Antimicrobial activity and acetilcolinesterase inhibition of oils and Amazon fruit extracts

Antimicrobial activity and acetilcolinesterase inhibition of oils and Amazon fruit extracts Ismael Montero Fernández*, Edvan Alves Chagas, Selvin Antonio Saravia Maldonado Jacqueline Aparecida Takahashi, Ricardo Santos Alemán, Antonio Alves de Melo Filho, Ricardo Carvalho dos Santos, Pedro Rômulo Estevam Ribeiro, Jhunior Abrahan Marcia Fentes, Pollyana Cardoso Chagas and Ana Cristina Gonçalves Reis de Melo


INTRODUCTION
In Brazil there are ten thousand plants considered medicinal, aromatic and useful, but around 99.6% of these plants are little known about the chemical composition (Silva et al., 2002). Many of these plant species have in their chemical composition secondary metabolites with a defensive function when they are attacked by certain microorganisms such as bacteria, fungi, parasites or virus among others. The compounds with antibacterial action usually are terpenoids, phenolic compounds, alkaloids, polypeptics, coumarins and camphors, being extremely numerous and at the same time, their chemical structures present high selectivity and specificity (Simões, 2003;Reschke et al., 2007;Chen et al., 2015). Fungi and bacteria present in plants environment make the latter to act in the fight against these phytopathogens, as well as against insect pests and herbivores (Peixoto et al., 2009). Since natural products have high biological activity, it is increasingly common to use extracts as an alternative, for example against certain diseases such as candidiasis treatment (Reis et al., 2011). Infectious diseases represent an important cause of morbidity and mortality in humans, especially in developing countries, and pharmaceutical industries have been motivated in recent years for the development of new antimicrobial drugs, especially due to the occurrence of microbial resistance to such diseases as the bacteria possess genetic ability and acquire resistance to drugs used as therapeutic agents (Nascimento et al., 2000).
On the other hand, there are compounds of natural origin that have the property of inhibiting acetylcholinesterase (AChE), a key enzyme in Alzheimer's disease. These compounds can be either isolated from plants or from microorganisms (dos Santos et al., 2017). Inhibition of AChE in vitro is attributed to several reasons, including the structure of phenolic compounds, considering the metabolism suffered by phenolic compounds after their ingestion at gastrointestinal tract and liver level (Roseiro et al., 2012).
In this context, the objective of this work was to perform a bioassay to evaluate inhibition of Gram-positive and Gram-negative bacteria, yeasts and the inhibition of the acetylcholinesterase enzyme by different oils and fruit extracts (abiu, acerola, camu-camu, bacupari, graviola, araçá, biribá and taperebá) of Northern Amazon and to correlate the different results through multivariate analysis techniques (PCA and HCA), aiming to collaborate to future pharmaceutical applications.

Sample preparation
Samples of the different fruits studied were collected in randomized points of the State of Roraima (Brazil) to guarantee the Fernández et al. 89 representativeness of the sample. Each of the fruits was collected in the corresponding production period and were collected in the ripening stage suitable for consumption. From all the samples collected at the different sampling points, a single composite sample was prepared for each of the fruits where they were taken to the Environmental Chemistry Laboratory of the Federal University of Roraima, where those that presented an optimum conservation status were selected. washed with 1% sodium hypochlorite solution and again with distilled water. Subsequently, a representative sample of each fruit was selected according to the following criteria: acerola, camu-camu and taperebá was selected 1 kg of fresh fruit, abiu, araçá and bacuparí was selected 2 kg of fruit and for biribá, fruta-do-conde and graviola were selected 10 units according with NTON 17002-02 (2002). All of them were separated into pulp, barks and seed and were placed in Ultrafreezer at -80ºC and then lyophilized in lyophilizer LIOTOP model L 101 for 48 hours until complete drying of the material and subsequently ground in LABOR model SP31 punch mill and stored material in airtight bags in the absence of light until the moment of performing the different analyzes.

Preparation of the extracts to realize the bioassays
The oil and extracts were obtained by extraction from hexane solvent in a Soxhlet apparatus for 6 h. The hexane was evaporated on a rotaevaporator and the oil and extracts were properly packaged in amber vials under nitrogen atmosphere and stored in a freezer (Jorge and Luzia, 2012).

