Triterpenes from Pouteria ramiflora (Mart.) Radlk. Leaves (Sapotaceae)

Food and Chemical Toxicology xxx (2017) 1e6

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Triterpenes from Pouteria ramiflora (Mart.) Radlk. Leaves (Sapotaceae) ~o Victor Dutra Gomes a, Claudia Masrouah Jamal b, Patrícia Marques Rodrigues a, Joa c  Alvaro Cunha Neto , Maria Lucilia Santos d, Christopher William Fagg e, rola de Oliveira Magalha ~es a, Paloma Michelle de Sales f, Yris Maria Fonseca-Bazzo a, Pe a , * ^maris Silveira Da Faculdade de Ci^ encias da Saúde, Universidade de Brasília - UnB, DF, Brazil Departamento de Ci^ encias Farmac^ euticas e CCS, Universidade Federal do Espírito Santo eUFES, ES, Brazil Departamento. de Química e CCE, Universidade Federal do Espírito Santo eUFES, ES, Brazil d Instituto de Química, Universidade de Brasília e UnB, DF, Brazil e ^ndia, Universidade de Brasília - UnB, DF, Brazil Faculdade de Ceila f rio Central -LACEN, Secretaria de Saúde do Distrito Federal -SES/DF, DF, Brazil Laborato a

b c

a r t i c l e i n f o

a b s t r a c t

Article history: Received 14 January 2017 Received in revised form 11 May 2017 Accepted 13 May 2017 Available online xxx

Pouteria ramiflora (Mart.) Radlk. (Sapotaceae) is a species used by inhabitants from the Cerrado for its edible fruits and medicinal value. Hexane crude extracts from leaves and fractions were evaluated for in vitro a-amylase inhibitory activity and antioxidant potential. The fraction with the highest a-amylase inhibitory activity was submitted to a phytochemical study. Three triterpenes were isolated, friedelin, epi-friedelanol, and taraxerol. This is the first report of these compounds isolated from P. ramiflora. Moreover, this is the first report of friedelin isolated from Pouteria sp. Epi-friedelanol was present in significant amounts, suggesting that this compound could be a candidate marker for this species. © 2017 Published by Elsevier Ltd.

Keywords: Pouteria ramiflora Sapotaceae Friedelin Epi-friedelanol Taraxerol Cerrado

1. Introduction The Pouteria genus (Sapotaceae) is widely distributed in tropical and subtropical regions and is well represented in the Cerrado biome. Triterpenes and flavonoids are the most common constituents found in this genus (Montenegro et al., 2006; Silva et al., 2009). However, little or no information is available on the chemical composition of several species, such as Pouteria amapaensis and Pouteria atlantica. Pouteria ramiflora (Mart.) Radlk. is a Brazilian native species commonly found in the Cerrado. This species is popularly known as curriola (curiola), brasa-viva (Lorenzi, 2002), ~o-de-galo (Almeida et al., figo-do-cerrado (Coelho et al., 2009), gra 1998), fruta-do-veado (Medeiros, 2011), massaranduba or maçaranduba, pessegueiro-do-cerrado, abiu-cutite, and pitomba-de-leite

rio de Produtos Naturais, Faculdade de Cie ^ncias * Corresponding author. Laborato da Saúde, Universidade de Brasilia, Asa Norte, Brasilia CEP 70910900, DF, Brazil. E-mail address: [email protected] (D. Silveira).

