Antioxidative and hepatoprotective Effects of Vitamin-A Enriched Cassava / Wheat Flour Composite Bread in Rats Fed High- Fat and Induced with Streptozotocin

Antioxidative and hepatoprotective Effects of Vitamin-A Enriched Cassava / Wheat Flour Composite Bread in Rats Fed High- Fat and Induced with Streptozotocin | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Antioxidative and hepatoprotective Effects of Vitamin-A Enriched Cassava / Wheat Flour Composite Bread in Rats Fed High- Fat and Induced with Streptozotocin Richard Akinlolu Ajani, Stephen Adeniyi Adefegha, Isiaka Adekunle Amoo, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6828908/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background Diabetes is a chronic metabolic disease characterized by hyperglycemia and could lead to several complications including such as hepatopathy, neuropathy and nephropathy, as well as increased risk of cardiovascular disease. Functional foods development has become a cheap and an effective strategy towards the management of diabetes and its complication. Hence, the supplementation of vitamin-A enriched cassava flour in bread production could provide additional nutritional, bioactive constituents and health benefits. Purpose The aim of this study is to assess the effect of vitamin-A enriched cassava /wheat flour composite bread on the in vivo antioxidant status and liver function biomarkers in order to evaluate the hepatoprotective effect of the bread. Methods Forty male Wistar rats, each weighing between 170 and 200 grams, were randomly assigned to eight groups with five rats per group. The control group received a standard basal diet, while the remaining groups were fed a high-fat diet (containing 30% fat) for two weeks before being administered streptozotocin (STZ) at a dose of 25 mg/kg body weight to induce type 2 diabetes. After induction, the diabetic rats were treated with various formulated breads for two weeks. A positive control group received acarbose (25 mg/kg body weight) along with a composite bread during the treatment period. Blood glucose levels were monitored every four days. At the end of the study, the rats were euthanized via cervical dislocation. Blood was collected through cardiac puncture, plasma was promptly separated, and the liver was removed, rinsed, and homogenized for analysis of antioxidant activity and liver function. Results The results revealed that the composite bread had a good antioxidative potential in rats fed with 100% vitamin-A enriched cassava bread plus cocoa powder (100% CF bread + FL) having a significant increase (p < 0.05) in superoxide dismutase, glutathione peroxidase, glutathione-S-transferase, and catalase activities in the liver when compared to the control rats. The value of total thiol and non-protein thiol also revealed a significant (p < 0.05) increase in the diabetic rats fed composite bread as compared with the control. Also, the activities of AST, ALT, and ALP were significantly (p < 0.05) reduced in diabetic rats fed composite bread with 100% CF bread + FL, showing the highest hepatoprotective effect. Conclusion The findings of this study indicate that bread made from a blend of vitamin-A enriched cassava and wheat flour may serve as a safe and effective functional food, offering notable antioxidant and liver-protective benefits. Vitamin-A enriched cassava antioxidant hepatoprotective high-fat diet diabetes Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 1.0 Introduction The risk of liver damage has recently increased due to higher exposure to environmental toxins, pesticides, a high level of alcohol intake, pharmaceuticals, and frequent use of chemotherapeutics (Sai Sruthi Arige et al. 2017; Nagappan et al. 2020). Liver damage is always associated with cellular necrosis, an increase in tissue lipid peroxidation, the depletion of tissue glutathione levels, and many other factors (de la Rosa et al. 2022, Shen-ping et al. 2022). Hepatic disease is a main threat to public health, indicating problems with the hepatic tissue or liver functions, which can also be caused by different factors, such as viruses, or bacteria, autoimmune disease, or the external action of different chemicals (drugs or toxic compounds) (Gan et al. 2025). Mechanisms that underlie the hepatoprotective activity are strongly related to the capacity of antioxidants to scavenge reactive oxygen species (ROS) that are produced by the metabolic conversion of xenobiotics, induce oxidative stress and damage to the tissue (Lelciu et al. 2021; Vicidomini et al. 2024). The liver is the second largest organ in our body, weighing approximately 1.5kg in adults, representing 2% of the total body weight (TBW) (Arige et al. 2017). It plays a remarkable function in our body, like bile production and excretion, the excretion of bilirubin, cholesterol, hormones, drugs, and the metabolism of proteins, fats, and carbohydrates. It is involved with almost all the biochemical pathways for growth, fighting against disease, nutrient supply, energy provision, and reproduction (Kalra et al. 2023). And it functions as a center of metabolism for the nutrients such as carbohydrates, proteins, and lipids and the excretion of waste metabolites (van de Graaf et al. 2024). The liver also removes harmful substances from our blood. It is called detoxification. Consequently, the renovation of a healthy liver is important for the overall well- being of a character (van Hul et al. 2024). Nowadays, modern medicine offers alternatives for the treatment of these pathologies, but despite the advances, few effective drugs that offer protection and regeneration of hepatic cells exist (Gan 2025). Moreover, existing treatments can cause adverse effects, which make the therapy of these pathologies even harder (Ahmed et al. 2024). Thereby, the need for identifying novel alternatives for the treatment of hepatic diseases and for the protection of the liver appears to be important, in order to develop novel agents with high efficiency and a superior safety profile (Gan 2025; Hora et al. 2023; Roehlen et al. 2020). Reactive oxygen species (ROS) in high concentration can cause cellular dysfunction, and, in the worst circumstances, cell death. Ros are extremely damaging agents that cause significant damage to lipids, proteins, and DNA (Nagappan et al. 2020). Oxidative stress is caused by an imbalance between free radicals and antioxidants, which endangers human life by increasing the occurrence of cancer, diabetes, cardiac disease, and neurological problems (Pal et al. 2021). As a result, there is a need to establish defensive mechanisms in order to survive and maintain a healthy lifestyle. Antioxidants are generally an effective approach to preventing oxidative stress by inhibiting ROS concentration by the breaking of chain reactions, free radical scavenging, and chelating transition metals that catalyze free radical formation (Sharma et al. 2019) Numerous synthetic antioxidants are commercially available to alleviate oxidative stress, but they have adverse side effects (Hassanpour and Doroudi 2023; Mukherjee et al. 2024). Plant-derived compounds may be effective as antioxidants because they do not have detrimental side effects because of their natural origin and composite character (Chaachouay et al. 2024; Pal et al. 2022). Plants are a major source of polyphenols. A class of compounds with a phenolic hydroxyl structure, they attract research attention for their potent antioxidant activities (Pal et al. 2021; Pal et al. 2022). Medicinal plants play a key function in human fitness care. Approximately 80% of the world population is predicted to use conventional medicine that is predominantly based on plant materials (Sharma et al. 2021; Devi et al. 2024). A large variety of medicinal plants have been tested and determined to include actives standard with a curative house towards ramification of sickness. A large range of plants and formations have been claimed to have hepatoprotective activities, so the improvement of plant based totally hepatoprotective drugs has been given significance within the global market place (Sharma et al. 2021; Devi et al. 2024). Cassava, being a major source of carbohydrate in local diets, when biofortified with increased levels of carotenoids, provides many other nutritional health benefits, such as improvement of visual ability and immune function (Eyinla et al. 2019; IITA 2017). The new variety of vitamin-A cassava was introduced by the Federal Government of Nigeria under Harvest Plus, which is 25% richer in β-carotene (a pro-vitamin A) than those earlier released in 2011 (IITA 2017; Omodamiro et al. 2012; Eyinla. et al. 2019). The characteristic yellow color associated with the vitamin-A cassava variety is due to its higher vitamin A content (Alos et al. 2016). Vitamin A deficiency is a major public health challenge in sub-Saharan countries (Arlappa et al. 2016). Bread is universally accepted as a very convenient form of food that is important to all populations. It is a good source of nutrients, such as macronutrients (carbohydrates, protein, and fat) and micronutrients (minerals and vitamins), that are essential for human health (Oluwajoba et al. 2012; Kaim et al. 2023). Efforts have been made to promote the use of flour from high protein seeds and locally grown crops to replace wheat flour used in baking bread, thereby decreasing the demand for imported wheat and leading to the production of protein enriched bread (Olaoye and Onilude 2012; Kaim et al. 2023). Most tropical cereal grains and some tubers have been used to make composite flour for bread making (Ayo et al. 2014). The nutritional importance arising from cassava/wheat flour bread’s blends will be harnessed to promote well-being and healthy living for the consumer with associated health challenges. The research may serve as an eye opener to possible solutions to the ravaging effects of cardiovascular challenges, hyperlipidemia, liver damage, and other disease conditions that arise from daily consumption of diets that pose a threat to human wellness and life. Therefore, this research was designed to investigate the effect of the composite bread blend’s blend produced from vitamin-A enriched cassava flour (CF)/ wheat flour (WF) flavoured with cocoa powder on endogenous antioxidant assays (SOD and Catalase, GST, GPx), total thiol and non-protein thiol liver homogenate, MDA level in the livers of HFD/STD-induced Type-2 diabetes in normal rats, and also, liver function biomarkers (AST, ALP, and ALT). More so, histopathological assays were equally carried out on the liver sections of the Wistar rats. 2.0 Materials and Methods. 2.1 Materials. Vitamin-A enriched Cassava, also known as yellow cassava was obtained from the International Institute of Tropical Agriculture, IITA Ibadan, Oyo State, Nigeria. It was peeled, washed, cut into smaller pieces, and oven dried in a cabinet dryer at 45 0 C for 72 hours before it was milled into flour. While wheat flour (Golden Penny), cocoa powder, yeast, and eggs were purchased from Waso market, a major market in Ogbomoso City, Oyo State. Aspartame was purchased from a chemical supplier in Ilorin, Kwara State. All the chemicals used were analytical grades while the water was glass distilled. 2.1.1 Production of cocoa powdered vitamin-A enriched cassava/wheat composite flour bread. Vitamin-A enriched cassava was peeled, washed clean, cut into smaller pieces, and then dried in the cabinet dryer at 45 o C for 72 hours. The dried cassava was milled into cassava flour (CF). The composite mixture of an improved variety of cassava flour (CF) and wheat flour (WF) is in the ratios of 100:0 (100% CF and 0% WF), 50:50 (50% CF and 50% WF), 20:80 (20% CF and 80% WF), 10:90 (10% CF and 90% WF), and 0:100 (0% CF and 100% WF). Sweetening with Aspartame, Egg Albumin was used as an emulsifier, cocoa powder as the flavouring agent (FL), and yeast and water to taste in the production of composite breads. It was baked in a gas oven at 250 o C for 30 minutes, or till it turned golden brown. 2.1.2 Diabetic experiment Forty (40) albino rats weighing 170 -200 g were used in the experiment. The animals were acclimatized for two weeks, given commercial feed and water ad libitum . The animals were grouped into two groups after acclimatization. Group I (the control) consists of five animals, and Group II consists of other rats. Animals in group I (control) were fed a basal diet, and those in group II were fed a high fat diet (30% fat) for two weeks. (Basal diet compositions are; corn flour, skimmed milk, mineral and vitamin premixed, and vegetable oil while High fat diet compositions are; Corn flour, skimmed milk, mineral and vitamin premixed and lard). Then, at the expiration of the second week, the group II animals were induced with 25mg/kg/body weight of streptozotocin (STZ) according to the method described by Adefegha et al. (2014). After 72 hours of induction with STZ, the blood glucose levels of animals were checked using the Fine Test Glucometer. Animals with blood glucose (≥250 mg/dl) were regarded as diabetic. Then, the diabetic group was divided into seven groups, groups II-VIII, of five rats each in a group. The rats were fed with the formulated composite breads for two weeks. Furthermore, group III was treated with acarbose (25mg/kg/body weight) in addition to the composite bread for the period during which the experiment lasted, and the blood glucose of all the rats was examined at four-day intervals using a fine test glucometer by collecting the blood through the tails of the animals. The animals were subjected to a night fast a day prior to the sacrifice by removing composite bread and water. The animals were sacrificed using cervical dislocation, blood was rapidly collected through a heart puncture, plasma was rapidly separated from the blood, and organs (liver, pancreas, and small intestine) were excised, rinsed in cold saline (0.9%), and homogenized in 0.1M phosphate buffer (pH 7.4) for further biochemical analyses. 