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NUTRITIONAL, ANTIOXIDANT, ANTICHOLINESTERASE AND

NEUROPROTECTIVE POTENTIAL OF THE HYDRO-ETHANOLIC EXTRACT OF
GUDDAIM FRUITS

Authors: Abdel Hamid Moustapha, Kuate Dieudonné, Njapndounke Bilkissou, Djuiné

Vanessa, Woumbo Cérile, Tekou Florian, Bendounga Rodrigue

Abstract

Introduction

Change in feeding Food consumption habits and lifestyle in general are nowadays
recognised to be the main cause of many non- communicable diseases, including
neurodegenerative disorders (Juliette, 2015). Neurodegenerative diseases mainly affect the
elderly, with an updated prevalence of 46.8 million cases (Martin et al. 2015). Even if the
clear mechanisms leading to these disorders are still unexplained, it is well accepted that
aggregation of B-amyloid and tau proteins, reduction brain acetylcholine concentrations,
glutamatergic deficiency, oxidative stress and/or inflammatory processes play an important
role in their development (Foyet et al., 2015). It should be highlighted that, Ooxidative stress,
which account as major one of the main causes of these neurodegenerative diseases, is
generally due to prolonged exposure to heavy metals, pesticide residues, patients' lifestyle
(Juliette, 2015), smoking and drugs (Pastre, 2005; Juliette, 2015),). To treat these diseases,
In their fight against these illnesses, many efforts have been made by the scientists are trying
to prolong the bioavailability of acetylcholine in the brain (reference). Drugs such as
donepezil, rivastigmine, galantamine and, memantine, and known as acetylcholinesterase
and/or gamma secretase inhibitors have been designed (reference). However, their
expensiveness, adverse effects, non-selectivity, limited efficacy and low bioavailability are
some problems still to be solvedpose problem to the patient (reference). Also, the precise and
early diagnosis of the disease, the heaviness of clinical trials and the stage of the pathology,
the irreversibility of neuronal loss make the treatment difficult (Adefegha, 2012). Thus there
is need to search for alternative methods for the treatment of such disorders.
Therefore, the ideal alternative would be a good prevention of such disorders. This can
bealternative methods have focus on done by fighting against their main cause which is
oxidative stress either by consumption of fnctional drinks or functional foods rich in
polyphenolic compounds and some vitamins known to possess high antioxidant and anti-
inflammatory power (Desport, 2002; Zhao, 2007). In this wayit is in this token that, several
studies have been made in order to promote alternative approaches through the
consumption of fruits and vegetables which are available and accessible at low cost.
Foyet et al. (2015) demonstrated the effectiveness of the hydroalcoholic extract of
Emilia coccinae in significantly improving the memory of rats, reversing amnesia and
decreasing the activity of acetylcholinesterase while increasing the acetylcholine levels
in the brains of rats treated with scopolamine. Also,; Zerrouki (2017) demonstrated the
effectiveness of extracts from plants of the genus Hypericum in significantly improving
behavior, memory as well as a clear reduction in nerve tissue damage in rats exposed to
aluminum chloride.
Grewia tenax (gudaim, in Arabic), is an edible fruit tree, of the Tiliacea family
(FAO, 1988). It’s fruits (gudaim) are small, round, sweet berries that can be eaten fresh
or dried (Dod, 1978), or made into a fermented drink. ((Dod, 1978; FAO/WHO, 1988).
This plant has been the subject of several studies, such as work done by Al-Said et al.
(2011) and Jose et al. (2017) which respectively focused on the efficacy of extracts of
these fruits in combating hepatic toxicity, induced in wistar rats by carbon tetrachloride
(CCl4), skin whitening and aging induced by radicals. The previous authors stated that
fruits of Grewia tenax was rich in polyphenolic compounds
Endowed with immense therapeutic virtues, can we say that the hydro-ethanolic extract
of the fruits of guddaim have an inhibiting activity of acetylcholinesterase and a
neuroprotective activity in rats induced by scopolamine? This study was based on the
assumption that the hydro-ethanolic extract of guddaim fruits exerts a neuroprotective
activity in scopolamine treated wistar rats. So, the present work has the general
objective to study the antioxidant, the anticholinesterase and the neuroprotective
properties of the hydro-ethanolic extract of the fruits of Guddaim in scopolamine
treated wistar rats.
A la fin de introduction faut nous dit c manquement de ton travail pr k’on puis connaitre
que c que tu veux faire est nouveux
Abdel regard les article que je t’avais donne toi et rodrigue tu vois comment a la fins ils
s’ont aborde leur objective specific
I. Material
I.1.Plant material
Guddaim fruits were harvested in January 2021 in Abéché, in the Ouaddaï rRegion in eastern
Chad. After a survey of the said fruit among the rural population, the fruits have been
identified and identification confirmed by Professor Ngaryo Fidèle Tonalta, Professor of
Systematic Botany, Dean of the Faculty of Science and Technology of the Adam Barka
University of Abéché (UNABA), Chad. The fruits were then wrapped in an airtight plastic
bag and taken transported to the Research Unit of Biochemistry of Medicinal Plants, Food
Sciences and Nutrition (URBPMAN) of the University of Dschang, Cameroon.
I.2.Experimental animals
Thirty (30) albino wistar rats of the Wistar strain, each with an average mass of 300g at the
start of the experiment, were used. obtained from the biochemistry animal facility of the
University of Dschang, Cameroon. The animals were housed in basins covered with small
mesh netting (1 rat/cage). They were raised under a nychthemeral cycle (12 hours of light and
12 hours of darkness) said animal house.
Conduct of the trial and experimental device
The animals were fasted for 8 hours before each the treatment. However, all animals were
allowed to drink water ad libitum (Foyet et al., 2015). Efforts were also made to minimize
animal suffering and reduce the number of animals used in the experiment. Each animal was
tested in a single behavioral test. The experiments were carried out in the morning. Cages,
drinkers, feeders and the experimental room were cleaned daily to ensure effective hygiene of
the place and the animals. Rats were treated according to the guidelines of the Cameroon
Bioethics Committee (reg N°.FWA IRB00001954) and according to the NIH-Care and Use of
Laboratory Animals manual (8th Edition).

