TABLE OF CONTENTS
CHAPTER ONE 
1.0   INTRODUCTION
1.1 Background of the Study
1.2 Aims and Objectives of the Study
1.3 Justification for the Study
CHAPTER TWO 
2.0   LITERATURE REVIEW 
2. Plantation of Cashew
2.2 Harvesting of Cashew
2.3 Pest and Diseases of Cashew Apple
2.4 Nutritional Composition of Cashew Fruit
2.5 Types of Cashew Apple
2.6 Red and Yellow Cashew Apple
2.7 Biochemistry of Cashew Apple
2.8 Medicinal Uses of Cashew Apple
2.9 Processing of Cashew Apple
2.10 Antioxidants
2.10.1 Type of Antioxidants
2.10.2 Non-enzymatic Antioxidant
2.10.3 Enzymatic Antioxidants
2.10.4 Oxidative Stress
2.10.5 Markers of Oxidative Stress
2.11 Free Radicals
2.11.1 Types of Free Radicals
2.12 Animal Model for Antioxidants Properties
2.12.1 Albino Rats
CHAPTER THREE 
3.0   MATERIALS AND METHODS
3.1.1 Plant Material
3.1.2 Chemicals and Reagents
3.2 Methods
3.2.1 Preparation of the Sample
3.3.2 Drying
3.3.1 Sun Drying
3.4.2 Solar Drying
3.3.3 Mechanical Drying
3.4 Schematic Processing of Cashew Apple/Pulp into Dried Form
3.5 Biochemical Analysis
CHAPTER FOUR 
4.0 RESULTS
4.1 Antioxidant Present in Red-Yellow Cashew Apple
CHAPTER FIVE 
5.0 DISCUSSION
5.1 Antioxidant Present in Red-Yellow Cashew Apple
CONCLUSION
RECOMMENDATION FOR FUTURE RESEARCHERS
REFERENCE
APPENDIX

CHAPTER ONE
1.0 INTRODUCTION
1.1 Background of the Study
Cashew (Anacardium occidentale) is an evergreen perennial plant of afforestation and soil conservation, belonging to the family Anacardiaceae and genus Anacardium. This family consists of 400-600 species. Among the eight species in the genus Anacardium, the only cashew is valuable due to its nutritious kernel. Cashew is a tropical originally found in South America. Plant height varies from 5 to 14 m. It was discovered by the Portuguese who introduced it into their colonies in Africa and Asia. The trunk is usually short and irregular, starting branches close to the ground. Leaves are green that are placed in a spiral pattern towards the end of the stem. Leaves become mature after 20-25 days. Flowering can occur at any time; individual flowers are short in size consisting of five yellowish-green sepals and five white to reddish petals (Lacroix, 2003).
The cashew tree can grow up to about 10 meters and its trunk diameter can vary between 1.2 and 1.5 meters. It is hardy, but grows best in hot tropical areas with alternating dry and wet seasons. It adapts to a variety of soils, but prefers light, sandy, deep, well-drained soils with a clay content of 25%. It can be grown for fruiting or for reforestation and has a life span of about 30 years. The main producing countries are Nigeria, Côte d’Ivoire, Vietnam, India and Brazil. This tree produces fruits composed of two parts: the cashew nut called “true fruit” and the cashew apple “false fruit” which is about 9 times the weight of the nut (Abreu, 2012).

Figure 1: Cashew tree and Cashew fruit
Source: Abreu, (2012).

Cashew nuts and cashew apples are the two main products of Cashew tree. Cashew nuts (kernels) are the main commercial product of this tree. It consists of a kernel and a shell. The roasted kernel can be used in a variety of ways (confectionery, pastries, and cashew butter) or roasted, salted and eaten alone or mixed with other nuts or dried fruits. While the shell yields cashew nut shell liquid (CNSL), also a high value-added product, highly acidic and corrosive, used in the manufacture of friction elements (Lautié et al., 2001).
After the nut develops, its stalk becomes enlarged and changes color to give a “false fruit” known as the cashew apple. When ripe, this cashew apple is red, yellow or orange in color. Whatever the color, the flesh is yellow. The shape of this false fruit is round or pear-shaped, hence the name Anacardium, which means “heart-shaped”, and it can be up to 10 centimeters long and 5 centimeters wide. The weight of a cashew apple is generally between 70 and 90 grams (Soro, 2012).
The cashew apple has a high nutritional potential. Indeed, it has great antiscorbutic qualities due to its vitamin C content (200 and 300 mg per 100 grams of fresh material) which is about five times higher than that of an orange. This is used to “fortify” juices of other fruits low in ascorbic acid. It is also rich in polyphenolic compounds (tannins), some of which are responsible for its characteristic astringency, carotenoids, dietary fibre, vitamins, sugars and mineral elements essential for human nutrition. The intensity of sweetness and astringency is quite variable depending on the variety and the stage of ripeness. The ripe cashew apple has a very particular flavour due to its content of aroma compounds (Soro, 2012).
Cashew apples are soft, juicy, slightly fibrous, astringent in taste, and covered with thin, waxy skin that easily bruises. They are mostly heart-shaped, 3-6 or more times larger than the nut, and when fully ripe, they are bright red, yellow, or a mixture of the two colours. The fruit has about six times more vitamin C per 100 ml than orange juice, with an average vitamin C content of 200–269 mg/100 ml. also contains anti-oxidants, sugar, amino acids, and phenolic compound. At the same time, there is an increase in cashew nut production and utilization worldwide and huge postharvest loss of its apple persists. For example, in Nigeria, Kwara State has reported a loss of about 467 kg of cashew apple per hectare. Similarly, Tanzania has reported approximately cashew apple production of 3,138,260.4 tones in season 2017/2018, of which the majority was left to rot in the farms. Globally, the loss of cashew apples is about 95 %, approximated from global production of cashew apples of about 30 million tones per annum. Despite their nutritional value and massive production, cashew apples are considered as waste, hence a non-targeted fruit during cashew nut production in less technological countries, including Nigeria and Tanzania (Akinwale, 2000).
After the nuts are harvested, more than 80% of the apples are left to rot in the farms, and only a small proportion is consumed during the season as snacks to quench thirst and hunger during farming activities. Moreover, cashew apples are neither harvested for family consumption nor marketed locally or internationally as they are too perishable to transport in places with no cold chain. Consequently, including cashew apples in value added products such as juice, jam, and wine will help decrease postharvest loss and could help Nigeria and Tanzania economy and small-scale farmers' quality of life (Akinwale, 2000). Cashew apple utilization is mainly hampered by its high perishability leading to speedy deterioration and inadequate skills and technologies for processing and preservation to extend shelf life. Additionally, astringent taste and negligence due to insufficient knowledge of their importance in food and nutrition security have contributed to this critical lack of attention (Akinwale, 2000).
Therefore, abandoning cashew apples in the farms leads to a massive loss of nutrient dense food that could otherwise improve food/feed and nutrition security, and ultimately well-being and social-economic transformation; if consumed fresh, processed alone, or incorporated into other food products (Deenanath et al., 2015). Additionally, processing cashew apples can generate employment possibilities in rural areas, notably for women and young people. This may benefit the nation's efforts to reduce poverty and foster sustainable economic growth to fully unleash the potential of cashew apples in Nigeria, addressing will be essential. This can be accomplished by investing in infrastructure, assisting small-scale farmers, and creating efficient distribution and marketing networks (Deenanath et al., 2015).
The processing of cashew apples is limited by the characteristics of the fruit. Indeed, its tannin content gives it a particularly strong astringency. In addition, the very high content of reducing sugars (glucose and fructose) in it, which are precursors of the Maillard reaction and non-enzymatic browning, poses a problem of color when the fruit is processed into juice. Also, the very short shelf life of cashew apples at room temperature, between 24 hours and 48 hours maximum, is the main limiting factor for quality, both for fresh sales and for processing (Delima et al., 2015). However, the cashew apple, despite its great fragility, has many advantages. In addition to its nutritional quality and medicinal properties, the apple has technological advantages: the edible part of the fruit (between 85% and 100%) is higher than that of other traditional tropical fruits, and its juicy and sweet flesh is free of seeds or pits. Moreover, very large volumes are available (Delima et al., 2015).  
Thus, the valorization of this false fruit, often considered as a by-product of nut exploitation. Cashew apples are used in various fields, the main ones being: food chemistry, bio-industrial chemistry, nutrition, electrochemical energy technology, animal nutrition, microbial biochemistry, biochemical methods and toxicology. however, have revealed that about 90%–95% of cashew apples go waste due to limited knowledge about their health benefits, high perishability, lack of appropriate harvesting techniques, high cost of processing equipment, lack of government support, poor post-harvest management, and non-adoption of processing technologies (Delima et al., 2015).
1.2 Aims and Objectives of the Study
      The main aim of this study was to extract and Investigate the impact of nutrition and antioxidant capability on Albino Rat using different drying methods (Sun, Solar AND Mechanical Dehydrator) for Red-Yellow Cashew Apple. 
While the specific objectives were to:
i.	To dry red-yellow cashew apple using Sun, Solar and Mechanical Dehydrator.
ii.	To Evaluate the Phytochemical and antioxidant properties of dried Red-Yellow Cashew Apple.
iii.	To feed the dried Red-Yellow cashew Apple to the Albino Rat.
iv.	To Investigate the Biochemical properties of Albino Rat fed with dried Red-Yellow Cashew Apple.
1.3 Justification for the Study
Due to increase in demand of cashew Apple Product and the medical awareness of the health properties, there is growing needs to evaluate the quality of Cashew Apple, how they are being extracted and their various Nutritional Compositions and health advantages to human consumptions and used. Cashew Apple have attained superstardom in the health industry, food industry, environmental sustainability, job creation and its many virtues (Mai, 2007).
A number of health benefits have been attributed to Cashew Apple, which includes; Addressing micronutrient deficiencies due to Cashew Apple is rich in Vitamins, minerals, and antioxidants. Chronic disease prevention such has cardiovascular disease, cancer and diabetes. Improved Nutrition and health vulnerable (Mai, 2007). However, this Study investigates the Nutritional Value potentials of juice extracted from Cashew Apple.

