Seasonal Variation in the Content of Condensed Tannins in Leaves of Xylopia emarginata Mart. (Annonaceae) in Response to Phenology and Climate

Variations in the concentrations of plant secondary metabolites can occur due to the phenological stages of the plants, combined with environmental variations. Plants rich in tannins are used in folk medicine for different purposes. Xylopia emarginata Mart. (Anonaceae)-“Pindaíba” has been used to treat skin edema, bronchitis and malaria. We evaluated variations in condensed tannin (CTs) contents in relation to phenological variables in leaves of Xylopia emarginata during one year. The study took place in a Vereda in northern Minas Gerais State, Brazil. Monthly phenological observations as well as quantifications of the contents of condensed leaf tannins in ethanol and aqueous extracts were performed. The production of X. emarginata leaves occurred throughout the study, with greater budding and leaf fall in the dry season. Phenological observations were correlated with CT levels and climatic data of precipitation and temperature. There was a significant correlation (p < 0.05) between fruiting and CT levels in the extracts, which were higher during the dry season, 13.2% in the ethanol extract and 7.8% in the aqueous extract.


Introduction
Phenology can be defined as the study of the occurrence of vegetative and reproductive events of a species (Lieth, 1974), being an indicator of the ecosystem's response to climate change (Andrew et al., 2013, Abernethy et al., 2018. The influence of seasonality for plant growth and performance has been the subject of research to elucidate how phenology responds to environmental factors (Chuine & Regniere, 2017).
The phenological stages of plants, combined with environmental variables, can modulate the biosynthesis of secondary compounds (Castro-Moreno et al., 2013). Studies indicate that substance concentrations are influenced by plant phenological stages and environmental factors such as precipitation and temperature (Pirbalouti et al., 2013;Djerrad et al., 2015;Pretti et al., 2018). The ecological implications that contribute to changes in the production levels of secondary metabolites are numerous and poorly understood (Schweitzer et al., 2008). Existing data indicate that climatic differences in precipitation and temperature are directly related to the phenological cycles of plants, and alter the levels of production of secondary metabolites (Simón, 1999;Ma et al., 2003).
Phenolic compounds are the most abundant secondary metabolites in plants, and receive more attention because of their distinct bioactivities (Alu'datt, 2017). The compounds of secondary plant metabolism have considerable value in the interactions between the plant and its ecosystem (Sant'ana & Assad, 2002). The accumulation of these compounds varies over the seasons and depends on genetic and environmental factors (Fratianni et al., 2007). These compounds have a common chemical structure that comprise an aromatic ring with one or more hydroxy substituents, whose main groups include flavonoids, phenolic acids, and tannins (Alu'datt, 2017).
Tannins are substances identified as plant defensive compounds (Zucker, 1983) exposed to environmental stresses such as drought (Getachew, 1999), found in several species of the Cerrado-Brazilian savanna (Morais-Costa et al., 2015) and have economic and ecological relevance (Monteiro et al., 2005). Due to their functional properties, added to the potential to add value and conserve the biodiversity of the Cerrado, the species of this biome have aroused growing interest (Rocha et al., 2011).
Plants of the genus Xylopia (160 species) have also stimulated interest in the search for the synthesis of new bioactive compounds, due to the numerous secondary compounds present and their respective bioactivities (Silva et al., 2015). The genus Xylopia is known as an antidiuretic in Brazilian popular medicine, and is used in the treatment of skin edema, and bronchitis (Macedo & Ferreira, 2004;Nascimento et al., 2006), in the treatment of malaria and other infectious diseases (Mesquita et al., 2007), and in the isolation of larvicidal compounds against Aedes aegypti (Sousa et al., 2020). Xylopia emarginata extracts show antiplasmodic activity against malaria and other infectious diseases (Fischer et al., 2004;Mesquita et al., 2007), and larvicidal action against Aedes aegypti (Sousa et al., 2020). Ethanol extracts of Xylopia emarginata leaves having different tannin contents were also evaluated in in vitro tests against ticks, with high activities being observed that were related to tannin contents (Cruz et al., 2021).
Among the species of this genus, Xylopia emarginata Mart. (Annonaceae) stands out; "pindaíba" or "pindaíba do brejo", is a tree species native to Brazil, occurring from the state of Bahia to São Paulo (Lorenzi, 1992). It is typical of riparian or gallery forests, showing wide dispersion in the Cerrado biome in humid environments, such as Veredas-palm swamps and swamp forest (Lorenzi, 1992;Nunes et al., 2015). It is a medium-sized evergreen tree, 10 to 20 m tall and has a trunk 30 to 40 cm in diameter (Lorenzi, 1992). Xylopia emarginata has also been the subject of studies, in which terpenoids, flavonoids, and alkaloids were isolated from leaves (Moreira et al., 2003) and fruits (Moreira et al., 2006).
Few studies have verified the accumulation of low polarity metabolites for X. emarginata (Moreira et al., 2007) and the influence of environmental variables on the production of phenolic compounds (Castro-Moreno et al., 2013). Thus, this study aimed to evaluate the phenological behavior and the variation of the condensed tannin (CT) content of the aqueous and ethanolic extracts of the leaves of X. emarginata over a year, in a Vereda in northern Minas Gerais, Brazil.

