Microsoft Word - 30-Sau_45927 1599 Bioscience Journal Original Article Biosci. J., Uberlândia, v. 35, n. 5, p. 1599-1613 , Sep./Oct. 2019 http://dx.doi.org/10.14393/BJ-v35n5a2019-45927 ANTI-INFLAMMATORY, ANTINOCICEPTIVE AND ANTIOXIDANT ACTIVITIES OF THE HYDROMETHANOLIC FRACTION FROM ANNONA NUTANS LEAVES ATIVIDADE ANTI-INFLAMATÓRIA, ANTINOCICEPTIVA E ANTIOXIDANTE DA FRAÇÃO HIDROMETANÓLICA DAS FOLHAS DE ANNONA NUTANS Nathalia L. SILVA1; Aline A. SALDANHA2; Denise B. SILVA3; Carlos A. CAROLLO3; Ângela L. B. SARTORI4; Adriana C. SOARES5; João M. de SIQUEIRA6 1. Doutoranda, Laboratório de Farmacognosia/Química de Produtos Naturais, Campus Centro-Oeste, Universidade Federal de São João Del Rei, Divinópolis, MG, Brazil; 2. Doutoranda, Laboratório de Farmacologia da Dor e Inflamação, Campus Centro-Oeste, Universidade Federal de São João Del Rei, Divinópolis, MG, Brazil; 3. Doutor, Laboratório de Produtos Naturais e Espectrometria de Massas, Universidade Federal de Mato Grosso do Sul, Campo Grande, MS, Brazil; 4. Doutor, Centro de Ciências Biológicas e da Saúde, Universidade Federal de Mato Grosso do Sul, Campo Grande, MS, Brazil; 5. Doutor, Laboratório de Farmacologia da Dor e Inflamação, Campus Centro-Oeste, Universidade Federal de São João Del Rei, Divinópolis, MG, Brazil; 6. Doutor, Laboratório de Farmacognosia/Química de Produtos Naturais, Campus Centro-Oeste, Universidade Federal de São João Del Rei, Divinópolis, MG, Brazil. jmaximo@ufsj.edu.br ABSTRACT: Annona nutans (Annonaceae) is a plant species found in Bolivia, Paraguay, Argentina, and the Brazilian Cerrado, specifically in the states of Mato Grosso and Mato Grosso do Sul (Brazil). Its common names are Araticû-Mi and Araticû-Ñu. The research contributions regarding the chemical composition and biological activities of extracts from A. nutans are rare, with only four articles being found in the literature. Therefore, the present study evaluated the anti-inflammatory and antinociceptive activities of the hydromethanolic fraction (FHMeOH) using carrageenan-induced paw edema and hot-plate tests. In addition, the antioxidant activity was evaluated by DPPH radical scavenging, total phenolic, flavonoid and tannin content assays and quantification of the major metabolites by LC-MS were performed. Oral treatment with the FHMeOH (at a dose of 300 mg.kg-1) significantly reduced paw edema 2 h and 4 h after the inflammatory stimulus. The intraperitoneal (i.p.) treatment with the FHMeOH (50 and 100 mg.kg-1) proved to be most effective, and the inhibition of acute inflammation was still visible 6 h after carrageenan injection. At doses of 50 and 100 mg.kg-1 (i.p.), FHMeOH exhibits central antinociceptive effects by increasing the latency of the reaction in the hot-plate model. The FHMeOH showed antioxidant potential, and the metabolites quercetin-3- O-galactoside, quercetin-3-O-glucoside, isorhamnetin-3-O-galactoside, quercetin-3-O-β-D-apiofuranosyl- (1→2)-galactopyranoside, and chlorogenic acid were identified and quantified by LC-MS. Our results indicate, for the first time, that FHMeOH has anti-inflammatory, antinociceptive and antioxidant potential, and it is a promising source of studies for new herbal medicines KEYWORDS: Annona nutans. Flavonoids. Anti-inflammatory. DPPH. Antinociceptive. HPLC quantification. INTRODUCTION Annona is among the 27 most important genera of the Annonaceae family. This genus, containing approximately 162 species distributed among the tropics, is represented mainly in South and Central America, with 110 native tropical species in the Americas and Africa (CHATROU et al., 2012; COUVREUR et al., 2011). Initially, plants belonging the Annonaceae family were believed to predominantly contain alkaloids (LEBOEUF et al., 1982); however, a great diversity of chemical constituents was recently acknowledged. Studies of species belonging to the Annonaceae family intensified following the isolation of a chemical group known as “annonaceous acetogenins”, which presented a wide variety of biological activities such as cytotoxic, antitumor, pesticidal, and antimicrobial (BERMEJO, 2005; CAVÉ et al., 1997). For many years, the interest in Annona species, and other genera in the Annonaceae family, was a result of the presence of acetogenins, which showed promising pharmacological activities (BERMEJO, 2005). However, epidemiological data from the island of Guadalupe (Caribbean) have associated the consumption of Annona species (A. muricata, A. reticulata, and A. squamosa) with the development of atypical parkinsonism, suggesting that acetogenins and quinoline alkaloid derivatives are directly related to its etiology, because the Received: 18/09/18 Accepted: 01/05/19 1600 Anti-inflammatory, antinociceptive… SILVA, N. L. et al Biosci. J., Uberlândia, v. 