Antinociceptive and antioxidant effects of extract enriched with active indole alkaloids from leaves of Tabernaemontana catharinensis A. DC.

Journal of Ethnopharmacology 239 (2019) 111863

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Antinociceptive and antioxidant effects of extract enriched with active indole alkaloids from leaves of Tabernaemontana catharinensis A. DC.

T

Dáfiner Perghera, Aline Picolottoa, Pauline Fagundes Rosalesa, Keilla Gomes Machadob, Aline Fagundes Cerbaroc, Raqueli Teresinha Françad, Mirian Salvadorc, Mariana Roesch-Elyb, Leandro Tassoe, Jozi Godoy Figueiredoa, Sidnei Mouraa,∗ a

Laboratory of Biotechnology of Natural and Synthetic Products, University of Caxias do Sul, Brazil Laboratory of Genomics, Proteomics and DNA Repair, University of Caxias do Sul, Brazil c Laboratory of Oxidative Stress and Antioxidants, University of Caxias do Sul, Brazil d Laboratory of Veterinary Diagnostics, University of Caxias do Sul, Brazil e Laboratory of Pharmacology, University of Caxias do Sul, Brazil b

ARTICLE INFO

ABSTRACT

Keywords: Antinoception Tabernaemontana catharinensis A. DC. Indole alkaloids Etnopharmacology

Ethnopharmacological relevance: Ethnopharmacological knowledge is important for the identification of active compounds from natural products. Pain may have different aetiologies with complex mechanisms. Tabernaemontana catharinensis A. DC. is well known for indole alkaloids, being used empirically in folk medicine, with antimicrobial and anti-inflammatory as well as antiofidic actions among others. Aim of the study: This work aims to evaluate the antinociceptive and antioxidant effect in mice of the alkaloids extract from leaves of Tabernaemontana catharinensis A. DC. (AITc). Materials and methods: The AITc was produced by ultrasound and acid-base extraction, and the chemical composition was evaluated by high resolution mass spectrometry. Male mice (Mus musculus), Swiss, were used for in vivo tests. The AITc was administrated at doses of 1.0, 5.0, and 10.0 mg/kg in acetic acid model, formalin, tail-immersion, hot plate, and open field tests, and compared to saline, morphine, or diazepam controls, depending on the test. The toxicological, biochemical, haemogram and antioxidant effect were evaluated in mouse organs such as liver, brain, kidneys, spleen and stomach. Results: In total, 10 compounds were identified in the AITc, being from the indole alkaloids from the ibogan and corynanthean classes. The extract in doses ranging from 5.0 to 10.0 mg/kg showed an antinociceptive effect for acetic acid, inhibiting by 47.7% and 61.6%. In the same line, reductions of 47.1% (first phase) and 43.6% (second phase) were observed for the 5.0 mg/kg dose in the formalin test. However, tail-immersion and hot plate tests did not show considerable modifications in the latency period, while in the open field test there was an inhibition of only 5.1%. It was observed no differences in NO levels and total antioxidant status of the mice in any of the studie tissues. Conclusions: The results justify the use of this plant in traditional medicine. in vivo tests indicate that these compounds possess central and peripheral mechanisms of action. This is study that reports the nociceptive action of these alkaloids, also including toxicity tests, which are intended to guarantee the safety of use of extracts of this plant.

Abbreviations: AITc, Alkaloids extract from leaves of Tabernaemontana catharinensis A. DC.; ALT, Alanine aminotransferase; ANOVA, Analysis of variance for repeated measurements; AST, Aspartate aminotransferase; Cbc, Full blood count; CEUA, Animals at the University of Caxias do Sul; COBEA, Brazilian College of Animal experimentation; CNS, central nervous system; DCG, Diazepam control group; HRMS, High Resolution Mass Spectrometry; I.P., Intraperitoneal; MCG, Morphine control group; MCHC, Mean corpuscular hemoglobin; MCV, Mean corpuscular volume; NO, nitric oxide; Q-TOF, Quadrupole/Time-of-Flight; S.C., Subcutaneous; SCG, Saline control group; TBARS, Substances reactive to thiobarbituric acid; TEAC, Assay antioxidant capacity equivalent to Trolox; UFLC, Ultra-fast liquid chromatograpy; V.o., orally route ∗ Corresponding author. University of Caxias do Sul, 1130, Francisco Getúlio Vargas st., CEP 95070-560, Caxias do Sul, Brazil. E-mail addresses: [email protected], [email protected] (S. Moura). https://doi.org/10.1016/j.jep.2019.111863 Received 13 January 2019; Received in revised form 2 April 2019; Accepted 3 April 2019 Available online 09 April 2019 0378-8741/ © 2019 Elsevier B.V. All rights reserved.

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1. Introduction

crude extract was kept in the dark and refrigerated (approximately 4 °C). In the next step, the crude extract was dissolved in acidic water (100 mL 0.1% HCl), which was extracted with dichloromethane (3 × 30 mL), and the organic phase was discarded. Afterwards, the remaining aqueous phase was neutralised with NaOH (aqueous 10%) until pH 11, and this solution was extracted again with dichloromethane (3 × 30 mL). The organic solvent was evaporated under pressure yielding the AITc extract.

Natural products with specific features have been used for a long time as essential components in traditional medicine (Fang et al., 2005). In this way, medicinal plants are considered as the main accessible resource in the treatment of diseases by many people around the world. Adams and Wang (2015) reported a series of tropical plants used for pain control. Pain is characterised as a prominent symptom of multiple diseases, affecting people's quality of life, which can be either acute or chronic (Antunes and Monico, 2015; Luo et al., 2015). In the same line, the traditional knowledge acquired from the use of plants in folk medicine indicates new active molecules, which can be isolated and modified to be prototypes of new drugs (Newman and Cragg, 2016; Salgueiro et al., 2016). The indole alkaloids have been highlighted as active compounds, examples being vinblastine and vincristine derived from Catharanthus roseus, which is currently used in cancer and Hodgkin’s disease (Leonti et al., 2017; Moon et al., 2018). Similarly, indole alkaloids from Aspidosperma cuspa, a shrub used in folk medicine for pain in South America, showed antinociceptive activities (Perez et al., 2012). Tabernaemontana catharinensis A.DC. is an arboreal species belonging to the Apocynaceae family (Matozinhos and Konno, 2011), which is popularly known as "jasmine-vane", "dairy-two brothers", "jasmine", "forquilheira", and "snake bark" (Matozinhos and Konno, 2011; Boligon et al., 2014). This plant is currently found in the north, northeast, southeast, central-west, and southern regions of Brazil, Argentina, Paraguay, Uruguay, and Bolivia (Marinho et al., 2016). Infusions and alcoholic extracts from this plant have been used in folk medicine as an antiofidic, vermifuge, antidote for tooth pain (Almeida et al., 2004), anti-inflammatory drug (Taesotikul et al., 2003), and analgesic (Boligon et al., 2014). In addition, T. catharinensis extracts showed in vitro antioxidant activity (Nicola et al., 2013), without antioxidant studies results in vivo reported. Rates et al. (1993) and Brum et al. (2016) reported that extacts derived from seeds and leaves of T. catharinensis produced antinocepitive actions in mice. However, there is controversial information about the active compounds from this plant. In this way, this study aims the evaluation of the antinoceptive and antioxidant activity, toxicological, biochemical and hemogram, as well as the histopathological in mice treated with T. catharinensis extracts enriched in indolic alkaloids. The AITc was prepared by ultrasound followed by a liquid-liquid extraction with pH control. The chemistry composition was determinated by High Resolution Mass Spectrometry (HRMS), with the extract solution injected directly or with aid of the ultra-fast liquid chromatograpy (UFLC) in qualification and quantification respectively.

