Isolation of active antiphytopathogen compound from extracts of Anadenanthera colubrina var. cebil

Several microorganisms are responsible for great economic losses in world agriculture. Preventive and treatment methods are applied to avoid contamination of crops by these microorganisms, however, the use of chemical antimicrobials damages health and the environment. Secondary plant metabolites are safe natural sources of antimicrobials for this application. Fabaceae family has its history described in the literature as a potential source for obtaining antimicrobial bioactive. The objective of this work was to isolate bioactive compounds guided by antimicrobial assays against bacteria and fungi in vitro. Organic extracts were prepared by eluotropic series of leaves of Anadenanthera colubrina var. cebil and were tested against six bacteria and six fungi phytopathogenic. The antimicrobial assays of minimum inhibitory concentration (MIC) and minimum microbicidal concentration (MMC) were performed at each purification step that occurred through HPLC-DAD, Flash Chromatography and HPLC-preparative analysis, to confirm the isolation of the bioactive. Through bioguided isolation, the compound p-hydroxybenzoic acid was obtained, which showed activity against the phytobacteria Xanthomonas campestris pv. campestris and Acidovorax citrulli.


Introduction
Vegetable diseases, caused by several pathogenic microorganisms, are one of the main problems faced by world agriculture [1] . Bacteria and fungi are among the main pathogens that cause a great decrease in productivity and consequently economic losses in this sector [2] . Among the methods applied to control diseases in the field are preventive methods, which have low efficiency, and treatment methods, such as the application of synthetic antimicrobials [3] . These have caused problems related mainly to damage to human and animal health, in addition to the accumulation of environmental contamination [4] .
Secondary plant metabolites are natural sources of substances with antimicrobial properties, which can be used as an alternative in substitution to synthetics [5] . This strategy minimizes the negative impacts associated with being considered safe for health and the environment, in addition to being economically advantageous due to the low cost, they are also capable of reducing the impact of microorganisms on agriculture. In addition, plant extracts reduce the possibility of causing microbial resistance, as they are complex mixtures of metabolites [6] .
Several studies have demonstrated how the species of the Fabaceae family have potential as sources of antimicrobials for a great diversity of pathogens [7][8][9] . Anadenanthera colubrina var. cebil (Griseb.) Altschul, popularly known as Angico, belonging to this family, it is widely used in folk medicine because it is related to the antimicrobial properties of its leaves [10,11] . This work aimed to conduct a bioguided study on organic extracts of A. colubrina var. cebil, to obtain compounds with antimicrobial activity against phytopathogenic bacteria and fungi.

Plant material
Leaves of A. colubrina were collected in the Catimbau National Park (08°34′30,96″ S e 37°14′51,76″ W), in the Northeast of Brazil. The collected material was oven dried at 45ºC for 72 h, then ground to obtain a thin powder, stored in an airtight container and kept at 4ºC until use. One specimen identified and registered by the Herbarium Dárdano de Andrade-Lima of the Instituto Agronômico de Pernambuco (IPA), under voucher IPA -80350. The plant material was registered in the Sistema Nacional de Gestão do Patrimônio Genético e do Conhecimento Tradicional Associado (SisGen) at number A08E18B.

Organic Extracts
A hundred grams of the powder of the leaves de A. colubrina subjected to eluotropic series of organic solvents: cyclohexane (CHX), chloroform (CHL), ethyl acetate (EtOAc) and methanol (MeOH) in Soxhlet, respecting the boiling temperature of each solvent, and each was kept under reflux for 24 h. The extracts obtained were then filtered (Whatman n°1), and the solvents were entirely removed on a rotary evaporator at 45°C under reduced pressure. The dry extracts were stored at 4°C hermetically sealed until use.