35, n. 5, p. 1599-1613 , Sep./Oct. 2019 http://dx.doi.org/10.14393/BJ-v35n5a2019-45927 acetogenin annonacin was shown to cause neurodegeneration in rats (CAPARROS- LEFEBVRE; STEELE, 2005). Apart from acetogenins, plants belonging to the Annonaceae family possess a wide variety of metabolites that are responsible for important pharmacological activities, such as anti-inflammatory, antinociceptive, and antioxidant (BENITES et al., 2015; FORMAGIO et al., 2013a, 2013b). Annona nutans (or Annona spinescens var. nutans) is a plant species found in Bolivia, Paraguay, Argentina, and the Brazilian Cerrado, specifically in the states of Mato Grosso and Mato Grosso do Sul (Brazil). Its more common names are aratico, chirimoya del campo, and sinini de la pampa; and those in the Guarani language are Araticû-Mi and Araticû-Ñu (CORRÊA, 1926; TROPICOS.ORG, 2017). The research contributions regarding the chemical composition and biological activities of the extracts from A. nutans are rare, with only four articles found in the literature (GLEYE et al., 2000, 1998; GONÇALVES et al., 2014; SILVA et al., 2015). The acetogenins were found in A. nutans roots (GLEYE et al., 2000, 1998), with studies in the literature showing the absence of acetogenins in the leaves (SILVA et al., 2015; SILVA, 2013). Thus, the present study identified and quantified some metabolites by LC-MS and quantitated the total phenolic, tannin, and flavonoid content, in addition to assessing the antioxidant potential as DPPH radical scavenging ability from the hydromethanolic fraction (FHMeOH) of A. nutans. Moreover, the anti-inflammatory and antinociceptive efficacy of the FHMeOH was evaluated in vivo. MATERIAL AND METHODS Plant material The leaves of A. nutans were collected in Porto Murtinho, the state of Mato Grosso do Sul, Brazil. The plant was previously identified by Renato de Mello-Silva, and a voucher specimen was deposited at the CGMS Herbarium (MS, Brazil) under number 27648. The present study obtained a Certificate of Registration from the National System for the Management of Genetic Heritage and Associated Traditional Knowledge (A90D499). Preparation of fractions Dried and powdered leaves (160 g) were percolated with methanol:water (9:1) (5.0 L) for 72 h at room temperature, yielding 32 g hydromethanolic extract. 28 g was dissolved in methanol:water (9:1) (500 mL), followed by sequential partitioning with n-hexane, chloroform, and ethyl acetate (200 mL, 5x of each solvent). The final hydromethanolic fraction (FHMeOH) was dried under a vacuum in a rotatory evaporator at 45 °C, and subsequently lyophilized, yielding 21 g. For the determination of total phenolic, tannin, and flavonoid contents, a stock solution in ethanol:water (1:1) was initially prepared from the FHMeOH solution at a concentration of 5 mg.mL-1, and successive dilutions at concentrations of 5.0; 2.5; 1.25; 0.625; 0.3125 mg.mL-1 were prepared. Determination of the total phenolic content The total phenolic content was determined by spectrophotometric quantitation using the Folin- Ciocauteu reagent in 96-well microplates, as described previously (ZHANG et al., 2006). Briefly, a 20 μL sample (prepared as previously described) and 100 μL Folin-Ciocauteu reagent (Imbralab®) were added to each well, shaken, and incubated for 5 min. Subsequently, 80 μL 7.5% Na2CO3 (Alphatec®) solution was added. A calibration curve was generated using gallic acid (Cromato Produtos Químicos Ltda®) at the standard at concentrations of 1; 0.5; 0.25; 0.125; 0.0625; and 0.0312 mg.mL-1. For the reagent blank, 20 μL methanol, 100 μL Folin-Ciocauteu reagent, and 80 μL 7.5% Na2CO3 were used. The plates were incubated in the dark at room temperature for 2 h. Readings were performed using a microplate spectrophotometer (SpectraMax®Plus384, Molecular Devices, Sunnyvale, CA, USA, Gen5 software) at λ = 750 nm. For the instrument blank, 20 μL CH3OH:H2O (1:1) and 180 μL distilled water were used. The total phenolic content (TPC) was determined by interpolation of the absorbance of the samples against the calibration curve obtained for the standard and are expressed as µ g of gallic acid equivalents.mg-1 for respective fractions. All analyses were performed in triplicate. Determination of the total tannin content Sample solutions were prepared at the same concentrations as described for the total phenolic quantitation; however, prior to the addition of Folin- Ciocauteu reagent, 0.01 g.mL-1 hide powder (Sigma- Aldrich) was added, and the solution was shaken for 60 min on an orbital shaker. The filtrates (20 μL) were added to each well of a 96-well plate with 20 μL methanol:water (1:1) and 100 μL Folin- Ciocauteu reagent, shaken, and incubated for 5 min. Subsequently, 80 μL of 7.5% Na2CO3 