BIOCHEMICAL AND BIOMEDICAL STUDIES ON AFRICAN WALNUT (TETRACARPIDIUM CONOPHORUM -MULL. ARG.) – A POSTHARVEST PERSPECTIVE

Thesis Overview

<p><b>1.0 Introduction&nbsp;</b></p><p><b>1.1 Background of study</b></p><p>Nuts have remained a major part of the diet for humans since pre-agricultural times (King, Blumberg, Ingwersen, Jenab &amp; Tucker, 2008). They are consumed either as snacks or part of a meal. They can be eaten whole (fresh or roasted), in spreads (peanut butter, almond paste) or part of commercial products such as sauces, baked goods, oils etc. (Rajaram &amp; Sabate, 2006). Some of the recognised and popular edible nuts include almonds, cashew nuts, peanuts, hazel nuts, macadamias, pecans, pistachios, Brazil nuts and English walnut (Bolling, Chen, McKay &amp; Blumberg, 2011; Gonzalez 2006). In many developing nations such as Nigeria, there is a continuous search for alternatives to animal protein which is fast getting out of reach for many citizens as a result of poor governance, inflation, political turbulence, ethnic and guerrilla wars. Great attention is being turned to plant sources of which nuts are of paramount interest. Several nuts exist in the wild in African forests and many are yet to be documented. Some that have been brought to the lime light are still to be thoroughly researched. Many of these nuts are consumed daily either raw or processed. The nuts are also often sold in the open markets (Figure 2) by table display or hawking. This practice exposes them to high temperatures and relative humidity which may be detrimental to food quality and may trigger fungal growth or even Maillard reactions leading to rancidity. Despite concerns over mycotoxin contamination, there is a scarcity of research on potential contamination of these nuts and how risks can be accentuated by postharvest handling. African Walnut (Tetracarpidium conophorum- Mull. Arg) is a perennial climbing shrub which grows mainly in the Western region of Africa. It is found mainly in Nigeria, Gambia, Sierra Leone, Gabon, Equatorial Guinea and Cameroon (Amaeze, Ayoola, Sofidiya, Adepoju-Bello, Adegoke &amp; Coker, 2011). The nuts are encased in pods which may contain between 2 to 5 nuts. The seed is made of two cotyledons which are enclosed in a hard shell-like case. The nuts mature and are harvested between the months of June and September. In Nigeria, the nuts are basically processed by boiling in water or roasting in hot sand before consumption as snacks (Nkwonta, Ezeokonkwo, Obidoa &amp; Joshua, 2010). The flour is used as a soup thickener especially by the Yoruba tribe. In ethnobotanical medicine, the nut is extensively used in decoctions for treatment of several ailments such as malaria, male sterility dysfunctions, dysentery, constipation, abdominal cramps and general fever, as well as in management of chronic diseases such as diabetes, cancer and high blood pressure (Aladeokin &amp; Umukoro, 2011; Amaeze, Ayoola, Sofidiya, Adepoju-Bello, Adegoke &amp; Coker, 2011). However, it is not certain as to the main role of the nut extract in the decoctions; whether it is to affect a direct impact on the disease causing agent either by preventive or curative processes, or simply to contribute to general body nourishment and hence boost the immune system of the patient. Another key challenge is that the nut is very susceptible to fungal infestation. It grows mouldy within few days of harvesting as the pods decay to release the nuts. Although the nuts may be processed, the fungal contamination is noticed when the shells are cracked as the cotyledons become slimy. This affects the sensory qualities of the nut as well as the sale by retailers. In spite of the promising potentials of the African walnut in the field of nutrition and medicine, very limited peer reviewed study reports have been documented. <br></p><p> <b>1.1.1 Nutrient content of nuts&nbsp;</b></p><p>Nuts are nutrient-dense foods since they contain a high total fat content which is associated with high caloric index. They are generally perceived by the public as having fattening potentials which is a risk for certain health conditions, however, the perception has been changed as research reports show that the fatty acid composition of nuts is more beneficial than harmful (Bes‐Rastrollo, Sabaté, Gómez-Gracia, Alonso, Martínez &amp; Martínez-González, 2012). The saturated fatty acid content is generally low, and they contain much of mono- (oleic acids) and poly-unsaturated fatty acids such as linoleic acid, α-linolenic acid, as well as ω–3 (omega-3) fatty acids (Ros &amp; Mataix, 2006). Furthermore, Bes-Rastrollo, Wedick, Martinez-Gonzalez, Li, Sampson &amp; Hu, (2009) showed that nut consumption is associated with reduced body mass index (BMI). Ros (2009) noted that, fatty fraction of nuts also contains sizeable amounts of phytosterols, which are known to have both antioxidant and cholesterol-lowering properties. Nuts are good sources of protein with high L-arginine content which is important in vasodilation processes. They also contain several other bioactive macronutrients such as flavonoids (Huynh &amp; Chin, 2006). Nuts are also a good source of dietary fibre, contain sizeable amounts of folate and are rich sources of antioxidant vitamins such as tocopherols and phenolic compounds (Blomhoff, Carlsen, Frost &amp; Jacobs, 2006; Ros, 2009). Almonds and English walnuts are rich in α-tocopherol, although the later contain significant