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European Journal of Applied Sciences – Vol. 11, No. 1
Publication Date: January 25, 2023
DOI:10.14738/aivp.111.13704.
Kouamé, E-C., Kra, A. K.M., Kouassi, K. A. M., Gnahoué, G. & Djaman, A. J. (2023). Acute and sub chronic toxicity of forastero
cocoa from Côte d'Ivoire. European Journal of Applied Sciences, 11(1). 185-201.
.
Services for Science and Education – United Kingdom
Acute and Sub chronic Toxicity of Forastero Cocoa from
Côte d'Ivoire
KOUAME Estelle-Clemence
Department of Biochemistry, Laboratory of biology and health,
UFR of Biosciences, Félix Houphouet-Boigny University,
Abidjan, 22 B.P. 582 Abidjan 22 Côte d’Ivoire
KRA Adou Koffi Mathieu
Department of Biochemistry, Laboratory of biology and health,
UFR of Biosciences, Félix Houphouet-Boigny University,
Abidjan, 22 B.P. 582 Abidjan 22 Côte d’Ivoire
KOUASSI Konan Armand Marcelin
Department of Biochemistry, Laboratory of biology and health,
UFR of Biosciences, Félix Houphouet-Boigny University,
Abidjan, 22 B.P. 582 Abidjan 22 Côte d’Ivoire
GNAHOUE Goueh
Superior Normal School of Abidjan, Côte d’Ivoire
DJAMAN Allico Joseph
Department of Biochemistry, Laboratory of biology and health, UFR of Biosciences,
Félix Houphouet-Boigny University, Abidjan, 22 B.P. 582 Abidjan 22 Côte d’Ivoire.
Department of Clinical and Fundamental Biochemistry,
Pasteur Institute, Abidjan, Côte d’Ivoire
ABSTRACT
In Côte d'Ivoire, forastero cocoa is used by traditional healers to treat metabolic and
chronic diseases like diabetes. This study aims to assess the toxicity of extracts from
forastero cocoa. Serial extraction was performed from cocoa powder using ethanol,
distilled water and hexane. The phytochemical screening of aqueous and
hydroethanolic extracts and crude cocoa powder was carried out using appropriate
specific reagents. Acute and sub chronic oral toxicity studies were performed in rats
according to OECD guidelines 423 and 408. The phytochemical screening reveals
the presence of polyterpenes, polyphenols, alkaloids and saponosides in the
aqueous, hydroethanolic extracts and crude cocoa powder. Regarding acute
toxicity, the LD50 value is higher than 5000 mg/kg body weight. In sub chronic
toxicity, results show that for all the doses, there is a significant increase in the
weight of male and female rats. Furthermore, no signs of intoxication or mortality
were observed during 90 days. Analysis of hematological and biochemical
parameters reveals that there is no significant change in blood parameters values
and liver, kidneys and heart serum markers. On the whole, the results show that
extracts from forastero cocoa are not toxic.
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European Journal of Applied Sciences (EJAS) Vol. 11, Issue 1, January-2023
Services for Science and Education – United Kingdom
Keywords: cocoa forastero, phytochemical screening, toxicity.
INTRODUCTION
Some plants are both nutritionally and therapeutically useful. Cocoa is one of them. Indeed,
according to Grassi et al. [1,2] and Ala’a et al. [3] cocoa is prized for its many nutritional
qualities and several investigations have already revealed its many beneficial properties for
cardiovascular system as well as certain metabolic diseases [4] [5] [6]. In addition, recently, an
ethnobotanical survey permitted to know that traditional healers in the Abengourou area (Côte
d'Ivoire) propose remedies based on forastero cocoa to treat diabetes. However, no toxicity
studies have been carried out on this variety. But this step is important. Indeed, toxicity study
of a substance is the set of pharmacological tests, which determine its degree of toxicity or its
harmful nature in order to regulate its use [7] [8]. The aim of this study is to deliver results
from our investigations on the acute and subchronic toxicity of aqueous and hydroethanolic
extracts from forastero cocoa.
MATERIAL AND METHODS
Plant Material
The plant material consists of cocoa beans (Theobroma cacao) of the forastero type. The cocoa
pods were harvested in Abengourou, an area located in the East of Cote d’Ivoire.
Animal Material
Albino rats of the Wistar strain, weighing 150 to 200 grams, were used in this study. These rats
were reared under adequate conditions of hygiene, ambient temperature of 25°C, and in
sufficient ventilation. They were housed in cages lined with wood chips where they had free
access to water and food. The rats were alternately subjected to 12 hours of light and 12 hours
of darkness.