solution was added, and the plates were incubated in the dark at room temperature for 2 h. Readings were performed using a microplate spectrophotometer at λ = 750 nm. 1601 Anti-inflammatory, antinociceptive… SILVA, N. L. et al Biosci. J., Uberlândia, v. 35, n. 5, p. 1599-1613 , Sep./Oct. 2019 http://dx.doi.org/10.14393/BJ-v35n5a2019-45927 For the instrument blank, 20 μL of methanol:water (1:1) and 180 μLof distilled water were used. The total tannin content (TTC) was determined from the standard curve by subtracting the calculated content of the sample solution for non-adsorbed polyphenols from the calculated TPC content (sample solution for total polyphenols). The expression is shown below. The results are expressed as μg.mL-1 of sample. All analyses were performed in triplicate. TTC = content of the sample solution for total polyphenols – content of the sample solution for non-adsorbed polyphenols in hide powder (BRASIL, 2010a; VERZA et al., 2007). Determination of the total flavonoid content The total flavonoid content (TFC), equivalent to quercetin, present in the fractions was determined using a previously described method (BANOV et al., 2006; BRASIL, 2010b), and expressed as μg quercetin . mg-1 fraction. For the calibration curve, quercetin (Sigma- Aldrich) was used as the standard reference flavonoid, of which a stock solution of 0.01 μg.mL-1 was prepared. From this stock, dilutions were prepared at concentrations of 20, 10, 5, 4, 3, 2, 1, 0.75, 0.5 and 0.3 μg.mL-1, to which 500 μL 5% (w/v) AlCl3 solution was added. Following a 30-min incubation, the absorbance was read at 425 nm using a UV-Vis spectrophotometer (QUIMIS®) and the calibration curve was constructed. To 100 μL 50% methanol, 100 μL FHMeOH and 500 μL 5% (w/v), AlCl3 solution was added. Following a 30-min incubation, the absorbance was read at 425 nm using a UV-Vis spectrophotometer (QUIMIS®). All analyses were performed in triplicate. Determination of the DPPH radical-scavenging capacity The radical-scavenging capacity of FHMeOH was determined according to the method described by Burda and Oleszek (2001) (BURDA et al., 2001). BHT (2,6-di-tert-butyl-4-methylphenol) was used as the reference compound. FHMeOH was prepared in triplicate for each concentration (1, 10, 100, 250, and 500 μg.mL-1). Each sample (75 µ L) was added to three wells of a 96-well plate containing 150 μL 0.002% (w/v) DPPH-methanol solution, shaken vigorously, and incubated in the dark for 30 min. The control was prepared as above without any extract or BHT. The absorbance was measured at λ = 517 nm using a UV-Vis spectrophotometer (Biotek Power Wave XS2/US, U.S.A) and methanol was used for baseline correction. The radical-scavenging activity is expressed as the inhibition percentage and was calculated as: [1 − (Abscontrol - Abssample)]× 100 where Abscontrol = absorbance of DPPH radicals in methanol and Abssample = absorbance of fraction in methanol + DPPH. The scavenging activity is expressed as µ g.mL-1. IC50 values (µ g.mL-1) were calculated using Probit analysis (FINNEY, 1980). Identification and quantitative UFLC-DAD- ESI- QTOF-MS analysis The identification and quantification of phytocompounds was performed on a Shimadzu Prominence UFLC TM system, equipped with a LC20AD, coupled to a diode array detector and mass spectrometer (MicroOTOF-Q III Bruker Daltonics, Billerica, USA) with an electrospray ion source. The analyses were monitored between 210 and 800 nm and the mass spectrometer operated in a negative ionization mode (m/z 120-1300). Chromatographic analyses were performed in a Kinetex C-18 column (Phenomenex, 2.6 μ, 150 x 2.1 mm). For quantification, FHMeOH was dissolved in methanol and water (1:1, v/v) at a concentration of 1 mg.mL-1. Standard solutions at a concentration of 1 mg.mL-1 were prepared with chlorogenic acid (5-O-caffeoylquinic acid), the flavonoids quercetin-3-O-galactopyranoside, quercetin-3-O-glucopyranoside, and isorhamnetin- 3-O-galactopyranoside (Sigma Aldrich®). For the analytical curve, dilutions were prepared at the concentrations from 0.0488 to 200.00 µ g.mL-1 (0.0488, 0.0976, 0.195, 0.390, 0.781, 1.56, 3.12, 6.25, 12.50, 25.00, 50.00, 100.00, and 200.00 µ g.mL-1). For the isorhamnetin-3-O- galactopyranoside, concentrations of 12.5, 25.0, 50.0, 100.0, and 200.0 µ g.mL-1 were prepared. About 3 μL of sample was injected in to the column by the auto sampler. The samples were eluted through the column with a gradient mobile phase consisting of A (water 0.1% (v/v) formic acid) and B (acetonitrile: formic acid 0.1% (v/v)). The gradient elution was programmed as follows: 0–2 min B (3%); 2–25 min B (25%); 25–26 min B (80%); 26–28 min B (80%); 28–29 min B (3%); and 29–35 min B (3%). The analyses were carried out in triplicate at a