Dereplication of Siparuna brasiliensis extract with in vivo anti-inflammatory activity

Abstract Siparuna brasiliensis is a medicinal plant widely used by indigenous communities of the Amazon rainforest to treat inflammatory diseases and related pathologies. Considering its ethnopharmacological application, it constitutes an important source of biologically active molecules in the development of anti-inflammatory drugs. This study describes a dereplication methodology of the bioactive extract from S. brasiliensis leaves and the evaluation of the anti-inflammatory potential in an in vivo inflammatory model with mice of the BALB/c lineage and in vitro using cell lines, as well as determining the production of an inflammatory mediator. From their charge-to-mass ratios (m/z) and elemental composition obtained through Ultrahigh-resolution mass spectrometry analysis by ESI(-)-Orbitrap MS and chromatographic profile by RP-HPLC-PDA, it was possible to annotate polyphenols with anti-inflammatory properties classified as flavonoids and organic acids. The administration of the extract significantly inhibited carrageenan-induced paw edema and showed effects similar to those of drug dexamethasone without affecting cell viability. Graphical Abstract


Introduction
Siparuna brasiliensis Spreng., known as 'negramina', is widely distributed in Brazil and frequent in some regions of occurrence. It is used as a medicinal plant, including by indigenous from Amazon rainforest, to treat inflammatory diseases, hypertension, fungal and viral infections, cutaneous and intestinal ulcers (Carmona and Guarim 2001;Renner and Hausner 2005;Souza and Felfili 2006;Valentini 2010;Conegundes et al. 2021).
Dereplication approach combined with liquid chromatography-tandem mass spectrometry have been applied and recognized as a strategy of choice in exploring the chemical diversity of medicinal plants since it allows the rapid identification of biologically active molecules and the efficient analysis of complex matrices (Sharma et al. 2021;Kumar et al. 2022). Few studies were found in the literature showing the antiinflammatory potential of S. brasiliensis. Hence, this study describes the in vitro and in vivo anti-inflammatory assessment of S. brasiliensis leaves aqueous extract, and the application of chromatography associated with Ultrahigh-resolution mass spectrometry in the metabolic analysis of the bioactive extract.

Dereplication of the bioactive extract by RP-HPLC-PDA and MS analysis
The chemical structure annotation of all metabolites was performed by HPLC-PDA ( Figure S1) and MS ( Figure S2) analysis through evaluations from UV spectrum, Massto-Charge ratio (m/z) and elemental composition obtained through Orbitrap mass analyzer, and using online dereplication platforms (MetFrag, PubChem and CFM-ID). The chromatographic profile of S. brasiliensis revealed a large production of polyphenols, mainly flavonoids and organic acids. Fourteen polyphenols classified as flavonoids and organic acids through UV spectrum and retention time analysis were detected. Most of the annotated metabolites were flavonoids ( Figure S1).
Concerning the metabolic profiling assessment employing mass spectrometry and chromatographic analysis, the compounds 1 and 2 were annotated as organic acids. The compound 1 (Rt ¼ 16.47 min) showed bands in the UV spectrum with absorption peaks at k 232 and 282 nm, whereas the compound 2 (Rt ¼ 17.52 min) showed bands in the UV spectrum with absorption peaks at k 232 and 282 nm. The analysis of their respective mass spectra showed the molecular ions of m/z 203.05250 [M þ H] of empirical formula C 8 H 11 O 6 , and m/z 219.02640 [M þ H] of empirical formula C 11 H 7 O 5 , which were annotated as derivative molecules from ulopyranosonic acid and oxochromene-3-carboxylic acid, respectively. Through spectrometric and chromatographic profiling, the compounds 3-10 were annotated as metabolites belonging to flavonoids and organic acids class. The flavonoids showed UV spectra with absorption peaks ranging from k 264-266 and 342-350 nm compatible with flavonoid structure, and they presented very similar retention times (retention times between 24 and 31 min) in the chromatographic analysis. From their mass-to-charge ratio (m/z), and elemental composition obtained through ultrahigh-resolution mass spectrometry analysis by ESI(þ)-Orbitrap MS analysis, it was possible to infer the chemical class as organic acid of the

In vitro and in vivo anti-inflammatory activity assessment of siparuna brasiliensis
The cell viability of the leaves aqueous extract of S. brasiliensis was evaluated in J774A.1 and RAW 264.7 macrophage lines by the MTT method at concentrations of 200, 100, 50, and 10 mg mL À1 (Figure S3A and S3B). The in vitro anti-inflammatory potential was evaluated through the production of the inflammatory mediator nitric oxide ( Figure S3C and S3D) in J774A.1 and RAW 264.7 macrophage lines by the Griess method at concentrations of 200, 100, 50, and 10 mg mL À1 . Nitric oxide (NO) is an inflammatory mediator with pro-inflammatory properties and controlling excessive NO production is important to the development of anti-inflammatory drugs ( Subedi et al. 2021). The S. brasiliensis extract reduced the production of NO in the highest concentrations, mainly in the J774A.1 cells, reaching a 50% of reduction ( Figure S3C and S3D). Therefore, phytochemicals that inhibit the synthesis of inflammatory mediators may be promising candidates for the development of anti-inflammatory drugs (Yunes and Calixto 2001).
Regarding the in vivo experiments, we applied experimental models using carrageenan as an inducer product of inflammation. In this work, we evaluated the effects of the administration of leaves aqueous extract of S. brasiliensis (500 mg kg À1 ) and dexamethasone on mouse paw edema. The administration of polyphenolic-rich extract of S. brasiliensis significantly reduced carrageenan-induced paw edema when compared to the positive control group, at 4 hours after the injection of carrageenan ( Figure S4). When the production of inflammatory mediators in the mouse footpad was examined, an increase in IL-10 was observed ( Figure S5), but non-significant alterations in the others cytokines, IL-1b, IL-6, IL-12, IFN-c and TNF-a (data not shown). Previous studies have already demonstrated that flavonoids present potential to modulate inflammatory modulators in murine models and accelerate wound healing (Fitzmaurice et al. 2011;Pereira et al. 2011;Carito et al. 2015). Some annotated metabolites such as gallic acid and kaempferol derivatives are reported in the literature with anti-inflammatory properties (Alam et al. 2020;Bai et al. 2021). The annotated galloyl glucopyranoside derivative showed anti-inflammatory action in monocytes (Bobrowska et al. 2018). However, further studies are necessary to identify the metabolites responsible for the anti-inflammatory activity of S. brasiliensis.

Conclusion
Our results reported that S. brasiliensis presents a large biosynthesis of polyphenols such as flavonoids and phenolic acids, which may be responsible for its anti-inflammatory potential. Hence, S. brasiliensis can be a scientific alternative to obtaining more effective anti-inflammatory compounds and developing of drugs with greater therapeutic efficacy and fewer side effects. The chromatographic techniques associated with mass spectrometry and a robust database have been successfully applied to obtain the molecular profile of complex plant extract. Further work need to be performed on the isolation and identification of the bioactive components.