Dereplication of polar extracts from Croton antisyphiliticus Mart. roots and its anti-inflammatory and antinociceptive potential

Abstract Croton antisyphiliticus Mart. is a plant popularly used in folk medicine by traditional communities from Brazilian savannah to treat general inflammation. According to ethnopharmacological data, this specie can be considered a source of biologically active molecules for the development of new drugs. Thereby, this study reports the results of the dereplication approach of C. antisyphiliticus roots extracts and the in vivo evaluation of its potential antinociceptive and anti-inflammatory in albino Swiss mice. Based on HPLC coupled to Q-Exactive Orbitrap Mass Spectrometer and using GNPS, a total of thirteen polyphenolic compounds were noticed, including four compounds that have been reported for the first time in the genus Croton. Ethanolic and aqueous roots extracts demonstrated a dose-dependent inhibition for the number of writes, reduced pain induced by formalin and hyperalgesia induced by carrageenan. These extracts also reduced paw edema, cell migration, and myeloperoxidase activity, with effects similar to indomethacin and dexamethasone drugs. Graphical Abstract

physical sciences; chemistry; natural products 1 Introduction the genus Croton belongs to the Euphorbiaceae family, which comprises about 1300 species distributed around the tropic regions of the world, of which, in Brazil is found around 252 endemic species (ndhlala et al. 2013).Several reports describe the biological potential and classes of compounds with medicinal effects, such as diterpenoids, alkaloids and flavonoids for this genus (Xu et al. 2018). in an ethnobotanical study carried out by Gomides et al. (2018), C. antisyphiliticus mart.species, popularly known as 'pé-de-perdiz' , was indicated for the treatment of the flu and general infection.Furthermore, decoctions and roots and leaves extracts are used for colic, rheumatism, syphilis, ulcers, and inflammation, especially in the genital tract (Reis et al. 2014).
in this work, it is showed a chromatography profile of two extracts of C. antisyphiliticus.therefore, this study presents the dereplication of the ethanolic (EE) and aqueous (aE) root extracts of C. antisyphiliticus by High performance Liquid Chromatography coupled to High Resolution tandem mass Spectrometry (HpLC-mS/ mS) employing Featured Based molecular networking (FBmn).Furthermore, the anti-inflammatory and antinociceptive effects of theses extracts were evaluated in albino Swiss mice.

Identification of phenolic compounds from C. antisyphiliticus
diterpenoid is the main chemical class reported for Croton roots (Xu et al. 2018).However, in this study the chemical investigation of EE and Ea roots extracts, by FBmn technique, showed a different perspective, with compounds mainly related to polyphenols.Furthermore, molnetEnhancer tool revealed an overview of the chemical classes.thereby, five subclasses of polyphenol molecules were notices from the samples, including benzoic acids and their derivatives, flavans, flavones, flavonoids glycosides, and hydrocinnamic acids and their derivatives.previous studies presented a wide diversity of polyphenol molecules in leaves (Sun et al. 2014), stems (Zou et al. 2010) and, roots (ndunda et al. 2015) of Croton species.a total of 188 nodes were organized in different spectral families, with 63 nodes without a correlation mass spectrum to each other.thirteen metabolites were perceived from C. antisyphiliticus roots extracts (supplemental table S1).Compounds annotation from FBmn was confirmed by standard, evaluated under identical experimental HpLC-mS/mS conditions, comparing the precursor mass, retention time, and main fragments, of which being classified as Level 1 in mSi level annotation (Sumner et al. 2007).