flow rate of 0.3 mL.min-1, at a temperature of 50 °C, with the detector set at λ= 340 nm. Calibration curves were plotted showing a linear relationship between concentrations versus peak areas for all reference compounds. The 1602 Anti-inflammatory, antinociceptive… SILVA, N. L. et al Biosci. J., Uberlândia, v. 35, n. 5, p. 1599-1613 , Sep./Oct. 2019 http://dx.doi.org/10.14393/BJ-v35n5a2019-45927 attribution of the chromatographic peak was based on the retention times and confirmed by the injection of standards. The concentration of each peak was calculated from the experimental peak areas by analytical interpolation in a standard calibration line. Peak areas were calculated at 340 nm. The limit of detection (LOD) was determined as a signal-to-noise ratio of 3:1 and the limit of quantification (LOQ) was determined as a signal-to- noise ratio of 10:1 (GARCÍA-SALAS et al., 2015). The precision was calculated by relative standard deviation (%RSD), and the selectivity was evaluated by comparing the chromatograms of the individual reference standards and the degree of interference between the peaks when injected simultaneously; the degree of purity of these peaks was also investigated (BRITO, 2014; RIBANI et al., 2004). In vivo assays Chemicals Indomethacin (Ind) and carrageenan λ type IV were purchased from Sigma-Aldrich Inc. (St. Louis, MO, USA). Fentanyl citrate (Fent) was purchased from Cristália (SP, Brazil). DMSO 2% in physiological saline was used as the control, and FHMeOH was prepared in this vehicle for oral or intraperitoneal treatments to mice. Animals Adult male Swiss mice (28–30 g) were obtained from the Bioterium of Universidade Federal de São João del-Rei, Brazil, and were housed in temperature-controlled rooms (22–25 °C), under a 12–12 h light–dark cycle, with access to food and water ad libitum. The mice were acclimated for one week prior to the experiment. Twelve hours prior to the beginning of oral treatments, the mice were fasted and received only water ad libitum. For the intraperitoneal experiment, the food and water were retained. The number of mice and the intensity of noxious stimuli used were the minimum necessary to demonstrate consistent effects of the drug treatments. All procedures were carried out in accordance with the guidelines set forth by the Brazilian National Council for the Control of Animal Experimentation and International Association for the Study of Pain and were approved by the Ethics Committee in Animal Experimentation of the Federal University of São João Del-Rei, Brazil (CEUA/UFSJ, protocol 034/2015). Assessment of the anti-inflammatory activity The anti-inflammatory activity was assayed using the carrageenan-induced paw edema model (LEVY, 1969). Mice were randomly divided into five groups (n=6), and orally (p.o.) received vehicle (10 mL.kg-1, control group), FHMeOH (at doses of 30, 100, and 300 mg.kg-1), or indomethacin (10 mg.kg-1). Moreover, FHMeOH, at doses of 25, 50 and 100 mg.kg-1, were intraperitoneally (i.p.) administered. Following 30 (i.p.) or 60 (p.o.) min following treatments, carrageenan (400 µ g, 30 µ L) was injected into the plantar side of the left hind paw. Paw volume was measured using a plethysmometer (Insight®, Brazil) prior to treatment (basal value) and at 1, 2, 4, and 6 h after the injection of the inflammatory stimulus. The volume of edema was calculated by the difference between the prior basal paw volume and the one after carrageenan injection. Evaluation of the antinociceptive activity Mice were tested on a hot-plate (Insight®, Brazil) kept at a constant temperature of 55 ± 0.50 °C for 24 h before the assay, and animals that remained on the apparatus for less than 15s were selected. Thus, the selected animals were randomly divided into five groups (n=7) and received (i.p.) vehicle (10 mL.kg-1, control group), FHMeOH (at doses of 25, 50, and 100 mg. kg-1), and Fentanyl (Fent 200 µ g.kg-1). Reaction times were recorded when the mice licked their paws or jumped at intervals of 30 min up to 120 min after treatments. A cut-off of 30s was chosen to avoid tissue lesions (MUHAMMAD; SAEED; H., 2012). Statistical analysis Microsoft Excel 2010 (Microsoft Corporation) was used for the quantitation of the total phenolic, tannin, and flavonoid content. In the evaluation of the anti-inflammatory and antinociceptive activities, results are expressed as the mean ± SEM. The statistical significance between groups was assessed using one-way analysis of variance (ANOVA) followed by the Bonferroni multiple comparison post-hoc test. All calculations for anti-inflammatory, antinociceptive activities and DPPH radical-scavenging capacity were performed using GraphPad Prism™ version 5.01 (GraphPad® Software Inc., San Diego, CA). A level of significance (p < 0.05) was considered for each experiment. 