Journal of Agricultural Science; Vol. 10, No.

3; 2018
ISSN 1916-9752 E-ISSN 1916-9760
Published by Canadian Center of Science and Education

Bromatological Analysis, Chemical Composition and Bioassays from

the Genipa americana L. (Rubiaceae)
Oneil Valerio Ávila1, Ismael Montero Fernández2, Habdel Nasser Rocha da Costa1,5,
Antonio Alves de Melho Filho1,2,5, Ricardo Carvalho dos Santos3 & Pedro Rômulo Estevam Ribeiro4
1
Post-Graduate Program in Chemistry, Center for Research and Graduate Studies in Science and Technology,
Federal University of Roraima, Paricarana Campus, Boa Vista, RR, Brazil
2
Postgraduate Program in Biodiversity and Biotecnology (BIONORTE), Campus Cauamé, Boa Vista, RR, Brazil
3
National Postdoctoral Program of CAPES, Associated to the Postgraduate Program in Agronomy of
Universidade Federal de Roraima, Campus Cauamé, Boa Vista, RR, Brazil, and to the Brazilian Agricultural
Research Corporation, Embrapa, Boa Vista, RR, Brazil
4
Post-Graduate in Natural Resources Program, Federal University of Roraima, Paricarana Campus, Boa Vista,
RR, Brazil
5
Department of Chemistry, Federal University of Roraima, Paricarana Campus, Boa Vista, RR, Brazil
Correspondence: Oneil Valerio Ávila, Postgraduate Program in Chemistry, Federal University of Roraima,
Campus Paricarana, CEP 69304-000, Boa Vista, RR, Brazil. E-mail: dragonquest_fly88@hotmail.com

Received: December 26, 2017 Accepted: January 15, 2018 Online Published: February 15, 2018
doi:10.5539/jas.v10n3p244 URL: https://doi.org/10.5539/jas.v10n3p244

Abstract
Genipa americana L. well known as genipap, is a tree that is widely distributed throughout the Brazilian territory.
The communities appreciate the genipap fruits, since they are used as food; in addition to that, their fruits have
numerous seeds that can be used in the production of vegetable oil. This being, in this work inclined to a
bromatological study of the genipap fruits and chemical composition and bioassays from vegetable oil seeds.
Obtaining the highest percentage of lipids in the seeds that was of 7.08%, the highest percentage of humidity
obtained was 74.66% present in the pulp, the highest amount of carbohydrates was found in the seeds, with a
percentage of 79.37%, the highest percentage of ash present was 3.99% found in the pulp, another parameter
analyzed were the proteins mostly present in the seeds with a percentage of 4.45% and finally the energy value
was calculated, being the majority in the seeds with 398.98 Kcal/100 g. The fatty acid profile showed the highest
percentage for the Linoleic Acid with 61.5%. The greatest inhibition in the antimicrobial assays was for S.
typhimurium with 42.12% inhibition. In the tests performed for the inhibition of the enzyme acetylcholinesterase
was 14.95%.
Keywords: acetylcholinesterase, GC-FID, genipap, microorganisms, nutrients
1. Introduction
The Amazon is considered and known as one of the regions suitable for the production of fruits, for its great
diversity of native plants that grow and develop (De-Souza, Rossi, Varella, Silveira, & Souza, 2015). The G.
americana is a tree belonging to the family of the Rubiaceae, is present throughout Brazil, naturally, or
cultivated, from the Amazon to São Paulo and Mato Grosso, in various forest formations (Lorenzi, 2008; Santana,
2014).
According to Rabbani, Silva-Mann, and Ferreira (2012), G. americana presents an economic importance for the
production of food, since its consumed in natura or used for the production of sweets, jellies and liqueurs
(Lorenzi, 1992). The fruits have numerous seeds, which are found together in the innermost part of the nucleus
(Figueiredo, Maia, Monteiro, & De-Figueiredo, 1991).
Besides its use as food according to Soares, Sousa, Garrido, and Lima (2012), fruits (peel, pulp and seeds),
leaves, roots and stem are used in folk medicine. The fruits have medicinal properties known as inhibitor of
bacteria, fungi, algae and protozoa, in addition to presenting high content of mannitol, which is recommended in

