Chemical Constituents of Psychotria nemorosa Gardner and Antinociceptive Activity

Table S1. Fractions from silica gel 60 (70-230 mesh) column chromatography from EE P. nemorosa leaves Number Fraction Elution Mass / g 1 1 (1-3) hexane 0.112 2 5 (4-5) hexane/EtOAc 5% 0.150 3 8 (6-8) hexane/EtOAc 10% 0.132 4 9 (9-10) hexane/EtOAc 10% 0.050 5 11 (11-13) hexane/EtOAc 10% 0.010 6 15 (14-15) hexane/EtOAc 20% 0.003 7 16 hexane/EtOAc 20% 0.035 8 18 (17-18) hexane/EtOAc 1:1 0.082 9 19 (19-21) hexane/EtOAc 1:1 0.067 10 22 (22-25) hexane/EtOAc 1:1 0.005 11 26 (26-29) EtOAc 1.691 12 32 (30-35) MeOH 2.826   Total 5.081


Introduction
Herbal drugs have been used since ancient times as medicines, for the treatment of a range of diseases.Many modern drugs have been evolved from ethnopharmacological approaches. 1,2Discovery of novel bioactive compounds from plants is a challenging, laborious, expensive process and requires long time to isolate and characterize an active compound.If the isolated bioactive is a known compound, then the time and resources spent on it will be quite a wasted effort. 3To overcome these issues, new techniques for direct identification of bioactive natural products from extracts were developed.Recent high throughput assays technologies to study plant extracts for biological activity has intensified the need for appropriate dereplication strategies. 4Dereplication is the process that allows a rapid identification of secondary metabolites in crude extracts by distinguishing previously identified compounds from novel ones.The dereplication process involves separation of single metabolites by chromatographic methods, identification by spectroscopic methods, bioassay for evaluation of the biological activity, and search in databases to verify the novelty of these compounds. 5yphenated chromatographic techniques possess characteristics that include efficient separation and rapid identification, among several other advantages in comparison with other analytical methods.These techniques bring significant improvements in the identification of novel natural compounds in complex matrices. 6,7sychotria comprises an important genus of Rubiaceae family and contains approximately 1700 species of flowering plants with tropical and subtropical distribution. 8he polyphyly of the genus has been demonstrated through recent worldwide molecular phylogenetic studies.Razafimandimbison et al. 9 established that Psychotria includes all its allied genera, rendering the tribe Psychotrieae monogeneric.][16][17] Psychotria nemorosa Gardner (Rubiaceae), popularly known as "casca d'anta", is a native terrestrial substrate shrub measuring 1-2 m in height with endemic growth in Brazil, found in Rain Forests in the states of Bahia, Paraíba (Northeast), Goiás (West-Central), Espírito Santo, Minas Gerais, Rio de Janeiro, São Paulo (Southeast), Paraná, Santa Catarina and Rio Grande do Sul (South). 18It is characterized by persistent bilobed stipules containing 8 to 10 arched secondary ribs on each side and visibles cross linked tertiary ribs.Flowering and fruiting occurs in January and March, respectively. 19iterature review showed that the extracts of many Psychotria species have anti-inflammatory and analgesic activity, and preliminary tests pointed to alkaloids as the major responsible for the effect, besides other compounds. 20,21Pain is a protective mechanism, triggered by potentially injurious stimuli, that influences the quality of life and is directly related to a high amount of medical licenses all over the world. 22,23Usually, the therapeutic profile is based on analgesic and anti-inflammatory drugs that present several side effects as ulcers, vomiting, tolerance or dependence. 24Due to these problems, there is continuous interest in the search of new substances that could act with lesser side effects.
As far as we know, no record on phytochemical studies nor biological approach of P. nemorosa is described in scientific literature.So, in this work, our objective was to investigate the chemical composition of the P. nemorosa leaves on a metabolomic approach and its antinociceptive property in mice.

General experimental procedures
All solvents were high-grade purity purchased from Tedia (Brazil).Infrared (IR) spectra were recorded on a Nicolet Magna IR 760 spectrometer using KBr pellets.Column chromatography was performed over silica gel (25 g, 200-300 mesh, Silicycle, Canada) and thin layer chromatography (TLC) on precoated silica gel GF 254 aluminum plates (Merck, Germany).Spots were visualized by spraying with acidic cerium (IV) sulfate reagent followed by heating at 110 °C and usual specific reagents for different classes of natural product.

