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J Food Biochem. 2020;00:e13318. wileyonlinelibrary.com/journal/jfbc
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https://doi.org/10.1111/jc.13318
© 2020 Wiley Periodicals LLC
1 | INTRODUCTION
Mistletoes (Tapinanthus bangwensis L.) are hemi-parasites which
are attached to the branches and stems of host plants via special-
ized absorbing organ called haustorium; this translocates water and
mineral nutrient s from the host plant to the parasite. They always
bear evergreen leaves that photosynthesize and they belong to the
Loranthaceae family (Edagbo et al., 2012; Milius, 2000). They grow
on a wide range of host trees of medicinal and economic importance
such as moringa, cashew, orange, mango, grape, kola nut, cocoa,
rubber, and others; different host species may supply a parasite
with different resources and vice-versa. Mistletoe leaves from dif-
ferent host plants are traditionally used in the treatment of a num-
ber of ailments like nervousness, sterility, rheumatism, psychosis,
diabetes, cancer, epilepsy and hypertension (Adodo, 200 4; Jadhav
et al., 2010). Several reports have suggested possible fundamental
changes in the metabolic profile and biological activities of host
plant (Cuevas-Reyes, Pérez-López, Maldonado-López, & González
Received:22January2020
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Revised:2 0March2020
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Accepted:12May2020
DOI : 10.1111 /jf bc .13318
SPECIAL ISSUE ORIGINAL ARTICLE
Modulatory effects of moringa (Moringa oleifera L.) leaves
infested with African mistletoe (Tapinanthus bangwensis L.)
on the antioxidant, antidiabetic, and neurochemical indices in
high sucrose diet-induced diabetic-like phenotype in fruit flies
(Drosophila melanogaster M.)
Olubukola H. Oyeniran1,2 | Adedayo O. Ademiluyi1 | Ganiyu Oboh1
1Functional Foods, Nutraceutic als and
Phytomedicine Unit, Department of
Biochemistry, Federal University of
Technology, Akure, Nigeria
2Department of Biochemistry, Federal
University Oye , Oye, Nigeria
Correspondence
Olubuko la H. Oyen iran, Function al Foods,
Nutraceuticals and Phytomedicine Unit,
Department of Biochemistry, Federal
University of Techno logy, Aku re, 340001,
Nigeria.
Email: oyeniranolubukola@gmail.com,
olubukola.oyeniran@fuoye.edu.ng
Abstract
Moringa is a common medicinal plant tree with mistletoe infestation and its leaf is
widely used as food and traditional medication in alleviating several metabolic and
neurodegenerative diseases. Hence, this study investigated the influence of African
mistletoe on the antioxidant, antidiabetic, and neuroprotective activities of infested
moringa leaf in sucrose induced diabetes in Drosophila melanogaster model. Glucose
and triglycerides were evaluated in the flies’ hemolymph and all other parameters
were evaluated in the tissues. A significant (p < .05) decrease in survival rate and in-
crease in the level of glucose and triglycerides in flies fed with 30% of sucrose when
compared with control was obtained. Treated flies had significant (p < .05) positive
alteration in the level of glucose, triglycerides, antioxidants (both enzymatic and non-
enzymatic), and enzyme activities when compared with normal and sucrose control
flies. This study suggests that mistletoe infestation did not alter the antioxidant, an-
tidiabetic, and neuroprotective effects of the moringa leaf.
Practical applications
This present study has shown that mistletoe infestation did not alter the protective
activities of moringa leaf, hence, moringa with or without mistletoe infestation could
be taken as functional food to mitigate several metabolic diseases.
KEYWORDS
diabetes, Drosophila melanogaster, mistletoe, Moringa oleifera
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Rodríguez, 2017; Edagbo et al., 2012). Moringa (Moringa oleifera L.)
is a common medicinal plant tree with mistletoe infest ation. They
are classified as Moringaceae and several reports has shown that
the leaves are natural sources of antioxidants and essential amino
acids (Ahmed, Alazzouni, Hassan, & Mohamed, 2016). The leaves
of moringa are used in folk medicine due to their antitumor, anti-
pyretic, antiepileptic, anti-inflammatory, antiulcer, antihypertensive,
antioxidant, antidiabetic, antimicrobial, hepatoprotective and neuro-
protective activities (Ademiluyi, Aladeselu, Oboh, & Boligon, 2018;
Chatchada, Jintanaporn, & Wipawee, 2013).
Diabetes mellitus (DM) is a metabolic disorder signaled by hyper-
glycemia which results from decrease insulin secretion or sensitivity
(Maher & Schubert, 2009). The global incidence of DM has been es-
timated to affect more than 366 million individuals by 2030 (Fradkin,
Cowie, Hanlon, & Rodgers, 2013). The morbidity and mortality of
DM results from its attendant complications (i.e., its resultant effects
on blood vessels, eyes, kidney, and the nerves) (Rathmann, Giani, &
Wild, 2004). Studies have also shown that DM affects the central
nervous system (CNS) where it induces another disease condition in
the region of the brain called diabetic encephalopathy signaled by
mild to severe cognitive deficits (Bauduceau et al., 2010; Zhou, Luo,
& Dai, 2013). The symptoms of diabetic encephalopathy are the at-
tributes of brain aging which includes: withering and weakening of
brain tissues and the brain blood vessels (Baquer et al., 2009; Biessels,
Heide, Kamal, Bleys, & Gispen, 2002). It has also been suggested
that rapid onset of hyperglycemia is linked with impairment in cog-
nition in both type-1 and type-2 DM patients and that there is have
an increased risk of developing Alzheimer's disease (AD) in diabetic
patients (Biessels, Staekenborg, Brunner, Brayne, & Scheltens, 2006;
Gispen & Biessels, 2000).
