All the chemicals and reagents used in this study were of analytical grade. For thin layer chromatography (CCD), silica gel PF254 plates (Whatman plc, UK) were used, which were observed under UV light 254 nm and developed with solutions of ferric chloride and the Liebermann-Burchard reagent, followed by heating. Chromatographic column separations (CC) were performed using silica gel 60 (70 - 230 mesh, Vetec, Brazil).

Leaves of P. caimito were collected on the campus of the Federal University of Tocantins, Gurupi, Tocantins, Brazil. An exsicta was deposited in the herbarium HUTO-UNITINS. Dry leaves (23.0 g) were comminute and extracted with EtOH by maceration at room temperature. The resulting solution after filtration was concentrated under reduced pressure to give the crude extract in EtOH (PFE, 3.8 g). Part of the PFE extract (3.5 g) was solubilised in a MeOH/H₂O solution (7:3) and submitted to liquid/liquid partition to yield the fractions in n-hexane (PFH, 0.9 g) and AcOEt (PFAE, 1.3 g), respectively.

The phytochemical screening of leaf extracts and fractions was assessed by standard phytochemical methods [18] . Phytochemical screening was carried out by the qualitative chemical composition of leaf extracts using different solvents and using colour change, foaming and precipitate to identify the major natural chemical groups, such as tannins, saponins, flavonoids, catechins, alkaloids, xanthones, triterpenes and steroids. General reactions in this analysis revealed the presence or absence of these classes of compounds in the tested leaf extracts.

Part of the ethyl acetate fraction (PFAE, 1.0 g) was subjected to fractionation using silica gel column chromatography with hexane, chloroform, ethyl acetate and methanol in isocratic mode as eluents. The four sub-fractions obtained were analysed by thin layer chromatography. The sub-fractions eluted with chloroform and ethyl acetate were selected for analysis by ultra-high performance liquid chromatography (UPLC) with a photodiode array detector (PDA) coupled to quadrupole time-of-flight mass spectrometry (QTOF-MS) to obtain the chemical profile, which allowed the identification of triterpenes, namely, euscaphic acid (1), hyptadienic acid (2), betulinic acid (3), ursolic acid (4), oleanolic acid (5), 3β-(O-p-coumaroyl)-alphitolic acid (6), 3β-(O-p-coumaroyl)-maslinic acid (7) and 3β-(O-p-coumaroyl)-2-hydroxy-urs-12-en-28-oic acid (8).

Chromatographic separation of compounds was performed on an ACQUITY UPLC system (Waters, Milford, MA, USA) with a conditioned auto sampler at 4˚C, using an Acquity BEH C18 column (50 mm × 2.1 mm i.d., 1.7 μm particle size) (Waters, Milford, MA, USA). The column temperature was maintained at 40˚C. The used mobile phase was water with 0.1% formic acid in water (solvent A) and acetonitrile (solvent B), which was pumped at a flow rate of 0.4 ml・min⁻¹. The gradient elution program was as follows: 0 - 5 min, 5% - 10% B; 5 - 9 min, 10% - 95% B. The injection volume was 10 μl. MS analysis was performed on a Xevo G2 QTOF (Waters MS Technologies, Manchester, UK). Source conditions as follows: capillary voltage, 2.0 kV; sample cone, source temperature, 100˚C; desolvation temperature 250˚C; cone gas flow rate 20 l・h⁻¹; desolvation gas (N₂) flow rate 600 l・h⁻¹. All analyses were performed using a lockspray, which ensured accuracy and reproducibility. Leucine enkephalin (5 ng・ml⁻¹) was used as a reference compound to calibrate mass spectrometers during analysis and introduced by a lockspray at 10 μl・min⁻¹ for accurate mass acquisition. All the acquisition and analysis of data were controlled using Waters MassLynx v 4.1 software.

Each experiment was performed at least three times. All values are expressed as means ± standard deviation. All statistical analyses were performed with GraphPad Prism 5 DEMO.

In phytochemical prospecting, it was verified that only triterpenes and steroids were present in the hexane fraction (PFH) of leaves of P. caimito. However, triterpenes, steroids, saponins, flavonoids and catechins were detected in the ethanolic extract (PFE) and the ethyl acetate fraction (PFAE) (Table 1). The study evidenced the presence of secondary metabolites, which may be related to the medicinal properties of P. caimito leaves.

UPLC-PDA-MS is a newly developed technique that provides a great amount of information rapidly and efficiently compared with other techniques. The high selectivity and sensitivity of UPLC-QTOF-MS makes it a widely applied technique in quantitative and qualitative analysis, as well as in metabolite analysis and the identification of complex compounds in natural products [21] .

Table 2 lists the tentatively identified compounds in PFAE. A total of eight compounds were identified by UPLC-QTOF-MS (Figure 1) based on database interrogation and references, as shown in Figure 2. Peaks 1, 2, 3, 4, 5, 6, 7 and 8 were identified as euscaphic acid, hyptadienic acid, betulinic acid, oleanolic acid, ursolic acid, 3β-(O-p-coumaroyl)-alphitolic acid, 3β-(O-p-coumaroyl)-maslinic acid and 3β-(O-p-coumaroyl)-2-hydroxy-urs-12-en-28-oic acid, respectively, based on UV, MS and MS/MS fragment ions [21] [22] . This is the first report of the occurrence of triterpenes 1, 2, 6, 7 and 8 in the Sapotaceae family. Euscaphic acid (1) isolated from Cecropalyratiloba presented cytotoxic activity against leukemic cell lines [23] and 3β-(O-p-coumaroyl)-alphitolic acid (6) leads to apoptotic cell death in human leukemia cells [24] . Betulinic acid (3) was previously identified in the species P. gardnerii, P. tomentosa and P. torta [5] . Oleanolic acid (4) was isolated from P. gardnerri [5] . Ursolic acid (5) was isolated from the species P. gardnerii, P. venosa, P. tomentosa and P. torta [5] . The triterpenes derived from oleanans and ursans present antitumor, anti-inflammatory and antioxidant activities [25] . Thus, the identification of these triterpenes in leaf extracts of P. caimito may contribute to the pharmacological potential of this species.

The Folin-Denis assay is a fast and simple method for determining the phenolic compound contents in plant samples. The fraction in AcOEt of the leaves of P. caimito presented the highest phenolic content in comparison to the other fractions of the leaves analysed (11.37 ± 0.3 mg GAE g⁻¹), followed by the extract in EtOH (Table 3). Extracts from leaves of P. caimito presented total phenolic contents similar to other species of this genus [12] [26] [27] .

The scavenging activity for free radicals of DPPH (1,1-Diphenyl-2-picrylhydrazyl) has widely been used to evaluate the antioxidant activity of natural products from plant and natural sources [28] . The results of the DPPH radical scavenging activity of the extract, fractions and ascorbic acid are presented in Table 3. PFE and PFAE had higher antioxidant activity than PFH (p < 0.05), but the antioxidant activity was significantly lower than the standard ascorbic acid (p < 0.05). The ethanol extract and ethyl acetate fraction showed a higher DPPH radical scavenging activity than that reported for the acetone extract of the pulp of P. caimito [12] . Part of the observed antioxidant activity can be attributed to phenolic compounds detected by the phytochemical analysis and triterpenes identified by UPLC-QTOF-MS.