Ethnobotanical, phytochemical and nutritional characterization of Bixa Orellana Linn. seeds of Benin Ecology

46 Akakpo et al. Int. J. Biosci. 2020 RESEARCH PAPER OPEN ACCESS Ethnobotanical, phytochemical and nutritional characterization of Bixa Orellana Linn . seeds of Benin Ecology Ezéchiel Akakpo1, Marius Eric Badoussi1,2, Claude Kouassi Gnacadja1, Hermance Houngbo1, Alphonse Dossou1, Fernand Gbaguidi3, Paulin Azokpota1* 1Laboratoire de Sciences des Aliments (LSA) Faculté des Sciences Agronomiques, Université d’Abomey-Calavi; 03 BP 2819 Jéricho, Cotonou, Bénin 2Ecole Nationale Supérieure des Biosciences et Biotechnologies Appliquées (ENSBBA), Université Nationale des Sciences, Technologies, Ingénierie et Mathématiques (UNSTIM), BP: 14 DassaZoumé, Bénin 3Laboratire National de Pharmacognosie et des Huiles essentielles, Centre Béninois de la Recherche Scientifique et Technique, Porto-Novo, BP 03-1665, Benin Key words: Biocolorant, food industries, socio-cultural group, pharmacological properties, Bénin. http://dx.doi.org/10.12692/ijb/17.1.46-56 Article published on July 17, 2020 Abstract Bixa orellana seeds, providers of biocolorant, are found in some localities of Benin. However, its local names, its uses such as the phytochemical and nutritional characteristics are not documented. This paper focuses on characterizing the ethnobotanical, phytochemical, and nutritional plan of Bixa orellana seeds of Benin ecology. An ethnobotanical investigation was first carried out close to 148 seeds users in two agro-ecological zones. The seeds collected at the investigation time were characterized through their phytochemical screening and evaluation of their nutritional value by reference methods. Various designations are associated to Annatto according to the socio-cultural groups of Benin such as "timati winiwini", "timati kouinon" or "Kpokpo" (Fon and Mahi, in Zou and the Collines); "Kpararou", "Dodokpara", "Kparara" or "Timati borou" (Bariba and Dendi, in the North); "Disonri", "Dipersonri" or "Monsonri" (Ditamari, in the North). These designations refer to its use in sauces as a substitute for tomatoes due to its red color (64.78%). Ripe fruit (10.81%) and seeds powder (8.10%) is used to treat anemia. Phytochemical screening of seeds revealed gallic tannins, flavonoids, anthocyanins, leucoanthocyanins, saponins, triterpenoids, steroids, mucilage, reducing compounds, and C-heterosides. Nutritionally, the seeds contain protein (12.83%), fats (4.64%), Potassium (14.59 mg/g), Sodium (6.93 mg/g), Phosphorus (4.79 mg/g), Calcium (2.89 mg/g), Magnesium (1.04 mg/g), Manganese (0.19 mg/g), Copper (0.19 mg/g), and Iron (0.01 mg/g). In addition to being an excellent source of dyes usable for various purposes, Annatto is a potentially nutritious product. It is therefore of paramount importance in the improvement of the quality of traditional food. * Corresponding Author: Paulin Azokpota  [email protected] International Journal of Biosciences | IJB | ISSN: 2220-6655 (Print), 2222-5234 (Online) http://www.innspub.net Vol. 17, No. 1, p. 46-56, 2020 47 Akakpo et al. Int. J. Biosci. 2020 Introduction Global food resources consist of a limited number of species that represent the basis of global nutrition. This globalization of food resources has led to the gradual disappearance of many cultivated species while others remain known only in restricted geographical areas (Djè et al., 2006). Such species occur, especially in tropical regions, where secular beliefs and traditions contribute to the maintenance of several crops designated as minor crops (Djè et al., 2006). This is the case of Annatto (Bixa orellana L.) which is a species native to central and south American, but now grown in the tropics (Periyasamy and Kumar, 2016) including Benin (Akoègninou et al., 2006; Fagbohoun, 2014; Akakpo et al., 2019). From a botanical point of view, Bixa orellana L. is an evergreen shrub or a small tree, measuring about 3-10 meters in height and belongs to the family of Bixaceae. It is cultivated not only because of its beautiful thorny ornamental flowers and red fruits but also for its socio-economic value (Akakpo et al., 2019). Different parts of the species are used in agrofood as well as for certain treatments, particularly by people in developing countries (Russell et al., 2005; Lans, 