannin contents, and extrusion was most effective in reducing tannins than the other processes.In addition, El-hady and Habiba (2003) have reported significant reduction in tannin content after extruding legume seeds at different moisture contents.
Derangements in cholesterol metabolism have been associated with the etiology of most human diseases.It is widely reported that high dietary cholesterol intake occasioned by a defect in cholesterol transportation, biosynthesis or catabolism is a risk factor in coronary heart disease (CHD) and atherosclerosis (30).Hence, prevention of hypercholesterolemia will be a positive step in the right direction for the management and treatment of cardiovascular diseases.The results of the present investigation show that rats fed with cholesterol rich diet developed hypercholesterolemia with a significant elevation in serum total lipid, total cholesterol (TC), triglycerides and low density lipoprotein (LDL-C) while there was a significant decrease in the HDL-C compared to control (Tab.2).These results are in agreement with earlier reports on dietary hyperlipidemia (Abdulazeez, 2011) and hypercholesterolemia (Olorunnisola et al., 2012).Moreover, prolonged administration of a high-cholesterol diet a,b, Means within same column followed by different letters are significantly different at (P<0.05),Values are means of three replicates (± SE).Raw→ raw soy flour, R1→irradiated at dose 5kGy, R2→ irradiated at dose 10kGy, H→ extruded, HR1→ irradiated (5KGY) +Extruded, HR2→ irradiated (10KGY) +Extruded.
a,b,c, Means within same column followed by different letters are significantly different at (P<0.05),Values are means of three replicates (± SE).Raw→ raw soy flour, R1→irradiated at dose 5kGy, R2→ irradiated at dose 10kGy, H→ extruded, HR1→ irradiated (5KGY) +Extruded, HR2→ irradiated (10KGY) +Extruded.Tab. 3. Effects of dietary supplementation by raw and treated soy flour to hypercholesterolemic rats on kidney function of kidney damage in soy isoflavones pretreatment groups shows that antioxidants of soy is effective in improving kidney function.

Conclusions
The results of our study showed that the soy flour can play a role in the prevention of risk of developing high blood cholesterol and a significant decrease in the concentration of total lipid, triglyceride, total cholesterol, lowdensity lipoprotein, risk factor.Along with a marked increase in the concentration of high-density lipoprotein, in addition to a marked improvement in kidney function of rats fed a high cholesterol diet supplemented with raw and processed soybean flour compared to rats fed on HCD only.
to animals will accelerate the synthesis of TG, inhibit the metabolism of fatty acids and diminish the secretion of TG from the liver to blood by decreasing the β-oxidation of fatty acids.It leads to the accumulation of excess TG in the liver and the content of TC in mouse serum increased significantly, compared with the normal control group rats (Feng et al., 2011).
On the other hand, the present study showed that consumption of HCD containing either raw or processed soy flour have benefit effects on lipid profile which lowered the levels of total lipids, cholesterol, triglycerides and low density lipoprotein (LDL), and significantly increased HDL-C concentration compared to HCD fed rats.Moreover, results of rats fed HCD with γ-irradiated (10kGy) +extruded soy flour provided marked effect on serum lipid and lipoprotein concentrations than the other groups.That refers to the both effect of gamma irradiation and extrusion which cause increase in the contents of essential amino acids and phenolic compound as well as removal of large portion of the antinutritional contents.
The mechanism for the cholesterol lowering effects of soy protein is that peptides resulting from the digestion of soy protein up regulate hepatic LDL receptors (Manzoni et al., 2003).Isoflavones may work by making the liver more efficient to remove the bad cholesterol from the blood by increasing LDL-receptor densities in the liver (Baum et al., 1998).In addition, Lee et al. (2012) reported that soy flour decreased low density lipoprotein-cholesterol and increased high density lipoprotein-cholesterol. High density lipoprotein (HDL) may enhance the removal of cholesterol from peripheral tissue to the liver for catabolism and excretion.
As a result of HCD intake in this study, urea, createinine and uric acid concentrations were increased in the serum of hypercholesterolemic rats (Tab.3).The elevated urea level in hypercholesterolemic rats is likely due to increased amino acid catabolism, impaired kidney function or liver damage (Pedraza et al., 2004).Yang et al. (2012) observed an increased serum creatinine levels and induced severe renal tubular necrosis in rats fed the high-cholesterol diet for 8 weeks but not in rats fed the normal diet or high-cholesterol diet for 2 weeks.The authors concluded that long-term hypercholesterolemia appeared to be a risk factor for contrast-induced acute kidney injury (CI-AKI), which might be associated with disorders in intrarenal prostaglandins and abnormalities in renal nitric oxide system induced by lipid peroxidation.