Download impact of selenium fortification in fenugreek

Indo American Journal of Pharmaceutical Research, 2015
ISSN NO: 2231-6876
IMPACT OF SELENIUM FORTIFICATION IN FENUGREEK (TRIGONELLA FOENUMGRAECUM)
D. Nancy, P. Thribuvana& P. Indra Arulselvi*
Plant and Microbial Biotechnology Lab, Department of Biotechnology, Periyar University, Salem, Tamil Nadu.
ARTICLE INFO
Article history
Received 06/01/2015
Available online
28/02/2015
Keywords
Fenugreek,
Sodium Selenate,
Human Nutrition,
Phenolic Compounds.
ABSTRACT
Two-third of the world’s population lack essential mineral elements at all stages of life.
Supplementation of these micronutrients during crop cultivation serves to be the ultimate goal
of modern agriculture. Among the micronutrients, Selenium (Se) has been proved to be an
essential element and has received considerable attention for its antioxidant activity,
anticancer effect and other physiological functions. The Recommended Dietary Allowance
(RDA) of selenium is 55- 200 µg/day for an adult. Agronomic fortification by foliar
application was carried out to investigate the effects of selenium on growth, selenium
accumulation, photosynthetic pigments, antioxidant activity and the micronutrients variations.
Plants were grown in greenhouse condition amended with sodium selenate. Selenium
enrichment enhanced the growth parameters and abridged the levels of chlorophyll at higher
concentrations. The current results suggested the beneficial effects of Se on fenugreek growth
and also its contribution to improve the nutritional value of fenugreek for human nutrition. To
our best knowledge, this was the first report on selenium supplementation in fenugreek plant,
which contains 29µg g-1DW; indicates intake of ~10-20 leaves /day will drive to maintain the
regular selenium level. The biochemical characterization proved the nutritional quality
enhancement by its increase in total phenolic and total protein contents. It indirectly indicates
the increase in antioxidant enzyme production. Thus, selenium fortified fenugreek leaves can
serve as a better nutraceutical food to improve the overall human health. In wrapping up,
further studies are needed to assess the functionality of high-Se that measures changes in
genome stability and immunocompetence.
Copy right © 2015 This is an Open Access article distributed under the terms of the Indo American journal of Pharmaceutical
Research, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
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Please cite this article in press as D. Nancy et al. Impact of selenium fortification in fenugreek (Trigonella foenum-graecum. Indo
American Journal of Pharm Research.2015:5(02).
635
Corresponding author
Dr. P. Indra Arulselvi
Assistant Professor, Department of Biotechnology,
Periyar University,
Salem- 636 011, Tamil Nadu, India.
[email protected]
Vol 5, Issue 02, 2015.
D. Nancy et al.
ISSN NO: 2231-6876
INTRODUCTION
The mankind needs nutritious food to stay healthy and to maintain life. More and more nutritional studies linked today’s most
prevalent, life threatening chronic diseases. The simple truth may be that susceptibility to disease is linked to either toxicity or
nutritional deficiency. Increasingly, scientific research has shown that the secret to life-long health is good nutrition. Much of our
agricultural soils have been exhausted of the minerals and organic material needed to grow nutritious food. Soils depleted of critical
minerals cannot grow healthy, nutrient rich crops. Crops require minerals and organic materials to change nutrients into forms that
plants can use for growth. Additional inputs, in the form of mineral nutrients and enhanced organic matter are needed to produce foods
with high nutritional values. Moreover, majority of research has reported numerous beneficial effects of supplemental Se on plant
growth and performance [1- 3]. However, Se is not a very abundant element in soil nor in greenhouse production, most plants being
cultivated using soilless substrates where Se availability is limited [4].On the other hand, fortification of plants with selenium could be
an effective way of producing selenium rich foodstuffs and thereby increase health benefits [5].
Among various micronutrients, selenium (Se) had been selected, for its essentiality for both people and plants. Increasing
scientific evidence indicates that selenium has many beneficial effects for human beings and many other forms of life [6].There is
evidence that less overt Se deficiency can affect human health in number of ways, such as immune function, viral infection,
reproduction (especially male fertility), thyroid function, asthma and inflammatory conditions [7].Selenium also has its role in the
prevention of cardiovascular disease [8].It is a constituent of selenoproteins, many of which have important functions, including
antioxidant protection, energy metabolism and redox regulation during transcription and gene expression [9].A weakened immune
system leads to many ailments and diseases. Selenium being a trace element has significant role in maintenance of regular immune
cells functioning and activation. Thus, selenium fortification will ensure the health improvement of the humans.