I.3.Chemicals
Ici pour chaque reactive faut dit son utilite je te conseil de regarde les article que je te donne
kan tu manipulais

Acettylcholinesterase; Scopolamine hydrobromide trihydrate; Tacrine; Acetylthiocholine

iodide; DPPH; 5, 5-dithiobis (2-nitro-benzoic acid) (DTNB) were purchased from Sigma-
Aldrich, Germany. The extracts and reagents were prepared daily. Scopolamine (dose) as a
disease inducer was given to the rats intraperitoneally (ip) unlike the extract, saline and
tacrine which were given to the latter by gavage to the different animals in the different
groups.
.

II. Methods
II.1. Obtaining powdersProduction of guddiam fruit powder
The powders wasere obtained by the method described by Elmurez et al., (2014) with a slight
modification to the drying conditions. Breily, Gguddaim fruits were placed in a tall glass and
washed in tap water, then in distilled water. They were sorted, then dried in an air oven at
45°C until constant weight. The dried fruits were then ground into a powder using a blender
(power, time), then sieved to finally have a good G. tenax fruit powder. The latter was placed
in plastic bags and stored until use.
Production of fruit extract and evaluation of yieldExtraction
The extraction was done according to the method described by Iqbal et al. (2007). Ten grams
(10g) of powder were macerated in 100 ml in (hydo-ethanolic) solvent placed at room
temperature for 48 hours. The mixture was then filtered using whatman N˚1 paper then. After
filtration, the filtrates were placed in an oven at 45°C. until a constant mass was obtained. The
extracts were then stored in the freezer at -4°C for later use.
The extract was is then weighed to calculate the yield.

Yield (%) = Mass of extract×100 / Mass of powder

Evaluation of the Physico-chemical analysisNutrititional composition

The bromatological composition was determined according to the AOAC (1990) method.

Extraction
The extraction was done according to the method described by Iqbal et al. (2007). 10g of
powder were macerated in 100 ml in solvent (hydo-ethanolic) placed at room temperature for
48 hours. The mixture was then filtered using whatman N˚1 paper then. After filtration, the
filtrates were placed in an oven at 45° C. until a constant mass was obtained. The extracts
were then stored in the freezer at -4°C for later use.
The extract is then weighed to calculate the yield.

