Chemical profile , antimicrobial activity , toxicity on Artemia salina and anti-acetylcholinesterase enzyme essential oil from Bauhinia ungulata L . ( Fabaceae ) leaves

Chemical profile, antimicrobial activity, toxicity on Artemia salina and anti-acetylcholinesterase enzyme essential oil from Bauhinia ungulata L. (Fabaceae) leaves Sandra R. N. A. Medeiros*, Antonio A. de Melo Filho, Habdel N. R. da Costa, Francisco dos Santos Silva, Ricardo C. dos Santos, Jacqueline A.Takahashi, Vany P. Ferraz, Ana C. G. R. de Melo, Pedro R. E. Ribeiro, Andreina G. de Andrade Laranjeira, Angela K. Kamezaki, Regildo M. G. Martins and Flavio da Silva Paulino


INTRODUCTION
Among many medicinal plants, there is the Bauhinia ungulata L. species, belonging to the genus Bauhinia belonging of Fabaceae family (Caesalpinioideae subfamily) one of the largest of angiosperms, which can be found mainly in tropical areas, with about 250-300 species (Souza and Lorenzi, 2008;Joly, 1993Joly, , 1998)).
In Brazil this species is popularly known pata de vaca, unha de boi, pé de boi, escada de macaco, unha de jaboti and mororó (Silva and Cechinel, 2002;Lorenzi and Matos, 2002).It is applied in the treatment of diabetes mellitus, cholesterol control as diuretic and expectorant (Correa, 1998).It was also reported the use of B. ungulata by Tapebas Indians in Ceará state, Brazil for the treatment of diabetes, and this species most frequently studied for its hypoglycemic action (Silva et al., 2002;Pepato et al., 2002;Morais et al., 2005).
In addition to the genus activities, aforementioned, there are the major compounds and compounds highlighted, folow.The essential oil from the leaves of another species, B. acurana, has as main constituents spathulenol, sesquiterpenes, epi-α-cardinol and caryophyllene oxide (Gois et al., 2011).The major constituents of essential oil from B. ungulata are spathulenol and caryophyllene oxide (Gramosa et al., 2009).
Essential oils are a lipophilic moiety of the chemical composition of a plant.Generally, consisting of sesquiterpenes, monoterpenes and phenylpropanoids (Cunha et al., 2004).Aromatic drugs are frequently used to destroy infection causing agents such as bacteria and pathogenic fungi (Costa, 2002).They are also used in industries, food and cosmetics (Bizzo, 2009).
This study aims to characterize the main volatile chemical constituents using gas chromatography and biological activities by essential oil from B. ungulata leaves.

Plant material and essential oil extraction
The leaves of B. ungulata were collected along the Água Boa River, near the BR 174, at 11 Km in Boa Vista, Roraima, Brazil, in January 2016 (dry season).The plant material was identified by José Ferreira Ramos (National Institute for Research in the Amazon, INPA), and a voucher specimen (272558) was deposited at the INPA Herbarium.
Fresh leaves (600 g) were cut into smaller pieces with scissors and putting in a Clevenger apparatus to obtain the essential oil by Medeiros et al. 443 hydrodistillation.Water droplets were removed from the essential oil by anhydrous sodium sulfate and stored at -20°C before analysis (Rubiolo et al., 2010;Sefidkon, 2002).

Determination of toxicity on Artemia salina
The essential oil was solubilized in Tween 20 (1%) and saline supplemented with water to give concentrations (1000, 500, 250 e 125 μL mL -1 ).They were transferred to tubes (3 mL) and added 10 organisms (nauplii Artemia salina).The tests were performed in triplicate for each concentration.Saline without extract was used as negative control also in triplicate and was subjected to the same experimental procedure.This system was incubated at room temperature for 24 h, with aeration and other tubes kept under illumination.After 24 h, the number of dead and live larvae in each tube was counted.Thereafter, the probability of mortality was calculated according to the formula: Where: r = number of dead nauplii; n = total number of A. salina in each tube.

Acetylcholinesterase (AChE) inhibition assay
Aliquots of a working solution (25 μL) (sample in Tween/DMSO/30%) was added to microplate wells, positive and negative controls were also prepared.To the first five wells of *Corresponding author.E-mail: antonio.alves@ufrr.br.
Author(s) agree that this article remain permanently open access under the terms of the Creative Commons Attribution License 4.0 International License a column (positive control), 25 μL of an eserine solution prepared at 10 mg mL -1 (in Tris/HCl at pH 8.0) was added.Thereafter, 25 μL of acetylthiocholine iodide (ATChI, Sigma A5751); the reaction mixture, 125 μL of 5'.5-dithio-bis (2-nitrobenzoate) (DTNB, Sigma D8130) and 50 μL of Tris/HCl (pH 8) containing 0.1% (m/v) bovine serum albumin were added to each well.Absorbance was measured at 405 nm every 1 min for 8 times.Then 25 μL (0.226 U mL -1 ) of Electric eel AChE (type VI-S) provided by Sigma (C3389-500UN) in Tris/HCl were added to each well.Absorbance was measured at 405 nm by 9 times (Frank and Gupta, 2005;Ellman et al., 1961).Percentual inhibition was calculated using the formula: Where PCA = (absorbance of the sample with enzymeabsorbance of the sample without enzyme); PSA = (absorbance of negative control with enzyme -absorbance of negative control without enzyme).