Higher plants are thought not to require Se and to have a low tolerance to it, but there are increasing indications that Se may
also have beneficial biological functions in higher plants. Selenium was shown to affect several physiological and biochemical
processes in plant species [10].In plants, Se may serve a role in antioxidative mechanisms and it can exert beneficial effects on plants at
low concentrations. Trace amounts of Se stimulated growth in a variety of plants e.g. tobacco, lettuce as well as potato [1-3, 11].
Spraying leaves with Se, increased seed yield in soybean due to a better partitioning efficiency, as evidenced by a greater number of
pods per plant, seeds per pod and seed weight [12]. Similarly, selenium supplementation to plants enhances the production and quality
of edible plant products, by increasing antioxidant activity of the plants, as shown in tea leaves and in rice [13].Se treatment has been
reported to improve growth in plants subjected to various abiotic stresses such as UV, salt and drought [14, 15].
Fenugreek as chemurgical crops widely used for industrial purpose and are widely cultivated as a drug plant. For medical
purpose the seeds were used to cure mouth ulcer, stomach irritation, gastrointestinal problems, diabetes management, fever, sore
throats and cancer treatment. Biochemical characterization on selenium fortified plants will indicate the metabolic activities of the
plant and its nutritional profile. The fortification methods will also play a crucial role for enhanced selenium accumulation. Hence,
determination of better method will result in maximum selenium accumulation with optimal concentration in fenugreek plants. The
present work includes analysis of optimum level of selenium for this fenugreek plants to be concluded with the selected concentration
of sodium selenate based on the biochemical and antioxidant activity analysis. Biochemical characterization on selenium fortified
plants will indicate the metabolic activities of the plant and its nutritional profile. Accumulation of organic forms majorly is strongly
suggestive of nutraceutical and dietary applications, to improve the human nutrition. The form of selenocompounds accumulated plays
an important role for its usage. Accumulation of organic forms majorly is strongly suggestive of nutraceutical and dietary applications,
to improve the human nutrition.
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Total selenium content and ionome variation
The total selenium content in leaf and root were determined in oven dried ground samples after digestion with nitric
and perchloric acids and reduction by hydrochloric acid. The wet digested sample was evaporated to dryness at 135-180ºC. Then, the
residue was dissolved in distilled water, boiled and filtered through No.42 whatmann filter paper [18].The digests were analyzed along
with the blank by hydride generation atomic absorption spectrophotometry (Department of Marine Science, Bharathidasan University,
Tiruchirappalli, Tamil Nadu).
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MATERIALS AND METHODS
Fortification and treatment conditions
To identify the level of selenium assimilation in the fenugreek plants; supplementation was done with sodium selenate in the
greenhouse condition without chemical fertilization in the Periyar University Campus, Salem, Tamil Nadu. Soil Nature was tested in
Tamil Nadu Government Agricultural Department, Chemistry Division in Soil Testing Laboratory, Salem. The soil pH was 7.9 with
146, 71 and 20kgh-1 of nitrogen, Potassium and Phosphorus respectively. There was no evidence of Calcium carbonate and EC value
was 0.9 d/sm. The chemicals and reagents used in this study were of analytical grade.
The fenugreek seeds were physically analyzed and the disease free, good condition seeds were selected for this fortification
protocols. The diseased and damaged seeds were removed. The temperature was 25-30ºC during the daytime and 18-24ºC during the
night time. Throughout the growing cycle, the fenugreek plants received different doses of sodium selenate (20, 40 and 50µgml -1) and
control plants were maintained without sodium selenate supplementation. The supplementation was given as foliar application at
regular intervals. Then the plantlets were uprooted for the physical parameter (Relative water content, index of tolerance, leaves
number and secondary roots number) analysis and the values were recorded, to check the changes in growth conditions due to
selenium assimilation in the plantlets under study[16, 17].
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Pigment content
The pigment (chlorophyll and carotenoid) analysis tends to depict the accumulation of selenium which is directly correlated
with the chlorophyll content. Fresh leaf tissue (100mg) was homogenized with ice cold 80% (v/v) acetone in a pre-chilled mortar and
pestle. The extract was centrifuged till the clear supernatant appears and analyzed at 665 and 640 nm spectrophotometrically for
chlorophyll a and b respectively and carotenoid at 470 nm. The result was expressed in mg g -1 of fresh weight [19].