Screening Antimicrobial Activity
To evaluate the antimicrobial activity of A. colubrina leaves, the organic extracts, fractions, and isolated compound were solubilized in an aqueous solution at a concentration of 100 mg.ml -1 with 10% dimethyl sulfoxide (DMSO) and were sterilized by filtration through a microfilter 0.22 µm (GV-Millipore).
The minimum inhibitory concentration (MIC) was determined by the microdilution method (CLSI, 2011) with modifications. A serial dilution of the extract/fractions was prepared in NYD or BD and 15 μl (Absorbance 600 nm = 0.150 ± 0.05) of bacteria or fungi suspension was added. The concentration of the samples ranged from 50 mg.ml -1 to 100 µg.ml -1 for organic extracts, from 6 mg.ml -1 to 22 µg.ml -1 for fractions and 500 to 1 ug.ml -1 for purified compound. The samples were incubated for 24 h for bacteria and 48 h for fungi, at 30°C for both. As a positive control, chloramphenicol was used for bacteria, and cercobin for tested fungi and sterile water with DMSO (10%) was used as a negative control. All tests were performed in triplicate.
To determine the minimum bactericidal or fungicidal concentration (CMB or CMF) after the microplate incubation period, 5 µL of the solution from each well was transferred to NYDA plates and incubated again for the same period. The complete absence of growth on the agar surface with the lowest concentration of the sample was defined as the MBC or CMF, respectively for bacteria and fungi.

Flash Chromatography
The active organic extract was fractionated by flash chromatography (Biotage Isolera one TABLE 1 shows the results obtained about the antimicrobial power of organic extracts from A. colubrina leaves against six phytopathogenic bacteria, according to the extraction solvent used. It is observed that all extracts showed reduced growth in the tested phytobacteria when compared to the control. However, it is observed that MIC ≤ 1.56 mg.ml -1 is registered in 66.6% of the phytobacteria in the ethyl acetate extract, followed by 33.3% in the chloroform and methanol extracts, and the cyclohexane extract showed no activity for any of the species. When evaluating the MBC, it is observed that only the ethyl acetate extract has bactericidal activity in concentrations ≤ 1.56 mg.ml -1 for A. citrulli and X. campestris pv. campestris.