(Carneiro et al., 2014). This plant produces edible fruits (Lamounier, 2012; Silva et al., 2009) and is used in folk medicine as an antihyperlipidemic agent and as a remedy for worms, dysentery, pain, and inflammation (Fontes Junior et al., 2009; Silva et al., 2010). The ethanol crude extract from P. ramiflora roots has antiinflammatory and anti-nociceptive activities in vivo, by inhibiting cell migration, nitric oxide synthesis, and adenosine deaminase activity (Fontes Junior et al., 2009). Antioxidant activities have been reported in ethanol, hydro-ethanol, and aqueous extracts from leaves (Castro et al., 2006; Costa, 2014). These extracts also inhibit a-amylase and a-glucosidase activities (Souza et al., 2012). The ethanol extract improves the glycemic index, increases glutathione peroxidase activity, decreases superoxide dismutase activity, and reduces lipid peroxidation and antioxidant status in rats with streptozotocin-induced diabetes (Costa et al., 2013). This extract exerts a neuroprotective effect against oxidative damage and myosin-Va expression and prevents hippocampal neuronal loss in diabetic rats (Costa et al., 2013). Additionally, ethanol extracts from stem and root bark reduce blood glucose levels in mice and

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Please cite this article in press as: Rodrigues, P.M., et al., Triterpenes from Pouteria ramiflora (Mart.) Radlk. Leaves (Sapotaceae), Food and Chemical Toxicology (2017), http://dx.doi.org/10.1016/j.fct.2017.05.026

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P.M. Rodrigues et al. / Food and Chemical Toxicology xxx (2017) 1e6

decrease salivary amylase activity (Gouveia et al., 2013). The ethanol extract also has antimicrobial activity against several bacterial strains, such as Staphylococcus aureus, Bacillus subtilis, Escherichia coli, Pseudomonas aeruginosa, Salmonella setubal, and Candida albicans (Nogueira, 2012). P. ramiflora extracts have been evaluated for insecticidal action against Dipetalogaster maxima but did not present any activity (Coelho et al., 2009). P. ramiflora exerts interesting allelopathic effects by inhibiting germination and growth of several species (Dale and Andrade, 2013; Fernandes et al., 2015; Oliveira et al., 2014). On the other hand, hexane and ethanol crude extracts from leaves have been evaluated by the Ames test and a micronucleus assay and showed potential genotoxicity (Sousa, 2011). Although P. ramiflora extracts are potentially useful, few data are available about their chemical composition, except for markers of the genus, such as myricetin-3-O-a-L-glucopyranoside, myricetin3-O-a-L-arabinopyranoside, and myricetin-3-O-b-D-galactopyranoside, isolated from the a hydroalcoholic extract of leaves (Costa, 2014). Therefore, the hexane extract from P. ramiflora leaves was subjected to a bioguided phytochemical study during our continuing our effort to chemically characterize Pouteria sp. 2. Materials and methods 2.1. General Infrared spectra (IR) were recorded on a PerkinElmer Spectrum 100 equipment (PerkinElmer, Waltham, MA, USA), using attenuated total reflectance (ATR). Nuclear magnetic spectra (NMR) spectra were obtained with 1H (600 MHZ) and 13C (125 MHz) in deuterated chloroform (CDCl3; Sigma-Aldrich, St. Louis, MO, USA) on a Bruker Ascend™ 600 instrument (Billerica, MA, USA). Chemical shifts (d) are reported in parts per million (ppm) about 2H. CDCl3 was used as an internal reference and tetramethylsilane was the internal standard. The gas chromatography-mass spectrometry (GC-MS) conditions were: GC-MS- QP2010 Plus Shimadzu (IE 70 eV; Tokyo, Japan); column: Rtxe5MS (30 m
0.32 mm
0.25 mm (5% diphenyl, 95% dimethylpolysiloxane; Restek Corp., Bellefonte, PA, USA). Mobile phase: helium; flow rate (column): 0.6 mL/min; injector temperature: 250  C; mode: splitless; injection volume: 2 mL; temperature program: from 100 to 300  C at 5  C/min. 2.2. Plant material Leaves of P. ramiflora were collected on the UnB campus in July 2013, and a voucher specimen (CW Fagg 2296) was deposited in the UnB Herbarium. All pertinent authorization was obtained to carried out for this study (CGEN 010,337/2014e8 and SISBIO-ICMBio 24,634). 2.3. Extraction and fractionation The plant was dried and powdered, and the material (1.840 g) was macerated at room temperature for 7 days using hexane as the solvent (1:10 ratio). After filtration, the extract solution was subjected to evaporation at <40  C to yield 103.12 g of crude extract (5.6% yield). A white amorphous solid precipitate appeared during evaporation of the solvent (P1, 0.8% yield from the crude extract). The hexane crude extract, without the precipitated substance, was filtered through silica gel (8.5 cm diameter and 5.3 cm height) using hexane; hexane: AcOEt (1:1); ethyl acetate; AcOEt:MeOH (1:1); and methanol as the eluents (Fig. 1). The hexane: AcOEt fraction was subjected to chromatographic techniques (column chromatography [CC] and thin-layer chromatography) to isolate