Antioxidant activities
PAL activity was measured in 300mg of fresh leaves with 50mM Tris HCl buffer (pH=8) and determined based on the rate of
cinnamic acid production. Ethyl acetate was used as the extracting solvent and solid residue was suspended in 0.05M NaOH. The
absorbance was read at 280nm (UV) and the reduction in the enzyme activity depicts the selenium assimilation [20].
Total antioxidant activity was estimated with the sample extract and the reagent solution (0.6M H2SO4, 28mM
sodium phosphate and 4mM ammonium molybdate). The reaction mixture was incubated at 95ºC for 90 min. After cooling to room
temperature, the absorbance was read at 695nm spectrometrically against methanol as blank with ascorbic acid as standard [21].
Nitrate and Total Phenolic Content (TPC)
The oven- dried ground leaf material500 mg fresh plant materials was used to determine the nitrate content by salicylic acid
method with 96% sulphuric acid and 1.5molL-1 NaOH was slowly added to the tubes. Yellow color appears when the samples were
cooled down to room temperature & absorbance was read spectrophotometrically at 410nm (Note: If the leaf material used, add 0.5g
MgCO3/CaOH2 to the supernatant, to remove the chlorophyll) [22].
Total phenolic content was determined by the folin-ciocalteau based method, using Gallic acid (GA) as a standard for the
calibration curve (10- 100 µgml-1). One ml of the diluted extract (0.1 g in 5 ml distilled water) was oxidized with 0.1 ml of folinciocalteau reagent (0.5 N). The mixture was neutralized with 2.5 ml 10% saturated sodium carbonate after 15 min incubation at room
temperature and incubated at room temperature for 30 min; the blue color formed was read spectrophotometrically at 760 nm [23].
RESULT AND DISCUSSION
The influence of diet in health is an active area of research and substantial evidences indicates that they can influence
physiological processes in humans. Cultivated plants generally are a better source of dietary selenium. Biofortification is the remedy,
which is a process by which the necessary daily micronutrients are delivered directly to staple crops to increase its nutritional value.
As a consequence, fortified foods are of increasing interest in the prevention and treatment of various diseases. Plants are versatile
biochemical factories, capable of synthesizing nearly full complement of essential micronutrients. The similarity of Se-aminoacids to
their S-analogues of cysteine and methionine can disrupt the normal biochemical reactions and enzyme functions within the cell [5].
The data presented here provides new insight into selenium fortification in non-accumulator plants. Interest in Se fortification
was escalated due to its nutrition and health benefits. The fenugreek plants were supplemented with sodium selenate by foliar
application with20, 40 and 50 µg ml-1 concentrations in the greenhouse condition. The fresh weight had been enhanced. The shoot
length was improved upto 37%; whilst the secondary root number had been increased to 10, 22 and 46% in final concentration (Table
1). Fortification of fenugreek plant depicts that, low selenium concentration had increased the plants’ tolerance towards abiotic stress
conditions and subsoil salinity. The increase in secondary root number and restricted root elongation might be caused by the damage
of the root plasma membrane during selenium penetration into the root cells and due to the disturbance of mineral balance of the plant.
Table 1: Growth parameters in Selenium fortified Fenugreek plants
Shoot Length
(Mean ± SE)
Root Length
(Mean ± SE)
Number of leaves
(Mean ± SE)
C
T1
T2
T3
12.8±1.33
15.2±1.02
16.4±0.75
16.0±0.80
6.4±1.02
6.8±1.17
4.8±0.75
4.6±0.75
8.50±0.50
8.75±0.45
8.20±0.70
8.00±1.12
Number of
2º roots
(Mean ± SE)
15.75±0.83
16.25±1.30
22.50±2.69
28.00±3.64
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The amount of selenium assimilated was 46.97, 53.98 and 58.80% higher in leaves respective to increasing concentrations of
sodium selenate. The maximum amount of selenium accumulation in roots and leaves by foliar application method, were 16.50 and
24.82 µg g-1 in 40 µg ml-1concentration (Figure 1).The leaves turned yellow at the final growth stage in the final concentration.
