Effect of Nitrogen Fertilizer on the Levels of Some Nutrients, Anti-nutrients and Toxic Substances in Hibiscus sabdariffa
Emmanuel O. Ogbadoyi
The presence of antinutrients and toxic substances in vegetables limits the derivable benefits from vegetables. The levels of these substances in vegetables are influenced by the nature of soil in which the vegetables are grown. The effect of applied nitrogen fertilizer on the levels of some antinutrients and toxic substances is investigated with a view to determine the appropriateness or otherwise of the application of nitrogen fertilizer in growing vegetables. Pot experiments were conducted to determine the effect of soil nitrogen levels on soluble and total oxalates, cyanide, nitrate and some micronutrients namely, vitamin C, β-carotene (precursor of vitamin A) and mineral elements (Fe, Mg, Zn, Cu, Ca, Na and K) in Hibiscus sabdariffa. The leaves of the vegetable were harvested and analysed at market maturity (vegetative phase) and fruiting (reproductive phase) of the plant development. Results obtained showed that the applied nitrogen fertilizer significantly elevated nitrate and β-carotene contents, while it decreases the levels of vitamin C, soluble and total oxalates in the vegetable. The levels of cyanide and mineral elements were not significantly affected by the applied nitrogen fertilizer.
Received: November 05, 2011;
Accepted: February 08, 2012;
Published: February 23, 2012
Vegetables are rich sources of micronutirents, vitamins and minerals with leafy
vegetables being the most important source of minerals (Lola,
2009). Roselle (Hibiscus sabdariffa) popularly called Yakuwa in Hausa
belongs to the family of Malvaceae and is a popular vegetable in Indonesia,
India, West Africa and many tropical regions (Fbabatunde,
2003; Tindal, 1986). The vegetable is widely grown
in the North-eastern and middle belt regions of Nigeria (Akanya
et al., 1997). This plant has been found to thrive on a wide range
of soil conditions. It can perform satisfactorily on relatively infertile soils
but for economic purposes, a soil well supplied with organic materials and essential
nutrients is important in the productions (Adanlawo and
Ajibade, 2006; Tindal, 1986). It can tolerate relatively
high temperature throughout the growing and fruiting periods. The plant requires
an optimum rainfall of approximately 45-50 cm distributed over a 90-120 day
growing period (Tindal, 1986). In Nigeria, two botanical
varieties are recognised, the red variety in which the calyx is used for the
preparation of sobo drink and the green variety which calyx and leaves are used
in stew and sauces (Adanlawo and Ajibade, 2006; Duke,
1985; Ojokoh, 2006). The leaves and calyx of the
green variety are very rich in β-carotene, vitamin C and riboflavin with
some major mineral elements (Babalola, 2000). Apart
from the nutritional values, the plant is also of therapeutic value as extract
of the calyx is used in the treatment of cardiovascular diseases and hypertension
(Abo-Baker and Mostafa, 2011). Roselle like other leafy
vegetables also contains appreciable levels of some antinutrients and toxic
substance which have been shown to have negative effects on animal and human
health at high concentrations (Ojokoh et al., 2002;
Morton, 1987). For instance oxalate and phytate chelate
minerals form complexes with proteins and thereby affects their nutrive value
(Evans and Bandemer, 1967). Oxalate in combination with
calcium form calcium oxalate which form precipitate in the kidney to form kidney
stone (Aletor and Omodara, 1994; Osagie,
1998; Prohp et al., 2006). Higher levels of
nitrate are responsible for methaemoglobineamia and cancer formation in human
(Anjana et al., 2007). Cyanogenic glucosides
also found in the vegetable are inhibitors of cytochrome oxidase enzyme and
thereby acts as respiratory poison (Aletor, 1993).
The amount of nutrients, antinutrients and toxic substances in the vegetable
beside other environmental factors, to a great extent depend on the soil nutrients
content. Nitrogen and phosphorus are major limiting nutrients for plant growth
and this explains the improvement in plant yield by external supply of these
elements to soils deficient in them (Tena and Beyene, 2011).
Thus this research was designed to study the effect of soil nitrogen levels
as it affects the bioaccumulation of some nutrients (β-carotene, vitamin
C, Fe, Cu, Mg, Zn, Ca, Na and K), antinutrients (soluble and total oxalates)
and toxic substances (cyanide and nitrate) in H. sabdariffa.
MATERIALS AND METHODS
The study area: The pot experiment was carried out between 6th June
and 18th December 2005 in the nursery of the School of Agriculture and Agricultural
Technology, Federal University of Technology, Minna, Niger State of Nigeria.