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Agricultural Sciience Vol. 10, No. 3; 2018

the Caribbbean countriess in the reducttion of high bblood pressuree (Cordeiro & Felix, 2014; Lorenzi & Matos,
M
2008).
Nutrients are chemical substances, ppresent in foodd, these proviide energy andd are used byy the body for the
mong them we have
functioninng of several viital bodily proocesses and theerefore, need too be obtained from food, am
minerals, pproteins, carboohydrates, lipidds and vitaminns (Tortara & D
Derrickson, 2017).
In the humman body, the nutrients fulffill essential fuunctions such as forming paart of the enzyymatic systemm that
catalyzes m metabolic reacctions involvinng proteins, ccarbohydrates and lipids. Thhe hardness off certain struc
ctures
such as teeeth and bones and many othher important ffunctions to keeep the body hhealthy, the neccessary amoun nts of
these nutriients can vary in grams, milligrams and evven microgram ms (Marzzoco & Torres, 20077).
Fruits are important souurces of many nutrients and serve as an inntegral part off the human diiet, as they pro ovide
vitamins, minerals, prooteins, carbohyydrates, lipidss and other vvital constitueents essential for human health h
(Coolbornn, Esther, Akkinsola, & Affolabi, 2016).. Therefore, tthe objective of this workk is to perforrm a
bromatological study of the different pparts of the gennipapo fruit, as well as to deteermine the fattyy acid profile in
i the
oil extractted from its seeeds, to characcterize the funnctional groupss by infrared aand biologicall activity studies of
antibacteriial and inhibitoory activity of the acetylchollinesterase enzzyme due to itss importance fo
for the development
of new druugs or pesticides.
2. Method
d
2.1 Plant M
Material and Extraction
E of V
Vegetable Oil
The genipaap fruits were harvested matture, accordingg to Figure 1, iin the municippality of Boa V Vista-RR (Braz zil) in
April 2016 at the folloowing coordinnate’s 2°50′01.5″N 60°42′21.6″W. They were exsiccatted, deposited d and
identified in the herbariium of the Fedderal Universiity of Roraimaa (UFRR), wiith a voucher number 8797. The
fruits weree taken to thee Environmental Chemistry Laboratory ((LQA), Paricarrana campus, in the Nucleu us of
Research aand Postgraduaate in Science and Technologgy (NPPGCT)) of the UFRR,, which were w washed and cleeaned
with distillled water, thenn dried and theen separated innto shell, pulp and seeds. Theese were weigghed and stored
d in a
freezer at -20 °C for lyophilization. T The lyophilizaation of the seeeds was carrieed out in a LÍÍOTOP lyophiilizer,
model L 101, for 48 hours h until coomplete dryinng of the matterial in the P Physics and L Land Management
Laboratoryy at the Agricuultural Sciences Center (LF FMSCSA), Cauuamé campus of the UFRR. Subsequently y, the
material wwas ground in MARCONI® ® knife mill, ssieved until hoomogenizationn of the particcles between 20-40
2
Mesh, andd then stored unnder cover of llight.

Figure 1. Ripee fruit of G. am

mericana

2.2 Bromaatological Anallysis

The physiical chemical parameters
p evvaluated to dettermine the nuutritional com
mposition weree the percentag ge of
moisture aand ash and thhe other nutritiional parameteers evaluated w
were the deterrmination of tootal proteins, lipids
l
and carbohhydrates to dettermine the tottal energy conttent.