Recently, fruit fly (Drosophila melanogaster M.) has become an
approved and widely alternative organism to animal models for re-
search into the various diseases of human (Bharucha, 2009; Morris
et al., 2012). About 70%–75% disease causing genes of human is con-
served in fruit fly. Therefore, they have also been used as a model for
the study of diabetes. Their brain possesses insulin-producing cells
(IPCs) which secretes seven insulin-like peptides (dILPs). The IPCs of D.
melanogaster are the equivalent of the mammalian islets of Langerhans
of β pancreatic cells (Broughton et al., 2005; Garelli, Gontijo, Miguela,
Caparros, & Dominguez, 2012). High sucrose diet has been reported
to induce observable features of insulin-resistant in fruit fly which is
the major patholog y of type-2 DM in humans (Murillo-Maldonado, Bou
Zeineddine, Stock, Thackeray, & Riesgo-Escovar, 2011; Teleman, 2010).
Dietary supplements from plants contain various phytoconstit-
uents which reduces the risk of several metabolic and neurodegen-
erative diseases. The leaves of moringa and mistletoes are being
consumed by man from time immemorial. Studies have provided a
clear link between the consumption of these common plant foods
and the prevention of diabetes (Chatchada et al., 2013; Ekhaise,
Kayode, & Sylvester, 2011). Hence, this study investigated the influ-
ence of African mistletoe on development and memory parameters,
antioxidant, antidiabetic, and neuroprotective activities of moringa
host leaf in sucrose-induced DM in D. melanogaster model.
2 | MATERIALS AND METHODS
2.1 | Leaf preparation
The fresh leaves of moringa with and without mistletoe infesta-
tion and the mistletoe growing on the moringa tree were harvested
at a farm plantation in Akure. It was authenticated at the Centre
for Research and Development (CER AD), Federal University of
Technology, Akure, Nigeria with Futa herbarium numbers 0151 and
0154. The leaves were sor ted, washed, and subsequently freeze
dried. The freeze-dried samples were pulverized using an electric
blender. The samples were kept in an air tight container prior to sub-
sequent analysis.
2.2 | Chemicals and reagents
Acetylthiocholine iodide, trichloroacetic acid, thiobarbituric acid,
5,5-Dithiobis-(2-nitrobenzoic acid) (DTNB), hydrogen peroxide,
acetic acid, hydrochloric acid, sucrose, and ferrous sulfate were ob-
tained from Sigma-Aldrich (Merck KGA, Darmstadt, Germany). All
other chemicals and reagents used were of analytical grade and the
water used was glass distilled.
2.3 | Fruit fly stock and culture conditions
D. melanogaster (Oregon wild fly strain) was obtained from the
National Species Stock Center (Bowling Green, OH, USA). The flies
were reared on corn meal medium containing brewer's yeast (1%),
powdered milk (1%), agar (1%), and methyl-p-hydroxybenzoate
(0.08%) at constant temperature (23 ± 1°C) and 60% relative humid-
ity in an incubator with 12-hr dark/light cycle. The first instar larvae
(L1) obtained from this strain was used due to their increased feed-
ing and rapid growth rate in comparison with adult flies.
2.4 | Experimental design
Forty (40) adult female flies of five (5) days old were allowed to lay
eggs in twelve (12) glass jars containing basal diet for a period of
12 hr. After 24 hr of laying eggs, the emerged first instar lar va (L1)
were divided into 12 groups containing 50 flies (L1) each. Groups
1 was placed on basal diet alone, groups 2–4 were placed on diet
containing noninfested moringa, infested moringa, and the parasite
whole leaf samples alone, groups 5 and 6 were placed on basal diet
containing 15% and 30% w t/vol sucrose while groups 7–12 were
placed on diet containing 15% and 30% wt/vol sucrose; treated
with noninfested moringa, infested moringa, and parasite whole leaf
samples (1% wt/vol inclusion) and reared until 5 days of adulthood.
The percentage dietary inclusion was based on previous toxicologi-
cal test s performed in adult flies for 20 days (data not shown). The
treatment lasted for 14 days and the newly emerged 5 days old adult
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flies were collected and used for the various analysis. This consisted
of three main studies of five vials per group.
2.5 | In vivo assays
2.5.1 | Survival rate
The survival rate of the larva was monitored by counting daily the
number of adult flies that emerged from viable larva from the 9th
to 14th day of treatment. The data obtained were analyzed and ex-
pressed as percentage of flies’ emergence (Adedara, Abolaji, Rocha,
& Farombi, 2016).
