Extraction and Identification of Flavonoids from the Leaves of Pilocarpus microphyllus: Focus on Antioxidant Activity and Neuroprotective Profile

Abstract

This work is based on research aiming to extract and identify flavonoids from jaborandi (Pilocarpus microphyllus) leaves and investigate their antioxidant and acute antinociceptive capacity. Characterization of the constituents of the ethyl acetate fraction (EtOAcF) obtained from the methanolic extract (ME) was performed by UV-Vis spectrophotometry, infrared spectroscopy, high-performance liquid chromatography (HPLC), mass spectrometry (MS), and cyclic voltammetry, demonstrating the possible majority component of this fraction, the flavone chrysin. Its solubility properties in HPLC are very close to those of the flavonol quercetin, revealing the characteristic presence of this group. An MS spectrum of the fraction revealed a major protonated molecule of m/z 254.9 [M+H]⁺. The EtOAcF fraction showed three oxidation processes at 0.32 V, 0.54 V, and 0.73 V vs. Ag/AgCl. Three reduction processes at the respective potentials: 0.60 V, −0.03 V, and -0.24 V vs. Ag/AgCl, indicating potential antioxidant activity. At DPPH and ABTS antioxidant radical capture assay, The IC₅₀ obtained was 0.5 mg/mL and 0.81 mg/mL, respectively. In vivo test to determine the mechanical nociceptive threshold in the von Frey test, the dose of 100 mg/kg of the EtOAcF was able to cause inhibition of behavioral changes in neuropathy. The results obtained in this study demonstrate the biological potential of an EtOAcF derived from jaborandi leaves.

1. Introduction

Nowadays, interest in natural products is increasing, but an integrated approach and using networking to investigate natural products represent a great challenge. Medicinal plants are used directly in pharmacological therapy, through the preparation of herbal products as extracts, or are a source of precursors for the synthesis of active principles [1,2,3].

Many current drugs are derived, directly or indirectly, from the chemical constituents of higher plants. Scientific studies on natural products (either the native form or active metabolites) are often guided by ethnobotanical knowledge and can substantially contribute to pharmaceutical innovation, providing new chemical structures and mechanisms of action, mainly after the improvement of extraction methods and the development of bioinformatics tools [4,5,6,7]. Extracts that are industrially obtained must be prepared from plant material with guaranteed quality through a process in which the solvents and extraction methods are standardized, as well as the concentration, drying, and homogenization steps [8,9].

The importance of natural products is particularly evident in research fields dedicated to chronic and degenerative diseases. These ailments are characterized by pathophysiological changes in inflammatory mechanisms, which are associated with imbalances in free radical metabolism [2,10,11,12]. Several studies associate the antioxidant substances derived from plant metabolism, especially phenolic compounds, with a protective effect against diseases such as cancer, cardiovascular and coronary diseases, and diabetes, among others [11,13,14].

The species Pilocarpus microphyllus Stapf ex. Wardeworth, popularly known as jaborandi, is heavily exploited as a source of the alkaloid pilocarpine. A shrub-type species with a preference for those climates that are typical of the north/northeast regions (hot and humid), it is endemic to Brazil, which is the only pilocarpine-producing country. Moreover, other groups of secondary metabolites, such as coumarins, flavonoids, and terpenes, have also been reported for Pilocarpus species that are native to Brazil [4,8,15,16].

Flavonoids have been highlighted by researchers due to their wide range of biological and therapeutic actions, which are demonstrated both in experimental conditions and in humans, including anti-inflammatory activity, antimicrobial activity, enzymatic inhibition associated with COVID-19, antiallergic activity, antioxidant activity, neurodegenerative pathology, and antitumor activity [17,18,19,20,21].

This large class of secondary metabolites is widely distributed in higher-level plants [22,23,24]. The long-term interaction between flavonoid-producing plants and different animal species may have contributed to the diversity of their biochemical and pharmacological activities. Among these activities, the ability of flavonoids to act as antioxidant agents is the most important and widely studied property, followed by their antimicrobial properties [17,25,26].

In this context, the present work aims to describe the bioprospecting, characterization, and investigation of the antioxidant potential in different in vitro models and the acute antinociceptive capacity in vivo of a fraction rich in flavonoids, extracted from jaborandi (P. microphyllus) leaves. Given the importance of these metabolites in therapy and the lack of studies on the flavonoids from this commercially valuable plant, this research seeks to fill that gap and contribute to understanding their potential applications.

