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

Project Abstract

<p>&nbsp;            <b>ABSTRACT&nbsp;</b></p><p>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 as well. The nuts are encased in pods which may contain between 2 to 5 nuts. The seed is enclosed in a hard shell-like case. The nuts are commonly processed by boiling or roasting and consumed as a snack or used as soup thickener. In ethnobotanical medicine, the nut extract is extensively used in decoctions for treatment and/or management of common and chronic ailments such as malaria, dysentery, high blood pressure, diabetes and cancer. The nuts are generally exposed to high temperatures (25 – 37 °C) and relative humidity (RH) which increases susceptibility to fungal contamination and nutrient degradation, hence, raising concerns over product quality and safety. Experiment simulating the common retail postharvest storage and processing practices was conducted to (i) determine the effects on the fatty acid profile; (ii) assess the impact on the fungal population contaminating the nut shells at different maturity stages, and potential mycotoxigenic implications; (iii) evaluate the cytotoxicity of four extract of the nut on lung cancer (A549) cells; and finally (iv) assay the total phenolic content and profile potential individual phenolic components of the nut. Results indicated the presence of essential and non-essential fatty acids namely; palmitate, oleate, stearate, linoleate, arachidate and α-linolineate with α-linolineate being the most abundant (1.1 – 8.2 mg/g freeze-dry weight). Boiling and roasting generally improved the concentration of the fatty acids best when nuts are cold stored at 5 °C for maximum of 10 days. Potential mycotoxigenic species - Aspergillus section Nigri, Aspergillus flavus/Parasiticus, Fusarium spp. and Penicillium spp. - were frequently isolated from cultured shell pieces of stored nuts. When compared with unprocessed nuts, roasting completely prevented fungal contamination in shell pieces from nuts in the non-stored (NSN) group at early maturity stage, while boiling significantly reduced the level of contamination to about 58 % (P &lt; 0.05). Simulating open market conditions caused 100 % fungal contamination in all boiled samples and roasted samples at early maturity. Mycotoxin analysis using Yeast Extract agar (YES) and High Performance Liquid Chromatography (HPLC) - Fluorescence detector (FLD) showed that Aflatoxins - G1 (AFG1), B1 (AFB1), G2 (AFG2), and B2 (AFB2) were produced by 20 isolates with both AFG1 and AFB1 being predominant at concentration ranges 4 – 32,200 and 4 – 22,700 ng/g plug weight, respectively. No Ochratoxin A (OTA) was detected. Phenolic component analysis indicated unprocessed (20.79 ± 1.0 mg gallic acid equivalent per gram freeze-dry weight – GAE/g FDW) samples showed the highest value for total phenolics while both boiling (9.90 ± 1.8 mg GAE/g FDW), and roasting (9.32 ± 2.7 mg GAE/g FDW) reduced the amount by more than 50 % when compared with unprocessed. Potential individual phenolic compounds were unambiguously separated using high performance liquid chromatography – diode array detector (HPLC-DAD). There were no differences between chromatograms of defatted and non-defatted unprocessed, roasted and boiled samples. Cytotoxicity evaluation showed no decrease in cell densities in plates treated with extracts from unprocessed nuts at all concentrations. Diethyl ether-ethyl acetate (10 µg/mL) and n-butanol (1000 and 500 µg/mL) extracts of roasted nuts as well as dichloromethane and water (1, 10 µg/mL) of boiled nuts caused a non-significant decrease of &lt; 10 % in cell densities when compared with the phosphate buffered saline-media control. However, all extracts showed no cytotoxic effect on the A549 cells African walnut is basically produced at subsistence level in Nigeria, but considering the presence of desirable fatty acid profile and phenolic compounds, need for increased industrial scale production is herein recommended. Although fungal attack and potential mycotoxin risk on the nut may be high, retail processing by roasting has prospects to greatly accentuate the risk. Cold storage of the nut may help to improve the shelf life although it may not be cost effective for local farmers in Nigeria and Africa, however, it provides opportunity for export business. Although the nut extracts showed no cytotoxic effect on A549 lung cancer cell lines, there is need to investigate further to confirm it non-cytotoxicity activity on other cancer lines and normal cell lines <br></p>

Project 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>