2.2. Chemical analysis The AITc and internal standard, 98% ajmalicine, were dissolved individualy in a solution of 50% (v/v) chromatographic grade acetonitrile, 50% (v/v) ultrapure water, and 0.1% formic acid. The solutions were infused directly into the ESI source by means of a syringe pump (Harvard Apparatus), at a flow rate of 180 μL/h for qualification, or assisted by an UFLC, for quantification. The solution of AITc (5.00 μg/L) was separated by an UFLC Shimadzu model 20 AD (Kyoto, JP), which was equipped with a binary pump system (LC-20AD), automatic injector (SIL-20A), and controller (CBM-20A). An isocratic method was performed using an NST C18 column (250 mm × 4.6 mm × 5 μm) with ultrapure water and 0.1% acetic acid in pump A and acetonitrile in pump B at a total flow of 0.5 mL/min (1:1) for a total of 30 min. A calibration curve of ajmalicine, ranging from 18.75 to 600.00 μg/L, was determined. The quantification was performed using the internal standardisation method, correlating the compound peak area of the IS with the linear response. In both cases, HRMS with ESI(+)-MS and tandem ESI(+)-MS-MS was used for compound identification using a hybrid high-resolution and high accuracy microTOF (Q-TOF) mass spectrometer (Bruker® Scientific, Billirica, USA). The equipment conditions were as follows: capillary and cone voltages were set to + 3500 V and + 40 V, respectively, with a desolvation temperature of 100 °C. For ESI(+)-MS/ MS, the energy for the collision induced dissociations (CID) was optimised for each component. Diagnostic ions in different fractions were identified by the comparison of their ESI(+)-MS/MS dissociation patterns with compounds identified in previous studies or standards. For data acquisition and processing, Q-TOF-control data analysis software (Bruker® Scientific) was used. The data was collected in a m/z range of 70–1100 at a speed of two scans per second, providing a resolution of 50,000 (FWHM) at 200 m/z. No important ions were observed below 100 m/z or above 800 m/z; therefore, ESI (+)-MS data is shown in the 100–800 m/z range. 2.3. Antioxidant activity of AITc

2. Materials and methods

The in vitro antioxidant activity of the AITc was determined by DPPH• radical reduction assay according to Yamaguchi et al. (1998) method. The AITc was diluted to different concentrations (ranging from 0,01–2 mg/mL) and added to Tris-HCl buffer (100 mM, pH 7.0) containing 250 μmol of DPPH• dissolved in ethanol. These mixture were stored in the dark for 20 min and then the absorbance was measured at 517 nm in a spectrophotometer (UV-1700, Shimadzu, Kyoto, Japan). The results were expressed as IC50 (mg/mL of AITc needed to scavenge 50% of the radical DPPH•).

2.1. Plant collection, extraction, and fraction preparation Leaves of T. catharinensis were collected in Ijuí, Rio Grande do Sul, Brazil, in November 2016 (28°26′06.4″S and 53°56′15.7″W), and a voucher specimen has been identified by Felipe Gonzatti and deposited in Caxias do Sul University Herbarium (HUCS 34038–34057/g 1669) at Caxias do Sul/RS, Brazil. The aerial parts of the plant (leaves and stems, around 300 g) were dehydrated in a greenhouse under forced air circulation, whose temperature was 32 °C, for a period of seven days. The dried material was milled with Willey TE 650 type knives and stored in light-protected containers. Ultrasound was used for the extraction process in accordance with Orio et al. (2012) followed by a specific method for indoles alkaloids as Guida et al. (2005), with modifications, briefly: to about 5 g of dry and ground leaves of T. catharinensis was added 50 mL of ethanol PA. This study used an Ultrasonic Processor Sonics Vibra-Cell YOU 505–750, operated at 40% amplitude and potency of 500 W, with 30 min of total extraction time. In the next step, the mixture was filtered, the solvent evaporated under pressure, and the

2.4. Animals For the experiments, male mice were used of the species Mus musculus, Swiss (30 g). During the whole experiment, the animals remained in the testing lab at the University of Caxias do Sul (UCS). The mice were packed in appropriate boxes, polypropylene filled with shavings of pinus (Granja RG, São Paulo, Brazil), inserted in ventilated shelving with air exhaustion, with controlled temperature (22 ± 2 °C) and humidity (55 ± 15%), and a light-dark cycle of 12 h. The mice received water ad libitum and "pellets" ration in excess for every test (mouse 2

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Nuvilab brand industrial CR-1), except on days of the procedures, in which they remained in food restriction for a period of 4 h. All procedures were in accordance with the ethical principles of animal testing adopted by the Brazilian College of Animal Experimentation (COBEA), and rules of the Ethics Committee on use of Animals at the University of Caxias do Sul (CEUA), whose experimental protocol number is 005/ 2017. Were delineated four groups, with n = 7 animals, for the development of chemical (acetic acid and formalin), thermal (tail-immersion and hot plate), locomotive (open field), toxicity (acute and subchronic), and antioxidant (TBARS, nitric oxide, TEAC, DPPH•, and IC50) tests. The animals received the composed AITc orally in doses of 1.0, 5.0, or 10.0 mg/kg. In the Saline Control Group (SCG), the animals received oral saline solution. In Morphine Control Group (MCG), the animals received morphine by subcutaneous injection (s.c.) in the dose of 5.0 mg/kg; and in the Diazepam Control Group (DCG), the animals received diazepam by intraperitoneal (i.p.) route at a dose of 2.0 mg/ kg.

animal on the hot plate for a maximum time of 45 s (to avoid injury to the legs).

2.5. Nociceptive model against acetic acid: abdominal contortions

2.10. Acute and subchronic toxicity study

Writhing and the extension of hind limbs was evaluated after induction by 0.6% acetic acid administered by intraperitoneal route (Oliveira et al., 2009). The extract of AITc was administered in doses of 1.0, 5.0 or 10.0 mg/kg orally 30 min before the start of test observations. The SCG received saline by the same route as the extract and the MCG at a dose of 5.0 mg/kg, through subcutaneous administration. Writhings were recorded simultaneously after the 10 min of administration of acetic acid, for a period of 20 min. The summations of the contortions during this period represent the nociceptive response, as per the methodology of de Queiroz et al. (2010).

To evaluate the possible toxic effects of the extract of AITc, acute and subchronic toxicological studies were conducted, being delimited into two experimental groups. One group received the AITc extract at a dose of 5.0 mg/kg given orally, obeying a dosage daily outlet only. The other group of mice (SCG) received saline the same way and dosage as the extract without the occurrence of any kind of fasting before the administrations. Blood was collected and separated into two aliquots, one containing EDTA anticoagulant (Tetrasodium) and the other heparin. Samples containing EDTA anticoagulant were used for realisation of the full blood count (Cbc), and to quantify the crit, total plasma proteins (PPt g/dL), erythrocytse, haemoglobin, mean corpuscular haemoglobin (MCHC), and mean corpuscular volume (MCV), to evaluate possible pathological changes. The counts of red blood cells, white blood cells, platelets, and haemoglobin concentration were determined by electronic cell counter (BC-2800vet) (Souza and Ferreira, 1985). Blood smears were prepared and stained with the Romanowski dye and used in differential leukocyte counts, as well as in morphological evaluation of erythrocytes, leukocytes, and platelets. The MCV and the MCHC were determined through a mathematical calculation (Al-Habori et al., 2002). Serum samples were used to assess kidney and liver profiles, and were determined in an automated biochemical analyser (BS-120 Mindray) using commercial kits (LABTEST) (Oliveira et al., 2014).

2.9. Open field test This test was described by Hall (1934), whose objective is based on introducing the animal to an unknown, enclosed environment. The animals received the AITc extract at a dose of 5.0 mg/kg administered by v.o., the SCG by the same route of administration as the extract, and the DCG at a dose of 2.0 mg/kg through i.p. injection. Mice were taken individually, for a period of 1 min for the recognition of the place, and observed for a time of 4 min. The response time was evaluated by the number of recorded fields of locomotion (number of entries of the animal with all four legs on any of the squares), peripheral locomotion (number of entries with all four legs in the squares next to the wall of the apparatus) and locomotion (number of entries with all four legs in any unit away from the wall of the apparatus) (Capaz et al., 1981).

2.6. Nociception against formalin The protocol was conducted in accordance with Hunskaar and Hole (1987). After 30 min of pretreatment, the mice received 1.2% formalin, s.c. intraplantar on the right hind leg, and the number of kicks were counted (Milano et al., 2008). The extract of AITc was used at a dose of 5.0 mg/kg through (v.o). The SCG had saline administered the same way as the extract and the MCG had morphine, at a dose of 5.0 mg/kg, administered through s.c. injection. The indication of nociception was considered based on the number of times the mouse licked the paw that received the chemical agent, during 0–5 min (neurogenic phase) and 15–30 min (inflammatory phase). 2.7. Tail-immersion test

2.11. Redox metabolism assays

The extract of AITc was used at a dose of 5.0 mg/kg by v.o. The SCG had saline administered the same way as the compound and the MCG received morphine, at a dose of 5.0 mg/kg, applied through s.c. injection. Later, 30 min after the administrations to the groups, the response time was evaluated with the immersion of the lower third of the tail of the mouse (3 cm tail, animal immobilised with tape) in a water bath, with a temperature of 56 ± 1 °C for a period of up to 20 s (Spencer and Sewell, 1976). The latency period of submersion of the tail was recorded at 30, 60, and 90 min after administration of AITc, SCG, or MCG. This latency period was taken as tolerance index (Fischer et al., 2008).