Niger state has a Savanna climate characterised by maritime air and rainfall
is between April and October. During harmattan, dry desert wind blows between
November and mid February while night temperature is very low. The geographical
location of Minna is longitude 90°40 N and latitude 6° 30
E. Minna lies in the Southern Guinea Savanna zone of Nigeria and has a sub-humid
semi arid tropical climate with mean annual precipitation of 1200 and 1300 mm.
About 90% of total annual rainfall occurs between the months of June and September.
Temperature rarely falls below 22°C with peaks of 40°C and 30°C
in February/March and November/December, respectively. Wet season temperature
average is about 29°C (Osunde and Alkassoum, 1998).
Soil sampling and analysis: The soil used in this study was collected
from Minna. The soil has been classified as Inseptisol (FDALR,
1985). The bulked sample was collected during the drying season from the
field which has been under fallows for about four years. The bulked soil sample
was passed through 2 mm sieve. Sub-sample of the soil was subjected to routine
soil analysis using the procedure described by Juo (1979).
The soil particle sizes were analyzed using hydrometer method, pH was determined
potentiometrically in water and 0.01 M CaCl2 solution in a 1: 2
soil/liquid using a glass electrode pH meter and organic carbon by Walkey-Black
method. Exchange acidity (E.A H+ and Al3+) was determined
by titration method.
|| Some physical and chemical properties of the soil (0-20 cm)
used for pot experiment
|Values represent means of triplicate determinations
Exchangeable Ca, Mg, K and Na were leached from the soil sample with neutral
1 N NH4OA solution. Sodium and potassium were determined by flame
emission spectrophotometry while Mg and Ca were determined by EDTA versenate
titration method. Total nitrogen was estimated by Macro-Kjedal procedure and
available phosphorus by Bray No. 1 method. The results of soil analyses are
presented in Table 1.
Seeds: The seeds of roselle (Hibiscus sabdariffa) were obtained from School of Agriculture and Agricultural Technologys Farm/Nursery of Federal University of Technology, Minna.
Planting, experimental design and nursery management: About ten seeds of roselle were planted in a polythene bag filled with 10 kg of top soil. Following emergence, the seedlings were thinned to two plants per pot. The Complete Randomised Design (CRD) was adopted, using two treatments namely; two levels of soil fertility. Each treatment had 10 pots replicated three times. This gave a total of 60 pots. The seedlings were watered twice daily (mornings and evenings) using watering can and weeded regularly. The experimental area and the surroundings were kept clean to prevent harbouring of pest. The pots were lifted from time to time to prevent the roots of the plants from growing out of the container. Insects were controlled using Sherpa plus four weeks after planting at the rate of 100 mL per 100 L of water.
Fertilizer treatment: The fertilizer treatment for the vegetable was
N fertilization at two levels. The first level was control (no N fertilization)
and the second level was N fertilization at the recommended dose specific for
the vegetable. Basfal application of P2O2 and K2O
in form of single super phosphate and muriate of potash were applied at recommended
rate, respectively. The details of the fertilizer treatments are as follow:
F1 (control): 0 N, 40 mg P2O5 kg-1
soil and 40 mg K2O kg-1 soil
F2: 40 mg N kg-1 soil, 40 mg P2O5
kg-1 soil and 40 mg K2O kg-1 soil
Plant tissues analysis: Both soluble and total oxalates content in the
vegetable leaves were determined by titrimetric method of Oke
(1966). The nitrate content in the test samples was determined by the colourimetric
method as decribed by Sjoberg and Alanko (1994). Alkaline
picrate method of Ikediobi et al. (1980) was
used to analyse the cyanide content in the test samples. The mineral elements
(Fe, Cu, Mg, Na and K) in samples were determined according to the method of
Ezeonu et al. (2002) while ascorbic acid content
in the samples was determined by 2, 6-dichlorophenol indophenols method of Jones
and Hughes (1983). β-carotene content on the other hand was determined
by ethanol and petroleum ether extraction method as described briefly. Two gram
of Na2SO4 was added to 10.0 g of vegetable leaves and
ground in mortar. The ground vegetables were extracted with 100 cm3
of hot 95% ethanol for 30 min in hot water bath. The extract obtained was filtered
and measured. Water was added to the extract to bring the percentage of the
ethanol extract to 85%. The 85% ethanol extract was cooled in a cold water bath
for some minutes. After cooling, the ethanol extract was put inside separating
funnel and 30 cm3 of petroleum ether was added and the mixture shaken.
