Determination of concentration of some essential and heavy metals in roots of Moringa stenopetala using flame atomic absorption spectroscopy

This study is aimed at determination of concentration of essential (Ca, K, Na and Mg) and heavy (Cd, Cr, Pb, Hg, Cu and Zn) metals using Flame Atomic Absorption Spectroscopy (FAAS) of samples taken from fresh roots of Moringa stenopetala. A wet digestion method involving use of mixture of (HCl and HNO3, ratio 3:1) and 10 ml of 30% H2O2 at an optimum temperature and time duration was deployed during sample preparations. Results show that concentration of essential elements Mg, Ca, Na, and K range from 10.12 to 10.99 mg/kg, 2.6 to 5.64 mg/kg, 4.3 to 5.26 mg/kg, and 1.26 to 1.77 mg/kg, respectively. Among the heavy metals, concentration of Cu fall in the range of 0.81 _ 1.44 mg/kg while that of zinc fall in the range of 0.37 _ 2.34 mg/kg, both lying below toxic level. The levels of toxic metals (Cd, Cr, Pb and, and Hg) were not detected. The results of the study indicate that the concentration of the entire essential and heavy metals are below the range of WHO/FAO limits.


INTRODUCTION
Moringa tree is a multi-purpose miracle tree with tremendous potential for food and medical uses (Agena, 2009).It belongs to the genus of family Moringaceae.Moringa requires an annual rainfall of 250-3000 mm to grow.It is drought resistant tree that grows best at altitude up to 600 m but still grows at altitude of 1000 m.Worldwide, some 14 species of the Moringa tree have been reported (Diriba et al., 2017).Among these, the best studied species with regard to potential nutritive, medicinal uses and the identification of compounds of potential therapeutic importance is Moringa oleifera which is native to the Indian subcontinent.Moringa stenopetala species is endemic to East Africa (Bosch, 2004) and grows widely in southern parts of Ethiopia.
Almost all parts of Moringa tree can be used as a source of nutrition for human and livestock as well as for other purposes (Bosch, 2004).Moringa tree is a deciduous plant with tuber-like root at the earlier stage.Moringa oleifera roots are important agents of healing and nourishment.The roots are used to make medicines, perfumes, natural pesticides, fertilizers, cleaning agents, animal feeding and many other important products.When *Corresponding author.E-mail: anaxorma@gmail.com.
Author(s) agree that this article remain permanently open access under the terms of the Creative Commons Attribution License 4.0 International License Moringa oleifera seedlings are 60 cm tall or shorter, their roots can be used to make special sauce that can serve medicinal purposes.Individuals suffering from malnutrition are encouraged to consume sauce made from the Moringa oleifera roots sauce as it contains high levels of fiber, protein, vitamins and minerals, which are known to bring about a quick recovery (Fahey, 2005).
Moringa roots are popular in medicine both traditionally and in modern life.Traditionally, the restorative and healthful benefits of the Moringa root have been used by Ayurvedic practitioners in India for centuries to treat a wide variety of ailments and in controlling disorders of the circulatory system including minor cardiovascular complaints.In small doses, Moringa tree roots can be used to stimulate appetite and improve function of digestive tract, making it useful for individuals with gastric upset and irritable bowel syndrome.Additionally, roots of this tree have been used in controlled doses to treat impotence, sexual dysfunction, female reproductive tract issues and to bring on menstruation.In poultice form, Moringa roots are used for cramps and arthritis pains (Karuna and Rajni, 2014).
In modern medical uses, Moringa tree roots play great role.As stated in Bosch (2004) and Karuna and Rajni (2014), Moringa tree roots contain elements that can combat epithelial ovarian cancer and provide new hope for cancer sufferers.Roots extracts of Moringa tree can help to reduce or eliminate kidney stones by allowing body to flush calcium and phosphates from the kidneys more efficiently.This tree root is also used as antiinflammatory agents with solid results in laboratory rats showing reduced swelling and improved healing in Edema and other artificially induced inflammations.The analgesic and soporific effects of Moringa tree root compounds have undergone rigorous scientific testing and have been found to be useful in supplementing pharmaceutical remedies, allowing patients to experience longer, less interrupted sleep when taking pain medications.
Metal concentration in human diets are classified into essential like Na, K, Cu, Zn, Ca, Mg, Fe and Mn and nonessential or toxic such as Hg, Cd, Pb and Cr metals (Longhurst, 2010).Low intake of essential metals produces deficiencies, while higher consumption may cause toxicity.However, non-essential metals are lethal and toxic even at low concentrations to human and environment.Non-essential metals are ranked among the most hazardous toxic substances owing to their persistence in the environment and absorption in food chain (Khan et al., 2013;Muhammad et al., 2013).Toxic effects of metals include vomiting, diarrhea, headache, irritability, hypertension, heart, lung, kidney, liver and intellectual problems and cancer (Shah, 2012).
The term heavy metal refers to any metallic element that has a relatively high density and is toxic or poisonous at low concentration.Heavy metals include Cadmium (Cd), Copper (Cu), Lead (Pb), Zinc (Zn), Mercury (Hg), Arsenic (As), Silver (Ag), Chromium (Cr), Iron (Fe) and Platinum.Metal elements like copper and zinc are essential trace elements for living organisms at low concentration (< 10 mg/L).The characteristics of heavy metals are described by Loannidou et al., (2005) and Wang et al. (2005).Toxicity that can last for long time in nature cannot be degraded including bio-treatment and are very toxic even at low concentration (1.0 -10.0 mg/L).With respect to their toxicity, heavy metals can be divided into two groups: micronutrients like Fe, Mn, Mo, Cu, Cr, Ni and Zn that are essential in small amounts and the only toxic ones are As, Cd, Hg and Pb without any known biological importance.Toxic metals are extremely persistence in the environment even at low concentrations and have been reported to produce damaging effects on human and animals because there is no good mechanism for their elimination from the body (Loannidou et al., 2005;Adah et al., 2013).
As deduced from different studies, Moringa tree have got different essential and non-essential, heavy as well as trace metals at different levels.According to Fagbohun et al. (2014), the following minerals were analyzed from Moringa oleifera tree roots in Nigeria using spectroscopic techniques FTIR and XRD.The research found zinc (Zn), iron (Fe), chromium (Cr) and copper (Cu) with concentration values (in ppm) 10.00, 21,510.00and 20.00, 2.755, respectively.
Karuna and Rajni ( 2014) have analyzed M. oleifera tree roots using spectroscopic techniques FTIR and XRD for its elemental composition in India and found zinc (Zn), iron (Fe), calcium (Ca), potassium (K), sodium (Na) and magnesium (Mg) with concentration values (in ppm) 47. 84, 5.04, 286.07, 860.59, 17.17 and 43.79, respectively.In addition to the nutritionally important minerals abovementioned, the study conducted by Karuna and Rajni (2014) had analyzed heavy metals such as Pb, Se, Hg and As, however, only Pb was detected (0.19 ppm) from the root part of Moringa oleifera using inductively coupled plasma atomic emission spectroscopy.Studies conducted in Nigeria have assessed concentration of heavy metals in Moringa oleifera and found lead, iron, copper and zinc with mean (mg/kg) of 0.9471±0.0173,55.60±0.012,0.1762±0.0230and 3.225±0.022,respectively though atomic absorption spectrometer analysis (Abdulkadir et al., 2016).
Most of the data reported in research account for M. oleifera.However, all the plant parts of M. stenopetala, which is highly cultivated in southern part of Ethiopia, have not been analyzed for their metal concentration and nutritive values.The present study showcases a comprehensive investigation on the elemental constitutes of M. stenopetala tree roots.People in the study areas, especially Konso people, use M. stenopetala tree roots for abdomen pain relief just by digesting it and swallowing water component only.The usage is without any scientific dosage and presence of toxic and heavy metals could result in cause of health problems.Thus, the purpose of this study was to determine the concentrations level of six heavy and four essential metals in M. stenopetala roots that are cultivated in urban and rural gardens in Southern part of Ethiopia.