and characterize the compounds. An additional amount of P1 (65 mg), was obtained by CC on silica gel 60 from the Hex: EtOAc (1:1) fraction. The precipitate (P1) was submitted to purification by CC on silica gel 60 G (1:120 sample: silica) using hexane: acetate (99:1) mixture as the eluent. 2.4. Evaluation of a-amylase inhibitory activity Inhibition of porcine pancreatic a-amylase enzyme activity was evaluated by a colorimetric method to determine reducing sugars as described previously with modifications (Bernfeld, 1955). In the preincubation step, 20 mL of the extract solution (50 mg/mL in ethanol: DMSO 3:1) and 50 mL of enzyme solution (40 UI/mL, dissolved in 0.02 M sodium phosphate buffer buffer) were added to a tube containing 930 mL of 0.02 M sodium phosphate buffer (pH 6.9 containing 0.0067 M sodium chloride) and were maintained at 25  C for 30 min. A 250 mL aliquot of this mixture was added to a tube containing 250 mL of buffer solution and 500 mL of 1% starch solution. The mixture was incubated at 40  C for 20 min. At the end of the incubation period, 500 mL of DNS reagent (350 mM of sodium hydroxide; 770 mM of potassium sodium tartrate; 33 mM of 3,5dinitrosalicylic acid) was added to all tubes. Then, the tubes were heated in boiling water bath for 5 min, and cooled in an ice bath for 15 min. After cool down, the samples were diluted by adding 4.5 mL of distilled water, and absorbance was obtained by a UV spectrophotometer (Shimadzu, UV-1601) at 540 nm in a cuvette with a 1 cm optical path against a blank. Inhibitory activity was determined by comparing the enzyme activity in the absence and presence of the evaluated inhibitor. Acarbose was used as a positive control (6.25 mg/mL). 2.5. Evaluation of total antioxidant capacity by the phosphomolybdenum complexation method Total antioxidant capacity was evaluated using a previous method (Prieto et al., 1999) with modifications. The assay was based on the reduction of Mo(VI) to Mo(V) by the extract, and subsequent formation of a green phosphate/Mo(V) complex in acid medium (Konyalioglu et al., 2005). A 0.1 mL aliquot from the extract solution (1 mg/mL) in ethanol was mixed with 1 mL of reagent solution (0.6 M sulfuric acid, 28 mM sodium phosphate, and 4 mM ammonium molybdate), and the tubes were incubated at 95  C for 90 min. The mixture was cooled to room temperature, and absorbance of the solution was measured at 695 nm against a blank. The blank solution contained 1 mL of reagent solution, and the same volume of the solvent used for the sample was incubated under the same conditions as the rest of the samples. This method is quantitative and expresses antioxidant activity as the number of equivalents of the standard compound. Ascorbic acid was used as the reference substance (positive control). The experiment was carried out in triplicate. 2.6. Statistical analysis The assays were conducted in triplicate, and data are expressed as mean values. Differences were identified by analysis of variance followed by Tukey's test. A p-value < 0.05 was considered significant. The following formula was used to calculate enzyme inhibition activity:

% inhibition ¼ ½ðC  AÞ=C
100 where C is absorbance of the negative controleabsorbance of the starch blank and A is absorbance of the sampleeabsorbance of the