Yield (%) = Mass of extract×100 / Mass of powder

II.2. Evaluation of the phytochemical composition
The macro and microelements were determined by the AOAC 1990 method.

-Determination of the total phenolic content

The total phenolic content of the extracts was determined by the spectrophotometric method
using the Folin-Ciocalteu reagent as described by Dohou et al. (2003). The experimental
protocol is summarized as follows:Briefly, in a test tube, 10 μl of the extract a solution of at
extract with a concentration of 2 mg/ml were introduced. Then, 1.39 ml of distilled water and
0.2 ml of Folin-Ciocalteu reagent were successively added. After 3 min of rest, 400 μl of
sodium carbonate (Na2CO3, 20%) were added. The tubes were vortexed and incubated for 20
min in a 40°C water bath. Absorbance was read against a blank at 760 nm using a
spectrophotometer. Calibration was carried out using a prepared aqueous solution of gallic
acid (0.2 g/l).

II.3. -Determination of the total flavonoid content

The flavonoids content were assayed by colorimetry. Flavonoids in the sample react with
aluminum trichloride and potassium acetate to form a pinkish colored solution. The flavonoid
content was determined according to the method described by Bahorun et al. (2006) and
summarized as follows: 100 μl of extract with a concentration of 2 mg/ml, 1.4 ml of distilled
water and 30 μl of a 5% sodium nitrite solution were successively introduced into a tube
( NaNO2). After 5 min, 200 μl of a 10% aluminum trichloride (AlCl3) solution was added,
followed by 200 μl of 10% concentrated sodium hydroxide (NaOH) solution and 240 μl of
distilled water. The whole was stirred using a vortex and the absorbance was read at 510 nm.

II.34. Measurement of antioxidant and anti-radical parameters

DPPH radical inhibitory assay
The antioxidant activity of the different extracts was evaluated by the methodthrough the
using DPPH (2, 2-diphenyl-1-picryl1-hydrazyl) scavaging activity, as described by Mensor et
al. (2001). The ability to trap the hydroxyl radical of the extract was compared with that of
vitamin C. The extracts were prepared at different concentrations (in μg/ml). 100μl of ethanol
were introduced into the tubes except the first line. 900 μl of DPPH solution were introduced
into the first two tubes. 900μl of pure ethanol were introduced into the tubes except the blank.
For each concentration, the test was repeated twice. The whole was placed at room
temperature and in the dark for 30 minutes. The reading was made by measuring the
absorbance at 517 nm by a spectrophotometer. For each dilution, blanks were prepared under
the same conditions. The result is expressed as follows:

absorbance du contôle−absorbance de l ' échantillontest

%I= ×100
absorbance du contrôle ( DO DPPH )

FRAP assay
The assay was realized using the protocol described by Oyaizu (1986). In test tubes
previously containing 100 μl of extract solution at different concentrations (in μg/ml),
prepared in the hydro-ethanolic solution (25:75), 500 μl of a phosphate buffer solution (0.2 M
pH 6.6) were added, followed by 500 μl of aqueous solution of potassium hexacyanoferrate
[K3Fe (CN)6] (1%). The mixture was incubated for 30 minutes at 50°C in a water bath.
Afterwards,This was followed by the addition of 500 μl (10%) of 10% trichloroacetic acid.
solution were added. The mixture was centrifuged at 3000 rpm for 10 minutes using a
centrifuge. A standard metal reducer (vitamin C) was prepared under the same conditions to
compare the reducing power of the different extracts. The blank consisted of all reagents
except the extracts. The absorbance of the reaction mixture was read at 700 nm against that of
the blank and the tests were carried out in triplicate. An increase in absorbance corresponds to
an increase in the reducing power of the extracts tested (Hubert, 2006). The result is was
expressed in mg/ml of extract. A standard metal reducer (vitamin C) was prepared under the
same conditions to compare the reducing power of the different extracts. The blank consisted
of all reagents except the extracts. The absorbance of the reaction mixture was read at 700 nm
against that of the blank and the tests were carried out in triplicate. An increase in absorbance
corresponds to an increase in the reducing power of the extracts tested (Hubert, 2006). The
result is expressed in mg/ml of extract.