Extraction and isolation
Dried leaves (60 g) were exhaustively extracted with a 90% aqueous ethanol (EtOH) solution at room temperature for 7 days.The resulting extracts were combined and concentrated under low pressure at 40 °C in a rotaevaporator to yield a dark green liquid (crude ethanolic extract (EE), 7 g).EE (1.0 g) was dissolved in MeOH and subjected to silica gel column chromatography (CC) (21 × 2.5 cm) eluted with hexane, a gradient of hexane/ethyl acetate (EtOAc) (5, 10, 20, 50 and 100%) and methanol (MeOH) to afford thirty-five fractions, which were combined in twelve resulting fractions according to TLC analysis (Table S1, Supplementary Information).

Gas chromatography-mass spectrometry (GC-MS)
GC-MS analyzes were performed on a gas chromatograph coupled to a mass spectrometry (GCMS-QP 2010 Ultra, Shimadzu, Kyoto, Japan) equipment with a quadrupole mass analyzer, electron impact ion source (at 70 eV), auto sampler AOC-20s and auto injector AOC-20i.Analyzes were carried out using a DB-1 capillary column of 30 m × 0.25 mm × 0.25 µm, helium as carrier gas at 1.0 mL min -1 .The GC temperature was programmed as: 60 °C (2 min), 60-120 °C (6 °C min -1 ), 120-290 °C (15 °C min -1 ) then 290 °C for 17 min, according to Patitucci et al. 25 Injector, ion source and interface temperatures were 280, 230 and 290 °C, respectively.Data acquisition was performed by Lab Solutions Software and data analysis by NIST MS search database.The sample injection volume was 1.0 µL, all diluted with EtOAc.The triterpenes were identified by comparison of the retention time with authentic standards.N-Octadecane was used as internal standard in gas chromatography coupled to a flame ionization detector (GC-FID) with the same conditions described above.
Similar UFLC equipment and chromatographic conditions were used, as described above, coupled to a mass spectrometer ESI-IT (Bruker Daltonicx, Billerica, MA, USA), fitted with an electrospray ionization source operating in positive and negative mode and ion-trap analyzer.Tandem mass spectrometry (MS 2 ) analyzes were developed in this equipment.The mass spectrometer parameters used were: capillary voltage, 3.5 kV; dessolvation temperature, 330 °C; gas flow, 10 L min -1 and pressure at 60 psi.Nitrogen was used as both drying and nebulizing gas.Amplitude fragmentation energy for MS/MS experiments was 0.70 V. Data analysis was carried out with Bruker Daltonics software version 4.1.

Animals
All experiments were performed with male Swiss Webster mice (20-25 g) obtained from our own animal facilities.Animals were kept in a room with controlled temperature 22 ± 2 °C for 12 h light/dark cycle with free access to food and water.Twelve hours before each experiment, the animals received only water, in order to avoid food interference with substances absorption.Animal care and research protocols were in accordance with the principles and guidelines adopted by the Brazilian College of Animal Experimentation (COBEA), approved by the Biomedical Science Institute/UFRJ, Ethical Committee for Animal Research, and received the number DFBCICB015-04/16.
Psychotria nemorosa fractions administration P. nemorosa (leaves) fractions were dissolved in dimethyl sulfoxide (DMSO) in order to prepare a stock solution at a concentration of 100 mg mL -1 .In all experiments, the final concentration of DMSO did not exceed 0.5% at which it has no effect per se.The crude extract (EE), hexane, hexane/ EtOAc 5%, hexane/EtOAc 10%, EtOAc and MeOH fractions were administered by oral gavage at 10, 30 or 100 mg kg -1 in a final volume 0.1 mL.The control group was composed by vehicle (ultrapure water), which was the same used to solubilize the EE and fractions that were administered on the day of the experiment.

Formalin-induced licking response
This procedure was similar to the method described by Gomes et al. 29 Mice received an injection of 20 µL of formalin (2.5% v/v) into the dorsal surface of the left hind paw.The time that the animal spent licking the injected paw was immediately recorded.The nociceptive and inflammatory response consists of the following two phases: the first phase lasts until 5 min after the formalin injection (first phase, neurogenic pain response), and the second phase occurs 15-30 min after the formalin injection (second phase, inflammatory pain response).The animals were pre-treated with oral doses of EE, hexane, hexane/ EtOAc 5% and hexane/EtOAc 10% fractions, EtOAc and MeOH 60 min before the administration of formalin.

