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Comparative Effect of Arbuscular Mycorrhizal
Fungi and Biostimulants on the Antioxidant and
Nutritional Potential of Moringa oleifera
Fowe Michelle Carole Djouhou1,2*, Dieudonné Nwaga2 and Elie Fokou1
1Department of Biochemistry, Laboratory for Food Science and Metabolism, University of Yaoundé I, Cameroon
2Department of Microbiology, Laboratory of Soil Microbiology, Biotechnology Centre, University of Yaoundé I, Cameroon
Submission: April 24, 2019; Published: August 13, 2019
*Corresponding author: Fowe Michelle Carole Djouhou, Department of Biochemistry, Laboratory for Food Science and Metabolism, University of Yaoundé I, Cameroon
How to cite this article: Djouhou Fowe Michelle Carole, Nwaga Dieudonné, Fokou Elie. Comparative Effect of Arbuscular Mycorrhizal
Fungi and Biostimulants on the Antioxidant and
Nutritional Potential of Moringa oleifera. Nutri Food Sci Int J. 2019. 9(2): 555758. DOI:10.19080/NFSIJ.2019.09.555758.
The influence of arbuscular mycorrhizal fungi (AMF) and biostimulants was evaluated on the antioxidant and nutritional potential of Moringa oleifera grown in the botanical garden of the University of Yaoundé I. Four treatments (Control or without fertilization (N0), 20g of AMF (N1), 10 g biostimulants (N2) and 20g AMF + 10g biostimulants (N3)) were made up and the experiment was designed in randomized block design with twenty-two replications. Moringa seeds were sown and four months after planting, leaves and roots were collected for the determination of their antioxidant and nutritional potential. Obtained data were analyzed using ANOVA at p < 0.05. It appeared that the arbuscular mycorrhizal fungi and biostimulants modulated the antioxidant and nutritional potential of Moringa oleifera with a remarkable influence of arbuscular mycorrhizal fungi on protein synthesis. Total phenolic content varied from 220mgEAG/100g (AMF + biostimulants) to 253mgEAG/100g (biostimulants) in leaves and from 164mgEAG/100g (biostimulants) to 245mgEAG/100g (control) in roots. Biofertilization increased the activities of both catalase and peroxidase in leaves and roots. Compared to control, AMF and biostimulants combination increased the nutritional content of about 90%, 2%, 26% in leaves and 6%, 3% and 14% respectively for lipids, sugars and proteins. AMF + biostimulants treatment was proved to be the best because of the greatest radical scavenging activities and larger quantities of chemical compounds in their plants.
The world has become aware of environmental issue in recent years affecting crop production, human nutrition and wellbeing. In tropics, the major limiting factor of crops is soil nutrient deficiency resulting from land degradation; and affecting the growth, nutrient content and uptake of the plant . To restore the productive potential of soils, some techniques can be used; fertilization is one of them .
Fertilizers are any organic or inorganic material of natural or synthetic origin that are added to soil to supply one or more plant nutrients essential for plant growth. Since synthetic compounds are highly polluting, hazardous and costliest, researchers are extensively working to develop natural products as they are less hazardous, cheaper and easily available.
In recent decades, agricultural growing practices have been evolving towards organic, sustainable or environmental
friendly systems. Modern agriculture implicates reduction of
chemical inputs without reducing the crop yield and quality.
Actually, research activity in the matter of agricultural systems has for years been oriented to increase yield without considering the quality of the products and the rational use of resources. In contrast, attention now is mainly focused on product quality and the sustainability of the cultivation systems. Moreover, cultivation management pays more attention to the reduction of production costs by lowering inputs .
This leads to the development of organic-based fertilizers and microorganisms which enhance the bioavailability and mobility of plant nutrients and, thus, improve the uptake
of nutrients by plant roots. Good fertility is fundamental to
successful plant growth, and the application of fertilizers
and manures is an essential growing activity. Arbuscular
mycorrhizal fungi (AMF) and biostimulants are products that
can achieve the balance between contributions and needs of
AMF constitute a group of roots obligate biotrophs that
exchange mutual benefits with about 80% of plants and
allow them to explore larger volumes of soil to absorb more
water and nutrients, enhance their transport, and increase
absorption of immobile mineral elements. Their action leads to
the stimulation of plant growth .
