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In vitro Evaluation of Antioxidant and Anti-sickling properties of Theobroma

Cacao (Sterculiaceae) extracts from East and South regions in Cameroon

Article · May 2018

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Academia Journal of Medicinal Plants 6(5): 079-090, May 2018
DOI: 10.15413/ajmp.2018.0114
ISSN 2315-7720
©2018 Academia Publishing

Research Paper

In vitro evaluation of the antioxidant and anti-sickling potential of cocoa

(Theobroma cacao) from two regions in Cameroon (South west and Littoral)

Accepted 14th May, 2018

ABSTRACT

Sickle cell anaemia is a genetic disorder which causes the expression of defective
haemoglobin resulting to irregular and shapeless red blood cells, known as “sickle
cells”. Sickle cells cause problems in the body, often blocking blood flow, causing
painful attacks, stroke and constitute a main cause of oxidative stress process.
Many treatments have been developed to improve the management of this disease.
Moreover, the high cost of these therapies and several other disadvantages does
not allow the patients to benefit from them. The present study aims to evaluate the
anti-sickling potential of Theobroma cacao (T. cacao) extract from South West /
Littoral regions of Cameroon. T. cacao was collected from Penja and Manfe and
then subjected to both hydroethanolic extraction at pH 3 and water, following
extracts (PH, PA, MH and MA). The antioxidant potential of the extract was
evaluated by measuring free radical scavenging potential (DPPH and ABTS), the
Biapa Nya Prosper Cabral1*, Jesse ability of iron reduction (FRAP) as well as, the quantification of total polyphenols
Juenkou Tekumu1, Chetcha Bernard2 and flavonoids. Moreover, the determination of the activities of catalase, SOD,
Tammo Floriane1, Yembeau Lena glutathione peroxidase, reduced glutathione and malondialdehyde help in
Natacha1, Nya Nkwikeu Prudence Josela1,
Kengne Fotsing Christian1 and Pieme
studying the protective property of T. cacao against oxidative stress. The anti-
Constant Anatole2 sickling activity, the osmotic fragility tests of these extracts and the principal
component analysis (PCA) was furthermore studied. PH, PA, MH and MA showed a
1Laboratory of Medicinal Plant high content of polyphenols and flavonoids. Moreover, these extracts excerpt a
Biochemistry, Food Science and Nutrition,
Department of Biochemistry, Faculty of
very strong radical scavenging activity with respect to the DPPH and ABTS
Science, University of Dschang, PO Box radicals, with IC50s between 3.24 and 6.35 µg/ml and between 4.87 and 19.29 µg/
67 Dschang – Cameroon. ml respectively, with an accent when cocoa extract from Manfe. The PCA revealed
2Laboratory of Biochemistry, Department
that the extract of Manfe showed a better ability to reduce sickling, to protect
of Biochemistry and Physiological
Sciences, Faculty of Medicine and
erythrocyte membrane from haemolysis and to regulate the enzyme activities
Biomedical Sciences, University of following the stress induced. These results suggest that cocoa bean extracts from
Yaoundé I – Cameroon, PO Box 1364 these localities particularly from Manfe could be used against sickling. More
Yaoundé-Cameroon. studies such as toxicological, in vivo antioxidant and antisickling are needed to
*Corresponding author. E-mail:
complete these data for the use of cocoa in the management of sickle cell anaemia.
prbiapa@yahoo.fr. Tel: 00237
675965283. Keywords: Sickle cell anaemia, oxidative stress, antioxidant, Theobroma cacao.

INTRODUCTION

Sickle cell anaemia (SCA) is an illness which results from a transforms normal adult haemoglobin (Hb A) into
mutation in a genetic code such that a single amino acid abnormal haemoglobin (Hb S). Under conditions of low
(glutamic acid) is replaced by another (valin) in the beta oxygen tension, Hb S molecules undergo aggregation and
globin chain of haemoglobin (Hb). This substitution polymerize and the red blood cells acquire a “sickle” or
 Academia Journal of Medicinal Plants; Cabral et al. 080