CHAPTER TWO
2.0	LITERATURE REVIEW OF CASHEW
       Cashew (Anacardium occidentale) belongs to the order sapindalas, family	Anacardiaceae and genus Anacardium. The Anacardiaceae family consists of about 75 genera and 700 species (Nakasone and Paull, 1998). Botanically, the Anacardiaceae includes primarily trees and shrubs with resin canals, resinous bark and clear to milky exudates. The trees or shrubs have alternate, often trifoliate or pinnate leaves. Flowers are generally not highly conspicuous and can either be unisexual or bisexual. Only one carpel matures, forming a drupe (a fleshy fruit with a stoney seed). In some cases, the drupy fruits produce an irritant called urushiol. Considering the significance of cashew in the livelihood and economy of the nation, a concerted research effort on improving nut quality and increasing production per land area is key. Attempts have also been made to develop rapid methods of propagation, including budding, grafting, marcotting and tissue culture protocols for mass multiplication (Aliyu, 2000). Efforts in these directions have broadened know ledge on cashew as a crop and contributed to enhancing its productivity. Presently however, increase in cashew production in Nigeria is attributed to increase in cultivated area rather than to increase in yield per hectare (Aliyu, 2000).
2.1 Plantation of Cashew
	The grasted plant obtained from the superior mother plant are usually planted at the onset of monsoon. It is essential to provide stakes ant temporary shade with the locally available materials wherever necessary (especially in the south west aspects in case of forest plantation) to reduce the mortality rate and achieve quicker establishment. If the monsoon rains are inadequate, one or two pot irrigation can be done during the initial stages to ensure establishment (Obiaha et al., 1992). The cashew is generally planted on the waste lands and hence availability of soil moisture is always low, hence mulching is essential. Mulching with black polythene is beneficial to increase the growth and yield of cashew (Shuklajash et al., 2006). It is observed that there are root borer and fruit on a fruit and nut borer are the major pests, which are reported to around 30% loss in yield (Shuklajash et al., 2006).
2.2 Harvesting of Cashew
	Harvesting is to be done during hours when temperatures are milder. In this stage, when touched apples easily detach from the tree. Moreover, due to cashews being climacteriz (Ripening does not continue after harvested). Apples need to be harvested when they are fully ripe, when they have their best taste and aroma (Maximum sugar content, lowest acidity and astringency) (Shuklajash et al., 2006).
Therefore harvesters (from January to march) must walk the orchard everyday during production season for the fact that ripe apple spontaneously datch from the tree, thus becoming useless for consumption (Price et al., 1980). For the purpose of producing cajuina, cashew apple must be completely healthy and ripe with soluble solids content preferably between 10.5 and 11.5 and must not be the  sour type. Their color may be red or yellow, no requirements related to this regard (Kumar et al., 1991). Fruits must neither be soiled in the sands or soil matter and non be contaminated with micro organisms (mold and bacteria) when directly picked ripe off the ground. For correct harvesting procedures, fruits are to be slightly turned from side to side, so that they detach from the panicle branch. In case apples are a bit hard to be harvested, such fact evidences early ripening stages, unsuitable for harvesting so as to avoid contaminating apples, harvesters must keep their nails clean (Kumar et al., 1991).
2.3 Pest and Diseases of Cashew Apple
	The insect species have been implicated with economic losses estimated between 52% and 75% of the production level (Ojelade, 1998). Analeptes trifasciata was reported to produce a significant damage to cashew in Nigeria while low level of Helopeltis incidences is recorded (Topper, 2002). Although, they are the main insect pests of cashew in East Africa and India (Boma et al., 1998, Topper et al., 1998,Topper, 2002). A survey in Nigeria showed a wide spread of Analeptes trifaciata infestation in almost all cashew producing states; making it an economic pest (Igboekwe, 1984; 1985; Asogwa et al.,2011). The Entomological unit of Cocoa Research Institute of Nigeria has archived the collection and identification of insect pests of cashew since 1971. Moreover, information on their symptoms was harnessed and protocols to reduce their menace were formulated (Asogwa et al., 2008).
2.4 Nutritional composition of cashew fruit   
With recent upsurges in non-communicable diseases and food insecurities, interest in healthier and safer foods is increasing. The health concerns of the populace, with reference to what they consume, have sparked up studies into nutritious foods. Fruits are known to be rich sources of minerals, phytochemicals that can help mitigate the prevalence of non-communicable diseases (NCDs), promote healthy living as well as ensuring food security. Cashew apple contains good amounts of vitamin C, sugars (fructose and sucrose), avonoids, carotenoids, total polyphenols, volatile components, avanols, amino acids and minerals such as potassium, and has currently been given much attention. Table 1 presents the major biological molecules or elemental components found in cashew apples 
Table 1.1  Nutritional Composition of Cashew Apple.                              
Composition 	Constituents 	Range 	Units                  	
Sugars 	Total sugar (Sucrose, maltose, raffinose and higher saccharides (unidentified) reducing 
sugar (glucose and fructose)	6.3-9.9

6.24-9.8 	g/ 100g                                              

 
  
g /100g	
Vitamins 		Vitamin C 	126 -372 	M g/ 100ml 	
Minerals 	Ca, P, Fe 
K 
Mg, Zn, Na	0.9 - 21.4
1.53
16- 105	g/L 
g/L  
mM	
Amino Acids 	Ala, Phe, Ser, Leu, Glu, Asp, Pro, Tyr	0.88- 3.36	Mm	
Polyphenols 	Gallic acid, Protocatechuic acid 	215.1 – 412.8	Mg/ 100Ml	
Organic acids 	Malic Acid, Citric Acid, Lactic Acid 
Tannins 
Proteins
Carotene
PH
TSS
Moisture 
Ash 
Specific 
Refractive index	0.1 – 0.36

0.22 – 0.58
0.1 – 1.04
0.03 – 0.74
3.67 – 4.53
7.4 – 12.8
90.4%
0.19 – 0.45
1.030
1.339	g/100ml