1603 Anti-inflammatory, antinociceptive… SILVA, N. L. et al Biosci. J., Uberlândia, v. 35, n. 5, p. 1599-1613 , Sep./Oct. 2019 http://dx.doi.org/10.14393/BJ-v35n5a2019-45927 RESULTS Total phenolic, tannin and flavonoid content The total phenolic, tannin, and flavonoid content of the FHMeOH extract from A. nutans was calculated based on the interpolation of the absorbance values of the samples from the calibration curve of gallic acid (TPC and TTC), which presented the following equation of the line, y = 0.2352x + 0.0044, R² = 0.9982, and from the calibration curve of quercetin (TFC), which presented y = 0.0228x + 0.0027, R2 = 0.9986. The total phenolic, tannin, and flavonoid content was 62.96 ± 3.73 μg.mg-1, 31.14 ± 3.11 μg.mg-1, and 18.07 ± 0.10 μg.mg-1, respectively. Determination of DPPH radical-scavenging capacity The DPPH radical-scavenging activity of the FHMeOH extract from A. nutans is presented in Figure 1. FHMeOH showed a dose-dependent inhibitory effect with an IC50 of 4.89 μg.mL-1, which was comparable to that of the commercial antioxidant, BHT (IC50 = 16.36 ± 3,63 μg.mL-1). The FHMeOH at doses of 1, 10, 100 and 250 µ g.mL-1 presented a scavenging effect on the DPPH radical that was statistically significant compared to the BHT standard. Figure 1. The DPPH radical-scavenging ability of the hydromethanolic fraction (FHMeOH) and 2,6-di-tert- butyl-4-methylphenol (BHT) at five different concentrations (µ g.mL-1). p < 0.001 as compared with BHT. Identification and quantitative UFLC-DAD- ESI- QTOF-MS analysis For the analytical curve and linearity, the regression equations were y = 12222x - 18557 (R² = 0.9992) for chlorogenic acid; y = 10087x - 13571 (R² = 0.9992) for quercetin-3-O-galactopyranoside; y = 9454.4x - 13865 (R² = 0.9991) for quercetin-3- O-glucoside; and y = 8597.5x + 12942 (R² = 0.9995) for isorhamnetin-3-O-galactopyranoside. The limit of detection (LOD) for all flavonoids was 4.88 x 10-2 µ g.mL-1 and chlorogenic acid was 9.77 x 10-2 µ g.mL-1. The limit of quantification (LOQ) was 1.95 x 10-1 µ g.mL-1 for flavonoids and 3.91 x 10-1 µ g.mL-1 for chlorogenic acid. Relative standard deviations (%RSD) were in the range of 0.11% to 3.49% and they were calculated as a mean of the three replications. The estimated concentration of metabolites was calculated in μg.mg-1 of FHMeOH measuring 3.04 μg.mg-1 of chlorogenic acid, 6.74 μg.mg-1 of quercetin-3-O-galactopyranoside, 3.62 μg.mg-1 of quercetin-3-O-glucopyranoside, 2.12 μg.mg-1 of isorhamnetin-3-O-galactopyranoside, and 4.86 μg.mg-1 of quercetin-3-O-β-D-apiofuranosyl-(1→2)- galactopyranoside. Since the metabolite, quercetin- 3-O-β-D-apiofuranosyl-(1→2)-galactopyranoside, had no reference standard, its content was calculated based on the analytical curve of quercetin-3-O-β- galactopyranoside with a correction factor based on the corresponding molecular weight, because they have the same aglycone and thus the same chromophore group. This compound (quercetin-3- O-β-D-apiofuranosyl-(1→2)-galactopyranoside) was identified in studies conducted in our laboratory using H1 and C13 NMR, COSY and DEPT techniques (SILVA et al., 2015; SILVA, 2013) The FHMeOH was also analyzed by UFLC- DAD-MS to identify its chemical constituents. The compounds were identified by the comparison of UV spectra and retention time with applied patterns and subsequent confirmation of their molecular weights and their fragmentation in MS2. The compounds identified are listed in Table 1 and illustrated on the chromatogram in Figure 2. Twenty-three compounds could be detected and identified from FHMeOH. Chlorogenic acids derivatives identified were 3-O-E-caffeoylquinic 1604 Anti-inflammatory, antinociceptive… SILVA, N. L. et al Biosci. J., Uberlândia, v. 35, n. 5, p. 1599-1613 , Sep./Oct. 2019 http://dx.doi.org/10.14393/BJ-v35n5a2019-45927 acid (3) and 4-O-E-caffeoylquinic acid (7), as well as the O-glycosylated flavonols 14-22 and alkaloids 4, 6, 8-11 and 23. These compounds were identified by comparing the spectral data reported for them in the literature (CLIFFORD et al., 2003; LIU et al., 2018) and data were also reported from Annona species for the alkaloids (FERRAZ et al., 2017; SHANGGUAN et al., 2018). Figure 2. Base peak chromatogram obtained in negative and positive ion modes from FHMeOH of A. nutans. Table 1. Constituents identified by UFLC-DAD-MS from FHMeOH Peak RT (min) Compound MF UV (nm) Negative mode (m/z) Positive mode (m/z) MS [M-H]- MS/MS MS [M+H]+ MS/MS 1 1.2 O-dihexoside C12H22O11 - 341.1086 191 365.1068Na 2 1.7 NI C11H22O8 - 279.1087 - 281.1252 3 6.4 3-O-E-caffeoylquinic acid C16H17O9 299, 323 353.0885 191, 179 355.1043 163 4 7.5 norcoclaurine- O- hexoside C27H35NO12 - - - 566.2268 272, 255, 