II.5. In Vitro Acetylcholinesterase inhibitory Assay

Acetylcholinesterase inhibitory activity of the extract was evaluated using the
modified method of Rahman and Choudhary (2001). The tests were carried out in triplicate.
The extracts (2mg/ml) were freshly prepared at 4 different concentrations; the working
solution was kept on ice throughout the manipulation; the work equipment has been sterilized
after each manipulation; the tests were carried out under aseptic conditions. The experiment
was carried out as described in table 1.
Table 1: In Vitro Acetylcholinesterase Inhibition Protocol
 Extract concentrations (μg/ml)

Reagents 25 50 100 200

ATCI (12mM) 10μl 10μl 10μl 10μl

DTNB (12mM) 10μl 10μl 10μl 10μl

Extract (2mg/ml) 10μl 10μl 10μl 10μl

Tris HCl buffer (0.1 mM,

950μl 950μl 950μl 950μl
pH=8)

AChE (0.1 UI/mL) 20μl 20μl 20μl 20μl

Read at 412 nm after every 60 seconds 5 times in a row

AchE: Acetylcholinesterase ; ATCI: Acetylthiocholine Iodide; DPPH : 2,2-diphenyl-1-picrylhydrazyl; DTNB:

5,5'-dithio-bis-(2-nitrobenzoic acid)

The cholinesterase inhibitory percentage was calculated using the following formula:

Inhibitory activity ( % )= ( 1−sample

control absorbance )
absorbance
X 100

II.6. Evaluation of Sspontaneous alternation behaviour

The Y-maze analysis was found to be a reliable and non-invasive test for determining
cognitive changes in Wistar rats by measuring spontaneous alternation behaviour in the Y-
maze task. In this study, the maze used consists of three arms (35 cm long, 15 cm high and 10
cm wide) covered with mesh and an equilateral triangular central area. The different groups
were tested in sequence throughout the experiment. Rats were treated once daily with the
most active extract of G. tenax (200 and 400 mg/kg), tacrine (3 mg/kg), or saline (10 ml/kg)
for 14 days consecutive. Thirty minutes after administration of the aforementioned drug; the
rats were placed at the end of one arm and were allowed to move freely through the maze for
8 min. An arm entry was counted when the rat's hind paws were completely within the arm.
Spontaneous alternation behavior was defined as three consecutive entries into three different
arms (i.e., A, B, C or B, C, A, etc.). The percentage of alternation was calculated using the
following formula:
total number of alternations
Alternation (%)¿ X 100
number of entries−2
In Additionally, the total number of arm entries was used as measurement of animal activity.
The maze was cleaned with 70% ethanol between each animal to minimize cue odors.

II.7. Behavioural test

The modified method of Foyet et al. (2015) was used.
The rats were randomly divided into five groups of 6 rats each as follows: Group I
received saline and serve as the normal control; Group II received scopolamine (1.5
mg/kg) administered intraperitoneally and served as positive control. Both groups
received normal saline for 14 days. Group III received tacrine (3 mg/kg), group IV and V
were treated with the extract at respective doses of 200 and 400 mg/kg, for 14 days.
Scopolamine (1.5 mg/kg) was administered at single dose 30 min on days 1, 4, 8 and 14
after drug or extract administration to all groups except the normal control group, to
induce memory impairment. Behavioural tests were carried out on days 8 and 14 after
the administration of the extract, 30 min after the injection of scopolamine.
Euthanasia, collection of blood samples and organ (brain)

Twenty-four hours after the last administration, After 14 day, the animals were sacrificed
euthanized, blood and organs (brain) collected .under light anesthesia using ketamine and blood
collected in dry tubes by cardiac puncture.

II.8. preparation Serum preparation and organ homogenates

thAfter sacrificing the rats, the brains of the different animals were removed and then ground in a
cold environment (+4°C). The collected blood were also centrifuged at 3000 rpm for 10 min to
obtain the serum For the brain, 0.5 g of the brain was weighed and introduced into a lab mortar
containing fine sand. The phosphate buffer (0.1 M; pH 7.4) at 10% (mass/volume) was gradually
added to the mortar during the grinding until a ground material was obtained. The ground material
was then centrifuged at +4°C at 3500 rpm for 30 min. The supernatant was collected and kept at -
18°C for subsequent assay of the biochemical analysis. Blood were also centrifuged at 3000 rpm
for 10 min and the serum collected.