2.1.3 Feed formulation and group treatments Group 1: Normal rats fed a basal diet + 100% WF bread Group 2: Diabetic rats fed a high-fat diet + 25mg/kg/ body weight STZ +100% WF bread. Group 3: Diabetic rats fed a high-fat diet +25mg/kg/ body weight STZ + 25mg/kg/body weight Acarbose + 100% WF bread Group 4: Diabetic rats fed a high-fat diet + 25mg/kg/ body weight STZ + 100% CF bread + cocoa powder. Group 5: Diabetic rats fed a high-fat diet + 25mg/kg/ body weight STZ + 50% CF + 50% WF bread + cocoa powder. Group 6: Diabetic rats fed a high-fat diet + 25mg/kg/ body weight STZ + 20% CF + 80% WF bread + cocoa powder. Group 7: Diabetic rats fed a high-fat diet + 25mg/kg/ body weight STZ + 10% CF + 90% WF bread + cocoa powder. Group 8: Diabetic rats fed a high-fat diet + 25mg/kg/ body weight STZ + 100% WF bread + cocoa powder. Table 1: Diet Formulation for both normal (control) and Type-2-diabetic rats. Composition Basal Diet (%) High Fat Diet (%) Skimmed milk 36 16 Corn flour/Starch 50 50 Mineral and Vitamin premix 4 4 Lard - 30 Vegetable oil 10 - Note: Skimmed milk = 36% protein; 7500i.u Vitamin A, 3000i.u Vitamin D 3 , 623.12g Calcium, 150g Choline, 125g Antioxidant equivalent, 62g Manganese, 50g Zinc, 44g Iron, 20g Niacin, 15g Vitamin E, 12g Phytase Enzyme, 10g Copper, 7g D plant acid, 5g Vitamin E equivalent, 4.5g Vitamin B 2 , 2.5G Vitamin K 3 , 1.3g Iodine, 1g Vitamin B 6 , 1g Apo-8-Ester, 1g Canthaxanthin, 1g Antioxidant, 0.5g Vitamin B 12 , 0.5g Folic acid, 0.25g Selenium, 0.02g Vitamin B 12 . 2.1.4 Preparation of Liver Homogenates The rats were sacrificed, and the liver was rapidly isolated, placed on ice, and weighed. These tissues were subsequently rinsed in cold 0.9% normal saline and homogenized in 0.1M phosphate buffer (pH 7.4). The homogenates were centrifuged at 3000 rpm for 10 minutes to yield a pellet that was discarded, and a low-speed supernatant (SI) was kept for lipid peroxidation assay (Belle et al. 2004) and total protein determination. 2.1.5 Preparation of Plasma At the end of the feeding trial, the rats were sacrificed, and the whole blood was collected into an EDTA bottle. The blood sample was centrifuged at 3000 rpm for 10 minutes to separate the plasma. The plasma was decanted into a plain sample bottle and stored at 4 o C for further analysis. 3.0 Results 3.1. Determination of in-vivo antioxidant enzymes activity 3.1.1 Determination of Liver Superoxide Dismutase (SOD) Activity Superoxide dismutase was carried out as described by the method of Misra and Fridovich (1972). Briefly, 0.1ml of the sample was diluted in 0.9ml of distilled water. An aliquot of 0.2ml of the diluted sample was added to 2.5ml of 0.05M carbonate buffer pH 10.2 to equilibrate in a cuvette and 0.3ml of 0.3M of adrenaline was added. The reference cuvette contained 2.5ml of carbonate buffer, 0.3ml of substrate (adrenaline), and 0.2ml of distilled water. The increase in absorbance at 480nm was monitored every 30 seconds for 150 seconds. 3.1.2 Determination of Liver Catalase Activity This was carried out according to the modified method of Beers and Sizers (1952). Briefly, 50 µl of the test sample was added to a reacting mixture containing 500 µl of 59 mM H 2 O 2 and 950 µl of 50mM phosphate buffer (pH 7.0). The reaction was carried out at 25 o C and the decrease in absorbance at 240nm was monitored for 3 minutes at 15 seconds intervals. A unit of the enzyme activity is defined as the amount of enzyme catalysing the decomposition of H 2 O 2 per minute at 25 o C and pH 7.0 under specified condition. 3.1.3 Determination of Liver Glutathione-S-Transferase Activity (GST) This was carried out according to the method of Mannervik and Guthenberg (1981). Briefly, 30 µl of the test sample was added to a reacting mixture containing 150 µl of 1 mM 1-chloro-2,4-dinitrobenzene (CDNB) and 2.79ml 0.1 M phosphate buffer (pH 6.5). The reaction was carried out at 28 o C and the increase in absorbance at 340nm was monitored for 3 minutes at 15 seconds interval. A unit of the enzyme’s activity is defined as the amount of enzyme catalyzing the formation of 1µmol of s-2, 4-dinitrophenyl glutathione from CDNB and GSH per minute at 28 o C and pH 6.5 under specified conditions (e 340 = 9.6 mM -1 cm -1 ). 3.1.4 Determination of Liver Glutathione Peroxidase (GPx) GPx activity was determined according to the method adopted by Rotruck et al. (1973). 200µl of the tissue was added to the mixture, which contain 200µl of 0.4M phosphate buffer pH 7.0, 100µl of 10mM sodium azide, subsequently, 200µl of 10mMGSH and 100µl 0.2mMH 2 O 2 were added. The whole reaction mixture was incubated at 37 o C for 10 minutes after which 400µl of 10% TCA was added, centrifuged at 3200 × g for 20 minutes. 500µl of Ellman’s reagent (19.8mg of 5,5’dithiobisnitrobenzoic acid in 100ml of 0.1% sodium citrate) and 3ml 0.2M phosphate buffer pH 8.0 were added to 1ml of the supernatants. The absorbance was read at 412nm in spectrophotometer and GPx activity was calculated. 3.1.5 Determination of Total Thiol Content Determination of the total thiol content in the tissue homogenate was done by the method of Ellman et al. (1959). The reaction mixture was made up of 270 µL of 0.1 M potassium phosphate buffer (pH 7.4), 20 µL of homogenate, and 10µL of 10 mM DTNB. This was followed by a 30-minute incubation at room temperature, and the absorbance was measured at 412 nm. The total thiol content was subsequently calculated and expressed as (µmol/mg protein). 3.1.6 Determination of Non-Protein Thiol Content Determination of the Non-protein thiol content in the tissue homogenate was determined by the method of Ellman et al. (1959). The reaction mixture was made up of 270 µL of 0.1 M potassium phosphate buffer (pH 7.4), 20 µL of homogenate, then, 10 % TCA was added and centrifuged or allowed to settle, after which 100µL was taken from the supernatant and 300µL of 10 mM DTNB. This was followed by 30-minute incubation at room temperature, and that absorbance was measured at 412 nm. The total thiol content was subsequently calculated and expressed as (µmol/mg protein). 3.2.5 Determination of Aspartate Aminotransferase (AST) Activity in Plasma The quantity of AST in plasma was determined as described in the manufacturer’s manual (Randox Laboratories Ltd.). Briefly, 0.1 ml of the sample (plasma) was mixed with 0.5 ml of Buffer R1, comprising 100 mmol/l, pH 7.4 phosphate buffer, 100 mmol/l L-aspartate, and 2 mmol/l α-oxoglutarate. Then, the mixture was incubated for 30 minutes at 37 o C. Thereafter, 0.5 ml of reagent 2 (2mmol/l 2, 4-dinitrophenylhydrazine) was added and allowed to stand for 20 minutes at 25 o C, after which 5 ml of 0.4 mol/l sodium hydroxide was added and thoroughly mixed. The absorbance of the sample (A sample ) was read against the reagent blank after 5 minutes at 546 nm using a spectrophotometer. The activity of AST in the plasma was subsequently calculated. 3.3.1 Determination of Alanine Aminotransferase (ALT) Activity in Plasma The quantity of ALT in plasma was determined as described in the manufacturer’s manual (Randox Laboratories Ltd.). Briefly, 0.1 ml of the sample (plasma) was mixed with 0.5 ml of Buffer R1, comprising 100 mmol/l, pH 7.4 phosphate buffer, 200 mmol/l L-alanine, and 2.0 mmol/l α-oxoglutarate. Then, the mixture was incubated for 30 minutes at 37 o C. Thereafter, 0.5 ml of solution R2 (2.0 mmol/ 2, 4-dinitrophenylhydrazine) was added and allowed to stand for 20 minutes at 25 o C, after which 5 ml of 0.4 mol/l sodium hydroxide was added and thoroughly mixed. The absorbance of the sample (A sample ) was read against the reagent blank after 5 minutes at 546 nm using spectrophotometer. The activity of ALT in the plasma was subsequently calculated. 3.3.2 Determination of Alkaline Phosphatase (ALP) Activity in Plasma The quantity of ALP in plasma was determined using the colorimetric method according to the recommendations of the Deutsche Gessellschaft fur Klinische Chemie, (1972). The principle of the method is as shown below: Procedure Briefly, 0.02 ml of the sample (plasma) was mixed with 1 ml of reagent R1 comprising 1 mol/l, pH 9.8 Diethanolamine buffer, 0.5mmol/l MgCl 2 and 10mmol/l p-nitrophenylphosphate (substrate). After mixing the sample and the reagent, the initial absorbance was read and read again after 1, 2 and 3 minutes at 405 nm using a spectrophotometer. The ALP activity was subsequently calculated. 3.3.3. Histopathological examination of the liver using h ematoxylin and eosin stains methods. Histopathological assay of Liver was carried out according to the method of Bancroff and Gamble (1996). The procedures of Heamatoxylin and Eosin stains were carried out; the Liver sections of the normal and HFD/STZ induced Type-2 diabetic rats were dewaxed and dehydrated. Paraffin wax was also removed from the section by immersing the slides in xylene for 2-3 minutes. This process was intensified by first warming the sections over a 60 o C flame until the paraffin wax began to melt. After being dewaxed, the slides were rehydrated by immersing them in descending grades of alcohol (2 changes of absolute alcohol, 90%, and 70% alcohol) and finally in a distilled water. The slide was further stained thoroughly in Harris hematoxilin for 10 minutes, which was later washed in running tap water for 5-10 minutes. Then the slides were stained with eosin for 1-2 minutes, which were later washed in running water until excess Eosin was removed. The slides were further dehydrated with ascending grades of alcohol. The absolute alcohol was finally removed and the slides were cleared in xylene. The slides were then mounted and cover slipped using Dibutylpthalate xylene (DPX). 4. 0 DISCUSSION The effect of vitamin-A enriched cassava/ wheat flour composite bread on the superoxide dismutase (SOD) activity in the liver of the normal control and HFD/STZ induced Type-2 diabetic rats in µ/mg protein is represented in Figure 2. The result shows that there was a significant (p<0.05) reduction in the SOD activity in the liver of the Type-2 diabetic control rats (STZ + 100% WF bread) as compared to the normal control. However, there was a significant (p<0.05) increase in SOD activity when the Type-2 diabetic Acarbose group was treated with Acarbose, but not as high as the Type-2 diabetic rats fed with 100% CF bread + FL. The order of the increase in SOD activity is as follows; STZ + 100% CF bread + FL > STZ + 50% CF + 50% WF bread + FL > STZ + Acarbose + 100% WF bread > STZ + 20% CF + 80% WF bread + FL > STZ + 10% CF + 90% WF bread + FL > Basal diet + 100% WF bread > STZ + 100% WF bread + FL > STZ + 100% WF bread. Moreover, the effect of vitamin-A enriched cassava/ wheat flour composite bread on the catalase activity of the liver of the normal control and HFD/STZ induced Type-2 diabetic rats is presented in Figure 3. The result shows that there was a significant (p<0.05) reduction in the catalase activity in the liver of the Type-2 diabetic control rats (STZ + 100% WF bread) as compared to the normal control. However, treatment of the Type-2 diabetic Acarbose group with Acarbose brought a significant (p<0.05) increase in its activity, but not as high as in Type-2 diabetic rats fed with 100% CF bread + FL. The trend in the increase in the activity of the catalase is as follows; STZ + 100% CF bread + FL > STZ + 50% CF + 50% WF bread + FL > STZ + Acarbose + 100% WF bread > STZ + 20% CF + 80% WF bread + FL > Basal diet + 100% WF bread > STZ + 10% CF + 90% WF bread + FL > STZ + 100% WF bread + FL > STZ + 100% WF bread. Figure 4 represents the effect of vitamin-A enriched cassava/ wheat flour composite bread on the glutathione-s-transferase (GST) activity in the liver of the normal control and HFD/STZ induced Type-2 diabetic rats. The result showed that there was a significant (p<0.05) reduction in the GST activity in the liver of the Type-2 diabetic control rats (STZ + 100% WF bread) as compared to the normal control rats. However, treatment of the Type-2 diabetic Acarbose group with Acarbose brought a significant (p STZ + 50% CF + 50% WF bread + FL > STZ + Acarbose + 100% WF bread > Basal diet + 100% WF bread > STZ + 20% CF + 80% WF bread + FL > STZ + 10% CF + 90% WF bread + FL > STZ + 100% WF bread + FL > STZ + 100% WF bread. In addition, the effect of vitamin-A enriched cassava/ wheat flour composite bread on the glutathione peroxidase (GPx) activity in the liver of normal and HFD/ STZ Type-2 diabetes is presented in units/mgprotein in Figure 5. The result revealed a significant (p<0.05) reduction in the GPx activity of the Type-2 diabetic rats’ control group as compared to the normal control. However, there was a significant (P<0.05) elevation in its activity in the Type-2 diabetic Acarbose group that was treated with Acarbose but not as high as the Type-2 diabetic rats fed with 100% CF bread + FL. The rate of increase in the GPx activity is in this order: STZ + 100% CF bread + FL > STZ + 50% CF + 50% WF bread + FL > STZ + 20% CF + 80% WF bread + FL > STZ + 10% CF + 90% WF bread + FL > STZ + Acarbose + 100% WF bread > Basal diet + 100% WF bread > STZ + 100% WF bread + FL > STZ + 100% WF bread. Catalase is a key enzyme that uses hydrogen peroxide, a non- radical ROS and its substrate. The enzyme is responsible for neutralization through the decomposition of hydrogen peroxide, thereby maintaining an optimum level of the molecule in the cell, which is also essential for cellular signalling processes (Ankita 2019). SOD S form the front line of defence against reactive oxygen species (ROS)-mediated injury. Due to its role in limiting the formation of ROS and RNS and the consequent oxidative stress damage, the availability of SOD as an