“holly leaf” shape which tend to block blood flow in the properties (NUTRA NEWS, 2008). Is the relationship
blood vessels. Hb S is considered as a pro-oxidant machine between cocoa compounds and the biological activity state
responsible for free radical formations which cause an extended in the case of sickle cell anaemia? The hypothesis
imbalance between the free radical and antioxidants and of the present study is that T. cacao extract possesses
then an oxidative stress in the body (WHO, 2010). antioxidant and anti-sickling properties. The general
SCA is predominantly found among African people, objective is to evaluate the anti-sickling potential of T.
Mediterranean, Arabic and Asian in relation to the survival cacao extracts from South West / Littoral regions of
advantage against malaria (Mabiala et al., 2005). Studies Cameroon in order to contribute to the new and safe
also indicate that approximately 1 in 12 African-Americans therapeutic strategy against sickle cell anaemia. More
are heterozygous for the disorder, and approximately 1 in specifically, it is to evaluate the antioxidant activity of cocoa
500 African-American newborns are diagnosed positive extract from two different regions of Cameroon (Littoral
annually (Boyd et al., 2005). The life expectancy of people and South West), to determine anti-sickling and the anti-
living with this pathology in developing countries is less osmotic fragility potential of extracts as well as, to
than 50 years of age. Annually, about 300,000 children are demonstrate the protective effect of extracts against the
born with it and about 75% of these cases occur (Makani et induced oxidative stress.
al., 2011). In Cameroon particularly, with respect to the
statistic given by World Health Organization and in
conformity with the national prevalence, 4000 children are MATERIALS AND METHODS
given birth annually with SCA and youths of about 10 to 29
years of age represent 89.2% of those suffering from this Plant collection and authentification
illness. This pathology leads to 4000 death each year in
Cameroon (DREPAVIE, 2013). Owing to the high rate at T. cacao beans were harvested in two towns in Cameroon:
which this disease is affecting the world, researchers are Manfe and Penja in February, 2017 and identified at the
still trying to investigate strategies in order to limiting the National Herbarium under the reference number
crisis state, complications as well as, to definitely cure it. 60071/HNC where the voucher specimen was deposited.
This led to a series of treatment such as the use of Beans were fermented and dried by specific methods
hydroxyurea which protects the body against oxidative according to each locality process. Once dried, they were
stress by increasing the amount of reduced glutathione, sorted, pulped and crushed in a blender to obtain the cocoa
stimulates the induction of haemoglobin F (Hb F) in order powder. The obtained powder was directly extracted.
to reduce Hb S formation and its effects (Flaurentino et al.,
2011).
Piracetam drug reduces the incidence of sickling crises by Preparation of T. cacao extracts
reversing the sickling process of erythrocytes (Al Hayeri et
al., 2011). However, the use of hydroxyurea in a long period The modified method of Benhammou et al. (2008) was used
can be toxic and produces some side effects such as leg for the extraction. 195 g of the cocoa beans powder were
ulcer, mouth ulcer and squamous cell carcinoma macerated in the mixture of ethanol-water (70v/30v) for
(Flaurentino et al., 2011). Stem cell transplant is another 48 h at pH 3 by the addition of a few drops of acetic acid.
opportunity to manage SCA complications but the cost is The mixture was stirred several times daily to maximize
very high and it faces also a problem of compatibility. Due extraction. After 48 h, the mixture was filtered using
to the aforementioned limitation and inconvenience, new Whattman paper No. 4 and 1. The obtained residue was re-
and safe therapeutics need to be checked. Most researchers extracted as earlier mentioned and the total filtrate dried in
have now turned on plant for their numerous life saving an oven for 24 h at 45°C.
and therapeutic properties. This is the case of Fagara
zanthoxyloides (Sofowora et al., 1971), Terminalia catappa
(Mgbemene et al., 1999), Carica papaya (Ogunyemi et al., Blood sample collection
2008), Pterocarpus santolinoides and Aloe vera which are
traditionally used against SCA. These plants revealed both The blood samples used in this study were obtained from
anti-sickling and antioxidant properties (Ugbor, 2006). homozygote SS patients between 18 and 30 years old, in the
Most a time the anti-sickling activity of plant extracts are Central hospital of Yaoundé. The blood samples were
related to their antioxidant capacity due to their polyphenol collected in the sodium EDTA tubes and stored at 4°C not
content (Nanfack et al., 2013). more than 24 h for the experiment. A written informed
Cocoa (Threoboma cacao) is one of the richest dietary consent was read and signed by all the patients
sources of polyphenols such as caffeic acid and epicatechin participating in the study. All the research procedures
which quantity is directly linked to its geographic received the approval of Research Ethics for Human Health
distribution (Caprioli et al., 2016). High antioxidant at the center (CRERSH / Ce) under the reference number
properties of cocoa confer to its numerous therapeutic 00255 / CRERSHC / 2017.
 Academia Journal of Medicinal Plants; Cabral et al. 081

Determination of antioxidant activity of T. cacao (DPPH•) radical. To 0.3 ml of extract of a series of