Description of the study area
The study was conducted in South Nation Nationalities and Peoples Region of Ethiopia.Three Woredas were selected: Gamo-Gofa Zuria, Konso and Derashe.Specifically, two places were taken: Karat and Dara from Konso, Gidole and Gato from Dirashe and Shara and Lante from Gamo-Gofa.Figure 1 displays study areas covered in the survey of this work.

Sampling protocol
Matured healthy and fresh M. stenopetala tree roots were collected from the farmlands of three areas mentioned in the section of study area; namely Gamo Gofa, Konso and Dirashe.The study areas were selected purposefully based on the productivity and traditional use of roots parts of M. stenopetala for medicinal purpose.To collect the representative sample from each sampling sites, two subsamples were taken from each site.In total, six samples were collected and put in clean polyethylene plastic bags, labeled and brought to laboratory for further pre-treatment.

Sample preparations
Roots samples collected from farmlands were washed with a running tap water to remove absorbed soil particulates and then rinsed with deionized water.The samples were chopped into pieces by stainless steel axe Teflon knife and Chopping board to facilitate drying and subsequently dried in drying oven at 70°C for constant weight.The dried samples were powdered using electronic blender and the powder was sieved through 2 mm sieve to prepare fine powder for digestion.

Acid digestion procedure
A gram of sieved powder of the samples was weighed out into acid washed glass beaker.Thereafter, the powder was digested with addition of 20 cm 3 of aquaregia (mixture of HCl and HNO4, ratio 3:1).10 ml of 30% H2O2 was added to avoid any possible overflow leading to loss of material from the 100-ml conical flask.Hydrogen peroxide is also used to enhance the reaction speed and to ensure complete digestion.The analyte was digested for 2 h in 100-ml conical flask covered with watch glass and reflex over a hot plate at 90°C for 2 h.The volume was adjusted to 100-ml with distilled water.Blank solution was handled as detailed for the samples.Working standard solutions of all metals were prepared from stock standard solution (1000 ppm).