2.5.2 | Locomotor performance
The newly emerged flies were subjected to climbing analysis (Abolaji
et al., 2015; Feany & Bender, 200 0). After the 14 days treatment
period, the newly emerged flies were tapped softly to the bottom of
a marked glass column and the flies that flew up above the 6 cm mark
of the column in 6 s were recorded. This process was repeated three
times at 1-min inter val. The data was expressed as the percentage
locomotor performance.
2.5.3 | Aversive phototaxis analysis
Short memory taxis of the newly emerged flies were also assessed.
After the 14 days treatment period, the flies were exposed to a
source of light in which there was a bitter substance (quinine) in the
light path. The number of flies along the light path within 15 s was
counted. Af ter 6 hr, the ability of these flies to remember the bitter
substance in the light path by monitoring their withdrawal within
15 s. The data were expressed as percentage of flies’ withdrawal
at 0 hr and 6 hr. This analysis was repeated three times at 1-min
interval.
2.5.4 | Weight of flies
Five (5) flies of three days old hatched from the lar va (L1) were sepa-
rated and weighed on an AMPUT high precision microbalance. This
is expressed as weight of flies in μg.
2.6 | Ex vivo assays
2.6.1 | Hemolymph collection
The hemolymph of five (5) days old adult flies was collected accord-
ing to Sigma-A ldrich method, with few modi fications. The f lies’ wings
were cut off in order to create an opening, these flies were then
placed in 500 μl Eppendorf tubes which contain small holes and was
centrifuged within 2,00 0 μl Eppendorfs in Kenxin refrigerated cen-
trifuge Model KX3400C (4°C) for 5 min at 3,000g. The hemolymph
obtained was diluted with 50 times anticoagulant buffer which
contains 186 mM of NaCl, 98 mM of NaOH, 1.7 mM of EDTA, and
41 mM of citric acid (Stoepler, Castillo, Lill, & Eleftherianos, 2012;
Teleman, Chen, & Cohen, 2005).
2.6.2 | Glucose and triglycerides levels
The glucose and triglycerides levels in the flies’ hemolymph were de-
termined using Sigma Randox kit and absorbance reading was taken
at 546 nm in an ultraviolet double beam spectrophotometer and ex-
pressed as μmol/L.
2.6.3 | Preparation of tissue homogenates
The flies (or head of flies) were immobilized on ice and homogenized
in sodium phosphate buffer (0.1 M, pH 7.4) using a micro Teflon
Homogenizer. The homogenates obtained were centrifuged at
10,000g, 4°C for 10 min in an ice-cold centrifuge. The supernatants
were later decanted into labelled Eppendorf tubes and used for the
various analysis.
2.6.4 | Enzymatic and nonenzymatic
antioxidant assays
Determination of total protein
The Coomassie blue method which uses bovine serum albumin as
standard was used to determine total protein content of the flies
(Bradford, 1976). The reaction mixture consisted of 20 µl of sodium
phosphate buffer (0.1 M, pH 7.4), 20 μl of tissue homogenate, and
200 μl of Coomassie blue. This was incubated at 25°C for 30 min and
absorbance was measured at 595 nm in a SpectraMax plate reader
(Molecular Devices, CA, USA). The protein concentration was cal-
culated from standard calibration curve versus absorbance graph.
Estimation of reactive oxygen species (ROS) produced
The method of Hayashi et al. (2007) with slight modifications was
used to estimate the intracellular level of ROS by quantifying hydro-
gen peroxide (H2O2; an important indicator of oxidative stress). The
reaction was made up of 10 μl of flies’ tissue homogenate and 183 μl
of N-N-diethyl-para-phenylenediamine (DEPPD). The absorbance was
taken at 505 nm using a SpectraMax plate reader (Molecular Devices,
CA, USA). The levels of H2O2 was calculated from H2O2 standard cali-
bration curve and expressed as mmol H2O2 produced/mg protein.
Determination of thiobarbituric acid reactive species
The meth od of Puntel, Nogue ira, and Rocha (20 05) was used to meas-
ure the level of thiobarbituric acid reactive substances (TBARS). The
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reaction mixture contained 50 µl flies’ tissue homogenate, 300 µl
8.1% of SDS, 500 µl of acetic acid and 500 µl of 0.8% TBA . This
was boiled at 100°C for 60 min and allowed to cool. They were later
centrifuged at 8,000g for 10 min and the absorbance of the superna-
tants were measured at 532 nm in a spectrophotometer. The TBARS
level were calculated and expressed as mmol/mg protein.
Determination of the total thiol content
The level of total thiol in flies’ tissue homogenate was carried out
by the method of Ellman (1959). The reaction contained 150 µl so-
dium phosphate buffer (0.1 M, pH 7.4), 40 μl tissue homogenate and
10 μl of DTNB (10 mM). This was incubated in dark for 30 min and
the absorbance was taken at 405 nm in a SpectraMax plate reader
(Molecular Devices, CA, USA). The tot al thiol level was subsequently
calculated from GSH standard calibration curve and expressed as
mmol/mg protein.