To assess redox metabolism it was collected the spleen, brain, stomach, liver and kidney of the mouses. The tissues were weighed and homogeneizados using a ground-glass-type Potter-Elvehjem homogenizer with cold PBS (pH 7.4) buffer in ice. Thiobarbituric acid reactive species (TBARS) was assayed according the methodology describe by Wills and Rotblat (1966), onde the tissue samples were combined with 5% trichloroacetic acid, sulfuric acid (3 M) and centrifugated at 3000 rpm for 10 min. After, the pellet were resuspended in thiobarbituric acid solution, mixture and incubated in a boiling water bath for 15 min and cooled at room temperature. Then, n-butanol were added and centrifugated at 3000 rpm for 5 min. The supernatant fraction was isolated and its absorbance was measured at 532 nm in a spectrophotometer (UV-1700, Shimadzu, Kyoto, Japan). The 1,1,3,3tetrametoxipropano (TMP) was used as standard, and the results were expressed as nmol of TMP/mg of protein. The NO content was assayed throught Griess reaction describe by Green et al. (1981). The tissue samples were added to Griess reagent (1:1) mixed and incubated in the dark for 10 min after reading at 550 nm by microplate reader. Sodium nitroprusside was used as the standard. The results were expressed as

2.8. Hot plate test As proposed by Woolfe and MCDonald (1944), this test evaluated the nociceptive action by the time the animal stays on a hot plate (55 °C ± 1.0 °C). The extract of AITc was administered (v.o.), at a dose of 5.0 mg/kg, the SCG by the same route as the extract and the MCG, at a dose of 5.0 mg/kg, administered through s.c. injection. After the course of 30 min from the administrations to the groups, the response time was evaluated at 30, 60, 90, and 120 min, with the insertion of the 3

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nmol of nitrite/mg of protein. The total antioxidant capacity (TEAC) was performed according to Re et al. (1999), the tissue samples were incubated to ABTS•+ 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) radical solution for 6 min and then read at 734 nm in a spectrophotometer (UV-1700, Shimadzu, Kyoto, Japan). The Trolox was used as the standard, and the results were expressed as mM of TEAC. Total proteins content was determined by Bradford method (1976) using albumin (BSA) as protein standard.

(1984), at the time when this plant was still called Peschiera catharinensis (ancient nomenclature of T. catharinensis). While, conodurine and voacorine are dimers of ibogan and corynanthean alkaloids (Araujo et al., 1984). Meanwhile, for quantification, a method using UFLC-ESI(+)-QTOF was adapted (Frozza et al., 2016), using ajmalicine as IS. A calibration curve ranging from 25.75 to 500.00 μg/L of the standard resulted in an R2 of 0.9988 with LOD and LOQ of 8.50 and 25.75 μg/L, respectively. The AITc showed voacristine, coronaridine hydroxyindoline, voachalotine, affinisine, 16-epi-affinine, and ibogamine at 812.78, 349.78, 78.99, 45.96, 26.88, and 25.89 μg/L, respectively. However, coronaridine, 12-methoxy-Nb-methyl-voachalotine, conodurine, and voacorine were present below the levels of detection of the method.

2.12. Histology The whole organs (stomach, right and left kidneys and liver) were removed from mice and weighed. Small pieces of control and AITc exposed in mice were fixed in 10% neutral formalin solution for 48 h. Tissues were further embedded in paraffin and solid sections were cut at 5 μm. Samples were further stained with haematoxylin and eosin (Monday and Uzoma, 2013).

3.2. Acetic acid-induced abdominal writhing test Initially, the antinociceptive activity was evaluated through the test of abdominal contortions induced by acetic acid. In this model, the AITc extract was shown to have a significant antinociceptive effect at all doses tested, thus expressing an inhibition considered in the number of abdominal writhes after its administration. The doses tested with greater effectiveness were those of 5.0 and 10.0 mg/kg, showing inhibitions of 47.7% and 61.6%, respectively. The MCG group also showed inhibition of 92.7%, as shown in Fig. 3.

2.13. Statistical analysis Data are expressed as mean ± E.P.M. (standard error of the mean) or median, according to the variables evaluated. For the evaluation of thermal and chemical tests and related locomotor antinociceptive activities, ANOVA (analysis of variance for repeated measurements) was used followed by the Bonferroni test. In rehearsals for acute and subchronic activity, validation biochemical tests and Cbc was used with ANOVA and Student’s t-test. For oxidative stress activities, ANOVA and Student’s t-test were used. For all analyses, the level of significance was delimited to p < 0.05.

3.3. Formalin test Subsequently, the formalin test was performed, and the results can be observed in Table 2. The AITc extract at the dose of 5.0 mg/kg was shown to have a significant antinociceptive effect, reducing the number of paw licks in both phases of the test, when compared to SCG. The AITc extract significantly reduced number of licks in the first phase (inhibition of 47.1%), as well as in the second phase (inhibition of 43.6%). MCG showed efficacy in both phases tested, presenting an inhibition threshold of 92.0% in the first phase and 88.2% in the second phase.

3. Results 3.1. Chemical composition and antioxidant activity of AITc The AITc extract were carried-out from T. catharinenses leaves and stems by sonication with subsequent acid-base extraction in accordance with Orio et al. (2012) and Guida et al. (2005) respectively, which had a yield of 2.0% (from the dry plant weight). The chemical composition qualification was by HRMS, where the AITc extract was diluted with acetonitrile/ultrapure water (1:1) with the addition of 0.1% formic acid. The solution was directly injected into the ESI source. Fig. 1 shows the HRMS spectrum in full mode and Table 1 shows the compounds identified. After, the AITc extract was evaluated for the scavenging ability of the DPPH• radical, showing significant in vitro antioxidant activity, with an IC50 of 0.09 μg/mL. In this way, ten indole alkaloids were indentified, which had previously been described for this species, Fig. 2. Three of these extracted are of the ibogan class (ibogamine, coronaridine, and voacristine); meanwhile three other (affinisine, voachalotine, and 12-methoxy-Nbmethylvoachalotine) are alkaloids belong to the class of corynanthean, usually subclassified into the akuammidine group. The oxidole alkaloid coronaridine is a T. catharinensis marker identified by Araujo et al.

3.4. Tail-immersion test and hot plate test In this thermal test, we used tail-immersion, because it is classified as a drug-sensitive model acting on the central nervous system. The AITc extract at the dose of 5.0 mg/kg showed no significant change in the latency period compared to the SCG at times of 30, 60, and 90 min. The MCG showed an antinociceptive effect at all times tested. The hot plate test demonstrated that the 5.0 mg/kg of AITc extract administered did not significantly increase the latency time of the mice, evaluated at 30, 60, 90, and 120 min, when compared to SCG. On the other hand, the MCG showed latency at all times after drug administration (Table 3). The animals were given oral saline (SCG), morphine (MCG) 5.0 mg/ kg via the s.c., and the extract AITc(alkaloids extract from leaves of Tabernaemontana catharinensis) at a dose of 5.0 mg/kg, v.o. A: After 30 min of group administrations, the response time was analysed by

Fig. 1. Spectrum of AITc by ESI(+) Q-TOF. 4

325.1894

339.2063

355.2005

3ç67.1971

385.2115

411.2213

705.4006

721.3962

4

5

6

7

8

9

10

309.2000

3

2

281.2016

1

1

Precursor íon m/ z

Entry

5

C43H53N4O6

C43H53N4O5

C24H32N2O4

C22H29N2O4

C22H27N2O3

C21H26N2O3

C21H26N2O2

C20H25N2O2

C20H24N2O

C19H25N2

Elementar composition

0.2

0.8

4.0

1.8

4.9

2.5

0.7

2.7

3.8

0.3

Error (ppm)

Table 1 Chemical composition of AITC by HRMS ESI(+) Q-TOF.

