Chemical composition, antioxidant and cytotoxic activities of extracts from the pericarp of Tecoma stans (L.) Juss. Ex Kunth (Bignoniaceae)

Abstract Tecoma stans (L.), popularly known as ipê-mirim, is used in traditional medicine for the treatment of diabetes and digestive problems. The components of the hexane (HE) and methanol (ME) extracts obtained from the pericarp of T. stans were identified by gas chromatography-mass spectrometry (GC-MS) in their methyl ester forms (FAME). The antioxidant and cytotoxic activities of extracts, fatty acids, and methyl esters were evaluated. Methyl linolenate, methyl linoleate, and methyl palmitate were the major compounds in the HE, while methyl hexacosanoate was the main component in the ME. The samples exhibited significant antioxidant potential by DPPH assay. In the Artemia salina larvae bioassay, FAME (HE) and FAME (ME) were considered toxic. This study showed, for the first time to our knowledge, the chemical composition of the hexane and methanol extracts from T. stans pericarp, as well as the antioxidant and cytotoxic activities of the extracts, fatty acids, and methyl esters. Graphical Abstract


Introduction
The universalization of access to health services, together with improvement in the performance of surveillance of health aggravations and risks, plays a fundamental role in improving the population's health and quality of life, in addition to contributing to democratization of the process of providing health services to the population of a country (Teixeira et al. 2018). It is socially and economically interesting and viable to invest in disease prevention strategies as well as in effective and low-cost treatments (Amorim et al. 2019;Paula et al. 2020).
Antioxidants have the ability to protect an organism from damage caused by free radicals, preventing or protecting them from the onset of various pathologies such as cardiovascular, chronic (cancer, atherosclerosis, rheumatic arthritis, and muscle hypertrophy), and neurodegenerative (Alzheimer's Disease) diseases (Oliveira 2015). Thus, there is growing interest in plant extracts from aromatic and medicinal plants, cereals, and fruits. These extracts are considered to be sources of natural antioxidants, which can be of great benefit in improving the quality of life of people (Oliveira 2015;Silva et al. 2018).
In addition, to ensure the safe use of natural products, it is important to assess their toxicity. The Artemia salina test is widely used for this. The bioassay with A. salina serves both as an indicator of toxicity and the bioactivity of several substances, including plant extracts. This assay has advantages such as speed, practicality, simplicity, and low cost. Also, it requires a small amount of sample. In addition, it presents a good correlation with in vivo tests, suggesting that it is a useful and reliable method (Lagarto Parra et al. 2001).
Tecoma stans (L.) Juss. Ex Kunth, popularly known as 'ipê-mirim' and 'ipê-de-jardim', belongs to the family Bignoniaceae. It is a native species of Mexico and the southern United States, and it is considered to be an exotic species in Brazil and commonly used as an ornamental plant. It is cited as an invader in Nicaragua, Indonesia, South Africa, Argentina, Polynesia and Brazil (Passini and Kranz 1997;Ren o et al. 2007). Bioactive substances, such as saponins, flavonoids, monoterpene alkaloids, tannins, steroids, anthraquinones, terpenes, hydrocarbons, volatile oils and glycosides have been reported in the species T. stans. Therapeutic effects as antimicrobial, antioxidant, antidiabetic, antitumor, antinociceptive, anti-inflammatory, and cytotoxic are attributed to leaves, flowers, roots and bark of T. stans (Gonçalves et al. 2020).
Due to the scarcity of studies with the pericarp of T. stans, the objective of this work was to determine the chemical composition of the extracts as well as to evaluate the antioxidant activity and the toxicity of the extracts, fatty acids, and methyl esters.