Determination of reduced glutathione (GSH) content
The method of Ellman (1959) was used in determining the level of
nonthiols (reduced GSH) in flies’ tissue homogenate. Briefly, 50 μl
of tissue homogenate was precipitated with 25 μl of 10% TCA, and
then, centrifuged at 4,00 0 r.p.m for 30 min. The supernatant ob-
tained was used to replace the tissue homogenate in the method
used for the determination of total thiol. The nonthiol content was
subsequently calculated from GSH standard calibration curve and
expressed as mmol/mg protein.
Determination of glutathione S-transferase activity
The method of Habig and Jakoby (1981) with slight modifications
(Abolaji et al., 2014) was used. The assay mixture consisted of 250 µl
of sodium phosphate buffer (0.1 M, pH 7.4), 10 µl of 25 mM GSH,
30 µl of flies’ tissue and 10 µl of 25 mM CDNB. The mixture was
monitored for 30 min (30 s intervals) at 340 nm in a SpectraMax
plate reader (Molecular Devices, CA, USA) and the enzyme activities
were calculated and expressed as mmol–1 · min–1 · mg–1 protein.
Determination of catalase activity
The method of Shina (1972) with slight modifications was used in
determining the catalase ac tivit y. The mixture was made up of 100
μl of flies’ tissue homogenate, 200 μl of 2 M H2O2, 400 μl of 10 μM
sodium phosphate buffer (pH 7.0) and 800 μl of dichromate acetic
acid. This was incubated at 100°C for 5 min and absorbance was
taken at 620 nm in a spectrophotometer. The enzyme activities were
calculated and expressed as mmol H2O2 consumed/mg protein using
an H2O2 standard calibration curve.
Determination of superoxide dismutase (SOD) activity
The SOD activity was measured according to the method of Jewett
and Rocklin (1993). The reaction mixture consisted of 50 μl of flies’
tissue homogenate, 100 μl of sodium carbonate buf fer (0.1 M, pH
10.2) and 50 μl of adrenaline. The absorbance was measured at 30 s
interv als for 5 min at 480 nm in a Sp ectraMax pl ate reader (Molecul ar
Devices, CA, USA) and the enzyme activities were calculated and ex-
pressed as μmol–1 · min–1 · mg–1 protein.
2.6.5 | Enzyme assays
Determination of α-amylase activity
The activity of α-amylase was determined in the flies’ tissue ho-
mogenate according to the method described by Worthington,
Biochemical Corp (1978). The reaction mixture consisted of 25 μl
of flies’ tissue homogenate and 100 μl of sodium phosphate buffer
(0.02 M, pH 6.9, 6 mM NaCl) containing pancreatic α-amylase
(0.5 mg/ml), this was incubated at 25°C for 10 min. Then, 25 μl of
1% starch solution was added and incubated at 25°C for 10 min.
Thereafter, 100 μl of dinitrosalicylic acid was added and incubated
at 100°C for 5 min. This was allowed to cool followed by addition
of 1 ml of distilled water. Absorbance was taken at 540 nm and the
enzyme activity was calculated and expressed as mmol/mg protein.
Determination of α-glucosidase activity
The method described by Apostolidis, Kwon, and Shet ty (20 07) was
used, this contained 15 μl of flies’ tissue homogenate, 215 μl of phos-
phate buffer (0.02 M, pH 6.9), and 3 mM of glutathione (in the same
buffer); incubation was done for 20 min at 37°C . Thereafter, 40 μl of
10 mM p-nitrophenyl-α-d-glucopyranoside (PNPG) solution (in the
same buf fer) was added and subsequently incubated for 20 min at
37°C. About 1% of Na2CO3 was finally added and absorbance was
taken at 40 0 nm. The α-glucosidase activity was c alculated and ex-
pressed as mmol/mg protein.
Determination of acetylcholinesterase (AChE) activity
AChE activity was determined in the head of the flies (Ellman, 1959).
The reaction mixture consisted of 40 µl of flies’ tissue homogenate,
20 µl of 25 mM DTNB, 40 µl of phosphate buffer (0.1 M, pH 7.4) and
20 µl of 8 mM acetylthiocholine as a substrate. The absorbance was
measured at 30 s intervals for 30 min at 412 nm using a SpectraMax
plate reader (Molecular Devices, CA, and USA) and the enz yme
activities were calculated and expressed as mmol–1 · min–1 · mg–1
protein.
Determination of monoamine oxidase (MAO) activity
The MAO activity was determined in the head of the flies (Green
& Haughton, 1961). Briefly, the reaction mixture contained 200 µl
of phosphate buffer (0.1 M, pH 7.4), 50 µl of flies’ tissue homoge-
nate, 25 µl of benzylamine (10 mM) and 500 µl of distilled water.
This was incubated for 30 min and 10% of perchloric acid was finally
added. This was centrifuged at 1, 500g for 10 min, the supernatant
was decanted and the absorbance was taken in a spectrophotometer
at 280 nm. The MAO activity was finally calculated and expressed
as μmol/mg protein.