Results and Discussion
The antifungal activity of the extracts against phytopathogenic fungi shown in TABLE 2. It observed that there was a reduction in growth in the tested concentrations when compared to the control, however, no extract was considered to have relevant inhibitory or antifungal activity due to all results being higher than 6.25 mg.ml -1 . In both microbiological tests, it was observed that the DMSO used to solubilize the organic extracts in an aqueous medium, did not affect the bacterial or fungal growth in the negative controls in the concentration used.  From the results obtained in the screening of antimicrobial activity, bioguided purification of the bioactive compound was continued. Given the observed, the EtOAc extract was selected to be analyzed on HPLC-DAD (FIGURE 1). This analysis allowed the detection of six main peaks (≥ 500 mAU), between the retention time (Rt) 3 and 4 min, peak 1 (λ max 228, 260 and 294), between Rt 4 and 5 min, peak 2 (λ max 256), between 7 and 8 min, peak 3 (λ max , 265, 354) peak 4 (λ max 256, 352) and peak 5 (λ max 256, 356), and between 10 and In the image we can see the six main compounds: peak 1 (λ max 228, 260 and 294), peak 2 (λ max 256), peak 3 (λ max , 265, 354), peak 4 (λ max 256, 352), peak 5 ( λ max 256, 356) and peak 6 λ max 256, 370).
The EtOAc extract was subjected to Flash Chromatography (Biotage™), this semi purification generated six fractions grouped according to the UV absorption spectrum (FIGURE 2). These were tested against the microorganisms Acc and Xcc according to the activity recorded for the crude EtOAc extract (TABLE 3).
Fraction 3 for the Xcc bacteria showed activity, as it had a MIC less than 1 mg.ml -1 , so it was selected for the purification of the active compound.  Thus, flash fraction 3 was analyzed and was subfractionated by HPLC-Preparative, giving rise to six subfractions that were again evaluated for their antimicrobial activity (TABLE 4). Subfraction 3 showed activity with MIC and MBC of 0.5 mg.ml -1 for Xcc. In view of its antibacterial potential, subfraction 3 was analyzed by HPLC-DAD, and a pure compound was detected. The UV spectrum associated with data available in the literature indicates that this compound is p-hydroxybenzoic acid (FIGURE 3), tracked through its UV absorption spectrum using flash chromatography (Biotage™) and HPLC-Preparative (Autopurification System™).  Given the importance of developing a biopesticide, several studies have been carried out, in search of this objective, thus, a screening carried out by Silva et al. [11] with several plants of medicinal importance against several phytopathogens pointed to A. colubrina as an important source of antimicrobial compounds. From this result, a bioguided purification approach was carried out, starting with an extraction following the eluotropic order of solvents and using an in vitro anti-phytopathogenic bioassay. According to Santos et al. [12] to fight bacteria, an extract with MIC > 2.0 mg.ml -1 is considered inactive, therefore, the active and promising extract for the isolation of bioactive compound was the one with the lowest MIC and MBC values. According to this criterion, the EtOAc extract was the most active, with MIC and MBC ≤ 1.56 mg.ml -1 against the bacteria A. citrulli and X. campestris pv. campestris.
HPLC-DAD analysis of the crude EtOAc extract revealed a phenolic acid as the major (peak 2), with λ max 256 nm, it's ultraviolet (UV) absorption spectrum being very similar to p-hydroxybenzoic acid [13][14][15][16][17] . This acid was previously isolated and identified in Anadenanthera colubrina in the works of Gutierrez-Lugo et al. [18] and Weber et al. [19] . It was also possible to verify the presence of four flavonoids ( (λ max 257,358) among others [20] . Peak 6 λ max 256 and 370 identified through the standard, as already mentioned, is quercetin.
The purification of the bioguided EtOAc extract by the assay against phytopathogens in vitro resulted in the isolation (FIGURE 2)  of the extract and the isolated substance may be due to the presence of nutritional components, common in extracts, such as proteins and sugars, which can contribute to the development of the microorganism [21] .
Our data are in agreement with the findings of Araújo et al. [22] which isolated and identified by nuclear magnetic resonance (NMR) p-hydroxybenzoic acid the major compound of the ethyl acetate extract of aerial parts of A. colubrina.
Phenolic acids are part of the group of phenolic compounds, rarely occur as free acids, are divided into benzoic, cinnamic acids and their derivatives, p-hydroxybenzoic acid is the simplest form found in nature [23] .
Despite data on the antimicrobial effects of phenolic acids [24,25] , studies dealing with the anti-phytopathogenic properties of its metabolites or derivatives are still scarce. Literature data indicate that p-hydroxybenzoic acid inhibits the growth of plant pathogens. Cho et al. [26] found that the acid inhibits the growth of X.
campestris with an IC50 of 0.136 mg.ml -1 . However research indicates that the phytopathogen Xcc may have developed a functional degradation pathway of 4-HBA (4-hydroxybenzoate) that plays a role in detoxifying phenolic metabolites in the host during infection, however, the mechanistic details and the biological significance of this phenomenon have yet to be elucidated [27] .
In fungi assays, all extracts were ≥ 6.25 mg.ml -1 , however, the CHX extract was the most active with MIC and MBC of 6.25 mg.ml -1 for Aspergillus flavus, Rhizopus sotolonifer, and Verticillium lecanii, the rest of the extracts presented MIC and MFC ≥ 12.5 mg.ml -1 . Campos et al. [28] perform a bioguided assay with A.
colubrina against fungi and identify or extract hexane as the most active, assign an antimicrobial activity to three substances, among them: β-sitosterol and β-sitosterol linoleate, both in fruits and leaves and with MICs of 0.25 and 0.5 mg.ml -1 in front of Alternaria alternata respectively. Due to the similarity with ergosterol, steroidal substances can compete with fungal proteins involved in the synthesis of this metabolite and can be lethal to the fungus [28] .

Conclusion
The bioguided approach carried out with the extracts of the leaves of A. colubrina through in vitro antimicrobial tests led to p-hydroxybenzoic acid which, in addition to being the major component of the active extract, showed antimicrobial activity against the phytopathogens Xanthomonas campestris pv. campestris with MIC and MBC of 0.5 mg.ml -1 . The data highlight the potential of the species as an alternative source of this compound to fight diseases of economic importance in agriculture.