Statistical analysis
Each experimental group consisted of 6 mice.The results are presented as mean ± standard deviation (S.D.).The area under the curve (AUC) was calculated using Prism Software 5.0 (GraphPad Software, La Jolla, CA, USA).Statistical significance between groups was determined using the application of analysis of variance (ANOVA) followed by Bonferroni's test.p-Values less than 0.05 were considered to be significant.

Results and Discussion
The crude ethanolic extract (EE) from leaves of P. nemorosa was fractionated in a silica gel open column chromatography affording twelve fractions, all analyzed by TLC.Each fraction was evaluated by GC-MS or liquid chromatography coupled to mass spectrometry (LC-MS) on a metabolomic perspective based on their polarities.Mass spectrometry (MS) allows a direct screening of the compounds, providing structural information without the need of laborious isolation since each family of metabolites may have a characteristic mass fragmentation pattern and retention times in GC-qMS and typical UV absorption and MS/MS profile in LC-DAD-MS/MS systems. 30 addition, fractions and EE were evaluated for antinociceptive properties on formalin-induced licking response in mice due the occurrence of two distinct phases of nociceptive behavior: one phase immediately after the injection, lasting for about 5 min, and the other phase starting approximately 20 min after the injection. 31he fractions resulting from the CC silica gel can be seen in Table S1 (Supplementary Information).Only those with sufficient amount to access biological activity were investigated in the phytochemical approach.To identify the less polar compounds, GC-MS was used and their profiles are shown in Figures 1A-1D.
GC-MS chemical compounds present in fractions 1, 5, 8 and 9 can be seen in Tables S2 and S3 (Supplementary Information).The MS of the compounds showed more than 90% of similarity with NIST database.These results attested to the major occurrence of cinnamic acid, sterols (campesterol, stigmasterol, γ-sitosterol and stigmast-4-en-3-one) and pentacyclic triterpenes such α/β-amyrone, α/β-amyrin acetate, 3,11-dioxoα/β-amyrene, lupeol and α/β-amyrin, besides some fatty acids, diterpene alcohols and hydrocarbons.Co-injection with authentic materials additionally confirmed the identification.It is important to report that fractions 15-22 were not investigated by GC-MS due to their increased polarity and also due to the small amounts for biological assays.Figure 2 shows the chemical structures of the compounds identified in this work.
Although Psychotria genus is characterized mainly as a source of alkaloids, the occurrence of terpenoids is well known.In P. yunnamensis, norisoprenoids and the monoterpenoid (6S)-menthiafolic acid were isolated, in addition to the ancorane sesquiterpene psycacoraone A. 32,33 Volatile compounds were also detected in P. leiocarpa. 34][38] Lupeol was found in P. vellosiana and P. mariniana. 17,35Phytochemical studies of P. adenophylla allowed the caracterization of other triterpenes such as bauerenol, bauerenol acetate, friedelin, betulin, ursolic acid, traces of αamyrin and betulinic acid. 39Ursolic acid was isolated from P. serpens. 40Besides P. adenophylla, αamyrin was also found in P. stachyoides. 37It should be emphasized that the occurrence of α/β-amyrone, α/β-amyrin acetate and 3,11-dioxo-α/β-amyrene is being reported for the first time in Psychotria genus.The mixture of α/β-amyrin (31a, 31b), previously purified from Protium sp.resin, 42 and lupeol (19), derived from Vellozia sp., 43 were subjected to classic esterification and oxidation reactions to give derivatives in good yields (Schemes 1 and 2).These reference compounds were used in co-injection assays or for comparison of retention time as an additional tool to confirm GC-MS results (Supplementary Information).
As α-amyrin acetate (20b) and lupeol acetate exhibit the same retention times in this condition (34.8 min), the mass fragmentation profile was determined to confirm the presence of α-amyrin acetate in fraction 5 (Supplementary Information).
Besides the triterpenes confirmation, GC-MS allowed us to detect a further series of hydrocarbons, long chain fatty acids and their esters.
Nowadays, ultra fast liquid chromatography/tandem mass spectrometry (UFLC-MS/MS) is one of the most convenient techniques for online characterization, due to its superior sensitivity, high selectivity, short run time and resolution power which allow direct screening of natural products. 44UFLC coupled to quadrupole-time-of-flight (Q-TOF) is widely applied in herb research and has brought a big convenience for qualitative analysis, allowing quick and effective data acquisition.In terms of the accurate mass measurement, it gives characteristic ions for the identification of molecular formula and the gas phase decomposition reactions activated by collision induction dissociation (CID) may furnish key information for a safe structural elucidation. 45,46he most polar fractions eluted with EtOAc and MeOH from the silica gel column chromatography of EE were analyzed by UFLC coupled with a Q-TOF and a quadrupole ion trap MS apparatus, to obtain high mass accuracy measurements of both parent and fragment ions, as also to deliver sufficient information to detect and reliably identify  [26][27][28] compounds in this complex mixture.Tables 1 to 4 show retention times, formulae, UV features, compound names, mass spectrum features in both positive and negative ions modes, and fragment ions results produced by MS 2 .
In the EtOAc fraction, it was possible to identify some compounds previously described in other Psychotria species, such as: loliolide, in P. cadigensis 47 and P. yunnanensis; 33 butin, also in P. yunnanensis; 33 epiloganin, as part of the alkaloid brachycerin in P. brachyceras 48 and deacetylasperuloside, in P. leiocarpa. 15The fraction eluted with MeOH yielded rutin and kaempferol-7-Oglucopyranoside, as in P. haianensis, 36 and epiloganin.Phenolic compounds closely related with syringol/vannilic alchool/hydroxytyrosol were described in P. yunnanensis. 33haracteristic UV spectra existed in different types of compounds, and can be an important tool to precisely identify isomers with distinguishable chromophores.Data from mass spectra, such as  49 MS and UV data are according to literature 50,51 (Table 1).Loliolide (m/z 197.1163, [M + H] + ) was identified by comparison of its mass spectra with previously reported values.Strong UV-Vis absorption at 218 nm suggested an α-β unsaturated ester/lactone. 52 1).
The occurrence of hordenine, resveratrol and ferulic acid are being reported for the first time in Psychotria genus.
This preliminary phytochemical screening by GC-MS and LC-MS proved to be an useful tool to identify secondary metabolites from P. nemorosa leaves, both polar and nonpolar bioactive compounds successfully mapped (Figure 2).