Relative to biostimulants, they are biological derived
fertilizer additives and similar products that are used in crop
production to enhance plant growth, health and productivity.
They help plants to tolerate abiotic stresses, improve
nutrient-use efficiency and improve quality attributes . As
environmental changes can have an impact on the chemical
and biochemical composition of plant’s organs, the objective of
this study was to evaluate the effect of AMF and biostimulants
as substrate amendment on the antioxidant and nutritional
potential of M. oleifera.
The present work was conducted at the Faculty of Science,
University of Yaoundé I from January to June 2013. The area
lies between latitude North 3 degrees 52 minutes 0 seconds
(3°52’0’’) and longitude East 11 degrees 31 minutes 0 seconds
Soils that provided support for the experiment were
basically a forest topsoil collected in Yaoundé (Centre region),
Cameroon. It consisted of a mixture of an Oxisol soil and sand
(proportion 3-1) that was sieved using a mason sieve (0.5cm)
and sterilized in an autoclave at 121 °C for 30 min. Once the
mixture cooled, it was introduced into perforated plastic bags
(20 × 20cm).
Inoculum, provided by the Laboratory for Soil Microbiology,
University of Yaoundé I consisted of two local selected strains of
AMF: Scutellospora gregaria and Gigaspora margarita (20spores/g
of sterilized soil). Bio-stimulants were a mixture of volcanic material
(podzolan), fish waste and animal bones finely ground.
The 3 litres containing plastic bags according to treatments
were ranged on shelves. Four treatments (Control: without
fertilization (N0), 20g of AMF (N1), 10g biostimulants (N2)
and 20g AMF + 10g biostimulants (N3)) were made up and the
experiment was designed in a factorial design with twentytwo
replications. AMF and biostimulants were applied in pots
and Moringa oleifera seeds sown. Biostimulants were applied
in two steps. The first dose was 5g at sowing stage and the
second dose, 5g five weeks after sowing. Watering was done
twice a day with tap water in the absence of rain.
Four months after planting, M. oleifera leaves and roots
were collected, cleaned and air-dried at room temperature.
They were kept away from high temperatures and direct sun
light to avoid destroying active compounds. They were then
pounded with an electric mill.
The process followed the established extraction procedure
of plant samples, using hydro-methanol (30:70 v/v) as solvents.
Serial extractions were done using 5g of plant powder, in 50ml
of solvent in Erlenmeyer flask. The mixture was stirred at two
hourly intervals for 24 hours. Rotavapor was used to recover
The amounts of phenolic compounds in leaves and roots
were estimated by using Folin-Ciocalteu reagent . Using
the standard (Gallic acid) curve, the total phenolic compound
content was calculated and expressed as Gallic acid equivalent
in mg/g of sample.
The free radical scavenging activity of the extract was
measured in terms of hydrogen donating or radical scavenging
ability using the stable free radical DPPH. EC50 value (the
concentration required to scavenge 50% DPPH free radicals)
was calculated. The ability to scavenge the DPPH radical was
calculated using the following equation:
DPPH scavenging effect (%) = [(A0 - A1)/A0 x 100].
Where A0 is the absorbance of the control reaction and A1
the absorbance in the presence of the sample.
Catalase activity was measured according to the method
described by Anderson et al.  where one unit of catalase
was defined as the amount of enzyme required to decompose
1μmol of H2O2 in 1min. The reaction was initiated by the
addition of 1.0ml of freshly prepared 20mM H2O2. The rate of
decomposition of H2O2 was measured spectrophotometrically
at 240nm for 1min. The enzyme activity was expressed as
units/g. One unit is the amount of enzyme activity required to
decrease the absorbance at 240nm by 50% units.
The peroxidase activity was measured using the method
described by . This method is based on the measurement
of the enzymatic activity with an electron donor (guaiacol),
which is oxidized to tetraguaiacol in the presence of hydrogen
peroxide. A peroxidase unit is defined as the amount of enzyme
that produces 1μmol/min of oxidized guaiacol.