2. Results

2.1. Extraction Yield

After an exhaustive maceration process, 18.4 g of methanolic extract (ME) was obtained from 250 g of plant material. The extraction yield for the ME was 7.4%, while the ethyl acetate (EtOAc) fraction yielded 4.8%, based on the mass of the plant material.

2.2. Analysis by UV-Vis Spectrophotometry

The spectral graph of the quercetin pattern (Figure 1B) in MeOH displayed absorption bands corresponding to the A-ring at 255 nm (band II) and the B-ring at 373 nm (band I). The absorbance spectrum of the EtOAc fraction (Figure 1B) exhibited the spectral characteristics typical of flavonoids. This observation was based on the visible absorption bands within the ranges associated with cinnamoyl and benzoyl, specifically between 269 and 405 nm. These similarities suggest that the compound may be a flavonoid, prompting further studies to confirm this hypothesis.

2.3. HPLC Profile of Secondary Metabolites of the EtOAcF

The EtOAcF chromatogram revealed an intense and well-defined peak with a retention time (Rt) of 53 min (Figure 2), corresponding to the predominant substance in the sample. The absorption of the 53 min peak of the EtOAcF (Figure 2B) was approximately 720 mV for a 2 mg/mL injection solution, which is directly related to the concentration of the compound in the fraction. Quercetin showed a peak with the same Rt (53 min) as the EtOAcF, as seen in Figure 2. Thus, it can be inferred that there is a great similarity between the major compound present in the fraction and the standard since these were subjected to the same chromatographic conditions. The major peak of EtOAcF was collected for further analysis by mass spectrometry and cyclic voltammetry.

2.4. Analysis by Mass Spectrometry (ESI-MS) of the Major Compound of the EtOAcF

The mass spectrum of the fraction revealed a major protonated molecule of m/z 254.9 [M+H]+ (Figure 3C). Analysis of the quercetin standard, which was diluted in MeOH, by ESI-MS showed a protonated molecule with an m/z of 302.9 [M+H]+ (Figure 3A). Among the studied flavonoids was quercetin. Such results overlap the fragmentation of the quercetin protonated molecule (m/z = 302.9), instead of the ESI-MS/MS spectrum of Figure 3B, in which fragments with m/z values of 229, 257, and 285 were observed.

2.5. Study Using Cyclic Voltammetry (CV) of the Major Compound of the EtOAcF

Using the CV technique, it was possible to discriminate significant differences in the redox profile of quercetin from the EtOAcF isolate. Figure 4A shows the cyclic voltammograms obtained from either a quercetin solution or the EtOAcF compound at 0.7 mg/mL. The voltammogram of the SCE (saturated calomel electrode), which was recorded in pure electrolyte, is also shown for comparative purposes. Under these conditions, the SCE did not show any redox process in the analyzed potential range and still showed low capacitive current, which boosted its use in later experiments. In the next step, the SCE voltammogram was recorded when in the presence of quercetin. In this case, two well-defined redox pairs were observed, which can be directly attributed to the electrochemical response of quercetin under the established experimental conditions (Figure 4A).

During the anodic scan, the first peak observed at −0.11 V vs. SCE corresponds to the charge transfer associated with the oxidation of the hydroxyl groups at the 3′ and 4′ positions of the B-ring of quercetin (Figure 4B). The second oxidation peak (at +0.25 V) was attributed to the oxidation of the hydroxyl groups at the 5 and 7 positions of the A-ring of the molecule, as illustrated in Figure 4B. In the cathodic scan, the respective reductions of these processes were observed at +0.17 V and −0.14 V vs. SCE (Figure 4A). In contrast to quercetin, only one irreversible oxidation peak was detected at around +0.2 V vs. SCE for the EtOAcF compound.

The differences between the B-rings of the two substances were once again evidenced since the redox processes of these groups were not observed in the voltammogram of the EtOAcF compound, only in quercetin. This reinforces the theory that the compound is chrysin since it does not have hydroxyls in the 3′ and 4′ positions of the B-ring but does have an A-ring with oxygenation at carbons 5 and 7. Considering that the maximum current intensity results from the oxidation reaction at the anodic peak current, this isolated compound is expected to have antioxidant activity, which involves an irreversible charge transfer process. It is a noteworthy possibility that the EtOAcF substance is a variation of quercetin obtained in a different oxidation state that was induced by the solvent used during chromatography. However, a deeper investigation must be carried out in order to elucidate these preliminary results.