Antinociceptive activity
As shown in Figure 3, the EE of P. nemorosa leaves exhibited a signicant antinociceptive activity on formalininduced licking response test in mice, but the EE from branches did not (data not shown).The injection of formalin (2.5%) leads to a biphasic licking response of the injected paw.The first phase lasts until 5 min after injection due to a direct stimulation of nociceptors, and the second phase occurs between 15 and 30 min after formalin injection due to a combination of an inflammatory reaction in the peripheral tissue and changes in central processing. 31e anti-inflammatory non-steroidal drug acetylsalicylic acid (ASA) reduced the time by 13.0% and the opioid analgesic morphine by 44.8%.Ethanol extract (EE) inhibited in 64.4,62.5 and 67.2% the licking time at the doses of 10, 30, and 100 mg kg -1 , respectively (Figure 3), when compared with the vehicle group (42.4 ± 7.7) in the first phase.Analysis of the 2 nd phase of the response to formalin showed a significant inhibition at doses of 30 and 100 mg kg -1 , with the following inhibition values: 44.2 and 62.4%, respectively.The positive control groups ASA and morphine showed 46.7 and 33.3% of reduction in the licking response, respectively.In the search for compounds exhibiting antinociceptive activity, the fractions obtained from silica gel column chromatography were tested on formalin-induced licking response in mice, and all of them were able to inhibit the phases of response to formalin (Figures 4 and 5).Only hexane/EtOAc 10% (fraction 9 that contains the sterols) and hexane/EtOAc 20% were not tested due to the small amount.
Figure 4A shows that the hexane fraction (fraction 1) was able to significantly inhibit the first phase at the dose of 100 mg kg -1 (41.3% of inhibition) and in the second phase at the dose of 30 mg kg -1 (41.7% of inhibition).The hexane/ EtOAc 5% fraction (fraction 5) was able to significantly inhibit only the first phase at the dose of 30 and 100 mg kg -1 (45.7 and 40.1% of inhibition, respectively) (Figure 4B).The hexane/EtOAc 10% fraction (Fraction 8) was able to significantly inhibit the first phase at the doses of 30 and 100 mg kg -1 (47.2 and 55.6% of inhibition, respectively) and in the second phase at the doses of 10 and 30 mg kg -1 (50.0 and 55.3% of inhibition, respectively) (Figure 4C).
][83][84] Figure 5A shows that EtOAc fraction was able to significantly inhibit the first phase at the doses of 10, 30 and 100 mg kg -1 (61.7, 46.7 and 63.7% of inhibition, respectively) and in the second phase at the dose of 100 mg kg -1 (36.7% of inhibition).On the other hand, MeOH fraction was able to significantly inhibit the first phase and the second phase at the doses of 10, 30 and 100 mg kg -1 (71.2, 59.7 and 52.4%; 41.7, 63.5 and 59.5% of inhibition, respectively) (Figure 5B).This effect may be attributed, at least partially, to the phenolic compounds butin, 85 resveratrol, 86 ferulic acid 87 and the glycosilated iridoid loganin, 88 present in EtOAc fraction.Next, in the MeOH fraction, the great inhibition may be due to the presence of alkaloids, 21,89,90 flavonoids 91,92 and phenolic compounds. 79,93We also tested whether the extracts could cause some effect on motor activity.The results obtained after treatment of the mice with doses of 100 mg kg -1 did not show any effect on motor performance of mice (data not shown).
The model used to start the studies was the formalininduced licking response, as it can discriminate pain in its central and/or peripheral components; this test studies moderate and tonic pain. 94It has two distinct phases, which reflect two different types of pain.The first phase (neurogenic pain) is characterized by direct chemical stimulation of nociceptors, afferent fibers predominantly type C and, in part, type Aδ, while the second phase (inflammatory pain) is characterized by the appearance of a local inflammatory process, where various proinflammatory chemical mediators are released and that can be inhibited by nonsteroidal anti-inflammatory drugs. 95,96he results showed that the EE and MeOH fraction (10,