Bioassays of fungi and yeasts
The extracts of different parts of the fruit studied were tested against the following microorganisms: yeast Candida albicans (ATCC 18804), Gram-positive bacteria Staphylococcus aureus (ATCC 29212) and Bacillus cereus (ATCC 11778), Gram-negative bacteria Escherichia coli (ATCC 25922) and Salmonella typhimurium (ATCC 14028). A pre-inoculum was prepared in which the microorganisms were transferred from the culture medium where they were stored into test tubes containing 3.0 mL of culture medium (BHI for bacteria and Sabouraud Broth for yeast). The tubes were then incubated in an oven at 37.5°C for 24 to 48 h. With the aid 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 adjusted to 600 nm (bacteria) and 530 nm (yeast), until obtaining transmittance between 74 and 75% (bacteria) and 75 and 76% (yeast), corresponding to the 0.5 McFarland standard turbidity, thus obtaining the suspensions of the inocula used in the bioassay. To prepare the working solution, the samples were previously solubilized in 12.5 mg.mL -1 dimethylsulfoxide (DMSO). From this solution, an aliquot of 40 μL was added to 960 μL of the culture medium used in the bioassay, obtaining a solution with concentration of 500 μg.mL -1 . The bioassays were run in 96-well plates in triplicate, adding 100 μL of the working solution at the concentration of 500 μg.mL -1 in three wells. Then, 100 μL of standardized microorganism inoculum was added to each well. Four controls were performed: growth control of the microorganism (to verify cell viability); the blank, which consists of the sample solution at the same concentrations evaluated, replacing the inoculum with sterile distilled water; positive control (the working *Corresponding author. E-mail: ismonterof@unex.es. Author(s) agree that this article remain permanently open access under the terms of the Creative Commons Attribution License 4.0 International License 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 an oven at 37.5°C and after 24 h the plate reader was read at 490 nm. The antibiotics used for the quality control of the assays were: ampicillin, for bacteria and miconazole, for yeast, previously prepared as described for the samples tested (CLSI, 2012).

Inhibition bioassay of the enzyme acetylcholinesterase (AChE)
The extracts of the different parts of Amazonian fruits were tested for inhibition of AChE enzyme using UV-visible molecular spectrophotometry method in 96-well microplates. A negative control of the assay was performed without the presence of inhibitors. The tests were performed in quintuplicate. An aliquot of 25 μL of acetylcholine iodide (15 mM) was pipetted into each well followed by 125 μL of 5,5'-dithiobis (2-nitrobenzoic acid) (DTNB); 50 μL of 0.1% w/v tris-HCl pH 8 bovine serum albumin and 25 μL of the samples (10 mg.mL -1 ) solubilized in DMSO with 10% v/v tween. The plates were read at 405 nm for 9 times over a period of 8 min. Immediately after the first reading, 25 μL of the enzyme acetylcholinesterase (Electrophorus electricus, Sigma Aldrich) (0.222 U.mL -1 ) was added and 9 readings were performed over a period of 8 min at 405 nm, the percentage inhibition of the enzyme shown in Equation 1. Eserine (10 mg mL -1 ) was used as positive standard control (Ellman et al., 1961;Rhee et al., 2001).
(1) where C = Control containing enzyme and substrate and A = Assay containing sample, enzyme and substrate.

Statistical analysis
Principal component analysis (PSA) and component hierarchy analysis (HSA) for total phenolic compounds and antioxidant activity by the two methods for the different parts of the fruit were evaluated using InfoStat software version 2016 (Rienzo et al., 2016).