antidote for xenobiotic toxicity would be a therapeutic advantage (Ros et al. 2021; Younus 2018). Glutathione S-transferase is a group of multifunctional proteins that partake in several roles in detoxification, and their biochemical processes may serve a crucial role in defence mechanisms (Alnasser 2024). It is worth nothing that the diabetic group in STZ + 100% CF + FL had the highest level of endogenous antioxidant (Catalase, SOD, GST, and GPx). This may be a result of increased supplementation of vitamin-A enriched cassava in composite bread. The result is in agreement with the one earlier reported by Semiz and Sen 2007 on the antioxidant and chemoprotective properties of Momordica charantia fruit extract. Furthermore, figure 6 reveals the effect of vitamin-A enriched cassava/ wheat flour composite bread on the total thiol level in the liver of normal and STZ/HFD Type-2 diabetic rats in µmol/mgprotein. From the result, it was observed that there was a significant (p<0.05) decrease in the level in the total thiol of Type-2 diabetic rats’ control group compared to that of the normal control group. However, there was a significant (p<0.05) increase in the level of total protein of the Type-2 diabetic Acarbose group treated with acarbose but not as higher as the Type-2 diabetic group fed with 100% CF bread + FL and 20% CF + 80% WF bread + FL. The sequential increase of the total thiol level in the levers of the treated rats is as follows: STZ + 100% WF bread < STZ + 100% WF bread + FL < STZ + 10% CF + 90% WF bread + FL< STZ + 20% CF + 80% WF bread + FL < STZ + Acarbose + 100% WF bread < Basal diet + 100% WF bread < STZ + 50% CF + 50% WF bread + FL < 100% CF bread + FL. The effect of vitamin-A enriched cassava / wheat flour composite bread in the non-protein thiol on the liver of the normal control rats and Type-2 diabetic rats is presented in Table 7 in µmol./mgprotein. It was observed from the result that there was a significant (p<0.05) decrease in the level of total thiol in the of Type-2 diabetic rats’ control group as compared to the normal control group. However, there was a significant (p<0.05) elevation in the level of non-total protein of the Type-2 diabetic Acarbose group treated with acarbose but not as high as the Type-2 diabetic group fed with 100% CF bread + FL and 20% CF + 80% WF bread + FL. The trend in the increase in the level of non-protein thiol is in this order; 100% CF bread + FL > STZ + 50% CF + 50% WF bread + FL > Basal diet + 100% WF bread > STZ + 20% CF + 80% WF bread + FL > STZ + Acarbose + 100% WF bread > STZ + 10% CF + 90% WF bread + FL > STZ + 100% WF bread + FL > STZ + 100% WF bread. Among all the antioxidants that are available in the body, thiols constitute the major portion of the total body antioxidants and play a significant role in defence against reactive oxygen species. (Kukurt et al. 2021). Although many cellular thiol functions have been shown to be mediated via the cysteine group found in enzyme or protein structures, the main players in cellular metabolism are thought to be the mobile low molecular weight thiols, often known as non-protein thiols (NPSH) (Toohey and Cooper 2014; Gronow 2020). An increase in the level of total and non- protein thiols in the diabetic rat groups resulted from the increased supplementation of vitamin-A enriched cassava in composite bread. Table 1 revealed the biochemical effect of vitamin-A enriched cassava/wheat composite bread on the liver function biomarkers; aspartate aminotransferase (AST), alanine aminotransferase (ALT), and alkaline phosphatase (ALP) in the plasma of diabetic rats. From the result, the value of AST was significantly (p<0.05) higher in the plasma of the HFD/ STZ induced Type-2 diabetic control group (STZ + 100% WF bread) as compared to the normal control group. However, treatment of the Type- 2 diabetic Acarbose group with Acarbose brought a significant (p<0.05) reduction to the value of AST but not as lower as the Type-2 diabetic group fed with 100% CF bread. The trend of the results in these groups is as follows: basal diet + 100% WF bread (22.11 a ± 0.91 U/L) < STZ + 100% CF bread +FL (25.61 b ± 0.72 U/L) < STZ + 50% CF +50% WF bread + FL (30.88 c ± 2.06 U/L) < STZ + 20% CF + 80% WF bread + FL (36.84 d ± 0.45 U/L) < STZ + Acarbose + 100% WF bread (37.89 e ± 0.18U/L) < STZ + 10% CF+ 90% WF bread + FL (40.35 f ± 2.06 U/L) < STZ + 100% WF + FL (62.10 g ± 1.68U/L) < STZ + 100WF bread (117.37 h ± 2.65 U/L). Moreover, the effect of vitamin-A enriched cassava/ wheat flour composite bread on ALT in the plasma of the normal and HFD/ STZ induced Type-2 diabetic rats is also presented in Table 1. The value of ALT was significantly (p<0.05) higher in the plasma of the HFD/ STZ induced type-2 diabetic control group as compared to the normal control group. However, treatment of the STZ- induced Type- 2 diabetic Acarbose group with Acarbose brought a significant (p<0.05) decrease to the value of ALT but not as lower as the Type-2 diabetic group fed with 100% CF bread. The result of the ALT showed what was revealed in the AST. The order of decrease in the value of the ALT in the plasma of the normal and treated rats is as follows; Basal diet + 100% WF bread < STZ + 100% CF bread + FL < STZ + 100% WF bread < STZ + 50% CF + 50% WF bread + FL < STZ + 20% CF + 80% WF bread + FL < STZ + 10% CF + 90% WF + FL < STZ + 100% WF bread + FL < STZ + 100% WF with these corresponding results; 21.40 a ± 0.31 U/L, 21.58 b ± 0.59 U/L, 27.89 c ± 2.09 U/L, 32.10 d ± 1.17 U/L, 36.49 e ± 1.11 U/L, 45.39 f ± 1.17 U/L, 61.40 g ± 1.21 U/L, 75.79 h ± 1.33 U/L. In addition, the effect of vitamin-A enriched cassava/ wheat flour composite bread on the value of ALP is presented in Table 2. It was shown from the result that the value of ALP was significantly (p<0.05) higher in the plasma of the HFD/ STZ induced Type-2 diabetic control group as compared to the normal control group. However, the values were significantly (p<0.05) lower in Type-2 diabetic control group treated with Acarbose but not as lower as Type-2 diabetic groups fed with composite bread. The values of the ALP in both the normal and treated rats are as follow; STZ + 100% WF bread (642.55 h ± 3.01 U/L) > STZ + 100% WF bread + FL (557.74 g ± 2.80 U/L) > STZ + 10% CF + 90% WF + FL (460.24 f ± 1.81 U/L) > STZ + 20% CF + 80% WF bread (410.31 e ± 2.78 U/L) > STZ + Acarbose + 100% WF bread (401.30 d ± 2.69 U/L) > Basal diet + 100% WF bread (388.98 c ± 2.42 U/L) > STZ + 50% CF + 50% WF + FL bread (360.98 b ± 3.03 U/L) > STZ + 100% CF bread + FL (243.47 a ± 2.40 U/L). Table i: AST, ALT and ALP of the plasma of normal control and Type-2-diabetic rats fed with the composite bread. AST ALT ALP GROUP U/L U/L U/L I 22.11 a ± 0.91 21.40 a ± 0.31 388.98 c ± 2.42 II 117.37 h ± 2.65 75.79 h ± 1.33 642.55 g ± 3.01 III 37.89 e ± 0.18 27.89 c ± 2.09 401.30 d ± 2.69 IV 25.61 b ± 0.72 21.58 b ± 0.59 243.47 a ± 2.40 V 30.88 c ± 2.06 32.10 d ± 1.17 360.98 b ± 3.03 VI 36.84 d ± 0.45 36.49 e ± 1.11 410.31 d ± 2.78 VII 40.35 f ± 2.06 45.39 f ± 1.17 460.24 e ± 1.81 VIII 62.10 g ± 1.68 61.40 g ± 1.21 557.74 f ± 2.80 Values carrying the same superscripts in the same column are not significantly different (p>0.05). Values represent the mean ± standard deviation (n = 5). KEY: Group 1-Normal Rats fed with Basal diet +100% WF bread without cocoa powder; Group II- Diabetic Rats fed with High fat diet + 25mg/kg/ body weight STZ + 100% WF bread without cocoa powder; Group II-Diabetic Rats fed with High fat diet + 25mg/kg/ body weight STZ + 25mg/kg/body weight Acarbose + 100% WF bread without cocoa powder; Group IV-Diabetic Rats fed with High fat diet + 25mg/kg/ body weight STZ + 100% CF bread + cocoa powder; Group V-Diabetic Rats fed with High fat diet + 25mg/kg/ body weight STZ + 50% CF + 50% WF bread + cocoa powder; Group VI-Diabetic Rats fed with High fat diet + 25mg/kg/ body weight STZ + 20% CF + 80% WF bread + cocoa powder; Group VII-Diabetic Rats fed with High fat diet + 25mg/kg/ body weight STZ + 10% CF + 90% WF bread + cocoa powder; Group VIII-Diabetic Rats fed with High fat diet + 25mg/kg/ body weight STZ + 100% WF bread + cocoa powder. An increase in these marker enzymes in the serum/plasma has been reported to be leakage from the liver to the blood stream, indicating hepatopathy (Lala et al. 2023). Normally, AST and ALP are enzymes located in the liver cells and leak out, making their way into the general circulation as liver cells are damaged (Ndrepepa 2021). The results showed that cassava / wheat flour composite bread has a hepatoprotective effect, and this may not be farfetched from its antioxidant properties, which was in agreement with the previous findings of Adefegha et al. 2014 on the ability of spices to restore the level of liver biomarker enzymes in diabetic rat models and Ajani et al. 2022b on the hypolipidemic effect and antioxidant properties of cassava/ wheat flour composite bread. The result revealed that an increased supplementation of vitamin-A enriched cassava in composite bread led to a significant reduction of the liver function biomarkers (AST, ALT, and ALP) in the plasma of normal and diabetic rats. Histopathological results in Figure 8 reveal the liver micrographs of the normal and HFD/ STZ-induced type- diabetic rats fed with vitamin-A enriched/ wheat flour composite bread. From the result, it was observed that the hepatic cells and profile in the diabetic control group (CC 2 ) were characterized by a severe loss of liver parenchyma, some mild derangement in the cellular profiles, haemorrhage and the presence of inflammatory red cells within and around the central vein, including the sinusoids as, well as distorted hepatic vessels (red thick arrows) as compared with the control group (CC 1 ) which showed no altered panoramic morphological presentation accompanied by a- outlined cellular profile. However, the effect of Acarbose (a diabetic drug) on the Acarbose group (CC 3 ) and the ameliorative effect of vitamin-A enriched cassava/ wheat flour composite bread reversed the damage that has been caused as a result of induction of streptozotocin on the experimental rats (A, B, C, D, and E) fed composite bread by showing a similar outline relative to the control group (CC 1 ) with a yellow arrow. It is worthy of note that vitamin-A enriched cassava/wheat flour composite bread has an hepatoprotective effect. 5.0 CONCLUSION In this study, vitamin-A cassava supplementation of wheat flour in composite bread manufactured showed a strong antioxidative impact with potential hepatoprotective benefits in rats with Type-2 diabetes induced by HFD/STZ. These products might be used as functional foods and offer protection against oxidative stress, hepatotoxicity, and other potentially pathological conditions. It is noteworthy, therefore, that the bread with 100%CF + FL had the highest level of HDL- cholesterol and the best potential for antioxidants. To confirm the impact of vitamin-A supplemented cassava/wheat composite bread, more research from human clinical trials is required. Declarations Ethical approval The study was approved by the Research Ethics Committee, Centre for Research and Development (CERAD), Federal University of Technology, Akure, Nigeria, with approval no. ERCFMBSLAUTECH:038/06/2024 Author Contribution R. A. wrote the manuscript being my Ph.D workS. A. reviewed the manuscript being my co-supervisor.I. A. reviewed the work being my co-supervisor.G. reviewed the work being my major supervisor and he was the designer of the work. Acknowledgement The authors express their profound gratitude to the Tertiary Education Trust Fund (TETFund) for given the grant for the work. 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Younus H (2018) Therapeutic effect of SOD. Inter J Hlth Sci 12(3):88-93. Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6828908","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":472980109,"identity":"15d45661-3a8f-4f9b-81f3-ff19ac5db60f","order_by":0,"name":"Richard Akinlolu Ajani","email":"","orcid":"","institution":"Ladoke Akintola University of Technology","correspondingAuthor":false,"prefix":"","firstName":"Richard","middleName":"Akinlolu","lastName":"Ajani","suffix":""},{"id":472980110,"identity":"7b1d0968-5571-4a1c-a3da-ab8907fcd1a0","order_by":1,"name":"Stephen Adeniyi Adefegha","email":"","orcid":"","institution":"Federal University of Technology","correspondingAuthor":false,"prefix":"","firstName":"Stephen","middleName":"Adeniyi","lastName":"Adefegha","suffix":""},{"id":472980111,"identity":"4b2d81fc-1b35-484e-affc-afdcc32371bb","order_by":2,"name":"Isiaka Adekunle Amoo","email":"","orcid":"","institution":"Federal University of Technology","correspondingAuthor":false,"prefix":"","firstName":"Isiaka","middleName":"Adekunle","lastName":"Amoo","suffix":""},{"id":472980112,"identity":"b0ccc9ed-d858-4d50-9a63-7fb8c75b1a8c","order_by":3,"name":"Ganiyu Oboh","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAx0lEQVRIiWNgGAWjYFACHgZmhgo5GJtoLWeMIaqJ18LYRooWeffeg48L5xkk7mc/wPjgbRtDnrwDAS2GZ84lG8/cZpDYw5PAbDi3jaHY8AAhLTNyzKR5t/1J7GFIYJPmbWNI3NhAlJY5QFv4H7D/JkqLvARISwNQi0QCGzNIy3wCOhgMeIB+4TlmYNxz42Gz5JxzEokbCGmRbweGGE+NgWx7f/LBD2/KbBLnE3KYwQE4kxGkVgJZBIctGGZiioyCUTAKRsFIBwCgBzxrvFFWmAAAAABJRU5ErkJggg==","orcid":"","institution":"Federal University of Technology","correspondingAuthor":true,"prefix":"","firstName":"Ganiyu","middleName":"","lastName":"Oboh","suffix":""}],"badges":[],"createdAt":"2025-06-05 11:53:28","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6828908/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6828908/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":89489436,"identity":"b89b2c4f-8846-481b-af89-0d9786b3e930","added_by":"auto","created_at":"2025-08-20 13:29:34","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":216895,"visible":true,"origin":"","legend":"\u003cp\u003e(a) Flow Chart representing the production of vitamin-A cassava-wheat flour composite breads and \u003cstrong\u003ePlate i\u003c/strong\u003e (b) Vitamin-A Cassava-wheat composite bread.