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

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) 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) 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 (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-3O-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


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 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., 2013aFORMAGIO et al., , 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(GLEYE et al., , 1998;;GONÇALVES et al., 2014;SILVA et al., 2015).The acetogenins were found in A. nutans roots (GLEYE et al., 2000(GLEYE et al., , 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.

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).

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).
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 µg of gallic acid equivalents.mg - 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.
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.

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, 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 µL) 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: where Abscontrol = absorbance of DPPH radicals in methanol and Abssample = absorbance of fraction in methanol + DPPH.The scavenging activity is expressed as µg.mL -1 .IC50 values (µg.mL -1 ) were calculated using Probit analysis (FINNEY, 1980).
For the isorhamnetin-3-Ogalactopyranoside, concentrations of 12.5, 25.0, 50.0, 100.0, and 200.0 µg.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 http://dx.doi.org/10.14393/BJ-v35n5a2019-45927attribution 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-tonoise 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).

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).

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 µg.kg - ).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.

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, R 2 = 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 µg.mL -1 presented a scavenging effect on the DPPH radical that was statistically significant compared to the BHT standard.

O-β-D-apiofuranosyl-(1→2)-galactopyranoside)
was identified in studies conducted in our laboratory using H 1 and C 13 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 MS 2 .The compounds identified are listed in Table 1 and illustrated on the chromatogram in Figure 2.

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 - (Figure 5).

DISCUSSION
The UFLC analysis in FHMeOH of A. nutans leaves revealed that quercetin-3-Ogalactopyranoside 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).
A direct relationship among antioxidant activity, phenolic compounds, and antiinflammatory efficacy has been demonstrated in the literature (FORMAGIO et al., 2013a(FORMAGIO et al., , 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 antiinflammatory 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 antiinflammatory 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  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 antiinflammatory 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.

Figure 2 .
Figure 2. Base peak chromatogram obtained in negative and positive ion modes from FHMeOH of A. nutans.

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.

Figure 4 .
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.

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.