extracts concentrations (0, 10, 15, 25, 75 and 100 μg/ml) was added
0.3 ml of (DPPH 0.1 mM) solution. The control consists of
Determination of the total polyphenols and flavonoids 0.3 ml of the DPPH solution and 3 ml of distilled water.
content Ascorbic acid was used as standard and prepared at the
same concentration. After homogenization they were
The total phenol content was assessed using Folin-Ciocalteu incubated in the obscurity at room temperature for 30 min.
colorimetric method. Briefly to 0.1 ml of plant extract (1 The absorbance was measured at 517 nm against the blank
mg/ml), was added 0.4 ml of the folin-Ciocalteu reagent ten (Molyneux, 2004). The percentage of inhibition of the DPPH
times diluted and 1 ml of sodium carbonate Na2CO3 (7.5%). radical by the extract was calculated according to the
The mixture was homogenized and incubated at room formula:
temperature in the obscurity for 2 h (Singleton et al., 1999).
The absorbance was measured at 765 nm and the total % DPPH radical scavenging activity= [(OD control - OD
polyphenol content determined from the standard curve and sample)/OD control] × 100
was expressed in mg equivalent of caffeic acid / g of extract
(ECA/g E). The IC50 values were calculated from the graphs
For the total flavonoids determination, the method of representing the variations of the percentage of inhibition
Zhishen et al. (1999) was used. according to a function of the different concentrations.
To 500 μl of the extracts (100 μg/ml), were added 300 μl of
distilled water and 30 μl of sodium nitrite (5%). After
incubating for 5 min at room temperature, 30 μl of Determination of free radical scavenging activity of
aluminum trichloride (10%) were added to the mixture. 2,2'-azino-bis- [3-ethylbenz-thiazolone-6-sulfonic acid]
After 1 min of incubation at ambient temperature, 200 μl of (ABTS °)
sodium hydroxide (1 mM) and 1000 μl of distilled water
were added. The absorbance of the solution was By reacting with a strong oxidant such as potassium
determined at 510 nm. A calibration curve was performed permanganate (KMnO4) or potassium persulfate, ABTS
in parallel under the same operating conditions using forms the radical ABTS•+, from blue to green. Adding an
quercetin as a positive control. The total flavonoid content antioxidant to this mixture will reduce the radical and
of the extracts was determined using a standard curve and cause discoloration. The ABTS•+ stock solution consisted of
expressed in milligram (mg) standard equivalent / g of 200 ml of ABTS (7 mM) and 200 ml of ammonium
extract (mg ES/g E). persulfate (2.45 mM). The reaction mixture was incubated
in the dark for 12 h at room temperature. To 40 μl of
extracts at 6 different concentrations (0, 25, 75, 100, 150
Determination of antioxidant potential of the extracts and 300 μg/ml) were added 2000 μl of the ABTS•+ solution.
by the method of Ferric Reducing Antioxidant Power The control consisted of 40 μl of distilled water and 2000 μl
(FRAP) of ABTS+ solution. Ascorbic acid was used as standard and
prepared at the same concentration. After homogenization,
This method measures the ability of samples to reduce iron the whole was incubated at room temperature for 6 min.
at pH 3.6 in the presence of an antioxidant. The FRAP Absorbance was measured at 734 nm against the blank (Re
reagent is a mixture of acetate buffer (300 mM, pH 3.6), et al., 1999). The percentage inhibition of the ABTS radical
TPTZ (10 mM) and FeCl3 (10 mM) in the proportion 10:1:1. using extracts was calculated according to the formula:
75 μl of plant extract of 1 mg/ml concentration was added
to 1000 μl of FRAP reagent. The mixture was homogenized Percentage ABTS radical scavenging activity= [(OD control -
and incubated for 12 min, and then the absorbance read at OD sample)/OD control] × 100
539 nm. Ascorbic acid concentration (100 μg/ml) was used
as the standard and the final results were expressed as mg The IC50 value will be defined as the concentration of the
equivalence of ascorbic acid per g of dry matter (mg EAA/g extract that causes the loss of 50% of the ABTS activity.
E), using the regression equation of the calibration curve
(Benzie and Strain, 1996).
Determination of anti-sickling activity of T. cacao bean
extracts
Determination of the scavenging activity of the DPPH
radical of plant extracts In vitro evaluation of inhibitory activity of extracts on
sickle cells
This method is based on measuring the ability of
antioxidants to trap the 2,2-dipheny-l,1-picrylhydrazil The evaluation of the anti-sickling activity of the T. cacao
 Academia Journal of Medicinal Plants; Cabral et al. 082

extracts was done according to the N'Draman-donou et al. Evaluation of the protective properties of the plant
(2015) protocol, slightly modified. The Malassez cell helped against oxidative stress
counting RBCs. A dilution of 1:250 (blood / physiological
saline) was performed. In order to obtain pure RBCs, Preparation of the homogenate of organs
plasma was isolated from the whole blood by pre-washing
them using 0.9% NaCl solution followed by a centrifugation Liver, kidneys and heart of two normal Wistar albino rats
for 5 min at 3000 rpm. This process was repeated twice to were isolated and then lodged in phosphate buffer (0.1 M,
completely liberate RBCs from impurities. Each extract pH 7). They were weighed and milled until a paste was
stock solution was prepared by mixing each plant extract in obtained and thereafter, homogenized in the same
0.85% NaCl solution for a final concentration of 10 mg / ml. phosphate buffer. The organ homogenate of 10% (w/v)
From these, different dilutions helped preparing obtained was centrifuged at 3000 rpm for 10 min. The
corresponding concentrations (500, 1000 and 2000 μg / supernatant was kept at – 20°C until use.
ml).

