Effects of Rainfall on the Antimicrobial Activity and Secondary Metabolites Contents of Leaves and Fruits of Anadenanthera colubrina from Caatinga Area

Background: Anadenanthera colubrina (Vell.) Brenan var. cebil (Griseb) is a plant widely used for medicinal proposes in Brazilian Northeast. Objective: This study aimed to analyze the influence of rainfall indexes (RI) in antimicrobial activity and phytochemical constituents of extracts from leaves and fruits of A. colubrina. Material and Methods: Samples were collected in Catimbau National Park (Buíque, Pernambuco, Brazil) at September 2010 (RI: 75 mm) and January (RI: 65 mm), April (RI: 162 mm) and June 2011 (RI: 73 mm). The extracts were prepared by Soxhlet extraction using cyclohexane, chloroform, ethyl acetate and methanol. The antimicrobial activity was determined by MIC and MBC values. Results: All extracts showed antimicrobial activity, but ethyl acetate extracts (from all periods) were more active. Strong correlations were found between the RI and the average MIC of MLE (ρ: -0.99), EALE (ρ: -0.81), CHFE (ρ: -0.81), EAFE (ρ: -0.80); while moderate and weak correlations were found for other extracts. Through a HPLC analysis was possible to reveal that the samples collected from dry periods had more chemical diversity (as they presented more peaks). Gallic acid and quercetin (and derivative compounds) were identified. The levels of quercetin were enhanced in extracts from dry months. Conclusion: Our results showed that the rainfall has a positive effect on the antimicrobial activity of leaves and fruits of A. colubrina, however these extracts showed more chemical diversity during dry months.


INTRODUCTION
The World Health Organization (WHO) has been drawing attention to the increasing problem of microbial resistance to conventional antibiotics for more than a decade. 1 This scenario of antimicrobial resistance has encouraged research using medicinal plants, especially those from under-exploited biomes, such as the Caatinga.This is a unique biome from Northeast of Brazil where traditional communities use hundreds of plant species form medical purposes, such as the control of microbial infections. 2,35][6][7] In recent years, the ethnomedicinal knowledge has stimulated several studies about the pharmaceutical potential of natural products of northeastern Brazil. 8This is justified by the fact that the intrinsic features of Caatinga area (drought, high solar radiation rates and other environmental stresses) influence the synthesis of secondary metabolites of its plants, 9 making them attractive targets for bioprospecting programs. 10In traditional medicine, Anadenanthera colubrina (Vell.)Brenan var.cebil (Griseb) Alstchull (1964), popularly known as Angico (synonyms: Acacia cebil, Peptadenia macrocarpa, Anadenanthera macrocarpa), 11 is frequently used by inhabitants of the Caatinga biome to treat anemia, cancer and inflammatory diseases. 2,36]12 In this context, the present study aims to analyze the effects of rainfall on the antimicrobial activity and secondary metabolites contents of leaves and fruits of Anadenanthera colubrina.With this information, the best collection time to maximize the bioactive compounds extraction for medicinal purposes may be determined.Samples (100g) from each tissue were subjected to Soxhlet extraction using an eluotropic series of solvents in the following order: cyclohexane, chloroform, ethyl acetate and methanol.All samples were subjected to saturation at reflux for 24 hours.After this time, the extracts were filtered (Whatman filter paper No 1).Solvent was completely removed from all extracts from leaves (L) or fruits (F) (cyclohexane: ChLE and ChFE; chloroform: CLE and CFE; ethyl acetate: EALE and EAFE: methanol: MLE and MFE) on a rotating evaporator at 45 o under reduced pressure and stored for later antimicrobial and phytochemical analyses.