RESULTS AND DISCUSSION
Results of the activity of pulps, barks and seeds of the different fruits studied in the microbiological inhibition and  AChE inhibition tests, respectively are shown in Tables 2 to 4. The inhibitory potential of pulp oils and extracts against S. aureus was relatively low for the oils tested, reaching only 28.80% for camu-camu pulp oil, and 25% inhibition for abiu and araçá, compared to amipicillin, the standard utilized, which presented 98.8% inhibition. The other Gram-positive bacterium tested, B. cereus, was less inhibited than S. aureus bacterium by the oils and pulps extracts from the Amazonian fruits. The highest percentages of inhibition were observed for abiu oil with 16.75% inhibition and araçá (15.97%), being very low values in relation to the standard ampicillin that showed 96.69% inhibition. According to Cordeiro (2011) S. aureus is present in both skin and mucous membranes, being considered as a causal agent of 7.7% of outbreaks of food poisoning in Brazil (Brazil, 2015). Morais et al., (2018) developed a study of biological activity for peppers from species Capsium species from the Amazonian region finding percent inhibition of S. aureus bacteria (13.71%) in line with the values found in this work.
B. cereus is present in food since it is resistant to the process of pasteurization. This bacterium reaches the environment easily contaminating food when the appropriate processing conditions are not used. It produces different toxins that shave human health when consumed in foods contaminated by this microorganism in concentrations of 105 to 108 CFU per gram of food (Granum and Lund, 1997).
For the two Gram-negative bacteria that were tested for the oils, E. coli was more inhibited, however in low percentages in relation to the tested standard ampicillin (inhibition = 96.03%). E. coli causes different types of diarrheogenic diseases (Kuhnert et al., 2000). The highest inhibition values were presented for the oil obtained from camu-camu pulp with 30.68% and abiu pulp with 25.33% inhibition. The other Gram-negative bacterium utilized in this screening was S. typhimurium which was also low inhibited by the oils tested. This bacterium is implicated in food poisoning problems causing gastrointestinal problems (Morpeth et al., 2009). Pulp of camu-camu was the best inhibitor of S. typhimurium (28.22% inhibition).
From the microorganisms tested in this study, the yeast C. albicans was the most susceptible microorganism to the oils tested. For instance, taperebá oil presented 85.22% inhibition, a percentage higher than that obtained for miconazole, the standard tested (82.82%), followed by abiu oil (76.71%) and aracá oil (64.28%). From the health point of view, it is interesting to look for new natural compounds with the capacity to inhibit this yeast since it causes candidiasis in the human body, an infection that can manifest in both oral and vaginal mucosa. C. albicans is the yeast from Candida genus predominant in the infection of candidiasis with 50%. Candida glabrata, Candida parapsilosis and Candida tropicalis are the minor yeats present in that infection. The worldwide mortality rate due to diseases due to yeasts from Candida genus is between 15 and 25% in adults and 10 and 15% in children (Alangaden, 2011).
As for the potential inhibition of acetylcholinesterase enzyme (AChE) by the oils and fats of the nine pulps, acerola pulp was the one with the most potent inhibition of AChE. Biribá, graviola and taperebá presented moderate inhibition potential and the other samples presented weak AChE inhibition potential (Vinutha et al., 2007) since it was considered that, for crude vegetable extracts, values above 50% mean potent inhibitors; between 30 and 50% are the moderate inhibitors and below 30% are weak inhibitors on AChE.