2006). Also, the roots and leaves of Bixa orellana are used to treat epilepsy, diarrhea, dysentery, fever, jaundice, parasitic diseases, cough and urinary infections, and some gastrointestinal and pulmonary diseases (Giorgi et al., 2013; de Araújo Vilar et al., 2014) in several countries including Brazil, Peru, Philippines, Nigeria, and Côte d'Ivoire (Omonhinmin et al., 2013; de Araújo Vilar et al., 2014). The different medicinal properties of these organs are linked to their phytochemical composition (Akakpo et al., 2019). However, of all the organs of the tree, the red seeds called roucou or rocou (French), annatto (English), and achiote (Spanish) are the most used (Venugopalan et al., 2011). Furthermore, apart from their traditional use as a condiment or seasoning due to their richness in nutrients (Senthil et al., 2007; Akakpo et al., 2019), annatto seeds provide the second natural colorant of economic importance (after caramel) (Venugopalan et al., 2011). Also, the seed extract, rich in tannins, contains a mixture of eight dyes from the carotenoid group. However, the main dyes are bixin and norbixin (Gulrajani et al., 2002; Das et al., 2007). These dyes are used in the textile, cosmetic, and craft industries to dye fibers (basketry), fabrics, and various masks. They are also used in the food industry, particularly in the coloring of ice creams, dairy products, juices and liqueurs, sauce, and cakes (Venugopalan et al., 2011; Fagbohoun, 2014; Akakpo et al., 2019). There are types of Bixa orellana with white flowers, but trees with pink flowers are much more common. Although there are many cultivated types, there is no official classification for cultivars (Jansen, 2005). However, despite the presence of the species in Benin, endogenous knowledge related to its uses is not yet documented. Also, the phytochemical and nutritional characteristics of the seeds of the cultivar(s) of Bixa orellana found in Benin are not yet known. A directory of information concerning the plant, the cultivar present in Benin localities, the seeds and their virtues, the zones of production and consumption as well as the evaluation of its nutritional compounds is, therefore, necessary for a better valorization of the resource. Thus, this study focuses on characterizing ethnobotanically, nutritionally, and phytochemically the Bixa orellana seeds of Beninese ecology. Materials and methods This study consisted, firstly, in inventorying endogenous knowledge relating to the use of different parts of the species. Seed samples were collected and characterized through phytochemical screening and evaluation of their nutritional value. Ethnobotanical characterization of Bixa orellana The study was conducted among the populations of the townships of NIKKI and N'DALI in agroecological zone III and the township of OUESSE in agro-ecological zone V. These different localities were chosen based on the presence of the species and its use by the population made up of several sociocultural groups. The reasoned choice method as described by Kpètèhoto et al. (2017) was used. Thus, the interviewees were selected taking into account their proximity to the identified plants, the presence 48 Akakpo et al. Int. J. Biosci. 2020 of the species in their field, and the frequency of seed harvesting as indicated by some owners of fields/hut gardens with the species. A total of 148 people (102 women and 46 men) were surveyed and distributed as follows: 50 respondents at NIKKI, 44 at N’DALI, and 54 at OUESSE. The information collected concerns the local names of the species, its availability (dry season or rainy season), the different parts of the plant used, the different uses that these parts constitute, and the places of collection of the organs of the plant. The table 1 presents the summary of the survey areas as well as the numbers registered in each locality. Characterization of Bixa orellana seeds Sampling plan: Seed samples were taken from three different fields, one field per municipality. At each field, the seeds were extracted from ripe and dried fruit picked from three different trees. The seeds collected from the three trees in the same field were combined in a bottle that was labeled, and this made up a sample. The samples thus collected were kept in the laboratory for the evaluation