\u003c/p\u003e\n\u003cp\u003eKey: A = 100% vitamin-A enriched cassava bread + cocoa powder; B = 50% vitamin-A enriched cassava + 50% wheat flour bread + cocoa powder; C = 20% vitamin-A enriched cassava + 80% wheat flour bread + cocoa powder; D = 10% vitamin-A enriched cassava + 90% wheat flour bread + cocoa powder; E = 100% wheat flour bread + cocoa powder; X = 100% vitamin-A enriched cassava bread without cocoa powder; Y = 100% wheat flour bread without cocoa powder;\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-6828908/v1/f79d57f01d9846d2d0041959.png"},{"id":89489788,"identity":"f20f5c9f-0482-4290-a3ab-6c927ab9c3bb","added_by":"auto","created_at":"2025-08-20 13:37:34","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":27499,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe effect of vitamin-A enriched cassava / wheat flour composite bread on SOD activity of the liver of normal control and Type- 2-diabetic rats.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eValues represent mean ± standard deviation (n=5)\u003c/p\u003e\n\u003cp\u003e*Value are significantly (p\u0026lt;0.05) different from normal and control group\u003c/p\u003e\n\u003cp\u003eKEY:\u003c/p\u003e\n\u003cp\u003eGroup 1-Normal Rats fed with Basal diet +100% WF bread without cocoa powder; Group II- Diabetic Rats fed with High fat diet + 25mg/kg/ body weight STZ + 100% WF bread without cocoa powder; Group II-Diabetic Rats fed with High fat diet + 25mg/kg/ body weight STZ + 25mg/kg/body weight Acarbose + 100% WF bread without cocoa powder; Group IV-Diabetic Rats fed with High fat diet + 25mg/kg/ body weight STZ + 100% CF bread + cocoa powder;\u003c/p\u003e\n\u003cp\u003eGroup V-Diabetic Rats fed with High fat diet + 25mg/kg/ body weight STZ + 50% CF + 50% WF bread + cocoa powder; Group VI-Diabetic Rats fed with High fat diet + 25mg/kg/ body weight STZ + 20% CF + 80% WF bread + cocoa powder; Group VII-Diabetic Rats fed with High fat diet + 25mg/kg/ body weight STZ + 10% CF + 90% WF bread + cocoa powder; Group VIII-Diabetic Rats fed with High fat diet + 25mg/kg/ body weight STZ + 100% WF bread + cocoa powder.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-6828908/v1/875d39e210d72d854fd6965a.png"},{"id":89489439,"identity":"614120dd-a5ff-4443-9cf8-fa377eeea28f","added_by":"auto","created_at":"2025-08-20 13:29:34","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":32233,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe effect of vitamin-A enriched cassava / wheat flour composite bread on the catalase activity of the liver of normal control and type- 2-diabetic rats.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eValues represent mean ± standard deviation (n=5)\u003c/p\u003e\n\u003cp\u003e*Value are significantly (p\u0026lt;0.05) different from normal and control group\u003c/p\u003e\n\u003cp\u003eKEY:\u003c/p\u003e\n\u003cp\u003eGroup 1-Normal Rats fed with Basal diet +100% WF bread without cocoa powder; Group II- Diabetic Rats fed with High fat diet + 25mg/kg/ body weight STZ + 100% WF bread without cocoa powder; Group II-Diabetic Rats fed with High fat diet + 25mg/kg/ body weight STZ + 25mg/kg/body weight Acarbose + 100% WF bread without cocoa powder; Group IV-Diabetic Rats fed with High fat diet + 25mg/kg/ body weight STZ + 100% CF bread + cocoa powder;\u003c/p\u003e\n\u003cp\u003eGroup V-Diabetic Rats fed with High fat diet + 25mg/kg/ body weight STZ + 50% CF + 50% WF bread + cocoa powder; Group VI-Diabetic Rats fed with High fat diet + 25mg/kg/ body weight STZ + 20% CF + 80% WF bread + cocoa powder; Group VII-Diabetic Rats fed with High fat diet + 25mg/kg/ body weight STZ + 10% CF + 90% WF bread + cocoa powder; Group VIII-Diabetic Rats fed with High fat diet + 25mg/kg/ body weight STZ + 100% WF bread + cocoa powder.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-6828908/v1/fd2e5b266bc8968460cfd4e6.png"},{"id":89489440,"identity":"1e7f3a3a-bf75-443a-8a86-1578a36f4490","added_by":"auto","created_at":"2025-08-20 13:29:34","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":37521,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe effect of vitamin-A enriched cassava / wheat flour composite bread on the GST activity of the liver of normal control and type- 2-diabetic rats.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eValues represent mean ± standard deviation (n=5)\u003c/p\u003e\n\u003cp\u003e*Value are significantly (p\u0026lt;0.05) different from normal and control group\u003c/p\u003e\n\u003cp\u003eKEY:\u003c/p\u003e\n\u003cp\u003eGroup 1-Normal Rats fed with Basal diet +100% WF bread without cocoa powder; Group II- Diabetic Rats fed with High fat diet + 25mg/kg/ body weight STZ + 100% WF bread without cocoa powder; Group II-Diabetic Rats fed with High fat diet + 25mg/kg/ body weight STZ + 25mg/kg/body weight Acarbose + 100% WF bread without cocoa powder; Group IV-Diabetic Rats fed with High fat diet + 25mg/kg/ body weight STZ + 100% CF bread + cocoa powder;\u003c/p\u003e\n\u003cp\u003eGroup V-Diabetic Rats fed with High fat diet + 25mg/kg/ body weight STZ + 50% CF + 50% WF bread + cocoa powder; Group VI-Diabetic Rats fed with High fat diet + 25mg/kg/ body weight STZ + 20% CF + 80% WF bread + cocoa powder; Group VII-Diabetic Rats fed with High fat diet + 25mg/kg/ body weight STZ + 10% CF + 90% WF bread + cocoa powder; Group VIII-Diabetic Rats fed with High fat diet + 25mg/kg/ body weight STZ + 100% WF bread + cocoa powder.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-6828908/v1/8d721be57c373e9003292073.png"},{"id":89489442,"identity":"68930770-f386-4ff9-a215-8d849ca45e9c","added_by":"auto","created_at":"2025-08-20 13:29:34","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":32258,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe effect of vitamin-A enriched cassava / wheat flour composite bread on glutathione peroxidase (GPx) of the liver of normal control and Type-2-diabetic rats.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eValues represent mean ± standard deviation (n=5)\u003c/p\u003e\n\u003cp\u003e*Value are significantly (p\u0026lt;0.05) different from normal and control group\u003c/p\u003e\n\u003cp\u003eKEY:\u003c/p\u003e\n\u003cp\u003eGroup 1-Normal Rats fed with Basal diet +100% WF bread without cocoa powder; Group II- Diabetic Rats fed with High fat diet + 25mg/kg/ body weight STZ + 100% WF bread without cocoa powder; Group II-Diabetic Rats fed with High fat diet + 25mg/kg/ body weight STZ + 25mg/kg/body weight Acarbose + 100% WF bread without cocoa powder; Group IV-Diabetic Rats fed with High fat diet + 25mg/kg/ body weight STZ + 100% CF bread + cocoa powder;\u003c/p\u003e\n\u003cp\u003eGroup V-Diabetic Rats fed with High fat diet + 25mg/kg/ body weight STZ + 50% CF + 50% WF bread + cocoa powder; Group VI-Diabetic Rats fed with High fat diet + 25mg/kg/ body weight STZ + 20% CF + 80% WF bread + cocoa powder; Group VII-Diabetic Rats fed with High fat diet + 25mg/kg/ body weight STZ + 10% CF + 90% WF bread + cocoa powder; Group VIII-Diabetic Rats fed with High fat diet + 25mg/kg/ body weight STZ + 100% WF bread + cocoa powder.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-6828908/v1/3679f80046be4be064b6870e.png"},{"id":89489789,"identity":"28fc4d07-72d8-439b-acbb-f4a7dfa64373","added_by":"auto","created_at":"2025-08-20 13:37:34","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":28176,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe effect of vitamin-A enriched cassava / wheat flour composite bread on the total thiol of the liver of normal control and type- 2-diabetic rats.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eValues represent mean ± standard deviation (n=5)\u003c/p\u003e\n\u003cp\u003e*Value are significantly (p\u0026lt;0.05) different from normal and control group\u003c/p\u003e\n\u003cp\u003eKEY:\u003c/p\u003e\n\u003cp\u003eGroup 1-Normal Rats fed with Basal diet +100% WF bread without cocoa powder; Group II- Diabetic Rats fed with High fat diet + 25mg/kg/ body weight STZ + 100% WF bread without cocoa powder; Group II-Diabetic Rats fed with High fat diet + 25mg/kg/ body weight STZ + 25mg/kg/body weight Acarbose + 100% WF bread without cocoa powder; Group IV-Diabetic Rats fed with High fat diet + 25mg/kg/ body weight STZ + 100% CF bread + cocoa powder;\u003c/p\u003e\n\u003cp\u003eGroup V-Diabetic Rats fed with High fat diet + 25mg/kg/ body weight STZ + 50% CF + 50% WF bread + cocoa powder; Group VI-Diabetic Rats fed with High fat diet + 25mg/kg/ body weight STZ + 20% CF + 80% WF bread + cocoa powder; Group VII-Diabetic Rats fed with High fat diet + 25mg/kg/ body weight STZ + 10% CF + 90% WF bread + cocoa powder; Group VIII-Diabetic Rats fed with High fat diet + 25mg/kg/ body weight STZ + 100% WF bread + cocoa powder.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-6828908/v1/05a92648c83d49e8bb675248.png"},{"id":89489790,"identity":"107579ac-0a9a-4ecc-b361-b0b823f9d0bb","added_by":"auto","created_at":"2025-08-20 13:37:34","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":27640,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe effect of vitamin-A enriched cassava / wheat flour composite bread on the non-protein thiol of the liver of normal control and type- 2-diabetic rats.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eValues represent mean ± standard deviation (n=5)\u003c/p\u003e\n\u003cp\u003e*Value are significantly (p\u0026lt;0.05) different from normal and control group\u003c/p\u003e\n\u003cp\u003eKEY:\u003c/p\u003e\n\u003cp\u003eGroup 1-Normal Rats fed with Basal diet +100% WF bread without cocoa powder; Group II- Diabetic Rats fed with High fat diet + 25mg/kg/ body weight STZ + 100% WF bread without cocoa powder; Group II-Diabetic Rats fed with High fat diet + 25mg/kg/ body weight STZ + 25mg/kg/body weight Acarbose + 100% WF bread without cocoa powder; Group IV-Diabetic Rats fed with High fat diet + 25mg/kg/ body weight STZ + 100% CF bread + cocoa powder;\u003c/p\u003e\n\u003cp\u003eGroup V-Diabetic Rats fed with High fat diet + 25mg/kg/ body weight STZ + 50% CF + 50% WF bread + cocoa powder; Group VI-Diabetic Rats fed with High fat diet + 25mg/kg/ body weight STZ + 20% CF + 80% WF bread + cocoa powder; Group VII-Diabetic Rats fed with High fat diet + 25mg/kg/ body weight STZ + 10% CF + 90% WF bread + cocoa powder; Group VIII-Diabetic Rats fed with High fat diet + 25mg/kg/ body weight STZ + 100% WF bread + cocoa powder\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-6828908/v1/1224f00899e464b31be5ba4a.png"},{"id":89490413,"identity":"bf0ea7da-cf1d-4b81-acc4-c2f9396f483d","added_by":"auto","created_at":"2025-08-20 13:45:34","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":1094145,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePhotomicrographs of liver section.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePlate ii: \u003c/strong\u003ePhotomicrographs of the liver micromorphological presentations in adult Wistar rats across the study groups. Hematoxylin and eosin stain (\u003cem\u003eX100 magnification\u003c/em\u003e). The portal triad as well as the well distributed hepatocytes (H) are well focused across the general cytoarchitecture, as demonstrated across study groups.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eKEY:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eGroup CC\u003csub\u003e1\u003c/sub\u003e-Normal Rats fed with Basal diet +100% WF bread without cocoa powder; Group CC\u003csub\u003e2\u003c/sub\u003e- Diabetic Rats fed with High fat diet + 25mg/kg/ body weight STZ + 100% WF bread without cocoa powder; Group CC\u003csub\u003e3\u003c/sub\u003e-Diabetic Rats fed with High fat diet + 25mg/kg/ body weight STZ + 25mg/kg/body weight Acarbose + 100% WF bread without cocoa powder; Group A-Diabetic Rats fed with High fat diet + 25mg/kg/ body weight STZ + 100% CF bread + cocoa powder;\u003c/p\u003e\n\u003cp\u003eGroup B-Diabetic Rats fed with High fat diet + 25mg/kg/ body weight STZ + 50% CF + 50% WF bread + cocoa powder; Group C-Diabetic Rats fed with High fat diet + 25mg/kg/ body weight STZ + 20% CF + 80% WF bread + cocoa powder; Group D-Diabetic Rats fed with High fat diet + 25mg/kg/ body weight STZ + 10% CF + 90% WF bread + cocoa powder; Group E-Diabetic Rats fed with High fat diet + 25mg/kg/ body weight STZ + 100% WF bread + cocoa powder.\u003c/p\u003e","description":"","filename":"8.png","url":"https://assets-eu.researchsquare.com/files/rs-6828908/v1/aa327d9c7a139eb8dcb94194.png"},{"id":100420839,"identity":"af28e456-f12c-4b10-9d57-6ab8f57a4563","added_by":"auto","created_at":"2026-01-16 13:29:56","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2883695,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6828908/v1/e412daf3-73af-49a1-bc5e-d135b3bb202d.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Antioxidative and hepatoprotective Effects of Vitamin-A Enriched Cassava / Wheat Flour Composite Bread in Rats Fed High- Fat and Induced with Streptozotocin","fulltext":[{"header":"1.0 Introduction","content":"\u003cp\u003eThe risk of liver damage has recently increased due to higher exposure to environmental toxins, pesticides, a high level of alcohol intake, pharmaceuticals, and frequent use of chemotherapeutics (Sai Sruthi Arige et\u003cem\u003e \u003c/em\u003eal. 2017; Nagappan et al. 2020). Liver damage is always associated with cellular necrosis, an increase in tissue lipid peroxidation, the depletion of tissue glutathione levels, and many other factors (de la Rosa et al.