Bromatological Analysis , Chemical Composition and Bioassays from the Genipa americana L . ( Rubiaceae )

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%.


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 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): 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: 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: 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): 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).
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.

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 BF 3 /methanol (14%) by 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).

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, 10 8 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): %Inhibition = 100 -

Absorbance of the sample with enzyme -Absorbance of the sample without enzyme
Absorbance of the negative control with enzyme -Absorbance of the negative control without enzyme × 100 (7)

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±0.13Kcal/100 g, highlighting that in this study they accounted for total dietary fibers.

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 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.

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.

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%.

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 applicability of the enzyme acetylcholinesterase, related to Alzehimer's disease.

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

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

Table 4 .
Percentage inhibition values of acetylcholinesterase