Selenium deficiency in humans has been associated with various diseases, the risks of which can be reduced through dietary supplementation. Selenium accumulating plants may provide a beneficial nutrient for avoiding such illnesses. Thus, leafy vegetables such as
Selenium is a semimetallic, biologically active trace element that forms bonds with carbon whose strength is in the range of common covalent bonds. Commonly occurring in selenoamino acids and selenoproteins [
Whereas selenium is essential as trace element, it can cause acute or chronic toxicity at higher serum concentrations [
Humans and animals commonly obtain selenium from cereals, grains, and vegetables grown on seleniferous soils and from animal products such as meat, milk, fish, and eggs [
(a)
The selected leafy vegetable species were grown on selenium enriched soil (except for control purpose) at the nursery site of the Botany Department, University of Dar es Salaam, following standard procedures [
Vegetable samples were harvested at early and late stages of their growth. Early sampling was done at the age of three weeks after seed germination for
Soil samples were collected in three stages that involved random sampling using shovel. The first collection was done immediately after nursery preparation before sowing seeds and application of selenium. This was necessary in order to determine any remaining traces of selenium left in the soil as 10 g Se/ha was applied to the area three years earlier before this study. Following application of selenium, on the next day, random sampling to collect a second set of soil samples was done. The soil samples were then air-dried, packed in plastic bags, and stored in the freezer (at −4°C) until the analysis. After all vegetable samples and seeds were harvested; the third set of soil sample was collected to determine the amount of selenium left in the soil.
Analytical grade reagents and HPLC grade solvents were used as received from commercial suppliers, without further purification: nitric acid (HNO3; 70%), hydrogen peroxide (H2O2; 30%), toluene, concentrated ammonia (NH3) solution, 0.5 mol L−1 sulphuric acid, and 3,3′-diaminobenzidine hydrochloride (DABH) solution that was freshly prepared by dissolving 0.125 g 3,3′-DABH in 25 mL acetone and then stored in amber-coloured bottle. A solution of sodium selenate (Na2SeO4) was used both for soil application and as a standard. Deionized water was used for preparing reagent solutions and samples throughout.
To dry vegetable samples, a Genlab Ltd. oven was used. During sample digestions and reaction of 3,3′-DABH and selenium in the sample a microwave oven (Sonash Co.) was applied. A Shimadzu-240 UV spectrophotometer equipped with 1 cm high quartz and silica cuvettes was used for reading the absorbance of the Se-3,3′-DABH complex (piazselenol). All laboratory glassware was kept overnight in 10% nitric acid solution to remove contaminants and was rinsed with deionized water and dried in a dust-free oven ahead of use. Whatman cellulose filter papers were used for filtration of the digested samples in order to remove the remained silica.
For determination of the selenium content of extracts the method established by Katamto and Al-Zehouri was adopted [
A 1 g portion of each of the powdered dry leaves, stems, seed, and soil sample was digested with 12 mL concentrated nitric acid (70% HNO3) and 4 mL hydrogen peroxide (30% H2O2) in 100 mL conical flasks. The mixture was subjected to heat at
Cytotoxic activity of the aqueous vegetable extracts was evaluated against the MDA-MB-231 human cancer cell line as previously reported by Irungu et al. [
Comparison of the concentrations of Se accumulated in different leafy vegetable species was achieved using Kruskal-Wallis Analysis of Variance (ANOVA) and Dunn’s Multiple Comparison test. Statistical analyses for cytotoxicity assay were performed using a two-tailed Student’s
The vegetables shown in Figure
The selenium concentration of the vegetable extracts was determined by UV spectrophotometric analyses with detection of the chromogen 3,3′-diaminobenzidine hydrochloride- (DABH-) selenium complex at
Method validation: the concentration of extracts possessing known amounts of selenium determined by gravimetric analyses and by UV spectrophotometry.