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Humidity was determined by weighing 5.0 g of fresh sample and placing in pre-weighed porcelain capsules and
placed in an air circulation oven for 6 hours at 105 °C until constant weight, being cooled in a desiccator to room
temperature (Instituto Adolfo Lutz [IAL], 2008) and the amount of water was calculated by Equation (1):
Humidity (g/100 g) = (P' – P'')/(P' – P) × 100 (1)
Where, P = weight of the porcelain capsule (g); P' = weight of the porcelain dish + fresh sample (g); P'' = weight
of the capsule + sample after drying (g).
For the determination of ashes, the methodology proposed for the food analysis of the (IAL, 2008) with
modifications was used, where 5 grams of the lyophilized material placed in porcelain pots previously heated in
a 110 °C air circulation oven were weighed. one hour to remove the moisture and then cooled in a desiccator to
room temperature, then the samples were placed in the crucibles and taken to a muffle for calcination of the
samples at a temperature of 600 °C for 12 hours, then the total content was calculated of ash (amount of total
minerals) by Equation (2):
N
%Ashes = × 100 (2)
M
Where, N = mass in grams of ashes; M = mass of the sample in grams.
The vegetable oil was obtained using a Soxlhet extractor with cartridges lined with cotton containing 47 g (seeds)
30 g (peel) and 32 g (pulp) crushed and separated plus 500 mL of solvent hexane for each extraction (Jorge &
Luzia, 2012 ). The extraction was carried out in a period of 3 hours under reflux of the solvent, then the obtained
mixture passed through a separation process using a vacuum roto-evaporator, separating the vegetable oil from
the solvent and for calculating the oil yield in accordance with the norm of the Adolfo Lutz Institute (IAL, 2008),
Equation (3) was used:
N
Vegetable oil (%) = × 100 (3)
P
Where, N corresponds to the number of grams of lipid; P corresponds to the number of grams of the sample.
According to the methodology of the Adolfo Lutz Institute, the determination of proteins is obtained from the
determination of total nitrogen by Kjeldahl distillation, because the organic matter is decomposed and the
existing nitrogen is transformed into ammonium, the content being of nitrogen of the different proteins
approximately of 16%, introduces the empirical factor 5.75 (conversion factor for vegetable protein) that will
transform the number of grams of nitrogen found with the number of grams of protein (IAL, 2008). Equation (4)
presents the calculation to determine the percentage of proteins in the samples:
Proteins = % N × 5.75 (4)
The determination of the carbohydrate content was made by the difference of the value 100 subtracted from the
sum of the already obtained values of humidity, ashes, lipids and proteins expressed in Equation (5):
Carbohydrates = 100 – (%Humidity + %Ash + %Lipids + %Proteins) (5)
The determination of the energy value was made through the results obtained by the contents of proteins (P),
lipids (L) and carbohydrates (C) using Equation (6) that expresses the calculation in Kcal/100 g according to the
methodology proposed by the Instituto Adolfo Lutz method (IAL, 2008).
Energy Value (Kcal/100 g) = (P × 4) + (L × 9) + (C × 4) (6)
Where, P = protein value (%); L = lipid value (%); C = value of carbohydrates (%); 4 = conversion factor in Kcal
determined in calorimetric pump for proteins and carbohydrates; 9 = conversion factor in Kcal determined in
calorimetric pump for lipids.
2.3 Oil Analysis by GC-FID
The determination of the fatty acids present in the oil of genipap seeds was made by Gas Chromatography in the
chromatography laboratory of the Federal University of Minas Gerais (UFMG), where the samples were
hydrolyzed and methylated. A quantity of 12 mg of oil sample in 100 μL of a solution of ethanol
(95%)/potassium hydroxide 1 mol/L (5%) was dissolved in a cryogenic tube of 2 mL. After vortexing for 10 s,
the oil hydrolyzed in a domestic microwave oven (Panasonic NN-ST254W), at a power of 60% (420 W), for 6
minutes. After cooling, 400 μL of 20% hydrochloric acid and one tip of the NaCl spatula (20 mg) and 600 μL of
ethyl acetate were added. After vortexing for 10 s and resting for 5 minutes, an aliquot of 300 μL was removed
from the organic chamber, placed in microcentrifuge tubes and dried by evaporation, thus obtaining free fatty
acids (Christie, 1989). Subsequently, the free fatty acids were methylated with 100 μL BF3/methanol (14%) by