Determination of total protein

Study of the anti-sickling activity of the extract
The total protein was determined using Biuret method.
50 μl of washed SS blood previously diluted with Briefly 20 μl of each homogenate were added 1000 μl of the
physiological saline (1:250 blood/ physiological saline) and Biuret reagent and incubated at room temperature for 30
50 μl of 2% sodium metabisulphite solution were mixed in min. The standard protein (70 g/L) was used. The contents
an Eppendorf tube. 15 μl of the mixture was placed on a were homogenized and absorbance obtained at 546 nm
Malassez cell covered by a cover slip and observed under (Gornall et al., 1949). The concentrations of the samples
light microscope to determine the percentage of sickle cell. were determined using the formula:

C = [DO test / DO standard] × n (standard concentration)

Study of the anti-sickling activity of the extract

To 50 μl of washed SS blood previously diluted with Preparation of the pro-oxidative solution

physiological saline (1:250 blood/ physiological saline),
were added 50 μl of each extract at different concentrations The pro-oxidative solution prepared consisted of mixing in
(500, 1000 and 2000 μg/ml) and 50 μl of sodium an equimolar volume two solutions R1 and R2. R1
metabisulfite 2%. The microscopic observation of 15 μl of containing 0.14 M iron trichloride (FeCl 3) in hydrogen
this mixture was done using Malassez cell under light chloride (1N) and 0.16 M nitriloacetic disodium while R2
microscope (40X objective) after 30 min, 1 h, 1 h 30 min, 2 h containing 0.2 M hydrogen peroxide (H2O2). The resulting
and 2 h 30 min. The percentage of sickle cells was then mixture was used as oxidant to determine the protective
obtained. A control group was performed in the same way properties of the plant against oxidative stress.
but the extract was replaced by phenylalanine (standard)..

Determination of antioxidant parameters

Osmotic fragility test of erythrocytes
An aliquot containing 0.58 ml of phosphate buffer, 200 μl of
The osmotic fragility of the erythrocytes is based on the each extract or standard, 200 μl of liver, heart or kidney
measurement of the stabilizing effect of their membrane homogenate and 20 μl of oxidizing solution were
after 24 h of incubation with the extract. Cell lysis is introduced into different tubes and the mixture obtained
determined by observing the turbidity shift using was then incubated for 1 h at 37°C to form the test
spectrophotometric method at 540 nm. Briefly, a serial solutions. Three other tubes were prepared under the same
concentration of extracts (500, 1000 and 2000 μg/ml) were conditions to serve as normal, negative and positive
diluted in NaCl (0.85%). In 800 μl of different NaCl control; in these tubes, the extracts were respectively
concentrations, were added respectively 200 μl of extracts replaced by the extraction, solvent and quercetin. These
and 10 μl of blood. The supernatant absorbance from the test solutions and control were used to determine
mixture was read at 540 nm against the blank made up of enzymatic and non-enzymatic parameters such as
NaCl after 24 h of incubation (Jaja et al., 2000). Heamolysis malondialdehyde (Wilbur et al., 1949), reduced glutathione
was expressed as: (Ellman, 1959); catalase activity (Sinha, 1972); glutathione
peroxidase activity (Sigma-Aldrich, 2016) and superoxide
% Haemolysis = DOsample × 100 dismutase (SOD) activity (Misra and Fridovich, 1972).
DOcontrol
 Academia Journal of Medicinal Plants; Cabral et al. 083

Table 1: Total polyphenol content, flavonoids and ferric reducing antioxidant power (FRAP) of different extracts.

50% Inhibitory
Polyphenol content (mg Eq Flavonoids content (mg Eq FRAP (mg equivalent of
Extracts concentration (IC50 in µg/ml)
of Quercetin/g of extract) of caffeic acid/g of extract) ascorbic acid /g of extract)
DPPH ABTS
MH 200.2 ± 0.02a 10.01 ± 1.12a 110 ± 1.12a 4.16 5.17
PH 130 ± 2.10b 02.01 ± 0.10b 100 ± 1.12b 4.51 6.28
MA 150.2 ± 0.02b 05.01 ± 1.12bc 90 ± 1.12c 6.35 9.29
PA 95 ± 2.10bc 1.01 ± 0.10bcd 50 ± 1.12d 5.98 6.74
The statistical analysis includes Kruskal-wallis followed by Dunnet. Values with different letter represent significant difference at 0.05. PH: Penja hydroethanolic cocoa extract. MH: Manfe
cocoa hydroethanolic extract. PA: Penja cocoa aqueous extract. MA: Manfe cocoa aqueous extract.