Preparation of Cocoa Powder, Aqueous and Hydroethanolic Extracts
The defatted cocoa powder (CoPw) was prepared according to the method of Jinap et al. [9],
with slight modifications in this experiment. The non-fermented and dried cocoa beans were
roasted for 20 minutes at 140°C in an oven, then cooled and shelled. The obtained almonds
were ground using a blender and the gotten cocoa mass was defatted with hexane using a
Soxhlet. The cake obtained was dried at 50°C for 1 hour 30 minutes, finely ground and then
sieved in order to obtain the defatted cocoa powder.
The aqueous extract (AqE) was prepared by homogenizing 100 g of defatted cocoa powder in
1 L of distilled water using a blender according to the method of Zihiri et al.[10]. After six cycles
of homogenization, the obtained homogenate was first wrung out in a fabric square and then
filtered thrice successively with absorbent cotton and twice with Whatman 3 mm filter paper.
The resulting filtrates were concentrated under vacuum at 60°C using a Büchi rotary
evaporator. Hydroethanolic extract (HEE) was prepared following the same process using a
mixture of ethanol (70%) and water (30%). These two crude extracts were weighed to
determine the yield of each extraction. Extracts were stored separately in hermetically sealed
jars and kept away from heat, moisture and light.
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Kouamé, E-C., Kra, A. K.M., Kouassi, K. A. M., Gnahoué, G. & Djaman, A. J. (2023). Acute and sub chronic toxicity of forastero cocoa from Côte
d'Ivoire. European Journal of Applied Sciences, 11(1). 185-201.
URL: http://dx.doi.org/10.14738/aivp.111.13704
Phytochemical Screening
Phytochemical screening was carried out using appropriate specific reagents whose
colorations generated by the tests indicate the presence or absence of the sought compounds.
The sought compounds were: saponosides, polyphenols, alkaloids, flavonoids, quinones,
polyterpenes, steroids and cardiotonic glycosides.
Assessment of Acute Toxicity
Acute oral toxicity of the extracts was performed in rats according to OECD guidelines 423 [11]
for the testing of chemicals. This method is a sequential process using three animals of one sex
by step. Depending on the mortality and/or moribund state of animals, two to four steps are
needed to assess the average acute toxicity of the test substance. Non-pregnant and nulliparous
female rats of 3 to 4 months and weighing 150 to 155 grams on average were used for
experimentation. Cocoa is a product already consumed by man and known as a non-toxic
substance. Therefore, the starting dose of the extracts chosen for this study was 2000 mg/kg
body weight. Three rats were deprived of food during the night. They were weighted and given
orally a single dose of 2000 mg/kg bw of AqE, HEE and CoPw from cocoa. After administration
of this substances, the three rats were again fasted for three hours before giving them food.
They were observed individually for the first 30 minutes and 24 hours after treatment, then
daily for 14 days. Twenty-four (24) hours after administration of AqE, HEE and CoPw from
cocoa at a dose of 2000 mg/kg bw, no mortality and no moribund state were observed. Then,
three other rats were treated in the same way. After this step, the immediately higher dose
(5000 mg/kg bw) of AqE, HEE and CoPw from cocoa, were respectively administered to three
new groups of 3 rats. During this study, the animals were weighed every two days and were
observed for the same times as before. Observations were made on coat modification, motility,
tremors, body mass, grooming, salivation, respiration, sensitivity to noise after a metal shock,
stool appearance, mobility as well as sleep and coma.
Assessment of Subchronic Toxicity
In preliminary tests, HEE from cocoa, that produced in vitro the best activity, was selected for
the in vivo pharmacological study. The sub-chronic toxicity study was conducted according to
OECD guidelines 408 [12]. Male and female albino rats aged 12 weeks were weighed and
distributed in 5 groups of six rats, consisting of 3 males and 3 females per group.
• Group 1 (control) was given 1 mL/100 g of distilled water for 90 days,
• Groups 2, 3 and 4 were treated for 90 days with HEE from cocoa at doses of 250, 500 and
1000 mg/kg bw respectively.
• Group 5 (satellite) was constituted to monitor reversibility of HEE at 1000 mg/kg bw.
During this study, rats were fed and hydrated ad libitum and weighed every week. At the end of
the treatment, the rats were fasted for 12 hours and euthanized. Their blood was then collected
in anticoagulant tubes (EDTA) and immediately used to determine white blood cell, red blood
cell, platelet and hematocrit levels using standard method.
The blood collected in the dry tubes was used for the determination of the biochemical
parameters such as transaminases (alanine aminotransferase, aspartate aminotransferase),
gamma glutamyl-transferase, lipids (triglycerides, total cholesterol, HDL cholesterol), proteins,
urea, creatinine, total and direct bilirubin.