g/100ml
g/100ml
mg/ 100g
_
Brix
g/10 0g
g/100g
_
_	
The cashew apple is highly acidic with a pH value of 3.5–4.8 and contains about 85% moisture, which contributes largely to its high perishability, which occurs usually a day or two after harvesting of the nuts. The cashew apple can therefore serve as a good food source with potential utilization in refreshing drinks, smoothies, and juices. The fruit is rich in carbohydrates, mainly reducing sugars (glucose and fructose), that are readily absorbed by the body. The reducing sugar content of cashew apples on a wet basis is 10.5%. This makes it a good caloric option for use in fruit juices or meals for children and the elderly as a source of energy, if the astringency is minimized. Its utilization in energy booster drinks for sportsmen will be a groundbreaking intervention in the food and beverage industry. Cashew apple contains protein (0.5–1.09%) although in very minute quantities on a dry matter basis, the cashew apple was found to contain 61.21% of dietary, of which 13.25% are soluble and 47.96% are insoluble, with appreciable potassium, phosphorus, sodium, magnesium, calcium, copper, zinc, iron, and phenolic contents. The cashew beer is being used as a fat replacer in burgers, in cereal-based extrudates and cookies to increase beer content and nutritional value. 
Thus, the use of this natural source from the cashew apple may confer health benets to humans; however, studies have shown that the phytic acid and insoluble beer content of the cashew apple might limit the bioaccessibility of the nutrient contents. An in vitro digestion of cashew apple juice and its beer (pulp) showed that the juice is highly bio-accessible than the beer, even though the raw, undigested beer had a higher copper content (12.20 mg/L) than the juice (2.10 mg/L). The bioaccessibility was attributed to the phytic acid (0.25%) present in the beer (peels and insoluble beer). The ash content of the cashew apple (1–1.5%) is an indication of its high mineral content. Cashew apple is a very rich source of vitamin C compared to most tropical fruits. The content of vitamin C (between 200–241 mg/100 g) in the cashew apple is about 3–5 times higher than citrus. Studies have revealed that the vitamin C content (241.13 mg/100 g) of the yellow cashew apple is generally higher than that (221.60 mg/100 g) of the red variety, while the latter has more amino acids and tannins than the former. 
The juice is a good source of vitamin C but not so with provitamin A. Thus, the cashew apple can be utilized as a source of Vitamin C in Africa, especially in Ghana, where the fruit is underutilized and usually left to rot. Cashew apple also has substantial quantities of B vitamins (thiamine, niacin, riboavin, pantothenic acid, pyridoxine, and folate). The apple juice is a rich source of minerals and is high in potassium, magnesium, phosphorus; but low in sodium, calcium, iron, copper with the least being zinc. The high mineral (potassium, magnesium, and phosphorus) content could be a good food source for incorporation into the DASH diet in the management of hypertension.         
2.5 Types of Cashew Apple
The cashew apple, a pseudofruit (accessory fruit) on the cashew tree, comes in various colors including yellow, red, pink, and orange. It's a fleshy, pear-shaped fruit that subtends the cashew nut and is often greenish when immature and matures to a brighter hue. The cashew apple is edible and has a juicy, slightly tart or sweet flavor (Russel, 1979). 
2.6 Red and Yellow Cashew Apple
Red cashew apple is nutritious and versatile fruits with sweet taste, crunchy texture and potential health benefits. Yellow cashew apple is sweet and tart, making them a great snacks (Russel, 1979). 
2.7 Biochemistry of Cashew Apple
The cashew apple is biochemically rich, containing significant amounts of sugars, vitamins (especially C), minerals, organic acids, and bioactive compounds like tannins and flavonoids. It's known for its high vitamin C content, antioxidants, and astringency due to polyphenols. The fruit also contains dietary fiber, amino acids, and various minerals (Russel, 1979). 
Composition 	Constituents 	Range 	Units                  	
Sugars 	Total sugar (Sucrose, maltose, raffinose and higher saccharides (unidentified) reducing 
sugar (glucose and fructose)	6.3-9.9

6.24-9.8 	g/ 100g                                              

 
  
g /100g	
Vitamins 	Vitamin C 	126 -372 	M g/ 100ml 	
Minerals 	Ca, P, Fe 
K 
Mg, Zn, Na	0.9 - 21.4
1.53
16- 105	g/L 
g/L  
Mm	
Amino Acids 	Ala, Phe, Ser, Leu, Glu, Asp, Pro, Tyr	0.88- 3.36	Mm	
Polyphenols 	Gallic acid, Protocatechuic acid 	215.1 – 412.8	Mg/ 100Ml	
Organic acids 	Malic Acid, Citric Acid, Lactic Acid 
Tannins 
Proteins
Carotene
PH
TSS
Moisture 
Ash 
Specific 
Refractive index	0.1 – 0.36

0.22 – 0.58
0.1 – 1.04
0.03 – 0.74
3.67 – 4.53
7.4 – 12.8
90.4%
0.19 – 0.45
1.030
1.339
	g/100ml

g/100ml
g/100ml
mg/ 100g
_
Brix
g/10 0g
g/100g
_
_
	
2.8 Medicinal Uses of Cashew Apple
Composition                    Constituents                          Range                    Units   	
Sugars                  Total sugar (Sucrose, maltose,            6.3-9.9                    g/ 100g                                              
                              raffinose and higher saccharides 
                               (unidentified) reducing 
                                sugar (glucose and fructose)              6.24-9.8                   g/ 100g                                              
Vitamins                 Vitamins C                                          126 -372                 M g/ 100ml
Minerals

           	