Skinner reported that water lettuce cultivated in the solution containing 60 to 80mg Se L -1 exhibited the yellow leaves and died in day
2 and 3, the appropriate concentration (20 and 40 mg SeL-1)contained 11.14-13.50 and 21.06-29.55mg Se kg-1, respectively [24]. But, a
contrary result was observed by Fargosova in chicory plants. In which, smaller increase of selenium content in root was observed, 73
and 46 mg kg- 1in Sinapis alba L [25].
637
Treatment
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Figure 1: Selenium Content in Selenium fortified Fenugreek plant.
The greater leaf surface area contributed to a relatively higher respiration rate and increased movement of selenium to the
transpiring leaves of plants [26].The index of tolerance was reduced with increasing concentration and Cu, Fe and Zn content
sequentially. The magnesium content decreased at lower concentration and increased at higher concentration of sodium selenate (50
µg ml-1).In assessment of micronutrient concentration (ionome variation), no alteration depicts that all the metabolic pathways are
normal, with regular transporters. The suitable plants for selenium Fortification should contain high protein, absorb and accumulate
selenium markedly and convert effectively inorganic selenium to organic selenium.
The chlorophyll content was comparatively reduced in treatment, when compared with the control plants. Carotenoid content
was 0.596 mg g-1of FW in 20 µg ml-1concentration (Figure 2). The decrease in chlorophyll content with sodium selenate application is
in agreement with the positive effects of Se treatment in delaying the loss of chlorophyll [27, 21]. An increase in PAL activity leads to an
accumulation of soluble phenolic compounds, which are sequestered in vacuoles, but the increase also generates browning reactions
when membrane disruption occurs, that occurs due to oxidative reactions. The total antioxidant activity gradually increased in
concordant with the growth of the plant and the sodium selenate concentration. The maximum activity was recorded in highest (50 µg
g-1) concentration of sodium selenate. The nitrate content had not much variation among the treated plants, when compared with the
control plants. The increase in nitrate content on Se fortification might be due to the mineral balance of the plants.
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Total phenolic content increased gradually in respect to increasing concentration of sodium selenate and was higher at of 40
days growth stage and reduced later. The TPC estimation in selenium fortified fenugreek plants was concordant with the previous
reports in selenium fortified spinach (increased 66%) tea [28, 2 and 29].Increase in total phenolic content on selenium fortification
represents the enhanced nutritional value. It is in agreement to previous reports on Se enriched potato sweet basil and tomato [30, 14 and
31]
. Among the phytochemicals, the phenolic compounds act on the enzymes such as oxidase, xanthine and thereby prevents the
enzymatic and non- enzymatic formation of ROS (Reactive Oxygen Species), thus enhanced its production [32].
However, effect of selenium on phyto-hormones balance and/or polyamine content could not be excluded. High selenium
levels may inhibit photosynthesis, impair nutrient uptake and transport [33]. Therefore, further study should focus on factors affecting
selenium absorption and accumulation such as levels and source of selenium, age of the plant and the efficiency of conversion of
638
Figure 2: Biochemical analysis in Selenium fortified Fenugreek plant.
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ISSN NO: 2231-6876
inorganic selenium to organic selenium. The recommended dietary allowance (RDA) for humans is 55- 200 µg/day for an adult.
Hence, this selenium supplemented fenugreek leaves contains 29 µg of selenium per gram of leaves; intake of ~10-20 leaves/day is
adequate for the regular immune system functioning for a healthy life.
CONCLUSION
From the biochemical characterization of the selenium fortified fenugreek; the higher selenium accumulation was reported in
foliar spray method at the 40 µg ml -1concentration studied. Among all the results; the total phenolic, total protein, nitrate contents and
total antioxidant activity increased in concordant with increasing concentrations. The selenium accumulation was supported by
reduced chlorophyll contents, based on its site of accumulation. It proved the health status of the fortified plant. Further research on
molecular level, specific to genes involved in enhancing organic selenoforms accumulation, with analysis of distribution and stability
of selenocompounds will shade a new light on selenium fortification in fenugreek.
ACKNOWLEDGEMENT
We gratefully acknowledge the Periyar University, Salem, Tamil Nadu for the support rendered through the University
Research Fellowship. We express our gratitude to the institutional incharges for providing space and the lab mates, for their immense
help in this research work.
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Author’s statements
Competing interest
The authors declare no conflict of interest.
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