161 5 7.9 NI C19H28O11 275 431.1555 - 433.1728 205, 187, 175 6 10.1 NI C18H19NO4 288 312.1239 - 314.1406 178, 163 7 10.7 4-O-E-caffeoylquinic acidst C16H18O9 299, 325 353.0886 191, 173 355.1027 163 8 11.3 di-O-methoxyl di- hydroxyl aporphine alkaloid C18H19NO4 285 312.1254 - 314.1409 298, 284, 270, 151 9 12.6 NI C17H19NO3 285 - - 286.1456 254, 237, 209, 191, 175, 165 10 13.0 Stepharine C18H19NO3 285 - - 298.1451 254, 238, 223, 161, 146 11 14.5 Magnoflorine C20H24NO4+ 280 - - 342.1712* 297, 282, 265, 237, 192 12 15.1 NI C19H23NO4 283 328.1566 - 330.1708 284, 267, 192, 177 13 17.4 NI C19H32O8 - 387.2021 - 389.2180 227, 209, 191, 173 14 17.5 Quercetin-O-hexosyl- deoxyhexoside C27H30O16 270, 350 609.1443 300, 271, 255 611.1605 303 15 17.8 Quercetin-O-hexosyl- C27H30O16 270, 609.1453 300, 271, 255 611.1623 303 1605 Anti-inflammatory, antinociceptive… SILVA, N. L. et al Biosci. J., Uberlândia, v. 35, n. 5, p. 1599-1613 , Sep./Oct. 2019 http://dx.doi.org/10.14393/BJ-v35n5a2019-45927 deoxyhexoside 350 16 18.0 Quercetin-3-O-β-D- apiofuranosyl-(1→2)- galactopyranoside C26H28O16 265, 350 595.1305 300, 271, 255, 243, 179 597.1473 465, 303 17 18.2 Quercetin-O-hexosyl- deoxyhexoside C27H30O16 270, 350 609.1441 300, 271, 255, 179 611.1630 465, 303 18 18.3 Quercetin-O-pentosyl- hexoside C26H28O16 265, 350 595.1289 300, 271, 255, 243, 179 597.1485 465, 303 19 18.5 Quercetin-3-O-β- galactopyranoside st C21H20O12 265, 352 463.0872 300, 271, 255, 243 465.1044 303 20 19.0 Quercetin-3-O-β- glucopyranoside st C21H20O12 266, 348 463.0881 300, 271, 255, 243, 179 465.1044 303 21 21.4 Quercetin-O-methyl-O- hexoside C22H22O12 268, 350 477.1031 314, 299, 285, 271, 257, 243 479.1196 317, 302, 285 22 21.8 Quercetin-O-methyl-O- hexoside C22H22O12 265, 350 477.1027 314, 299, 285, 271, 257, 243 479.1201 317, 302, 285 23 24.5 Xylopine C18H17NO3 284 - - 296.1288 279, 264, 249, 234, 221, 206, 178 25 26.2 NI C59H87N5O15 - - - 553.8154** 482, 416, 359, 237, 209 RT: retention time, MF: molecular formula; NI: non-identified; Na: [M+Na]+; *: [M]+; ** [M+2H]+2; st:confirmed by the injection of authentic standard Anti-inflammatory effect of the FHMeOH With respect to the anti-inflammatory activity, at 2 and 4 h post-carrageenan injection, the p.o. administration of 300 mg.kg-1 FHMeOH promoted a significant reduction in paw edema by 48.57% (p < 0.05) and 52.0% (p < 0.05), respectively, as compared with the control group (Figure 3). Figure 3. The effect of p.o. administration of the FHMeOH from A. nutans on carrageenan-induced paw edema in mice. Data were analyzed by ANOVA followed by Bonferroni’s multiple comparison post-hoc test. Values are expressed as the mean ± SEM (n = 6). *p < 0.05 and **p < 0.01 compared with the control group. In contrast, i.p. treatment with FHMeOH at minor doses (50 and 100 mg.kg-1) exerted long- lasting anti-inflammatory effects that remained significant 6 h after inflammatory stimulus (Figure 4). The inhibitory values of paw edema at 2 and 4 h post-carrageenan-induced acute inflammation were 95.45% (p < 0.001) and 74.29% (p < 0.001), respectively, for 50 mg.kg-1 of the FHMeOH. 1606 Anti-inflammatory, antinociceptive… SILVA, N. L. et al Biosci. J., Uberlândia, v. 35, n. 5, p. 1599-1613 , Sep./Oct. 2019 http://dx.doi.org/10.14393/BJ-v35n5a2019-45927 Figure 4. The effect of i.p. administration of the hydromethanolic fraction (FHMeOH) from A. nutans on carrageenan-induced paw edema. Data were analyzed by ANOVA followed by Bonferroni’s multiple comparison post-hoc test. Values are expressed as the mean ± SEM (n = 6). *p < 0.05, **p < 0.01 and ***p < 0.001 as compared with the control; #p < 0.05 and ##p < 0.01 as compared with the 25 mg.kg-1 FHMeOH group. Antinociceptive activity of the FHMeOH FHMeOH at doses of 50 and 100 mg.kg-1 (i.p.) induced a significant increase in the latency of reaction, and the central antinociceptive effects began 90 min post both treatments and were still observable after 120 min for the 50 mg.kg-1 (Figure 5). Figure 5. Effects of i.p. administration of the hydromethanolic fraction (FHMeOH) in the hot-plate model. Data were analyzed by ANOVA followed by Bonferroni’s multiple comparison post-hoc test. Values are expressed as the mean ± SEM (n = 7). *p < 0.05, **p < 0.01 and ***p < 0.001 as compared with the control. DISCUSSION The UFLC analysis in FHMeOH of A. nutans leaves revealed that quercetin-3-O- galactopyranoside is the identified phenolic compound which is the major constituent. The quantification by the UFLC-DAD method was validated and showed linearity, selectivity, and precision (BRASIL, 2003; DE AMORIM et al., 2014; LANDIM; FEITOZA; DA COSTA, 2013). From the Annonaceae family, reports including the quantitative determination of metabolites are uncommon, in particular for flavonoids (GARCÍA- SALAS et al., 2015), and most of the studies in the literature have evaluated the alkaloid and acetogenin content, because these two classes are the main ones in the species of the family (ALMEIDA, J. R. G. S.; JUNIOR, R. G. O; DE OLIVEIRA, 2015). 