Please cite this article in press as: Rodrigues, P.M., et al., Triterpenes from Pouteria ramiflora (Mart.) Radlk. Leaves (Sapotaceae), Food and Chemical Toxicology (2017), http://dx.doi.org/10.1016/j.fct.2017.05.026

P.M. Rodrigues et al. / Food and Chemical Toxicology xxx (2017) 1e6

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Fig. 1. Extraction and fractionation of hexane extract from Pouteria ramiflora leaves.

starch blank. A linear regression was applied to evaluate total antioxidant capacity (y ¼ 0.0056
0.0245) from the ascorbic acid standard curve (r ¼ 0.99), and the results are given in equivalents of ascorbic acid. Statistical analyses were performed using GraphPad Prism Version 5.0 computer software (GraphPad Software Inc., La Jolla, CA, USA). 3. Results and discussion Fractionating P1 by CC on silica gel 60 G (1: 120 sample: silica) led to the isolation of three compounds: 1 (friedelin, 13.4 mg, 6.7% yield); 2 (epi-friedelanol, 112.5 mg, 56.3% yield); and 3 (taraxerol, 20 mg, 10% yield). 3.1. Friedelin (1) White amorphous solid. IR cm1 (ATR): 2923 and 2852 cm1 1718 cm1 (C¼O), 1,456, 1385 cm1 (C-H). EIMS m/z: 426 [M]þ, 411 [M-CH3], 369, 355, 341, 327, 315, 302, 287, 273, 257, 246, 231, 218, 205, 191, 179, 163, 149, 137, 123, 109, 95, 81, 69, 55 and 41. m/z 341, 273, 205, and 123. The fragmentation pattern was characteristic of a friedelane skeleton (Hirota et al., 1975), with a mass compatible with the molecular formula C30H50O (Costa et al., 2015). The 1H NMR (CDCl3, 600 MHz), showed signals at d ¼ 2.4 ppm (ddd, J ¼ 13.9; 5.0 and 2.0 Hz 2H), d ¼ 2.3 ppm (m 2H), d ¼ 2.3 ppm (q, J ¼ 6.7 Hz 4H) and d ¼ 2.0 ppm (m 1H); eight intense signals were observed between d ¼ 1.2 ppm and d ¼ 0.7 ppm, corresponding to methyl groups. The 13C NMR spectrum revealed the presence of 29 signals in the region between d ¼ 0.0 ppm and d ¼ 70.0 ppm and a signal at d ¼ 212.2 ppm, which was characteristic of a ketone carbonyl group. A comparison of chemical shifts with the literature showed signals identical to those presented by the already isolated

friedelin triterpene in other species (Quintans et al., 2014; Trevisan et al., 2012; Van Chau et al., 2005) (Table 1). 3.2. Epi-friedelanol (2) White amorphous solid. IR cm1 (ATR): weak band in 3625 (O-H off) 3468 cm1 (O-H), 2928 and 2869 cm1 (stretch, O-H), 1,457, 1385 and 1355 cm1, characteristic of CH bond deformation of a cyclic hydrocarbon. EIMS m/z: 428 [M]þ, 413 [MeCH3], 304, 257, 205, 191, 177, 165, 149, 135, 125, 109, 95, 81, 69, 55 and 41, also with the characteristic fragmentation pattern of the friedelane skeleton, and a mass compatible with the molecular formula C30H52O and (Hirota et al., 1975). The 1H NMR (CDCl3, 600 MHz) analysis showed signals at d ¼ 3.7 ppm (J ¼ 2.0 Hz 3H), corresponding to a hydrogen carbonyl; a signal at d ¼ 1.7 ppm, characteristic of hydrogen in methine group (CH), and signals at d ¼ 1.2 ppm; d ¼ 1.1 ppm; d ¼ 1.0 ppm; d ¼ 0.9 ppm; d ¼ 0.8 ppm, and d ¼ 0.7 ppm, from methyl groups (CH3). The 13C NMR spectrum showed 30 signals in the region between d ¼ 0.0 and d ¼ 70.0. The distortionless enhancement by polarization transfer (DEPT)-135 NMR experiment identified eight carbon signals from methyl groups, 11 carbon signals from methylene groups, and five carbon signals corresponding to methine groups; six other carbons did not appear in the DEPT spectrum and, therefore, were not attached to hydrogens. Comparison of chemical shifts presented by 2 with the literature (Kundu et al., 2000) suggested that this compound was epi-friedelanol (Table 1). 3.3. Taraxerol (3) White amorphous solid. The GC-MS analysis showed two peaks. The major one (35.71%), at a retention time of 51.19 min, presented mass spectra with EIMS m/z: 426 [M]þ, and the fragments with