The nutritious nature and high polyphenolic content have various implications for the use of the cashew apple in maintaining and promoting health. The signicant amount of essential minerals found in cashew apples make them a good choice for maintaining strong immunity, promoting proper uid balance, nerve transmission and muscle contraction, and also for the management of micronutrient deciencies. The high mineral content can aid bones, cardiovascular health, and assist in metabolism, and bioactive pathways (Rani et al., 2014).
Due to its high caloric content and amount of reducing sugars, the cashew fruit is and can be used as an instant energy booster. A cohort study discovered that cashew apple juice enhanced fat oxidation and proposed that it might increase endurance during exercise. The high vitamin C content of the fresh juice promotes its usage in treating sore throat, maintaining good gum/oral health, serving as cofactors for enzyme and bioactive compounds activity, and as an antioxidant (Rani et al., 2014).
The fruit has various traditional and medicinal uses, due to its rich phytochemical prole. Thus, the cashew fruit has the potential to be used as a nutraceutical and pharmaceutical ingredient. Traditionally, cashew juice is used for the treatment of sore throats, colds, and coughs, and gastric disorders, including diarrhea, dysentery, and ulcer. Pharmacological studies have demonstrated the ability of the anacardic acids in the fruit to protect against ulcers by inhibiting the growth of Helicobacter pylori. Hence, the cashew apple is gastro protective. The signicant amounts of polyphenolic compounds in the apple make it an excellent source of natural antioxidants, such as anthocyanins, avonoids, avones, carotenoids, gallic acid, protocatechuic acid, conjugate cinnamic acid, and free cinnamic acid. These compounds are very prominent in scavenging reactive oxygen species (ROS) and inhibiting free radical formation, thus preventing cardiovascular diseases and damage to cellular components. Pascal et al. also demonstrated the anti-radical scavenging activity of the cashew apple. Cashew apple is classied as a functional food ingredient due to its high carotenoid content, which is very potent in maintaining good health and acting as antioxidant (Rani et al., 2014).
Phytochemicals like avonoids, tannins, and other acids have been found to contribute signicantly to the anti-inammatory, anti-microbial, and wound-healing properties of human cells upon consumption of cashew apples. The presence of bioactive compounds and polyphenols inhibits microbial growth and activity, as the cashew apple proved eective against the growth of Streptococcus spp., Micrococcus luteus, Salmonella typhimurium, Entrococcus faecalis, Streptococcus spp., and Bacillus cereus. A similar study in Cote D'Ivoire also revealed the inhibitory potential of apple juice against Staphylococcus aureus. The anti-tumor properties of cashew apple have also been conrmed using mice models. Studies have indicated that phenolic compounds like anacardic acid and gallic acid have a cytotoxic eect and inhibit the proliferation of cancer cells. However, the cashew apple extract from the residue had no eect on human tumor and non-cancerous cell lines (Rani et al., 2014).
Studies have also reported the lowering eect of consumption of meals rich in avonoids (myricetin and quercetin) on the risk of developing type 2 diabetes, obesity, and insulin insensitivity in mice fed with a high-fat and high-sucrose diet. Beejmohun et al. demonstrated the eectiveness of the cashew apple in the management of obesity and diabetes through the use of obese experimental mice (Rani et al., 2014). The study revealed that cashew apple extracts, which are rich in derivatives of myricetin and quercetin, inhibited the accumulation of fat and insulin resistance in obese mice. The study also established the reductive eect of cashew apples on body weight gain, fat storage, hyperglycemia, hyperinsulinemia, and insulin resistance in the obese mice (Rani et al., 2014).
2.9 Processing of Cashew Apple
Cashew apple is a fruit that is normally used for brewing alcoholic beverages after separation of the nut. However, it can be processed to produce titbits in sugar syrup, jams and pulp in honey. The project aims to produce cashew apple bits in sugar syrup, cashew apple jam, and pulp in honey on a small scale (Devasagayam et al., 2004). Cashew apple in sugar syrup The fruit is washed, cut into cubes and kept separately. In the kettle, sugar is converted into syrup and boiled to around 75 degrees brix. The cubes are added to the sugar syrup and boiled for a few minutes. The syrup is cooled and then transferred into bottles. Each bottle of 500 grams will contain 250 grams of cashew apples In 250 ml of sugar syrup. Necessary preservatives and colours are added in the kettle at the time of cooking (Devasagayam et al., 2004) 
Cashew apple jam The cashew apple after washing is passed through the pulper when the pulp and juice are extracted. The extracted mass is taken to the kettle where it is cooked under the influence of jacketed steam for ten minutes. Sugar is then added in desired quantities and the mass further cooked with constant stirring till a thick mass is formed with a reading of 65 to 70 degrees brix on the brix meter. After cooking, the required quantities of citric acid, pectin, flavours and colours are added and the mass stirred thoroughly. The mass after mixing is emptied into steel containers from where they are poured into bottles of 400 grams capacity. On cooling the jam sets. The bottle is capped after placing a foil paper at its top. The bottles are placed in cartons, strapped and dispatched (Devasagayam et al., 2004). Cashew apple in honey The cashew apple after washing is passed through the pulper when the pulp and juice are extracted. The extracted mass is taken to the kettle where it is cooked under jacketed steam to form a thick mass and almost all the water has evaporated. The steam is switched off and the mass cooled. Honey is then added to the desired extent and the contents mixed till a homogenous mass is formed. Finally the desired quantities of powdered cardamom and preservatives are added, stirred and the final product packed (Devasagayam et al., 2004) 
2.10 Antioxidants  
Antioxidants are those molecules that inhibit, decrease, delay, or completely scavenge the action of free radicals and oxidants, and protect the body from oxidative damage. (Phatak, and Chandra, 2010). The antioxidant defense is a universal mechanism that is present inside the cells and tissues of both plants and animals though their types and concentration vary. Thus, effective antioxidants have the ability to delay oxidation reaction or obstruct the development of free radicals or break the generation of the autoxidation chain reaction that generates free radicals/oxidants. They also act as reducing agents and metal chelators which convert hydroperoxides into stable compounds. Some antioxidants act as metal chelators that transform metal prooxidants into stable form. Oxidative/nitrosative stress results from disequilibrium in oxidant-antioxidant balance in the favor of reactive species with the increase of reactive oxygen and/or RNS production, respectively (Kurutas, 2015). The body’s defense mechanisms against oxidative damage are operative in two main systems. The first one includes removal of free radicals and reactive species by enzymes such as superoxide-dismutase, CAT, GPx, etc. and second one is scavenging of free radicals by electron donors, such as glutathione (GSH), tocopherols, ascorbic acid, thioredoxin, etc (Devasagayam et al., 2004).
The antioxidants in foods help to prevent oxidative reactions that decrease their quality. Many dietary supplements have been promoted as antioxidants, but only a few, few antioxidant dietary supplements have been shown to promote health. The health effect of an antioxidant depends on the systemic bioavailability, the concentration of the compound that can be delivered to specific organ sites, and whether this antioxidant can perform the expected function. With the exception of antioxidant nutrients, many dietary antioxidants are generally less effective in combating ROS compared with the antioxidant and cytoprotective enzyme systems (Devasagayam et al., 2004) 
2.10.1 Type of Antioxidants
2.10.2 Non-enzymatic Antioxidant
Dietary micronutrients constitute many water and lipid soluble nonenzymatic antioxidants founds in the mammalian system. Important antioxidants include vitamin A (retinol and β-carotene as provitamin A), vitamin C (ascorbic acid), and vitamin E (tocopherol and tocotrienol). These antioxidants work as defense barrier against free radicals and non-radical oxidants and prevent the assault to DNA, proteins, lipids, and other molecules which are induced by oxidants. The most abundant cellular antioxidant is tripeptide GSH which prevents oxidation of thiols groups inside the cell. Another endogenous protein called thioredoxin which is involved in antioxidative reactions inside the cell is also discussed here (Noctor and Foyer, 1998).
1. Ascorbic Acid 
Vitamin C is commonly called as ascorbic acid and it is mainly divided into two compounds, L-ascorbic acid and L-dehydroascorbic. Both possess antioxidant activity and can be enzymatically converted in vivo and may be absorbed through the gastrointestinal tract. Ascorbic acid is readily oxidized to dehydroascorbic acid. It is a vital water-soluble antioxidant found in biological fluids and required for normal metabolic functions of the body (Jaffe, 1984). It is a very potent free radical scavenger and conserves the integrity of low density lipoprotein (LDL) and also maintains the level of vitamin E in cell membranes. It prevents the macromolecules such as DNA, lipids, and proteins from oxidative damage by scavenging reactive oxygen and nitrogen species (Noctor and Foyer, 1998). Ascorbic acid mainly scavenges ONOO− , NO, and HOCl but also quenches O−⋅ 2 , ·OH, and O2, and reduces H2O2 to H2O via ascorbate peroxidase reaction (Noctor and Foyer, 1998).
2. Tocopherols 
Naturally occurring four tocopherols are designated as alpha (α), beta (β), gamma (γ), and delta (δ)-tocopherols and out of these isoforms α-tocopherol (αT) being the most abundant is also called vitamin E. All the isoforms of tocopherols are composed of a chromanol ring and a 16-carbon phytyl like side chains (Jiang, 2014). While α-tocopherol was the first vitamin E analoge to be recognized, eight chemically distinct analoges are now known, consisting of α, β, γ, and δ-tocopherols (T) and α, β, γ, and δ-tocotrienols (T3), all of them are now referred to as vitamin E. The tocopherols are saturated forms of vitamin E, whereas the tocotrienols are unsaturated and possess an isoprenoid side chain (Ahsan et al., 2015).
3. β-carotene 
β-Carotene has numerous biological functions in body but humans are not able to synthesize it, hence, it is necessary to supply these compounds with food or pharmaceuticals. Originally β-carotene was recovered from plants by physicochemical extraction mainly from carrots but nowadays β-carotene is mainly produced through chemical synthesis (Bogacz-Radomska and Harasym, 2018). Monaghan and Schmitt (1932) first described β-carotene as a fat-soluble vitamin A. This antioxidant protects lipids against rancidity and is known as the most efficient singlet oxygen radical scavenger (Bogacz-Radomska and Harasym, 2018).
4. Thioredoxin 
Thioredoxin is a pervasive cellular protein disulfide reductase which serves as an electron donor for several enzymes including ribonucleotide reductase, thioredoxin peroxidase, and methionine sulfoxide reductase. Thioredoxin is a small (12 kDa) multifunctional protein consisting of 105 amino acid residues and acts as a powerful antioxidant (Masutani, and Yodoi, 2007). It is responsible for maintaining proteins in the reduced state thus regulating redox reactions in signal transduction pathways. It functions as an intracellular reductase through a dithiol–disulfide exchange reaction using two cysteine residues (Holmgren, 1985). Reduced thioredoxin forms dithiols and the oxidized form incorporates disulfide bonds in the active site. Reduced thioredoxin transfers a hydrogen ion (H+ ) to the disulfide in the targeted oxidized protein to reduce it and itself becomes oxidized. Thioredoxin is also susceptible to nitrosylation. Thioredoxin levels are high in some cancers such as hepatocelluler (Miyazaki et al., 1998). 
2.10.3 Enzymatic Antioxidants 
Human system possesses a battery of enzymes that neutralize the reactive species formed. The important ones include CAT, SOD, GPx, and glutathione reductase (GRx). The activity of SOD, CAT, and GPx constitute the first line of antioxidant defense which plays a key role in the total defense mechanisms of the host biological system (Ighodaro and Akinloye, 2018).
Superoxide Dismutase 
Superoxide Dismutase was discovered by Irvin Fridovich in 1968. This enzyme catalyzes the transition O−⋅ 2 into H2O2 (McCord and Fridovich, 1968). SODs are located in cytosol and mitochondria and belong to the family of multimeric metalloenzymes. They are categorized into different families: Cu-SOD, Mn-SOD, Cu-Zn-SOD, Fe-SOD, and Ni-SOD. These enzymes are found at different places, for example, Cu-Zn-SOD is predominantly present in the chloroplast and cytosol of eukaryoic cells, while MnSOD is mostly present in the matrix of mitochondria and cytosol of bacteria, and FeSOD is found in prokaryotes and some plants (Duke and Salin, 1985). Among these enzymes, Cu-Zn-SOD is known to provide a strong defense against oxygen toxicity inside the cell, and according to studies it is accepted that Cu-Zn-SOD is an important enzyme for aerobic life and is irreplaceable (Peskin and Zbarsky, 1977). Studies have shown that SODs play a role in protecting enzymes and proteins against oxygen toxicity in both prokaryotes and eukaryotes including higher plants (Peskin and Zbarsky, 1977).
Catalase 
Catalase is present in most cells and is considered as a major antioxidant enzyme. Catalase is an enzyme which is present mainly in the peroxisomes of mammalian cells. Though found in mitochondria, animal CAT is largely localized in peroxisomes. It is a tetrameric enzyme consisting of four identical, tetrahedrally arranged subunits of 60 kDa each containing an active heme group and NADPH (Kirkman and Gaetani, 1995). Catalase catalyzes the decomposition of H2O2 to H2O and O2 (Aslani and Ghobadi, 2016). Catalase has two enzymatic activities depending on the concentration of H2O2. If the concentration of H2O2 is high, Catalase acts catalytically, that is, removes H2O2 by forming H2O and O2. However, at a low concentration of H2O2 and in the presence of a suitable hydrogen donor, for example, ethanol, methanol, phenol, and others, it acts peroxidically, removing H2O2, but oxidizing its substrate (peroxidatic reaction) (Aslani and Ghobadi, 2016).
Glutathione Peroxidase and Glutathione Reductase 
Glutathione peroxidase and glutathione reductase GPx is a family of enzymes that require reduced GSH as a substrate. They provide a second line of defense against oxidative stress. It is made up of four identical subunits of 21 kDa and each subunit contains a selenocysteine (Sec) residue (Lubos and Handy, 2011). GPx catalyzes the reduction of organic and inorganic H2O2 to H2O and corresponding alcohols, using GSH as a cofactor (Birben, Sahiner, Sackesen, Erzurum, & Kalayci, 2012) It plays an important role in the protection of cell membrane polyunsaturated fatty acids, where it functions as a multicomponent antioxidant defense system. GPx has a higher affinity for substrate than CAT and is known to reduce fatty acid hydroperoxides (Gathwala and Aggarwal, 2016). Most animal cells have both CAT and GPx (Gathwala and Aggarwal, 2016).
Glutathione or γ-glutamylcysteinylglycine is the major nonenzymatic antioxidant, involved in many cellular functions. It is a ubiquitous tripeptide that regulates intracellular redox homeostasis and is present either in reduced (GSH) or oxidized form (GSSG) (Figure 3). GSH is a tripeptide (cysteine, glycine, and glutamic acid) found in relatively high concentrations in many bodily tissues. It is found in almost all types of cell compartments: cytosol, ER, mitochondria, and vacuoles at millimolar concentration (Jimenez, Hernandez, Pastori, Rio, & Sevilla, 1998; Meister and Anderson, 1983). GSH scavenges many ROS such as H2O2, O−⋅ 2 and · OH (Misak et al., 2018). One of the basic role of GSH as an antioxidant is its ability to restore ascorbic acid via the ascorbate-GSH cycle. GSH is a potent detoxifier of xenobiotics and acts as a barrier against hydroperoxide induced oxidation. 
It plays a pivotal role in reducing oxidative stress, maintaining redox balance, enhancing metabolic detoxification, and regulating the immune system. Various chronic, age-related diseases such as those related to neurodegeneration, mitochondrial dysfunction, and even cancer, have been related to suboptimal or deficient GSH level. There is increasing awareness of its ability in mitigating toxin load through its ability to enhance hepatic conversion and excretion of compounds such as mercury and persistent organic pollutants (Minich & Brown, 2019).
2.10.4 Oxidative Stress
Oxidative stress is a condition that arises from an imbalance between the production of reactive oxygen species (ROS), also known as free radicals, and the body's ability to neutralize them with antioxidants. This imbalance can lead to cell and tissue damage, contributing to various diseases and aging. 
2.10.5 Markers of Oxidative Stress
Oxidative stress can be measured indirectly by measuring the levels of DNA/RNA damage, lipid peroxidation, and protein oxidation/nitration, rather than a direct measurement of reactive oxygen species. These oxidative stress markers are more enduring than reactive oxygen species (Alugoju et al., 2015).
Lipid Peroxidation
Malondialdehyde (MDA) is the most commonly used lipid marker of oxidative stress. It is formed via peroxidation of polyunsaturated fatty acids and is typically quantified using the TBARS assay. Competitive ELISA assays for MDA are also available. Other lipid peroxidation markers include 4-HNA, 8-isoprostane, lipid hydroperoxides, and oxidized LDL (Alugoju et al., 2015).
Protein Oxidation / Nitration
Oxidative damage to proteins can take the form of protein carbonylation and protein nitration (3-nitrotyrosines). Reactive oxygen species can also cause the formation of advanced glycation end products (AGE) and advanced oxidation protein Products (AOPP). All of these markers can be measured by standard assays (Alugoju et al., 2015).
2.11 Free Radicals
A free radical is defined as an atomic or molecular species containing one or more highly reactive unpaired electrons in atomic or molecular orbitals. The most important class of radical species derives from oxygen (O2).
O2 itself is a free radical: it has two unpaired electrons and is able to accept electrons to its antibonding orbitals, becoming reduced and acting as a strong oxidizing agent. O2 is the basis of a host of other free radicals, the O2 free radicals, more generically called reactive oxygen species (ROS). There is also a second group of radicals, generated in the presence of nitrogen or nitrogen-containing groups, called reactive nitrogen species (RNS). Both ROS and RNS are normally generated during cellular metabolism and can be both harmful and beneficial for living system depending on the concentration, specific cellular conditions, and timing of their generation; low concentrations of ROS and, to a lesser extent, RNS are in general beneficial and act as a cellular defense against infectious agents or contribute to mitogenic responses (Ahsan et al., 2014).
At high concentrations, ROS/RNS cause oxidative/nitrosative stress; increase of ROS/RSN is due to both overproduction and deficiency of enzymatic or nonenzymatic antioxidants. The balance between beneficial and harmful effects of free radicals is a key homeostatic mechanism of cells/tissues and organisms, and it is obtained by several mechanisms in a ‘redox regulation’ systems (Ahsan et al., 2014).
2.11.1 Types of Free Radicals
There are two types of radicals: the sigma radicals and the pi radicals. An unpaired electron in the sigma-radical is in the sigma orbital and an unpaired electron in the pi radical is in the pi orbital respectively. Therefore, the radicals above are pi radicals, t-Butyl radical is also pi radical since this radical is stabilized by the hyperconjugation. However, the phenyl radical and the vinyl radical are typical sigma radicals (Ahsan et al., 2014). Normally, pi radicals are stabilized by the hyperconjugation effect or the resonance effect. However, sigma radicals are very reactive because there is no such stabilizing effect (Ahsan et al., 2014).
2.12 Animal model for Antioxidants properties
Animal models, particularly rodent models like rats and mice, are frequently used to study the antioxidant properties of various substances, including dietary supplements and natural compounds. These models allow researchers to investigate the in vivo effects of antioxidants and their potential impact on various physiological processes and diseases (Vandenbergh, 2000).
2.12.1 Albino Rats
Laboratory rats or lab rats are strains of the rat subspecies Rattus norvegicus domestica (Domestic Norwegian rat) which are bred and kept for scientific research. While less commonly used for research than laboratory mice, rats have served as an important animal model for research in psychology and biomedical science,[1] and "lab rat" is commonly used as an idiom for a test subject (Vandenbergh, 2000).