Antibacterial and yeast assay
Two Gram-negative: Salmonella typhimurium (ATCC 13311) e Citobacter Freundii (ATCC8090), two bacterium Gram-positive: Staphylococcus aureus (ATCC 25923), and Bacillus cereus (ATCC 11778) and one fungus (yeast) Candida albicans (ATCC 18804) yeast were used in the assay.Concentrations assayed were 250, 125, 62.5, 31.25,15.6, 3.9 and 1.95 μg mL -1 (Zacchino and Gupta, 2007).Samples were weighed and dissolved in DMSO to 500 mg mL -1 .124 μL of this solution was added to a flask containing 2976 μL of BHI (Brain Heart Infusion) broth (working solution) for bacterium and 2976 μL of Sabouraud for yeasts.A pre-inoculum was prepared in which the bacteria and the yeast, stored under refrigeration, were transferred with a platinum loop to test tubes containing 3 mL of freshly made BHI broth.The tubes were incubated at 37°C for 24 h.Then, the pre-inoculum (500 μL) was transferred to tubes containing 4.5 mL of sterile distilled water.The tubes were homogenized and the concentration adjusted to 0.5 of McFarland turbidity standard (108 CFU mL -1 ), thereby obtaining the inocula used in the bioassays.
Assays were performed in 96-microwell plates in triplicate.100 μL of BHI broth was added to each well.In the first well, 200 μL of working solution were also added.The solution was homogenized and 100 μL transferred to the next well and so on until the last well, from where 100 μL was discarded.Then, 100 μL of microorganism inocula were added to wells.Eight different concentrations of each sample were tested.A positive control devoid of the working solution allowed us to examine microorganism growth.A negative control, which lacked the inoculum made it possible to discount the color coming from the working solution.A control plate containing 100 μL of BHI culture medium and 100 μL of sterile distilled water were added to the experiment as a control of BHI broth sterility.
Another control was also prepared, containing the standard antibiotics Ampicillin (antibacterial), miconazole and nystatin (antifungals) to observe the activity of these antibiotics over the microorganisms.Microorganism growth was measured in ELISA plate reader (492 nm) immediately after ending the experiment (0 h).They were incubated at 37°C and read again after 24 h of experiments, ending the test.Results were calculated as percentual inhibition using the formula: AC1 = absorbance of the sample; AC2 = absorbance of control sample; AH = absorbance of microorganisms in the control control and AM = absorbance of culture medium control.