Anti-inflammatory activity of C. antisyphilicus roots extracts
EE and aE demonstrated a dose-dependent effect with maximum response using the dosage of 250 mg/kg of the EE and 1200 mg/kg of the aE. the anti-inflammatory effect was evidence by paw edema, pleurisy, and polymorphonuclear leucocyte-cell migration after carrageenan injection.
according to supplemental Figure S1-a, EE (250 mg/kg) and aE (1000 mg/kg) shown antiedematogenic effect in the four hours tested, when compared to control group.at 1h after injection of carrageenan, EE and aE caused suppression of paw edema formation (20%, 26%, 21% and 32% to EE, and 38%, 43%, 41% and 38% to aE).dexamethasone (1 mg/kg) showed pronounced significant reduction of paw edema, presenting a superior inhibition activity on carrageenan-induced paw edema assay than C. antisyphiliticus roots extracts.However, the paw edema reduction promoted by EE and aE might be related to the reduction of endogenous mediators, such as prostaglandins (pGE2 and pGE2α), serotonin, histamine (Rege et al. 2021), consequently reducing cell migration which inhibits edema formation.
in pleurisy induced by carrageenan model, EE (250 mg/kg) and aE (1000 mg/kg) promoted reduction of total leucocyte-cell migration to pleural cavity by 64% and 51%, respectively, when compared to control group (supplemental Figure S1-B).Whereas, dexamethasone (1 mg/kg), an agent used as a standard drug, showed strong inhibition of total leucocyte-cells migration (supplemental Figure S1-B).Furthermore, in a differential counting of leucocyte-cell mononuclear (mono) and polymorphonuclear (pmn), EE and aE reduced mainly of pmn, which are cells characteristics of acute inflammation, by 78% and 62%, respectively, in which dexamethasone produced suppression of pmn and mono by 73% and 22%, respectively (supplemental Figure S1-C).
Similarly, EE (250 mg/kg) and aE (1000 mg/kg) reduced myeloperoxidase enzyme activity, which is predominantly present in active leucocyte cells, whose levels are directly proportional to the cells concentration in inflamed tissue, confirming the result of the total/differential count of cells (supplemental Figure S1-d).thus, EE and aE might reduce myeloperoxidase enzyme activity, which is predominantly present in active neutrophils, obtaining levels directly related to the neutrophil concentration in inflamed tissue (odobasic et al. 2016).Considering the importance of these cells to inflammatory response, the antiedematogenic effects of EE and aE might be presumed to ability to inhibit the release of inflammatory mediators in the mice organisms.

Antinociceptive activity of C. antisyphilicus roots extracts
acetic acid-induced abdominal writhing, formalin, tail-flick tests and, mechanical hyperalgesia evaluation were carried out in vivo antinociceptive activity.
oral treatment with EE (250 mg/kg) and aE (1200 mg/kg) in acetic acid-induced writhing model decreased the number of writhes when compared to the control group.though, EE and aE doses showed an efficient antinociceptive response was reduced by 54% (supplemental Figure S2-a) and 44% (supplemental Figure S2-B) the number of writhes, respectively, when compared to nonsteroidal indomethacin drug (10 mg/kg) by 47% and, 42%, respectively (Figure S2).tail-Flick assay was employed to assess the thermal stimulus effects of C. antisyphiliticus roots extracts, specifically evaluating their impact on central mechanism.EE (250 mg/kg) and aE (1000 mg/kg) treatment did not increase latency by thermal stimulation in the measurement intervals when compared to control group.However, morphine, a known antinociceptive agent used as a reference drug, significantly prolonged the latency, and exhibited strong anti-nociceptive properties (supplemental Figure S3).these results corroborate with the nociceptive effects found in the abdominal writhing induced by acetic-acid model, suggesting an antinociceptive effect of both C. antisyphiliticus roots extracts showing a peripheral nociceptive response.therefore, C. antisyphiliticus roots extracts can be capable of blocking nociceptive receptor binding or blocking the release of endogenous mediators that excite pain nerve endings (Rege et al. 2021).
aiming further investigation of C. antisyphiliticus roots extracts nociception properties, reduction of paw licking time by formalin-induced test was performed.EE (250 mg/kg) and aE (1000 mg/kg) were able to reduce the time of animal took licking injected paws in neurogenic (Figure S4-a, 37% and 34% respectively) and inflammatory (Figure S4-B, 31% and 56% respectively) phase, when compared to control group.indomethacin (10 mg/kg) decreased the licking time only in the inflammatory phase, where EE showed effect comparable to indomethacin, while aE presented as more efficient in both phases.in contrast, administration of morphine demonstrated a significant antinociceptive response in both phases (95% and 99%, respectively).mechanical test was employed to evaluate the sensitivity to increased nociceptors by hyperalgesia stimuli.EE (250 mg/kg) and aE (1000 mg/kg) treatments reduced the intensity of hyper-nociception inflammatory between the paws (uninflamed and inflamed) in all hours of the test when compared with the control group (supplemental Figure S5).Furthermore, EE and aE doses showed antinociception responses comparable to indomethacin.thus, C. antisyphiliticus roots extract might be capable of decreasing prostaglandin levels and directly acting on nociceptive sensitization in inflammatory pain.

Conclusion
the use of a mass spectrometry-based molecular networking as dereplication tool enabled the annotation of 13 phenolic compounds, assigned in five metabolites subclasses.though several phenolic compounds were reported previously for this genus, herein the compounds 4-hydroxycinnamic, protocatechuic, gentisic acids, and naringenin are described for the first time within the many Croton species.additionally, administration of EE and aE are associated with the reduction of paw edema and pmn leucocyte-cell migration inhibition, and these activities may contribute to peripheral antinociception responses during inflammatory pain.therefore, our findings corroborate the traditional uses of C. antisyphiliticus, that provide a scientific basis for the species' ethnopharmacology usage for alleviating pain and treating inflammatory disorders.

Disclosure statement
no potential conflict of interest was reported by the author(s).