Determination of minimal inhibitory concentration (MIC) and minimum bactericidal concentration (MBC)
The minimal inhibitory concentration (MIC) was determined by the microdilution method. 12Twofold serial dilutions of each extract (initial concentration: 50 mg/mL) were prepared in Müller-Hinton broth (MHB) and 10 μL of bacterial suspension (approximately 1.5 × 10 8 CFU/ mL) were added.The samples were incubated for 24 h at 37°C.Resazurin solution (0.01%) was used as an indicator by color change visualization: any color changes from purple to pink were recorded as bacterial growth.The lowest concentration at which no color change occurred was taken as the MIC.Afterwards, cultures were seeded in MHA and incubated for 24 h at 37°C to determine the minimum bactericidal concentration (MBC), which corresponds to the minimum concentration of the sample that eliminated the bacteria.

High-performance liquid chromatography (HPLC) analysis
Investigations of phenolic content were performed by a High-performance liquid chromatography system (HPLC; ProStar, Varian) which is comprised by a quaternary pump, diode array detector, auto-sampler.The reagents used were acetonitrile HPLC grade (Panreac®) and acetic acid (Vetec®).Water was purified through a milli-Q (Merck®) system.Phenolic compounds were analysed on a Phenomenex C18 column (250 x 4.6 mm, 5 µm), applying mobile phase gradient of acidified water (0.3% acetic acid) (solvent A) and acetonitrile (B) as follows: Linear gradient from 10 to 20% (B) from 0 to 10 min; and linear gradient from 20 to 28% (B) from 10 to 60 min.The flow rate was kept constant at 0.8 mL/min and detection was in the range of 190 to 450 nm.Phenolic compounds were identified by comparison of their retention times and their absorption spectra of ultraviolet light (UV).Gallic acid, chlorogenic acid, ellagic acid, rutin and quecertin were used as standard compounds (all purchased from Sigma-Aldrich).

Statistical analyses
Statistical analyses were performed by One-way analysis of variance (ANOVA).All analyses were carried out using software Graph Prism, version 4. The correlation indices were calculated using the Pearson coefficient (ρ).Only for correlation between Rainfall Index and MIC values a negative ρ -value is considered as direct correlation.

Effects of rainfall on the antimicrobial activity of Anadenanthera colubrina
All extracts from of A. colubrina showed antimicrobial activity, being more active against gram-positive organisms (Table 1).Most tested bacteria were more sensitive to ethyl acetate extracts (from all periods).Taking into account the results for Gram-positive bacteria (all S. aureus strains and B. subtilis), ethyl acetate extracts from leaves showed the lowest average MIC values (1.06 mg/mL), this value was significantly lower than those observed for other extracts (p <0.05).In the same way, among fruits extracts, the lowest average of MIC values was found to ethyl acetate extracts (17.50 mg/mL), however no significant differences were observed between the results.For this reason, the ethyl acetate extracts were selected for chemical analysis.Regarding the effect of the collection period on antimicrobial activity, the lowest MIC values were observed in the months with the highest rainfall index (RI) for extract from both tissues (Table 1).Strong correlations were found between the RI and the average MIC of MLE (ρ: -0.99), EALE (ρ: -0.81), CHFE (ρ: -0.81), EAFE (ρ: -0.80); moderate correlation was observed for CLE (ρ: -0.62); while weak correlations were found for CFE (ρ: -0.48), MFE (ρ: -0.42), and CHLE (ρ: -0.27).