12.5

9.8

8.7

5.5

3.4

9.2

10.4

11.2

8.5

9.1

Isotopic ratio (msig) Ibogamine

158.0955 [C11H12N]+; 144.0802 [C10H10N]+, 122.0964 [C8H12N]+ 291.1869 [C20H23N2]+, 172.1010 [C11H12N2]+; 158.0860 [C10H10N2]+; 146.0950 [C10H12N]+; 138.0911 [C8H12NO]+, 120.0791 [C8H10N]+ 307.1808 [C20H23N2O]+; 265.1683 [C18H21N2]+; 152.1064 [C9H14NO]+; 122.0973 [C8H12N]+ 307.1776 [C20H23N2O]+, 258.1108 [C16H22N2O]+, 198.0905 [C13H12NO]+; 158.0965 [C11H12N]+; 144.0811 [C10H10N]+, 122.0981 [C8H12N]+ 337.1870 [C19H23N3O2]+, 323.1717 [C20H23N2O2]+, 238.1103 [C15H14N2O]+; 160.0756 [C10H10NO]+; 144.0797 [C10H10N]; 122.0961 [ C8H12N]+ 335.1777 [C21H23N2O2]+, 307.1803 [C20H23N2O]+, 291.1869 [C20H23N2]+, 258.1161 [C16H22N2O]+, 200.1068 [C13H14NO]+; 172.1020 [C11H12N2]+; 146.0960 [C10H10N]+; 138.0911 [C8H12NO]+; 120.0788 [C8H10N]+ 367.2000 [C22H27N2O3]+, 335.1746 [C21H23N2O2]+, 307.1773 [C20H23N2O]+, 258.1106 [C16H22N2O]+, 198.0888 [C13H12NO]+; 174.0892 [C11H12NO]+; 136.1109 [C9H14N]+; 122.0961 [C8H12N]+ 383.1920 [C22H27N2O3]+, 365.1814 [C22H25N2O3]+; 353.1860 [C21H25N2O3]+; 335.1750 [C21H23N2O2]+; 307.1803 [C20H23N2O]+, 258.1161 [C16H22N2O]+, 200.1068 [C13H14NO]+; 138.0920 [C8H12NO]+; 120.0753 [C8H12N]+ 674.3835 [C42H50N4O4]+, 645.3802 [C41H49N4O3]+, 512.2940 [C35H36N4]+, 367.2022 [C22H27N2O3]+ 307.1793 [C20H23N2O]+;, 291.1872 [C20H23N2]+; 200.1072 [C13H14NO]+; 136.1158 [C9H14N]+; 122.0965 [C8H12N]+ 674.3845 [C42H50N4O4]+, 645.3852 [C41H49N4O3]+, 512.2932 [C35H36N4]+, 367.2020 [C22H27N2O3]+ 307.1795 [C20H23N2O]+;, 291.1892 [C20H23N2]+; 200.1045 [C13H14NO]+; 136.1162 [C9H14N]+; 122.0963 [C8H12N]+ Voacorine

Conodurine

12-methoxy-Nb-methylvoachalotine

Voacristine

Voachalotine

Coronaridine hydroxyindoline

Coronaridine

16-epi-affinine

Affinisine

Compound

Fragmentation pathway

Cava et al. (1968).

Araujo et al., 1984.

Batina et al. (2000); Nicola et al. (2013)

Nicola et al. (2013); Pereira et al. (2008).

Van Beek et al., 1984; Nicola et al. (2013).

Pereira et al. (2008).

Araujo et al., 1984; Cardoso and Vilegas, 1999.

Araujo et al., 1984.

Braga, 1984; Cardoso and Vilegas, 1999. Vieira et al., 2008; Nicola et al. (2013).

Reference

D. Pergher, et al.

Journal of Ethnopharmacology 239 (2019) 111863

Journal of Ethnopharmacology 239 (2019) 111863

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Fig. 2. Chemical structures of indoles alkaloids identified in AITc by HRMS.

above, showed the reaction time in seconds. * p < 0.05 indicates statistically significant difference when compared to saline (n = 7). The # indicates statistical significance compared to morphine. (ANOVA, Bonferroni test). 3.5. Open field test The AITc extract was administered before the animals entered into the open field test, and did not show a significant difference (inhibition of 5.1%) compared to the SCG. Concerning the DCG, it showed a significant loss of locomotor activity (inhibition of 84.5%) when compared to the SCG. In this way, the locomotor activity of the mice was not affected by the administration of the extract, as can be observed in Fig. 4. Fig. 3. Tabernaemontana catharinensis (AITc) reduces the number of abdominal writhes induced by acetic acid in mice. * p < 0.05 indicates statistically significant difference when compared to saline (n = 7). The # indicates statistical significance compared to morphine. (ANOVA, Bonferroni test).

3.6. Acute and subchronic toxicity study Mice treated with the AITc extract for acute and subchronic toxicological studies not expressed adverse effects, with no signs of mortality or morbid state. There were no physical signs of behavioural toxicity or changes in breathing of these animals during the duration of the experiments. Body weight was without significant changes. In the biochemical tests, when compared to the SCG at 1 and 14 days, treated mice did not show considerable differences in their results. In erythrogram tests, haematocrit, PPt g/dL, red blood cells, haemoglobin, MCV, and CHCM values did not express significant changes when

immersion of the mouse tail in water, whose temperature of 56 °C, for a period of up to 20 s. Data represented ± SEM at the time of up to 90 min. B: The reaction time of this, when the animals began to lick their hind legs, being recorded as a nociception sign after being inserted into the hot plate. Time was recorded before (T = 0) and 30, 60, 90 and 120 min after treatment of the groups. The values quoted in the table Table 2 AITc reduces the time of licking the formalin test in mice. Experimental Groups

Paw Licking (s) Neurogenic phase

SCG (v.o.) MCG (5.0 mg/kg; s.c) AITc (5.0 mg/kg; v.o.)

Inflammatory phase

First phase

Inhibition (%)

Second phase

Inhibition (%)

73.20 ± 7.52 5.83 ± 3.98# 38.71 ± 3.99*

– 92.03% 47.11%

59.60 ± 4.06 7.00 ± 1.71# 33.57 ± 2.67*

– 88.25% 43.67%

The, mice received oral saline (SCG) 5.0 mg/kg morphine (MCG) was administered s.c., and the extract AITc (alkaloids extract from leaves of Tabernaemontana catharinensis), at the dose of 5.0 mg/kg v.o. They received formalin 1,2% intraplantar, by administration, s.c. After the administration period, the nociception was evaluated in two moments: first phase (neurogenic) and second (inflammatory) phase. Data represented ± E.P.M for analgesia by formalin up to 30 min. The # indicates significant statistical difference when compared to morphine. * p < 0.05 indicates significant difference to the saline group (n = 7) (ANOVA, Bonferroni test). 6

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Table 3 Effect of AITc on tail-immersion, and Hot Plate test. A Experimental Groups

Time (min)

SCG (v.o.) MCG (5.0 mg/kg; s.c) AITc (5.0 mg/kg; v.o.)

30′

60′

90′

3.86 ± 0.10 3.54 ± 7.10# 3.80 ± 3.85*

4.13 ± 6.89 7.61 ± 3.36# 4.09 ± 0.59*

4.41 ± 2.61 5.86 ± 4.19# 4.68 ± 2.23*

B Experimental Groups

SCG (v.o.) MCG (5.0 mg/kg; s.c) AITc (5.0 mg/kg; v.o.)