III. In-vivo testing

III.1. Determination of brain acetylcholinesterase activity
Acetylcholinesterase activity was determined by the method of Ellman, adapted for use in
microtiter plates as described by Haigh et with slight modification al. and using ATCI as a
1mM final concentration substrate to measure acetylcholinesterase activity. Working
solutions were prepared and used the same day and kept on ice during use.
 Table 2: Protocol brain acetylcholinesterase activity assay

Reagents Trials Blank

Working Solution 1.2ml 1.3ml
Serum 0.1 0
Substrate (ATCI) [1mM] 0.2 0.2
Read the optical density against blank at 412 nm for 3min T0-T3 (T0 = 30 s; T3 = 210 s; ɛ
= 13.6mM-1 .Cm-1
Working solution: mixture
- 9.75 ml of 0.1M sodium phosphate, pH=8, containing 1Mm of EDTA
- 0.25 ml of 10 mM DTNB
(dT )×V
Results: Activity=
ε × b ×Vs × nbre de proteine total

With: V is the total volume of the solution and Vs is the volume of the blank

III.2. Evaluation of serum Pprotein estimation

Proteins were measured in all brain samples by the method of Lowry et al (25). Bovine serum
albumin (BSA) (1 mg/ml) was used as a standard and measured in the range of 0.01-0.10
mg/ml.

III.3. Malondialdehyde (MDA) assay

It is the best known and most widely used indicator of peroxidation. It results from the
degradation of hydroperoxides formed during the peroxidation of polyunsaturated fatty acids,
by cyclo-oxygenase, by the interaction of the hydroxyl radical with vitamin C or with
deoxyglucose. It was assayed according to the protocol of Yagi (1976). 100 μl of homogenate,
500 μl of 1% thiobarbituric acid (TBA reagent) prepared in 1% trichloroacetic acid and 500 μl
of 1% phosphoric acid were introduced into the test tubes. The mixture was incubated at
100°C in a water bath for 15 minutes, then cooled on ice for 30 minutes. The mixture was
centrifuged at 3000 rpm for 10 minutes after cooling and the absorbance of the supernatant
was read at 532 nm against the blank which contained 500 μl of TBA solution, 500 μl of
phosphoric acid and 100 μl of distilled water incubated under the same conditions. The
concentration of MDA was determined using its molecular extinction coefficient (ε =
1.53x105 M.cm-1).
III.4. Evaluation of Rreduced glutathione dosage
The reduced glutathione assay was carried out according to the method described by Ellman.
(1959). Briefly, 100 μl of homogenate and 900 μl of Ellman's reagent prepared in Tris-HCl
buffer (0.1 M, pH 6.5) were respectively introduced into the test tubes. After homogenization,
the mixture was incubated at room temperature for 30 min. The absorbances were read at 412
nm against the blank which contained 900 μl of the reagent solution and 100 μl of 0.9% NaCl
incubated under the same conditions. The concentration of thiol group (SH) was determined
using the molecular extinction coefficient of DTNB (ε=1.36×105 M-1.cm-1). The results
were expressed in μmol/g of total proteins.
IV. Statistical analysis
Parameters values were expressed as the mean ± standard error of the mean. After analysis of
variance, the comparison of means between different groups was carried out by the Waller-
Duncan test using SPSS version 20.0 software. P values < 0.05 were considered statistically
significant.
V. Results
V.1. Nutritional composition of Guddaim fruits
The proximate composition of Guddaim fruits is given in table 3. It shows that Guddaim is
rich in carbohydrates, fibre and calcium.