2.6. Characterization of the EtOAcF by Infrared Spectroscopy (FTIR)

The infrared spectrum in Figure 5, showing the functional bands of the EtOAcF, clearly exhibits the functional groups present in flavonoids: OH, C=O, C=C, and C-H. Absorption in the 3600–2700 cm⁻¹ region is associated with axial deformation vibrations in the hydrogen atoms bonded to carbon, oxygen, and nitrogen (C-H, O-H, and N-H). The 3600–3200 cm⁻¹ range refers specifically to the OH group associated with the molecule, which is neither free nor chelated. In EtOAcF, an intense band was observed at 3450 cm⁻¹, which probably corresponds to the hydroxyl group. Strong bands around 2900 cm⁻¹, like the one that appears in this spectrum, have been revealed in almost all the spectra of organic compounds, as they result from the presence of the C–H stretch.

It was possible to identify the C=C group in the aromatic rings of flavonoids that appear at 2300–1900 cm ⁻¹. Absorption in this region is related to (non-accumulated) double-bond axial strain vibrations. The absorption frequency ranges corresponding to the C=C group of aromatics are 1600–1650 and 1450–1500 cm⁻¹. In the vibrations of aromatic nuclei, the 1580 cm⁻¹ band is intense when the phenyl group is conjugated with unsaturation or is even bonded to atoms with lone pairs of electrons. The 1450 cm⁻¹ band is usually obscured, while the 1500 cm⁻¹ band is generally stronger. It was observed in the IR spectrum absorption peaks at 860, 750, and 692 cm ⁻¹, which may correspond to this band of aromatics.

The absorption band of the -CH=CH- group constituting the aromatic ring was identified in the spectrum within the expected range of 970–960 cm⁻¹, as well as in the region of 700–690 cm⁻¹. The carbonyl stretching region (Figure 5) was represented between 1850 and 1650 cm⁻¹. Intense bands in this interval relate to the presence of the carbonyl group of the various carbonyl compounds. These absorptions are shifted from 30–40 cm ⁻¹ to lower frequencies in α and β unsaturated systems. The region of 1720 cm ⁻¹ could be observed in the EtOAcF spectrum, which probably corresponds to the region of the carbonyl group of the ketone function that exists in the C-ring of flavonoids. Other characteristic bands are due to the vibration of the phenol group, with C-OH strain vibrations (1308–1370 cm ⁻¹) and C-OH stretching vibrations (1112–1172 cm ⁻¹).

2.7. EtOAcF Biological Assays

2.7.1. Antioxidant Activity by DPPH and ABTS Radical Capture

The absorption values expressed as mean ± SD, which are shown in the kinetic curve (Figure S1), indicate that the EtOAcF derived from jaborandi can sequester free electrons. This is evident in the significantly lower absorbance values obtained when the DPPH radical was reacted with different concentrations of the EtOAcF, compared to the negative control (DPPH + solvent). The IC₅₀ value obtained was 0.5 mg/mL. The control with vitamin C revealed an IC₅₀ of approximately 30 µg/mL.

The ethyl acetate fraction showed a TEAC (total Trolox equivalent antioxidant capacity) of 819 mM of Trolox/g of sample and IC₅₀ of 0.81 mg/mL. The EtOAcF of jaborandi was evaluated after 6 and 30 min of reaction time with the radical. The TEAC value was calculated using the linear regression equation obtained in the 6-min reaction Trolox test in Figure S2C. The 30-min time graph revealed that the EtOAcF continued to react with the ABTS radical throughout the reaction time (Figure S2C), showing higher absolute TEAC values with longer times.

2.7.2. Antioxidant Activity Observed in the Electrochemical Profile of the EtOAcF

The redox potentials of the EtOAcF fraction are shown in Figure 6 in a cyclic voltammogram obtained for the EtOAcF in PBS buffer 0.1 mol L⁻¹, with a pH of 6.4. A cyclic voltammogram of the electrode with only the pure support electrolyte is also shown for comparative purposes. As expected, no redox process was observed under the experimental conditions employed for the pure electrolyte. Conversely, in the voltammogram recorded for the EtOAcF, a set of redox processes was observed. Three oxidation processes (I, II, and III) were observed during the anodic scan, at 0.32 V, 0.54 V, and 0.73 V vs. Ag/AgCl, respectively. During cathodic scanning, 3 reduction processes were observed (processes IV, V, and VI) at the respective potentials of 0.60 V, −0.03 V, and −0.24 V vs. Ag/AgCl. The reversibility of each of these processes is still being investigated. In general, the various oxidation processes that were observed in the range between 0.3 V and 0.7 V indicate that the EtOAcF fraction has potential antioxidant activity.

