In vivo evaluation of analgesic and anti-inflammatory activity of hydroalcoholic extracts from Handroanthus impetiginosus and their chemical composition by UPLC/MS analysis

Abstract This study demonstrates in vivo analgesic and anti-inflammatory properties of hydroalcoholic extracts of leaves, bark and flowers from the Handroanthus impetiginosus (Bignoniaceae) plant, recognized as ‘Ipê roxo’ in Brazil. The extracts were evaluated in male Swiss albino mice via oral administration. Moreover, results of the in vivo paw oedema test induced by carrageenan revealed that extracts of leaves and bark displayed relevant anti-inflammatory activity potential at the dosage of 100 mg/kg, 300 mg/kg, and 500 mg/kg. Likewise, the results obtained for leaves and flowers extracts suggested potent analgesic action in the conventional hot plate test. UPLC/MS analysis of the hydroalcoholic extracts samples identified metabolites belonging to several classes, mainly naphthoquinones and iridoids derivatives as well as flavonoids. Thus, the obtained results indicate that the extracts of H. impetiginosus plant parts could be considered as a complementary herbal medicine for the treatment of pain and inflammation disorders. Graphical Abstract


Introduction
Pain is defined as an unpleasant sensory and emotional experience associated with potential or actual tissue damage (Ismail et al. 2021;Shimoji and Kurokawa 2021). The discovery of drugs that prevent pain, established the first step for medicine to advance in the modern era. Without anesthetics, the development of surgery would be inconceivable. Anaesthesia paved the way for the development of several surgical modalities, which also made it possible to treat and cure some of the most serious and fatal diseases (Ismail et al. 2021;Shimoji and Kurokawa 2021).
The occasional use of analgesics for the relief of severe pain is important, especially with pain such as nephrotic colic or toothache. However, it requires moderately constant supplies of analgesics and anti-inflammatories to sustain the relief of painful conditions. In these circumstances, patients still may experience some undesirable effects of these drugs (Ismail et al. 2021;Shimoji and Kurokawa 2021). Certain high-risk groups are more sensitive and prone to adverse reactions, such as young children, pregnant women, the elderly, the chronically ill or those with genetic abnormalities. The choice of analgesic demands careful consideration, especially when treating chronic and acute pain under conditions that does not threaten health (Besson and Chaouch 1987;Shimoji and Kurokawa 2021;Revand and Singh 2021). Thus, researching new natural products sources with analgesic and anti-inflammatory activity are relevant to increase the therapeutic arsenal of drugs for treating pain conditions. Therefore, the plants with anti-inflammatory and analgesic activity are widely accepted and used in folk medicine, and thus, it is necessary to intensify research and studies with plants based on ethnopharmacological reports (Machado et al. 2003). Certain plants are popularly used to reduce pain and inflammation, while the treatment can be local, in the form of poultices, or oral use in the form of infusion and hydroalcoholic extract (Zhang et al. 2020).
In this context, the Handroanthus impetiginosus popularly known as 'Ipê roxo' in Brazil, it is used in folk medicine to treat inflammation, toothache, joint pain, besides other medical conditions (Almeida et al. 1990;Zhang et al. 2020). In addition, the biological activities reported in the literature generally regards its main active principles, such as the naphthoquinone lapachol (2-hydroxy-3-(3-methyl.butenyl)-1-4-naphthoquinone), which has several biological activities reported, including analgesic and anti-inflammatory properties (de Miranda et al. 2001). Thus, herein we bring the possibility of using H. impetiginosus extracts as a potential complementary treatment for inflammation and pain.

Anti-inflammatory effects
In the search of the analgesic and anti-inflammatory effects of the hydroalcoholic extracts (HE) of the bark, leaves and flowers of H. impetiginosus, experiments were planned to best express the mechanisms of action of these extracts when compared with standard substances, which the mechanisms of action are known and widely proven. Moreover, herein the model used was the carrageenan-induced paw oedema test. This model involves the release of several mediators that induce an inflammatory reaction in two distinct phases: the initial and the late phases. The former occurs from zero to three hours after the injection of the phlogistic agent, and by the action of histamine, serotonin (5HT) and bradykinin (Di Rosa et al. 1971;Kassuya et al. 2021 Revand andSingh 2021). The late phases, depends on the production of tissue prostaglandins (Di Rosa et al. 1971;Kassuya et al. 2021).
Our results evidenced that the three evaluated doses of H. impetiginosus flowers showed little or almost no anti-inflammatory activity ( Figure S1). Although, a certain dose-dependence relationship was observed, suggesting that doses higher than the 500 mg/kg used in the experiment might display an anti-inflammatory effect. On the other hand, it was observed that the HE of bark and leaves of H. impetiginosus, in the three evaluated doses showed a significant reduction in paw oedema, when compared to indomethacin positive control ( Figure S1).
Thus, the obtained results suggest that both HE extracts of bark and leaves of H. impetiginosus could inhibit different inflammation mediators. Likewise, Liu et al. (2003) demonstrated the anti-oedematogenic effect of an enriched fraction of b-lapachol obtained from H. impetiginosus bark in acute lung oedema induced by E. coli lipoproteins. The authors' demonstrated the action of b-lapachol as a histamine antagonist, by inhibiting the action of cytokines, such as the TNF-a.

