Chemical constituents isolated from extracts of Annona vepretorum Mart. (Annonaceae) leaves

1 Center for Studies and Research of Medicinal Plants, Federal University of San Francisco Valley, Petrolina, Pernambuco, Brazil. 2 Department of Chemistry of Natural Products, State University of North Fluminense, Campos dos Goytacazes, Rio de Janeiro, Brazil. 3 Federal University of Paraíba, João Pessoa-PB, Brazil. 4 Departament of Chemistry, Federal University of Amazonas, Manaus, Amazonas, Brazil.


INTRODUCTION
The Anonnaceae family is composed of 135 genera and about 2500 species (Chatrou et al., 2004), distributed mainly in tropical regions.Among these genera, 34 can be found in South America.Annona L., Duguetia St. Hil., Guatteria Ruiz et Pavon, and Xylopia L. are the predominant genera of Annonaceae (Fechine et al., 2002).
Author(s) agree that this article remain permanently open access under the terms of the Creative Commons Attribution License 4.0 International License antimalarial (Dutra et al., 2012;Costa et al., 2011;Cruz et al., 2011;Santos et al., 2007;Di Stasi and Hiruma-Lima, 2002).
Annona vepretorum Mart. is a tree popularly known as "araticum", "bruteira", and "pinha da Caatinga", predominantly tropical, endemic to Brazil and distributed in the Caatinga biome (Maas et al., 2017;Dutra et al., 2014;Santos et al., 2012;Costa et al., 2011).A. vepretorum is widely used in human nutrition (Costa et al., 2012).Phytochemical studies have shown spathulenol as the major component of its essential oil (Araújo et al., 2015).Seven diterpenes, two sesquiterpenes and three steroids were reported in the stem bark of A. vepretorum (Dutra et al., 2014).Other studies described the isolation and identification of six alkaloids from the leaves of A. vepretorum (Teles et al., 2015).
Previous reports showed that the crude ethanol extract and fractions (hexane and chloroform) from the leaves of A. vepretorum exhibited cytotoxic activity against human ovarian, human colorectal and human glioblastoma tumor cell lines.These samples also exhibited strong antibacterial activity against Escherichia coli (Almeida et al., 2014).Studies performed by our research group have shown that the crude ethanol extract from the leaves of A. vepretorum had sedative activity without affecting the motor coordination in mice.In addition, significant antinociceptive and anti-inflammatory properties were demonstrated and other results suggested that the extract may be useful in the orofacial pain treatment (Diniz et al., 2013;Silva et al., 2015;Silva et al., 2016).
In this paper, we described for the first time the isolation and characterization of four chemical constituents obtained from the ethanol extract of A. vepretorum leaves.

General experimental procedures
1 H and 13 C NMR spectra were obtained on a Bruker NMR spectrometer (DRX 500), operating at a frequency of 200 MHz for 1 H and 50 MHz for 13 C.The samples were prepared in deuterated solvents CIL (Cambridge Isotopes Laboratories) (CDCl3 and CD3OD).Chemical shifts (δ) were referenced to the 1 H-NMR peaks characteristic of protons belonging to the non-deuterated solvents in relation to TMS: Chloroform (δH = 7.24, δC = 77.0)and CD3OD (δH = 4.84 and 3.30, δC = 49.0).For adsorption column chromatography (CC), it was used silica gel 60 (70-230 mesh, ASTM), with 0.063 to 0.200 mm particles (Merck ® ).For thin layer chromatography (TLC), silica gel 60 PF254 was used (Merck ® ).Fractions were monitored by analytical thin layer chromatography (TLC -Aluminum F254), determining the purity of the sample when a single spot was observed after staining under UV irradiation chamber (254 and 365 nm), eluted with at least three solvent systems.

Plant material
Leaves of A. vepretorum were collected in December 2010 and January 2012, in Jaguarari-BA and Petrolina-PE, respectively.The plant material was identified by the botanist José Alves de Siqueira Filho.The material of the first collection was compared to the voucher specimen #946 and the voucher specimen of the species of the second collection was deposited in the Herbarium of San Francisco Valley (HVASF) at the Federal University of San Francisco Valley (UNIVASF) under number #18350.