of the different phytochemicals and physicochemical parameters. Determination of phytochemical characteristics: The phytochemical compounds were determined through a phytochemical screening. This is a qualitative analysis based on coloring and/or precipitation reactions carried out on dry seeds. These were at first reduced into powder in a mortar using a pestle and then by standard reactions described by Houghton and Raman (1998) and Adjatin et al, (2013). The main secondary metabolites were sought using the methods grouped in Table 2. Evaluation of the nutritional value of Bixa orellana seeds The sample was analyzed for moisture, crude protein, crude fat, and ash content. Crude protein was determined by using the Kjeldahl method (Nair et al., 2013). The moisture and crude fat were determined according to the procedure of the Association of Official Analytical Chemists (AOAC, 1990). The percentage was calculated based on the dry weight. Ash was determined after incineration in a muffle furnace following Bangash et al. (2011). Mineral composition of the samples was determined according to methods recommended by the Association of Official Analytical Chemists (AOAC, 1990) and Badau et al. (2013). The samples were incinerated in the oven at a temperature of 550°C for 3 hours. The samples of Bixa orellana seeds were each digested using a mixture of concentrated nitric (HNO3), perchloric (HClO4), and sulphuric (H2SO4) acids in the ratio 9:2:1 (v/v) respectively (Nair et al., 2013). Copper (Cu), iron (Fe), sodium (Na), potassium (K), calcium (Ca), magnesium (Mg), and Manganese (Mn) were determined by Atomic Absorption Spectrophotometer (AAS) (PerkinElmer AAnalyst 700, England). Phosphorus contents of the samples were determined using a Flame photometer as specified in Alinnor and Oze (2011). The concentration of each element in the sample was calculated from the dry matter. The analysis was performed with triplicates for the needs of statistical analysis. Statistical data analysis methods The data obtained from the investigation and the physicochemical analysis were entered and processed with the Microsoft Excel spreadsheet. The different local designations as well as the uses of the various organs of the species have been grouped in the form of a table and figure. The different physicochemical parameters evaluated as a function of the sample collection area were compared using an analysis of variance. This was followed by a Fisher LDS test at the 5% threshold with STATISTICA 7.1 software. Results and discussion Ethnobotanical characteristics of Bixa orellana In the areas surveyed, there is only one type of Bixa orellana. This is the type with pink flowers that produces fruit during the rainy season (April-May) and the dry season (November-December). However, only the production of fruit during the dry season is often harvested because the seeds of this season do not rot under the effect of rainwater. Local people traditionally classify name, and group the species of 49 Akakpo et al. Int. J. Biosci. 2020 plants they use withthe introduction, ecology, morphology, and technological traits (Dansi et al., 2008). Although the local names for Bixa orellana vary from one socio-cultural group to another (Table 3), the majority of the names given to the species relate to its technological traits. This means that the names "timati winiwini" or "timati kouinon" given by Fon and Mahi or "Timati borou" by Bariba and Dendi or "Timati atchidjitô" by Adja brings out the use of seeds as tomato for various food preparations (64.18% of respondents) (Fig. 1). Similarly, "tomate mougou" in Côte d'Ivoire and "tomati enshee" in Burkina-Faso which relate to the use of seeds as tomato had been reported (Akakpo et al., 2019). On the other hand, the seeds are used in food preparations, either crushed alone and directly added to the sauce, or crushed in a mixture with cornflour to use as tomato powder. Also, the name Kpokpo given by Fon to the leaf sheath of Sorghum bicolor to bring out its coloring power is given to the seeds of Bixa orellana. Bixa orellana seeds, like leaf sheaths, could be used to color traditional foods such as local cheese wagashi. The use of Bixa orellana seeds has been reported in dairy technology for the production of butter, ice cream, margarine, and