Please cite this article in press as: Rodrigues, P.M., et al., Triterpenes from Pouteria ramiflora (Mart.) Radlk. Leaves (Sapotaceae), Food and Chemical Toxicology (2017), http://dx.doi.org/10.1016/j.fct.2017.05.026

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Table 1 13 C Nuclear magnetic resonance (NMR; 125 MHz, CDCl3) data of triterpenes from Pouteria ramiflora. C

1

friedelin

2

epi-friedelanol

3

taraxerol

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30

21.3 40.5 212.2 57.2 41.1 40.3 17.2 52.1 36.5 58.5 35.0 29.5 38.7 37.3 31.8 34.6 29.0 41.8 34.4 27.2 31.4 38.3 5.8 13.7 16.9 19.3 17.7 31.1 34.0 30.8

22.6 41.9 213.4 58.6 42.5 41.7 18.6 53.5 37.8 59.9 36.0 30.9 40.1 38.7 32.8 36.4 30.4 43.2 35.7 28.5 33.1 39.6 7.1 15.0 18.3 20.6 19.0 32.4 35.4 32.1

15.8 35.3 72.7 49.1 37.1 41.7 17.5 53.2 37.8 61.3 35.2 30.6 38.3 39.6 32.3 36.0 30.0 42.8 35.5 28.1 32.8 39.3 11.6 16.4 18.2 18.6 20.1 32.06 35.0 31.8

15.8 35.2 72.8 49.2 37.1 41.7 17.6 53.2 38.4 61.4 35.3 30.6 37.8 39.7 32.3 36.1 30.0 42.8 35.6 28.2 32.8 39.27 11.6 16.4 18.2 18.6 20.1 31.8 35.0 32.1

38.0 27.2 79.1 38.8 55.5 18.8 35.1 39.0 48.8 37.7 17.5 35.8 37.7 158.1 116.9 36.7 37.6 49.3 41.3 28.8 33.7 33.1 28.0 15.4 15.4 29.9 26.0 29.8 33.4 21.3

38.0 27.1 79.1 38.8 55.5 18.8 35.1 39.0 48.7 37.7 17.5 35.8 37.7 158.1 116.9 36.7 37.5 49.3 41.3 28.8 33.7 33.1 28.0 15.4 15.4 29.9 25.9 29.8 33.3 21.3

Friedelin (Van Chau et al., 2005); epi-friedelanol (Kundu et al., 2000); taraxerol (Swain et al., 2012).

1

2

mass of 411 [MeCH3], 393, 365,355, 341, 327, 311, 302, 287, 269, 257, 245, 231, 218, 204, 189, 175, 161, 147, 135, 121, 107, 95, 81, 69, 55, and 41, with characteristic fragmentation pattern of taraxerol and a mass compatible molecular formula C30H50O (Budzikiewicz et al., 1963). The second peak (40.22%), at a retention time of 53.47 min, showed a fragmentation profile identical to that of 2. The 1 H NMR (CDCl3, 600 MHz) analysis showed the following signals: d ¼ 5.5 (dd, J ¼ 8.3, 3.1 Hz, 15H), 3.20 (dd, J ¼ 11.2, 4.2 Hz, 1H), d ¼ 1.9 and signals between d ¼ 1.1. The 13C NMR (CDCl3) data are shown in Table 1. A comparison with the literature (Oladoye et al., 2015; Swain et al., 2012) revealed that 3 was identical to taraxerol. 3.4. a-Amylase inhibitory activity According to the World Health Organization, diabetes was the