1607 Anti-inflammatory, antinociceptive… SILVA, N. L. et al Biosci. J., Uberlândia, v. 35, n. 5, p. 1599-1613 , Sep./Oct. 2019 http://dx.doi.org/10.14393/BJ-v35n5a2019-45927 Currently, studies involving polyphenols such as flavonoids and chlorogenic acid derivatives have also gained great importance due to their diverse biological properties such as antioxidant, anti-inflammatory, antinociceptive, antimicrobial, and cardioprotective activity, among others (CALDERÓN-MONTAÑO et al., 2011; GALVÃO, STANLEY DE S. L. MONTEIRO et al., 2016; GARCÍA-LAFUENTE et al., 2009; GEORGIEV; ANANGA; TSOLOVA, 2014). A direct relationship among antioxidant activity, phenolic compounds, and anti- inflammatory efficacy has been demonstrated in the literature (FORMAGIO et al., 2013a, 2013b; HIRANO et al., 2001), including in certain Annona species. The methanolic extract from A. crassiflora, for instance, has been shown to have a high total phenolic and flavonoid content (BENITES et al., 2015), and it effectively reduced paw edema and leukocyte recruitment induced by carrageenan at doses of 100 and 300 mg.kg-1 (ROCHA et al., 2016). Similarly, the methanolic extract of A. dioica has high levels of total phenols and flavonoids, and in the concentration of 30 to 300 mg.kg-1 p.o., it exhibited an anti-edematogenic effect in carrageenan-induced paw edema in a time- and dose-dependent manner (FORMAGIO et al., 2013b). A. reticulata has been also shown to possess strong antioxidant ability and dose-dependent inhibition of paw edema following carrageenan injection in rats (KANDIMALLA et al., 2016). The present study demonstrated, for the first time, the antioxidant, anti-inflammatory and antinociceptive activities of the FHMeOH of A. nutans leaves. Moreover, the anti-inflammatory and antinociceptive activities of FHMeOH were demonstrated using acute inflammation and thermal hyperalgesia models in mice. Carrageenan injection into the paw provokes a biphasic response characterized by the initial phase (0 to 1 h) and the later phase (over 1 h). In the first phase, the release of histamine, serotonin, and bradykinins, and, to a lesser extent, prostaglandins occurs. The later phase is related to the overproduction of prostaglandins and polymorphonuclear leukocyte migration (CUZZOCREA et al., 1998). The p.o. treatment with FHMeOH failed to significantly inhibit paw edema formation with the lowest doses tested (30 and 100 mg.kg-1). Only the highest dose of 300 mg/kg FHMeOH significantly inhibited paw edema 2 and 4 h after carrageenan injection. Since this inhibition was no longer observed at t = 6 h, this experiment suggests a low bioavailability by this route. On the other hand, the FHMeOH administered via i.p. exhibited significant anti- edematogenic activity in both phases. According to the literature, in the some studies of the pharmacological effects of natural products, using these models and the intraperitoneal route, the treatments with plant extracts showed anti- inflammatory and antinociceptive activities at higher doses (75-500 mg.kg-1) (ALMEIDA et al., 2012; IBRAHIM et al., 2002; NARDI et al., 2003; SADANHA et al., 2016) than those used for FHMeOH in the present study. Regarding the antioxidant activity, the DPPH radical-scavenging potential was used and is often compared with that of butylated hydroxytoluene (BHT), a commercial antioxidant used as a food additive (BURDA et al., 2001). As for measured by DPPH radical scavenging, FHMeOH presented an IC50 of 4.89 μg.mL-1, which was superior to that shown by BHT (IC50 = 16.36 μg.mL-1). The IC50 of A. nutans is lower than the methanolic extract (17.84 μg.mL-1) of A. dioica leaves (FORMAGIO et al., 2013b), and A. dioica presented high rates of flavonoids (FORMAGIO et al., 2013b). Although flavonoids present known antioxidant activity, it seems that the diverse metabolic profile of A. nutans has better antioxidant activity than other Annona species with higher content in flavonoids. A possible cause for these data is the presence of other metabolites which presented an antioxidant action such as proaporphine alkaloids stepharine (AVULA et al., 2018; COSTA et al., 2015), aporphine magnoflorine (KUKULA-KOCH et al., 2016; NASEER et al., 2015), and oxoaporphine xylopine (COSTA et al., 2010). As soon as we identified the anti- inflammatory activity of FHMeOH, we tested its analgesic activity, because these properties are shared by several non-steroidal anti-inflammatory drugs. The hot-plate model is a specific central