Statistical analysis content, flavonoids and Ferric Reducing Antioxidant function of the salt concentration and the
Power (FRAP) of different extracts. In general, results concentrations of various extracts. In general, it is
Results were expressed as mean ± standard deviation revealed that all extracts (MH, PH, MA and PA) possess clear that the haemolysis percentage decreases with
and each experiment was performed in triplicates. The high polyphenol content and antioxidant power. both increase in the extract concentration (500, 1000
Kruskal-Wallis test was used, followed by post-hoc Polyphenol content, flavonoids and FRAP values vary and 1500 μg/ml) and the salt concentrations. Results
Dunnet to analyze the antioxidant potential and significantly (P<0.05) and ranged between 95 and also revealed that there are some significant
antiradical activity of each plant extract in order to 200.2 mg Eq of Quercetin/g of extract, 1 and 10 mg Eq differences (P < 0.05) between control (positive
determine significant differences (p<0.05). The Mixed of caffeic acid/g of extract and between 50 and 110 mg control) and extracts at each extract concentration
Linear Effect Model helped to study the interactions equivalent of ascorbic acid /g of extract respectively. from 0.35% salt concentration (MH, PH, and MA), from
between factors (extracts, concentration and the MH presented the best potential of both three studied 0.35% salt concentration (PA) and at each salt
repeated time factor) after data restructure. The parameters. IC50 is between 4.16 and 6.35 for DPPH, concentration for the standard (PHE). Furthermore,
Spearman correlation enabled us to establish 5.17 and 9.27 for ABTS. The IC50 of Manfe extract is when fixing the concentration of extracts, each extract
correlations between plant extracts and various anti- lower than that of Penja. influenced in decreasing significantly (P < 0.05) the
radical methods. IC50 were determined by the use of haemolysis percentage with salt concentration
multiple regression analyzes. The software SPSS dependent. MH and MA seem to present the best
version 16 for Windows 7 was used for statistical Radical scavenging activity of different extracts activity.
analysis.
Figure 1 shows the radical scavenging activity of
different extracts. In general, all the studied extracts Anti-sickling properties of the T. cacao extracts
RESULTS scavenged significantly (P<0.05) DPPH and ABTS
radicals with extract concentrations dependent. The Figure 3 shows the percentage of anti-sickling
Antioxidant actvity of the various extracts of T. inhibition is most accentuated when using quercetin activity of various extracts at different
cacao (standard).
concentrations as a function of time. This study
Total polyphenol content, Flavonoids, ferric revealed that the simple induction of cell sickling
reducing antioxidant power (FRAP) and IC50 of Properties of the T. cacao extracts against red without other any treatment (2% metabisulfite
different extracts blood cell membrane fragility control) increased significantly (P<0.05) the
sickling with time dependent (between 22.05 and
Table 1 shows results relative to the total polyphenol Figure 2 shows the percentage of haemolysis as a 56.67%). The presence of different extracts lowered
 Academia Journal of Medicinal Plants; Cabral et al. 084

80 80
Inhibitory percentage of DPPH (%) 45 45
Inhibitory percentage of DPPH (%)

Inhibitory percentage of ABTS (%)

Inhibitory percentage of ABTS (%)
A A 40 B B
70 70 40
60 60 35 35
50 50 PH PH 30 30 PH PH
25 25
40 40 MHMH MHMH
20 20
30 30 PA PA PA PA
15 15
20 20 MAMA 10 10 MAMA
10 10 5 5
querquer querquer
0 0 0 0
0 05 510 10 25 25
75 75100100 0 025 25
75 75
100100
150150
300300
Concentration of extracts
Concentration of extracts Concentration of extracts
Concentration of extracts
Figure 1: DPPH and ABTS scavenving activity of various extracts. Kruskal-wallis followed by Dunnet. PH: Penja hydroethanolic
cocoa extract. MH: Manfe cocoa hydroethanolic extract. PA: Penja cocoa aqueous extract. MA: Manfe cocoa aqueous extract.

120
Percentage of haemolysis (%)

100

80

60 Control
PHE
40 PH
MH
20 PA
MA
0
C1C2C3C1C2C3C1C2C3C1C2C3C1C2C3C1C2C3C1C2C3C1C2C3C1C2C3
HT S0 S 0.15 S 0.25 S 0.35 S 0.45 S 0.55 S 0.65 S 0.75 S 0.85

Concentration of
extracts(µg/ml)/Concentration of salt (%)
Figure 2: percentage of haemolysis as a function of the salt concentration and the concentration of the various extracts. The statistical
analysis include Kruskal-wallis followed by Dunnet. Values with different letter represent significant difference at 0.05. PHE:
phenylalanine (standard). PH: Penja hydroethanolic cocoa extract. MH: Manfe cocoa hydroethanolic extract. PA: Penja cocoa aqueous
extract. MA: Manfe cocoa aqueous extract. HT: total haemolysis. S0 – 0.85%: salt concentration. Control: red blood cell treated only with
salt solution at different concentration.
 Academia Journal of Medicinal Plants; Cabral et al. 085

70

60
Sickling percentage (%)

50

Control
40
PHE

30 PH
MH

20 PA
MA
10 Power (Control)

0
C1 C2 C3 C1 C2 C3 C1 C2 C3 C1 C2 C3 C1 C2 C3
0 h INDi 30 min 1h 1 h30 2h 2 h30

Concentration of extracts/Time
Figure 3: Anti-sickling percentage of different extracts. The statistical analysis include Kruskal-wallis followed by Dunnet. Values
with different letter represent significant difference at 0.05. PHE: phenylalanine (standard). PH: Penja hydroethanolic cocoa
extract. MH: Manfe cocoa hydroethanolic extract. PA: Penja cocoa aqueous extract. MA: Manfe cocoa aqueous extract. HT: total
haemolysis. C1 – C3: concentration of extracts (C1=500, C2=1000 and C3=2000 µg/ml). Control: red blood cell treated only with
2% sodium metabisulfite. 0 h: before sickling induction.