\u003cem\u003e \u003c/em\u003e2022, Shen-ping et al. 2022). Hepatic disease is a main threat to public health, indicating problems with the hepatic tissue or liver functions, which can also be caused by different factors, such as viruses, or bacteria, autoimmune disease, or the external action of different chemicals (drugs or toxic compounds) (Gan et al. 2025). Mechanisms that underlie the hepatoprotective activity are strongly related to the capacity of antioxidants to scavenge reactive oxygen species (ROS) that are produced by the metabolic conversion of xenobiotics, induce oxidative stress and damage to the tissue (Lelciu et al. 2021; Vicidomini et al.\u003cem\u003e \u003c/em\u003e2024). \u003c/p\u003e\n\u003cp\u003eThe liver is the second largest organ in our body, weighing approximately 1.5kg in adults, representing 2% of the total body weight (TBW) (Arige et al. 2017). It plays a remarkable function in our body, like bile production and excretion, the excretion of bilirubin, cholesterol, hormones, drugs, and the metabolism of proteins, fats, and carbohydrates. It is involved with almost all the biochemical pathways for growth, fighting against disease, nutrient supply, energy provision, and reproduction (Kalra et al. 2023). And it functions as a center of metabolism for the nutrients such as carbohydrates, proteins, and lipids and the excretion of waste metabolites (van de Graaf et al. 2024). The liver also removes harmful substances from our blood. It is called detoxification. Consequently, the renovation of a healthy liver is important for the overall well- being of a character (van Hul et al. 2024). \u003c/p\u003e\n\u003cp\u003eNowadays, modern medicine offers alternatives for the treatment of these pathologies, but despite the advances, few effective drugs that offer protection and regeneration of hepatic cells exist (Gan 2025). Moreover, existing treatments can cause adverse effects, which make the therapy of these pathologies even harder (Ahmed et al. 2024). Thereby, the need for identifying novel alternatives for the treatment of hepatic diseases and for the protection of the liver appears to be important, in order to develop novel agents with high efficiency and a superior safety profile (Gan 2025; Hora et al. 2023; Roehlen et al. 2020). \u003c/p\u003e\n\u003cp\u003eReactive oxygen species (ROS) in high concentration can cause cellular dysfunction, and, in the worst circumstances, cell death. Ros are extremely damaging agents that cause significant damage to lipids, proteins, and DNA (Nagappan et al. 2020). Oxidative stress is caused by an imbalance between free radicals and antioxidants, which endangers human life by increasing the occurrence of cancer, diabetes, cardiac disease, and neurological problems (Pal et al.\u003cem\u003e \u003c/em\u003e2021). As a result, there is a need to establish defensive mechanisms in order to survive and maintain a healthy lifestyle. Antioxidants are generally an effective approach to preventing oxidative stress by inhibiting ROS concentration by the breaking of chain reactions, free radical scavenging, and chelating transition metals that catalyze free radical formation (Sharma et al. 2019)\u003c/p\u003e\n\u003cp\u003eNumerous synthetic antioxidants are commercially available to alleviate oxidative stress, but they have adverse side effects (Hassanpour and Doroudi 2023; Mukherjee et al.\u003cem\u003e \u003c/em\u003e2024). Plant-derived compounds may be effective as antioxidants because they do not have detrimental side effects because of their natural origin and composite character (Chaachouay et al. 2024; Pal et al. 2022). Plants are a major source of polyphenols. A class of compounds with a phenolic hydroxyl structure, they attract research attention for their potent antioxidant activities (Pal et al.\u003cem\u003e \u003c/em\u003e2021; Pal et al. 2022).\u003c/p\u003e\n\u003cp\u003eMedicinal plants play a key function in human fitness care. Approximately 80% of the world population is predicted to use conventional medicine that is predominantly based on plant materials (Sharma et al. 2021; Devi et al. 2024). A large variety of medicinal plants have been tested and determined to include actives standard with a curative house towards ramification of sickness. A large range of plants and formations have been claimed to have hepatoprotective activities, so the improvement of plant based totally hepatoprotective drugs has been given significance within the global market place (Sharma et al. 2021; Devi et al. 2024). \u003c/p\u003e\n\u003cp\u003eCassava, being a major source of carbohydrate in local diets, when biofortified with increased levels of carotenoids, provides many other nutritional health benefits, such as improvement of visual ability and immune function (Eyinla et al. 2019; IITA 2017). The new variety of vitamin-A cassava was introduced by the Federal Government of Nigeria under Harvest Plus, which is 25% richer in \u0026beta;-carotene (a pro-vitamin A) than those earlier released in 2011 (IITA 2017; Omodamiro et al. 2012; Eyinla. et al. 2019). The characteristic yellow color associated with the vitamin-A cassava variety is due to its higher vitamin A content (Alos et al. 2016). Vitamin A deficiency is a major public health challenge in sub-Saharan countries (Arlappa et al. 2016). \u003csub\u003e \u003c/sub\u003e \u003c/p\u003e\n\u003cp\u003eBread is universally accepted as a very convenient form of food that is important to all populations. It is a good source of nutrients, such as macronutrients (carbohydrates, protein, and fat) and micronutrients (minerals and vitamins), that are essential for human health (Oluwajoba et al. 2012; Kaim et al. 2023). Efforts have been made to promote the use of flour from high protein seeds and locally grown crops to replace wheat flour used in baking bread, thereby decreasing the demand for imported wheat and leading to the production of protein enriched bread (Olaoye and Onilude 2012; Kaim et al. 2023). Most tropical cereal grains and some tubers have been used to make composite flour for bread making (Ayo et al. 2014). \u003c/p\u003e\n\u003cp\u003eThe nutritional importance arising from cassava/wheat flour bread\u0026rsquo;s blends will be harnessed to promote well-being and healthy living for the consumer with associated health challenges. The research may serve as an eye opener to possible solutions to the ravaging effects of cardiovascular challenges, hyperlipidemia, liver damage, and other disease conditions that arise from daily consumption of diets that pose a threat to human wellness and life. Therefore, this research was designed to investigate the effect of the composite bread blend\u0026rsquo;s blend produced from vitamin-A enriched cassava flour (CF)/ wheat flour (WF) flavoured with cocoa powder on endogenous antioxidant assays (SOD and Catalase, GST, GPx), total thiol and non-protein thiol liver homogenate, MDA level in the livers of HFD/STD-induced Type-2 diabetes in normal rats, and also, liver function biomarkers (AST, ALP, and ALT). More so, histopathological assays were equally carried out on the liver sections of the Wistar rats. \u003c/p\u003e"},{"header":"2.0 Materials and Methods.","content":"\u003cp\u003e\u003cstrong\u003e2.1 \u0026nbsp; Materials.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eVitamin-A enriched Cassava, also known as yellow cassava was obtained from the International Institute of Tropical Agriculture, IITA Ibadan, Oyo State, Nigeria. It was peeled, washed, cut into smaller pieces, and oven dried in a cabinet dryer at 45\u003csup\u003e0\u003c/sup\u003eC for 72 hours before it was milled into flour. While wheat flour (Golden Penny), cocoa powder, yeast, and eggs were purchased from Waso market, a major market in Ogbomoso City, Oyo State. Aspartame was purchased from a chemical supplier in Ilorin, Kwara State. All the chemicals used were analytical grades while the water was glass distilled.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.1.1 Production of cocoa powdered vitamin-A enriched cassava/wheat composite flour bread.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eVitamin-A enriched cassava was peeled, washed clean, cut into smaller pieces, and then dried in the cabinet dryer at 45\u003csup\u003eo\u003c/sup\u003eC for 72 hours. The dried cassava was milled into cassava flour (CF). The composite mixture of an improved variety of cassava flour (CF) and wheat flour (WF) is in the ratios of 100:0 (100% CF and 0% WF), 50:50 (50% CF and 50% WF), 20:80 (20% CF and 80% WF), 10:90 (10% CF and 90% WF), and 0:100 (0% CF and 100% WF). Sweetening with Aspartame, Egg Albumin was used as an emulsifier, cocoa powder as the flavouring agent (FL), and yeast and water to taste in the production of composite breads. It was baked in a gas oven at 250\u003csup\u003eo\u003c/sup\u003eC for 30 minutes, or till it turned golden brown.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.1.2 \u0026nbsp; \u0026nbsp;Diabetic experiment\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eForty (40) albino rats weighing 170 -200 g were used in the experiment. The animals were acclimatized for two weeks, given commercial feed and water \u003cem\u003ead libitum\u003c/em\u003e. The animals were grouped into two groups after acclimatization. Group I (the control) consists of five animals, and Group II consists of other rats. Animals in group I (control) were fed a basal diet, and those in group II were fed a high fat diet (30% fat) for two weeks. (Basal diet compositions are; corn flour, skimmed milk, mineral and vitamin premixed, and vegetable oil while High fat diet compositions are; Corn flour, skimmed milk, mineral and vitamin premixed and lard). \u0026nbsp;Then, at the expiration of the second week, the group II animals were induced with 25mg/kg/body weight of streptozotocin (STZ) according to the method described by Adefegha et al. (2014). After 72 hours of induction with STZ, the blood glucose levels of animals were checked using the Fine Test Glucometer.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAnimals with blood glucose (\u0026ge;250 mg/dl) were regarded as diabetic. Then,\u0026nbsp;the diabetic group was divided into seven groups, groups II-VIII, of five rats each in a group. The rats were fed with the formulated composite breads for two weeks. Furthermore, group III was treated with acarbose (25mg/kg/body weight) in addition to the composite bread for the period during which the experiment lasted, and the blood glucose of all the rats was examined at four-day intervals using a fine test glucometer by collecting the blood through the tails of the animals. The animals were subjected to a night fast a day prior to the sacrifice by removing composite bread and water. The animals were sacrificed using cervical dislocation, blood was rapidly collected through a heart puncture, plasma was rapidly separated from the blood, and organs (liver, pancreas, and small intestine) were excised, rinsed in cold saline (0.9%), and homogenized in 0.1M phosphate buffer (pH 7.4) for further biochemical analyses.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.1.3 \u0026nbsp; \u0026nbsp;Feed formulation and group treatments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eGroup 1:\u0026nbsp;\u003c/strong\u003eNormal rats fed a basal diet + 100% WF bread\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eGroup 2:\u0026nbsp;\u003c/strong\u003eDiabetic rats fed a high-fat diet + 25mg/kg/ body weight STZ +100% WF bread.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eGroup 3:\u0026nbsp;\u003c/strong\u003eDiabetic rats fed a high-fat diet +25mg/kg/ body weight STZ + 25mg/kg/body weight Acarbose + 100% WF bread\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eGroup 4:\u0026nbsp;\u003c/strong\u003eDiabetic rats fed a high-fat diet + 25mg/kg/ body weight STZ + 100% CF bread + cocoa powder.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eGroup 5:\u0026nbsp;\u003c/strong\u003eDiabetic rats fed a high-fat diet + 25mg/kg/ body weight STZ + 50% CF + 50% WF bread + cocoa powder.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eGroup 6:\u0026nbsp;\u003c/strong\u003eDiabetic rats fed a high-fat diet + 25mg/kg/ body weight STZ + 20% CF + 80% WF bread + cocoa powder.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eGroup 7:\u0026nbsp;\u003c/strong\u003eDiabetic rats fed a high-fat diet + 25mg/kg/ body weight STZ + 10% CF + 90% WF bread + cocoa powder.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eGroup 8:\u003c/strong\u003e Diabetic\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003erats fed a high-fat diet + 25mg/kg/ body weight STZ + 100% WF bread + cocoa powder.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 1:\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eDiet Formulation for both normal (control) and Type-2-diabetic rats.