Latency time (s) T0

T30

T60

T90

T120

17.41 ± 0.20 17.09 ± 0.10 16.85 ± 0.28

17.26 ± 13.75 38.87 ± 14.31# 17.67 ± 0.90

16.39 ± 13.45 37.53 ± 13.79# 18.11 ± 1.52

15.85 ± 12.23 35.08 ± 11.53# 16.45 ± 0.36

16.98 ± 5.46 25.55 ± 6.59# 15.58 ± 0.97

has been highlighted, with alkaloids being among the main responsible for these effects (Quintans et al., 2014). For identification of plants and, consequently, the active extract, ethnopharmacological knowledge is essential (Arome et al., 2014; da Silva et al., 2014). Indole alkaloids derived from leaves, stens and flowers of Tabernaemontana divaricata are among the recent examples of the association of popular knowledge that leads to the identification of active compounds from plants capable of being used for pain (Ali-Khan et al., 2018). In the same line, the traditional use of T. catharinensis as antiofidic, antidote for tooth pain (Almeida et al., 2004), anti-inflammatory (Taesotikul et al., 2003), and analgesic (Boligon et al., 2014), are based in infusions and alcoholic extracts of leaves and stens. The species T. catharinensis is known for the production of indole alkaloids of classes ibogan and corynanthean as secondary metabolites (Marinho et al., 2016). In this line, the first step was the production of an extract rich in these compound, using ultrasound followed by the acid-base processes of Orio et al. (2012) and Guida et al. (2005). Thus, 2.0% yield was obtained from dry T. catarinensis leaves, which was a result close to that determined by Adams and Wang (2015) for extraction of quinolizidine alkaloids from Sophora alopecuroides assisted by ultrasound with pH control. For chemical characterisation of plant metabolites, HRMS with (ESI) in MS and MS-MS mode is a powerful tool. It has been used for complex samples of plant extracts such as those enriched in flavonoids and terpenoids (Yang et al., 2007). In this work, the chemical characterisations by HRMS were conducted in positive mode with the introduction of 0.1% (v:v) of formic acid, identifying 10 compounds in AITc (Table 1, Fig. 1). The HRMS used a set of information such as exact mass, isotopic ratio, and fragmentation pathway for unequivocal identification and differentiation of isobaric interferences. In this line, Fig. 7 demonstrates the fragmentation for voacristine (385.2115 m/z), which was the main extract in AITc. This result was similar to those found by Nicola et al. (2013) and Marinho et al. (2016), who had revised the chemical composition of the Brazilian Tabernamontana genus, which is related to those found in AITc. The quantification, using chromatographic separation and detection by ESI-HRMS, identified that the main compounds present in the extract are voacristine, coronaridine hydroxyindoline, and voachalotine ranging from 813 to 45 μg/L. Animal models have been used for evaluation of the antonicociceptive activity of isolated compounds or mixtures, as for instance plant extracts (Silva et al., 2013). In this way, these studies can confirm the activities of medicinal plants used popularly for pain, supporting the search for new active compounds. The purpose of the acetic acid test is to evaluate the analgesic or anti-inflammatory properties of new agents, described as a typical

Fig. 4. AITc does not cause changes in the open field test in the mice. * p < 0.05 indicates statistically significant difference when compared to saline (n = 7). The # indicates statistical significance compared to morphine. (ANOVA, Bonferroni test).

compared to the control group. In the leukogram tests, the values of monocytes, lymphocytes, neutrophils, and eosinophils, when compared with SCG, did not present any relevant modifications (Table 4). 3.7. Effect of AITc on histopathology of different organs Morphologic analysis after hematoxylin and eosin staining showed that control samples stomach, right and left kidneys and livers exhibited normal histological architecture with prominent nuclei with well-arranged. Histopathological studies of the treated group after 14 days of exposure presented no substantial morphological changes in any sections of organs evaluated after exposition to AITc (Fig. 5). 3.8. Redox metabolism assays The redox metabolism was evaluated after 7 days (acute) and 14 days (subchronic) in spleen, brain, stomach, liver and kidneys of mice treated with AITc. Here, was observed an reduction of 41% in TBARS and 47% of reduction after 7 and 14 days respectively. There was no change in NO levels and total antioxidant capacity in any of the evaluated tissues, both acute and sub-chronic. Fig. 6. 4. Discussion The application of popular medicine in the cure/treatment of pain 7

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Table 4 The effects of acute and subchronic administration of AITc, do not alter the biochemical parameters, erythrogram and hemogram in mice. A Treatments

Biochemical parameters

Acute Toxicity SCG (v.o.) AITc (5.0 mg/kg; v.o.) Subchronic Toxicity SCG (v.o.) AITc (5.0 mg/kg; v.o.)

ALT

AST

Urea

Creatinine

57.20 ± 8.12 48.20 ± 5.56

190.10 ± 15.03 215.10 ± 22.34

36.00 ± 2.58 34.00 ± 3.36

0.41 ± 0.01 0.32 ± 0.06

53.50 ± 4.70 63.60 ± 5.60

204.50 ± 28.29 195.70 ± 12.74

42.50 ± 1.50 38.50 ± 3.40

0.32 ± 0.02 0.25 ± 00.4

B Treatments

Acute Toxicity SCG (v.o.) AITc (5.0 mg/kg; v.o.) Subchronic Toxicity SCG (v.o.) AITc (5.0 mg/kg; v.o.)

Erythrogram Parameters Hematocrit

PPt g/dL

Red Blood Cells

Hemoglobin

MCV

MCHC

33.40 ± 1.61 33.00 ± 1.11

4.97 ± 0.30 5.05 ± 0.31

7.21 ± 0.31 7.42 ± 0.17

10.72 ± 0.48 10.87 ± 0.25

45.51 ± 1.09 46.17 ± 0.86

32.68 ± 0.44 31.83 ± 0.69

30.00 ± 2.00 26.00 ± 1.47

5.60 ± 0.41 6.05 ± 0.99

6.22 ± 0.34 5.50 ± 0.28

10.86 ± 0.88 8.97 ± 0.67

48.67 ± 5.04 47.25 ± 1.38

36.30 ± 2.13 34.57 ± 2.15

C Treatments

Acute Toxicity SCG (v.o.) AITc (5.0 mg/kg; v.o.) Subchronic Toxicity SCG (v.o.) AITc (5.0 mg/kg; v.o.)

Parameters Leukogram Total Leukocytes

Monocytes

Lymphocytes

Neutrophils

Eosinophils

2.62 ± 0.42 4.11 ± 1.79

70.38 ± 26.00 83.33 ± 64.95

2.22 ± 0.45 2.16 ± 0.49

564.87 ± 94.90 542.00 ± 189.10

31.88 ± 11.50 28.20 ± 5.90

10.58 ± 8.24 10.95 ± 9.85

33.51 ± 15.57 22.33 ± 21.33

11.27 ± 10.04 12.89 ± 0.05

6.50 ± 2.76 4.24 ± 3.24

30.00 ± 6.00 18.50 ± 16.50

A: The mice received single doses daily in acute treatment and for fourteen consecutive days in subchronic treatment. Urea, creatinine and alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities. B: Hematocrit, total plasma protein (PPt), red blood cells, hemoglobin, mean corpuscular volume (MCV), and mean corpuscular hemoglobin concentration (CHCM). C: Leukocytes, monocytes, lymphocytes, neutrophils and eosinophils.One-way ANOVA followed by Student's ttest. Values were the mean ± SEM (n = 7). * p < 0.05 indicates a significant difference in the respective vehicle group.

model of visceral inflammatory pain (Tjølsen and Hole, 1997). The AITc extract, given at doses of 1.0, 5.0, and 10.0 mg/kg, was able to reduce abdominal writhing in the mice (Fig. 4). In a similar study, Brum et al. (2016) established as active the ethyl acetate extracts of the same plant at doses of 200.0 500.0 or 800.0 mg/kg, which are up to 80 times

greater than the most effective dose found in this study. In the same way, Gomes et al. (2009) used ethanolic extracts of T. catharinensis stem bark at doses of 37.5, 75.0, and 150.0 mg/kg administered intraperitoneally, which reduced the number of abdominal writhings of mice. The differences are directly related to the chemical composition

Fig. 5. Histopathological changes in different organs of Mice, stained with Hematoxylin and Eosin. A) Micrograph (4×) of the Normal Stomach in GSC mice; and B) Micrograph (4×) of the Stomach in mice treated with AITc 5.0 mg/kg for 14 days; C) Micrograph (4×) of the normal Left Kidney in GSC mice; and D) Micrograph (4×) of the Left Kidney in mice treated with AITc 5.0 mg/kg for 14 days); E) Micrograph (10×) of normal right Kidney in GSC mice; and F) Micrograph (10×) of Right Kidney in mice treated with AITc 5.0 mg/kg for 14 days; G) Micrograph (20×) of normal liver in GSC mice; and H) Micrograph (20×) of the Liver in mice treated with AITc 5.0 mg/kg for 14 days. 8

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Fig. 6. TBARS (1) in 7 (A) and 14 (B) days; Nitric Oxide (2) in 7 (A) and 14 (B) days; TEAC (3) in 7 (A) and 14 (B) days.