significantly (P<0.05) the aforementioned observations as a decreased significantly (P<0.05) in all studied organs when
function of time and the extract concentrations compared treated only with the pro-oxidative solution (positive
to the 2% metabisulfite control. It is noticeable that from all control). Enzyme activities significantly increased (P<0.05)
the studied extracts, cocoa extracts from Manfe-Cameroon as compared to positive control in all studied organs when
presented best values of anti-sickling activity (between extracts were administered. Among extracts, MH and PH
22.05 and 15.04). presented the activity especially in liver homogenate. As far
as, the GSH is concerned (Figure 4D), the same observation
as earlier mentioned is presented in this case. GSH level
Protective properties of T. cacao extracts on some decreased in the positive control group whereas the
organ homogenates administration of various extracts increased the GSH
concentration in all studied organ homogenates. MH
Figure 4A, B, C and D shows both enzymatic and non- showed the best potential to increase GSH followed by PA
enzymatic antioxidant potential as well as, the lipid as the aforementioned increase in GSH is more pronounced
peroxidation status as the protective effect of T. cacao in liver homogenate. The reverse situation is observable in
extracts on heart, liver and kidney homogenates. Figure 5A, Figure 4E where lipid peroxidation (MDA) increased
B and C represent the enzymatic antioxidant potential significantly (P<0.05) in positive control compared to the
(Catalase (CAT), superoxide dismustase (SOD) and normal control. As earlier mentioned, the extract
Glutathion peroxidase (GPX) respectively). In general, administration regulated the situation in decreasing the
compared to normal control, these enzyme activities MDA level with significant difference compared to the
 Academia Journal of Medicinal Plants; Cabral et al. 086

A B C

D
E

Figure 4: Enzymatic and non enzymatic antioxidant potential as well as, the lipid peroxidation status and the protective effect of
T. cacao extracts on studied organs homogenates. Mixed Linear Effect Model helped studying interactions between factors
(extracts, organs) after data restructure. Kruskalwallis and Dunnett when fixing factors. C.Pos = Positive Control (oxidant). Cnor:
normal control. PHE: phenylalanine (standard). PH: Penja hydroethanolic cocoa extract. MH: Manfe cocoa hydroethanolic extract.
PA: Penja cocoa aqueous extract. MA: Manfe cocoa aqueous extract.

Variables (Axes: F1 and F2: 64.50%) Biplot (Axes: F1 and F2: 64.50%)
Variables (Axes: F1 and F2: 64.50%) Biplot (Axes: F1 and F2: 64.50%)
A
B
A
BC

AB
F2 (25.70%)

F2 (25.70%)
F2 (25.70%)

F2 (25.70%)

F1 (38.81%)
F1 (38.81%) F1 (38.81%)
F1 (38.81%)

Figure 5: Principal component analysis between protective tests. PH: Penja hydroethanolic cocoa extract. MH: Manfe cocoa
hydroethanolic extract. PA: Penja cocoa aqueous extract. MA: Manfe cocoa aqueous extract. MDA: malondialdehyde, GSH:
reduced glutathion, GPX: Glutathion peroxidase, SOD: superoxide dismustase, CAT: Catalase, H: from heart, L: from liver, K: from
kidney. DPPH: 2,2-diphenyl-dipicryl phenyl hydrazine, ABTS: 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid), FRAP:
ferric reducing antioxidant power. (a) distribution of tests around the F1 and F2 axes; (b) Pojection of the heads and extracts
around the F1 and F2 axes.
 Academia Journal of Medicinal Plants; Cabral et al. 087

Table 2: Correlation matrix of the protective and antioxidant parameters.

MDAH MDAL MDAK GSHH GSHL GSHK GPXH GPXL GPXK SODH SODL SODK CATH CATL CATK DPPH ABTS PolyP Flavd FRAP
MDAH 1
MDAL 0.955 1
MDAK 0.955 0.995 1
GSHH -0.300 -0.234 -0.234 1
GSHL -0.136 -0.054 -0.054 0.891 1
GSHK -0.396 -0.393 -0.393 0.793 0.757 1
GPXH 0.009 0.108 0.108 0.464 0.627 0.541 1
GPXL -0.342 -0.286 -0.286 0.613 0.613 0.893 0.775 1
GPXK -0.073 0.054 0.054 0.573 0.573 0.487 0.918 0.721 1
SODH 0.054 0.071 0.071 0.757 0.937 0.607 0.631 0.464 0.523 1
SODL -0.418 -0.378 -0.378 0.291 0.491 0.108 0.164 -0.036 0.055 0.559 1
SODK -0.523 -0.679 -0.679 0.234 0.306 0.393 -0.036 0.143 -0.198 0.393 0.667 1
CATH -0.306 -0.214 -0.214 0.793 0.577 0.643 0.631 0.714 0.847 0.429 -0.054 -0.107 1
CATL -0.306 -0.214 -0.214 0.793 0.577 0.643 0.631 0.714 0.847 0.429 -0.054 -0.107 0.998 1
CATK -0.200 -0.072 -0.072 0.873 0.745 0.577 0.655 0.595 0.845 0.631 0.173 -0.090 0.937 0.937 1
DPPH 0.745 0.847 0.847 -0.273 0.055 -0.288 0.455 -0.036 0.291 0.180 -0.118 -0.541 -0.180 -0.180 -0.036 1
ABTS 0.600 0.703 0.703 -0.309 0.018 -0.108 0.491 0.180 0.273 0.072 -0.245 -0.505 -0.180 -0.180 -0.127 0.927 1
PolyP -0.393 -0.334 -0.334 -0.075 -0.075 0.445 0.168 0.593 0.056 -0.296 -0.337 -0.037 0.074 0.074 -0.150 -0.112 0.262 1
Flvd -0.393 -0.334 -0.334 -0.075 -0.075 0.445 0.168 0.593 0.056 -0.296 -0.337 -0.037 0.074 0.074 -0.150 -0.112 0.262 0.985 1
FRAP -0.617 -0.445 -0.445 0.037 -0.150 0.148 0.206 0.408 0.374 -0.408 -0.187 -0.296 0.445 0.445 0.262 -0.224 -0.037 0.615 0.615 1
Spearman Correlation. Correlation coefficients in bold character are significantly different (p<0.05). MDA: malondialdehyde, GSH: reduced glutathion, GPX: Glutathion peroxidase, SOD: superoxide
dismustase, CAT: Catalase. H: from heart, L: from liver, K: from kidney.