Study Area
The survey was carried out in a Vereda vegetation (phytophysiognomie of Cerrado), called Vereda Almescla, in the municipality of Bonito de Minas, in the north of the state of Minas Gerais, Brazil (44°53′46.5″W and 15°20′54.8″S). According to the Köppen classification, the predominant type of climate in the region is Aw (savanna climate), which is characterized by two well-defined seasons, dry and rainy (Alvares et al., 2013). To characterize the region's climatic norms, total precipitation and average temperature over the last 30 years  were considered. The rainy season runs from November to March and the dry season runs from April to October (Figure 1  Note. * Significant value p < 0.05.

Discussion
The variation in CT production in response to phenology and seasonality was verified in this study. The species X. emarginata showed a variation in the production of CT, according to seasonality, which caused an increase in the levels quantified in the dry season, mainly in the months of June and July, where the highest values of CT occurred in the AE and EE.
Previous studies on leaves of other Cerrado plant species, which used a similar extraction method with water as a solvent, also quantified CT during the dry season and reported a variation of 7.4% (Casearia sylvestris), 6.4 % (Paullinia sp.), 0.83% (Caryocar brasiliense), 0.3% (Ximenia americana), and 0.2% (Piptadenia viridiflora) (Fonseca, 2014;Morais-Costa et al., 2015). Note that the species Casearia sylvestris and Paullinia sp. showed high levels of CT, even so, they were below the levels found for the species under study, X. emarginata, when collected and extracted in the dry period in the AE (highest value of 7.8%).
The increase in CT levels during the dry season is consistent with the increase in other secondary metabolites during drought in other species. Water stress increased the amount of secondary metabolites found in the species Papaver somniferum (Szabó et al., 2003), Catharanthus roseus (Jaleel et al., 2007), and Annona lutescens (Castro-Moreno et al., 2013). Studies of other species, such as Murraya euchrestifolia (Wu et al., 1996) and Hydratis canadensis (Douglas et al., 2010), also pointed out the variation of secondary metabolites in leaf extracts according to seasonality. This confirms that seasonality with changes in temperature and precipitation may influence the production of secondary metabolites. What can be proven in this study, is that X. emarginata had its CT levels with variation according to seasonality with greater production in the dry period and greater quantification in EE (13.2%) in relation to AE (7.8%).
Plant species are dynamic organisms, whose accumulation of phenolic compounds varies with seasonality (Lima et al., 2009), depending on intrinsic (genetic) and extrinsic (environmental) factors (Frantianni et al., 2007) and varies with different seasons (Bussotti et al., 1998;Ma et al., 2003;Brooks & Feeny, 2004). Thus, studies on the seasonal variation of phenolic compounds indicate that the climatic changes that occur during an annual cycle, notably those related to precipitation and temperature, influence the phenotypic changes in plants that alter their production pattern of phenolic compounds to suit situations of water stress (Ma et al., 2003;Brooks & Feeny, 2004).
The vegetative phenological behavior of X. emarginata showed that the species is evergreen, with some variations in production and leaf fall throughout the year. However, only budding was influenced by climatic variables. The species showed low intensity of reproduction, which was restricted to dry months, but fruiting also responded to climatic factors. The manifestations of these phenological events responded negatively to the increase in precipitation and temperature. In addition, levels of CTs in the leaves of X. emarginata, both in the aqueous and in the ethanolic extracts, were higher in the period of fruiting of the species.
Budding during the dry season is an indication that the production of X. emarginata leaves is not limited by precipitation. This can be explained by the characteristics of the species, which occupies humid phytophysiognomies in the Cerrado (Mendonça et al., 2008). The study area is a Vereda, with vegetation associated with peat soils, sodden, with outflow of ground water .
The vegetative phenological behavior of the species, despite varying between seasons, did not show abrupt variations, with budding, expansion, and leaf fall throughout the year. Low percentage values of intensity of reproductive phenophases were observed for X. emarginata in the present study. Flowering occurred during a short period, in April and May. Xylopia emarginata in the central region of the Amazon, also presented short flowering, (September and October), and only once in the period of two years of study, indicating that this species seems to have a supra-annual pattern and with irregular flowering (Webber & Gottsberger, 1999). study. During two years of observation in the Amazon, Weber and Gottsberger (1999) did not notice fruiting for X. emarginata. Studies carried out by Ragusa-Netto (2008) in the Cerrado vegetation and in humid areas also noticed fruiting during the rainy season and for X. emarginata other fruiting peaks occurred in the middle of the dry season. For X. emarginata, further studies are needed to find out whether reproduction (flowering and fruiting) always occurs in the same season in subsequent years. Variation in flowering and fruiting periods related to precipitation are frequent in humid tropical forests (Bendix et al., 2006).

Conclusion
The study demonstrated that there was variation in the CT content of the AE and EE of the leaves of X. emarginata in response to phenology and climate (precipitation, temperature). The leaves of X. emarginata have the potential to extract CT in the dry period, and were higher in the period of fruiting of the species. The EE showed a higher quantification of CT contents in relation to the AE.
The production of X. emarginata leaves in the Cerrado/Vereda occurred throughout the study period, registering a higher rate of budding and leaf fall during the dry season and less rainfall. The occurrence of the species in a humid habitat shows a behavior similar to that presented in humid Amazonian forests.