of the AITc, rich in indole alkaloids, which have potentiated the nociception effects, in comparison with the extracts tested by the other authors. The formalin test is recognised as a model of acute and chronic pain (Trongsakul et al., 2003), making it possible to differentiate between central or peripheral actions (Murray et al., 1988; Melo et al., 2013). The effectiveness of the AITc at 5.0 mg/kg indicates that it acts in both phases, showing superior inhibition in the inflammatory phase (2nd stage) (Table 2). These results are in agreement with the work of Brum et al. (2016), although at higher doses. The central effect reported is related to the high doses used by these authors. In the same line, Florentino et al. (2016) had used extracts of Memora nodosa, rich in

indole alkaloids, at doses of 500.0 and 100.0 0 mg/kg, for inhibition of both phases. The tail-immersion test gives information about the mechanism of noception, if peripheral or central acting (Florentino et al., 2016). In this test, the antinociceptive action of AITc at 5.0 mg/kg confirmed its peripheral action, since it did not exhibit considerable modifications in the latency period (30, 60, and 90 min) (Table 3A). Brum et al. (2016) reported that a dose of 800.0 mg/kg of the same plant extract was sufficient to cause antinociceptive activity. The AITc at the dose of 5.0 mg/kg did not present significant influence in the hot plate, which confirms the peripheral action of this alkaloidal extract, Table 3. The same effects were reported by Regalado et al. (2017), who worked with 9

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Fig. 7. Analysis of ion m/z 381.2115 in MS-MS mode in A], and expand for analysis of isotopic ratio in B].

alkaloidal extracts of the Tabebuia hypoleuca stems. The authors indicated that doses of 300 and 500 mg/kg presented a prolongation in the test latency period. In order to verify the effect of T. catharinensis alkaloids on spontaneous locomotor activity, the open field test was performed. The results had demonstrated that the AITc extract has no effect on locomotor andthe mice sleep (Fig. 4). Brum et al. (2016) reported similar results using morphine as control. In this way, this indicates that at high doses indole alkaloids can cause injury to the locomotor system, with sedation as a side effect. AITc showed antioxidant activity in vitro (IC50 = 0.09 μg/mL), which was significantly higher than that observed for ethanolic extract and fractions of T. catharinenses (Nicola et al., 2013). This result is related to the present indole alkaloids, which corroborated with in vivo assays. The brain is exceptionally vulnerable to the cytotoxic effects of free radicals derived from oxygen (Kontos, 1989; Haliwell and Gutteridge, 1992) and therefore probably more adequate to detect the inadequacy of antioxidant defense mechanisms. These data are similar to the study by Katalinic et al. (2005), in which it sought to elucidate the differences between the genera in the susceptibility to oxidative stress, verifying activity to thiobarbituric acid male mice. Boligon et al. (2014), tested T. catharinensis crude extract, which exhibited an inhibition of thiobarbituric acid reactive species. In the same line, no differences in total antioxidant capacity were observed in any of the tissues studied. However, has been reported that the alkaloid ibogaine is able to increase the activity of the antioxidant enzyme superoxide dismutase (Chuang and Chen, 2013; Nikolic et al., 2015). This enzyme disrupts the superoxide radical in oxygen and hydrogen peroxide, which in turn is disrupted by the enzyme catalase (Halliwell, 2010). Modulation of superoxide dismutase enzyme activity may be associated with a reduction in TBARS levels observed in our study. Brain is a highly oxygenated tissue and it derives most energy from oxidative metabolism. Plants used in traditional medicine should be safe to use. In this work, the toxicological safety was determinated by comparison of biochemical and haematological parameters of animals receiving doses of 5.0 mg/kg AITc with the SCG (1 and 14 days) (Table 4). In both groups, the AST remained unchanged, indicating absence of toxicity in organs such as the liver and kidneys. In the same line, the urea did not present alterations, which is indicative that the AITc does not affect renal function. Brum et al. (2016) had obtained similar results for the same test, working with extracts of T. catharinensis in doses up to 80

times greater. The AITc did not cause any toxic effects on the bone marrow or circulatory system homeostasis, as indicated by the erythrogram exams (Table 4). The erythrocyte, hemoglobin, hematocrit, MCV and CHCM concentration values in the AITc groups were similar to the control groups. Brum et al. (2016), in assays with extracts of T. catharinensis in larger doses (200 mg/kg) and for 28 days, reported alteration of immunological and haematopoietic systems. The histopathological tests of animals treated with AITc at 5.0 mg/kg in the acute and subchronic treatments did not show significant alterations, with no degree of impairment detected in any of the areas analysed (Fig. 5). This extract, at the dose of 5.0 mg/kg, was able to present antinociceptive activity in the acetic acid and formalin tests, demonstrating a mechanism of peripheral action and antioxidant in the tests performed. These actions are certainly mediated by the presence of ibogan and corynanthean indole alkaloids, which do not cause toxicological, locomotor, or depressive damages. These results differ from those reported by Brum et al. (2016) and Boligon et al. (2014), which indicated flavonoids as responsible for the noceptive action of this plant. 5. Conclusion In summary, this is the first study that reports in vivo tests with an extract rich in T. catharinensis indole alkaloids, with smaller doses showing antinociceptive and modulation of oxidative stress without damaging toxicological safety and without altering locomotor behaviour. This work indicate the traditional use of this plant for pain and antioxidant effects, in addition to generating data for future studies with focus on ibogan and corynanthean type alkaloids, which can be prototypes for new drugs for pain control and also assist in combating free radicals. For this, a more complete modulatory study is necessary at these doses, with a design for the mechanism of action and correlation with the chemical compounds present in future studies, elucidating the action of the substances responsible for antinociceptive and antioxidant activity. Conflicts of interest The authors declare no conflict of interest.