positive control. kidney) and GSH (Heart), GSH from heart and liver, considering the position of studied parameters on
flavonoids and polyphenol and between ABTS and both F1 and F2 axes. It is clear from the Figure 5b
DPPH. that the extract contributions are as followed:
Correlation between protective and antioxidant The Principal Component Analysis (PCA) shows MH˃MA˃PH˃PA.
parameters both the contribution of each parameter on F 1 and
F2 axes (Figure 5a) as well as, the impact of each
Table 2 shows correlation matrix of the protective extract to that contribution (Figure 5b). MDAH, DISCUSSION
and antioxidant parameters. The studied MDAL, MDAK, DPPH, ABTS, SODK, Polyphenol,
parameters were MDA (malondialdehyde), GSH flavonoids and FRAP contribute more on the F 2 axis In Cameroon, 4000 children are given birth
(reduced glutathione), GPX (Glutathion peroxidase), whereas the rest of parameters (CAT, GPX, GSH annually with SCA and youths of about 10 to 29
SOD (superoxide dismustase) and CAT (Catalase) in from all studied organs, SODH and SODL) contribute years of age represent 89.2% of those suffering
three organs (Heart, liver and kidney). There are more on the F2 axis. The biplot presented on Figure from this illness. Existing treatments such as the use
strong correlations between CAT (Heart, liver and 5b deduces the role or contribution of each extract of hydroxyurea and piracetam, etc are still effective
 Academia Journal of Medicinal Plants; Cabral et al. 088

