X-ray fluorescence analysis of selected micronutrients in ten African indigenous leafy vegetables cultivated in Nairobi, Kenya

Introduction There is a growing interest on vital roles of micronutrients in the maintenance of good health and enhancement of the immune system for prevention of diseases. Methods Energy dispersive X-Ray fluorescence (EDXRF) spectrometer was used for qualitative and quantitative analysis of selected micronutrients Zinc (Zn), Iron (Fe), Magnesium (Mg), Calcium (Ca) and Potassium (K) in ten African indigenous leafy vegetables (AILVs) (Corchorus olitorius, Cucurbita moschata, Amaranthus blitum, Brassica carinata , Cleome gynandra, Solanum scabrum , Crotalaria ochroleuca, Urticadioica, Manihot esculenta, Vigna unguiculata) which are cultivated, marketed and consumed in Kenya and across East and West Africa. Results In this study, the highest levels of Calcium, Zinc and Iron were found in Urticadioica leaves (75.0 mg/g), Manihot esculenta leaves (0.1 mg/g) and Cucurbita moschata leaves (1.0 mg/g). Amaranthus blitum leaves exhibited the highest content of Magnesium (9.5 mg/g) and Potassium (36.5 mg/g) respectively. This study demonstrated that based on weight and bioavailabilty, 10 g of Cucurbita moschata (dried weight) provides between 57% to 136% daily value of iron for children age between 7 and 10 years old and can provide up to 41%, 81% and 27% daily value of iron for female aged 18+, lactating females aged 18+ and males aged 18+ respectively. A 10 g portion of Urticadioica leaves (dried weight) will provide 75% or 58% of the daily value of calcium based on the North American or western European recommendation respectively while the same amount of Amaranthus leaves provides between 37% to 50 % of daily value of magnesium for adults of nineteen years and older based on their weight. The daily value of zinc despite its dependency with age, weight and Zinc bioavailability can be supplied by 10 g of Manihot esculenta leaves (dried weight) at a percentage ranged from 8% to 39%. Based on the 3510 mg daily recommendation, 10 g of Amaranthus, Brassica carinata, Cleome gynandra and Cucurbita moschata (dried weight) will provide 10.4%, 10.0%, 9.8% and 9.3% daily value for potassium respectively. Conclusion The research findings are scientific evidences of the nutritional contribution of African indigenous leafy vegetables.


Introduction
African indigenous leafy vegetables (AILVs) are part of the African indigenous vegetables (AIVs) whose natural habitat originated in Africa [1][2][3] as opposed to the traditional African leafy vegetables that were introduced over a century ago and due to long use, have become part of the food culture in the continent. Both African indigenous leafy vegetables and traditional African leafy vegetables have long been important components in African diets. They are indispensable ingredients of soups or sauces that accompany carbohydrate staples [4]. In the Plant Resources of Tropical Africa (PROTA), 6,376 plants are reported as African indigenous plants with information available on cultivation practices for 280 African indigenous leafy vegetables [5]. Even though a few studies have assessed AILVs' consumption [1,6,7], their share in a diet at the household level has not been adequately evaluated in Africa.
Consumption of different varieties is influenced by the cultural backgrounds, hence some varieties are only found in certain communities [8]. It is interesting to recall that, our ancestors in Africa lived well for many years as they mainly relied on traditional foods that included the African indigenous leafy vegetables. Scientific study results show that such a population that relies on traditional food is less likely to suffer from cancer and other ailments [9]. Unfortunately changes in dietary patterns and food systems have led to increasing consumption of highly processed foods in many countries in Africa.
Readily available and accessible, these products are often high in fat, sugar and salt and signal a shift away from traditional diets [10].
These changes have affected the consumption of African indigenous leafy vegetables as they are stigmatized and often associated with poor rural lifestyles and low status. There is a dire need currently to strategically reposition indigenous vegetables in the horticultural sector so that their potential can be fully exploited for food nutrition and income generation [6]. Scientific [3,7,11] using Energy Dispersive X-Ray Fluorescence (EDXRF) spectrometer.

Methods
The vegetables were sampled in a peri-urban farm 20 km away from the city center of Nairobi and met the following criteria: the leaves are the primary edible parts, they were under cultivation but can also be gathered wild and are used by several ethnic groups. Table 1 present the different African indigenous leafy vegetables selected for the study. The leafy part of each type of the vegetables was washed with distilled water to remove soil and other earthy materials before being damped on a tissue to remove excess water and dried in an oven for 24h at a temperature of 90 0 Celsius.
Hand gloves were used during sample collection and preparation to minimize contamination. Dried vegetables were grinded and sieved using a standard laboratory sieve of 150 microns. For each vegetable, pellets were made in triplicate by mixing 2.4g of powder with 0.3g of starch as a binder. The mixture was milled in a pestle using a mortar for 10 min to maximize homogeneity and pressed into pellets of 25 mm to give reproducible irradiation and counting geometry. The pellets were then analyzed using Energy Dispersive X-ray Fluorescence Spectrometer (EDXRFS). EDXRFS is a multi-elemental technique for simultaneous analysis of samples (e.g biological samples) based on measurement of characteristic X-ray intensities emitted by the elements in the sample [12] following excitation with high energy X-ray beams. The experimental measurements were conducted using the Rigaku NEX CG Energy Dispersive X-Ray Spectrometer equipped with Rh X-ray tube source with Pd anode target, 50 W maximum power, 50 kV maximum voltage. NEX CG is powered by a new qualitative and quantitative analytical software, RPF-SQX, that features Rigaku Profile Fitting (RPF) technology. The software allows semi-quantitative analysis of micronutrients without standards and rigorous quantitative analysis with standards [13]. were then calculated to determine the precision.