The separating funnel was clamped to the retort stand for some time to allow
the solution to settle down into layers. The bottom layer containing ethanol
was collected into the beaker while the top layer of the petroleum ether was
stored in 250 cm3 conical flask. The ethanol layer in the beaker
was re-extracted twice with 10 cm3 of petroleum ether. The ether
layers of re-extraction was added to the original petroleum extract in the conical
flask and re-extracted with 50 cm3 of 85% ethanol in order to remove
any xanthophylls which may be present. The top petroleum ether layer which contained
β-carotene was collected, measured and the volume noted.
Lastly, the optical density (OD) of the final petroleum ether extract was determined at the wave length of 450 nm with spectrophotometer using petroleum ether as blank.
The concentration of β-carotene was calculated thus:
||Absorbance of the sample
|| Extinction coefficient of β-carotene
|| Path length (usually 1.0 cm)
Statistical analysis: T-test was used to determine the effect of soil
fertility using two levels of nitrogen fertilizer on the level of the parameters
Physical and chemical properties of soil: Result of analyses of the
soil used for pot experiment is presented in Table 1. The
texture class of the soil is sandy loam indicating that the water holding capacity
is moderate. The organic matter content, total nitrogen and available phosphorus
are low. Sodium and calcium contents are moderate while magnesium and potassium
contents are high. The CEC (Cation Exchange Capacity) is moderate while base
saturation percentage is high. Soil pH indicates that the soil is slightly acidic
(FAO, 1984; Black, 1985; FDALR,
Effect of soil nitrogen levels on antinutrients and vitamins content:
The determination of the effects of soil nitrogen levels on cyanide concentrations
in Hibiscus sabdariffa showed that the applied nitrogen fertilizer had
no significant effect on cyanide content of the studied vegetable irrespective
of the stage of plant development. The mean values for controls at market maturity
(459.60±21.00 mg kg-1) and fruiting (390.20±32.00 mg
kg-1) were not significantly different from the values (419.50±21.00
and 410.60±26.00 mg kg-1, respectively) for vegetables grown
on nitrogen fertilized soil (Table 2).
The mean nitrate contents in the vegetable grown on nitrogen treated soils at market maturity (101.90±26.00 mg kg-1) and (344.40±29.00 mg kg-1) were significantly higher when compared to the level of controls (85.00±28.00 and 285.20±23.00 mg kg-1, respectively) as shown in Table 2.
The soluble oxalate concentrations of control and nitrogen fertilized Hibiscus sabdariffa at market maturity were 1.62±0.05 g/100 g and 1.37±0.05 g/100 g while at fruiting the values obtained were 2.93±0.15 g/100 g and 1.77±0.07 g/100 g. The results showed that the application of nitrogen fertilizer significantly (p<0.05) decreased the soluble oxalate content of the vegetable irrespective of stage of plant development (Table 2). Similarly the applied nitrogen fertilizer significantly (p<0.05) decreased the total oxalate content of the vegetable in both stages of plant development. The mean values of the antinutrient recorded in the control and test Hibiscus sabdariffa at market maturity were 2.08±0.07 g/100 g and 1.92±0.04 g kg-1 while the corresponding values recorded at fruiting were 4.04±0.27 g/100 g and 3.22±0.20 g/100 g, respectively (Table 2).
The mean β-carotene contents of control and test plants at market maturity were 5405.00±433.00 and 7067.00±266.00 μg/100 g while at fruiting the values obtained were 6123.00±379.00 and 6481.00±406.00 μg/100 g. Data analysis showed that with application of nitrogen fertilizer there is a significant elevation (p<0.05) in the provitamin content at market maturity while no significant variation was recorded at fruiting (Table 2).
The determination o f vitamin C content in Hibiscus sabdariffa showed that the applied nitrogen fertilizer significantly decreased the vitamin content at both stages of plant development. The mean values recorded in the test and control vegetable at market maturity were 12.51±1.10 mg/100 and 15.39±1.30 mg kg-1 while at fruiting the values recorded were 13.08±0.77 mg/100 g and 16.08±0.82 mg/100 g, respectively (Table 2).
|| Effect of soil nitrogen levels on antinutrients and vitamins
content in Hibiscus sabdariffa
|DW = Dry weight, FW = Fresh weight. Values represent means
of nine determinations. Mean values carrying the same superscripts within
a row do not differ significantly from each other (p>0.05)
|| Effect of soil nitrogen levels on mineral content in Hibiscus
|Values represent means of tripple determinations. Mean values
carrying the same superscripts within a row do not differ significantly
from each other (p>0.05)
Effect of soil nitrogen levels on mineral elements content: Analysis of Fe Mg, Zn, Cu, Ca, Na and K were conducted in control and in Hibiscus sabdariffa grown on soil treated with nitrogen with a view of determining the effect soil nitrogen levels on the mineral contents of the vegetable. The results obtained showed that the applied nitrogen fertilizer had no significant effect on the mineral contents of the vegetable irrespective of the stage of plant development (Table 3).