against SCA, but recent research have shown some of their may be attributed to the high production of ROS (Alsultan et
limitations as earlier mentioned in the introduction. Plants al., 2010). The excess production of MDA has additional toxic
constitute a rich natural source of bioactive chemicals effects leading to alterations of the proteins, including
which are currently used to fight this disease (Williamson antioxidant enzymes and protein receptors (Bruno et al.,
et al., 1992). The present study aims to evaluate the anti- 2015). Alsultan et al. (2010) added that GSH is an essential
sickling potential of T. cacao extract from South West and cofactor for GPX activity. It was reported that GSH
Littoral regions of Cameroon. concentration decreased in erythrocytes of sickle cell
Results obtained from the antioxidant tests showed that disease (SCD) individuals due to the excess production of
T. cacao extracts from different sources (MH, MA, PH and ROS which consume GSH leading to the reduction in the
PA) and especially the hydroethanolic extracts from Manfe activity of GPX. Excess production of ROS may also have a
followed by Penja (MH) contain high total polyphenols, serious adverse effect on RBCs membrane leading to protein
flavonoids and possess a capacity to reduce ferric iron and lipid peroxidation enhancing production of carbonyl
(FRAP). This could be explained either by the geographical and MDA.
difference of cocoa beans or by the type of extraction Gelation of HbS was observed to occur rapidly with
solvent. This result corroborates the report of Vicaş et al. sodium metabisulphite 2%. Metabisulphite has a strong
(2009) on the study of Viscum album collected at different reducing power capable to remove oxygen from the
seasons which attributed the aforementioned results to immediate environment of the haemoglobin, thereby,
environmental factors such as climate, soil and eliciting gelation of the haemoglobin and the consequent
temperature. The observed antioxidant potential is directly sickling of erythrocytes. Reduction of sickling was dose-
linked to the presence of polyphenols in the studied dependent with increasing concentration of extract. Infact,
extracts. Previous study of N'guessan et al. (2007) revealed in the presence of cocoa bean extracts 30 min after
that the different amount of polyphenols could also induction, sickling was reduced, showing that the extracts
contribute to different level of antioxidant capacity which is of T. cacao contain anti-sickling substances. This result is in
beneficial to human health. The hydroethanolic solvent at line with previous works of Mpiana et al. (2010) and
pH 3 is commonly used in maximizing polyphenol Ngbolua (2012) which revealed that some plant
extraction and could explain the aforementioned result. metabolites could interact with haemoglobin S in inhibiting
The radical scavenging activity of extracts translated by polymerization and thus, prevent the sickling of
the DPPH and ABTS tests increased with the extract erythrocytes. In addition, they recently demonstrated that
concentration dependency. The IC50 of extracts varies from phenolic compounds have a great importance in fighting
4.16 at 6.35 for DPPH, 5.17 and 9.27 for ABTS. The IC 50 of against SCA due to its capacity in either reducing the
Manfe extract is lower than that of Penja. This anti-radical polymerization of HbS or stabilizing the erythrocyte
potential of extracts could be related to the high polyphenol membrane.
content observed. Indeed, N'guessan et al. (2007) previously In addition, Tabassum et al. (2011) showed that
showed a correlation between the total phenol content and flavonoids have the ability to improve endothelial function
the anti-radical activity of some Ivorian plants. According to and vasodilation (by stimulation of NO production). It
Chen and Ho (1995), the functional groups present in the would appear that they also act on platelet aggregation and
phenolic compounds of green and black tea in general can cytokine production (Buijsse et al., 2006).
easily give up an electron or a proton to neutralize the free Principal Component Analysis (PCA) showed some
radicals. significant correlation between protective parameters in
In this study, enzymatic and non-enzymatic parameters liver, heart and kidney and antioxidant tests. This result can
were measured in order to evaluate the protective effect of furthermore be explained by the high content on bean
extracts against oxidative stress. Activities of CAT, SOD and cocoa extracts in polyphenols which in turn protects liver,
GPX; GSH and MDA concentrations increase in the presence heart and kidney against oxidative stress. PCA also allowed
of extracts. Due to the high production of free radicals, the MH to be chosen as the best extract.
severity of a disease could be explained by the imbalance The effect of T. cacao on the membrane stability of RBCs
between antioxidants and pro-oxidants in favour of pro- can be evaluated by
oxidants. Thus, more antioxidants need to be either comparing the haemolysis rates of untreated and treated
synthesized or absorbed to regulate that balance. The sickle RBCs with beans of cocoa. The stability test as the
antioxidant molecules of plant presenting free-radical osmotic fragility assay is one of the possible mechanism in
scavenging properties could be of interest as therapeutic establishing potential anti-sickling substances (Mpiana et al.,
agents in several diseases related to oxidative stress 2010). A decrease in the percentage of haemolysis as a
(Ramchoun et al., 2009). These results are in agreement function of the salt concentration and that of the extracts
with those of Chirico and Pialoux (2012) which showed that was generally noted. This decrease is related to the
the increase of the activities of such parameters could appreciable protective effect of the extracts on the
explain the severity of the disease. Moreover, it has been erythrocyte membrane, hence, their resistance against
demonstrated that decrease in SOD, CAT, and GPX activities haemolysis. This indicates that the cocoa beans extract
 Academia Journal of Medicinal Plants; Cabral et al. 089

regulated the ability of sickle cells to take up water by a YN (2015). In Vitro Ion Chelating, Antioxidative Mechanism of Extracts
from Fruits and Barks of Tetrapleura tetraptera and Their Protective
mechanism of re-hydration without lysis (Mpiana et al.,
Effects against Fenton Mediated Toxicity of Metal Ions on Liver
2010). This stabilization effect could be explained by the fact Homogenates. Evid. Based Complement. Alternat. Med. 2015; 2015:
that flavonoids reduce the effect of the high concentration of 423689. doi: 10.1155/2015/423689
NaCl solution (Elekwa et al., 2005). In fact, the low Buijsse B, Feskens E, Kok F (2006). Cocoa Intake, Blood Pressure, and
Cardiovascular Mortality. The Zutphen Elderly Study. In : Archives of
concentration of NaCl solution (hypotonic solution) in the
Internal Medicine 166 (4) : 411 -417.
presence of red cells is responsible for the increased volume Buijsse B, Feskens EJ, Kok FJ, et al. (2006). Cocoa intake, blood pressure,
of the RBCs and then its haemolysis due to the entrance of and cardiovascular mortality: the Zutphen Elderly Study. Arch. Intern.
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to shrink in size. In consequence, antioxidants prevent red inflammation in vascular diseases. Human health and pathology,
cells dehydration in maintaining both membrane integrity Université Claude Bernard - Lyon I, France. Hal. archives-ouvertes pp. 1-
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DREPAVIE (2013). Association DREPAVIE. www.drepavie.com. Accessed
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Ellman G
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Ibegbulem CO, Eyong EU Essien EU (2011). Polymerization inhibition
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against sickle cell disease. In order to complete these data, Jaja S., Kehinde M., Ghenebitse S. and Mojiminyi A. (2000). Effect of vitamin
C on arterial blood pressure, irreversible sickled cells and osmotic
other studies such as in vivo antioxidant capacity,
fragility in sickle cell anaemia subjets. Nigerian J. Physiol. Sci. 16(2): 14-
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