Extraction and isolation
The plant material was dried in an oven with circulating air at an average temperature of 40°C for 72 h, obtaining the dried and pulverized plant material for the first (1400 g) and the second sample (431 g).The dried and powdered plant material was submitted to exhaustive maceration with 95% ethanol.The extractive obtained solution was concentrated on a rotatory evaporator (50°C) to give the crude ethanol extract of the first sample (Av-EEB1, 600 g) and ethanol extract of the second collection (Av-EEB2, 135 g).The material was partitioned to isolate the chemical constituents.
Av-EEB2 had a precipitate which was washed with chloroform to give a yellow amorphous powder, soluble in methanol, identified as compound 1 (36.0 mg).
Av-CHCl31 fraction (4.0 g) was subjected to column chromatographic using silica gel 60 as stationary phase and hexane, chloroform and methanol as eluents, alone or in binary solvent mixtures, in an ascending concentration gradient.Fraction 68 was washed with methanol to achieve a soluble phase (supernatant) and an insoluble phase (precipitate).The precipitate, white in color, was subsequently identified as a mixture of two substances, 2 and 3 (12.3mg).
Fraction 110 to 133 was purified by preparative thin layer chromatography (TLC preparative), using a mixture of hexane/chloroform (50:50) as eluent, and the procedure was performed twice, in succession, resulting in the isolation of compound 4 (61.3 mg).
In studies conducted by Afanas'ev et al (1989), it was observed the effect of rutin on the smooth muscle without promoting potential toxicity.Several other routine activities have been also elucidated, such as the effectiveness of rutin in treating arthritis and anti-candida activity (Han, 2009), antihyperlipidemic activity (Santos et al., 1999), anticonvulsive (Nassiri-Asl et al., 2008), and anti-inflammatory effects (Guardia et al., 2001).
The mixture of β-sitosterol (2) and stigmasterol (3) was obtained as white crystals soluble in chloroform.The 13 C-DEPTQ NMR spectrum showed the presence of three non-hydrogenated carbons (δ C 140.7, 36.5, 42.2), fourteen methine carbons, twelve methylene carbons and nine methyl carbons.Among them, the presence of the oximethinic carbon at δ C 71.8 related to C-3 and four olefinic carbons were highlight, of which two are common for both compounds: δ C 140.7 and δ C 121.7 (corresponding to C-5 and C-6, respectively).Besides, two olefinic signals at δ C 138.2 (C-23) and δ C 129.2 (C-22) were attributed to the double bond of the side chain present in stigmasterol structure (Table 2).The 1 H-NMR spectrum revealed signals in the region of δ H 0.8 to 2.0 related to methine, methylene and methyl groups.A multiplet at δ H 3.51 and signals between δ H 5.00 and 5.40 indicated the presence of the olefinic protons.In comparison with literature data (Chaturvedula and Prakash, 2012), the sample was identified as a mixture of β-sitosterol (2) and stigmasterol (3).
Lupeol acetate (4) was obtained as white crystals with R f value of 0.95 (hexane:chloroform 1:1).The 13 C-NMR spectrum using the APT technique (CDCl 3 ) revealed characteristic signals of terpenes: seven nonhydrogenated carbons, seven methine carbons, ten methylene carbons, and eight methyl carbons.In addition, it was observed the signal at δ C 168.4 corresponding to a carbonyl carbon (C-1') as well as signals at δ C 148.4 and 106.8 that are characteristic of double bonds between a carbon non-hydrogenated (C-20) and other methylene (C-29).NMR data led to the characterization of Av-4 as a pentacyclic triterpene lupane type (Table 2).The 1 H-NMR spectrum showed signals in the region of δ H 0.77 to 1.67 ppm related to  (Silva et al., 1998), it was possible to identify the substance as lupeol acetate (4).

Conclusion
Phytochemical investigation of Annona vepretorum extracts led to the isolation and identification of four compounds, a triterpene (lupeol acetate), two steroids (βsitosterol and stigmasterol) and a glycosylated flavonoid (rutin).These compounds have been reported for the first time in this species.

Table 2 .
Cont'd.and methyl groups.In addition, a singlet at δ H 4.56, a doublet at δ H 4.67 compatible with H-2' and olefinic hydrogen (H-29), and the signal at δ H 2.02 compatible with H-30 were observed.After NMR analyses and subsequent comparison with the literature data