RESULTS AND DISCUSSION
The yield of essential oils of B. ungulata in this study was 0.066%, higher than that obtained by Gramosa et al. (2009) who in their study developed this species in the Northeast, which was 0.007%.The yields of essential oils obtained from the genus Bauhinia varies between bauhinia rufa with 0.37% (Silva and Camara, 2014) and B. acuruana with 0.01% (Gois et al., 2011).
Table 1 shows the chemical components identified by chromatographic analysis GC-FID and GC-MS with their Kovats index and mass and Figure 1 shows chemical structures of the major constituents identified in the essential oil in B. ungulata.
Figure 2 shows the chromatographic profile for B. ungulata oil.18 components corresponding to 65.5% of essential oil composition were identified.Among the compounds identified, nine are majority corresponding to 61.1%.
In Boa Vista, Roraima, Brazil compared to studies Gramosa et al. (2009), the composition varies mainly in the major constituents.Secondly, according to Oliveira et al. (1998), plant species, in general, may present variations in relation to the yield and chemical composition of essential oils according to the part of the plant studied as well as their interactions with the environment, climate, micro-organisms and also genetic factors.Gramosa et al. (2009) identified 13 compounds, which represent 95.9% of the essential oil content, the majority were spathulenol (47.7%), caryophyllene oxide (18.3%), humulene epoxide II (5.2%), β-caryophyllene (4.2%), αhumulene (3.5%) and α-copaene (2.9%).
It is observed so spathulenol presented itself as the main constituents for the essential oil of B. ungulata in studies of Gramosa et al., (2009), with a percentage of 47.7.However in the present study this compound presents a lesser amount (2.1%).
According to Figueiredo et al. (2008) and Simões et al. (2004), depending on the part of the plant, type of collection, climatic conditions and extraction methods, essential oils, may suffer variations in their income.
In this study it was found that the highest percentage constituent was the β-caryophyllene (15.9%), a fact equivalent to the study of Neto (2006), where this substance was also the majority with 25.65%.
Oils with high concentrations of β-caryophyllene  (Ferreira, 2014).The α-bisabolol (4.7%) was not identified in other regions for the species B. ungulate, indicating that it may have been the formation of a new chemotype for this species in Boa Vista, RR.
The α-bisabolol compound has correlation with activity against acetylcholinesterase.According to Nurulain et al. (2015), the compound binds directly to the receptor a7-nAChRs, leading to inhibition of acetylcholine.
It also has anti-inflammatory action (Kim et al., 2011) and biological activity against bacteria and fungi, as well as, Aedes aegypt, S. aureus, Pseudomonas aeruginosa, Microsporum gypseum, Trichophyton mentagrophytes and Trichophyton rubrum (Vila et al., 2010).The Figure 3 shows the bioactivity of essential oil from B. ungulata leaves for A. salina.
Interpretations of the results of toxicity were carried out taking into account the above literature, which can be classified as highly toxic LC 50 values between 0-500 µg mL -1 ; moderate toxicity between 500-1000 µg mL -1 and low toxicity or nontoxic values above 1000 µg mL -1 (Meyer et al., 1982;Lopes et al., 2002;Rodriguez et al., 2004).    .According to Amarante et al. (2011), the plant extracts and derivatives that have a high toxicity against A. salina are high potential indications for biological activities.This finding reinforces the importance of the method as it is very useful to use this bioassay, when you want to develop biological studies.The results of the inhibition tests for acetylcholinesterase essential oil B. ungulata are as shown in Table 2.
The oil showed a good inhibitory potential, corresponding to 95.96%, and can see that the standards used for Eserine and galantamine showed inhibition of 91.93 and 94.36%, respectively, which were lower than the inhibition of essential oil.Savalev et al. (2003) observed synergistic interactions among the components (1.8-cineole, camphor, α-pinene, β-pinene, borneol, caryophyllene oxide, linalool and bornyl acetate) which come from the species Salvia lavandulaefolia.Among those components mentioned above was found the essential oil of B. ungulata in the presence of α-pinene, βpinene and caryophyllene oxide; certainly there was synergism of compounds present in the oil of the species studied by high inhibition of the enzyme, highlighting the chemical components β-caryophyllene and α-bisabolol, which has a close relationship with neurodegenerative diseases and reported by Nurulain et al. (2015), Ferreira (2014), Alcantara et al. (2010) and Santos et al. (2015).
Another biological activity of essential oil of B. ungulata leaves deserves attention as well as the bioactivity against the pathogenic microorganisms (Table 3).Graphs were plotted with the aid of software origin 8.0, as can be seen in Figure 4 and using equation Y = A2 + (A1-A2) / (1 + (x/x0) ^p) was calculated from IC 50.By analyzing the percentages of inhibition and IC 50 , it can be seen that there has been satisfactory inhibition of four of the tested microorganisms, whereas inhibition was greater than 50%.Checking greater emphasis on the S. aureus which was inhibited in all eight concentrations tested and showed a IC 50 < 1.95 µg mL -1 as well as C. albicans to inhibit the microorganism concentration 15.62 μg mL -1 of essential oil, this value was found to be in agreement with the IC 50 (16.4µg mL -1 ).It is noteworthy that the three highest concentrations were those that had greater inhibition ranging from 80-85%.
The Figure 4 shows graphs of the minimum inhibitory concentrations of the essential oil to the front microorganisms studied.The bacterium C. freundii showed an inhibition of 46.16%, thus less than 50%, not showing satisfactory results.

Figure 2 .
Figure 2. Chromatogram of total ions essential oil from leaves of B. ungulata L.
Through the straight equation formula Y = A + BX, we can calculate the CL 50 .Considering Y = 50, A = 42.183e B = 0.054, is the value of X is equal to 144.75 µg mL -1.It can be considered that the essential oil of B. ungulata has high toxicity, based on the LC 50 value found to be less

Figure 3 .
Figure 3. Curve ahead activity of A. salina for essential oil from leaves of B. ungulata L.

Figure 4 .
Figure 4. Activity curve for essential oil from leaves of B. ungulata (L) front of the A) Candida albicans; B) Bacillus cereus; C) Staphylococcus aureus; D) Salmonella typhimurium.

Table 1 .
Chemical composition (%) of essential oils from fresh leaves of B. ungulata L. leaves.
Figure 1.Chemical structures of the major constituents from essential oil from leaves of B. ungulata L.

Table 2 .
Results of inhibition of acetylcholinesterase and their respective standard deviations.

Table 3 .
Antimicrobial activity of the essential oil from B. ungulata leaves