Effects of rainfall on phytochemical composition of Anadenanthera colubrina
It was observed by TLC analysis that the ethyl acetate extracts from both tissues exhibited flavonoids, cinnamic derivatives, terpenes, cyanogenic glycosides and proanthocyanidins.Whereas TLC assays did not show qualitative differences between the extracts obtained from leaves or fruits (Table S1), we were able to detect 15 phenolic compounds with different retention time (Rt) and UV spectra in HPLC chromatograms.The presence and concentration of these compounds varied with tissue and rainfall index.The extracts from fruits showed more compounds than those from leaves: peaks 2 and 5 were found at all fruits samples, EAFE1 had the highest chemical phenolic diversity as 13 peaks were detected and some compounds were only found in this sample (peaks 3 and 4).EAFE2, EAFE3 and EAFE4 showed 10 peaks.The concentration of some compounds also varied according to collection time.For example, peak 6 had maximum detection on September (EAFE1); while peaks 8, 12 and 15 apparently were more found in April and January (Figure 1).Regarding the qualitative results for leaf extracts, EALE2 showed the highest number of peaks (8).Only two peaks were specific for EALE2 (12 and 13) and peak 14 was only absent for EALE1; the other peaks were present in all extracts.However, quantitative differences for some compounds contents could also be observed, for example, peak 1 was larger in January, and peak 15 in September and January (Figure 1).The identity of gallic acid (peak 1) and quercetin (peak 15) were confirmed by co-injection of an internal standard for each compound (Figure S1).Some compounds (peak 10 and 11) are presumably quercetin derivatives due to the close similarity in the UV absorption spectra (Figure S2).Gallic acid, quercetin (and its derivatives) had already been  detected in the extracts from A. colubrina. 6,21[16]

DISCUSSION
This work analyzed the effects of rainfall on the antimicrobial action and phytochemical constituents of extracts from leaves and fruits of A. colubrina.6]12 In the present work we employed a different extraction method, by which we obtained extracts with ac-tivity against tested Gram negative bacteria.Anyway, the best activity was found against S. aureus and B. subtilis, and ethyl acetate extracts had the best activity.It is also important to highlight that some variation on antimicrobial activity of products derived from A. colubrina has been found according with area of cultivation.For example, extracts derived of samples from Cerrado biome did not show antagonist activity, 18 while others from Caatinga area were able to inhibit microbial grow. 19ince the production of secondary metabolites is influenced by environmental conditions (such as temperature, soil composition, solar irradiation, water availability), we attempted to evaluate the influence of rainfall on the antimicrobial action and chemical composition of extracts from both leaves and fruits.First we analyzed the variation on antimicrobial  activity of those extracts.We correlated the RI for each month where the collection was performed with the MIC average obtained.Strong correlations were found for most of extracts.There is no consensus on the effect of rainfall index in the biological activity of plants, both positive and negative correlations are reported. 20,21Our results corroborate with several reports which showed positive effects of rainfall on the biological activity of plant extracts. 20,21he influence of rainfall on the phytochemical composition of A. colubrina extracts was first analyzed by TLC based assay.In general, we observed the same composition reported in our previous work. 12It was not noted qualitative differences between the extracts prepared in each period.Thus, we attempted to perform a HLPC analysis which revealed some quantitative differences: the samples collected from dry periods had more diversity (as they presented more peaks).Some compounds were identified: gallic acid (peak 1), quercetin (peak 15) and two quercetin derivatives (peaks 10 and 11).In addition, quercetin levels increased in those extracts from dry months.Enhanced levels of quercetin have been reported to other plants during dry seasons. 22The production of quercetin and other flavonoids was shown to be up-regulated during stressful conditions such as excessive ultra-violet radiation and high salinity. 23These findings could explain the well-known photoprotective effect of quercetin. 24

CONCLUSION
Our results confirm that the rainfall levels have influence on the antimicrobial activity and chemical diversity of leaves and fruits of A. colubrina.Specifically, the antimicrobial activity was enhanced during months with higher rainfall levels, while the most chemical diversity was found in dry months.The increased levels of quercetin found in samples from dry months suggest metabolic alterations in order to produce compounds related to plant resistance during stressful conditions.These analyses may direct for the best collection time for obtain antimicrobial products derived from A. colubrina which can be applied for biomedical purposes.

Figure 1 :
Figure 1: HPLC-UV chromatograms of compounds from ethyl acetate extracts of leaves and fruits of Anadenanthera colubrina collected at September 2010 (A), January 2011 (B), April 2011 (C), June 2011 (D).I: Chromatogram from fruits detected at 280 nm; II: Chromatogram from fruits detected at 340 nm; III: Chromatogram from leaves detected at 280 nm; IV: Chromatogram from leaves detected at 340 nm.