CHAPTER THREE
3.0 MATERIALS AND METHODS
3.1 Plant Material 
The Red-Yellow cashew Apple used for this study were were harvested from the back of Kwara State Polytechnic medical along ara road village ilorin, kwara state, Nigeria.
3.1.2 Chemicals and Reagents
Sodium Benzoate (NaC6H5CO2)
3.2 Methods
3.2.1 Preparation of the Sample
The Cashew Apples were sorted, washed and the nut were removed. The Cashew Apple (1kg) were sliced to 2mm. 1kg of Cashew slices, 0.0008 L of sodium benzoate (NaC6H5CO2) were diluted into it for 5 minutes. The sliced Cashew Apple were then divided into three batches shows the process flow diagram for sample preparation. 
 3.3.2 Drying 
The drying of the Cashew Apple slices was done as described below
3.3.1 Sun drying
Freshly Cashew Apple was plunked from Cashew Tree, the cashew apple were pretreated and washed with clean water, the cashew apple was slice and 5g of sodium benzoate (NaC6H5CO2) were introduced to the sliced cashew apple of 2.0kg. sliced drained cashew apples were laid on the surface of a tray. There is fold Paper Lay on the tray before laying the sliced Cashew Apple on the fold paper, then Cover the Cashew Apple with net for 5 days until it is dried. this was achieved at an ambient temperature of 26-30°C. The samples are packaged into an air tight water resistant transparent Ziploc bag and labeled accordingly.	
3.4.2 Solar drying
Freshly Cashew Apple was plunked from Cashew Tree, the cashew apple were pretreated and washed with clean water, the cashew apple was slice and 5g of sodium benzoate (NaC6H5CO2) were introduced to the sliced cashew apple of 2.3kg. drained cashew  apples were spread on a tray mesh and placed on an existing solar dryer using a solar Collector as the heating Source alone. The solar energy from the panel also charge the battery, so that at night the stored energy in the battery powers the heater to generate heat to facilitate the drying process. It used 5 days in the solar dryer, the sample was packed into Ziploc bag after 5 days.  
3.3.3 Mechanical-drying
Freshly Cashew Apple was plunked from Cashew Tree, the cashew apple were pretreated and washed with clean water, the cashew apple was slice and 5g of sodium benzoate (NaC6H5CO2) were introduced to the sliced cashew apple of 2.75kg. The slice Cashew Apple were spread into dehydrator machine for 7 hours, The temperature (65°c) of the air in the drying chamber was controllable by a fan speed controller for 7hours. The dried cashew apple were allowed to cook and then packaged in an air tight water resistant transparent Ziploc bag and labeled accordingly. The dried sample was milled into flour for laboratory analysis using an electric blender. The blender was washed before and after blending the samples from each dried Red yellow cashew apple. Analysis was carried out on the dried samples.
3.4 Schematic Processing of cashew Apple/ pulp into dried form
The drying of the cashew apple slices was done as described below:
Cashew fruit pulp 
 	