snacks. It is also used in cosmetics (Guiliano et al., 2003; Akakpo et al., 2019). Table 1. Distribution of respondents by sex and locality. Communes Villages/Districts Sex Number of respondents Men Women Ouessè Odougba 9 21 30 Dokoundoho 8 16 24 Nikki Danri 9 18 27 Gourou 6 17 23 N’Dali Travo 8 11 19 Ouénou 6 19 25 Total - 46 102 148 The seeds of Bixa orellana are variously used within prospecting zones. Fig. 1 shows some uses of the various organs of the species of Beninese ecology.According to Fig. 1 which illustrates the proportions of the use of Bixa orellana organs in the study areas, it appears that the organs with great use in these zones are the dry seeds in food followed by the leaves and roots, ripe fruits, and dry seeds for medicinal uses. Table 2. Metabolites sought and research methods. Test N° Secondary metabolites sought Methods used 1 Alkaloids Mayer’s test 2 Quinone derivatives Borntrager‘s test 3 Catechic tannins Stiasny test 4 Gallic tannins Ferric chloride test after saturation with sodium acetate 5 Flavonoids Shinoda test and magnesium powder 6 Cyanogenic derivatives Picric acid test 7 Triterpenoids Acetic acid test + mixture of acetic anhydride-sulfuric acid 8 Steroids Kedde reaction 9 Saponins test index foam 10 Cardiac glycosides Raymond Marthoud reaction 11 Anthocyanins Test with hydrochloric acid and ammonia diluted to half 12 Leucoanthocyanes Shinoda test 13 Mucilages Test of absolute alcohol 14 Reducing compounds Test with Fehling’s solution 15 Coumarin Test with ether and ammonia 16 Free anthracene derivatives Test with chloroform and ammonia 17 Combined anthracene derivatives Test with chloroform and ammonia 50 Akakpo et al. Int. J. Biosci. 2020 Apart from their use as an ingredient for various food preparations, dry seeds are sometimes powdered and incorporated into the porridge to relieve people suffering from anemia, particularly children (8.10%). Non-food uses of seeds have also been identified. The seeds are also used not only in convents for the adornment of the followers of certain endogenous religions during different ceremonies but also for occult purposes. Also, the use of dye from seeds to color traditional masks has been reported (Fagbohoun, 2014). Other parts of the tree, especially the decoction leaves and roots are used to treat stomach aches, diarrhea, and urinary infections (14.86% of respondents). Similar uses of these organs have been reported in the Brazilian Pharmacopoeia (Venugopalan et al., 2011; Akakpo et al., 2019). Undried ripe fruit is used as a decoction in children to relieve anemia problems (10.81% of respondents). Table 3. Some local appellations of Bixa orellana according to socio-cultural groups. Socio-cultural groups Local names Meaning Mahi/Fon Timati winiwini, Small tomato Timati kouinon Seeds tomato Kpokpo - Bariba/Dendi Pkararou, Dodokpara, Kparara - Timati borou Powdered Tomato Ditamari Risonri, Disonri, Dipersonri ou Monsonri - Ayizô Sokplè - Kotafon Ebiahou dékou Which is redder than palm oil Adja Yovogbo, Yovogbo atchidjitô Tomato from tree Phytochemical Profile of Bixa Orellana Seeds Phytochemical screening revealed the presence of gallic tannins, flavonoids, anthocyanins and leucoanthocyanins, saponins, triterpenoids, steroids, mucilage, reducing compounds, and C-heterosides (Table 4). According to Table 4, it appears that Bixa orellana seeds samples have the same large groups of chemical compounds. Table 4. Secondary metabolites examined and detected in the Bixa orellana seeds powder. Chemical compounds E1(OUESSE) E2 (NIKKI) E3 (N’DALI) Alkaloids - - - Polyphenol compounds  Cathetic tannins - - -  Gallic tannins + + +  Flavonoids + + +  Anthocyanins + + +  Leucoanthocyanins + + + Quinone derivatives - - - Saponins + + + Triterpenoids + + + Steroïds + + + Cyanogenic derivatives - - - Mucilages + + + Coumarins - - - Reducing compound + + + Anthracene derivatives + + +  Free anthracene derivatives - - -  Combined anthracene derivatives O-heterosids - - -  Combined anthracene derivatives C-heterosides + + + Cardiac glycosides - - - (+): Presence; (-): Absence. 51 Akakpo et al. Int. J. Biosci. 