Phytochemical characterisation, antioxidant capacity, and in vitro toxicity of Richardia brasiliensis gomes crude extracts

Abstract Richardia brasiliensis is a species used in folk medicine and rich in active compounds. In this study, the extracts were submitted to UHPLC-ESI-MS/MS analysis and total polyphenols, tannins, and flavonoids assays. Besides, it was determined its antioxidant capacity, oxidative stress markers and toxicological profile. Fourteen polyphenols were found and, in the dosages, a slight change in the concentrations in each extract was observed. Regarding the antioxidant capacity, the responses were different in the methods used. There was an increase in lipid peroxidation, and NO, however total ROS remained unchanged. The cells remained more than 90% viable and the extracts did not cause damage to single strands of DNA, with the exception of the crude autumn and spring extracts at 500 µg/mL. The results found in this study suggest that extracts are potentially toxic to human leukocyte cells in high concentrations; however, more studies should be performed in different cell lines. Graphical Abstract


Introduction
Richardia brasiliensis Gomes (Rubiaceae) is an herb popularly known as 'poaia branca', is found mainly in the Southern and Midwestern regions of Brazil (Lorenzi 2008). There are reports of its use in traditional medicine as an antiemetic, hypoglycemic, anti-hemorrhoids, and in the treatment against worms and skin diseases (Figueiredo et al. 2009).
In its chemical matrix, there are secondary metabolites such as polyphenols, alkaloids, steroids, triterpenes, organic acids (Morais et al. 2013); however, little is known about biological activities and the pharmacological mechanisms.
Traditionally, medicinal plants have been used to treat and cure human diseases, but they are also a source for synthesising drugs in current clinical use. In this context, the aims of this study were to characterise the phytochemical matrix in plant material, to identify and quantify polyphenolic substances in the extracts of R. brasiliensis, as well as to determine the in vitro antioxidant capacity, and the cytotoxic and genotoxic effects of the four crude extracts obtained in the different seasons of the year.
In the measurements of secondary metabolites (Table S1), the highest content of total polyphenols was found in the spring, differing significantly from the other seasons. For flavonoids, the highest concentration was in autumn, but without significantly differing from the levels found in winter and spring. As for tannins, the highest content was in the summer, differing from other seasons. Through UHPLC-ESI-MS/MS, fourteen polyphenols were identified in the R. brasiliensis crude extracts. The predominant phenolic compounds were chlorogenic acid and rutin (Table S2).
Several studies have demonstrated these differences in the biochemical constitution of extracts, relating such changes mainly to environmental variables to which the plant is subject, extraction methods, plant organ or tissue used, solubility, and type of solvent used to extract the compounds (Hayat et al. 2020).
Through the DPPH method, the extracts showed low antioxidant capacity when observed about the ascorbic acid standard. In the anti-hemolysis assay (AAPH), although not significantly different from the standard, the four C.E. had a pronounced antioxidant capacity highlighted on the CES and the CESP (Table S3). The C.E. showed promising antioxidant activity obtained through in the ORAC analysis ( Figure S1). These results demonstrate that the antioxidant capacity is also related to the environmental conditions to which the plant is subjected. In addition, the amount and position of the hydroxyl groups, the molecular weight, the particle size, the solvent concentration, the contact time of the plant material/solvent, the temperature and the mass/solvent ratio also influence their ability to act as an antioxidant agent (Farag et al. 2020).
In the TBARS analysis, the extracts increased malondialdehyde (MDA) levels at 500 lg/mL, being statistically similar to the positive control H 2 O 2 (100 lM), causing increased lipid peroxidation in the tested cells ( Figure S2). In the Nitric Oxide assay, it was observed that there was a slight increase in NO at concentrations of 500 mg/mL, except CES. However, there is no significant difference to N.C. (Figure S3). In the assay evaluating reactive oxygen species, in general, the extracts maintained low ROS levels in all extracts, although showing some statistically different alterations from both controls ( Figure S4).
Despite their antioxidant capacity, bioactive compounds such as polyphenols can become highly reactive molecules, increasing oxidative stress since some natural substances have a biphasic action effect, being an antioxidant at low concentrations and pro-oxidant when in higher doses (Zhang and Tsao 2016;Chikara et al. 2018). In the evaluation of lipid peroxidation, our extracts seemed to be damage inducers when used at a higher concentration, increasing MDA levels in leukocyte membranes, which may culminate in important pathogenic events, since changes in polyunsaturated chains alter fluidity and compromise membrane integrity, resulting in cell damage and loss of function (Alch e 2019). It was also possible to observe that both ROS and NO levels slightly increase when evaluated at the highest concentration. Since they are regulatory molecules for many essential physiological functions, these changes and the excess of reactive species can often cause harmful effects on living cells, besides being associated with the development of various types of carcinomas, diabetes, aging, and inflammatory conditions (Fan et al. 2020).
In vitro genotoxicity and cytotoxicity assays were used as biological markers to determine possible mutagenic and genotoxic effects of the plant under study. In cell viability, the extracts could maintain cells at more than 90% viable ( Figure S5), and this critical parameter was a prerequisite for the development of other cytotoxicity assays (Carvalho et al. 2015). In the assay that detected the presence of doublestranded DNA exposed to the medium, an increase in the presence of free dsDNA in the solution was observed ( Figure S6).
In the alkaline comet assay, the extracts presented a dose-dependent behavior in which the concentrations of 500 mg/mL slightly increased the damage, decreasing as the tested concentration decreases ( Figure S7). However, only CEW (500 mg/mL) showed a significant difference. Compared to P.C. (39 D.I.), all seasons and concentrations evaluated presented a low damage index (D.I.). At the 500 mg/mL concentration, the damage indices for CES, CEA, CEW, and CESP were 9, 10, 13, and 8 D.I., respectively. In the same extracts, but at the concentration of 100 mg/mL, the D.I.s were 9, 7, 7, 7, and at the concentration of 10 mg/mL, they were 7, 4, 4, 7, respectively.

Conclusion
In summary, the chemical characterisation of R. brasiliensis extracts determined the presence of flavonoids, alkaloids, coumarins, organic acids, among others. Fourteen polyphenols were identified that differed in their concentrations in the different extracts, showing high concentrations of rutin and chlorogenic acid. In the dosages of total polyphenols, flavonoids, and tannins, variations were also observed in different seasons, confirming that environmental changes (temperature, humidity, precipitation, etc.) directly influenced the chemical constitution of the plant under study. Regarding the antioxidant capacity, the DPPH assay showed low activity; however, our results were entirely satisfactory in the anti-hemolysis and ORAC assay, demonstrating that the species has an excellent capacity to protect against oxidative damage. The in vitro assays showed that the extract was able to induce oxidation levels depending on the concentrations tested, besides the present specific cytotoxic potential for human leukocytes at the highest tested concentration. Our findings were an essential step towards a better phytochemical and biological knowledge of the species. Despite the varied phytochemical composition, emphasising polyphenols' presence, of polyphenols, in general, R. brasiliensis seems to have a certain degree of toxicity in vitro. However, the mode of action of the extract is still unclear, and further studies of toxicological characterisation are needed.