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2.7 | Data analysis
The results were expressed as mean ± standard deviation (SD). One-
way Analysis of Variance (ANOVA) followed by Tukey's post hoc test
was used in analyzing the results. Differences between the groups
were regarded significant at p < .05 and the analysis was done using
GraphPad Prism (V.5.0).
3 | RESULTS AND DISCUSSION
The adverse effects of excessive use of sucrose (commonly known as
table sugar) and some other sweeteners have been a subject of dis-
cussion in this part of the world. Consumption of diets that are rich in
sugar has been shown to induce diabetic-like phenotypes in fruit fly (D.
melanogaster) (Musselman et al., 2011; Pasco & Leopold, 2012). The
use of fruit f ly as an alternative non-mammalian model organism for re-
search has been widely approved (Bharucha, 2009; Morris et al., 2012).
Several reports have suggested that the growth of mistletoe alters the
metabolic and biological activities of the host plant (Cuevas-Reyes
et al., 2017; Edagbo et al., 2012). Moringa is a common medicinal plant
with mistletoe infestation and its leaf is the most widely used part
of the plant due to its several health promoting benefits (Ademiluyi
et al., 2018). Consequently, this present study investigated the influ-
ence of African mistletoe on development and memory parameters,
antioxidant, antidiabetic, and neurochemical indices of infested mor-
inga leaf in sucrose induced diabetes in D. melanogaster model.
3.1 | Effects of infested and noninfested moringa
leaves on development and memory parameters
Moringa leaf is one of the world most useful leaves as it is able to ex-
tend lifespan, improve health status, and promote antiaging ef fect s
(Zaku, Emmanuel, Tukur, & Kabir, 2015). The survival of flies was es-
timated by counting the number of adult flies that emerged from the
first instar larva (L1) from the 9th day to the 14th day of treatment
(Figure 1). The result revealed that the rate of emergence of the nor-
mal and 15% of sucrose groups treated flies were not significantly
(p < .05) different from the control. However, the normal flies that
was fed with infested and noninfested moringa leaf had the highest
(70%–75%) rate of emergence on the 14th day of treatment. This
study agrees with the repor t of Zaku et al. (2015) as moringa leaf
was able to improve the sur vival of the normal treated larvae (L1)
and their emergence to adult flies. There was a significant (p < .05)
decrease in the survival and emergence of flies fed with 30% of
sucrose when compared with control, this agrees with the report
of Lushchak et al. (2013), whereby high sucrose diet resulted in de-
crease lifespan of fruit fly. The result of the locomotor performance
of the emerged flies fed with sucrose, infested, and noninfested
moringa leaves (Figure 2a) revealed that there was no significant
(p < .05) difference obt ained but a slight reduction was observed in
the flies fed with only 15% and 30% of sucrose diet.
When fruit flies are exposed to a dark environment, they are ex-
pected to naturally fly toward a single introduced source of light in
few seconds; a signific ant reduction was observed in the response
of the sucrose fed flies to light at 0 hr and a little increase in their
withdrawal from the source of light in which quinine was present in
the light path after 6 hr (Figure 2b). This is indicative of the fact that
memory impairment might have occurred during the development
of the flies (L1) due to the presence of high level of sugar (15% and
30% of sucrose) in their diets. At 0 hr, the withdrawal of the flies
from the light path was higher in the normal treated control flies
when compared with the sucrose fed flies. After 6 hr, the normal
treated control flies had the highest withdrawal while the sucrose
treated flies had lit tle increase in their withdrawal. This report is
in agreement with previous studies in which moringa and mistle-
toe leaves has been shown to improve memory impairment in rats
FIGURE 1 Emergence of flies from larva fed with sucrose, infested and noninfested moringa leaves. Values represent mean ± SD (n = 5).
CTR, control; IM, infested moringa; NIM, noninfested moringa; P, parasite
6 of 14
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(Chatchada et al., 2013; Ekhaise et al., 2011). The result obtained
for the weight of the flies is shown in Figure 2c. This showed that
there was no significant (p < .05) difference obser ved but it should
be noted that the flies that was fed with the highest sugar-rich diet
(30% of sucrose) had the highest weight and this implies obesity.
There was a general reduction in the weight of the treated flies
when compared with the normal and sucrose control. This study is
consistent with earlier repor ts in which sugar-rich diets has been
reported to cause obesity (Musselman et al., 2011). The presence
of infested and noninfested moringa and the parasite leaves were
able to reduce the weight of both the flies fed with normal and
sucrose rich diets. This follows the trend obtained in earlier re-
ports in which moringa was shown to be able to ameliorate obesity
(Souravh, Guru, & Ramaica, 2014).