2020 A study carried out by Fleisher et al. (2003) had revealed, in the Bixa orellana seeds, the presence of flavonoids, saponins, and reducing compounds. It also revealed the absence of alkaloids, coumarins, free anthracenes, and quinone derivatives as shown by the present study. On the other hand, the study carried out by Prathima et al. (2016) identified in Bixa orellana seeds the presence of alkaloids and Cardiac glycosides. These differences observed could be related to the origin of the different seeds used. It also recognized flavonoids, antiviral, antitumor, anti-inflammatory drugs, anti-allergic, and anticancer activities (Morel, 2011). Tannins enable the reduction of the biodisponibility of iron and other micronutrients and present an antibacterial activity from weak to moderate. Besides, the bacteria as an anticryptococcus activity (yeasts), antiviral, antiinflammatory, anti-hypertensive, antimutagenic, immuno-stimulative, antitumor, and anti-diarrheal are relatively high (Feldman et al., 1999). Table 5. Content of organic compounds in Bixa orellana seeds. Seeds’ origins Parameters E1(OUESSE) E2 (NIKKI) E3 (N’DALI) Moisture (%) 2.7± 0.1 (a) 2.7± 0.1(a) 2.8±0.1(a) Crude protein (%) 12.5±0.2(a) 13.0±0.1(b) 13.0±0.2(b) Crude lipid (%) 4.3±0.1(a) 4.9±0.1(b) 4.6±0,1(b) The seeds also contain steroids and anthocyanins. These compounds could, therefore, be responsible for the antimicrobial activity of Bixa orellana seeds against Staphylococcus aureus, Baccilus Cereus, and Escherichia coli (Abhishek et al., 2010). Thus, the presence of gallic tannins, flavonoids, anthocyanins and leucoanthocyans, saponosides, triterpenoids, steroids, mucilage, reducing compounds, and Cheterosides could explain the use of seeds in the traditional pharmacopoeia for various medicinal treatments (Akakpo et al., 2019) as a purgative and against oral tumors (Fleischer et al., 2003). The seeds have anti-leishmanial, anticonvulsant, antidiabetic, and cardio-protective properties (Viuda-Martos et al., 2012; Akakpo et al., 2019). Furthermore, the absence of toxic compounds such as quinone derivatives, cardiac glycoside, and cyanogenic derivatives in the three samples studied could explain to some extent the consumption of these seeds without risk of food poisoning. The studies of toxicity indicated that Bixa orellana did not produce toxic effects in rats or mice if it is administered orally (26 mg/day for rats, and a drop of 10% in soya oil/day for mice). Its consumption is thus without risk for the human food (de Paula et al., 2009). Therefore, consumption is safe for human food. Table 6. Ash content (g / 100g DM), and individual minerals of the three seed samples. Seeds’ origins Composition E1 (OUESSE) E2 (NIKKI) E3 (N’DALI) Mean CV (%) Ash (g/100g) 6,2±0,07(a) 5,1±0,1(b) 5,2±0,1(b) 5,5 11,06 individuals Mineral (mg/g) Potassium (K) 18,17 13,02 12,59 14,59 0,21 Sodium (Na) 9,66 5,64 5,49 6,93 0,34 Phosphorus (P) 6,15 4,11 4,09 4,78 0,25 Calcium (Ca) 3,92 2,43 2,33 2,89 0,31 Magnesium (Mg) 1,39 0,94 0,79 1,04 0,29 Manganese (Mn) 0,24 0,17 0,17 0,19 0,21 Copper (Cu) 0,20 0,18 0,18 0,19 0,48 Iron (Fe) 0,02 0,01 0,01 0,01 0,01 DM = Dry Matter. 52 Akakpo et al. Int. J. Biosci. 2020 Also, the research results of Paumgartten et al. (2002) showed that the extract of Bixa orellana is neither maternally toxic nor embryotoxic. Shilpi et al. (2006) reported that Bixa orellana does not have any mortal effect 24 hours after the administration of its extract, even to the highest amount (4.000 mg/kg) for mice. According to Rajib et al. (2009), the extracts of Bixa orellana have a very high hepatoprotective activity. Nutritional characteristics of Bixa orellana seeds Table 5 shows the water, crude protein, and total lipid content of the Bixa orellana seed samples. The water content of the Bixa orellana seed samples is 2.7% and does not reveal any significant difference between the different samples (Table 5). This value is lower than those reported by Valério et al. (2015) and Dike et al. (2016) which are 6.74% and 4.89%, respectively. The low water content recorded for these seeds could allow their conservation for a relatively long period without altering their nutritional qualities. Thus, the high humidity generates a greater activity of watersoluble enzymes and coenzymes required for metabolic activities (Badau et al., 2013). The protein content of Bixa orellana seeds varies from 12.5 ± 0.2 to 13.0 ± 0.2% with a significant difference between the samples from the