Washing/Cleaning
 	
  					        Slicing  
Treatment used in (NaC6H5CO2)

   				 	         Drying 

   Dried Cashew

   				 	  Blended (Cashew Powder) 
   
Packaged (Ziploc bag)
3.5 Biochemical Analysis
Determination of Total protein 
The total protein concentration in the liver, kidney and serum of the animals was assayed, using Biuret reagent as described by Gornallet al (1949). 
Principle: The biuret reagent is an alkaline solution of copper potassium tartarate. Compounds containing two or more peptide bounds react with Cu2+ ion to give a violet colour. The buiret reaction is due to coordination of Cu2+ with the unshared electron pairs of peptide nitrogen and the oxygen of water which results into the coloured complex. 
Procedure: 4.0 ml of Biuret reagent was added to 1.0 ml of the sample (appropriately diluted). This was mixed thoroughly by shaking and left undisturbed for 30 minutes at room temperature for colour development. The blank was constituted by replacing the sample with 1.0 ml of distilled water. The absorbance was read against blank at 540 nm. 
Aspartate Aminotransferase Activity (AST)
The method described by Reitman and Frankel (1957) was used in the assay of the activity of aspartate aminotransferase. 
Principle: The enzyme catalyzes the reversible reaction involving α–ketoglutarate and L-aspartate to form L-glutamate and oxaloacetate. Aspartate aminotransferase was measured by monitoring the concentration of oxaloacetate hydrazone formed with 2, 4-dinitrophenyl hydrazine. 
Procedure: 0.5 ml of reagent 1 was added to each test tube containing 0.1 ml of the enzyme source (appropriately diluted) and incubated for 30 minutes at 370C. Then, 0.5 ml of reagent 2 was added and the assay mixture was mixed and left undisturbed for 20 minutes at 250C. The reaction was terminated immediately by adding 0.5 ml of 0.4N sodium hydroxide. The blank was constituted by replacing the enzyme source with 0.1 ml of distilled water. The solution was thouroughlymixed and absorbance read against blank after 5 minutes at 468 nm. The enzyme activity was obtained from the calibration curve (APPENDIX IV).
Acid phosphatase activity 
The method described by Wright et al (1972a) was employed in this assay. 
Principle: The amount of phosphate ester split within a given period of time is a measure of the phosphatase enzyme. Para-nitrophenylphosphate  was hydrolyzed to para-nitrophenol and phosphoric acid at a pH of 10.1. The para-nitrophenol confers a yellowish colour on reaction mixture and its intensity is read spectrophotometrically at 400 nm. 
Procedure: 2.2 ml of sodium acetate (0.1 M) and 0.1 ml of MgSO4.7H2O (0.1 M) were added in series to the test tubes. Then 0.2 ml of the enzyme source (appropriately diluted) was added and incubated at 37   for 10 minutes. 0.5 ml of of 10 mM p-nitrophenyl phosphate (substrate) was added and the assay mixture incubated again  for 30 minutes at 37 . The reaction  wasterminated immediately by adding 2.0 ml of 1N sodium hydroxide. The blank was constituted by replacing the enzyme source with 0.2  ml  of  distilled water. The  absorbance was read spectrophotometrically at 400 nm. 
Antioxidant assays
Superoxide dismutase
The method described by Misra and fridovich (1972) was used to assay for the activity of superoxide dismutase. 
Principle: Superoxide dismutase activity was measured as the inhibition of the rate of reduction of cytochrome c by the superoxide radical, observed at 550 nm:
2O2-. + 2H+                      SOD	         O2 + H2O2
Cytochrome c (oxidized) + O.-			Cytochrome c(reduced) + O2
The superoxide radical is produced enzymatically by the reaction:
Xanthine + O2 + H2O   Xanthine oxidase	 Uric acid + O2- + H+
Procedure:Tissue homogenate of 0.5ml was diluted in 4.5ml of distilled water (1:10) dilution factor. An aliquot of 0.2ml of diluted serum sample was added to 2.5ml of 0.05M carbonate buffer (pH 10.2) to equilibrate in a spectrophotometric cuvette and the reaction was started by adding 0.3ml of substrate (0.3mM Epinephrine) and 0.2ml of distilled water. The increase in absorbance at 480nm was monitored every 30 seconds for 150 seconds.
Catalase 
The method described by Beers and sizer (1952) was used to assay for the activity of catalase.
Principle: The UV light absorption of hydrogen peroxide can be easily measured between 230-250 nm. On decomposition of hydrogen peroxide by the catalase, the absorption decreases with time. The enzyme activity can be estimated by this decrease in absorption.
2H2O2	Catalase	2H2O + O2
Procedure
Hydrogen peroxide (0.036% w/w, 2.9 mL) was added and mixed with appropriately              diluted homogenate (0.1 mL).A blank was prepared containing potassium phosphate buffer (50 mM, pH 7.0, 3.0 mL). The time required for the A240nm of the reaction mixture to decrease from 0.45 to 0.40 absorbance units was recorded.
Reduced glutathione (GSH)
The level of GSH in the liver homogenate was determined using the procedure described by Ellman (Ellman, 1959). Briefly, 1.0 mL of liver homogenate was added to 0.1 mL of 25% trichloroacetic acid (TCA) and precipitate was removed by centrifugation at 5,000 g for 10 min. Supernatant (0.1 mL) was added to 2 mL of 0.6 mM DTNB prepared in 0.2M sodium phosphate buffer (pH 8.0). The absorbance was read at 412 nm.
Glutathione Transferase (GST)
GST activity in the liver was determined using the method of Habig et al., (1974).  1mM CNDB was added to buffer containing 1mM GSH and an aliquot of the sample. Upon addition of CNDB the change in absorbance at 340nm was measured as a function of time.   
Malondialdehyde
The concentration of MDA was quantified according to the method of Nelson, (2004) as outlined below:
 A portion of TBA reagent (2ml of 0.7% and 1ml of TCA) were added to 2ml of the sample. The mixture was thoroughly heated in a water bath at 100oC for 20minutes.  It was then cooled and centrifuged at 78g (4000rpm) for 10minutes. The absorbance of the supernatant was read at a wavelength of 540nm against a reference blank of distilled water after centrifuging for another 10 minutes.
Conc. Of MDA=                       Abs              
				Extinction coefficient
Extinction Coeff. Of MDA = 1.56 x 105 nm-1cm-1.
TBA: 0.7% 1.e 0.7g in 100ml.
TCA:  20% i.e. 20g in 100ml

Procedure for determination of Na+,K+-ATPase activity in tissue supernatant
	For the test, 400µlof 200 mM NaCl/40 mM KCl/60 mM Tris (pH 7.4)waspippetted into test tube. Thereafter, 20µl of MgCl2.6H2O (80 mM), 20µl of EGTA (20 mM), 240µl of distilled water and 20µl of appropriately diluted tissue supernatant were added. This was mixed and incubated at 370C for 5 minutes. Then, 100 µl of ATP (8 mM) was added. This was mixed and incubated at 370C for 30 minutes. Thereafter, 200µl of SDS (5%) and 2,000µl of reagent C were added. The mixture was allowed to stand at room temperature for 30 minutes for colour development. The blank was similarly prepared but 20µl of distilled water was used instead of the 20µl of tissue supernatant. The absorbance of the test was read against the blank at 820 nm. The absorbances obtained were then extrapolated from the calibration curve for phosphate (Appendix IX) to obtain concentration of inorganic phosphate.
Gluthatione Peroxidase (GPx)	
The method described by Pippenger et al., (1998) was used.
Principle:
GPx catalyses the oxidation of GSH by cumene hydroperoxide. The oxidized glutathione is converted to the reduced form in the presence of glutathione reductase and NADPH. In this reaction NADPH is oxidized to NADP+ simultaneously. The decrease in absorbance at 340nm is then measured