2.7.3. Neuropathy Induction Test by Sciatic Nerve Compression

CCI induced the development of thermal and mechanical hyperalgesia in saline-treated rats. Administration of the EtOAcF at doses of 50 and 100 mg/kg significantly (p < 0.0001) prevented these events, as shown in Figure 7A. During the experiment, neuropathy was observed from the fourth day and lasted until the seventh day. Only the animals in the sham control group did not exhibit this behavior. After the seventh day, the treatment was carried out in groups and evaluated at 0, 6, 120, and 180 min. The results of the tested controls, which consisted of morphine, sham, and vehicle groups, were satisfactory, as shown in Figure 7B. Only the higher dose of 100 mg/kg of the EtOAcF was statistically significant (p < 0.001) in the inhibition of behavioral changes from neuropathy (Figure 7B).

3. Discussion

Recent studies in the literature highlight that ethyl acetate (EtOAc) is recognized as one of the most effective solvents for fractionating and isolating flavonoids [27,28]. Importantly, EtOAc is associated with lower residual toxicity compared to more toxic solvents like methanol (MeOH). Studies indicate that after extraction, the levels of residual EtOAc in the final extract are significantly lower than those of MeOH, thereby minimizing the potential toxicity [29,30,31]. This makes EtOAc a preferred choice for the extraction of flavonoids, ensuring both efficacy and safety in the resulting extracts.

In this study, the absorption spectrum of the ethyl acetate fraction (AcOEtF) exhibited spectral characteristics typical of flavonoids. The absorption bands that were observed, specifically between 269 nm and 405 nm, align with those associated with the cinnamoyl and benzoyl systems, indicating the potential presence of flavonoids, likely including various classes such as flavonols, flavanones, or isoflavones. The chemical composition of each sample can influence the shift of these bands in the spectra of flavonoid complexes [32,33].

The positions and intensities of these absorption bands provide important information regarding the nature of flavonoids and their oxidation patterns. The spectral data suggest that the compound present in the AcOEtF may belong to the groups of flavonols, flavanones, or isoflavones, as indicated by the spectral characteristics reported in the literature [32,34]. These studies indicate that band I of the flavones occurs in the range of 304–380 nm, while that of flavonols appears at 352–400 nm [35,36] Additionally, flavonols with substituted 3-hydroxyl groups exhibit band I at 328–357 nm, reinforcing the relationship between chemical substitutions and spectral characteristics [32,35].

Despite exhibiting a similar retention time in HPLC, the quercetin and the major compound in the AcOEtF displayed distinct fragmentation profiles with ESI-MS. The mass spectrum, with a principal ion at m/z 254.9 [M+H]⁺ for the compound of interest (Figure 3), aligns with the literature values for the protonated molecule of chrysin in positive ionization mode [37,38,39]. This raises the hypothesis that this flavone may be the predominant compound in AcOEtF.

The electrochemical profile observed for quercetin corroborates the existing data in the literature. A strong band within this range means that its intensity depends on its concentration, showing that there is a considerable amount of this grouping in the sample [40,41]. According to Kainat et al. [42], substituent groups in meta positions or electron donors further decrease the aromatic stretching frequency of flavones. These can be seen at positions 900–860, 810–750, and 710–690 cm⁻¹ for the meta-disubstituted benzene ring because it exists in the quercetin and chrysin molecules [43,44]. Absorption bands appear for –CH out-of-plane angular deformation, which finding provides information regarding the position of substituents on the aromatic ring [42]. In general, the infrared spectrum obtained was similar to those reported in the literature for chrysin [34,45].

The IC₅₀ value obtained was 0.5 mg/mL. The control with vitamin C exhibited an IC₅₀ of approximately 30 µg/mL, which was similar to that reported in the literature [46]. Leite-Legatti et al. [47] detected very high TEAC values (945 mM of Trolox/g of the sample) in the peel of the jaboticaba fruit and related the effect to the high content of phenolic compounds, which are mainly flavonoids. It was observed that the reaction with ABTS radical, in general, tended to stabilize after 30 min, as observed in other studies [18,48,49].

Previous authors have reported that the compounds considered the best antioxidants are generally those that present oxidation at lower potentials and with greater current intensity, meaning that they oxidize more easily. In this sense, electroanalytical methods are relevant for determining the reducing potential of phenolic compounds, including the study of the antioxidant protection mechanisms of flavonoids [50].