amounts of its isomer - γ - tocopherol. Both α-tocopherol and its isomer are recognized as relevant anti-atherogenic molecules (Ros, 2009; Wagner, Kamal &amp; Elmadfa, 2004). When nuts are compared with similar food sources such as vegetables, they show optimal nutritional density with minerals such as calcium, magnesium, and potassium and their sodium content are usually infinitesimal (Ros, 2009; Segura, Javierre, Lizarraga &amp; Ros, 2006). <br></p><p> Although nuts are composed of healthy nutrients and bioactive compounds as enumerated above, it cannot be conclusively said that consumption of nuts in whole or as part of diet will guarantee healthy state of individuals. This is because the bioavailability of nutrients and bioactive substances are influenced by genetic variation. Lots of individuals and people groups are believed to process various nutrients in different ways depending on the information encoded in their genes as a result of polymorphism. In addition, the various methods of processing, storage and other postharvest activities before consumption, as noted by Terry and Thompson (2011), play significant roles in modifying the quality and quantity of these multipurpose nutrients. Extensive studies are therefore required to establish correlation between human genetic interactions and delivery of these nutrients from nut sources, as well as to ascertain the biophysiological changes that arise as a result of postharvest processes.<br></p><p> <b>1.2 Aim and objectives</b>&nbsp;</p><p>The aim of this research is to identify and quantify specific nutrients and bioactives contained in African walnut; study the impact of postharvest processing and storage on these substances; examine potential fungal infestation and mycotoxin contamination and finally investigate possible effects of these bioactives/nutrients as contained in the extracts on cell lines of chronic diseases such as cancer. <br></p><p> <b>1.2.1 Specific objectives</b></p><p>&nbsp;1) To identify and quantify fatty acids contained in African walnut and study the impact of postharvest processing and storage, as well as shelf-life on its profile using gas chromatography with flame ionization detector (GC-FID) and single - quadruple gas chromatography coupled mass spectrometer (GC-MS) techniques.&nbsp;</p><p>2) Examine potential fungal infestation and mycotoxin contamination on the nut shells of African walnut at different maturity stages using controlled temperature and relative humidity techniques, as well as classical culture methodologies&nbsp;</p><p>3) Investigate cytotoxic effects of sequential solvent extracts of the nut on cell lines of chronic diseases such as cancer (lung cancer cell line - A549 cells).&nbsp;</p><p>4) Identify possible phenolic bioactives in the nut extract and carryout targeted metabolomics studies on the compounds identified using ultra high definition accurate-mass quantitative time of flight liquid chromatography/mass spectrometry (UPLC-Mass QToF) techniques. <br></p><p> <b>1.3 Thesis Structure&nbsp;</b></p><p>This study report is grouped into seven chapters with different sections and sub-sections. Chapter one gives the background and considerations for undertaking the study as well as specifies the aim and objectives of the research. Chapter two sections 2.1 to 2.3 details on review of literature relating to nutritional and health benefits of nuts in relation to specific health conditions. Further discussions in section 2.3 were based on the main bioactive/phytochemicals commonly found in nuts. The review also elaborated in section 2.4, on African walnut description, agro-geographic distribution, ethnobotanical uses, and survey of various published research studies on the nut since its discovery. The methods normally used in processing and preserving nuts and their effects on nutrient contents were reviewed in sections 2.5 and 2.6. The last section of chapter reviews the factors that affect nut fungal invasion and mycotoxin contamination.</p><p> Chapter three explains the general experimental design, sampling regime, experimental factors considered, and the general materials and methods used in treatment of samples. The chapters begins with a brief introduction explaining the basic postharvest retail handling practice of local farmers on African walnut. It further explains the rational for conducting only one major experiment during one harvest season of the nut. The rest of the chapters are based on different analyses carried out on the nut samples whilst assessing the different effects of the experimental factors. They all follow similar pattern of brief introduction, specific materials and methods, results, discussion and conclusion&nbsp;</p><p> Chapter four presents quantitative and qualitative profiling of main fatty acids in oil extracted from African walnut and the effect of postharvest storage and retail processing methods on these fatty acids. Chapter five captures the study on the fungal infestation and mycotoxin contamination of African walnut which was a follow-up mini experiment on the main experiment. Chapter six focuses on the preliminary analysis of potential phenolic compounds in African walnut methanolic extract as well as the cytotoxicity test of four different extracts of the sample on A549 cancer cell line. It includes sections for both assay of total phenolic content and high performance liquid chromatography flame ionization detector. &nbsp; &nbsp;&nbsp;<br></p>