The water content was determined according to the
method described by Tee et al. . It is based on the mass loss
of the samples at 105 °C.
The total protein content was measured by the Kjeldahl
method based on the conversion of organic nitrogen to
ammonium sulphate. The nitrogen content (N) is converted
into protein using 6.25 as the converting factor.
The total lipid content was determined based on their
differential solubility in certain organic solvents such as
hexane. They are extracted in hot hexane by continuously
refluxing for 12 hours .
The sugar content was determined by the method
described by Dubois et al.  based on dehydration followed
by cyclization of C6 sugars (hexoses) and C5 (pentoses) in hot
acidic medium to give respectively hydroxymethylfurfural
and furfural. The latter react with phenol to give a complex
with a maximum absorption at 490nm.
The data were subjected to statistical analysis to verify
and evaluate the difference between the antioxidant and
nutritional potential of Moringa oleifera leaves and roots four
months after planting. Data were statistically analysed with
SPSS software, followed by post hoc analysis using Fischer’s
LSD. The difference was considered significant if P<0.05.
Results were expressed as mean ± S.D.
Hydro-methanol was chosen as extraction solvent for
both phenolic content and scavenging activity because of it
wide solubility properties for low molecular and moderately
polar substances, including the antioxidant-active phenolic
The influence of the different treatments on the leaf and
root phenolic content and effective concentration 50 (EC50) is
presented by Figure 1.
In leaves, values went from 219.83 ± 0.26 mgEAG/100g
(AMF + biostimulants) to 253.55 ± 0.17 mgEAG/100g
(biostimulants) while in roots, they ranged from 164.23 ± 0.02
mgEAG/100g (biostimulants) to 245.35 ± 0.69 mgEAG/100g
The sample concentration required to decrease the initial
concentration of DPPH to 50% (EC50) is a commonly used
parameter for determining the antioxidant activity. These
values are compared to a positive control (Gallic acid), which
has an EC50 of 26.90mg/g of DPPH.
The application of biostimulants decreased the total
phenolic compounds of both M. oleifera leaves and roots,
but AMF increased this parameter compared to the control
The leaves of M. oleifera have a better scavenging activity
than roots; this could be explained by their high secondary
metabolite content. The antioxidants react with DPPH, a stable
purple coloured free radical and convert it into a colourless
2.2-diphenyl-1-picryl hydrazine. Antioxidants, on interaction
with DPPH, either transfer an electron or hydrogen atom to
DPPH, thus neutralizing its free radical character . Moringa
oleifera l eaf a nd r oot e xtracts s ignificantly r educed D PPH
radicals. The degree of discoloration indicates the scavenging
potential of the antioxidant extract, which is due to the radical
Phenolic compounds because of their structure have the
ability to give a proton to free radicals which become more
stable. The DPPH scavenging ability of the extract may be
attributed to its hydrogen donating ability.
These results indicate that there is no correlation between
antioxidant activity and total phenolic content (P>0.05).
However, different results were reported on this aspect; some authors found correlation between phenolic content
and scavenging activity; whereas the others found no such
relationship, since other compounds are responsible for the
antioxidant activity [14,15]. The low antiradical activity of
roots compared to leaves could be explained by the existence
of chemical compounds that mask the groups of molecules
responsible for the scavenging activity.
Plants are continuously exposed to stress. It is therefore
important that they synthesize protective molecules or
compounds. The leaves and roots of Moringa oleifera were
analyzed for the enzymatic antioxidant levels/activities such
as catalase and peroxidase. The results (Tables 1) showed that
the biostimulants and AMF + biostimulants treatments present
the highest enzymatic activities.
Fertilization increased the activity of both enzymes.
In leaves, catalase activities reached from 3.61 x 103 ±
0.07 Mmoles/min (AMF) to 4.82 ± 0.53 x 103 Mmoles/min
(biostimulants) and peroxidase from 3.89 x 103 ± 0.37 Mmoles/
min (control) to 5.45 x 103 ± 0.02 Mmoles/min (biostimulants).