The electrochemical mechanism of flavonoids is discussed in several studies [41,51,52,53,54]. Such works mainly expose the oxidative process undergone by the OH groups, which results in the formation of the correlative o-quinone. They also propose the generation of this same product, which is a very unstable compound that is capable of undergoing homogeneous chemical reactions such as intramolecular rearrangements, forming polymeric compounds generated from dimers or oligomers that are similar to quinones [51,53,55]. These rearrangements must be considered, mainly when dealing with non-pure samples that contain several molecules or active principles, as is the case with theEtOAcF.

Considering chrysin as the major compound, according to the literature, at doses of 50 mg/kg, this flavone was able to protect against the depletion of reduced glutathione (GSH) and to reduce the levels of xanthine oxidase (XO), both of which are involved in the protection and generation of ROS, respectively [56,57].

Neuropathic pain (NPP) results in the onset of sensory changes, such as hyperalgesia and allodynia [58]. These symptoms can also be observed in animals subjected to experimental models, such as CCI (chronic constriction injury). Observing these responses allows for inferring the existence of pain [59,60,61]. The CCI proposed by Bennet and Xie [62], as used in this study, consists of unilaterally applying four ties to the common trunk of the animal’s sciatic nerve with bioabsorbable thread. This model allows for the detection of both spontaneous and evoked pain.

Alterations in different regions of the neuraxis are the result of nerve compression and can be observed from the first days after the injury. Locally, ischemia, the release of pro-inflammatory substances, intraneural edema [63], and the degeneration of myelinated and non-myelinated fibers [64] are observed. These peripheral events lead to changes in excitability and connectivity in the spinal and supraspinal regions [65,66], the mediation of which seems to involve the release/formation of cytokines, neurotrophins [63,67], and reactive oxygen species—ROS [68]. The subsequent behavioral changes, such as mechanical and thermal hyperalgesia, can be observed from the first days after the injury [69,70] and can persist for several weeks [71].

Barbosa et al. [72] evaluated the hydroethanolic extract of propolis at a dose of 1 and 10 mg/kg in rats. The administration of the extract at a dose of 10 mg/kg showed significant results in sciatic nerve recovery after its constriction. Many neurochemical modulators are involved in the onset and maintenance of NPP. Due to nerve injury, several inflammatory mediators are produced at the injury site and act both there and in the CNS.

In a previous study by Caraffa [73], the role of flavonoids in neuroprotection is discussed, pointing out that these natural molecules can act by regulating the pathways of p38 mitogen-activated protein kinases (MAPKs) and nuclear factor kappa B (NF-kB), inhibiting pro-inflammatory mediators, metalloproteinases (MMPs), and reactive oxygen species (ROS). The nerve ligation model used can cause an exacerbation in the amount of nitric oxide (NO) released, resulting in hyperalgesia. This mechanism can also be inhibited through the action of flavonoids [74,75]. Findings in the literature show the neuroprotective effect of chrysin, a possible major constituent of the EtOAcF, demonstrating the potential of this flavonoid to reduce the activation of NF-kB and the expression of inducible nitric oxide synthase (iNOS), in addition to reducing oxidative stress [76,77].

Regarding the toxicity of fractions derived from plants that are rich in flavonoids, studies indicate a protective potential against liver and kidney damage, acting on the pathways involved in the inflammatory process and the generation of free radicals, which are harmful [78,79]. Furthermore, other studies show the beneficial effects of chrysin, such as neuroprotective, hepatoprotective, nephroprotective, colon-protective, and anti-inflammatory effects, providing evidence that the fraction studied here is not toxic at the doses used [76,77,80,81,82]. Sulaiman et al. [83] proved the hemocompatibility of chrysin through in vitro testing. It is worth mentioning that an acute toxicity protocol must be carried out to ensure the safety of an EtOAcF derived from jaborandi leaves.

4. Materials and Methods

In the initial phase of this study, an ethyl acetate fraction (EtOAcF) was obtained from jaborandi leaves. A methanolic extract was first prepared, followed by the extraction of the EtOAcF using a conventional maceration method at room temperature [84]. Afterward, characterization assays for the EtOAcF and screening for antioxidant activity and neuroprotective properties were conducted. Throughout the experiments, the samples and standards were protected from light and heat to prevent oxidation and were stored under refrigeration to avoid microbial contamination and degradation.