Municipality of Ouèssè (agro-ecological zone V) and the Communes of N'dali and Nikki (agro-ecological zone III). This same observation was noted at the level of lipid content which varies from 4.3 ± 0.1 to 4.9 ± 0.1% (Table 5). These recorded differences could be linked to the effects of agro-ecological zones. Fig. 1. Different uses of Bixa orellana organs identified in the study areas. The photosynthetic reactions responsible for the production of organic compounds in plant organs are subject to sunshine, which is not the same in the different survey areas. Therefore, it can differentiate the content of organic compounds such as proteins and lipids in plant organs (Le Gall et al., 2015). Also, a variation in the biochemical composition of different parts (leaves, pulp, and seeds) of baobab of Beninese ecology has already been observed depending on the area of origin of the samples (Assogbadjo et al., 2012). Furthermore, the difference observed could presage the probable existence of two cultivars. The protein contents obtained are higher than those obtained by Valério et al, (2015), which is 11.5%. This is, however, similar to the value of 12.55% reported by Dike et al, (2016). The lipid contents are found in the range of reported values which varied from 2.23 to 7.20% (Valério et al., 2015; Dike et al., 2016; Akakpo et al., 2019). The seeds of Bixa orellana, therefore, contain significant amounts of protein and lipid which are organic compounds that are beneficial for the body of consumers. 53 Akakpo et al. Int. J. Biosci. 2020 The ash content of Ouessè sample is higher than that of Nikki and N’dali samples (Table 6). This difference could be linked to the types of soil in the areas where the samples come from. Ouessè is in an agroecological zone different from that of Nikki and N’dali. According to INSAE (2016), tropical ferruginous soils are dominant in the township of N'Dali where they are deep, not concreted, and subjected to leaching. Ferralitic soils are found in the township of Nikki while the township of Ouessè is characterized by tropical ferruginous soils on a crystalline base and colluvial soils. The differences observed could, therefore, be linked in particular to the mineral content of these soils. However, the values obtained are in the range of values reported in the literature which varies from 5.05% to 6.32% (Senthil et al., 2007; Valério et al., 2015; Dike et al., 2016). The ash content is a reflection of the mineral content in food products. Therefore, Bixa orellana seeds constitute a significant source of mineral elements which are important components of diets because of their physiological and metabolic function in the body (Adjatin et al., 2013). The average calcium content of Bixa orellana seed samples is 2.89 mg. Foods colored with the extract of these seeds can be considered as a good source of calcium because the dye brings its calcium supplement to the food it colored. Magnesium was determined and evaluated at 1.04 mg/g for the seed samples analyzed. This value is found in the range of the values reported by Akakpo et al. (2019). The manganese content of Bixa orellana seeds was evaluated at 0.19 mg, and this value is lower than 0.25 mg obtained by Senthil (2007). Copper is necessary for the body for the production of enzymes and the biological transport of electrons (Alinnor and Oze, 2011). The copper content, of the dry weight of the seeds, was 0.19 mg. The iron content of Bixa orellana seeds found in this study (0.01 mg/g) is lower than the value (0.03 mg/g) reported by Akakpo et al. (2019). These seeds can be considered as a significant source of mineral elements for various food formulations. Conclusion The local designations of Bixa orellana in the regions explored vary from one socio-cultural group to another and sometimes within the same sociocultural group. These seeds used mainly in the food sector are potential sources of proteins and mineral salts. They also contain certain active phytochemical conferring on the seeds and certain therapeutic properties which are usable in the treatment of certain human pathologies. These chemical compounds in Bixa orellana seeds vary from one agro-ecological zone to another. Because of these different results, it, therefore, becomes necessary to promote this species as a