GPx

GR

ROOH + 2GSH              ROH + GSSG + H2O
GSSG + NADPH + H+              NADP+ + 2GSH
Preparation of Working Reagent;
One vial of the GPx reagent R2 was reconstituted with 10ml of buffer (1ml). It was stabilized for eight (8) hours at 25ᶿC.
Dilution of Cumene Hydroperoxide 
10µl of cumene hydroperoxide was diluted with 10ml of distilled water.
Procedure:
To 0.05ml of the diluted sample in a test tube, 2.5ml of working reagent was added to it, the solution was incubated at 37ᶿC for ten (10) minutes. 0.1ml of the diluted cumene was then added to it. The initial absorbance was immediately taken after one (1) minute and simultaneously read again after one (1) and two (2) minutes at 380nm.
Glutathione Reductase (GR)
Elabscience revised edition (8th), 2018 was used to assay for GR analysis
Principle
With coenzyme as hydrogen donor, GSSG under the catalysis of GR. Then the GSH content increased and NADPH decreased. The activity of GR can be calculated by detecting the change of NADPH.
GSSG + NADPH + H+             NADP+ + 2GSH

Preparation of working solution
The working solution was prepared according to the ratio of Reagent 1: Reagent 2: Reagent 3 = 2300:60:30
Reagent 1 contains the Buffer solution
Reagent 2 & 3 contains the substrate and enzyme reagent respectively in powdered form (each reagent was dissolved with 1ml of distilled water).
Procedure
A couple of 1cm diameter quartz cuvette was pre-heated in the incubator at 37oc for 5 minutes, one is used for sample detection and the other one for setting to zero with distilled water. The UV spectrophotometer was set to 340nm. 65 µl of sample was pipette into the pre-heated cuvette, 3120 µl of working prepared reagents was added to it, and the solution was mixed thoroughly. The absorbance value A1 and A2 was immediately taken at 30 seconds and 3 minutes respectively.
ΔA= ΔA1 – ΔA2

CHAPTER FOUR
4.0     RESULTS
The table 1 includes 11 samples and measures several key enzymatic and non-enzymatic antioxidant markers, as well as liver function indicators and protein content. These parameters give insight into the oxidative stress status, antioxidant capacity, detoxification ability, and nutritional profile of the Red-Yellow cashew apple.
1. MDA (Malondialdehyde) Ranged from 0.55 to 2.25 nmol/mL, MDA is a marker of lipid peroxidation, indicating the extent of damage caused by reactive oxygen species (ROS) to cell membranes. Most samples in this table have low MDA values (<1.0), suggesting minimal lipid peroxidation and good protection against oxidative damage.
Sample D shows a peak MDA level of 2.25, which may indicate a higher degree of oxidative stress or cellular damage in that sample. The low overall MDA levels suggest that red-yellow cashew apples contain compounds that protect cell membranes from oxidative degradation.
2. SOD (Superoxide Dismutase) Ranged from 1.431 to 2.231 U/mg, SOD is a primary antioxidant enzyme that neutralizes superoxide radicals (O₂⁻), preventing chain reactions of oxidative stress. In the table 1, all samples are moderate-to-high SOD activity indicates strong enzymatic defense against ROS.
Sample E records the highest SOD activity (2.231), suggesting to have superior antioxidant content. High SOD activity implies that cashew apple extracts could help in mitigating cellular oxidative stress, particularly in early stages of free radical exposure.
3. GST (Glutathione S-Transferase) Ranged from 0.896 to 1.280 U/mg, GST plays a crucial role in detoxification by binding glutathione to harmful compounds, making them easier to eliminate.
Levels are relatively stable across all samples, showing that detoxification systems are active and efficient. Slightly higher GST in sample K (1.280) may indicate a stronger ability to detoxify oxidative by-products.
4. GSH (Reduced Glutathione) Ranged from 22.76 to 32.24 µmol/mL, GSH is a major intracellular antioxidant, essential for neutralizing free radicals and regenerating other antioxidants. 
GSH is highest in sample 3 (32.243), and consistently high in others, indicating strong non-enzymatic antioxidant reserves. Lower values (e.g., sample H at 22.757) may reflect mild depletion but are still within protective ranges.
5. CAT (Catalase) Ranged from 0.636 to 0.937 U/mg, CAT is another key antioxidant enzyme that breaks down hydrogen peroxide (H₂O₂), a reactive and damaging ROS.
All values are within moderate activity range, with sample E showing the highest CAT activity. Adequate CAT levels reflect the cashew apple's ability to neutralize peroxides, contributing to cellular protection.
6. GPx (Glutathione Peroxidase) Ranged from 1.260 to 2.116 U/mg, GPx works with GSH to remove peroxides and prevent lipid oxidation. The highest activity (2.116) in sample 3 indicates strong peroxide-reducing power. GPx complements the antioxidant network, and its robust presence supports the use of cashew apple in oxidative stress modulation.
7. GR (Glutathione Reductase) Ranged from 24.38 to 36.81 U/mg, GR regenerates GSH from its oxidized form (GSSG), keeping the antioxidant system operational. 
Sample A shows the highest GR activity (36.809) than other 10 samples, enabling more rapid GSH recycling. High GR values enhance the sustainability of antioxidant activity, making cashew apple a strong supporter of long-term oxidative balance.
8. ALP (Alkaline Phosphatase) Ranged from 3.09 to 7.57 U/mg, ALP is often associated with liver function and bone metabolism. In food-based studies, it may also reflect membrane transport activity or metabolic activity.
Sample H Having the Highest ALP (7.570). No concerning elevation; values suggest healthy cellular enzymatic activity, possibly influenced by Red cashew apple’s nutrient content.
9. AST (Aspartate Transaminase) Ranged from 4.83 to 9.86 U/mg, AST is a marker of cellular damage, particularly from the liver, heart, or muscle tissues. All values are within non-toxic physiological ranges, suggesting no harmful tissue effects from cashew apple consumption.
Sample E shows the highest AST (9.867) but still not in pathological range. Cashew apple shows no hepatotoxicity and may be safe for liver health.
10. Protein Content Ranged from 37.939 to 59.941 mg/mL, Protein content reflects the nutritional quality of the samples.
Sample F have the Highest in (59.941) and sample 7 (57.309). High protein values enhance the nutritional profile of the cashew apple, supporting its use in diets targeting malnutrition or recovery.
TABLE 1: Antioxidant Present in Red-Yellow Cashew Apple			
SAMPLE	MDA (nmol/Ml)	SOD (U/mg)	GST
(U/mg) 	GSH (µmol/mL) 	CAT 
(U/mg)	GPx
(U/mg)	GR
(U/mg)	ALP
(U/mg)	AST
(U/mg)	PROTEN
(mg/mL)	
A	0.55	1.927	1.056	27.022	0.894	1.748	36.809	4.146	8.285	44.152	
B	0.564	1.833	1.079	28.052	0.722	1.819	33.839	3.09	6.847	46.930	
C	1.558	1.837	1.019	32.243	0.695	2.116	33.893	3.942	7.879	46.784	
D	2.250	1.606	1.024	24.889	0.732	1.478	25.960	3.535	4.828	52.705	
E	0.558	2.231	0.896	26.765	0.937	1.929	36.290	4.794	9.867	37.939	
F	0.814	1.431	1.009	22.905	0.636	1.424	26.305	5.413	5.929	59.941	
G	0.715	1.467	1.193	24.485	0.666	1.356	24.385	5.802	8.816	57.309	
H	0.738	1.828	1.113	22.757	0.796	1.706	29.350	7.570	9.611	46.637	
I	0.590	1.532	1.066	24.633	0.734	1.260	28.666	3.555	5.015	55.337	
J	0.721	1.674	1.057	26.103	0.687	1.614	35.758	3.849	7.71	50.366	
K	0.801	1.499	1.280	23.639	0.636	1.453	29.415	3.469	6.585	56.798	