4.1. Obtaining Extracts and Fractions

4.1.1. Collection and Botanical Identification of Plant Material

Species identification was supervised by Dr. Ivanilza Moreira de Andrade (Department of Biology, Campus Ministro Reis Velloso, UFPI), and its specimen number (TEPB 27152) was deposited in the Herbarium of the Centro de Ciências da Natureza, UFPI, Teresina, Piauí, as well as being registered with the Genetic Heritage Management Council (SisGen) (AE263D6). The leaves were collected in the city of Parnaíba, PI, in June 2015.

4.1.2. Preparation of Plant Material

The leaves were properly cleaned to eliminate any contaminants. After drying the leaves in a ventilated oven at room temperature, they were ground down in a knife mill to obtain the raw plant material.

4.1.3. Obtaining Extracts

To obtain the methanolic extract, the ground leaves (250 g) were subjected to exhaustive maceration with 1 L of methanol (MeOH) 85% at room temperature for 5 days, with filtrate collection at the end. The MeOH from the extract was removed with the rotary evaporator, leaving an aqueous suspension. This suspension was then subjected to lyophilization, resulting in the crude extract. Yield was calculated as a percentage, according to the following formula: [crude extract weight (g)/plant material weight (g)] × 100.

4.1.4. Extract Fractionation

A portion of the methanolic extract (10 g) was resuspended in methanol:water (30:10, v/v) and then transferred to a separatory funnel. Then, 50 mL aliquots of ethyl acetate (EtOAc) were used for liquid–liquid partition fractionation. Eventually, the EtOAcF was obtained. Subsequently, the fraction was dried in a water bath (50 °C) and drying was followed by the subsequent tests.

4.2. Characterization of Extracts and EtOAcF

4.2.1. UV-Vis Spectrophotometry

The EtOAcF was analyzed in a Shimadzu UV spectrophotometer (UV-1800), using the UV-probe program, version 2.33. The sample dissolution, including the quercetin standard, was performed in MeOH at a concentration of 0.05 mg/mL, with 0.005 mg/mL for quercetin, in 1.0 mL quartz cuvettes with an optical path of 1 cm. The samples and standard were analyzed at similar temperatures (approximately 25 °C). The scan comprised the wavelengths (λ) between 800 and 200 nm. The results of the absorbance spectra of the samples determined the wavelengths that would be analyzed using HPLC.

4.2.2. High-Performance Liquid Chromatography (HPLC)

An HPLC device (Shimadzu, Kyoto, Japan) equipped with a UV/VIS detector, model SPD-20A, was used with a binary pumping system LC-6AD and a CBM-20A controller. The data acquisition and processing program used was LCsolution (version 1.24 SP1). The EtOAcF solutions (2 mg/mL), previously filtered through a 0.22 μm pore membrane, were submitted for HPLC in several different analyses, in order to define their composition profile. The analysis conditions are shown in Table S1. The MeOH used was obtained from Biosolve Chimie SARL; the ultrapure Milli-Q water (H₂O) and glacial acetic acid were from P.A. (Vetec).

In addition to the sample, a solution containing quercetin (0.025 mg/mL in MeOH) as a standard was injected under the same chromatographic conditions listed above. The wavelengths thus analyzed corresponded to those in the absorption evidence from the previously obtained absorbance spectra present in all samples, including the standard.

4.2.3. Mass Spectrometry (MS)

The major compound isolated by HPLC from the EtOAcF, together with the standard quercetin (0.1 mg/mL in MeOH), were subjected to analysis by an AmaZon SL mass spectrometer (Bruker Daltonics, Bremen, Germany) with electron-spray ionization, quadrupole, and a mass analyzer of the ion-trap type. Nitrogen was used as a nebulization gas and helium as a collision gas to carry out the fragmentation. Detection was performed in positive ionization mode under atmospheric pressure. The ESI-MS conditions were a capillary of +6000 V and a desolvation temperature of 50 °C, along with a Hamilton syringe pump with a flow of 3 µL min⁻¹. Data acquisition and processing were performed using the Trap control software (version 3.2).