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Author contributions

Fischer, L.G., Santos, D., Serafin, C., Malheiros, A., Delle Monache, F., Delle Monache, G., Cechinel Filho, V., de Souza, M.M., 2008. Further antinociceptive properties of extracts and phenolic compounds from Pliniaglomerata (Myrtaceae) leaves. Biol. Pharm. Bull. 31 (2), 235 239. Florentino, I.F., Silva, D.P.B., Galdino, P.M., Lino, R.C., Martins, J.L.R., Silva, D.M., de Paula, J.R., Tresvenzol, L.M.F., Costa, E.A., 2016. Antinociceptive and anti-inflamatory effects of Memora nodosa and allontoin in mice. J. Ethnopharmacol. 186, 298. 304. https://dx.doi.org/10.1016/j.jep.2016.04.010. Frozza, C.O. das Silva, Brum, E. da Silva, Alving, A., Moura, Silva, Henriques, J.A.P., Roesch-Ely, M., 2016. LC-MS analysis of Hep-2 and Hek-293 cell lines treated with Brazilian red propolis reveals differences in protein expression. J. Pharm. Pharmacol. 1, 13. https://doi.org/10.1111/jphp.12577. Gomes, R.C., Neto, A.C., Melo, V.L., Fernandes, V.C., Dagrava, G., Santos, W.S., Pereira, P.S., Couto, L.B., Beleboni, R.O., 2009. Antinociceptive and anti-inflammatory activities of Tabernaemontana catharinensis. Pharmaceut. Biol. 47 (4), 372–376. https:// doi.org/10.1080/13880200902753239. Green, L.C., Tannenbaum, S.R., Goldman, P., 1981. Nitrate synthesis in the germfree and conventional rat. Science 212, 56–58. Guida, A., De Battista, G., Bargardi, S., 2005. Actividad antibacteriana de alcaloides de Tabernaemontana catharinensis. ADC Times 10, 167–173. Hall, C.S., 1934. Emotional behavior in the rat. I. Defecation and urination as measures of individual differences In emotionality. J. Comp. Psychol. 18, 385–403. Haliwell, B., Gutteridge, J.M., 1992. Formation of thiobarbitutic-acid-reactive-substance from deoxyribose int he presence of iron salts: the role of superoxide and hydroxyl radicals. FEBS Lett. 128, 347–352. Hunskaar, S., Hole, K., 1987. The formalin test in mice: dissociation between inflammatory and non-inflammatory pain. Pain 30, 103–114. Katalinic, V., Modun, D., Music, I., Boban, M., 2005. Gender differences in antioxidant capacity of rat tissues determined by 2,2V-azinobis (3-ethylbenzothiazoline 6-sulfonate; ABTS) and ferric reducing antioxidant power (FRAP) assays. Comp. Biochem. Physiol. C Toxicol. Pharmacol. 140 (47), 52. https://doi.org/10.1016/j.cca.2005.01. 005. Leonti, M., Stafford, G.I., Cero, M.D., Cabras, S., Castellanos, M.E., Casu, L., Weckerle, C.S., 2017. Reverse ethnopharmacology and drug discovery. J. Ethnopharmacol. 23, 417–431. https://doi.org/10.1016/j.jep.2016.12.044. Luo, J., Feng, J., Liu, S., Walters, E.T., Hu, H., 2015. Molecular and cellular mechanisms that initiate pain and itch. Cell. Mol. Life Sci. 72, 3201–3223. https://dx.doi.org/10. 1007/s00018-015-1979-y. Marinho, F.F., Simões, A.O., Barcellos, T., Moura, S., 2016. Braziliam Tabernaemontana genus: indole alkaloids and phytochemical activities. Fitoterapia 127–137. https:// doi.org/10.1016/j.fitote.2016.09.002. Matozinhos, C.N., Konno, T.U.P., 2011. Diversidade taxonômica de Apocynaceae na Serra Negra, MG, Brasil. HOEHNEA 38 (4), 569–596. https://dx.doi.org/10.1590/S223689062011000400005. Melo, A.S., Monteiro, M.C., da Silva, J.B., de Oliveira, F.R., Vieira, J.L.F., de Andrade, M.A., Baetas, A.C., Sakai, J.T., Ferreira, F.A., Cunha Sousa, P.J., da, Maia, C., do, S.F., 2013. Antinociceptive, neurobehavioral and antioxidant effects of Eupatorium triplinerve Vahl on rats. J. Ethnopharmacol. 147, 293–301. https://dx.doi.org/10.1016/j. jep.2013.03.002. Milano, J., Oliveira, S.M., Rossato, M.F., Sauzem, P.D., Machado, P., Zanatta, N., Martins, M.A.P., Mello, C.F., Rubin, M.A., Ferreira, J., Bonacorso, H.G., 2008. Antinociceptive effect of novel trihalomethyl-substituted pyrazoline methyl esters in formalin and hot-plate tests in mice. Eur. J. Pharmacol. 581, 86–96. https://dx.doi.org/10.1016/j. ejphar.2007.11.042. Monday, O., Uzoma, A.I., 2013. Histological changes and antidiabetic activities of Icacina trichantha tuber extract in beta-cells of alloxan induced diabetic rats. Asian Pac. J. Trop. Biomed. 3, 628–633. https://doi.org/10.1016/S2221-1691(13)60127-6. Moon, S.H., Pandurangan, M., Kim, D.H., Venkatesh, J., Patel, R.V., Mistry, B.M., 2018. A rich source of potential bioactive compounds with anticancer activities by Catharanthus roseus cambium meristematic stem cell cultures. J. Ethnopharmacol. 217, 107–117. https://doi.org/10.1016/j.jep.2018.02.021. Murray, C.W., Porreca, F., Cowan, A., 1988. Methodological refinements to the mouse paw formalin test an animal model of tonic pain. J. Pharmacol. Methods 10, 175–186. https://doi.org/10.1016/0160-5402(88)90078-2. Newman, D.J., Cragg, G.M., 2016. Natural products as sources of new drugs from 1981 to 2014. J. Nat. Prod. 79, 629–661. https://doi.org/10.1021/acs.jnatprod.5b01055. Nicola, C., Salvador, M., Gower, A.E., Moura, S., Echeverrigaray, S., 2013. Chemical constituent’s antioxidant and anticholinesterasic activity of Tabernaemontana catharinensis. Sci. World J. https://dx.doi.org/10.1155/2013/519858. Oliveira, R.R.B., Rebecca, M., Góis, O., Siqueira, R.S., Jackson, R.G.S., Almeida, J.T., Lima, X.P., Siqueira, J.S., 2009. Antinociceptive effect of the ethanolic extract of Amburana cearensis A.C. Sm., Fabaceae, in rodents. Revista Brasileira de Farmacognosia 19 (3). https://dx.doi.org/10.1590/S0102-695X2009000500004. Oliveira, S.M., Silva, C.R., Wentz, A.P., Paim, G.R., Correa, M.S., Bonacorso, H.G., Prudente, A.S., Otuki, M.F., Ferreira, J., 2014. Antinociceptive effect of 3-(4fluorophenyl)-5-trifluoromethyl-1H-1-tosylpyrazole. A Celecoxib structural analog in models of pathological pain. Pharmacol. Biochem. Behav. 124, 396–404. https://doi. org/10.1016/j.pbb.2014.07.005. Orio, L., Alexandru, L., Cravotto, G., Mantegna, S., Barge, A., 2012. SFE-CO2 and classical methods for the extraction of Mitragyna speciosa leaves. Ultrason. Sonochem. 19, 591–595. https://doi.org/10.1016/j.ultsonch.2011.10.001. Pereira, P.S., Franca, S.D., de Oliveira, P.V.A., Breves, C.M.D., Pereira, S.V., Sampaio, A., Nomizo, D.A., 2008. Chemical constituents from Tabernaemontana catharinensis root bark: a brief NMR review of indole alkaloids and in vitro cytotoxicity. Quim. Nova 31, 20–24. https://dx.doi.org/10.1590/S0100-40422008000100004. Perez, M.N., Fatima, B., Torrico, Abelardo Morales, 2012. J. Ethnopharmacol. 143,