sector through the promotion of its culture and the development of its value chain. References Abhishek M, Rakshanda B, Prasad GBKS, Dua VK, Verma SK, Agarwal PK. 2010. Antimicrobial activity of plants traditionally used as medicines against some pathogens, Rasayan Journal of Chemistry 3, 615-620. Adjatin A, Dansi A, Badoussi E, Loko YL, Dansi M, Azokpota P, Ahissou H, Akoègninou A, Akpagana K, Sanni A. 2013. Phytochemical screening and toxicity studies of Crassocephalum Rubens (Juss. ex Jacq.) S. Moore and Crassocephalum crepidioides (Benth.) S. Moore consumed as vegetable in Benin. International Journal of Current Microbiology and Applied Sciences 2, 1-13. Akakpo E, Badoussi ME, Gnacadja KC, Houngbo H, Dossou A, Azokpota P. 2019. Le rocouyer (Bixa Orellana), une source de biocolorant pour les industries alimentaires: revue analytique. International Journal of Biological and Chemical Sciences 13, 2332-2351. https://dx.doi.org/10.4314/ijbcs.v13i4.36 Akoègninou A, Van der Burg WJ, Van der Measen LJG, Adjaèkidjè V, Sinsin B, Yédomonhan H. 2006. Flore Analytique du Bénin, 54 Akakpo et al. Int. J. Biosci. 2020 Backhuys Publishers, 1034 p. Alinnor IJ, Oze R. 2011. Chemical evaluation of the nutritive value of Pentaclethra macrophylla benth (African Oil Bean) Seeds. Pakistan Journal of Nutrition 10, 355-359. AOAC. 1990. Official Methods of Analysis. (13th edn). Association of Official Analytical Chemists: Washington DC. Assogbadjo AE, Chadare FJ, Glele-Kakaï R, Fandohan B, and Baidu-Forson JJ. 2012. Variation in biochemical composition of baobab (Adansonia digitata) pulp, leaves and seeds in relation to soil types and tree provenances. Agriculture, Ecosystems and Environment 157, 94– 99. Badau MH, Abba HZ, Agbara GI, Yusuf AA. 2013. Proximate composition, mineral content and acceptability of granulated maize dumpling (Dambu Masara) with varying proportions of ingredients. Global Advanced Research Journal of Agricultural Science 2, 320-329. Bangash JA, Arif M, Khan F, Khan F, Rahman AU, Iqbal H. 2011. Proximate composition, mineral and vitamin content of selected vegetables grown in Peshawar. Journal of the Chemical Society of Pakistan 33, 118-122. Dansi A, Adjatin A, Adoukonou-Sagbadja H, Faladé V, Yedomonhan H, Odou D, Dossou B. 2008. Traditional leafy vegetables and their use in the Benin Republic. Genetic Resources and Crop Evolution 55, 1239-1256. Das D, Maulik SR, Bhattacharya SC. 2007. Dyeing of wool and silk with Bixa orellana. Indian Journal of fibre & Textile Research 32, 366–372. deAraújoVilar D, de Araujo Vilar MS, de Lima e Moura TFA, Raffin FN, Oliveira MR, de Oliveira Franco CF, de AthaydeFilho PF, FormigaMeloDiniz MF, Barbosa-Filho JM. 2014. Traditional Uses, Chemical Constituents, and Biological Activities of Bixa orellana L.: A Review. The Scientific World Journal 2014, p11. http://dx.doi.org/10.1155/2014/857292. de Paula H, Pedrosa ML, Joamyr Victor RJ, Kenji Haraguchi F, dos Santos RC, Silva ME. 2009. Effect of an Aqueous Extract of Annatto (Bixa orellana) Seeds on Lipid Profile and Biochemical Markers of Renal and Hepatic Function in Hypercholesterolemic Rats. Brazilian Archives of Biology and Technology 52, 1373-1378. Dike IP; Ibojo OO, Daramola FY, Omonhinmin AC. 2016. Phytochemical and Proximate Analysis of Foliage and Seed of Bixa orellana Linn. International Journal of Pharmaceutical Sciences Review and Research 36, 247-251. Djè Y, Kouonon CL, Zoro Bi IA., Gnamien YG, Baudoin JP. 2006. «Etude des caractéristiques botaniques, agronomiques et de la biologie florale du melon africain (Cucumismelo L. var. agrestis Naudin, Cucurbitaceae)», Biotechnologie, Agronomie, Société et Environnement 10, 109 – 119. Fagbohoun L. 2014. Etude chimique de colorants naturels et matériaux résineux traditionnels du Bénin dans le domaine artisanal. Thèse de doctorat de l'université d'Abomey-calavi (Bénin) et de l'université d'Avignon et des pays de Vaucluse (France) Spécialité: Chimie organique et substances naturelles. p 295. Feldman KS, Sahasrabudhe K, Smith RS and Scheuchenzuber WJ. 1999. Immunostimulation by plant polyphenols. A relationship between tumor necrosis factoralpha production and tannin structure. Biorganic and medical chemistry letters 9, 985-990. Fleischer TC, Ameade EPK, Mensah MLK, Sawer IK. 2003. Antimicrobial activity of the leaves