4.2.4. Electrochemical Characterization of the Major Compound of the EtOAcF

In the electrochemical characterization, the cyclic voltammetry (CV) technique was used to evaluate the redox potentials of both quercetin and the EtOAcF isolate obtained by HPLC. To obtain cyclic voltammograms, an AUTOLAB potentiostat/galvanostat, model PGSTAT 128N, was used, coupled to an electrochemical cell with a 10.0 mL capacity of support electrolyte, a Teflon lid containing a slot for three electrodes, and an inlet and outlet for gas. A pyrolytic graphite (EGP) rod (A = 2.1 mm²) was used as the working electrode and a platinum plate (A = 2.0 cm²) acted as the counter electrode. The saturated calomel electrode (SCE) was used as the reference electrode. During the recording of the voltammograms, all electrodes remained immersed in a 0.7 mg/mL solution of quercetin or the EtOAcF, solubilized in potassium phosphate buffer (PBS) 0.1 mol L⁻¹, pH 5.6 (the supporting electrolyte). It is noteworthy that the working electrode underwent an electrochemical cleaning procedure, whereby the potential was repeatedly cycled between −0.9 V and +0.9 V to remove any impurities from the electrode until the registered current became constant (by around the tenth cycle).

4.2.5. Characterization of the EtOAcF by Infrared Spectroscopy

The EtOAcF was characterized by Fourier transform infrared spectroscopy (FTIR). The FTIR spectra were collected using a resolution of 1 cm⁻¹ with a KBr window on a Shimadzu IRAffinity-1 and a wavenumber in the spectral range of 4000–400 cm⁻¹, which is commonly used to characterize organic compounds. The main organic groups in the molecule were identified from the wavenumber vs. frequency graph.

4.3. EtOAcF Antioxidant Assays

4.3.1. Determination of Antioxidant Activity by the DPPH Radical Scavenging Method

This assay used the methodology employed by Lima et al. [49]. First, 0.3 mM ethanolic solution of DPPH was prepared (2.9574 mg of DPPH dissolved in ethanol in a 25 mL volumetric flask). The standard was obtained by reading the absorbance of the DPPH solution without the sample in a spectrophotometer at 517 nm. The measurement of the EtOAcF sample’s antioxidant activity by this method was carried out in triplicate, adding 170 µL of sample in 830 µL of DPPH for each fraction and reading the absorbance 30 min after the reaction started. We conducted the reading procedure using five different fraction concentrations (0.4; 0.8; 1.2; 1.6; 2.0 mg/mL), obtained from an initial solution of 4 mg/mL. Ethanol was used to calibrate the spectrophotometer. The drop in the reading of the optical density of the samples was correlated with a control that contained a DPPH solution without the sample, establishing the percentage of discoloration of the DPPH radical according to the formula below:

% protection = (Control Abs − Sample Abs) × 100/Control Abs

(1)

After calculating the percentage of protection, the results were plotted in a graph containing the concentrations used (mg/mL) on the X-axis and the calculated protection percentages on the Y-axis, along with the linear regression equation that was used to find the IC₅₀ value (the amount of sample needed to reduce the initial concentration of the DPPH radical by 50%). Therefore, the lower the IC₅₀ value, the greater the antioxidant activity of the analyzed sample. As a control, the determination of the IC₅₀ of vitamin C, a compound with well-known antioxidant properties, was performed using concentrations of 40, 50, 60, 70, and 80 µg/mL under the same analysis conditions as for the EtOAcF.

4.3.2. Determination of Antioxidant Activity by the ABTS Radical Scavenging Method

The ABTS radical 2,2′-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) was formed from the reaction of 7 mM ABTS with 2.45 mM potassium persulfate, after which it was incubated at room temperature and in the absence of light for 16 h. After this time, the solution was diluted in ethanol until a solution was obtained with an absorbance of 0.70 (± 0.01). To conduct the analyses, 30 μL of the diluted sample was added to 1970 μL of the solution containing the radical, and the absorbance was determined in a spectrophotometer (Shimadzu UV-1800) at 734 nm after 6 and 30 min of reaction. As a standard solution, the synthetic antioxidant Trolox was used at concentrations of 100, 200, 400, 800, and 1000 mM in ethanol. The following fraction concentrations were evaluated: 0.2, 0.4, 0.8, 1.0, and 1.2 mg/mL. All readings were performed in triplicate, and the results were expressed in mM of Trolox per gram of EtOAcF. Then, the IC₅₀ of the fraction was determined from the graph, using the relationship between the concentration (mg/mL) and the protection percentages.

4.3.3. Statistical Analysis of the DPPH and ABTS Tests

The results were expressed as mean ± standard deviation. To compare the arithmetic means, an analysis of variance (ANOVA) and Tukey’s tests were performed on Prism 4.0 software (GraphPad, La Jolla, CA, USA). A significance level of 5% of probability (p < 0.05) was adopted. For the study of the linear correlations of the antioxidant activities by the ABTS and DPPH capture method, the Origin 5.0 program was used.

