Evaluation of antioxidant activity and toxicity in Artemia salina of the ether extract and fractions from Tecoma stans seeds

Tecoma stans (L.) Juss. Ex Kunth is a species belonging to the Bignoniaceae family, popularly known as yellowing, yellow bell, and garden little ipe. Studies with T. stans seeds are scarce and it have potential in the search for natural compounds with biological activities. This study aimed to evaluate the toxicity and antioxidant activity of the ether extract (EE) and fractions from T. stans seeds. The EE was obtained in Soxhlet apparatus with petroleum ether and fractions were obtained by hydrolysis and esterification reactions. Phytochemical screening evaluated the presence of steroids, triterpenoids, alkaloids, coumarins and anthraquinones in EE. The toxicity was evaluated by the A. salina lethality test and antioxidant activity by DPPH method. It was observed the presence of steroids, triterpenoids and alkaloids in EE, which had no toxicity to A. salina (LC50 > 1000 μg/mL). The fractions of seeds exhibited toxicity on A. salina. All samples showed antioxidant activity with EC50 between 0.9 and 1.1 μg/mL. These results indicated potential applications for T. stans seeds.


Introduction
Tecoma stans (L.) Juss. Ex Kunth is a species belonging to the Bignoniaceae family, popularly known as yellowing, yellow bell and garden little ipe (Kranz & Passini, 1997). This species exhibits antibacterial, antioxidant, antinociceptive, antiinflammatory, antidiabetic and larvicidal activities; and these effects are correlated to presence of alkaloids, anthraquinones, phenolic compounds, steroids, glycosides, hydrocarbons, essential oils, tannins, terpenes and saponins (Alonso-Castro et al., 2010;Prasanna et al., 2013;Salem et al., 2013). Studies with T. stans seeds are scarce and it have potential in the search for natural compounds with biological activities.
An important problem that concerns researchers are diseases caused by the uncontrolled production of free radicals.
Many diseases, such as cardiovascular diseases, cancer, Alzheimer's and cataracts, are correlated to excess free radicals in the body (Neha et al., 2019;Pohanka, 2018). Various plants have polyunsaturated fatty acids (omega 3 and omega 6) and carotenoids, which may be applicable in the treatment of diseases associated with oxidative stress, that promote lipid peroxidation, damage to DNA, enzymes and proteins (Martin et al., 2006;Simopoulos, 2002).
Due to the scarcity of studies that prove the biological potential of plants used by the population, it is necessary to evaluation for the toxicological activity of these extracts to ensure their safe use. Tests with A. salina allow the assessment with sensitivity, being an indicative model of toxicity for animal species that make up the ecosystem (Favilla et al., 2006).
Thus, the objective of this work was to evaluate the antioxidant activity and toxicity of the ether extract and fractions from T. stans seeds.

Methodology
This work is an experimental research with a quantitative approach (Pereira et al., 2018), carried by data collection through the use of measurements of values, being the application of this method necessary to verify the results obtained from the objectives proposed.

Plant material
The plant material was collected in a Cerrado area located in Divinópolis, Minas Gerais State (20º10'44''S latitude and longitude 44º55'6" W GRW). The vouchers were identified as Tecoma stans (L) Juss. Ex Kunth by Andréia Fonseca Silva and deposited in the PAMG Herbarium, belonging to the Minas Gerais Agricultural Research Corporation (EPAMIG), under registration number 58284. This study has access permission to the components of plant genetic heritage and it is registered in the SisGen Platform (Register AEF6C95), according to Brazilian Biodiversity Law (13.123/2015).

Obtaining extract and fractions
To obtain ether extract (EE), the plant material was exhaustively extracted with petroleum ether at 40 °C in the Soxhlet apparatus for 40 cycles. For hydrolyzed fatty acids (FA), the ether extract was solubilized in KOH (1 mol/L) solution and kept under reflux for 30 minutes. After cooling, the solution was acidified with 1 mol/L hydrochloric acid (HCl) and extracted with hexane, which was removed in a rotary evaporator (Ika RV10). To obtain the fatty acids methyl esters (FAME), the fatty acids were solubilized in hexane, a 2% v/v methanolic solution of sulfuric acid (H2SO4) was added, which was kept under reflux for 1 hour. After cooling, the organic phase obtained with the addition of saturated sodium chloride solution (NaCl), and the hexane was removed in a rotary evaporator (Ika RV10) (Silva et al., 2015).

Phytochemical screening
Steroids and triterpenoids presence was evaluated by Lieberman-Burchard reaction, alkaloids by Dragendorff reaction and coumarins and anthraquinones by the addition of NaOH (1 mol/L) (Matos, 2009).

