Chemical composition, anti-inflammatory and antinociceptive effects of the butanolic fraction of Annona nutans (Annonaceae) leaves

Abstract The species Annona nutans (R. E. Fries) is a plant found in Bolivia, Paraguay, Argentina and the Brazilian cerrado. Considering the anti-inflammatory and antinociceptive activities of the hydrometanolic fraction (FHMeOH) of A. nutans leaves previously reported, the present study aimed to evaluate in vivo anti-inflammatory and antinociceptive activities of a subfraction obtained from FHMeOH, the butanolic fraction (FBuOHf). Intraperitoneal (i.p.) treatment with FBuOHf (50 and 100 mg · kg−1) inhibited paw edema induced by carrageenan. Moreover, FBuOHf (100 mg · kg−1, i.p.) also suppressed polymorphonuclear (PMN) leukocyte migration in the footpad. Regarding the antinociceptive activity, FBuOHf (50, 100, and 200 mg · kg−1, i.p.) inhibited acetic acid-induced abdominal writhing. In the formalin test, this fraction (200 mg · kg−1, i.p.) reduced licking time only in the inflammatory phase. The FBuOHf contents flavonoids and cinnamic acid derivatives, such as quercetin-3-O-galactoside, quercetin-3-O-glucoside, isorhamnetin-3-O-galactoside, quercetin-3-O-β-D-apio-furanosyl-(1→2)-galactopyranoside and chlorogenic acid, identified and quantified by LC-MS. The FBuOHf possesses anti-inflammatory and peripheral antinociceptive activities. Graphical Abstract


Introduction
The search for medicinal plants with analgesic and anti-inflammatory activity, for example, has been labeled as a promising source of new molecules and mechanisms with therapeutic potential (Murugesan and Deviponnuswamy 2014). Due to the complexity of the inflammatory process, and the side effects caused by long-term treatment of nonsteroidal (NSAIDs) and steroidal (glucocorticoid) antiinflammatory drugs, such as gastrointestinal damage and cardiovascular problems (Medzhitov 2010) there is a need to search for new compounds with potential therapeutic effects on acute or chronic inflammatory diseases, and natural products became a source of search of new drugs for the health care (Gonz alez et al. 2015).
Annona nutans it is a plant of the Brazilian cerrado commonly known as Aratico, and Araticû-Ñu. (Silva et al. 2015). Few chemical data were available in the literature, which described the isolation of acetogenin and cohibins from the bark (Gleye et al. 2000), and polyketide derivatives, flavonoids and cinnamic acid derivatives from the leaves (Silva et al. 2015). To date, only a few scientific studies have evaluated the chemical composition of A. nutans and concern the biological activities. In this continuous search for active natural products, it has reported that the hydromethanolic fraction (FHMeOH) of A. nutans leaves have anti-inflammatory and antinociceptive properties in acute local inflammation (Silva et al. 2019).
Since A. nutans leaves show anti-inflammatory and antinociceptive activities, the present study aimed to identify and quantify the chemical composition by ultra-fast liquid chromatography with an electrospray ion source and evaluate the anti-inflammatory and antinociceptive effects of the butanolic fraction obtained by the liquid-liquid partition, from FHMEOH of A. nutans leaves.