4.3.4. Determination of Antioxidant Potential by CV

The EtOAcF was evaluated using the CV technique to analyze the redox potentials. To obtain cyclic voltammograms, a Dropsens µStat 400 potentiostat/galvanostat (Metrohm Pensalab) was used. A volume of 50 µL of EtOAcF was added to 100 µL of PBS 0.1 mol. L⁻¹, pH 6.4, which was used as the supporting electrolyte. From this mixture, 100 µL was removed and spread over the surface of a screen-printed electrode, which used pyrolytic graphite as the working electrode, a platinum semicircle as the counter-electrode, and a part of Ag/AgCl as the reference. All measurements were taken at room temperature (25 °C).

4.4. Determination of Activity Against Neuropathic Pain Using a Method of Induction by Triggering the Sciatic Nerve

4.4.1. Surgical Procedure

Under the anesthetic action of xylazine hydrochloride (20 mg/kg, i.p.) associated with ketamine hydrochloride (120 mg/kg, i.p.), partial sciatic compression (CPC) was achieved. An asepsis and trichotomy of the posterior region of the right hind paw of female Wistar rats were initially performed in preparation for the surgical procedure. After initiating anesthesia, the sciatic nerve was located by separating the biceps femoris muscle using surgical equipment. The sciatic nerve was exposed and freed from the surrounding connective tissue. Subsequently, the nerve was wrapped with a silk suture thread (3-0, Shalon Medical, Teresina, PI)), thus compressing its original diameter. The procedure was completed with an approximation of the edges of the incision, suturing of the animal’s skin, and treatment with a topical solution of chlorhexidine (2%), which lasted until the healing of the surgical incision. The animals were placed in metabolic cages with daily asepsis. In one group (Sham), the procedure was only simulated; the nerve was exposed and touched by surgical instruments, but they did not connect.

4.4.2. Acute Antinociceptive Assessment

Before the surgical procedure, the von Frey test was performed to measure the mechanical nociceptive threshold (MNL) of the baseline response of these animals. In this test, the right hind paw was touched, in three repetitions, by different filaments starting from the lowest weight in grams to the highest. The von Frey test was also performed on the fourth day to monitor the onset of neuropathy, and on the seventh day, when the treatment and evaluation of the antinociceptive activity of the fraction were carried out.

To evaluate the effect of a single administration of EtOAcF, we performed the same procedure as described above. The animals were treated with 50 or 100 mg/kg of EtOAcF, morphine at 5 mg/kg, or the vehicle (distilled water with 1% DMSO, i.p.) on the seventh day. After the treatments, the von Frey test was performed after durations of 0, 60, 120, and 180 min. In this test, there was also a group of animals treated similarly to the vehicle group, where the surgical procedure was only simulated. The experiments were authorized by the local Ethics and Research Committee (Teresina, Piauí) with protocol number 082/2014.

4.4.3. Statistical Analysis

To compare the arithmetic means, an ANOVA was performed, using the Prism 6.0 software (GraphPad). A significance level of 5% of probability (p < 0.05) was adopted.

5. Conclusions

The pharmacognostic characterization of EtOAcF from the leaves of jaborandi (P. microphyllus) revealed the presence of flavonoids and pointed to the flavone chrysin as a probable major compound in the analyzed fraction. This same fraction showed moderate antioxidant action during methods based on radical scavenging, indicating better activity with the ABTS method than in the DPPH assay. The EtOAcF also revealed redox processes at low potentials in the cyclic voltammetry test, indicating antioxidant activity since it oxidized more easily. During the in vivo test on Wistar rats, compression of the sciatic nerve was used to induce neuropathy, and the results showed significant antinociceptive activity for the EtOAcF treatment. The observed effect may be due to the influence of flavonoids on the inflammatory pathways, which is important for neuropathy when established after nerve ligation, as well as for the balance of ROS and the release of nitric oxide. However, additional research is needed to identify the mechanism of action underlying the beneficial effect of this fraction on neuropathic pain in an animal model, in addition to elucidating the toxicity of an EtOAcF derived from jaborandi. It is important to highlight the commercial importance of the species and the scarcity of studies regarding derivatives rich in molecules other than alkaloids. Therefore, this study sheds light on the new biological potential of jaborandi leaf derivatives.