Flavonoids composition, antioxidant, anticholinesterase, and cytotoxic activities of flowers extract of Senna cearensis Afr. Fern

Abstract The Senna genus has a wide diversity of species, with an interesting chemical composition of secondary metabolites and applications as medicine. The folk medicine, mainly in developing countries, uses species of Senna to treat seizures, epilepsy and constipation. Though the recognized bioactivity, some Brazilian native species of Senna remain unexplored, such as Senna cearensis, a native species of Caatinga. This is the first report about the bioactivity of the flowers of S. cearensis Afr. Fern., a native species of northeast Brazil. In this communication, flavonoids and catechins are identified in S. cearensis flowers and the chemical composition is linked with its antioxidant, anticholinesterase, and cytotoxic activities. By UPLC-QTOF, some compounds that belong to the class of flavonoids were identified. They are: catechin, epigallocatechin, guibourtinidol-(4α-8)-catechin, and cassiaflavan-(Cat)-epicatechin. The cytotoxic activity is more noticeable for the HL60 (leukemia) cell line with a cell growth inhibition (GI%) of 81.65 ± 5.65. Graphical Abstract


Introduction
The species of genus Senna (Fabaceae) are numerous and widely distributed around the world, mainly in tropical and subtropical regions, and are used by various people as a medicine, mainly due to its purgative effect. There is use of several Senna species for other diseases, such as treating seizures, epilepsy, hypertension and dermatoses (Lorenzi and Matos, 2008). The Senna extracts have a wide pharmacological application such as laxative, antioxidant, antitumor, and anti-inflammatory agent, as it contains alkaloids, terpenes, anthraquinones, flavonoids and sterols in their composition (Oladeji, Adelowo, and Oluyori, 2021). Senna occidentalis, for instance, is reported in the literature as a great cytotoxic agent against tumor cell lines, such as human cancer cells of the central nervous system (SF-268), with an IG 50 of 3.9 mg.mL À1 (Calder on et al., 2006). Though the literature has a considerable number of studies about the chemical composition of Senna species and their uses for treating diseases, there are only a few studies concerning native species of Brazil, such as Senna cearensis Afr. Fern. This plant is native to Brazil's biome Caatinga and is commonly known as "flor-de-besouro" or "p e-de-bode" although its name is not resolved, there are several databases that recognize the species S. cearensis Afr. (Macedo, Alan e Silva and Silva, 2016).
Till date, only two reports about the chemical composition of S. cearensis are found in the literature. A study of allelopathic potential and phenolic composition of leaf extracts of S. cearensis was conducted by Torquato et al. (2020);Franca, Cavalheiro, and Silva (2021) described the chemical profile of seven different Senna species, including extracts of S. cearensis, with the authors identifying flavonoids and proanthocyanidins by UPLC-QTOF. The flavonoid content in the plants' composition has a great influence on their bioactivity as an antioxidant and antileukemic agent (Kopustinskiene et al., 2020). Considering the unexplored potential of S. cearensis bioactivities and chemical composition in the literature, this is the first study about Senna cearensis Afr. Fern. antioxidant, anticholinesterase, cytotoxic activities along with the investigation of its chemical composition.

Results and discussion
In our previous study, we could identify flavonoids glycosides in the composition of the S. cearensis flowers by UPLC-QTOF, named: quercetin 3,4 0 -diglucoside, rutin, quercetin-3-O-b-D-glucopyranoside, and mirificin (Franca, Cavalheiro, and Silva, 2021). The investigation of Senna species as natural therapeutic medicine is related to the presence of relevant secondary metabolites, such as flavonoids and phenolics compounds, which are beneficial for antioxidant activity (Oladeji, Adelowo, and Oluyori, 2021). In this report, putative identification of catechins and dimers of catechins by UPLC-QTOF was made. Detailed information of the fragments is displayed in Table 1. The same polyphenolic compounds were identified in the extracts of Senna macranthera var pudibunda by Maia et al. (2019), and in this communication for S. cearensis flowers. The catechins are a flavan-3-ol, a secondary metabolite commonly found in plants with great antioxidant properties (Grzesik et al, 2018). The antioxidant effect of the extracts of S. cearensis flowers by free radical-scavenging activity was approached by DPPH and ABTS assays. The DPPH and ABTS have similar results, as both methods are based on single-electron transference (Monteiro et al 2018). The results are expressed using IC 50 in Table 2, the flavonoid rutin was used as standard control. The high antioxidant activity is associated with the phenolic and flavonoid content of the methanolic extract, which is confirmed by the considerable total phenolic composition (TPC) of 400.00 ± 36.55 mg GAE/g, and the flavonoids identified in the methanolic flowers extract of S. cearensis by Franca, Cavalheiro, and Silva (2021).
The result of the qualitative test for the inhibition of the cholinesterase enzyme is positive, corroborating the results of antioxidant potential. The anticholinesterase (AchE) activity is important due to the decrease in the level of acetylcholine caused by the enzyme cholinesterase, which is important for brain functions. The results also corroborate with the findings of antioxidant and anticholinesterase activities for other species of Senna, for instance S. pendula and S. cana (Monteiro et al 2018). The phenolic content and the flavonoids have a great influence on the antioxidant and anticholinesterase action, flavonoids are known as radical oxygen scavengers that prevent oxidative stress diseases (Kopustinskiene et al, 2020). For example, quercetin and their glucosides, also identified in other species of the genus, as S. splendida, in which the quercetin DPPH IC 50 (mM) was 4.75 (Maia et al, 2018). The catechins identified also contribute for the antioxidant and anticholinesterase properties of the extract, since their antioxidant properties are interesting for their ability to cross the blood-brain barrier (Grzesik et al, 2018).  The cytotoxic effect of the S. cearensis flowers methanolic extract was evaluated by in-vitro assays for four cell lines. The results are displayed in Table 2. Amid the tested cell lines, a specific cell growth inhibition (GI) for the HL60 (leukemia) cell line was observed. The cytotoxic efficacy for the HCT-116 (colon-human) was the lowest. The flavonoids identified in the methanolic extract are part of this group of biomolecules with antileukemic potential. There are several studies in the literature that highlight the antitumor potential of natural flavonoids, such as quercetin and rutin, which are present in the extract of S. cearensis flowers (Kopustinskiene et al, 2020). The epigallocatechin has demonstrated growth inhibition against breast cancer cells models in animals. The catechins identified also have reports in the literature describing them as good tumoral inhibitor, including the synergic effect of a mixture of catechins, such as those in the extract (Cheng et al. 2020).

Conclusions
The methanolic extract of the flowers of S. cearensis, a native species of Caatinga, showed considerable antioxidant activity and antitumor growth inhibition for the in vitro tests. By UPLC-QTOF it was possible to identify the catechins: catechin, epigallocatechin, guibourtinidol-(4a-8)-catechin, and cassiaflavan-(Cat)-epicatechin. The positive results of the bioactivity of the S. cearensis flowers support that the species deserve a more detailed investigation of its chemical composition profile and role as an antitumor agent of the extract and its isolated compounds.