Conceptualization - A.P.; S.M.; J.G.F.; L.T. Investigation - A.P.; D.P.; G.P.P.; L.T.; M.R.E.; K.G.M.; Resources - S.M.; H.S.B.; Writing - Original Draft – A.P. D.P. J.G.F.; Writing – Review and Editing, S.M.: L.T.; M.R.E.; H.B.; Supervision - S.M.; Funding Acquisition – S.M.; H.S.B; M.R.E. Acknowledgements The authors would like to thank the colleague Fabiana Agostini and research fellow Paulo Henrique Ancilaggo for the important participation in the present work. The authors thank the FAPERGS, CAPES and CNPq for financial support. References Adams Jr., , Wang, J.D., 2015. Control of pain with topical plant medicines. Asian Pac. J. Trop. Biomed. 25 (4), 268–273. https://doi.org/10.1016/S2221-1691(15)30342-7. Al-Habori, M., Al-Aghibari, A., Al-Mamary, M., 2002. Toxicological evaluation of Catha edulis leaves: a long term feeding experiment in animals. J. Ethnopharmacol. 83 (3), 209–217. Ali-Khan, S.M., Ahmed, M.N., Arifuddin, M., Rehman, A., Ling, M.P., 2018. Indole alkaloids and anti-nociceptive mechanisms of Tabernaemontana divaricata (L.) R. Br. flower methanolic extract. Food Chem. Toxicol. 118, 953–962. https://doi.org/10. 1016/j.fct.2018.06.007. Almeida, L., Cintra, A.C.O., Veronese, E.L.G., Nomizoa, A., Franco, J.J., Arantes, E.C., Giglio, J.R., Sampaio, S.V., 2004. Anticrotalic and antitumoral activities of gel filtration fractions of aqueous extract from Tabernaemontana catharinensis (Apocynaceae). Comp. Biochem. Physiol. 137, 19–27. https://doi.org/10.1016/j.cca. 2003.10.012. Antunes, S.M., Monico, L.S.M., 2015. Depressão, ansiedade e stress em doentes deprimidos: Estudo com a EADS-21. Int. J. Dev. Educ. Psychol. INFAD Revista de Psícologia 1. https://doi.org/10.1590/s0066-782x2009001000007. Araujo, A.R., Kascheres, F., Fujiwara, A.J., Marsaioli, 1984. Catharinensine, an onxindole alkaloid from Peschiera catharinensis. Phytochemeristry 23, 2359–2363. https://doi. org/10.1016/S0031-9422(00)80552-X. Arome, D., Enedige, C., Ameh, S., Agbafor, A., Mbonne, E., 2014. Absence of anxiolytic activity of Sarcocephalus latifolius extract. Orig. Artic. 5, 4–7. https://doi.org/10. 4103/0976-9234.136772. Batina, M.F.C., Cintra, A.C.O., Veronese, E.L.G., Lavrador, M.A.S., Giglio, J.R., Pereira, P.S., Dias, D.A., Franca, S.C., Sampaio, S.V., 2000. Inhibition of the lethal and myotoxic activities of Crotalus durissus terrificus venom by Tabernaemontana catharinensis: identification of one of the active components. Planta Med. 66, 424–428. https://dx.doi.org/10.1055/s-2000-8577. Boligon, A., Piana, M.T., Kubiça, T.F., Mario, D.N., Dalmolin, T.V., Bonez, P.C., Weiblen, R., Lovato, L., Alvez, S.H., Campos, M.M.A., Athayde, M.L., 2014. HPLC analysis and antimicrobial, antimycobacterial and antiviral activities of Tabernaemontana catharinensis A. DC. J. Appl. Biomed. 13, 1–12. https://doi.org/10.1016/j.jab.2014.01.004. Bradford, M.M., 1976. Rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248–254. https://doi.org/10.1016/0003-2697(76)90527-3. Braga, R.M., Filho, H.F.L., Reist, F.A.M., 1984. NMR analysis of alkaloids from Peschiera fuchsiaefolia. Phytochemistry 175–178. https://doi.org/10.1016/0031-9422(84) 83102–7. Brum, E.D.S., Moreira, L.D.R., da Silva, A.H.R., Boligon, A.A., Carvalho, F.B., Athayde, M.L., Brandão, R., Oliveira, S.M., 2016. Tabernaemontana catharinensis ethyl acetate fraction presents antinociceptive activity without causing toxicological effects in mice. J. Ethnopharmacol. 19, 115–124. https://dx.doi.org/10.1016/j.jep.2016.06. 036. Capaz, F.R., Vanconcellos, L.E., de Moraes, S., Neto, J.P., 1981. The open field: a simple method to show ethanol withdrawal symptoms. Arch. Int. Pharmacodyn. Ther. 251, 228–236. Cardoso, C.A.L., Vilegas, W., 1999. Droplet counter-current chromatography of indole alkaloids from Tabernaemontana hilariana. Phytochem. Anal. 10, 60–63. https://doi. org/10.1002/(SICI)1099-1565(199903/04)10:23.0.CO;2-Y. Cava, M.P., Tjoa, S.S., Ahmed, Q.A., Da Rocha, A.I., 1968. Alkaloids of Tabernaemontana Riedelii and T. rigida. J. Organomet. Chem. 33 (3), 1055–1059. Chuang, C., Chen, J.H., 2013. Photooxidation and antioxidant responses in the earthworm Amynthas gracilis exposed to environmental levels of ultraviolet B radiation. Comp. Biochem. Physiol. 164 (3), 429–437. https://doi.org/10.1016/j.cbpa.2012.11. 006. da Silva, A.R.H., Moreira, L.D.R., Brum, E.D.S., de Freitas, M.L., Boligon, A.A., Athayde, M.L., Roman, S.S., Mazzanti, C.M., Brandão, R., 2014. Biochemical and hematological effects of acute and sub-acute administration to ethyl acetate fraction from the stem bark Scutia buxifolia Reissek in mice. J. Ethnopharmacol. 153, 908–916. https:// dx.doi.org/10.1016/j.jep.2014.03.063. Fang, X., Shao, L., Zhang, H., Wang, S., 2005. CHMIS-C: a comprehensive herbal medicine information system for cancer. J. Med. Chem. 48, 1481–1488. https://dx.doi.org/10. 1021/jm049838d.

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D. Pergher, et al. 599–603. Queiroz, A.C., de Lira, D.P., Dias, T. de L., de Souza, E.T., da Matta, C.B., de Aquino, A.B., Silva, L.H., da Silva, D.J., Mella, E.A., Agra, M. de F., Filho, J.M., de Araújo-Júnior, J.X., Santos, B.V., Alexandre-Moreira, M.S., 2010. The antinociceptive and anti-inflammatory activities of Piptadenia stipulacea Benth. (Fabaceae). J. Ethnopharmacol. 128 (2), 377–383. https://doi.org/10.1016/j.jep.2010.01.041. Quintans, J.S.S., Antoniolli, A.R., Almeida, J.R.G.S., Valter, J., Quintans-Junior, L., 2014. Natural products evaluated in neuropathic pain models. A systematic review. Basic Clin. Pharmacol. Toxicol. 114, 442–450. https://doi.org/10.1111/bcpt.12178. Rates, S.M.K., Schapoval, E.E.S., Souza, I.A., Henriques, A.T., 1993. Chemical constituents and pharmacological activities of Peschiera australis. Int. J. Pharmacogn. 31. https:// doi.org/10.3109/13880209309082955. Re, R., et al., 1999. Antioxidant activity applying and improved ABTS radical cation decolorization assay. Free Radic. Biol. Med. 26 (910), 1231–1237. Regalado, A.I., Mancebo, B., Paixão, A., López, Y., Merino, N., Sánchez, L.M., 2017. Antinociceptive activity of methanol extract of Tabebuia hypoleuca (C. Wright ex Sauvalle) Urb. Stems. Med. Princ. Pract. 26, 68–374. https://doi.org/10.1159/ 000478015. Salgueiro, A.C., Folmer, V., da Rosa, H.S., Costa, M.T., Boligon, A.A., Paula, F.R., Roos, D.H., Puntel, G.O., 2016. In vitro and in silico antioxidant and toxicological activities of Achyrocline satureioides. J. Ethopharmacol. 194, 6–14. https://dx.doi.org/10.1016/ j.jep.2016.08.048. Silva, J.C., Saraiva, S.R.G. de Lima, de Oliveira Junior, R.G., Almeida, J.R.G. da Silva, 2013. Modelos experimentais para avaliação da atividade antinociceptiva de produtos naturais: uma revisão. Revista Brasileira de Farmácia 94 (1), 18–23. Souza, G.E., Ferreira, S.H.H., 1985. Blockade by antimacrophage serum of the migration of PMN neutrophils into the inflamed peritoneal cavity. Agents Actions 17 (1), 97–103.

Spencer, P.S.J., Sewell, R.D.E., 1976. Antinociceptive activity of narcotic agonist and partial agonist analgesics and other agents in the tail immersion test in mice and rats. Neuropharmacology 15, 683–688. Taesotikul, T., Panthong, A., Kanjanapothi, D., Verpoorte, R., Scheffer, J.J.C., 2003. Antiinflammatory, antipyretic and antinociceptive activities of Tabernaemontana pandacaqui Poir. J. Ethnopharmacol. 84 (1), 31–35. Tjølsen, A., Hole, K., 1997. Animal models of analgesia. In: In: Dickenson, A., Besson, J. (Eds.), The Pharmacology of Pain, vol. 130. pp. 1–2. Trongsakul, S., Panthong, A., Kanjanapothi, D., Taesotikul, T., 2003. The analgesic, antipyretic and anti-inflammatory activity of Diospyros variegata Kruz. J. Ethnopharmacol. 85 (2–3), 221–225. https://doi.org/10.1016/S0378-8741(03) 00020-5. Van Beek, T.A., Verpoortem, R., Svendsenm, A.B., Leeuwenbergm, A.J., Bissetm, N.G., 1984. Tabernaemontana L. (Apocynaceae): a review of its taxonomy, phytochemistry, ethnobotany and pharmacology. J. Ethofarmacol. 10 (1), 1–156. Wills, E.D., Rotblat, J., 1966. The formation of peroxides in tissue lipids and unsaturated fatty acids by irradiation. Int. J. Radiat. Biol. Relat. Stud. Phys. Chem. Med. 8, 551–567. Woolfe, G., MCDonald, A.D., 1944. The evaluation of the analgesic action of pethidine hydrochloride (demerol). J. Pharmacol. Exp. Ther. 80 (3), 300–307. Yamaguchi, T., Takamura, H., Matoba, T., Terao, J., 1998. HPLC method for evaluation of the free radical-scavenging activity of foods by using 1,1-diphenyl-2-picrylhydrazyl. Biosci. Biotechnol. Biochem. 62 (6), 1201–1204. https://doi.org/10.1271/bbb.62. 1201. Yang, M., Wang, X., Guan, S., Xia, J., Sun, J., Guo, H., Guo, D.A., 2007. Analysis of triterpenoids in ganoderma lucidum using liquid chromatography coupled with electrospray ionization mass spectrometry. J. Am. Soc. Mass Spectrom. 18 (5), 927–939. https://doi.org/10.1016/j.jasms.2007.01.012.

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