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heating in a 60 °C water bath for 10 minutes. The methylated fatty acids were extracted with 500 μL of hexane
and analyzed by Gas Chromatography.
After the preparation of the fatty acids, the analyzes were performed in an HP7820A Gas Chromatograph
(Agilent) equipped with a flame ionization detector. Data acquisition program EZChrom Elite Compact (Agilent).
A Supelcowax-10 15 m × 0.2 mm × 0.2 μm column (Supelco) was used with a temperature gradient: 120 °C, 0
min, 10 °C/min up to 250 °C; injector (1/50 split) at 250 °C and detector at 260 °C. Hydrogen as a stripping gas
(3.0 mL/min) and injection volume of 1 μL. The identification of the peaks was made by comparing Supelco37
fame mix methylated fatty acid standards (Supelco cat no 47885-U).
2.4 Oil Analysis Bioassay
The bioassays in the oil of genipap seeds: antimicrobial activity and the bioassay of the inhibitory activity of the
enzyme acetylcholinesterase (ACHE) was in the biotechnology and bioassay laboratory of the Federal University
of Minas Gerais (UFMG). For the bacterial activity, a pre-inoculum was prepared, in which the microorganisms
were transferred from the culture medium where they were stored for test tubes containing 3.0 mL of culture
medium (BHI for bacteria and Sabouraud for yeast). Next, the tubes were incubated in a greenhouse at 37 °C for
36 h. With the help of a micropipette, 500 μL of this pre-inoculum were transferred to test tubes containing
sterile distilled water. The tubes were homogenized and the concentration was adjusted to 600 nm (bacteria) and
530 nm (yeast), until reaching a transmittance between 74-75% (bacteria) and 75-76% (yeast), corresponding to
scale 0 , 5 of McFarland of standard turbidity, that is, 108 CFU/mL, thus obtaining the suspensions of the inocula
used in the bioassay.
For the preparation of the work-solution, the samples were previously solubilized in dimethylsulfoxide (DMSO)
at a concentration of 12.5 mg/mL. From this solution, an aliquot of 40 μL was withdrawn, which was added to
960 μL of the culture medium used in the bioassay, obtaining the working solution in the concentration of 500
μg/mL.
The bioassays were performed in 96 microwell plates, in triplicate. In the first well, 200 μL of the working
solution in the concentration of 500 μg/mL was added. In the following wells, 100 μL of culture medium per
well was added, followed by serial microdilution (1:1) of the sample solution, so that the concentrations ranged
from 250 to 0.98 μg/mL. Next, 100 μL of the standardized microorganism inoculum was added to each well.
Four controls were carried out: growth control of the microorganism (to verify cell viability); white, which
consists of the solution of the sample in the same concentrations evaluated, replacing the inoculum with sterile
distilled water; (the work-solution is replaced by a commercial antibiotic) and the sterility control of the culture
medium, containing 100 μL of culture medium and 100 μL of sterile distilled water. The microplates were
incubated in a greenhouse at 37 °C and after 24 hours the plate reader was read at 490 nm (Zacchino & Gupta,
2007).
The antibiotics used for the quality control of the tests were ampicillin for bacteria and nystatin for yeast, whose
working solutions were prepared as described above for the samples tested.
The samples were tested against the following microorganisms:
• Candida albicans: ATCC 18804 (yeast)
• Staphylococcus aureus: ATCC 29212 (Gram-positive bacteria)
• Bacillus cereus: ATCC 11778 (Gram-positive bacteria)
• Escherichia coli: ATCC 25922 (Gram-negative bacteria)
• Salmonella typhimurium: ATCC 14028 (Gram-negative bacteria)
The bioassay for the inhibition of the activity of the acetylcholinesterase enzyme was carried out in 96-well
microplates, to which 50 μL of Tris-HCl buffer (50 mM, pH 8.0), 125 μL of 5.5'-were added. dithiobis
(2-nitrobenzoic acid)-DTNB (3 mM, 25 μL of the sample solution (10 mg/mL in DMSO) and 25 μL of
acetylcholine iodide-ATCI (15 mM) DMSO was used as a negative control and galantamine (10 mg/mL in
DMSO) as a positive control (standard inhibitor of the enzyme) The absorbance was measured at 405 nm using a
microplate reader, with intervals of 1 minute for eight times, after these readings were added to the wells 25 μL
acetylcholinesterase enzyme solution (0.22 U/mL in buffer) The absorbance were measured again at 1 minute
intervals at 10 to 405 nm (Frank & Gupta, 2005, Ellman, Courtney, Andres Jr., & Feathestone, 1961). The
percent inhibition was calculated by comparing the absorbance of the samples with the absorbance of the blank
using Equation (7):

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Absorbance of the sample with enzyme – Absorbance of the sample without enzyme
%Inhibition = 100 – × 100 (7)
Absorbance of the negative control with enzyme – Absorbance of the negative control without enzyme