Results and discussion
In this study, the extracts obtained from the pericarp were investigated by gas chromatography-mass spectrometry (GC-MS) in their methyl ester forms ( Table 1). The pericarp of T. stans was rich in fatty acids and revealed a high percentage of saturated fatty acids (40.28% in the hexane extract, HE, and 70.98% in the methanol extract, ME) as compared with unsaturated fatty acids (33.72% in HE and 13.78% in ME). In T. stans, methyl linolenate (16.89%), and methyl linoleate (16.83%) were detected as the major unsaturated esters, and methyl palmitate (21.00%) was the most abundant among saturated esters in HE. In the ME, the saturated ester methyl hexacosanoate (62.83%) was identified as the main component followed by methyl linolenate (6.93%), methyl linoleate (6.85%), and methyl palmitate (5.52%).
The compounds methyl linoleate, methyl linolenate, methyl palmitate, and methyl stearate have already been identified in the hexane fraction of flowers of T. stans (Gonc¸alves et al. 2022). The methyl palmitate, methyl margarate, methyl linoleate, methyl linolenate, methyl stearate, methyl eicosanoate, methyl heneicosanoate, methyl docosanoate, methyl tricosanoate, methyl tetracosanoate, and methyl hexacosoanoate esters have been reported in Tecoma Â smithii (Saleh et al. 2019). To the best of our knowledge, these compounds are being reported for the first time in T. stans.
The extracts, fatty acids (FA), and methyl esters (FAME) from the pericarp were evaluated for their antioxidant and cytotoxic activities. The samples and BHT had a concentration-dependent inhibitory effect in the DPPH and FRAP assays. At concentrations of 1 and 10 mg/mL, the samples of T. stans were more effective at scavenging DPPH radicals than BHT ( Table 2). The small IC 50 values (0.85-11.10 mg/mL) found in the DPPH-scavenging activity for extracts, FA, and FAMEs from the pericarp of T. stans, demonstrated a higher activity than BHT (IC 50 ¼ 16.36 mg/mL, p < 0.05). In the FRAP assay, ME demonstrated the best activity among the samples tested, with an IC 50 value (2.54 mg/mL) similar to BHT (1.19 mg/mL) ( Table 2).
The antioxidant activity by DPPH method of T. stans has been reported previously. The ether extract, FA, and FAME from seeds exhibited antioxidant potential with IC 50 values between 0.9 and 1.1 mg/mL (Azevedo et al. 2021). Gonc¸alves et al. (2022) also demonstrated the antioxidant effect of the ethanol extract and fractions of flowers, with IC 50 values ranging from 2.99 to 166.07 mg/mL.
Some reports of antioxidant activity by DPPH assay were also found in the literature for FA and FAME. Lima et al. (2012) demonstrated the antioxidant effect of fatty acid methyl esters (FAME) obtained from Annona cornifolia (Annonaceae) seeds, with IC 50 ¼ 3.83 mg/mL. Methyl esters obtained from commercial oils of soybean, corn, and  The results of antioxidant activity obtained in this study for HE, FA, and FAME were similar to those reported in the literature (Lima et al. 2012;Pinto et al. 2017;Amado et al. 2018;Azevedo et al. 2021). This fact can be explained by the similarity in the chemical composition of the samples tested. The main compounds present in the samples were methyl palmitate, methyl oleate, methyl linoleate and methyl linolenate (Lima et al. 2012;Pinto et al. 2017;Amado et al. 2018), this composition being similar to HE, FA (HE), and FAME (HE) of T. stans. These compounds are important for the food and pharmaceutical industries and can be used in the preparation of food and therapeutic supplements (Anand and Basavaraju 2021). No reports were found in the literature for the antioxidant activity of methyl hexacosanoate.
The results for brine shrimp (Artemia salina) lethality bioassay are presented in Table S1 which shows the mortality rate of larvae exposed to extracts, FA, and FAME from the pericarp of T. stans. The best result was for FAME (ME) which had mortality rates of 43.33%, 93.33%, 100% and 100% at concentrations of 125, 250, 500 and 1000 mg/mL, respectively. FAME (HE) also promoted the mortality of larvae at 10.00%, 63.33% and 83.33% for concentrations of 250, 500 and 1000 mg/mL, respectively. HE and FA (HE) showed little toxicity to the larvae, with little mortality of the larvae exposed to these samples.
The lethal concentration (LD 50 ) for A. salina assay was determined for samples. FA (HE), FAME (HE), and FAME (ME) from the pericarp of T. stans exhibited LD 50 values of 1804.01, 463.74 and 132.95 mg/mL, respectively (Table S1). The lethal doses of FAME (HE) and FAME (ME) were less than 1000 mg/mL, suggesting that these samples are toxic according to Meyer et al. (1982). The other samples were not considered cytotoxic, because they caused low lethality in the A. salina assay. Smilax brasiliensis (Smilacaceae) popularly known as 'salsaparrilha' or 'japecanga', a native species to the Brazilian Cerrado, and used in traditional medicine as a diuretic, diaphoretic, and stimulant, were evaluated by A. salina bioassay. The results showed that the petroleum ether extract was not toxic, but FA and FAME obtained from this extract, exhibited LD 50 values of 899.34 and 681.59 mg/mL, respectively (Amado et al. 2018). Our results corroborate this study, since FAME were the most toxic samples for A. salina, and HE and ME did not cause lethality. The proximity of the LD 50 values for FAME (HE) of T. stans and FAME of S. brasiliensis may be due to the similarity in the chemical composition of these samples.
Lethality studies on A. salina with the T. stans species are still scarce, with only two reports being found in the literature. Azevedo et al. (2021) evaluated the toxicity on A. salina, and the results showed that the FA and FAME of the ether extract from the seeds were toxic, with LD 50 values of 486.27 and 7.52 mg/mL, respectively. Thein and Oo (2019) evaluated the larvicidal activity of T. stans leaves on A. salina, with mortality ranging between 7% and 96% for the aqueous extract at concentrations between 187.5 and 3000 mg/mL. For the ethanol extract (70%), the mortality was between 3% and 91% at concentrations between 250 and 4000 mg/mL, with LD 50 values of 878 mg/ mL for the aqueous extract, and 1318 mg/mL for the ethanol extract (70%). No lethality studies were found on A. salina for samples of the pericarp of T. stans, and the contribution of this study is unprecedented, according to our knowledge.

Conclusion
Methyl linolenate, methyl linoleate, and methyl palmitate were the major compounds in the HE, while methyl hexacosanoate was the main component in the ME. The samples from the pericarp of T. stans exhibited significant antioxidant potential by DPPH assay. In the A. salina larvae bioassay, FAME (HE) and FAME (ME) were considered to be toxic. The identified compounds and the presence of antioxidant activity encourage further studies to evaluate the possibility of using extracts and fatty acids from the pericarp of T. stans by the food and pharmaceutical industries, as new sources of vegetable oils, natural antioxidants, and supplements therapeutics.