Chemical variability of Copaifera langsdorffii Desf. from environmentally contrasting populations

Abstract Copaifera langsdorffii Desf. is recognised as one of most famous medicinal and economic species of Copaiba, occurring in several distinct biomes. An untargeted metabolomic approach was used to evaluate the chemical variability of C. langsdorffii from contrasting climates biomes (Atlantic Rainflorest and the semiarid Cerrado). Metabolomic analysis enabled the identification of 11 compounds, including glycosylated flavonoids and galloylquinic acid derivatives. Multivariate analysis highlighted that Cerrado population had a significantly higher concentrations of galloylquinic acid derivatives in comparison to the rainforest biome. Meanwhile, Atlantic Rainforest populations presented higher content of flavonols. Semiarid biome, reduced the concentration of flavonoids, mainly concerning quercetin and kaempferol derivatives, however, in this biome flavonoids were more diverse. Both chemical classes presented relevance to be used as geographical origin chemical markers by qualitative and quantitative features. Graphical Abstract


Introduction
Plants are extremely complex organisms which can adapt depending on the environment in which they grow. Such adaptation process induces the formation of populations with substantially different morphology, chemical and genetic features (Christmas et al. 2016). For instance, excessive exposure to solar radiation can induce biosynthesis of phenolic compounds to prevent oxidative stress (Zahedi et al. 2021). Identifying intraspecific variability among populations contributes to the development of conservation and restoration practices, and to the prediction of which populations are more suitable for bioproducts exploitation.
The genus Copaifera is a famous group of medicinal plants that produce as oil resin with great value to medicine, cosmetic and perfume industry (Veiga and Pinto 2002). In addition, Copaifera non-volatile extracts also have interesting bioactive metabolites, such chlorogenic acid derivatives (Oliveira et al. 2020). Yet, few reports are focused on the metabolomic evaluation of these fractions, and far less discuss their intraspecific variability. In the present paper, we aim to compare the metabolomic profile of C. langsdorffii, one of the economic important Copaifera species, from contrasting biomes.

Results and discussion
C. langsdorffii is widely distributed in many different biomes. Within Brazil, it occurs in both, Cerrado and Atlantic Forest biomes. These biomes have highly contrasting environmental conditions, in which Atlantic Rainforest (AR) has at least twice the annual rainfalls of Cerrado (Oliveira-Filho and Fontes 2000).
The chromatographic profile of C. langsdorffii samples presented 55 distinct compounds, 11 of which were identified (Table S1). The major compound in all samples were the quercetin-3-O-rhamonside (retention time of 5.55 ± 0.01 min. on Figure S1 and S2). However, AR samples presented the highest relative concentrations among both evaluated biomes (Table S1). Overexpression of flavonols can be associated with low specialisation of biosynthetic pathways, associating AR samples to a less evolved variation of C. langsdorffii. Another flavonol identified within all samples was kaempferol-3-O-rhamnoside (retention time of 5.91 min. on Figure S1). The relative concentration of kaempferol-3-O-rhamnoside compared to quercetin-3-O-rhamonside was one of the differential features among the biomes. Kaempferol expression was a strong correlation with solar radiation (Mart ınez-Luscher et al. 2019), which could explain their distinct concentration in both biomes.
Atlantic Rainforest samples were also characterised by the presence of 3,4,5-tri-O-(3-O-methylgalloyl) quinic acid (retention time of 5.05 ± 0.01 min. on Figure S1). Galloylquinic acids are a common group of metabolites found within C. langsdorffii being recently quoted as promising chemomarkers (Brancalion et al. 2012;Furtado et al. 2015;Nogueira et al. 2015;Oliveira et al. 2020;Antonio et al. 2021). It is worth mentioning that the concentration of galloylquinic acid derivatives among the evaluated biomes were not significantly different (Table S1), corroborating this chemical class potential as a C. langsdorffii marker, since it is not influenced by intraspecific variability.
Multivariate analysis (Figure 1) presented two main clusters characterised by 14 chromatographic peaks (Table S1). Loading analysis evidences the chromatographic peaks at retention times 0.33, 4.06, 4.67 and 7.48 for Cerrado samples, and the retention times 0.42, 4.17, 4.58, 5.05 and 5.55 for Atlantic Forest samples. Multivariate analysis ( Figure 1) demonstrates that although all samples have galloyl quinic acid derivatives, their qualitative profile, associated within flavonoids can be used to assess the intraspecific variability of C. langsdorfii in different biomes. Such results also corroborates previous findings in which both chemical classes were relevant chemotaxonomic markers to distinguish organs and species of Copaifera (Antonio et al. 2021).
Cerrado population presented larger variability among samples, being characterised by the lowest concentrations of galloylquinic acids derivatives (retention time from 4,0 to 4,67) and the presence of velutin (retention time 7,48). Yet, velutin occurrence, along with the unidentified chromatographic peak at the retention time 0.34 were the major features producing variability within Cerrado samples, increasing the Euclidian distance of sample 444 (Figure 1).
Loadings analysis (Table S1, Figure 1) demonstrate that the major difference among the evaluated biomes is due to the occurrence of quercetin-3-O-rhamonside (AR samples) and velutin (Cerrado samples). It is worth mentioning that this is the first record of velutin within Copaifera. This flavone has been reported as a strong anti-inflammatory and antitumoral compound (Xie et al. 2012).
The production of phenolic compounds and chlorogenic acid derivatives is affected by drought stress (Zahedi et al. 2021). However, depending on the species, drought stress causes different effects. For instance, in Arabidopsis thaliana drought stress increases the accumulation of flavonoids (Nakabayashi et al. 2014), while in Achillea pachycephala, flavonoids content decreases (Gharibi et al. 2019). Within Copaifera, previous studies on Cerrado biome reported that galloylquinic acid derivatives concentrations increases in high temperatures seasons, along with small modifications on total phenolics (Franco et al. 2016;Motta et al. 2019). Our data corroborates those previous studies, and also highlight qualitative modifications on flavonoids profile due drought stress in Copaifera. In addition, such qualitative profile and distinction on multivariate analysis indicate that galloylquinic acid derivatives and flavonoids can be used to track geographical origin of samples.

Conclusions
C. langsdorffii chemical fingerprint is mainly characterised by its galloylquinic acid derivatives profile. Despite the low intraspecific variability observed among species, it was possible to differentiate distinct sampling locations based on its flavonoids qualitative profile and quantitative modification on the concentration of galloylquinic acid derivatives. The contrasting environmental conditions shows to produce distinct populations of C. langsdorffii.