Results
The typical EDXRF spectra of African indigenous leafy vegetable (Crotalaria Ochroleuca and Cleome Gynandra) samples overlaid are shown in Figure 1.

Method validation using NIST Bowen's Kale as certified
reference material: Table 2 shows the precision of EDXRF used in this study and the Instrument Limit of Detection (ILD) that represents the threshold above which a peak intensity can be distinguished from the background noise at a specified level of confidence [14]. The experimental values are all in good agreement with the certified values due to low calculated relative errors (%) for Bowen kale. This therefore clearly indicate a validated reproducible method for analysis of micronutrient levels in African indigenous leafy vegetables with guaranteed accurate results.

Mean (n=12) concentration (mg/g) of micronutrients in
African leafy vegetables Calcium: Figure 2 shows the concentration of Ca in ten African indigenous leafy vegetables. Urticadioica stands out with a level of calcium (75 mg/g) almost 11 times greater than the level of calcium in the Manihot esculenta leaves (7 mg/g) which is the lowest.
Amaranthus blitum and Vigna unguiculata have almost the similar amount of calcium, 28 mg/g and 29 mg/g respectively. Same apply to Solanum scabrum, Cleome gynandra and Cucurbita moschata leaves with 20 mg/g and 19 mg/g respectively. Although other related studies showed that calcium requirement may vary from culture to culture for dietary, genetic, lifestyle, and geographical reasons [15], calcium salts provide rigidity to the skeleton and calcium ions play a role in many, if not most, metabolic processes. Calcium deficiency is known to destroy bone, hence contributing to bone resorption. This study clearly demonstrates that people living in Kenya and in the African continent can rely on the AILVs as a source of calcium.
Unfortunately, there is very little literature on calcium daily requirement on data derived from African countries. Balk et al.
reported that countries in Africa and South America mostly have low calcium intake between about 400 and 700 mg/day [16].

Recommended calcium allowances for adults based on North
American and Western European data are between 1000 mg/day to 1300mg/day with the highest amount for post menopause females and males beyond 65 years old [15]. A 10 g dry weight of Urticadioica leaves with the highest concentration of calcium (75 mg/g), can provide 75% and 58% of the daily value of calcium based on the North American and western European recommendation. Corchorus olitorius are at the bottom with an equal zinc amount of 0.03 mg/g. Table 3 present the recommended nutrient intakes to meet the normative storage requirements from diets differing in zinc bio-availability and the corresponding percentage daily value of zinc in 10 g of Manihot esculenta leaves (dried weight). Bio-availability refers to the fraction of intake that can be absorbed into the blood system and used for physiological functions of the body [17]. For zinc, in healthy individuals, it is determined by three factors: the individual's zinc status, the total zinc content of the diet, and the availability of soluble zinc from the diet's food components [18].
Although green leafy vegetables are only modest sources of zinc < 10mg/g [15], they can contribute significantly to the recommended nutrient intake (RNI) for dietary zinc. 10g of Manihot esculenta leaves (dried weight) contains 39% of the daily value of zinc for females aged 19 to 65 and 28% for males in the same age range. Figure 4 shows the levels of iron in mg/g in the African indigenous leafy vegetables analyzed in the present study. Cucurbita moschata had the highest iron content of 1.01mg/g when Manihot esculenta leaves exhibited the smallest iron content of 0.13mg/g. The recommended nutrient intakes for iron varied with dietary iron bioavailabilities, age and body weight. Table 4 presents the % Daily Value of iron in 10 g of Cucurbita moschata leaves (dried weight).

Iron:
About 10g of Cucurbita moschata leaves (dried weight) provides between 57% to 136% daily value of iron for children age between 7 and 10 years old and can provide up to 41%, 81% and 27% daily value of iron for female aged 18+, lactating females aged 18+ and males aged 18+ respectively. This is very important knowing that worldwide the highest prevalence figures for iron deficiency (being defined as an absence of iron stores) are found in infants, children, teenagers, and women of childbearing age. With an overall 600-700 million people presenting a marked iron deficiency anemia [19]

Competing interests
The authors declare no competing interests.

Authors' contributions
Alix Dehayem-Kamadjeu: sample collection and preparation, data collection and analysis. Justus Okonda: manuscript drafting; data collection and manuscript review. All the authors have read and agreed to the final manuscript.