Concentration |
Mean absorbance | Measured concentration ( |
Recovery (%) | Recovery (%) ± SD |
---|---|---|---|---|
1 | 0.06 | 0.93 | 93.10 | 94.56 ± 1.44 |
3 | 0.17 | 2.88 | 95.97 | |
5 | 0.27 | 4.73 | 94.60 |
All seven investigated leafy vegetable species were found to accumulate selenium; that is, they possessed higher concentrations than the controls (Table
Selenium concentration (
Vegetable species | Mean concentration ( | |||||
---|---|---|---|---|---|---|
Leaves samples | Stems samples | |||||
Early harvest | Late harvest | Control | Early harvest | Late harvest | Control | |
|
5.86 ± 0.46 | 7.01 ± 0.21 | 0.75 ± 0.16 | 3.39 ± 0.41 | 4.37 ± 0.13 | 0.56 ± 0.09 |
|
6.49 ± 0.26 | 7.90 ± 0.40 | 1.30 ± 0.27 | 2.32 ± 0.32 | 5.35 ± 0.78 | 0.56 ± 0.13 |
|
2.37 ± 0.37 | 2.81 ± 0.09 | 0.28 ± 0.06 | 1.22 ± 0.09 | 1.97 ± 0.02 | 0.19 ± 0.02 |
|
1.95 ± 0.12 | 2.21 ± 0.09 | 0.22 ± 0.07 | 1.12 ± 0.10 | 1.83 ± 0.05 | 0.16 ± 0.16 |
|
3.05 ± 0.69 | 4.48 ± 1.61 | 0.38 ± 0.02 | 2.39 ± 0.40 | 2.57 ± 0.60 | 0.37 ± 0.01 |
|
3.25 ± 0.36 | 4.11 ± 0.99 | 0.33 ± 0.02 | 2.53 ± 0.05 | 2.73 ± 0.10 | 0.14 ± 0.02 |
|
4.13 ± 0.41 | 5.56 ± 1.42 | 0.43 ± 0.02 | 3.10 ± 0.21 | 3.08 ± 0.18 | 0.45 ± 0.04 |
The stems of some leafy vegetables, such as those of
Variance analyses indicated that the increased selenium concentration of the vegetables that were grown on selenium enriched soil was statistically significant, when compared to the control samples. The differences between late and early harvests of leaves and stems were, however, not significant (Table
The one-way ANOVA for the late, the early, and the control harvests of vegetables.
Test samples |
|
Average | Variances | Differences |
---|---|---|---|---|
LLH versus CS |
|
4.86943 | 4.3871 |
|
0.52871 | 0.14622 | |||
|
||||
LSH versus CS |
|
3.12829 | 1.65798 |
|
0.34643 | 0.03463 | |||
|
||||
ELH versus CS |
|
3.871 | 2.98636 |
|
0.52871 | 0.14622 | |||
|
||||
ESH versus CS |
|
2.29643 | 0.74049 |
|
0.34643 | 0.03463 | |||
|
||||
LLH versus ELH | 0.34985 | 4.86943 | 4.3871 | ns |
3.871 | 2.98636 | |||
|
||||
LSH versus ESH | 0.18075 | 3.12829 | 1.65798 | ns |
2.29643 | 0.74049 |
ELH: early leaf harvest, LLH: late leaf harvest, ESH: early stem harvest, LSH: late stem harvest, CS: control samples,
Statistical analyses (Supporting Information in Supplementary Material available online at
During the growth stage, selenium is usually found in the leaves whilst during the reproductive stage it is accumulated in the seeds [
Selenium concentrations observed for
Vegetable species | Concentration |
Mean concentration, ( | ||
---|---|---|---|---|
|
0.21 | 0.24 | 0.24 | 0.23 ± 0.02 |
|
1.32 | 1.21 | 1.27 | 1.26 ± 0.06 |
|
0.31 | 0.35 | 0.43 | 0.36 ± 0.06 |
|
2.07 | 2.31 | 2.43 | 2.27 ± 0.18 |
Soils ability to retain minerals may allow a single selenium enrichment to provide soil for functional foods for more than a single season and is thus of economic importance for farmers. Selenium concentrations of the soil prior to and following the enrichment, that is, immediately after the harvest, were measured, the results being summarized in Table
Selenium concentration of soil samples.
Soil samples | Concentration |
Mean concentration, ( | ||
---|---|---|---|---|
BSA | 0.43 | 0.40 | 0.47 | 0.43 ± 0.03 |
ASA | 18.43 | 29.43 | 22.78 | 23.55 ± 5.54 |
AVH | 0.88 | 0.93 | 1.35 | 1.05 ± 0.26 |
BSA: before selenium application, ASA: after selenium application, and AVH: after vegetable harvest.
The accumulated selenium concentrations of the investigated vegetable samples ranged from
To determine the anticancer potential of the selenium enriched vegetables, the aqueous extracts were studied for their ability to inhibit the growth of the human MDA-MB-231 cancer cell line (Table
Cytotoxicity evaluation of the water extracts against the human MDA-MB-231 cell line.
Vegetable leaves extract | Concentration | |||||
---|---|---|---|---|---|---|
100 |
<100 | |||||
A | B | C | A | B | C | |
SV | + | + | + | − | − | + |
SS | + | + | + | − | − | − |
IB | − | − | − | − | − | − |
AH | − | − | − | − | − | − |
AS | − | − | − | − | − | − |
CM | − | − | − | − | − | − |
VU | − | − | − | − | − | − |
SV:
Seven vegetable species were identified as selenium accumulators with their selenium content being within the WHO-determined safe, nontoxic concentration, <1400
The authors declare that there is no conflict of interests regarding the publication of this paper.
The authors amply appreciate the financial auspices from Dar es Salaam University College of Education (DUCE) through Science and Technology Higher Education Project (STHEP), SIDA/Sarec-UDSM Core-Support Funding Project (2011), and the Swedish Research Council/SIDA (Project no. 2012-6074).