\u003c/strong\u003e\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"619\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 229px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eComposition\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 198px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eBasal Diet (%)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 192px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eHigh Fat Diet (%)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 229px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eSkimmed milk\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 198px;\"\u003e\n \u003cp\u003e36\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 192px;\"\u003e\n \u003cp\u003e16\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 229px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eCorn flour/Starch\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 198px;\"\u003e\n \u003cp\u003e50\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 192px;\"\u003e\n \u003cp\u003e50\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 229px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eMineral and Vitamin premix\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 198px;\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 192px;\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 229px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eLard\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 198px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e-\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 192px;\"\u003e\n \u003cp\u003e30\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 229px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eVegetable oil\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 198px;\"\u003e\n \u003cp\u003e10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 192px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cstrong\u003eNote:\u0026nbsp;\u003c/strong\u003eSkimmed milk = 36% protein; 7500i.u Vitamin A, 3000i.u Vitamin D\u003csub\u003e3\u003c/sub\u003e, 623.12g Calcium, 150g Choline, 125g Antioxidant equivalent, 62g Manganese, 50g Zinc, 44g Iron, 20g Niacin, 15g Vitamin E, 12g Phytase Enzyme, 10g Copper, 7g D plant acid, 5g Vitamin E equivalent, 4.5g Vitamin B\u003csub\u003e2\u003c/sub\u003e, 2.5G Vitamin K\u003csub\u003e3\u003c/sub\u003e, 1.3g Iodine, 1g Vitamin B\u003csub\u003e6\u003c/sub\u003e, 1g Apo-8-Ester, 1g Canthaxanthin, 1g Antioxidant, 0.5g Vitamin B\u003csub\u003e12\u003c/sub\u003e, 0.5g Folic acid, 0.25g Selenium, 0.02g Vitamin B\u003csub\u003e12\u003c/sub\u003e.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cstrong\u003e2.1.4\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;Preparation of Liver Homogenates\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe rats were sacrificed, and the liver was rapidly isolated, placed on ice, and weighed. These tissues were subsequently rinsed in cold 0.9% normal saline and homogenized in 0.1M phosphate buffer (pH 7.4). The homogenates were centrifuged at 3000 rpm for 10 minutes to yield a pellet that was discarded, and a low-speed supernatant (SI) was kept for lipid peroxidation assay (Belle et al. 2004) and total protein determination. \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u003csub\u003e\u0026nbsp;\u003c/sub\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.1.5 \u0026nbsp; \u0026nbsp;Preparation of Plasma\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAt the end of the feeding trial, the rats were sacrificed, and the whole blood was collected into an EDTA bottle. The blood sample was centrifuged at 3000 rpm for 10 minutes to separate the plasma. The plasma was decanted into a plain sample bottle and stored at 4\u003csup\u003eo\u003c/sup\u003eC for further analysis.\u003c/p\u003e"},{"header":"3.0 Results","content":"\u003cp\u003e\u003cstrong\u003e3.1.\u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Determination of in-vivo antioxidant enzymes activity\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.1.1\u0026nbsp; \u0026nbsp;\u0026nbsp;Determination of Liver Superoxide Dismutase (SOD) Activity\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSuperoxide dismutase was carried out as described by the method of Misra and Fridovich (1972). Briefly, 0.1ml of the sample was diluted in 0.9ml of distilled water. An aliquot of 0.2ml of the diluted sample was added to 2.5ml of 0.05M carbonate buffer pH 10.2 to equilibrate in a cuvette and 0.3ml of 0.3M of adrenaline was added. The reference cuvette contained 2.5ml of carbonate buffer, 0.3ml of substrate (adrenaline), and 0.2ml of distilled water. The increase in absorbance at 480nm was monitored every 30 seconds for 150 seconds. \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.1.2\u003c/strong\u003e \u003cstrong\u003eDetermination of Liver Catalase Activity\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis was carried out according to the modified method of Beers and Sizers (1952). Briefly, 50 \u0026micro;l of the test sample was added to a reacting mixture containing 500 \u0026micro;l of 59 mM H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e and 950 \u0026micro;l of 50mM phosphate buffer (pH 7.0). The reaction was carried out at 25\u003csup\u003eo\u003c/sup\u003eC and the decrease in absorbance at 240nm was monitored for 3 minutes at 15 seconds intervals. A unit of the enzyme activity is defined as the amount of enzyme catalysing the decomposition of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e per minute at 25\u003csup\u003eo\u003c/sup\u003eC and pH 7.0 under specified condition.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.1.3\u0026nbsp; \u0026nbsp;\u0026nbsp;Determination of Liver Glutathione-S-Transferase Activity (GST)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis was carried out according to the method of Mannervik and Guthenberg (1981). Briefly, 30 \u0026micro;l of the test sample was added to a reacting mixture containing 150 \u0026micro;l of 1 mM 1-chloro-2,4-dinitrobenzene (CDNB) and 2.79ml 0.1 M phosphate buffer (pH 6.5). The reaction was carried out at 28\u003csup\u003eo\u003c/sup\u003eC and the increase in absorbance at 340nm was monitored for 3 minutes at 15 seconds interval. A unit of the enzyme\u0026rsquo;s activity is defined as the amount of enzyme catalyzing the formation of 1\u0026micro;mol of s-2, 4-dinitrophenyl glutathione from CDNB and GSH per minute at 28\u003csup\u003eo\u003c/sup\u003eC and pH 6.5 under specified conditions (e\u003csub\u003e340\u0026nbsp;\u003c/sub\u003e= 9.6 mM\u003csup\u003e-1\u003c/sup\u003e cm\u003csup\u003e-1\u003c/sup\u003e).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.1.4\u0026nbsp; \u0026nbsp;\u0026nbsp;Determination of Liver Glutathione Peroxidase (GPx)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eGPx\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eactivity was determined according to the method adopted by Rotruck et al. (1973). 200\u0026micro;l of the tissue was added to the mixture, which contain 200\u0026micro;l of 0.4M phosphate buffer pH 7.0, 100\u0026micro;l of 10mM sodium azide, subsequently, 200\u0026micro;l of 10mMGSH and 100\u0026micro;l 0.2mMH\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u0026nbsp;\u003c/sub\u003ewere added. The whole reaction mixture was incubated at 37 \u003csup\u003eo\u003c/sup\u003eC for 10 minutes after which 400\u0026micro;l of 10% TCA was added, centrifuged at 3200 \u0026times; g for 20 minutes. \u0026nbsp;500\u0026micro;l of Ellman\u0026rsquo;s reagent (19.8mg of 5,5\u0026rsquo;dithiobisnitrobenzoic acid in 100ml of 0.1% sodium citrate) and 3ml 0.2M phosphate buffer pH 8.0 were added to 1ml of the supernatants. The absorbance was read at 412nm in spectrophotometer and GPx activity was calculated. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u0026nbsp; \u003cstrong\u003e3.1.5 \u0026nbsp; Determination of Total Thiol Content\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eDetermination of the total thiol content in the tissue homogenate was done by the method of Ellman et al.\u003cem\u003e\u0026nbsp;\u003c/em\u003e(1959). The reaction mixture was made up of 270 \u0026micro;L of 0.1 M potassium phosphate buffer (pH 7.4), 20 \u0026micro;L of homogenate, and 10\u0026micro;L of 10 mM DTNB. This was followed by a 30-minute incubation at room temperature, and the absorbance was measured at 412 nm. The total thiol content was subsequently calculated and expressed as (\u0026micro;mol/mg protein).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.1.6 \u0026nbsp; \u0026nbsp;Determination of Non-Protein Thiol Content\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eDetermination of the Non-protein thiol content in the tissue homogenate was determined by the method of Ellman et al.\u003cem\u003e\u0026nbsp;\u003c/em\u003e(1959). The reaction mixture was made up of 270 \u0026micro;L of 0.1 M potassium phosphate buffer (pH 7.4), 20 \u0026micro;L of homogenate, then, 10 % TCA was added and centrifuged or allowed to settle, after which 100\u0026micro;L was taken from the supernatant and 300\u0026micro;L of 10 mM DTNB. This was followed by 30-minute incubation at room temperature, and that absorbance was measured at 412 nm. The total thiol content was subsequently calculated and expressed as (\u0026micro;mol/mg protein).\u003csup\u003e\u0026nbsp;\u003c/sup\u003e \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.2.5\u0026nbsp; \u0026nbsp;\u0026nbsp;Determination of Aspartate Aminotransferase (AST) Activity in Plasma\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe quantity of AST in plasma was determined as described in the manufacturer\u0026rsquo;s manual (Randox Laboratories Ltd.). Briefly, 0.1 ml of the sample (plasma) was mixed with 0.5 ml of Buffer R1, comprising 100 mmol/l, pH 7.4 phosphate buffer, 100 mmol/l L-aspartate, and 2 mmol/l \u0026alpha;-oxoglutarate. Then, the mixture was incubated for 30 minutes at 37 \u003csup\u003eo\u003c/sup\u003eC. Thereafter, 0.5 ml of reagent 2 (2mmol/l 2, 4-dinitrophenylhydrazine) was added and allowed to stand for 20 minutes at 25 \u003csup\u003eo\u003c/sup\u003eC, after which 5 ml of 0.4 mol/l sodium hydroxide was added and thoroughly mixed. The absorbance of the sample (A\u003csub\u003esample\u003c/sub\u003e) was read against the reagent blank after 5 minutes at 546 nm using a spectrophotometer. The activity of AST in the plasma was subsequently calculated.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.3.1\u0026nbsp; \u0026nbsp;\u0026nbsp;Determination of Alanine Aminotransferase (ALT) Activity in Plasma\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe quantity of ALT in plasma was determined as described in the manufacturer\u0026rsquo;s manual (Randox Laboratories Ltd.). Briefly, 0.1 ml of the sample (plasma) was mixed with 0.5 ml of Buffer R1, comprising 100 mmol/l, pH 7.4 phosphate buffer, 200 mmol/l L-alanine, and 2.0 mmol/l \u0026alpha;-oxoglutarate. Then, the mixture was incubated for 30 minutes at 37 \u003csup\u003eo\u003c/sup\u003eC. Thereafter, 0.5 ml of solution R2 (2.0 mmol/ 2, 4-dinitrophenylhydrazine) was added and allowed to stand for 20 minutes at 25\u003csup\u003eo\u003c/sup\u003eC, after which 5 ml of 0.4 mol/l sodium hydroxide was added and thoroughly mixed. The absorbance of the sample (A\u003csub\u003esample\u003c/sub\u003e) was read against the reagent blank after 5 minutes at 546 nm using spectrophotometer. The activity of ALT in the plasma was subsequently calculated.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.3.2\u0026nbsp; \u0026nbsp;\u0026nbsp;Determination of Alkaline Phosphatase (ALP) Activity in Plasma\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe quantity of ALP in plasma was determined using the colorimetric method according to the recommendations of the Deutsche Gessellschaft fur Klinische Chemie, (1972). The principle of the method is as shown below:\u003c/p\u003e\n\u003cp\u003e\u003cimg src=\"data:image/png;base64,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\" width=\"601\" height=\"69\"\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;Procedure\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBriefly, 0.02 ml of the sample (plasma) was mixed with 1 ml of reagent R1 comprising 1 mol/l, pH 9.8 Diethanolamine buffer, 0.5mmol/l MgCl\u003csub\u003e2\u003c/sub\u003e and 10mmol/l p-nitrophenylphosphate (substrate). After mixing the sample and the reagent, the initial absorbance was read and read again after 1, 2 and 3 minutes at 405 nm using a spectrophotometer. The ALP activity was subsequently calculated.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.3.3. Histopathological examination of the liver using h\u003c/strong\u003e\u003cstrong\u003eematoxylin and eosin stains methods.\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHistopathological assay of Liver was carried out according to the method of\u0026nbsp;Bancroff and Gamble (1996).\u0026nbsp;The procedures of Heamatoxylin and Eosin stains were carried out; the Liver sections of the normal and HFD/STZ induced Type-2 diabetic rats were dewaxed and dehydrated. Paraffin wax was also removed from the section by immersing the slides in xylene for 2-3 minutes. This process was intensified by first warming the sections over a 60 \u003csup\u003eo\u003c/sup\u003eC flame until the paraffin wax began to melt. After being dewaxed, the slides were rehydrated by immersing them in descending grades of alcohol (2 changes of absolute alcohol, 90%, and 70% alcohol) and finally in a distilled water. \u0026nbsp; The slide was further stained thoroughly in Harris hematoxilin for 10 minutes, which was later washed in running tap water for 5-10 minutes. Then the slides were stained with eosin for 1-2 minutes, which were later washed in running water until excess Eosin was removed. The slides were further dehydrated with ascending grades of alcohol. The absolute alcohol was finally removed and the slides were cleared in xylene. The slides were then mounted and cover slipped using Dibutylpthalate xylene (DPX).\u003c/p\u003e"},{"header":"4. 0 DISCUSSION","content":"\u003cp\u003eThe effect of vitamin-A enriched cassava/ wheat flour composite bread on the superoxide dismutase (SOD) activity in the liver of the normal control and HFD/STZ induced Type-2 diabetic rats in \u0026micro;/mg protein is represented in Figure 2. The result shows that there was a significant (p\u0026lt;0.05) reduction in the SOD activity in the liver of the Type-2 diabetic control rats (STZ + 100% WF bread) as compared to the normal control. However, there was a significant (p\u0026lt;0.05) increase in SOD activity when the Type-2 diabetic Acarbose group was treated with Acarbose, but not as high as the Type-2 diabetic rats fed with 100% CF bread + FL. The order of the increase in SOD activity is as follows; STZ + 100% CF bread + FL \u0026gt; STZ + 50% CF + 50% WF bread + FL \u0026gt; STZ + Acarbose + 100% WF bread \u0026gt; STZ + 20% CF + 80% WF bread + FL \u0026gt; STZ + 10% CF + 90% WF bread + FL \u0026gt; Basal diet + 100% WF bread \u0026gt; STZ + 100% WF bread + FL \u0026gt; STZ + 100% WF bread.