In the roots, these activities ranged from 1.00 ± 0.87 x 103
Mmoles/min to 2.43 ± 0.08 x 103 M moles/min (catalase) a nd
from 1.25 x 103 ± 0.44 Mmoles/min to 0.45 ± 2.2 x 103 Mmoles/
In Moringa oleifera l eaves, c atalase a nd p eroxidase
activities were important than in root and the activity is more
important in treated plants compare to control. On one hand,
this could be explained by the fact that the leaves exposed to
environmental conditions, face oxidative stress and need to
protect their selves. On the other hand, antioxidant enzymes
form the first line of defence against free radical-induced
damage, offer protection against free radicals. Peroxidase
is an enzyme that catalyses the reduction of hydroperoxide,
including hydrogen-peroxide and function to protect the cell
from peroxidative damage . It has been found that in crops,
pest and bio-aggressor attack level can be an indicator of the
nutritional composition. Actually, plant rich in some minerals
and nutrients are suitable for some bio-aggressors and pests
(article to be submitted) and they need to synthetize protective
compounds to fight again such situations.
The influence of the AMF and biostimulants on water,
protein, lipid and sugar contents of Moringa leaves and roots
is presented in the Table 2. Nutrient concentrations were
significantly (P < 0.05) influenced by soil amendment.
The water content of plant ranged from 66.65 ± 2.65 %
to 77.88 ± 2.06 % and from 75.09 ± 1.27 to 80.96 ± 0.16 %
respectively for leaves and roots.
The lipid values of the leaves varied from 4.0 ± 0.40 %
(Control) to 7.6 ± 0.40 % (AMF + Biostimulants). Roots values
ranged from 1.1 ± 0.10 % (Biostimulants) to 6 ± 0.40 % (AMF).
For the sugar content values ranged from 49.07 ± 0.13 %
(AMF) to 53.09 ± 0.09 % (Biostimulants) for leaves and from
41.89 ± 0.12 % (Control) to 44.06 ± 0.25 % (Biostimulants) for
The use of AMF and biostimulants increased the levels
of macronutrients (total sugars, lipids and proteins) of the
plant. Microorganisms, because of the mycorrhizal symbiosis
reduced the soluble carbohydrate content of the leaves and
roots compared to the control plants. This is understandable
because during mycorrhizal symbiosis, when providing the host
plant with nutrients, increasing the abiotic (drought, salinity,
heavy metals) and biotic (root pathogens) stress resistance,
AMF receive between 4 and 20 % of photosynthetically fixed
carbon from host plant .
From this study, we notice that AMF have a remarkable
influence in protein synthesis. These protein contents indicate
the best metabolism functioning of the plant; AMF highly
stimulate plant metabolism. The level of crude protein content
is of particular nutritional significance as it may meet animal’s
protein and energy requirements and boost the immune
system against diseases [18-20].
Crop nutrient deficiencies are now recognized as an
important contributor to the global burden of disease. Macro
and micronutrients are very important for a healthy diet and
prevent nutritional problems. The positive effect of AMF and
biostimulants has boosted Moringa oleifera biomass for leaves
and roots, and secondary metabolites quality, thus enhancing
its antioxidant capacity, making it more applicable for use in
Soil bio-fertilization positively influences the nutritional
and antioxidant potential of Moringa oleifera . Indeed, the
application of AMF and biostimulants significantly modify
the EC50 of leaves and roots. This latter is an indicator of
antioxidant potential of an extract. Due to the fact that tropical
soils gradually lose their fertility after a formal continuous
culture, it is important to find biological alternatives to
this problem in order to reduce the risk of environmental
destruction. Biostimulants enrich soil with nutrients and
substances when AMF promote their absorption. However,
mycorrhizal symbiosis reduces the sugar content. The increase
of enzymatic activity of catalase and peroxidase in Moringa
leaves compared to roots may be a plant response to oxidative
stress probably caused by environmental conditions such
as abiotic (drought, salinity, heavy metals) and biotic (root
The authors thank all the members of the Laboratory
for Food Science and Metabolism (LabSAM), Department of
Biochemistry, University of Yaoundé I and members of the
Laboratory for Soil Microbiology, Department of Microbiology,
University of Yaoundé I.
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