The insignificant effect of nitrogen fertilizer on cyanide and nitrate content
in Hibiscus sabdariffa is at variance with the report of Richard
(1991), Chweya (1993), Peter
and Birger (2002), Mozolewski and Smoczynski (2004),
Anjana et al. (2007), Boroujerdnia
et al. (2007) and Wobeto et al. (2007).
These authors reported that application of nitrogen fertilizer to the soil elevated
the nitrate and cyanide content of the plants that were grown on them. Worthington
(2001) stressed that plants require nitrogen for normal growth and for the
synthesis of proteins. Nitrogen is one of the major elements that are essential
for plant growth and development (Mostafa and Abo-Baker,
2010; Suriharn et al., 2011; Undie
et al., 2012). However, if nitrogen is applied in excess of what
the plant requires for protein production, the excess is accumulated as nitrates
and stored predominantly in the green leafy part of the plant. Thus the insignificant
difference in the nitrate content recorded between nitrogen applied Hibiscus
sabdariffa and the control, may infer that, the amount of nitrogen supplied
to the vegetable is adequate for optimum utilization for normal growth and protein
formation or that the control has enough nitrogen for normal growth and protein
formation for the particular species.
Similarly, Peter and Birger (2002) further stated that
increase in cyanide content following nitrogen fertilization is because the
applied nitrogen stimulates the enzymatic conversion of tyrosine to p-hydroxymandelonitrile
which ultimately lead to increase in the biosynthesis of cyanogenic glycoside.
It therefore, follows that the variation of this result from the findings of
these authors may suggest that the bioaccumulation of cyanide in response to
the applied nitrogen varies from one cultivar to another.
Significant higher β-carotene content obtained in response to the applied
nitrogen fertilizer than in controls in the vegetable is in line with the observations
of Chweya (1993) and Kansal et
al. (2005) that nitrogen fertilizer increased the provitamin content
of the vegetable. The reason for this increase may be probably due to elevation
in the content and activity of chlorophyll and associated light absorbing pigments
(including carotenoids) following the application of nitrogen fertilizer (Taiz
and Zeiger, 2002; Havling et al., 2006).
The generally lower levels of vitamin C in the vegetables, soluble and total
oxalates in Hibiscus sabdariffa grown in soil supplied with nitrogen
fertilizer compared with the control indicate that the applied nitrogen fertilizer
significantly reduced the levels of these compounds in the vegetables (Chweya,
1993; Worthington, 2001; Mozafar,
2005; Singh, 2005). Worthington
(2001) further stressed that the observed decrease in vitamin C content
resulted from the increase in protein production and decrease in carbohydrate
production following the application of nitrogen fertilizer. Because vitamin
C is formed from carbohydrates, its synthesis is also reduced. Singh(2005)
stated that not only nitrogen fertilizers decrease the oxalate content of the
vegetables but anions generally reduce the levels of the antinutrients since
they compete with oxalate for cations and depress the oxalate synthesis. The
decrease in oxalate concentration in the vegetables following nitrogen fertilization
may be attributed to decreasing effect of nitrogen on vitamin C content, since
oxalates are synthesised via vitamin C (Streeter, 2005.).
This present results also revealed that nitrogen fertilizer had no significant
effect on the studied mineral elements (Fe, Mg, Zn, Ca, Cu, Na and K). It may
be possible to increase the levels of these minerals in the vegetable through
integrated nutrient management using appropriate combinations of organic and
inorganic fertilizers as this is known to increase nutrient yield (Abd
El-Lattief, 2011; Singh et al., 2011).
Even though nitrate and β-carotene contents in the vegetable are elevated with nitrogen fertilizer, the nitrate contents in the test vegetable are still within the tolerable levels. The β-carotene contents in the controls are high enough to meet the adult recommended daily allowance of 900 μg of vitamin A. The level of vitamin C decreased significantly with nitrogen fertilizer, however, the vitamin content in both control and nitrogen treated vegetable are still lower than adult recommended dietary allowance of 60 mg. Thus in either case, supplementation of the vitamin from other sources is required. However, the significantly higher soluble and total oxalates content in controls compared with nitrogen treated vegetable may encourage the application of this recommended nitrogen fertilizer when growing Hibiscus sabdariffa on this soil type. This practice will reduce oxalate toxicosis associated with high level of oxalates in the food.
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