3. Results and Discussion

3.1 Bromatological Analysis of Genipap Fruit
In this study the highest percentage of moisture was found in the pulp and it was 74.66% and the lowest
percentage was found in the seeds, which was 5.63% according to Table 1. In similar studies carried out on
genipap pulps, collected in the state of Minas Gerais, reported a humidity percentage of 70.0% (Pacheco, Paz,
Silva, & Pascoal, 2014), in other studies with genipap pulps collected in the state of Ceará were reported 74.81%
(Figueiredo, Maia, Holanda, & Monteiro, 1986) and study made with genipap seeds presented a moisture
percentage of 5% (Carvalho & Nascimento, 2000).
The other nutritional parameter studied was the ashes, which their highest percentage was reported in the pulp
with a percentage of 3.99% and their seeds with a slightly lower percentage of 3.46% according to Table 1.
Studies made by Pacheco et al. (2014) with genipap pulp collected in the state of Minas Gerais had a percentage
of ash of 3.6%. Other studies made genipap seeds reported a percentage of ash of 1.69% (Luzia, 2012).
Lipids are natural substances, esters of fatty acids and of an alcohol or a polyol. These perform important
biological functions such as: component of the cell membrane, metabolic fuel, skin protective layer, etc.
(Bruneton, 2001). They are also used in the food industry, pharmaceutical, as a biofuel (Vianni & Braz, 1996). In
this study, the highest percentage of lipid was obtained from the seeds and was 7.08% according to Table 1, a
little lower, compared to studies done by other authors on genipap seeds that showed a lipid percentage of
10.39% (Luzia, 2012).
Proteins are considered to be the most important constituent of living cells, which can be of plant or animal
origin and represent the largest chemical group in the body of animals, among some of the main functions of
proteins are; essential constituent of all cells, in the growth and development of the body and in the production of
metabolic and digestive enzymes (FAO, 2002). In the present work, the highest percentage was found in the
seeds with 4.45% and the pulp the edible part was 3.97% according to Table 1, other authors in similar studies
with genipap pulps, collected in the state of Minas Gerais they reported a protein percentage of 1.7% (Pacheco et
al., 2014) and in studies made with genipap seeds they reported a protein percentage of 25.33% (Luzia, 2012).
Carbohydrates are compounds that contain carbon, hydrogen and oxygen in proportions of 6:12:6, these in the
metabolism are burned and produce energy, in the human diet are like starch and various sugar, carbohydrates
can be divided into monosaccharides, disaccharides and polysaccharides (FAO, 2002). According to the World
Health Organization, who suggests that carbohydrates should meet most of the energy needs and that represent
between 55% to 75% of daily intake and where the intake of fruits and vegetables should reach 400 g per day
(WHO, 2003). In this study, the highest percentage of carbohydrates for seeds was reported with 79.37%.
According to Thompson, Manore, and Vaughan (2008), energy is the fuel our body uses to develop its vital
functions, being the Kilocalories (Kcal) the unit in which it is expressed and its recommended daily values vary
between 2000 and 2500 calories, depending on age, sex, physiological status and physical activity. In the present
study the highest energy value is present in the seeds with 398.98 Kcal/100 g as can be seen in Table 1, higher
compared to the results of studies done by Luzia (2012), with genipap seeds that presented an energetic value of
205.91±0.13 Kcal/100 g, highlighting that in this study they accounted for total dietary fibers.

Table 1. Nutritional analysis of the fruit of G. americana

Nutritional Contribution
Genipa americana
Humidity (%) Ashes (%) Lipids (%) Carbohydrates (%) Proteins (%) Energetic Value (Kcal 100 g-1)
Pulp 74.66 ±1.41 3.99±0.25 0.44±0.02 16.93±1.20 3.97±0.03 87.53±4.83
Peel 58.69 ±2.41 3.24±0.36 0.72±0.12 35.64±2.71 1.70±0.01 155.86±1.25
Seed 5.63±0.09 3.46±0.09 7.08±0.38 79.37±0.42 4.45±0.02 398.98±1.71

3.2 Fatty Acids Analysis

According to Bruneton (2001), lipids play an essential role in living organisms as well as in the pharmaceutical
industry, food industry as food and other industries such as biofuel. Within nature, palmitic acid, stearic acid,
oleic acid and linoleic acid are the four fatty acids that make up 95% of the acids present in the various types of
lipids (Simoês et al., 2007). In this study, of the fatty acids identified linoleic acid (C18:2) was the one with the

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highest percentage of 61.5% and in the background palmitic acid (C16:0) with 13.1% according to Figure 2,
similar to studies conducted by Figuereido et al. (1991), who presented percentages of 60.5% and 10.3%
respectively, as can be seen in Table 2.