3.2 | Effects of infested and noninfested moringa
leaves on glucose and triglyceride level
The influence of mistletoe on moringa was further evaluated by in-
vestigating its ef fect on carbohydrate (glucose) and lipid (triglyceride)
contents of the flies’ hemolymph. The influence of treatment on the
glucose level of emerged flies is depicted in Figure 3a, this showed that
a significant (p < .05) rise was obtained in the 15% and 30% of sucrose
control and this was accompanied by a significant (p < .05) decrease
in the sucrose treated groups. The triglyceride level of the normal
and sucrose-induced diabetic flies is shown in Figure 3b. A significant
(p < .05) rise in the triglyceride level of flies that received 15% and
30% of sucrose with a concomitant significant (p < .05) decrease in the
treated fruit flies was observed. High sugar diets resulted in significant
FIGURE 2 (a) Locomotor per formance (b) Short memor y taxis and (c) Weight of emerged flies from larva fed with sucrose, infested and
noninfested moringa leaves. Values represent mean ± SD (n = 5). CTR, control; IM, infested moringa; NIM, noninfested moringa; P, parasite; #
represents significant difference at p < .05 from normal control; * represents significant difference at p < .05 from sucrose control
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OYENIRA N Et Al.
(p < .05) elevated levels of glucose and triacylglycerides. This is indica-
tive of metabolic disorders and biochemical signals of DM. This result
is in agreement with the report of Pasco and Leopold (2012) where
high sugar diet was reported to induce the observable features of
type-2 diabetes in flies. These effects were mitigated by the varieties
of moringa leaf in the diet, this might be due to the ability of moringa
to inhibit soluble epoxide hydrolase and inhibition of soluble epoxide
hydrolase has been reported to ameliorate diabetes and its complica-
tions (Goswami et al., 2016; Nathani, Rema, Bruce, & Sumanta, 2018).
3.3 | Effects of infested and noninfested moringa
leaves on nonenzymatic antioxidants level
The hyperglycemia and hypertriglyceridemia conditions which oc-
curs as a result of sugar-rich diet consumption in the flies activates
some oxidative events. Therefore, this study evaluated the influence
of mistletoe on the antioxidant activities of moringa. The first oxida-
tive stress biomarker investigated is the ROS level. When the level
of ROS exceeds the antioxidant defense mechanism, this results in
FIGURE 3 (a) Glucose and (b) Triglyceride levels of emerged flies from larva fed with sucrose, infested and noninfested moringa leaves.
Values represent mean ± SD (n = 5). CTR, control; IM, infested moringa; NIM, noninfested moringa; P, parasite; # represent s significant
difference at p < .05 from normal control; * represents significant difference at p < .05 from sucrose control
8 of 14
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oxidative stress with consequent cell damages which af fects DNA,
lipids, proteins, and carbohydrate contents of the affected living or-
ganism (Valko et al., 2007). The ROS level of the emerged flies from
larva fed with sucrose, parasitic, and nonparasitic moringa leaves is
depicted in Figure 4a. This revealed that the level of ROS increased
in the sucrose-induced diabetic flies with subsequent significant
(p < .05) reduc tion in all the flies that received infested moringa
leaves when compared with the normal and sucrose control. This
agrees with earlier reports where increased ROS levels were ob-
tained in living organisms fed with sugar rich diets (Folmer, Soares,
& Rocha, 20 02). The reduced levels of ROS obtained in the infested
moringa leaf treated flies could be attributed to the fact that the
growth of mistletoe on moringa plant could have resulted in the im-
proved activity. This trend is consistent with the reports of Anselmo-
Moreira, Teixeira-Costa, Ceccantini, and Furlan (2018) where they
reported that high flavonoid content was detected at the gall region
of mistletoe infested Tapirira guianensis plant and this could be its
mechanism of quenching ROS.
Thiobarbituric acid reactive species (TBARS) are products
formed during the free radic al degradation of polyunsaturated fatty
acids and it is one of the most frequently used biomarkers that mea-
sures the extent of lipid peroxidation in tissues (Davey, Stals, Panis,
FIGURE 4 (a) ROS (b) TBARS (c) Total thiol and (d) Nonthiol levels of emerged flies from larva fed with sucrose, infested and noninfested
moringa leaves. Values represent mean ± SD (n = 5). CTR, control; IM, infested moringa; NIM, noninfested moringa; P, parasite; # represents
significant difference at p < .05 from normal control; * represents significant difference at p < .05 from sucrose control
|
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OYENIRA N Et Al.
Keulemans, & Swennen, 2005). The TBARS level of the emerged
flies from larva fed with sucrose and the moringa leaves is shown in
Figure 4b. The TBARS level for the 15% and 30% of sucrose control
was significantly (p < .05) increased when compared with the normal
control and a significant (p < .05) decrease in the treated flies when
compared with the sucrose control was obta ined. The level of TBAR S
obtained was in agreement with the ROS result whereby significant
(p < .05) rise in TBARS levels were obtained in the sucrose fed flies
and a reduction in the treated flies to almost same level obtained in
the nonsucrose fed flies. This might be attributed to the action of
the various phytoconstituents present in the leaf as previous studies
has shown that the antioxidant activity of plant food is proportional
to its phytochemical constituents (Ademiluyi et al., 2018; Chu, Sun,
Wu, & Liu, 2002). The sulfydryl containing compounds, called thiols
make up a higher proportion of the total body antioxidants which
play significant role in providing defense against damage caused
by free radical generation (Prakash, Shetty, Tilak, & Anwar, 2009).