Table 3: Proximate composition of guddaim fruits

Macro-elements (g/100g)
Proteins fibers Ashes Lipids Carbohydrates
8.32 9.72 6.4 5.53 69.93
Minerals (mg/100g)
Sodium Calcium Magnesium Iron Zinc
110.56 1012.02 106.65 6.04 7.68

Nutritional parameters Value

Proteins 8.32
fibers 9.72
Ashes 6.4
Lipids 5.53
Carbohydrates 69.93
Sodium 110.56
Calcium 1012.02
Magnesium 106.65
Iron 6.04
Zinc 7.68
Ici c un exemple comment tes tableau dois etre
V.2. Phytochemical parameters tests; antioxidants and anti-radicals
The total phenolic and flavonoids contents of the hydro-ethanolic guddam fruits
extract were respectively 91.5±0.02 mg GAE/g and 3.39±0.95 mg CE/g.
The extract showed concentration-dependent DPPH radical scavenging activities
varying from 19.32% to 88.32% with an IC50 of 13.36 μg/ml.

This activity is strongly correlated with the phenol and flavonoid contents (in EAGT/g of
extract, 91.5 and 3.39 respectively). These analyzes indicate that there is a linear relationship
between the antioxidant activity and the total contents of phenols and flavonoids. Statistical
analysis shows that the scavenging activity of ascorbic acid (vitamin C) was significantly
stronger than that of the extract at all concentrations (Figure 1).

Ta pas interprete les figures

120 a
v d n
radical inhibition in

100 v d d
b s s d
80
a a b
percent

60
t t
40
20
0
0.125 0.25 0.5 1 2

extract concentration in mg/ml

GHE Vita C

Figure1: Variation in the anti-radical activity of the HE extract of G. tenax at different

concentrations compared to vitamin C.

Figure 2 shows the variations of the reducing power of guddam extract at different
concentrations compared to BHT. The extract presented low ferric reducing power.
a
n f
d
c
o
d m
a v a v d d
t s b i
t s c
s
 1.2
Absorbance at 700 nm 1
0.8
0.6
0.4
0.2
0
12.5 25 50 100 200

extract concentrations in µg/ml

GHE BHT

Figure 2: Variation of iron-reducing power of HE extracts of G. tenax at different concentrations

compared to BHT.

V.3. Acetylcholinesterase inhibitory capacity of guddaim fruit extract

Acetylcholinesterase inhibitors are currently an essential therapeutic approach for
neurodegenerative diseases. The hydro-ethanolic (HE) extract of G. tenax showed significant
acetylcholinesterase inhibitory activity. This activity varies from 87.70% to 94.37% for
concentrations ranging from 25 to 200 mg/ml. Guddam extract better inhibit
acetylcholinesterase than tacrine (10μM) which has a maximum inhibitory percentage of
87.29%.

96
b
inhibition percent (%)

94 ab
92 ab
90 a a
88 t t
86
84
82
25 50 100 200 tacrine
10µM
Extract concentrations

GHE (2mg/ml)

Figure 3: Inhibitory activity of the acetylcholinesterase of the HE extract of G. tenax.

V.4.Effect of the extract on the memorative memoring capacity

Figure 4 shows the memory testThe results obtained during the Y-maze reference memory
test are shown in the figure below, on days 8 and 15 of the test. No significant difference
emerges between the percentage of alternation of the different groups of rats on the 7th day of
treatment. On the other hand, it can be highlighted that,we note that on day 14, the animals
having received an untreated induced animals have a low rate of alternation compared to the
normal group and those having received the extract. Also, the group that received the high-
dose extract alternated more than the other groups.

spontaneous altternation (%)

23.5 b
23 ab
ab
spontaneous altternation (%)

35
22.5 a ab 30
22 25
t 20
21.5
15
21 10
20.5 5
e e e 00 00 0
qu in
cr
in
E2 E4
ogi l a m a H H
l T
io po G1 G1
hys s co
p
u
Ea
7 days of treatments 14 days of treatments

figure4: Activity of the hydro-ethanolic extract of Guddaim fruits and of tacrine on the locomotor activity
of animals in the Y-maze task.