The inhibitory potential of the oils and extracts of the barks against S. aureus was lower than for the oils extracted from the corresponding pulps, since the samples tested did not reach 25% inhibition. Taperebá presented the highest percentage of inhibition against S. aureus (23.26%) while the tested standard amipicillin presented 98.8%. For the other Gram-positive bacterium tested (B. cereus), the oils from barks also presented low inhibition, the major percentage of inhibition found for the oil extracted from bacuparí barks with 38.70% of inhibition.
Form the two Gram-negative bacteria tested, E. coli presented the highest percentage of inhibition, but the values found are still low in relation to the tested standard ampicillin. The better inhibition was presented by acerola extract with 24.65% of inhibition followed by abiu barks oil with 21.53% inhibition. For S. typhimurium, the highest percentage of inhibition was for the oil from camu-camu barks (28.04%) followed by oil from acerola barks oil (19.01% inhibition). Among the microbiological inhibition tests for fruits barks, the best results were obtained towards the yeast C. albicans. The oil from barks of taperebá presented a potent antimicrobial effect with 94.46% inhibition. This value is even higher than that obtained for miconazole, the standard tested that showed 82.82% inhibition. The oil from barks of acerola inhibited 87.12% of C. albicans growth, which was also superior to the inhibition presented by the standard tested. Another extract very active against this yeast was that from camucamu barks with 71.35% inhibition.
Concerning the barks of the studied fruits, the oil extracted from biribá barks presented good inhibition of AChE (86.39%), even higher than the value found for the positive standard serine, in the conditions utilized in this test. The oil of taperebá barks also presented potent inhibition while abiu barks and fruta-do-conde presented moderate inhibition. Mota et al. (2012) tested different ethanolic extracts of medicinal plants in Brazil for the inhibitory capacity towards the AChE enzyme. In that study, the potent inhibition of AChE by the aqueous extract of Vitex agnus-castus L. was highlighted (74%), a value slightly lower than that found in this work for biribá barks oil.
The inhibitory potential of the oils and extracts prepared from the seeds (Table 4) was low. The oil of camu-camu seeds presented the best behavior but inhibition of S. aureus was only 27.11% while ampicillin achieved 98.8% inhibition, while for B. cereus, bacuparí seeds inhibited 26.73% followed by the acerola seed with 17.28% (ampicillin with 96.69% inhibition).
The Gram-negative bacteria were again, only slighted inhibited by the extracts. E. coli was more susceptible to the oil of acerola seeds (27.99% inhibition) and aracá seeds (25.55%), values very low compared to positive control (96.63%). For S. typhmurium, inhibition by seeds extracts and oils were still lower (19.13% inhibition for camu-camu oil). Only four of the nine oils and extracts from seeds inhibited C. albicans. Aracá oil was slightly more active (85.23% inhibition) than miconazole (82.82% inhibition).
Biribá, taperebá and fruta-do-conde oils presented potent inhibitory potential towards AChE according to the classification proposed by Vinutha et al. (2007). Acerola, graviola and camu-camu seeds presented moderate potential and the remaining seed oils presented weak inhibitory potential or did not present any inhibition of enzyme AChE.
Dos Santos et al. (2015) studied the bioactive potential of Annona hypoglauca seeds, another species of Annona, finding 79.55% of AChE inhibition, which is higher than the results found for the Annonaceae seeds studied in the present work. The percentage of C. albicans inhibition reported for A. hypoglauca (90.11%) while for the bacteria E. coli and S. aureus, literature results agree with those presented in this work.