The total thiol and nonthiol level of the emerged flies from larva fed
with sucrose, infested, and noninfested moringa leaves is depicted
in Figure 4c,d. There was no significant (p < .05) dif ference obtained
for both the total thiol and nonthiol contents of the flies. The ob-
served depletion in the 15% and 30% of sucrose control group might
indicate the progress of diabetic condition. However, the increased
level of thiols obtained in the treated group could be a function of
the endogenous antioxidants present in the moringa leaves coupled
with its ability to attenuate oxidative stress (Ademiluyi et al., 2018).
The presence of mistletoe on the moringa leaf does not produce any
significant effect in the flies’ thiol and nonthiol content.
3.4 | Effects of infested and noninfested moringa
leaves on enzymatic antioxidants activities
Another important useful approach used by organism to circumvent
damage caused by ROS is through the action of enzymatic antioxi-
dants such as glutathione-S-transferase (GST), sodium dismutase
(SOD), and catalase. GST are antioxidant enzymes that facilitates the
binding of reduced glutathione (GSH) to electrophilic centre com-
pounds which result s in the formation of a thioether bond between
the sulfur atom of GSH and the substrate. They detoxify xenobiot-
ics, neutralizes intracellular peroxides, and electrophilic oxidants and
provides protection against oxidative stress; therefore, the intracel-
lular level of GST is used as important biomarker for monitoring in-
jury in tissues (Abolaji et al., 2014). The glutathione-S-transferase
(GST) activit y of emerged flies from larva fed with sucrose and the
leaves is shown in Figure 5a. An increase in GST activit y of the nor-
mal treated flies with a signific ant (p < .05) decrease in all the sucrose
fed flies when compared with the normal control. The low level of
GST activity obtained in the sucrose fed flies indicates that a high
level of damage might have taken place in the tissues of this flies due
to increased production of ROS. The slight increase obtained in all
the treated flies might be due to the ability of the moringa leaves to
quench ROS. The trend obtained in this result is in agreement with
previous studies where plant foods has been repor ted to increase
antioxidant activities in living organisms (Ademiluyi, Oyeniran,
Jimoh, Oboh, & Boligon, 2019).
Superoxide dismutase (SOD) converts superoxide anion to com-
pounds with lesser damaging effect s, and then, catalase converts
these lesser damaging compounds to water. Previous studies have
reported that this mechanism is important for lifespan ex tension and
survival of the fruit flies (Sharma, Gescher, & Steward, 2005; Suckow
& Suckow, 2006). Figure 5b depicts the SOD activity of the emerged
flies from larva fed with sucrose, parasitic, and nonparasitic moringa
leaves. The infested moringa leaves had significant (p < .05) ele-
vated level of SOD in the normal flies, this might be attributed to the
growth of mistletoe on the moringa leaf and thereby enhancing its
activity. A general reduction was obtained in the normal and sucrose
controls and this was upregulated in all the treated groups. This
might be due to the presence of the reported phytochemical con-
stituents in the moringa and mistletoe leaves (Ademiluyi et al., 2018;
Oboh, Omojokun, & Ademiluyi, 2015). This trend is in agreement
with the report of Akinyemi, Oboh, Ogunsuyi, Abolaji, and Udofia
(2017) whereby curcumin (a plant food) was found to increase SOD
activity in flies. The same trend of result was also obtained for the
activity of catalase in the flies (Figure 5c), this is expected as catalase
is responsible for the complete conversion of superoxide anion to
water.
3.5 | Effects of infested and noninfested moringa
leaves on enzyme activities
Since the flies used in this study was fed with diet supplemented
with sucrose, it is therefore paramount to assay for the activities of
carbohydrate hydrolyzing enz ymes. Report has shown that amylase
and sucrase are the major carbohydrate hydrolyzing enzymes found
in fruit fly (Haloi, Hasan, & Boro, 2014; Szyszka & Galizia, 2018).
α-amylase is involved in the hydrolysis of starch and other polysac-
charides to disaccharides and α-glucosidase is responsible for the
breakdown of disaccharides such as sucrose to absorbable monosac-
charides in living organisms. The result of α-amylase activity of the
emerged flies from larva fed with sucrose and moringa (infested and
noninfested) leaves is presented in Figure 6a. A significant (p < .05)
decrease was obser ved in the α-amylase activity of normal treated
flies when compared with the normal control but a slight decrease
was obtained in the diabetic treated fruit flies when compared with
the sucrose control. The α-glucosidase activity of the emerged flies
from larva fed with sucrose, infested, and noninfested moringa
leaves is shown in Figure 6b. A significant (p < .05) decrease in the
α-glucosidase activity of the treated normal flies with a significant
(p < .05) increase in the flies fed with 30% of sucrose was obtained
when compared with the normal control. A significant (p < .05) de-
crease was also obser ved in all the treated flies fed with sucrose
when compared with the sucrose control. The significant reduc-
tion (p < .05) obtained in α-amylase and α-glucosidase activities
of the normal and sucrose fed treated flies might be attributed to
10 of 14
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OYENIR AN Et Al.
the potent phytoconstituents present in these leaves as previous
reports has suggested that phytochemicals in many medicinal and
plant foods interacts with critical enzymes involved in carbohydrate
metabolisms (Ademiluyi & Oboh, 2012).