\u003c/p\u003e\n\u003cp\u003eMoreover, the effect of vitamin-A enriched cassava/ wheat flour composite bread on the catalase activity of the liver of the normal control and HFD/STZ induced Type-2 diabetic rats is presented in Figure 3. The result shows that there was a significant (p\u0026lt;0.05) reduction in the catalase activity in the liver of the Type-2 diabetic control rats (STZ + 100% WF bread) as compared to the normal control. However, treatment of the Type-2 diabetic Acarbose group with Acarbose brought a significant (p\u0026lt;0.05) increase in its activity, but not as high as in Type-2 diabetic rats fed with 100% CF bread + FL. The trend in the increase in the activity of the catalase is as follows; STZ + 100% CF bread + FL \u0026gt; STZ + 50% CF + 50% WF bread + FL \u0026gt; STZ + Acarbose + 100% WF bread \u0026gt; STZ + 20% CF + 80% WF bread + FL \u0026gt; Basal diet + 100% WF bread \u0026gt; STZ + 10% CF + 90% WF bread + FL \u0026gt; STZ + 100% WF bread + FL \u0026gt; STZ + 100% WF bread.\u003c/p\u003e\n\u003cp\u003eFigure 4 represents the effect of vitamin-A enriched cassava/ wheat flour composite bread on the glutathione-s-transferase (GST) activity in the liver of the normal control and HFD/STZ induced Type-2 diabetic rats. The result showed that there was a significant (p\u0026lt;0.05) reduction in the GST activity in the liver of the Type-2 diabetic control rats (STZ + 100% WF bread) as compared to the normal control rats. However, treatment of the Type-2 diabetic Acarbose group with Acarbose brought a significant (p\u0026lt;0.05) increase in GST activity but not as high as in Type-2 diabetic rats fed with 100% CF bread + FL. This is the trend of the sequential increase in GST activity: STZ + 100% CF bread + FL \u0026gt; STZ + 50% CF + 50% WF bread + FL \u0026gt; STZ + Acarbose + 100% WF bread \u0026gt; Basal diet + 100% WF bread \u0026gt; STZ + 20% CF + 80% WF bread + FL \u0026gt; STZ + 10% CF + 90% WF bread + FL \u0026gt; STZ + 100% WF bread + FL \u0026gt; STZ + 100% WF bread.\u003c/p\u003e\n\u003cp\u003eIn addition, the effect of vitamin-A enriched cassava/ wheat flour composite bread on the glutathione peroxidase (GPx) activity in the liver of normal and HFD/ STZ Type-2 diabetes is presented in units/mgprotein in Figure 5. The result revealed a significant (p\u0026lt;0.05) reduction in the GPx activity of the Type-2 diabetic rats\u0026rsquo; control group as compared to the normal control. However, there was a significant (P\u0026lt;0.05) elevation in its activity in the Type-2 diabetic Acarbose group that was treated with Acarbose but not as high as the Type-2 diabetic rats fed with 100% CF bread + FL. The rate of increase in the GPx activity is in this order: STZ + 100% CF bread + FL \u0026gt; STZ + 50% CF + 50% WF bread + FL \u0026gt; STZ + 20% CF + 80% WF bread + FL \u0026gt; STZ + 10% CF + 90% WF bread + FL \u0026gt; STZ + Acarbose + 100% WF bread \u0026gt; Basal diet + 100% WF bread \u0026gt; STZ + 100% WF bread + FL \u0026gt; STZ + 100% WF bread.\u003c/p\u003e\n\u003cp\u003eCatalase is a key enzyme that uses hydrogen peroxide, a non- radical ROS and its substrate. The enzyme is responsible for neutralization through the decomposition of hydrogen peroxide, thereby maintaining an optimum level of the molecule in the cell, which is also essential for cellular signalling processes (Ankita 2019). SOD\u003csub\u003eS\u0026nbsp;\u003c/sub\u003eform the front line of defence against reactive oxygen species (ROS)-mediated injury. Due to its role in limiting the formation of ROS and RNS and the consequent oxidative stress damage, the availability of SOD as an antidote for xenobiotic toxicity would be a therapeutic advantage (Ros et al. 2021; Younus 2018). Glutathione S-transferase is a group of multifunctional proteins that partake in several roles in detoxification, and their biochemical processes may serve a crucial role in defence mechanisms (Alnasser 2024). It is worth nothing that the diabetic group in STZ + 100% CF + FL had the highest level of endogenous antioxidant (Catalase, SOD, GST, and GPx). This may be a result of increased supplementation of vitamin-A enriched cassava in composite bread. The result is in agreement with the one earlier reported by Semiz and Sen 2007 on the antioxidant and chemoprotective properties of \u003cem\u003eMomordica charantia\u0026nbsp;\u003c/em\u003efruit extract.\u003c/p\u003e\n\u003cp\u003eFurthermore, figure 6 reveals the effect of vitamin-A enriched cassava/ wheat flour composite bread on the total thiol level in the liver of normal and STZ/HFD Type-2 diabetic rats in \u0026micro;mol/mgprotein. From the result, it was observed that there was a significant (p\u0026lt;0.05) decrease in the level in the total thiol of Type-2 diabetic rats\u0026rsquo; control group compared to that of the normal control group. However, there was a significant (p\u0026lt;0.05) increase in the level of total protein of the Type-2 diabetic Acarbose group treated with acarbose but not as higher as the Type-2 diabetic group fed with 100% CF bread + FL and 20% CF + 80% WF bread + FL. The sequential increase of the total thiol level in the levers of the treated rats is as follows: STZ + 100% WF bread \u0026lt; STZ + 100% WF bread + FL \u0026lt; STZ + 10% CF + 90% WF bread + FL\u0026lt; STZ + 20% CF + 80% WF bread + FL \u0026lt; STZ + Acarbose + 100% WF bread \u0026lt; Basal diet + 100% WF bread \u0026lt; STZ + 50% CF + 50% WF bread + FL \u0026lt; 100% CF bread + FL.\u003c/p\u003e\n\u003cp\u003eThe effect of vitamin-A enriched cassava / wheat flour composite bread in the non-protein thiol on the liver of the normal control rats and Type-2 diabetic rats is presented in Table 7 in \u0026micro;mol./mgprotein. It was observed from the result that there was a significant (p\u0026lt;0.05) decrease in the level of total thiol in the of Type-2 diabetic rats\u0026rsquo; control group as compared to the normal control group. However, there was a significant (p\u0026lt;0.05) elevation in the level of non-total protein of the Type-2 diabetic Acarbose group treated with acarbose but not as high as the Type-2 diabetic group fed with 100% CF bread + FL and 20% CF + 80% WF bread + FL. The trend in the increase in the level of non-protein thiol is in this order; 100% CF bread + FL \u0026gt; STZ + 50% CF + 50% WF bread + FL \u0026gt; Basal diet + 100% WF bread \u0026gt; STZ + 20% CF + 80% WF bread + FL \u0026gt; STZ + Acarbose + 100% WF bread \u0026gt; STZ + 10% CF + 90% WF bread + FL \u0026gt; STZ + 100% WF bread + FL \u0026gt; STZ + 100% WF bread.\u003c/p\u003e\n\u003cp\u003eAmong all the antioxidants that are available in the body, thiols constitute the major portion of the total body antioxidants and play a significant role in defence against reactive oxygen species. (Kukurt et al. 2021). Although many cellular thiol functions have been shown to be mediated via the cysteine group found in enzyme or protein structures, the main players in cellular metabolism are thought to be the mobile low molecular weight thiols, often known as non-protein thiols (NPSH) (Toohey and Cooper 2014; Gronow 2020). An increase in the level of total and non- protein thiols in the diabetic rat groups resulted from the increased supplementation of vitamin-A enriched cassava in composite bread.\u003c/p\u003e\n\u003cp\u003eTable 1 revealed the biochemical effect of vitamin-A enriched cassava/wheat composite bread on the liver function biomarkers; aspartate aminotransferase (AST), alanine aminotransferase (ALT), and alkaline phosphatase (ALP) in the plasma of diabetic rats. From the result, the value of AST was significantly (p\u0026lt;0.05) higher in the plasma of the HFD/ STZ induced Type-2 diabetic control group (STZ + 100% WF bread) as compared to the normal control group. However, treatment of the Type- 2 diabetic Acarbose group with Acarbose brought a significant (p\u0026lt;0.05) reduction to the value of AST but not as lower as the Type-2 diabetic group fed with 100% CF bread. The trend of the results in these groups is as follows: basal diet + 100% WF bread (22.11\u003csup\u003ea\u003c/sup\u003e \u0026plusmn; 0.91 U/L) \u0026lt; STZ + 100% CF bread +FL (25.61\u003csup\u003eb\u003c/sup\u003e \u0026plusmn; 0.72 U/L) \u0026lt; STZ + 50% CF +50% WF bread + FL (30.88\u003csup\u003ec\u003c/sup\u003e \u0026plusmn; 2.06 U/L) \u0026lt; STZ + 20% CF + 80% WF bread + FL (36.84\u003csup\u003ed\u0026nbsp;\u003c/sup\u003e\u0026plusmn; 0.45 U/L) \u0026lt; STZ + Acarbose + 100% WF bread (37.89\u003csup\u003ee\u003c/sup\u003e \u0026plusmn; 0.18U/L) \u0026lt; STZ + 10% CF+ 90% WF bread + FL (40.35\u003csup\u003ef\u0026nbsp;\u003c/sup\u003e\u0026plusmn; 2.06 U/L) \u0026lt; STZ + 100% WF + FL (62.10\u003csup\u003eg\u0026nbsp;\u003c/sup\u003e\u0026plusmn; 1.68U/L) \u0026lt; STZ + 100WF bread (117.37\u003csup\u003eh\u0026nbsp;\u003c/sup\u003e\u0026plusmn; 2.65 U/L). Moreover, the effect of vitamin-A enriched cassava/ wheat flour composite bread on ALT in the plasma of the normal and HFD/ STZ induced Type-2 diabetic rats is also presented in Table 1. The value of ALT was significantly (p\u0026lt;0.05) higher in the plasma of the HFD/ STZ induced type-2 diabetic control group as compared to the normal control group. However, treatment of the STZ- induced Type- 2 diabetic Acarbose group with Acarbose brought a significant (p\u0026lt;0.05) decrease to the value of ALT but not as lower as the Type-2 diabetic group fed with 100% CF bread. The result of the ALT showed what was revealed in the AST. The order of decrease in the value of the ALT in the plasma of the normal and treated rats is as follows; Basal diet + 100% WF bread \u0026lt; STZ + 100% CF bread + FL \u0026lt; STZ + 100% WF bread \u0026lt; STZ + 50% CF + 50% WF bread + FL \u0026lt; STZ + 20% CF + 80% WF bread + FL \u0026lt; STZ + 10% CF + 90% WF + FL \u0026lt; STZ + 100% WF bread + FL \u0026lt; STZ + 100% WF with these corresponding results; 21.40\u003csup\u003ea\u003c/sup\u003e \u0026plusmn; 0.31 U/L, 21.58\u003csup\u003eb\u0026nbsp;\u003c/sup\u003e \u0026plusmn; 0.59 U/L, 27.89\u003csup\u003ec\u003c/sup\u003e \u0026plusmn; 2.09 U/L, 32.10\u003csup\u003ed\u0026nbsp;\u003c/sup\u003e\u0026plusmn; 1.17 U/L, 36.49\u003csup\u003ee\u0026nbsp;\u003c/sup\u003e\u0026plusmn; 1.11 U/L, 45.39\u003csup\u003ef\u0026nbsp;\u003c/sup\u003e \u0026plusmn; 1.17 U/L, 61.40\u003csup\u003eg\u0026nbsp;\u003c/sup\u003e\u0026plusmn; 1.21 U/L, 75.79\u003csup\u003eh\u0026nbsp;\u003c/sup\u003e\u0026plusmn; 1.33 U/L. In addition, the effect of vitamin-A enriched cassava/ wheat flour composite bread on the value of ALP is presented in Table 2. It was shown from the result that the value of ALP was significantly (p\u0026lt;0.05) higher in the plasma of the HFD/ STZ induced Type-2 diabetic control group as compared to the normal control group. However, the values were significantly (p\u0026lt;0.05) lower in Type-2 diabetic control group treated with Acarbose but not as lower as Type-2 diabetic groups fed with composite bread. The values of the ALP in both the normal and treated rats are as follow; STZ + 100% WF bread (642.55\u003csup\u003eh\u003c/sup\u003e \u0026plusmn; 3.01 U/L) \u0026gt; STZ + 100% WF bread + FL (557.74\u003csup\u003eg\u0026nbsp;\u003c/sup\u003e \u0026plusmn; 2.80 U/L) \u0026gt; STZ + 10% CF + 90% WF + FL (460.24\u003csup\u003ef\u0026nbsp;\u003c/sup\u003e\u0026plusmn; 1.81 U/L) \u0026gt; STZ + 20% CF + 80% WF bread (410.31\u003csup\u003ee\u0026nbsp;\u003c/sup\u003e\u0026plusmn; 2.78 U/L) \u0026gt; STZ + Acarbose + 100% WF bread (401.30\u003csup\u003ed\u003c/sup\u003e \u0026plusmn; 2.69 U/L) \u0026gt; Basal diet + 100% WF bread (388.98\u003csup\u003ec\u0026nbsp;\u003c/sup\u003e\u0026plusmn; 2.42 U/L) \u0026gt; STZ + 50% CF + 50% WF + FL bread (360.98\u003csup\u003eb\u0026nbsp;\u003c/sup\u003e\u0026plusmn; 3.03 U/L) \u0026gt; STZ + 100% CF bread + FL (243.47\u003csup\u003ea\u003c/sup\u003e \u0026plusmn; 2.40 U/L).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable i: AST, ALT and ALP of the plasma of normal control and Type-2-diabetic rats fed with the composite bread.