antinociceptive assay, and the nociceptive response to thermal stimulus is supraspinally integrated (JULIUS, D; BASBAUM, 2001; WOOLFE; MACDNOALD, 1944). The FHMeOH produced central antinociceptive effects, verified by the increase in reaction time. However, further studies are needed to establish the possible mechanisms of the antinociceptive action of FHMeOH (SALDANHA et al., 2016). It can be stated that inflammation and nociception are correlated, because nociception is one of the cardinal signs of inflammation (LENARDAO et al., 2016; YIMAM et al., 2016). It is also known that flavonoids and cinnamic derivatives, for example, chlorogenic acids, have anti-inflammatory and antinociceptive activities 1608 Anti-inflammatory, antinociceptive… SILVA, N. L. et al Biosci. J., Uberlândia, v. 35, n. 5, p. 1599-1613 , Sep./Oct. 2019 http://dx.doi.org/10.14393/BJ-v35n5a2019-45927 (GARCÍA-LAFUENTE et al., 2009; GEORGIADES et al., 2014; RATHEE et al., 2009; SERAFINI; PELUSO; RAGUZZINI, 2010; ZHAO, 2015; ZHU et al., 2013). These secondary metabolites may act via the inhibition of prostaglandin synthesis, neutrophil degranulation, and histamine, phosphodiesterase and protein kinases release (BASTOS, D. H. M.; ROGERO, M. M.; AREAS, 2009; RATHEE et al., 2009). The flavonoids and cinnamic derivatives play an important role because they present several biological actions besides anti-inflammatory and antinociceptive activities, such as antioxidant and anti-microbial effects, as well as the modulation of metabolic disorders (NAVEED et al., 2018). The antinociceptive and anti-inflammatory activities of FHMeOH can be attributed, at least in part, to the metabolites found in the fractions, such as flavonoids and chlorogenic acid derivatives. CONCLUSION The present study demonstrates, for the first time, that the FHMeOH fraction obtained from the leaves of A. nutans possesses in vivo anti- inflammatory and antinociceptive activities. Furthermore, the combination of phenolics present in this fraction can explain, at least partially, the effects observed. These activities raise interest in the therapeutic potential of the FHMeOH for the treatment and/or management of inflammatory and painful conditions. ACKNOWLEDGEMENTS AND FUNDING This work was supported by the Coordenação de Aperfeiçoamento de Pessoal do Nível Superior (CAPES) in the CAPES/PNPD program, under the number 2833-2011. The authors acknowledge FAPEMIG and Federal University of São João del-Rei postgraduate fellowship and CNPq for an awarded research grant. RESUMO: Annona nutans (Annonaceae) é uma espécie de planta encontrada na Bolívia, Paraguai, Argentina e no Cerrado brasileiro, especificamente nos estados de Mato Grosso e Mato Grosso do Sul (Brasil). Seus nomes mais comuns são aratico e Araticû-Mi e Araticû-Ñu. As contribuições da pesquisa em relação à composição química e atividades biológicas dos extratos de A. nutans são raras, com apenas quatro artigos encontrados na literatura. Portanto, o presente estudo avaliou as atividades anti-inflamatória e antinociceptiva da fração hidrometanólica (FHMeOH) utilizando edema de pata induzido por carragenina e testes de placa quente. Além disso, a atividade antioxidante foi avaliada por meio de sequestro de radical DPPH, e foram realizados ensaios de quantificação de fenóis, flavonoides e taninos totais e quantificação dos principais metabólitos por CL-EM. O tratamento oral com a FHMeOH (na dose de 300 mg.kg-1) reduziu significativamente o edema da pata 2 e 4 h após o estímulo inflamatório. Por outro lado, o tratamento intraperitoneal (i.p.) com FHMeOH (50 e 100 mg.kg-1) provou ser mais eficaz e a inibição da inflamação aguda foi ainda visível 6 horas após a injeção de carragenina. Nas doses de 50 e 100 mg.kg-1 (i.p.), FHMeOH exibiu efeitos antinociceptivos centrais aumentando a latência da reação no modelo de placa quente. FHMeOH apresentou potencial antioxidante e os metabólitos quercetina-3-O-galactosídeo, quercetina-3-O-glicosídeo, isoramnetina-3-O-galactosídeo, quercetina- 3-O-β-D-apiofuranosil-(1 → 2)-galactopiranosídeo e ácido clorogênico foram identificados e quantificados por CL-EM. Nossos resultados indicam, pela primeira vez, que o FHMeOH possui efeitos anti-inflamatórios, antinociceptivos e antioxidantes, sendo uma fonte promissora de estudos para novos medicamentos fitoterápicos. PALAVRAS-CHAVE: Annona nutans. Flavonóides. Anti-inflamatório. DPPH. Antinociceptiva. Quantificação por CLAE REFERENCES ALMEIDA, J. R. G. DA S.; ARAÚJO, E. C. DA C.; RIBEIRO, L. A. DE A.; LIMA, J. T. DE; NUNES, X. P.; CARNEIRO LÚCIO, A. S. S.; AGRA, M. DE F.; BARBOSA FILHO, J. M. Antinociceptive Activity of Ethanol Extract from Duguetia chrysocarpa Maas (Annonaceae). 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