Studies have shown that patients with hyperglycemia have an
increased risk of developing neurodegenerative diseases when
compared with individuals without hyperglycemia (Biessels
et al., 2006; Brands, Biessels, de Haan, Kappelle, & Kessels, 2005).
It was therefore imperative to evaluate the effects of these leaves
on some key enzymes that are relevant to neurodegeneration. The
cholinergic system is poorly affected in hyperglycemic conditions
and this results in increase cholinesterase activity with subsequent
FIGURE 5 (a) Glutathione-S-transferase (b) Superoxide dismutase and (c) Catalase activities of emerged flies from larva fed with sucrose,
infested and noninfested moringa leaves. Values represent mean ± SD (n = 5). CTR , control; IM, infested moringa; NIM, noninfested moringa;
P, parasite; # represents significant difference at p < .05 from normal control; * represents significant difference at p < .05 from sucrose
control
|
11 of 14
OYENIRA N Et Al.
cognitive decline (Capiotti et al., 2014). Figure 6c shows the influ-
ence of treatment on AChE activit y of emerged flies from larva fed
with sucrose and the leaves. There was a significant (p < .05) rise in
the AChE activity of the flies fed with 30% of sucrose when com-
pared with the normal control and a significant (p < .05) decrease
was obtained in all the treated groups when compared with the
normal and sucrose control. Therefore, the significant (p < .05) in-
creased AChE activity obtained in the 30% of sucrose control might
be indicate to an ongoing neurodegenerative condition resulting
from hyperglycemia in the flies. The significant (p < .05) reduction
obtained in the cholinesterase activity of the treated flies suggests
the possible modulatory effects of these moringa leaves which
might occur via reduction in oxidative stress and thereby improving
the cholinergic system (Chatchada et al., 2013). The presence of mis-
tletoe infestation on the moringa leaf resulted in a slight decrease
in the AChE ac tivity in the three main groups. Report from previous
FIGURE 6 (a) α-Amylase (b) α-Glucosidase (c) Acetylcholinesterase and (d) Monoamine oxidase activities of emerged flies from larva
fed with sucrose, infested and noninfested moringa leaves. Values represent mean ± SD (n = 5). C TR, control; IM, infested moringa; NIM,
noninfested moringa; P, parasite; # represents significant difference at p < .05 from normal control; * represents significant difference at p <
.05 from sucrose control
12 of 14
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OYENIR AN Et Al.
studies has shown that neurodegeneration could be improved by fla-
vonoids, therefore, the improvement of cholinergic system by mo-
ringa and mistletoe leaves may likely be attributed to it s repor ted
phenolics and flavonoid content (Ademiluyi et al., 2018; Youdim &
Joseph, 2001).
MAO is involved in the mechanism of dopaminergic transmis-
sion and its regulatory role is critical to neuronal function. This en-
zyme system is involved in the oxidative deamination of biogenic
amines such as dopamine, serotonin, histamine and catecholamine
(i.e., dopamine, epinephrine, and norepinephrine) to their corre-
sponding aldehyde and free amines. Therefore, MAO inhibitors will
prevent the oxidative deamination of monoamines. These mono-
amines must be kept within physiologic levels as they are import-
ant for brain development and functions ( Thomas, 200 0; Ramsay
& Gravestock, 2003). The MAO activit y of the emerged flies from
larva fed with sucrose, infested and noninfested moringa leaves is
shown in Figure 6d. The significant (p < .05) elevated activit y of
MAO obtained in the flies fed with 3 0% of sucrose suggests brain
damage and a slight decrease was obtained in the groups treated
with the moringa leaves. This could likely be attributed to the
presence of phytochemical constituents such as caffeic acid, quer-
cetin and kaempferol in moringa and mistletoe leaves (Ademiluyi
et al., 2018; Oboh et al., 2015).
4 | CONCLUSION
This study shows that mistletoe infestation did not alter the anti-
oxidant, antidiabetic, and neuroprotective ef fect s of moringa leaf,
hence, infested or noninfested moringa leaf could be taken as func-
tional food to mitigate several metabolic diseases.
CONFLICT OF INTEREST
The authors declare that they do not have any conflict of interest.
ETHICAL APPROVAL
International, national, and/or institutional guidelines for the care
and the use of laboratory animals were followed.
ORCID
Olubukola H. Oyeniran https://orcid.org/0000-0001-5262-7487
Adedayo O. Ademiluyi https://orcid.org/0000-0001-8325-1304
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How to cite this article: Oyeniran OH, Ademiluyi AO, Oboh
G. Modulatory ef fect s of moringa (Moringa oleifera L.) leaves
infested with Afric an mistletoe (Tapinanthus bangwensis L.) on
the antioxidant, antidiabetic, and neurochemical indices in
high sucrose diet-induced diabetic-like phenotype in fruit
flies (Drosophila melanogaster M.). J Food Biochem.
2020;00:e13318. https://doi.org/10.1111/jfbc.13318
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