\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 156px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 156px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eAST\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 156px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eALT\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 156px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eALP\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 156px;\"\u003e\n \u003cp\u003eGROUP\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 156px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eU/L\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 156px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eU/L\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 156px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eU/L\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 156px;\"\u003e\n \u003cp\u003eI\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 156px;\"\u003e\n \u003cp\u003e22.11\u003csup\u003ea\u003c/sup\u003e \u0026plusmn; 0.91\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 156px;\"\u003e\n \u003cp\u003e21.40\u003csup\u003ea\u003c/sup\u003e \u0026plusmn; 0.31\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 156px;\"\u003e\n \u003cp\u003e388.98\u003csup\u003ec\u003c/sup\u003e \u0026plusmn; 2.42\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 156px;\"\u003e\n \u003cp\u003eII\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 156px;\"\u003e\n \u003cp\u003e117.37\u003csup\u003eh\u003c/sup\u003e \u0026plusmn; 2.65\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 156px;\"\u003e\n \u003cp\u003e75.79\u003csup\u003eh\u003c/sup\u003e \u0026plusmn; 1.33\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 156px;\"\u003e\n \u003cp\u003e642.55\u003csup\u003eg\u003c/sup\u003e \u0026plusmn; 3.01\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 156px;\"\u003e\n \u003cp\u003eIII\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 156px;\"\u003e\n \u003cp\u003e37.89\u003csup\u003ee\u003c/sup\u003e \u0026plusmn; 0.18\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 156px;\"\u003e\n \u003cp\u003e27.89\u003csup\u003ec\u003c/sup\u003e \u0026plusmn; 2.09\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 156px;\"\u003e\n \u003cp\u003e401.30\u003csup\u003ed\u003c/sup\u003e \u0026plusmn; 2.69\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 156px;\"\u003e\n \u003cp\u003eIV\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 156px;\"\u003e\n \u003cp\u003e25.61\u003csup\u003eb\u003c/sup\u003e \u0026plusmn; 0.72\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 156px;\"\u003e\n \u003cp\u003e21.58\u003csup\u003eb\u003c/sup\u003e \u0026plusmn; 0.59\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 156px;\"\u003e\n \u003cp\u003e243.47\u003csup\u003ea\u003c/sup\u003e \u0026plusmn; 2.40\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 156px;\"\u003e\n \u003cp\u003eV\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 156px;\"\u003e\n \u003cp\u003e30.88\u003csup\u003ec\u003c/sup\u003e \u0026plusmn; 2.06\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 156px;\"\u003e\n \u003cp\u003e32.10\u003csup\u003ed\u003c/sup\u003e \u0026plusmn; 1.17\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 156px;\"\u003e\n \u003cp\u003e360.98\u003csup\u003eb\u003c/sup\u003e \u0026plusmn; 3.03\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 156px;\"\u003e\n \u003cp\u003eVI\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 156px;\"\u003e\n \u003cp\u003e36.84\u003csup\u003ed\u003c/sup\u003e \u0026plusmn; 0.45\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 156px;\"\u003e\n \u003cp\u003e36.49\u003csup\u003ee\u003c/sup\u003e \u0026plusmn; 1.11\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 156px;\"\u003e\n \u003cp\u003e410.31\u003csup\u003ed\u003c/sup\u003e \u0026plusmn; 2.78\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 156px;\"\u003e\n \u003cp\u003eVII\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 156px;\"\u003e\n \u003cp\u003e40.35\u003csup\u003ef\u003c/sup\u003e \u0026plusmn; 2.06\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 156px;\"\u003e\n \u003cp\u003e45.39\u003csup\u003ef\u003c/sup\u003e \u0026plusmn; 1.17\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 156px;\"\u003e\n \u003cp\u003e460.24\u003csup\u003ee\u003c/sup\u003e \u0026plusmn; 1.81\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 156px;\"\u003e\n \u003cp\u003eVIII\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 156px;\"\u003e\n \u003cp\u003e62.10\u003csup\u003eg\u003c/sup\u003e \u0026plusmn; 1.68\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 156px;\"\u003e\n \u003cp\u003e61.40\u003csup\u003eg\u003c/sup\u003e \u0026plusmn; 1.21\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 156px;\"\u003e\n \u003cp\u003e557.74\u003csup\u003ef\u003c/sup\u003e \u0026plusmn; 2.80\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003eValues carrying the same superscripts in the same column are not significantly different (p\u0026gt;0.05).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eValues represent the mean \u0026plusmn; standard deviation (n = 5).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eKEY:\u003c/p\u003e\n\u003cp\u003eGroup 1-Normal Rats fed with Basal diet +100% WF bread without cocoa powder; Group II- Diabetic Rats fed with High fat diet + 25mg/kg/ body weight STZ + 100% WF bread without cocoa powder; Group II-Diabetic Rats fed with High fat diet + 25mg/kg/ body weight STZ + 25mg/kg/body weight Acarbose + 100% WF bread without cocoa powder; Group IV-Diabetic Rats fed with High fat diet + 25mg/kg/ body weight STZ + 100% CF bread + cocoa powder;\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eGroup V-Diabetic Rats fed with High fat diet + 25mg/kg/ body weight STZ + 50% CF + 50% WF bread + cocoa powder; Group VI-Diabetic Rats fed with High fat diet + 25mg/kg/ body weight STZ + 20% CF + 80% WF bread + cocoa powder; Group VII-Diabetic Rats fed with High fat diet + 25mg/kg/ body weight STZ + 10% CF + 90% WF bread + cocoa powder; Group VIII-Diabetic Rats fed with High fat diet + 25mg/kg/ body weight STZ + 100% WF bread + cocoa powder.\u003c/p\u003e\n\u003cp\u003eAn increase in these marker enzymes in the serum/plasma has been reported to be leakage from the liver to the blood stream, indicating hepatopathy (Lala et al. 2023). Normally, AST and ALP are enzymes located in the liver cells and leak out, making their way into the general circulation as liver cells are damaged (Ndrepepa 2021). The results showed that cassava / wheat flour composite bread has a hepatoprotective effect, and this may not be farfetched from its antioxidant properties, which was in agreement with the previous findings of Adefegha et al. 2014 on the ability of spices to restore the level of liver biomarker enzymes in diabetic rat models and Ajani et al. 2022b on the hypolipidemic effect and antioxidant properties of cassava/ wheat flour composite bread. The result revealed that an increased supplementation of vitamin-A enriched cassava in composite bread led to a significant reduction of the liver function biomarkers (AST, ALT, and ALP) in the plasma of normal and diabetic rats. \u003csup\u003e\u0026nbsp;\u003c/sup\u003e\u003c/p\u003e\n\u003cp\u003eHistopathological results in Figure 8 reveal the liver micrographs of the normal and HFD/ STZ-induced type- diabetic rats fed with vitamin-A enriched/ wheat flour composite bread. From the result, it was observed that the hepatic cells and profile in the diabetic control group (CC\u003csub\u003e2\u003c/sub\u003e) were characterized by a severe loss of liver parenchyma, some mild derangement in the cellular profiles, haemorrhage and the presence of inflammatory red cells within and around the central vein, including the sinusoids as, well as distorted hepatic vessels (red thick arrows) as compared with the control group (CC\u003csub\u003e1\u003c/sub\u003e) which showed no altered panoramic morphological presentation accompanied by a- outlined cellular profile.\u003c/p\u003e\n\u003cp\u003eHowever, the effect of Acarbose (a diabetic drug) on the Acarbose group (CC\u003csub\u003e3\u003c/sub\u003e) and the ameliorative effect of vitamin-A enriched cassava/ wheat flour composite bread reversed the damage that has been caused as a result of induction of streptozotocin on the experimental rats (A, B, C, D, and E) fed composite bread by showing a similar outline relative to the control group (CC\u003csub\u003e1\u003c/sub\u003e) with a yellow arrow. It is worthy of note that vitamin-A enriched cassava/wheat flour composite bread has an hepatoprotective effect.\u003c/p\u003e"},{"header":"5.0 CONCLUSION","content":"\u003cp\u003eIn this study, vitamin-A cassava supplementation of wheat flour in composite bread manufactured showed a strong antioxidative impact with potential hepatoprotective benefits in rats with Type-2 diabetes induced by HFD/STZ. These products might be used as functional foods and offer protection against oxidative stress, hepatotoxicity, and other potentially pathological conditions. It is noteworthy, therefore, that the bread with 100%CF + FL had the highest level of HDL- cholesterol and the best potential for antioxidants. To confirm the impact of vitamin-A supplemented cassava/wheat composite bread, more research from human clinical trials is required.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eEthical approval\u003c/h2\u003e\n\u003cp\u003eThe study was approved by the Research Ethics Committee, Centre for Research and Development (CERAD), Federal University of Technology, Akure, Nigeria, with approval no. ERCFMBSLAUTECH:038/06/2024\u003c/p\u003e\n\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\n\u003cp\u003eR. A. wrote the manuscript being my Ph.D workS. A. reviewed the manuscript being my co-supervisor.I. A. reviewed the work being my co-supervisor.G. reviewed the work being my major supervisor and he was the designer of the work.\u003c/p\u003e\n\u003ch2\u003eAcknowledgement\u003c/h2\u003e\n\u003cp\u003eThe authors express their profound gratitude to the Tertiary Education Trust Fund (TETFund) for given the grant for the work. Also, our appreciation go to the Functional Food and Nutraceutical Unit (FFNU) of the Department of Biochemistry, FUTA Akure, Ondo State and Ladoke Akintola University of Technology, LAUTECH Ogbomoso for allowing us to use their laboratories facilities.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAdefegha SA, Oboh G, Adefegha OM, Boligon AA, Athayde ML (2014) Antihyperglycemic, hypolipidemic, hepatoprotective and antioxidative effects of dietary clove (\u003cem\u003eszyzgium aromaticum\u003c/em\u003e) bud powder in a high-fat diet/streptozotocin-induced diabetes rat model. J Sci Food Agric 94(13):2726-2737. \u003c/li\u003e\n\u003cli\u003eAhmed SK, Hussein S, Qurbani K, Ibrehim RH, Fareeq A, Manmood KA, Mohamed MG (2024) Antimicrobial resistance: Impacts; challenges and future prospects. Journal of Medicine, Surgery and Public Health 2. Doi.org/10.1016/j.g/medi.2024.100081.\u003c/li\u003e\n\u003cli\u003eAlnasser SM (2024). 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Inter J Hlth Sci 12(3):88-93. \u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Vitamin-A enriched cassava, antioxidant, hepatoprotective, high-fat diet, diabetes","lastPublishedDoi":"10.21203/rs.3.rs-6828908/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6828908/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e\u003cp\u003eDiabetes is a chronic metabolic disease characterized by hyperglycemia and could lead to several complications including such as hepatopathy, neuropathy and nephropathy, as well as increased risk of cardiovascular disease. Functional foods development has become a cheap and an effective strategy towards the management of diabetes and its complication. Hence, the supplementation of vitamin-A enriched cassava flour in bread production could provide additional nutritional, bioactive constituents and health benefits.\u003c/p\u003e\u003ch2\u003ePurpose\u003c/h2\u003e\u003cp\u003eThe aim of this study is to assess the effect of vitamin-A enriched cassava /wheat flour composite bread on the \u003cem\u003ein vivo\u003c/em\u003e antioxidant status and liver function biomarkers in order to evaluate the hepatoprotective effect of the bread.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e\u003cp\u003eForty male Wistar rats, each weighing between 170 and 200 grams, were randomly assigned to eight groups with five rats per group. The control group received a standard basal diet, while the remaining groups were fed a high-fat diet (containing 30% fat) for two weeks before being administered streptozotocin (STZ) at a dose of 25 mg/kg body weight to induce type 2 diabetes. After induction, the diabetic rats were treated with various formulated breads for two weeks. A positive control group received acarbose (25 mg/kg body weight) along with a composite bread during the treatment period. Blood glucose levels were monitored every four days. At the end of the study, the rats were euthanized via cervical dislocation. Blood was collected through cardiac puncture, plasma was promptly separated, and the liver was removed, rinsed, and homogenized for analysis of antioxidant activity and liver function.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e\u003cp\u003eThe results revealed that the composite bread had a good antioxidative potential in rats fed with 100% vitamin-A enriched cassava bread plus cocoa powder (100% CF bread\u0026thinsp;+\u0026thinsp;FL) having a significant increase (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) in superoxide dismutase, glutathione peroxidase, glutathione-S-transferase, and catalase activities in the liver when compared to the control rats. The value of total thiol and non-protein thiol also revealed a significant (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) increase in the diabetic rats fed composite bread as compared with the control. Also, the activities of AST, ALT, and ALP were significantly (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) reduced in diabetic rats fed composite bread with 100% CF bread\u0026thinsp;+\u0026thinsp;FL, showing the highest hepatoprotective effect.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e\u003cp\u003eThe findings of this study indicate that bread made from a blend of vitamin-A enriched cassava and wheat flour may serve as a safe and effective functional food, offering notable antioxidant and liver-protective benefits.\u003c/p\u003e","manuscriptTitle":"Antioxidative and hepatoprotective Effects of Vitamin-A Enriched Cassava / Wheat Flour Composite Bread in Rats Fed High- Fat and Induced with Streptozotocin","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-08-20 13:29:29","doi":"10.21203/rs.3.rs-6828908/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"ca6856c9-2524-4c1f-a203-6b4cef024955","owner":[],"postedDate":"August 20th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-01-16T13:27:21+00:00","versionOfRecord":[],"versionCreatedAt":"2025-08-20 13:29:29","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6828908","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6828908","identity":"rs-6828908","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}