Analgesic activity
For the evaluation of the antinociceptive potential (central and peripheral analgesic activities) biological assays were performed, and the results are demonstrated in Figures S2 and S3. The test was performed using the hot plate to evaluate centrally acting acute pain. The results demonstrated that the HE of leaves from H. impetiginosus at all tested doses did not increase the latency time of mice, compared to the analgesic effects of opioids ( Figure S2). The results obtained with the different doses of the HE of bark and flowers exhibited a discreet central analgesic action. This action was slightly more intense in the HE of flowers when compared to the HE of bark.
Moreover, the HE of flowers showed slight dose-dependence at the concentration of 500 mg/kg, with results that were still incipient, however relatively stable from the first hour of the experiment ( Figure S2).
In addition, by evaluating the results of the acetic acid-induced contortion test for the HE of bark, a reduction in the contortion number was observed. Despite that, the number of contortions still was higher when compared to the positive control group (indomethacin) ( Figure S3). These results are close to those previously obtained by de Miranda et al. (2001), which performed analgesic assays with the aqueous extract of H. impetiginosus, and herein crude extract samples were hydroethanolic.
The results obtained for HE leaves and flowers of H. impetiginosus demonstrated potent analgesic action at the concentrations of 100, 300 and 500 mg/kg ( Figure S3). Subsequently, the evaluated doses were reduced to 20, 40 and 60 mg/kg ( Figure S3). A marked analgesic effect still was obtained for both extracts, with greater intensity when flower extract was applied. Such results lead us to believe that the antinociceptive effect might be similar to mechanisms of actions described for analgesic and antiinflammatory substances. As is the case with aspirin and indomethacin and other, whose analgesic effects are due to the inhibition of prostaglandin synthesis and by the decrease in the sensitivity of peripheral nociceptive receptors (Berkenkoff and Weichgman 1988;Ferreira 1978;Tiwary et al. 2021;Yemitan and Adeyemi 2017). This effect may be related to the inhibition of the cyclooxygenase enzymes, which catalyzes the conversion of arachidonic acid into endoperoxides. Endoperoxides provide intermediate substrates for tissue synthesis of a variety of biologically active prostaglandins (Deraedt et al. 1980;Barung et al. 2021).

Metabolic profile and dereplication
Satisfactory separation of the peaks from the extracts was obtained in the UPLC-ESI-qTOF chromatograms of H. impetiginosus crude extract samples. The data treatment allowed the detection of 1336 peaks (m/z and retention time -ID 1 to 1100) counting for both identification modes. The UPLC-ESI-qTOF analyses indicated a clear difference in the chemical composition of leaves, bark, seeds, flowers, and fruits from H. impetiginosus plant. The dereplication has evidenced that the most often class of metabolites present in the samples are flavonoids, triterpenoids, iridoid glycosides, hydroxyanthraquinones, naphtofurans, naphthoquinones, coumaric acids derivatives, lignans and phenolic compounds (Tables S1 and S2). In addition, lapachol and several naphthoquinone derivatives were annotated in the samples. Even though these metabolites were not isolated and individually tested in the in vivo experiments, the dereplication of all classes of compounds present in the H. impetiginosus crude extract may speculate the responsible metabolite classes for the observed bioactivities of the extracts, especially those present in larger amounts, such as O-methylated flavonoids, iridoid O-glycosides and naphthoquinones.
Several articles have discussed the effect of lapachol, b-lapachone and derivatives on inflammatory diseases. The results showed positive anti-inflammatory effects in various diseases such as pancreatitis (Mokarizadeh et al. 2020). However, analgesia studies with these naphthoquinone derivatives and extracts rich in these substances are few and limited. In addition, the lack of clinical trials in patients is one of the main points to research. Therefore, the present study sought to provide researchers with a perspective to design new studies using H. impetiginosus extracts. There are many therapeutic challenges associated with using naphthoquinone-rich extracts used against inflammatory diseases. We thus tried to elucidate the benefits of its consumption in these diseases in a rodent model. One of the essential characteristics of extracts with lapachol, b-lapachone and derivatives is probably the absence of side effects due to the natural nature of this substance, which can be of great help in the treatment of inflammatory diseases. In addition, studies indicate that a and b-lapachone increase wound healing rate by elevating Sirt3 expression, indicating the involvement of Sirt family proteins in the healing process of skin wounds and skin regeneration (Ahmad et al. 2020). However, using this material as herbal medicine (H. impetiginosus extract) in patients, needs further studies.

Conclusions
The HE of bark and leaves of H. impetiginosus showed antiedematogenic activity at the evaluated doses. Oppositely, anti-inflammatory activity was not observed in the flowers extract. For the results of centrally acting analgesic, none of the three extracts tested showed significant changes in latency time. For the evaluation of peripheral analgesia, the bark extract showed effects similar to those obtained with indomethacin, which was the positive control, whereas the extracts of flowers and leaves showed an interesting analgesic effect, even with reduced dosages. In addition, the UPLC/MS characterization of the chemical composition of the extracts revealed the presence of lapachol and naphthoquinone derivatives. As well, there are studies in the literature that carried out analgesia assays using enriched lapachol fractions from H. impetiginosus and obtained promissory results, and thus corroborating the analgesic and anti-inflammatory effects observed by our present study. Finally, the H. impetiginosus is widely used for pain relief purposes in folk medicine, and the results obtained in this research are extremely important to support the scientific knowledge related to H. impetiginosus and its potential as a medicinal plant to aid the treatment of inflammatory diseases and pain conditions.

Disclosure statement
No potential conflict of interest was reported by the authors.

Funding
The authors thank the Daniel Luiz Montagnini and to National Council for Science and Technology (Conselho Nacional de Desenvolvimento Cient ıfico e Tecnol ogico-CNPq) and Coordination for the Improvement of Higher Education Personnel (Coordenação de Aperfeic¸oamento de Pessoal de N ıvel Superior-CAPES) for financial support and the researchers from Natural Products and Organic Synthesis Research Group (GEAPS-CNPq-UFES).