Table 2. Profile of G. americana seeds oil fatty acids

Author Figueiredo et al., 1991
Fatty Acids
RT/min % %
Myristic Acid (C14:0) 3.9 0.1 -
Palmitic Acid (C16:0) 5.6 13.1 10.3
Palmitoleic Acid (C16:1) 5.7 0.1 -
Stearic Acid (C18:0) 7.2 9.1 9.7
Oleic Acid (C18:1) 7.4 10.7 19.5
Linoleic Acid (C18:2) 7.7 61.5 60.5
Linolenic Acid (C18:3) 8.2 0.4 -
Arachidonic Acid (C20:0) 8.9 0.2 -
Other - 4.8 -

Back Signal
D 003.dat
Name
750 750

C18:2
700 700

650 650

600 600

550 550

500 500

450 450

400 400
pA

pA
350 350

300 300
C16:0

250 250
C18:1

200 200
C18:0

150 150

100 100
C18:3

50 50
C20:0
C14:0

C16:1

0 0
1 2 3 4 5 6 7 8 9 10 11 12
Minutes

Figure 2. Fatty acids chromatogram

3.3 Microorganisms Assay

The bioassays for the antimicrobial activity with the microorganisms tested in this study, as can be seen in Table
3, had the highest percentage of inhibition for S. typhimurium with 42.12% inhibition, followed by B. cereus
with 35.87% and finally with the lowest percentage of inhibition S. aureus with 34.58%. Studies conducted by
Santos et al. (2015), with oil extracted from Annona hypoglauca in the same working conditions, reported a
percentage of inhibition of 7.23% for S. typhimurium and 28.93% for S. aureus.

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Table 3. Percentage of antimicrobial inhibition at 250 μg/mL

Microorganisms
Sample
S. aureus B. cereus S. typhimurium E. coli C. albicans
Seeds oil 34.58±11.46 35.87±11.64 42.12±2.54 0.00 0.00
Ampicillin 87.85±6.57 92.60±0.67 97.90±4.43 94.37±4.31 -
Nystatin - - - - 98.17±6.62

3.4 Inhibition of the Activity of the Acetylcholinesterase Enzyme Assay

The inhibition assay of the acetylcholinesterase enzyme for this study made with the oil extracted from genipap
seeds was weak with a percentage of inhibition of 14.95 as can be seen in Table 3, in comparison with studies
done by Santos et al. (2015), under the same conditions with oil extracted from Annona hypoglauca seeds, that
showed a percentage of inhibition of 79.55%. That according to Vinutha et al. (2007), who classifies the
potential of gross extracts in weak inhibitors if they present a value below 30%, moderate inhibitors those that
present 40 to 50% and potent inhibitors those that show more than 50%.

Table 4. Percentage inhibition values of acetylcholinesterase

Sample Seeds oil Galantamine
Inhibition (%) 14.95±2.91 91.71±0.38
Coefficient of variance 0.19 0.004

4. Conclusion
This work shows the nutritional importance of a fruit that is not yet very considered in the market. The three
parts of the same have energetic importance, being the one of smaller contribution the pulp being able to be used
for the consumption in natura, as well as for the preparation of jellies, sorbets and another type of snack. In
addition, the content of saturated fatty acids in the oil extracted from their seeds should be highlighted, especially
linoleic acid, which has the structural and functional importance in the cell, besides being one of the essential
fatty acids related to the prevention of certain cardiac diseases.
An important property of this fruit is that the oil of its seeds has antimicrobial and anti-inhibitory applicability of
the enzyme acetylcholinesterase, related to Alzehimer’s disease.
Acknowledgements
We are grateful to CAPES for the scholarship received, the Federal University of Minas Gerais, especially the
researcher Jacqueline Aparecida Takahashi for biological analysis and Vanny P. Ferraz for analysis of gas
chromatography of the oil and group Oleochemicals and laboratory of Physics and soil fertility (NUPAGRI)
management UFRR for supporting the development this research.
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