V.5. Influence of the extractEffect of Guddaim fruits extract on some biochemical

parameters
V.6. Malondialdehyde
The MDA concentrations in the brain of treated rats are shown on figure 5. The
positive control had a high MDA concentration while the extracts at different concentrations
decreased it.
MDA Concentration In µM

0.7 a
0.6 a t a
a a
0.5 t t
t t
0.4
0.3
0.2
0.1
0
Eau phys- Scopolamine( Tacrine GHE GHE
iologique 1,5mg/kg) (3mg/kg) 200(mg/kg) 400(mg/kg)

Figure 5: Variation in the concentration of malondialdehyde in the brain of rats

Kan je vois l’analysis statistic des groupe pour le parameter MDA ya pas de difference
significative? Pour dit tes extrait n’ont rien faire ?
V.7. Total protein
The total protein of the rat treated with Guddaim fruits extract are shown in From
figure 6, it can be see highlighted that scopolamine, induced a significant (P<5%) reduction
of thein the total protein concentration in the brain of rats. However, whiletreatment of rats
with tacrine and different concentration of the extracts at all doses had protein concentration
not significantly different from the significantly increased it in comparison to normal control.

0.3
b
Total protein level in g/dl

0.25

0.2 b b
a b
0.15
t
0.1

0.05

0
normal non traité tacrine GHE200 GHE400

tacrine concentration 10µM

extracts concentration: 2mg/ml

Figure 6: Variation in the level of total proteins in the brain of rats

V.9. Reduced glutathione

The figure 8 shows the level of glutathione (in µmol/g of tissue) in the brain of rats.
The untreated group hads the lowest a low rateglutathion level compared to the other groups.
The normal group hads a high level of glutathione in the normal group and then in both
groups treated with low and high dose extracts.
 100
glutathion level in µmol/g of
90
80
70
60
50
tissu

40
30
20
10
0
normal non traité tacrine G1HE200 G1HE400

tacrine Concentrations 10µM

extracts Concentration : 2mg/ml

V.8. invivo Acetylcholinesterase activity

The following Ffigure 7 shows the activities of acetylcholinesterase in the brain of
rats. From this figure, wet can be reveal that, notice that the untreated group hads a higher
activity compared to the other groups. Rats treated with the high-dose extract showed the
lowest activity.

0.9
v
0.8
Activity in µmoles/min/mg

s b
0.7
0.6
a
t ab a
of protein

0.5
0.4
t
0.3
0.2
0.1
0
NormalScopolamine Tacrine G1HE200 G1HE400

figure7: Variation in the enzymatic activity of acetylcholinesterase and tacrine in rats.

V.9. Reduced glutathione

The following figure shows the level of glutathione (in µmol/g of tissue) in the brain
of rats. The untreated group has a low rate compared to the other groups. The normal group
has a high level of glutathione in the normal group and then in both groups treated with low
and high dose extracts.
glutathion level in µmol/g of 100 v
90 s
80
70
60 b b
50
tissu

40 a ab
30
t
20
10
0
normal non traité tacrine G1HE200 G1HE400

tacrine Concentrations 10µM

extracts Concentration : 2mg/ml

figure8: Effect of extract on reduced glutathione levels in the brain of rats.

