Chemical composition of B. dracunculifolia extract
The chemical composition of the extract of B. dracunculifolia was analyzed by ESI-QToF-LC-MS (Fig. 1). The identification of substances was carried out using GNPS, MoNA, PubChem, ReSpect databases, and literature data (Kumazawa et al. 2003; Tomazzoli et al. 2021). Dereplicated compounds were listed in Table 1, as well as the following data: Retention time RT (min), molecular formula, m/z [M-H]−, and major fragments m/z (relative abundance).
Figure 1
The analysis of the UV spectra, together with MS and MS/MS spectra, allowed the identification of 18 compounds. Among them, four caffeoylquinic acids, derived from chlorogenic acid, with deprotonated ions [M-H]− m/z 515.1181, m/z 515.1199 (two isomers derived from di-caffeoylquinic acid), m/z 529.134 (caffeoylferuloylquinic acid, A), and m/z 677.1500 (3,4,5-tricapheoylquinic acid, B). The differentiation of di-caffeoylquinic acids using mass spectrometry can be found in the works of Clifford et al. (2003) and De Maria and Moreira (2004) where it is possible to carry out the assignment of molecules through the presence and abundance of available fragments after MS3 and MS4 sequential fragmentation experiments. The main fragments of these isomers were identified in our study; however, even though the ESI technique is efficient in identifying isomers, it was not possible to assign them to di-caffeoylquinic acids (3,4-; 3,5-; 4,5-). Some works that describe the chemistry of B. dracunculifolia show the 3,4- and 4,5-di-caffeoylquinic isomers, which may suggest its presence in the analyzed extract (Kumazawa et al. 2003; Tomazzoli et al. 2021).
The presence of prenylated derivatives of hydroxycinnamic acid (p-coumaric acid) is widely reported for the species, as well as for green propolis obtained from B. dracunculifolia (Kumazawa et al. 2003; Tomazzoli et al. 2021; Machado et al. 2016; Chang et al. 2008; De Sousa et al. 2009). Among these derivatives, it was possible to identify 3,4-dihydroxy-5-prenylcinnamic acid (m/z 247.0978, C), 3,5-diprenyl-4-hydroxycinnamic acid (m/z 299.1656; artepilin C, M), 3-prenyl-4-hydroxycinnamic acid (m/z 231.034; drupanin, D), and 3-prenyl-4-dihydrocinnamoyloxycinnamic acid (m/z 363.1601; bacarin, E). In addition, it was possible to identify some peaks with the UV spectrum (UVmax 312 nm) of prenylated derivatives. The UV spectrum profile corresponding to cumaric acid (UVmax 309 nm) was verified; however, a corresponding molecular ion was not observed. This leads us to suggest the presence of polar p-coumaric acid derivatives in the optimized extract sample.
Flavonoids were detected and possible tri-substituted flavones (UVmax 330 nm), kaempferol derivatives (UVmax 365 nm), apigenin (UVmax 336 nm), and naringenin (UVmax 288 nm) were identified. It was possible to identify nine molecular ions [M-H]− of the flavononols dihydrokaempferol (m/z 287.0561, F) and dihydrokaempferide or AME-aromadendrin-4'-methyl ether (m/z 301.0723, G); the flavonols kaempferide (m/z 299.0575, O) and velutin (m/z 313.0719, H); the flavones apigenin (m/z 269.0453, N); diosmetin (m/z 299.0566, I); 4',5,7-trihydroxy-3,6-dimethoxyflavone (m/z 329.0669, J); and the flavanones naringenin (m/z 271.0618, K) and isosakuranetin (m/z 285.0771, L). In addition to these flavonoids, the presence of other derivatives of flavones and flavonols were achieved, but the complete assigns were not possible to confirm. Many of these flavonoids have already been reported by Kumazawa et al. (2003) and Tomazzoli et al. (2021) and, in addition, other authors have already reported these same constituents in green propolis, which also serves as a guide when assigning the identity of B. dracunculifolia compounds (Tomazzoli et al. 2021; Machado et al. 2016; Xu et al. 2020). The Fig. 2 shows the main chemical compounds identified in B. dracunculifolia extract by ESI-QToF-LC-MS.
Table 1
Figure 2
S. cerevisiae photoprotective efficacy and antiphotomutagenic effect of B. dracunculifolia extract in AWP001 (yno1) strain against SSL irradiation-induced cytotoxicity and genotoxicity
Photoprotection potential of B. dracunculifolia extract against SSL irradiation-induced cytotoxicity was evaluated upon S. cerevisiae AWP001 (yno1) strain treated in comparison with untreated AWP001 (yno1) strain (control). Figure 3 displays cell survival of AWP001 (yno1) strain in the absence (control) and in the presence of the B. dracunculifolia optimized extract (100 µg/ml). This extract was able to protect the S. cerevisiae AWP001 (yno1) strain cells against induced cytotoxic lesions at the highest SSL irradiation exposure (1 h 44’ 9” and 2 h 18’ 52”), when compared to control (p ˂ 0.05) (Fig. 3). Therefore, it seems to be a promising photoprotective ingredient in case of long-term SSL exposure, where enzymatic DNA repair tends not to be able to deal with the amount of lethal induced lesions. Additionally, the use of an oxidative damage bioindicator strain, such as the AWP001 (yno1) strain, suggests that the B. dracunculifolia extract proved to be particularly promising due to its antioxidant property as described in literature (Vilas-Boas et al, 2023; De Sousa, 2011; França et al, 2022; Da Silva, 2022b).
Regarding the photomutagenic/antiphotomutagenic potential upon SSL irradiation of the B. dracunculifolia optimized extract, the S. cerevisiae AWP001 (yno1)-treated strain displayed a lower photomutagenicity frequency in terms of CanR/107 cells compared to control (p < 0.05) (Fig. 4). This result indicates that besides presenting photoprotective potential against SSL-induced lethal cytotoxic lesions, this extract also showed antiphotomutagenic potential by protecting cells against SSL-induced oxidative mutagenic lesions.
Figures 3 and 4
The combination of phenolic substances identified in the extract of B. dracunculifolia that has known absorption in the ultraviolet and visible spectrum (Vilas-Boas et al, 2023) could be related to the photoprotection provided by the extract. In the light of this study, it was also possible to verify that this particular combination of flavonoids, phenolic acids, and their derivatives protects cells even in conditions similar to those that occur in human skin, in which there is cumulative sun exposure, through redox therapeutic response which prevents the formation of DNA damage. Phenolic acids are related to the increase of antioxidant and anti-inflammatory mediators and the activation of pro-survival signals (Caruso et al, 2022). On the other hand, flavonoids modulate the cellular response by regulating the oxidative system, contributing to cellular homeostasis (Sarkar et al, 2022). Similar results were observed for B. dracunculifolia extract in rat hepatoma cell line culture (HTC). The researchers concluded that the extract has the ability to reduce DNA damage and has no mutagenic or genotoxic effects (Roberto et al, 2016). Thus, the use of this extract as an active ingredient would be an alternative with a dual mechanism: prevention of oxidative damage and absorption of sun rays.