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direct cause of 1.5 million deaths in 2012. Moreover, 9% of adults had diabetes in 2014 and type 2 diabetes comprised 90% of people with diabetes worldwide (WHO, 2015). It is estimated that the number of individuals with diabetes will increase 55% by in 2040, reaching 642 milion adults aged 20e79 years (IDF, 2015; WHO, 2016). One of the therapeutic strategies to control diabetes is using aamylase and a-glucosidase inhibitors, which reduce the digestion rate of complex carbohydrates, such as starch, leading to less glucose absorption. As shown in Fig. 2, the F1 and F2 fractions from the crude extract presented pronounced a-amylase inhibitory activities compared with that in the crude extract and other fractions, whereas fractions F4 and F5 were not different from the crude extract. A mixture of taraxerol (1), friedelin (2), and epi-friedelanol (3)

Please cite this article in press as: Rodrigues, P.M., et al., Triterpenes from Pouteria ramiflora (Mart.) Radlk. Leaves (Sapotaceae), Food and Chemical Toxicology (2017), http://dx.doi.org/10.1016/j.fct.2017.05.026

P.M. Rodrigues et al. / Food and Chemical Toxicology xxx (2017) 1e6

Fig. 2. a-Amylase inhibitory activity crude hexane extract of Pouteria ramiflora and fractions expressed in %. The result is expressed of mean values and were analyzed by ANOVA followed by Turkey with p < 0.05. * (p < 0.05
EBH); ** (p < 0.05
F3, F4 and F5) and *** (p < 0.05
F5). EBH ¼ crude hexane extract; and fractions F1 ¼ hexane; F2 ¼ hexane:ethyl acetate; F3 ¼ ethyl acetate; F4 ¼ ethyl acetate:methanol and F5 ¼ methanol.

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composition. Moreover, no information was found on metabolic interfering factors, such as seasonality, altitude, circadian rhythms, climatic conditions (temperature, rainfall, and UV radiation), atmospheric composition, herbivory, pathogen attack, water availability, macro and micronutrients, or plant age, which can influence the production of secondary metabolites (Gobbo-Neto and Lopes, 2007). This is the first time friedelin, epi-friedelanol, and taraxerol have been isolated from this species, in addition to other unidentified triterpenes and fatty acids. Epi-friedelanol (2) and taraxerol (3) have been isolated from other Pouteria species such as P. torta (2), P. venosa, and P. caimito (3) (Silva et al., 2009). However, this the first time friedelin (1) has been isolated from the genus Pouteria. The previously observed anti-inflammatory (Fontes Junior et al., 2009) and other biological activities of extracts from leaves of this species, such as anti-hyperglycemic activity (Gouveia et al., 2013), can be explained considering that P. ramiflora is rich in such compounds, mainly epi-friedelanol. Several reports are available about the anti-inflammatory and anti-nociceptive activity of friedelin and epi-friedelanol (Antonisamy et al., 2011; Biswas et al., 2009; Mahalakshmi et al., 2013), and taraxerol seems to be useful for managing hyperglycemia (Sangeetha et al., 2010). The results of this study show that while the non-polar crude extract did not present biological activity for the evaluated characteristics, fractionation seemed to favor this activity, probably by concentrating the active portion. The identification of these and other metabolites present in this extract will help to clarify the reported biological activities of this species. Financial support

Fig. 3. The total antioxidant capacity of crude hexane extract of Pouteria ramiflora and fractions expressed as equivalent of ascorbic acid. The results, expressed in mean values, were analyzed by ANOVA followed by Turkey with p < 0.05. * (p < 0.05
EBH,F1 and F2); ** (p < 0.05
F4 and F5). EBH ¼ crude hexane extract; and fractions F1 ¼ hexane; F2 ¼ hexane:ethyl acetate; F3 ¼ ethyl acetate; F4 ¼ ethyl acetate:methanol and F5 ¼ methanol.