VI. Discussion
Neurodegenerative diseases are a public health problem today. The however, drugs use
for treatment developed are not only ineffective to treat of these diseases but are alsoare
expensive and sometimes have many side effects. In the present study, we studied the
influence of the harvesting area on the nutritional properties on the one hand and the
antioxidant properties on the other hand.
During this study, we realized the macroelements and some microelements in these
fruits. We note a protein content of guddaim fruits of 8.32%. This agrees with the results of
Boutros (1986), Eltom (2006), which are 08%; 08.4% respectively. As for the ash content, it
is 6.5%. It is higher than the data of Sati and Ahmed (2018) which is 3.55%. This difference
would in this case be due to edapho-climatic factors (Rathore, 1970 and Gautreau, 1973). For
the carbohydrate content, we notice a significant content. This is in agreement with the results
of Sati and Ahmed (2018). Concerning the fibers, we find a content correlating with the data
of Mustafa et al., (2011); Eltom (2006). We estimated the lipid content of our samples.
However, this results in a content of 5.53%. Which is comparatively low compared to other
macronutrients. En general pr bien discute les resultat des parametre nutritionelle faut donne
l’important de chaque parametre en relation avec la maladie a traite et evite juste de corrobore
les resultat, et kan i faut corrobore chercher les auteur rescent sa ne sert a rien d’avoir de
corroboration avec un ancien auteur, donc ta discussion et a refaite , regard les autre article
sava t’aide
As far as micronutrients are concerned, dosages have been made (in mg/100g),
Guddaim fruits are rich in potassium and calcium respectively of 1214.53 and 1012.02. The
latter is the same with the results of Mustafa et al., in 2011. The estimated iron content in the
fruits is considerable. It is relatively lower than the results of Eltom (2006). Regarding the
zinc content, these fruits have an encouraging content. It can participate in cell division,
participate in the transfer of nerve impulses, etc..
The total phenolic and flavonoids contents obtained in this study greatly exceed that of
Li et al. (2012) and Gupta et al. (2006).
The guddaim fruits’ extract had a high DPPH radical scavenging activity with an IC 50
is 13.38μg/ml. According to Souri et al., (2008), which stated that extract with IC50 lesser than
20μg/ml are highly activite, the extract were reported as high scavenger.
The iron-reducing power of this extract can be attribute to the presence of the hydroxyl
group in phenolic compounds such as flavonoids which can serve as electron donors, thus
reducing ferric ions to ferrous ions. As a result, the increase in ferrous ion would thus reflect
an increase in the reducing power of the extract. However, antioxidants are considered
oxidant reducers and inactivators (Sellal and Becker, 2007). Thus, the evaluation of the In
vitro antioxidant activity of the various extracts of the fruit of guddaim allowed us to conclude
that these fruits are endowed with phenolic compounds known for their biological activities.
For this purpose, the hydro-ethanolic extract from the town of Abéché which shows
Acetylcholinesterase inhibition has become a widely used clinical approach to treat the
symptoms of Alzheimer's disease. Various plant extracts have been tested positive in in vitro
inhibition of acetylcholinesterase. They owe this power to their phenolic composition (Sanda,
2014). In this study, the hydro-ethanolic extract of the fruits of Grewia tenax from the sample
from Abéché was successfully tested in the in vitro inhibition of acetylcholinesterase. There is
indeed an inhibition percentage of at least 80%. This activity was compared to that of tacrine,
a reference drug often prescribed to people with Alzheimer's disease. From this comparison, it
appears that these extracts have an activity greater than that of the drug.
The induction of neurological disorders is made by scopolamine by intrapetonial
injection. Indeed, scopolamine, an anticholinergic, muscarinic receptor antagonist, readily
crosses the blood-brain barrier to induce antimuscarinic activity resulting in cholinergic
deficit and memory loss (Seihosseini et al., 2011). As an age-related decline in amnesic
function is believed to be primarily due to impaired cholinergic transmission, exposure to
scopolamine is experimentally applied to induce cholinergic disorder in animals to test the
level of medications for learning and memory disorders (Stone et al., 1988). In order to test
the capacity of the extract to fight against this disorder, our results showed that these extracts
improve the memory of rats in the Y maze test after, 7 and 14 days of administration. We do
not note a significant difference between the different groups after seven days of treatment.
On the other hand, on the 14th day, the rats having received the extract at different doses
show a more intense activity than on the 7th day of treatment. These results agree with those
of Foyet et al. (2015) and Foyet et al. (2019).
ROS are continuously generated in the nervous system during normal metabolism and
normal neuronal activity. The brain is regularly subjected to lipid peroxidation induced by
free radicals. It is also known that the protective system of the brain is weak against oxidative
stress, compared to other tissues (Jeong et al, 2014). Administration of scopolamine to rats
resulted in increased malondialdehyde and protein levels; and an increase in reduced
glutathione in untreated diseased rats. However, we note low levels in rats that received the
extract at low and high doses. This means that the administration of the hydo-ethanolic extract
of the fruits of Grewia tenax fights against lipid peroxidation in rats due to oxidative stress.
This is due to the aforementioned phytochemical composition of the extract. These results are
in line with those of Foyet et al. (2019).
The administration of scoplamin to rats significantly increased the level of AchE in their
brains. We note, however, a decrease in the activity of the latter in the brains of rats having
received the extracts. This result confirms the ability of this extract to inhibit the activity of
the acetylcholinesterase tested above.
Conclusion

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