Results and discussion
Twenty-one compounds were detected from FBuOH f , and identified based on the retention time values in UFLC analysis, combined with UV spectra, MS and MS/MS fragment data, by comparing the spectral data reported for them in the literature (Clifford et al. 2003;Liu et al. 2018). The compounds identified are present in Table S1.
In the quantification of flavonoids and chlorogenic acid, the calibration plots indicated a selective and linear method, with the regression coefficients, were higher than 0.99 in all cases. The LOD was 4.88 Â 10 À2 mg.mL À1 for the flavonoids and 9.77. 10 À2 mg.mL À1 for the chlorogenic acid. The relative standard deviations (%RSD) were in the range of less than 5%. The estimated content of metabolites in FBuOH f was 8.90 The chemical components are enriched in the FBuOH f , comparing it with the FHMeOH, which exhibited the 3.04 lg Á mg À1 , 6.74 lg Á mg À1 , 3.72 lg Á mg À1 , 2.12 lg Á mg À1 and 4.86 lg Á mg À1 , respectively (Silva et al. 2019).
The anti-inflammatory effect was evidenced by inhibition of paw edema and polymorphonuclear (PMN) leukocyte migration after carrageenan injection. Treatment with the FBuOH f (25, 50 and 100 mg Á kg À1 ) showed an antiedematogenic effect at all hours ( Figure S3). The carrageenan-induced mice paw edema is a biphasic model of acute inflammation. An initial phase (0-1 h) is mediated by the release of histamine, serotonin, and bradykinins, while the later phase (2, 4 and 6 h) has been linked to an overproduction of prostaglandins and PMN leukocyte infiltration (Cuzzocrea et al. 1998). FBuOH f showed a paw edema inhibitory effect more pronounced in the later phase, presenting effects similar to those found for NSAIDs drugs (Burke et al. 2006).
According to Figure S4A, rare PMN leukocytes and potential resident immune cells were observed in the footpad of the naive group. By contrast, the histopathological results of control group footpads ( Figure S4B) showed in the extensive dermis extravasation of mainly PMN leukocytes, which are cells characteristic of acute inflammation. Treatment with 100 mg Á kg À1 FBuOH f ( Figure S4C) reduced the tissue injury 4 h after carrageenan stimulus, being observed a notable inhibition of PMN leukocyte recruitment, similar to that promoted by the indomethacin group (10 mg Á kg À1 ) ( Figure S4D). The histological analysis, FBuOH f promoted a decrease in PMN leukocyte migration in comparison to the control group, 4 h after carrageenan stimuli, and its action was verified up to 6 hours after induction of inflammation.
The results depicted in Figure S5 show FBuOH f caused significant inhibition (p < 0.001) of the nociception induced by acetic acid. The reduction of the abdominal writhing was observed at doses of 50, 100, and 200 mg Á kg À1 by 80.35, 84.85, and 86.49%, respectively. Interestingly, standard drug Ind (10 mg Á kg À1 ) produced inhibition (p < 0.001) of the abdominal writhing by 71.75%.
As shown in figure S6, in the formalin-induced nociception, FBuOH f at a dose of 200 mg Á kg À1 significantly (p < 0.01) reduced licking time by 61.54% in the second phase (inflammatory phase). Regarding the antinociceptive effect, the abdominal writhing induced by acetic acid was used as a model (Le Bars et al. 2001). The administration of acetic acid promotes a local irritation that leads to an increase in the production of histamine, serotonin, bradykinin, cytokines, eicosanoids and other mediators in the peritoneal fluid (Deraedt et al. 1980). The formalin test, FBuOH f was able to inhibit the licking time only in the inflammatory phase, showing a peripheral antinociceptive action that is probably linked, at least partly, to the anti-inflammatory effect observed in the carrageenan-induced paw edema model.
We observed a huge enrichment of flavonoids and cinnamic derivatives such as 4-O-E-caffeoylquinic acid, quercetin-3-O-b-galactopyranoside, quercetin-3-O-b-glucopyranoside in BuOH f , that may be responsible for the inhibition of the inflammation and nociception by different mechanisms of action. We emphasize chlorogenic acid, such as 4-O-E-caffeoylquinic acid, with an increase of almost three times and flavonoid heteroside content that now represents at least 25% of the fraction (against approximately 2% in the FHMeOH). There is a description in the literature of these metabolites to have anti-inflammatory activity, especially 4-O-E-caffeoylquinic acid and quercetin-3-O-b-galactopyranoside, which act by decreasing the secretion of proinflammatory cytokines IL-8 and IL-6, reduced the neutrophil migration (Hebeda et al. 2011). Studies suggest that the flavonoid quercetin-3-O-b-galactopyranoside inhibited pro-inflammatory cytokines including IL-1b and TNF-a, as well as the expression of inducible nitric oxide synthase, attenuated the inflammatory responses via p38 and NFjB pathways (Fan et al. 2017).
They also may act, for instance, by attenuating adhesion and migration of CD8 cells, inhibition of lymphocyte proliferation, inhibiting prostaglandin synthesis, neutrophil degranulation, histamine release, phosphodiesterases and protein kinases activities (L€ attig et al. 2007;Coutinho et al. 2009;Rathee et al. 2009). Studies demonstrated that flavonoids play an important role in the inhibitory action of LTB4 formation. This inflammatory mediator exerts chemotactic activity for leukocytes, eosinophils, and monocytes, promoting the migration of these inflammatory cells to the affected site, which degranulate and produce superoxides (Loke et al. 2008).

Conclusion
The present study showed for the first time that the FBuOH f from A. nutans leaves have anti-inflammatory and peripheral antinociceptive effects. Furthermore, the antiinflammatory activity may be linked to inhibition of paw edema and PMN leukocyte recruitment, and its activity may contribute to peripheral antinociception promoted by FBuOH f . It is plausible that these effects are due to the enrichment of phenolic compounds such as flavonoids and chlorogenic acids identified in this fraction.

Disclosure statement
The authors declare no conflict of interest.

Funding
This work was supported by the Coordenac¸ão de Aperfeic¸oamento de Pessoal de N ıvel Superior (CAPES) under the number 2833-2011. NLS is grateful to Fundação de Amparo a Pesquisa do Estado de Minas Gerais (FAPEMIG) and AAS to the Federal University of São João del Rei for a postgraduate fellowship.