CHAPTER FIVE
5.0 DISCUSSION
The antioxidant profiling of Red-Yellow Cashew Apple was evaluated through a range of enzymatic and non-enzymatic parameters including Malondialdehyde (MDA), Superoxide Dismutase (SOD), Glutathione S-Transferase (GST), Reduced Glutathione (GSH), Catalase (CAT), Glutathione Peroxidase (GPx), Glutathione Reductase (GR), Alkaline Phosphatase (ALP), Aspartate Transaminase (AST), and Total Protein Content. The values across the 11 samples (A to K) provide insights into the oxidative balance and biochemical richness of the cashew apple variety under study.
5.1 Antioxidant present in Red-Yellow Cashew Apple
1. Lipid Peroxidation (MDA) as a Marker of Oxidative Stress.
MDA is a key indicator of lipid peroxidation and oxidative damage in biological tissues. The lowest MDA levels were recorded in samples A (0.55 nmol/mL) and B (0.564 nmol/mL), suggesting minimal oxidative damage and a potentially better antioxidant status. Conversely, sample D (2.250 nmol/mL) had the highest MDA value, indicating elevated lipid peroxidation, which could be due to lower antioxidant enzyme activity or higher reactive oxygen species (ROS) burden. This aligns with the findings of Akinwale et al. (2000) and Ishola et al. (2011), who reported low lipid peroxidation in cashew apple juice, attributing it to the fruit's rich antioxidant capacity. In a study by Akinmoladun et al. (2010), tropical fruit extracts with high polyphenol and vitamin C content—including cashew apple—were associated with suppressed MDA levels, confirming the role of natural antioxidants in inhibiting lipid peroxidation.
2. Superoxide Dismutase (SOD) Activity.
SOD plays a crucial role in converting superoxide radicals into less reactive species like hydrogen peroxide. The highest SOD activity was observed in sample E (2.231 U/mg), while sample F (1.431 U/mg) had the lowest. High SOD activity in samples A, B, and E suggests these samples are better equipped to neutralize superoxide radicals, reducing the risk of oxidative injury. Similarly, Ogunmoyole et al. (2014) documented high SOD and CAT activities in methanolic cashew apple extracts, especially in red and yellow varieties, supporting their ability to scavenge reactive oxygen species (ROS).
Additionally, Ogbonna and Ukaoma (2015) reported that cashew apple extracts increased CAT and GPx activity in oxidative stress models, suggesting cytoprotective effects through enzymatic modulation. These studies corroborate the strong antioxidant enzyme responses observed in our analysis.
3. Glutathione-S-Transferase (GST)
GST detoxifies reactive intermediates by conjugating them with glutathione. Sample K showed the highest GST activity (1.280 U/mg), indicating strong detoxification potential, while sample E had the lowest (0.896 U/mg). Samples A through D maintained GST levels around 1.0–1.08 U/mg, reflecting moderate antioxidant defense through conjugation pathways. GST activities also paralleled earlier observations by Jimoh and Adedapo (2013), who highlighted the role of cashew polyphenols in enhancing glutathione recycling and detoxification enzymes. This shows that Red-Yellow Cashew Apple contributes significantly to redox balance via the glutathione system.
4. Reduced Glutathione (GSH)
GSH is a non-enzymatic antioxidant and a cofactor in many enzymatic reactions. It was highest in sample C (32.243 µmol/mL) and lowest in sample H (22.757 µmol/mL). The elevated GSH levels in sample C suggest strong endogenous antioxidant capacity, which likely contributes to reduced oxidative stress, despite a relatively high MDA level in that same sample, hinting at a compensatory mechanism. These findings are consistent with the work of Okigbo and Mmeka (2006), who found elevated GSH levels in tropical fruit pulps, including cashew apple, due to the presence of flavonoids and vitamin C.
5. Catalase (CAT)
Catalase detoxifies hydrogen peroxide to water and oxygen. The CAT activity was highest in sample E (0.937 U/mg), which may explain its relatively low MDA value. The lowest CAT values appeared in samples F and K (0.636 U/mg). Low catalase may compromise the cell's ability to deal with hydrogen peroxide buildup, potentially leading to oxidative stress if not balanced by other enzymes like GPx. Akinmoladun et al. (2010): "Antioxidant activity of selected tropical fruits" Included cashew apple, guava, and pawpaw in evaluating MDA suppression, antioxidant enzymes, and non-enzymatic antioxidant compounds. CAT were significantly higher in cashew apple than in pawpaw.
6.0 Glutathione Peroxidase (GPx)
GPx complements CAT by reducing hydrogen peroxide and lipid peroxides using GSH. Sample C showed the highest GPx activity (2.116 U/mg), while sample I had the lowest (1.260 U/mg). The high GPx in sample C aligns with its high GSH level and may compensate for oxidative challenges reflected by its elevated MDA.
7. Glutathione Reductase (GR)
GR regenerates reduced glutathione from its oxidized form, maintaining the GSH pool. Samples A and E had the highest GR values (36.809 and 36.290 U/mg), promoting a steady GSH supply and supporting other GSH-dependent enzymes like GPx and GST. Low GR in samples G and D reflects potentially weaker redox recycling capacity. GR activities also paralleled earlier observations by Jimoh and Adedapo (2013), who highlighted the role of cashew polyphenols in enhancing glutathione recycling and detoxification enzymes. This shows that Red-Yellow Cashew Apple contributes significantly to redox balance via the glutathione system. 
8. Alkaline Phosphatase (ALP)
ALP is involved in phosphate metabolism and is sometimes considered a marker of cell turnover. Sample H had the highest ALP activity (7.570 U/mg), possibly reflecting higher metabolic or cellular activity. Samples B and D showed relatively low ALP levels, which may be associated with less active phosphate metabolism. Iweala and Okeke (2012) findings “Comparative biochemical effects of aqueous extracts of cashew apple and bark” ALP levels rose mildly in subjects treated with cashew extracts, especially in prolonged exposure. The increase was considered a marker of enhanced metabolic demand or a modest hepatic response to phytochemicals. No signs of tissue toxicity were associated with this increase, indicating adaptive physiological response.
9. Aspartate Transaminase (AST)
AST is a transaminase enzyme that may indicate tissue stress or damage when elevated. Sample E and H showed high AST values (9.867 and 9.611 U/mg), potentially reflecting a response to oxidative challenges or cellular turnover. Sample D, on the other hand, had the lowest AST value (4.828 U/mg), consistent with its poor antioxidant profile and high MDA level. These findings align with Ogunmoyole et al. (2014) who reported a moderate increase in AST in rats exposed to oxidative agents without cashew apple extract treatment. In groups treated with cashew apple extract, AST levels were significantly reduced, indicating a protective role of antioxidants.
10. Total Protein
Protein content indirectly reflects the metabolic and enzymatic richness of a sample. Sample F showed the highest protein concentration (59.941 mg/mL), while sample E had the lowest (37.939 mg/mL). Higher protein levels suggest better biochemical support for antioxidant activities. Sample D had high protein content (52.705 mg/mL) but also the highest MDA, possibly indicating oxidative damage to proteins or compromised regulation. The high protein values observed (e.g., 59.94 mg/mL in Sample F) are comparable to values reported by Ogunlesi et al. (2007), who analyzed cashew apple juice and found it rich in amino acids, especially glutamine, a precursor of GSH synthesis. Similarly, Morton (1987) described the cashew apple as a source of vitamin C and phenolics, which synergize with proteins in maintaining antioxidant defenses.

CONCLUSION
The comprehensive analysis of antioxidant parameters in Red-Yellow Cashew Apple reveals its significant potential as a natural source of both enzymatic and non-enzymatic antioxidants. The low levels of malondialdehyde (MDA) across most samples indicate minimal lipid peroxidation and strong protection against oxidative damage. Elevated activities of key antioxidant enzymes such as Superoxide Dismutase (SOD), Glutathione Peroxidase (GPx), Catalase (CAT), and Glutathione Reductase (GR) suggest the presence of active biological compounds capable of neutralizing reactive oxygen species (ROS) and maintaining redox homeostasis.
The presence of high levels of Reduced Glutathione (GSH) and moderate to high activity of Glutathione-S-Transferase (GST) further demonstrate the fruit’s capacity to support cellular detoxification and non-enzymatic antioxidant defense. In addition, the biochemical indicators of liver function (ALP and AST) remained within normal ranges across all samples. 
The total protein content was also notably high, enhancing the nutritional value of the Red-Yellow Cashew Apple and supporting its use in nutraceuticals or as a dietary supplement. 
RECOMMENDATION FOR FUTURE RESEARCHERS
Given the promising antioxidant properties demonstrated by the Red-Yellow Cashew Apple in this study, future researchers are encouraged to expand on this work in several key areas:
1. Isolation and Characterization of Specific Antioxidants:
          While this study assessed general antioxidant enzyme activity and non-enzymatic markers, future work should focus on isolating, identifying, and quantifying specific phytochemicals such as phenolics, flavonoids, and vitamins using advanced techniques like HPLC, LC-MS/MS, or NMR.
2. Formulation into Functional Foods or Supplements:
      Researchers should explore the development of nutraceuticals or antioxidant rich dietary supplements from cashew apple extracts, assessing stability, bioavailability, and shelf life.
3. Exploration of Synergistic Effects:
     Investigating how cashew apple antioxidants interact with other plant-based compounds or medications could uncover potential synergistic effects or contraindications.
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APPENDIX
Taxonomic Level	        Red-Yellow Cashew Apple	
Kingdom	       Plantae	
Phylum	       Tracheophyta 	
Class	       Magnoliopsida	
Order	       Sapindales	
Family	       Anacardiaceae	
Genus	       Anacardium	
Species	       Anacardium occidentale	

Taxonomic Level	      Albino Rat	
Kingdom	      Animalia	
Phylum	      Chordata	
Class	      Mammalia	
Order	      Rodentia	
Family	      Muridae	
Genus	      Rattus	
Species	      Rattus norvegicus