The effect of seasons on Brazilian red propolis and its botanical source: chemical composition and antibacterial activity

Abstract The aim of this study was to evaluate the effect of seasons on the chemical composition and antibacterial activity of Brazilian red propolis (BRP) and its plant source. BRP was collected from Maceio, Alagoas state, north-east of Brazil, during one year. Chemical composition was determined by physicochemical analyses and HPLC while antimicrobial activity was assessed against Streptococcus mutans, Streptococcus sobrinus, Staphylococcus aureus and Actinomyces naeslundii by determining the minimal inhibitory and bactericidal concentrations (MIC and MBC, respectively). The comparative chemical profiles varied quantitatively according to the collection period. Formononetin was the most abundant compound in both propolis and resin, while isoliquiritigenin, (3S)-neovestitol, (3S)-vestitol are suggested to be responsible for antimicrobial activity of Brazilian red propolis. MIC varied from 15.6 to 125 μg/mL, whereas MBC varied from 31.2 to 500 μg/mL. Therefore, season in which propolis and its botanical source are collected indeed influences their chemical compositions, resulting in variations in their antibacterial activity.


Introduction
Brazilian propolis is a non-toxic resinous substance from buds or exudates of plants collected by Apis mellifera bees (Silva et al. 2008). In folk medicine, it has been used by locals as a food additive and/or dietary complement because of its beneficial properties to human health such as antimicrobial, anti-inflammatory, antioxidant, anticancer and antileishmanial (Oldoni et al. 2011;de Groot 2013;Rosalen et al. 2014;Santana et al. 2014;Bueno-Silva et al. 2015;Franchin et al. 2016).
The chemical composition of propolis depends both on its harvest period and on the biodiversity of the region in which it is collected (Sforcin et al. 2000;Castro et al. 2007). In this way, Brazilian red propolis has been extensively studied and Dalbergia ecastophyllum is the major plant source (Silva et al. 2008). Moreover, some bioactive compounds such as isoliquiritigenin, neovestitol, vestitol (Piccinelli et al. 2005;Oldoni et al. 2011;, as well as others with pharmacological properties, were identified. Brazilian red propolis was recently sub-divided into three different types according to its geographical location and it was possible to find different types of red propolis in neighbouring Brazilian states (Frozza et al. 2013).
The time of harvest might influence the chemical composition and antimicrobial activity of the propolis obtained in north-east and south-east Brazil (Castro et al. 2007). In addition, variation was observed in the chemical composition of red propolis from Sergipe state in Brazil during one-year period (de Mendonça et al. 2015). However, no reports have systematically examined the potential influence of harvest time on other types of Brazilian propolis. Therefore, the aim of this study was to evaluate the influence of the seasons on the chemical profile and antimicrobial activity of both Brazilian red propolis (from Alagoas state, Brazil) and its plant source -D. ecastophyllum, over a one-year period.

Results and Discussion
Thin layer chromatography (TLC) is a time-saving qualitative approach to determine whether qualitative chemical changes occurred over the one-year study period in the chemical profile of both ethanolic extract of propolis (eeP) and ethanolic extract of resin (eeR) . Figure S1 shows the plaques used for the reverse phase-high performance thin layer chromatography (RP-HPTLC) revealed at 366 nm (UV-light) (A) and using the anisaldehyde reagent (B). RP-HPTLC for all samples was developed using UV light (366 nm, Figure S1A) and showed fluorescent bands in blue, green and orange, which are characteristic colours of flavonoids and isoflavanones. Thin layer chromatograms, developed with anisaldehyde reagent ( Figure  S1B), showed colourless spots that had no spectral response in the UV-vis range. These bands were in red and orange, which are characteristic colours of terpenes, essential oil and anticianine (Makin 1985). In this way, our results are in accordance with the literature that identified isoflavones in propolis using GC-MS and HPLC (Oldoni et al. 2011;. Moreover, this propolis originates from D. ecastophyllum, a member of the leguminosae family, whose typical compounds include isoflavones.
No qualitative variation in the UV-vis spectra or RP-HPTLC was observed in the chemical composition of samples collected over the one-year study period. Both techniques represent qualitative assays of the chemical composition; in contrast, HPLC allows quantitative analysis through the integration of relative peak areas.
The chromatograms obtained by HPLC ( Figure S2) analysis of all samples exhibited similar chemical profiles for both the eeP and eeR. The peaks (compounds) had similar retention times; however, the content of these compounds (percentage of relative area) varied among the samples. Thus, this analysis suggested that the season of collection influences the quantitative chemical composition of the eeP and eeR, but not the quality of the chemical profiles. The amount of compounds was calculated through the percentage of relative area and is shown in Table S1. Formononetin was the most abundant compound in both, eeP and eeR. However, there is no report in the literature about its antimicrobial activity. Nevertheless, the identification of (3S)-neovestitol is important because our previous articles suggested that this isoflavonoid is one of the main bioactive compound of BRP (Bueno- . Literature is quite extensive about propolis. However, there are few reports regarding seasonal effects on propolis. The Brazilian green propolis did not change its effects on survival curve of Staphylococcus aureus and Escherichia coli during one year (Sforcin et al. 2000). In addition, the authors did not evaluate the chemical composition of Brazilian green propolis as we have in the present work. On the other hand, the antioxidant activity of propolis from San Juan, in Argentina, was altered by seasonal effect. Again, chemical analyses during a year were not conducted (Isla et al. 2009). The lack of a chemical analysis in studies of propolis is a serious deficiency (although common) (Jeon et al. 2011) and because of that, the present study is noteworthy.
The results of the biological assays of samples collected during one year are shown in Table 1. For Streptococcus mutans UA159, the MIC varied from 31.2 μg/mL to 62.5 μg/mL; whereas the MBC varied from 62.5 to 250 μg/mL for both eeP and eeR. For Streptococcus sobrinus, the MIC varied from 15.6 to 31.2 μg/mL, and the MBC varied from 31.2 to 125 μg/mL. The MIC for the eeP and eeR against Staphylococcus aureus ranged from 31.2 to 125 μg/mL; whereas MBC ranged from 62.5 to 125 μg/mL. No variation was detected in the MIC or MBC values (31.2 and 125 μg/mL, respectively) of the eeP against Actinomyces naeslundii; however, MIC values for the eeR varied from 31.2 to 125 μg/mL, and the MBC varied from 125 to 250 μg/mL (Table 1).
The seasonal effect altered the biological activities of eeP and eeR, except for eeP against A. naeslundii, revealing constant MIC and MBC values over the assessed harvest period. This one-year quantitative variation could be explained by the detected variation in the concentrations of compounds( Figure S2 and Table 1). The highest antimicrobial activity was observed from January to May, a period characterised by a tropical climate in north-east Brazil, where there are constant rains and high relative humidity. It is therefore possible that the high relative humidity promotes an accumulation of greater quantities of the active compounds observed in both the propolis and resin. Also, in March and May, eeP showed the highest neovestitol and vestitol amount of the year (Table S1).

Conclusion
The time or season of collection quantitatively affects the chemical composition and antimicrobial activity of Brazilian red propolis due to variations in the concentrations of bioactive compounds present in both the propolis and resin of D. ecastophyllum.