Phytochemical screening
The presence of steroids, triterpenoids and alkaloids was observed in EE, which corroborates with the literature. The monoterpene alkaloids (tecomine and tecostanin), 5-β-hydroxyskitantine and boschniakina have already been identified in T. stans seeds and leaves (Costantino et al., 2003). Gonçalves, Parreira & Lima (2020) also related the presence of alkaloids and steroids in hexane fraction from flowers. Research, Society and Development, v. 10, n. 9, e12510917829, 2021 (CC BY 4.

Evaluation of antioxidant activity
As shown in Table 1, the EE and FA exhibited antioxidant activity greater than 50% in all concentrations evaluated.
The samples, at concentrations of 1 and 10 µg/mL, were more efficient in capturing the DPPH radical than the positive control BHT, with small values of EC50.  (Gonçalves, 2020). The fruits extract obtained with water/ethanol (1:4), under reflux at 70 °C, presented inhibition concentration (IC50) of 12.7 µg/mL (Marzouk et al., 2006). These results indicate that the increase in temperature does not influence the antioxidant activity (Simões et al., 2007).
Dry leaves extract, obtained by percolation with water/ethanol (1:1), and the fractions of petroleum ether, ethyl acetate and methanol showed antioxidant activity, being hydroethanolic extract the most active, with 64.32% of DPPH inhibition (Larbie, Nyarkoh & Adjei, 2019). Leaves and branches extracts obtained by maceration exhibited IC50 values between 10 and 100 µg/mL (Larbie, Nyarkoh & Adjei, 2019;Salem et al., 2013). The results obtained for the seeds were more promising than for the other plant parts.
The antioxidant activity of EE may be related to secondary metabolites observed. The cyclic carbons together with conjugated double bonds in steroids and terpenes make them potential reducing agents, because they can capture and stabilize Reactive Oxygen Species (ROS) and Reactive Nitrogen Species (RNS) from the environment. Nitrogen atoms in alkaloids molecules have free electron pairs that can also stabilize ROS and RNS. The steroidal alkaloids presented high antioxidant potential (Cerqueira, de Medeiros & Augusto, 2007;Simões et al., 2007).
The antioxidant activity of FA and FAME of T. stans corroborates with the literature. FAME obtained from the seeds of Annona cornifolia (Annonaceae) presented IC50 = 3.83 µg/mL (Lima et al., 2012), as well as FAME from commercial soy, corn and sunflower oils exhibited IC50 values between 1 and 10 µg/mL (Pinto et al., 2017). A study with FA and FAME of leaves of S. brasiliensis showed IC50 values < 1 µg/mL (Amado et al., 2018).

Artemia salina lethality assay
As shown in Table 2, the EE promoted low mortality of A. salina in all concentrations, causing lethality below 20% in the highest concentration tested. FA caused mortality below 50% at concentration of 500 µg/mL; however, at concentration of 1000, the mortality was 91.11%. FAME exhibited mortality higher than 50% in all concentrations evaluated. A. salina is considered a good model for assessing the toxicity of plant extracts since the results obtained in this assay can be extrapolated to other tests (Pimenta et al., 2003). However, studies with FA and FAME assessing lethality in this model are still scarce. FAME from A. cornifolia seeds promoted high mortality in A. salina nauplii, with LC50 = 8.77 µg/mL (Lima, 2006).
Another study evaluating the lethality of EE, FA and FAME of S. brasiliensis leaves in A. salina, it was observed that FAME exhibited higher lethality (LC50 = 681.59 µg/mL), followed by FA (LC50 = 899.34 µg/mL), while FAME did not present considerable toxicity (Amado et al., 2018).
Studies evaluating the activity of T. stans on A. salina are even scarce, with only one report in the literature. Thein & Oo (2019) evaluated the larvicidal activity of aqueous and ethanolic extracts (70%) from leaves. For the aqueous extract, the concentrations between 187.5 and 3000 µg/mL were tested, with mortality ranging between 7% and 96%; for the ethanolic extract (70%), the concentrations were from 250 to 4000 µg/mL, with mortality between 3% and 91%. The LC50 values obtained were 878 µg/mL for the aqueous extract and 1318 µg/mL for the ethanolic extract (70%). In our study, the LC50 values found for FA and FAME of the seeds demonstrated greater toxicity than the aqueous and ethanol extracts (70%) of the leaves. Thus, as FA and FAME have LC50 values less than 1000 µg/mL, they are considered toxic; while EE has no toxicity (Meyer et al., 1982).

Final Considerations
In this study, it can be concluded that EE, FA and FAME from T. stans seeds have antioxidant activity, with potential for future studies. This effect may be related to the presence of steroids, triterpenoids and alkaloids. Among the samples, the EE is safe for the environment, while FA and FAME have toxicological potential.
In order to improve this study, it is interesting to identify the compounds present in EE, FA and FAME, and evaluate their effect in other biological models.