Experimental setup
Flame atomic absorption spectrophotometer (FAAS) (Model: 210-VGP, USA) was used for absorbance recordings of the elements.Signal of each radiation for specific element was detected and were converted into concentration information for the analytics from calibration curves of each element.

Statistical analysis
Data entry management and preliminary summaries were done on Microsoft Office Excel spread sheet.Means and standard deviations of data collected were determined.All analyses were carried out in triplicates and data presented as means ± standard deviations.One-way analysis of variance (ANOVA) at p < 0.05 was used to determine statistically significant differences in the mean concentrations of metals among groups of root samples from different study areas.Data were further manipulated with ASA and SPSS 20.

RESULTS AND DISCUSSION
Concentration of four essential and two heavy metals were obtained form absorption intensity.Average results of the elemental analysis of the metals observed in this work are displayed in Tables 1 and 2. It is to be noted that each result is an average of three independent triplicate measurements (n = 3).Results show that essential (Ca, K, Na, Mg) and heavy (Cu, Zn) metals were detected and the heavy metals Pb, Cd, Cr and Hg were not found to be to the level of detection limit of the technique used in this experiment.Results indicate that samples have variable composition of each analyte metals with different concentration ranges among different sample sites.Table 1 presents concentration of essential elements while Table 2 presents mean concentration of heavy metals.

Calcium (Ca)
One-way analysis of variance (ANOVA) shows that mean concentration of calcium is statistically insignificant in all the areas of investigation, except the two Dirashe areas.
The average concentration of calcium in the studied sites ranges from 2.60±0.13 to 5.64±0.25 mg/kg.Relatively high concentration was observed in Dirashe areas (Table 1).The pattern of concentration of calcium in the studied sample sites is in the order; Derashe Gato > Derashe Gidole > Konso Karat > Gamo Gofa Lante > Gamo Gofa Shara > Konso Dara.

Potassium (K)
As can be seen from Figure 2, mean concentration of potassium in all areas is found to be lower.When sites are taken into consideration, there is statistical significant difference in the content of potassium concentration.The highest concentration of potassium is observed in Derashe-Gato (1.77±0.26mg/kg).While relatively the lowest concentration is obtained in Gamo Gofa-Shara (1.26±0.00mg/kg).Depending on the potassium levels in the studied samples, the order of the studied areas is Derashe-Gato > Derashe-Gidole > Konso-Karat > Gamo Gofa-Lante > Konso-Dara, Gamo Gofa-Shara.

Sodium (Na)
The concentration of sodium found in this work range from 4.33±0.80 to 5.31±0.28mg/kg in studied root samples.As can be observed from Table 1, the mean concentration of sodium is next to that of magnesium, except, in Dirashe.The pattern of sodium level in the samples is in the order of; Gamo Gofa-Shara > Derashe-Gato > Konso-Karat > Gamo Gofa-Lante > Derash-Gidole > Konso-Dara.One-way analysis of variance shows that the mean concentration of sodium does not show statistically significant difference among the six sample sites.

Magnesium (Mg)
As can be observed from Figure 1, in comparison to other elements, magnesium concentration is observed to be high in all areas covered in this work.Moreover, as can be seen from Table 1, its average is greater or equal to 10 mg/kg, which is far higher in content than all the metals determined in this work, in all the three study Woredas.Relatively, the concentration of magnesium is found to be higher in Derashe Gato (10.99±0.01mg/kg) and followed by other sample sites in the order of 10.78±0.00mg/kg, 10.53±0.05mg/kg, 10.43±0.13mg/kg, 10.43±0.01 and 10.12±0.22 mg/kg for Konso-Karat, Konso-Dara, Derashe-Gidole, Gamo Gofa-Shara and Gamo Gofa-Lante, respectively.Analysis of variance reveals that the mean concentration of magnesium is significantly different among the six sample sites.

Copper (Cu) and Zinc (Zn)
Of the heavy metals considered in this work, copper and zinc are to the limit of detection level of the spectroscopic technique deployed in this experiment.The level of the heavy metals copper and zinc in this study samples are in the range of 0.81±0.01-1.44±0.35mg/kg and 0.37±0.90-2.34±0.01mg/kg, respectively.One-way analysis of variance shows that the concentration of copper has shown significantly no difference among sampled sites, except Dirashe-Gato.The Derashe Gato (0.81±0.01 mg/kg) copper concentration is significantly lower than other sample sites while Konso-Karat relatively records more (1.44±0.35mg/kg) mean concentration.One-way analysis of variance shows that the concentration of zinc is significantly different among sample sites.Zinc concentration is observed to be higher than copper in Derashe and Konso-Karat.On the other

Table 1 .
Mean concentration of essential metals calculated in this work.Means with the same letter in a given column are not statistically significantly different. *

Table 2 .
Mean concentration of heavy metals calculated in this work.
*ND -Not Detected, *means with the same letters in a column are not significantly different.