Dereplication and evaluation of the antinociceptive and anti-inflammatory activity of hydroethanolic extract of leaves from Campomanesia xanthocarpa O. Berg

Abstract Campomanesia xanthocarpa popularly known as gabiroba is used as a medicinal plant for the treatment of inflammatory diseases, ulcers, among other uses, requiring studies to assist in proving these activities. In this study, the extract of leaves from C. xanthocarpa (CxHE) was submitted to assays of formalin-induced paw-licking, peritonitis induced by lipopolysaccharide and carrageenan-induced mechanical hyperalgesia tests. In chemical analysis, a preliminary phytochemical screening and the determination of phenol and flavonoid content were carried out, in addition to analysis by ESI-MS/MS system and HPLC-DAD system. The CxHE presented compounds such as tannins, triterpenoids, steroids and saponins and content of phenols (35.9 ± 1.3 GAE/g extract) and flavonoids (23.3 ± 2.1 mg EQ/g extract). Protocatechuic acid, gallic acid, ethyl gallate, quercetin, and 3-methyl epigallocatechin gallate, alpha and beta-amyrins were identified. CxHE at doses of 10-1000 mg/kg p.o. demonstrated anti-inflammatory and antinociceptive effects in all in vivo assays employed in this study.


Introduction
Campomanesia xanthocarpa O.Berg. (Myrtaceae), popularly known as 'guavirova', 'gabiroba' or 'guabiroba', is a species native to Brazil occurring in the Atlantic forest and in the cerrado (Klafke et al. 2010). The leaves of this plant are used as an infusion in folk medicine to treat gastrointestinal and anti-inflammatory diseases, hypercholesterolemia, besides antiplatelet and muscle relaxant (Alice et al. 1995). Flavonoids, saponins, terpenes and tannins have already been demonstrated in other studies with plants of the Myrtaceae family, as well was observed in the leaves of C. xanthocarpa (Cardozo et al. 2018).
Extracts of the leaves of C. xanthocarpa showed antimutagenic activity to cancer cells in vitro (Fernandes and Vargas 2003), besides antifibrinolytic, antiplatelet and antithrombotic activities (Klafke et al. 2010;Klafke et al. 2012), antiulcerogenic effects (Markman et al. 2004), furthermore showed anti-inflammatory activity in hypercholesterolemic animals (Klafke et al. 2016) and reduced oxidative stress in hypercholesterolemic individuals and improved the levels of nitric oxide (Viecili et al. 2014).
In this study, we evaluated the anti-inflammatory and antinociceptive potential of a hydroethanolic extract of C. xanthocarpa (CxHE) in different animal models of pain and inflammation. Additionally, we studied the chemical composition of the extract by physicochemical, spectrometric and chromatographic techniques, to better discuss the observed results, and contributing to chemical characterization of this species.

Results and discussion
Phenolic compounds are present in a high amount in the CxHE extract (Total phenols ¼ 35.9 ± 1.3 GAE/g; total flavonoids ¼ 23.3 ± 2.1 QE/g). Protocatechuic acid, gallic acid, ethyl gallate, quercetin and epigallocatechin gallate was identified in CxHE by direct infusion electrospray ionization tandem mass spectrometry by comparing the spectral data reported for them in the literature (Su et al. 2013;Chernonosov et al. 2017) in Table S1 (Supplementary material). Additionally, we identified beta-amyrin and alpha-amyrin by HPLC-DAD ( Figure 1).
All doses of CxHE (10, 100 and 1000 mg/Kg) produced a significant antinociceptive activity compared to the control group during the second phase of the formalin test (F5,58 ¼ 4.416; p < 0.01), showing an indomethacin-like effect (Meunier et al. 1998;Masocha et al. 2016), as shown Figure S1. CxHE 1000 mg/kg reduced the formalin-induced nociceptive response at the first phase (F5,58 ¼ 5.673; p < 0.001; Supplementary material Figure S1), such as morphine and morphine-like molecules (Sevostianova et al. 2003). These results show potent anti-inflammatory/antihyperalgesic effect and a possible central action of CxHE (Supplementary material Figure S2). In the mechanical hyperalgesia test, CxHE 100 and 1000 mg/Kg increased the nociceptive mechanical threshold in mice sensitized by carrageenan, reinforcing the anti-hyperalgesic effect previously suggested (Supplementary material Table S2).
CxHE 10, 100 and 1000 mg/kg significantly reduced the cell recruitment (p < 0.05) when compared to the group without pre-treatment in the LPS-induced peritonitis (Supplementary material Figure S2), which is a model for acute inflammation triggered by bacteria (Vilela et al. 2010). Group treated with indomethacin 10 mg/Kg also reduces (p < 0.01) the number of cells in the peritoneal cavity (Supplementary material Figure S2).
Phenolic compounds such as gallic acid and quercetin had already been identified in leaves (Sant'Anna et al. 2017) and ethyl gallate in fruits of C. xanthocarpa, while epigallocatechin gallate was founded in fruits of C. phaea (Azevedo et al. 2017).
Gallic acid and its esters can neutralize reactive oxygen species, which are involved in inflammatory response (Badhani et al. 2015). In the model of carrageenan-induced inflammation, protocatechuic acid reduced the edema and the related granuloma,  (2), ethyl gallate (3), quercetin (4), epigallocatechin gallate (5), beta-amyrin (6) and alpha-amyrin (7). furthermore inhibiting the release of NO and TNF-a by LPS-activated macrophages (Kakkar and Bais 2014). In turn, quercetin can inhibit the inflammation triggered by LPS-induced macrophages, by different mechanisms related to the release of cytokines, as well as inhibiting the expression of iNOS in murine macrophages of different strains, and COX-1 and 5-LOX from dose-dependent manner (Tuñ on et al. 2009;Li et al. 2016). Furthermore, epigallocatechin gallate inhibits the transfection of NF-B and AP-1 to downregulate the expression of iNOS and COX-2 which decreases the production of inflammatory factors (Chu et al. 2017).
The in vivo action of the phenolic compounds depends on their bioavailability, nevertheless, exist evidences that tannins, which were detected in CxHE, are absorbed normally, and larger tannins appear to facilitate absorption of smaller other phenolic compounds, without being absorbed themselves (Neilson et al. 2016). In another hand, alpha and beta-amyrins, which are ubiquitous triterpenes in nature, also present anti-inflammatory and analgesic activities with moderate or low potency (Rokicka and Wojciak-Kosior 2014), thus, may be contributing to the observed effects.

Conclusions
Oral administration of the CxHE produced antinociceptive effects in the first and second phase of the formalin test, in fact strongly relied on concentration, especially for the first phase. It had significant anti-inflammatory action in the peritonitis induced by LPS test, and increased the mechanical threshold in hyperalgesia induced by carrageenan test.