was isolated from the most active F2 fraction. Taraxerol stimulates Akt, a serine/threonine kinase, in vascular smooth muscle (Castellano et al., 2013). This compound was also identified as a PI3K-dependent dual activator of glucose transport and glycogen synthesis in 3T3-L1 adipocytes, and the activation levels are comparable to those of insulin (Sangeetha et al., 2010). 3.5. Total antioxidant capacity As shown in Fig. 3, only the most polar fractions had antioxidant potential according to the phosphomolybdenum method. The ethyl acetate fraction was the most active, and 1 mg of this fraction showed antioxidant potential equivalent to about 150 mg ascorbic acid. 4. Conclusion Although some studies on the biological activities of P. ramiflora extracts are available, there is a lack of research on micromolecular

 gico Conselho Nacional de Desenvolvimento Científico e Tecnolo ~o de Aperfei(CNPq), process number 564208/2010-8; Coordenaça ~o de çoamento Pessoal de Nivel Superior (CAPES); Fundaça  gicos (FINATEC); Decanato Empreendimentos Científicos e Tecnolo s-Graduaça ~o-UnB (DPP/UnB); and Fundaça ~o de de Pesquisa e Po  Pesquisa do Distrito Federal (FAP/DF), process number Apoio a 193.000.484/2011. Transparency document Transparency document related to this article can be found online at http://dx.doi.org/10.1016/j.fct.2017.05.026. References cies VegAlmeida, S.P., Proença, C.E.B., Sano, S.M., Ribeiro, J.F., 1998. Cerrado: Espe etais Úteis. Embrapa-CPAC, Planaltina. Antonisamy, P., Duraipandiyan, V., Ignacimuthu, S., 2011. Anti-inflammatory, analgesic and antipyretic effects of friedelin isolated from Azima tetracantha Lam. in mouse and rat models. J. Pharm. Pharmacol. 63, 1070e1077. Bernfeld, P., 1955. Amylases a and b. In: Colowick, S.P., Kaplan, N.O. (Eds.), Methods in Enzymology. Academic Press, Inc, San Diego, pp. 149e158. Biswas, M., Biswas, K., Ghosh, A.K., Haldar, P.K., 2009. A pentacyclic triterpenoid possessing anti-inflammatory activity from the fruits of Dregea volubilis. Phcog Mag. 5, 64e67. Budzikiewicz, H., Wilson, J.M., Djerassi, C., 1963. Mass spectrometry in structural and stereochemical problems. XXXII. 1 Pentacyclic triterpenes. J. Am. Chem. Soc. 85, 3688e3699. Carneiro, C.E., Alves-Araújo, A., Almeida Junior, E.B., Terra-Araújo, M.H., 2014. cies da Flora do Brasil. Jardim Bota ^nico do Rio de Sapotaceae, Lista de Espe Janeiro. Disponível em: http://floradobrasil.jbrj.gov.br/jabot/floradobrasil/ FB21020. Castellano, J.M., Guinda, A., Delgado, T., Rada, M., Cayuela, J.A., 2013. Biochemical basis of the antidiabetic activity of oleanolic acid and related pentacyclic triterpenes. Diabetes 62, 1791e1799. Castro, C., Silva, C., Perfeito, J., Santos, M., Resck, I., Paula, J., Silveira, D., 2006. cies de Pouteria. 29a Avaliaç~ ao da atividade antioxidante de algumas espe  ~o Anual da Sociedade Brasileira de Química, Aguas ia ([Links]). Reunia de Lindo

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Please cite this article in press as: Rodrigues, P.M., et al., Triterpenes from Pouteria ramiflora (Mart.) Radlk. Leaves (Sapotaceae), Food and Chemical Toxicology (2017), http://dx.doi.org/10.1016/j.fct.2017.05.026