Statistical analysis
Firstly, the coefficient was calculated to evaluate the consistency of the hierarchical groupings, obtaining a value of 0.973, and values close to the unit indicate a better representation (Ferreira et al., 2002;Cruz and Carneriro 2003;Moura et al., 2006).

Principal component analysis (PCA)
The main components analyses were carried out jointly for the evaluated systems (abiu, bacuparí, acerola, graviola, camu-camu, fruta-do-conde, araçá, biribá and taperebá) independently for each part of the fruits for C. albicans, S. aureus, B. cereus. E. coli, S. typhimurium and AChE in the different parts of the fruit in order to find variables (main components) which are not correlated to explain the structure of the variation. The weight of each variable analyzed in each component (axes) is represented. The main components blipot for the different parts of the evaluated fruit are as shown in Figures 1 to 3.
In blipot (Figure 1), the results of the principal component analysis (PCA) for the microbiological inhibition of the extracts of the different pulps studied are represented and 82.3% of the original variability of the data retained in these components is explained.
The arrangement of the sequence in Figure 1 shows that the systems can be grouped into two sets, the first major component (CP1) contributed 46.9% of the total variance explained, however most of the variables that were strongly affected contributed from all bacteria and yeasts (B. cereus, C. albicans, E. coli, S. aureus, and S. typhimurium) were positive for CP1; the other test was inhibition of the AChE enzyme.
The second main component (CP2) explained 35.4% of the total data appearing in this case, the percentage of AChE inhibition. Analysis of this component also showed that this attribute negatively projects on the bacteria and yeast tested being the extracts of the acerola, biribá, graviola and bacuparí pulps have been associated.
In blipot (Figure 2), the results of the principal components analysis (PCA) for the microbiological inhibition of the extracts of the different pulps studied are represented and 65.7% of the original variability of the data retained in these components. The arrangement of the sequence in Figure 2 shows that the systems can be grouped into two sets. The firstmajor component (CP1) contributed 38.3% of the total variance explained. However, most of the variables that were strongly affected that contributed to all bacteria and yeasts (B. cereus, C. albicans, E. coli, S. aureus and S. typhimurium) were positive and the other test was the inhibition of the AChE enzyme. These results indicate that CP1 allowed to distinguish the oils from fruits that are strongly associated, being taperebá, abiu, bacuparí and acerola barks. The second main component (CP2) explained 27.4% of the total data, appearing in this case, the percentage of inhibition of AChE. The analysis of this component also showed that this attribute projects  negatively on the tested bacteria and yeast, being the extracts of the biribá, fruta-do-conde and graviola barks, who have been associated.
In blipot (Figure 3), the results of the principal component analysis (PCA) for the microbiological inhibition of the extracts of the different pulps studied are presented explaining the 73.2% of the original variability of the data retained in these components.
The arrangement of the sequence in Figure 3 shows that the systems can be grouped into two sets, the first major component (CP1) contributed 48.6% of the total variance explained. However most of the variables that were strongly affected contributed from positive form to CP1 the bacteria and yeasts (S. aureus, C. albicans, E. coli and B. cereus) and inverse with the other test that was the inhibition of the enzyme AChE and S. typhimurium. These results indicate that CP1 allowed distinguishing oils from fruits that are strongly associated with them being camu-camu with araçá and acerola and abiu with bacuparí. The second main component (CP2) explained 24.6% of the total data, appearing in this case, the percentage of inhibition of AChE and S. typhimurium. The analysis of this component also showed that this attribute projects negatively on the other bacteria and yeast tested being the oils and extracts of the seeds of the fruta-do-conde and taperebá were associated.

Analysis of hierarchical groupings (HCA)
Through HCA, data can be displayed in a two-dimensional space in order to emphasize their natural groupings and patterns, relating the samples so that the most similar are related to each other presenting the samples in dendogram, grouping the samples and variables according to their similarity. Figures 4 to 6 show the dendrograms for HCA analysis of inhibition of the different strata studied.
For the percentages of inhibition of oils and extracts of the fruits studied, the trends observed through the analysis of PCA main components were observed through the HCA, mainly observing two large groups: one of them formed by the association of araçá with abiu that present a major contribution, smaller Euclidean distance and they are grouped together with taperebá for a major Euclidean distance, but are still associated. On the other hand, the other existing grouping just as happens in the HCA it is for acerola and graviola that they are strongly associated and increasing the Euclidean distance, they are associated with the fruta-do-conde. Finally, the two fruits whose extracts have opposing antimicrobial properties which are the bacuparí pulp with the camucamu pulp which are not related in the HCA joining, the elevated Euclidean distance of 21.58%.
For the percentages of inhibition of fruits oils and extracts studied, the trends observed through the analysis of PCA main components of the barks were observed by HCA where there is great dispersion between the grouping of the studied fruits where the main grouping to be highlighted with smaller Euclidean distance is for graviola with abiu.
For the seeds two large clusters are observed being in agreement with the results presented by PCA. The first association is that between taperebá and graviola with smaller Euclidean distance that are later associated with biribá. The other association with Euclidean distance of 4.09 is for the strata of the fruta-do-conde and camucamu. All these fruits are grouped together and the HCA shows that they do not have a relation with or another group of fruits that contribute in an opposite way (bacuparí, araçá, acerola and abiu) whose connection happens with elevated Euclidean distance of 16.38.

Conclusion
Given the potent results of the oils and extracts tested in this article against the potential inhibition of C. albicans  yeasts such as the barks and pulps of taperebá, acerola barks or araçá seed, this can be a starting point for the development of new drugs with specific action to minimize virulent factors making difficult the development of the infectious process of candidiasis caused by the yeast C. albicans.
On the other hand, the results found for the antibacterial action of the oils and extracts did not present high inhibition percentage to inhibit the bacteria of pathogenic action.
Finally, in relation to inhibition of AChE, several extracts demonstrate a strong inhibitory capacity of the enzyme such as biribá barks, taperebá seed or acerola pulp, but in most of the studies, the isolated compounds responsible for the AChE inhibitory activity have not been identified or characterized.