3.
Effects of EE extract from P. nemorosa on formalin-induced licking response in mice.Animals were pre-treated with different doses (10, 30 or 100 mg kg -1 , p.o.) of EE, ASA (200 mg kg -1 ), morphine (2.5 mg kg -1 ) or vehicle.The results are presented as mean ± S.D. (n = 6) of the time that the animal spent licking the formalin-injected paw.Statistical significance was calculated by ANOVA followed by Bonferroni's test.*p < 0.05 when compared to vehicle-treated mice; # p < 0.05 when compared to morphine-treated mice.30 and 100 mg kg -1 ) significantly inhibit both phases of the formalin-induced licking response.The fractions hexane (100 mg kg -1 ), hexane/EtOAc 5%, 10% and EtOAc (10, 30 and 100 mg kg -1 ) significantly inhibit the first phase.The results of the first phase suggest an antinociceptive activity that can result in both direct action on opioid receptors (predominantly type µ), present in primary afferent fibers (type C) or even an inhibition in the release of mediators such as serotonin, substance P, kinins, histamine and calcitonin gene-related peptide (CGRP).An inhibitory effect shown by EE (30 and 100 mg kg -1 ), hexane (30 mg kg -1 ) and hexane/EtOAc 10% (10 and 30 mg kg -1 ), EtOAc (100 mg kg -1 ) and MeOH (10, 30 and 100 mg kg -1 ) in the second phase of formalin may suggest an inhibition in the formation and/or release of arachidonic acid metabolites such as prostaglandins and leukotrienes and other inflammatory mediators such as bradykinin, histamine and serotonin, as well as cytokine, eicosanoids, kinins, glutamate and nitric oxide (NO). 94,95
Furthermore, to the best of our knowledge, this is the first work describing the phytochemical profile and antinociceptive properties of P. nemorosa leaves.The extract and fractions were able to produce an oral antinociceptive effect in acute pain model in mice (formalin-induced licking response).

Figure 5 .
Figure 5. Effects of P. nemorosa (A) EtOAc and (B) MeOH fractions on formalin-induced licking response in mice.Animals were pre-treated with different doses (10, 30 or 100 mg kg -1 , p.o.) offractions, ASA (200 mg kg -1 ), morphine (2.5 mg kg -1 ) or vehicle.The results are presented as mean ± S.D. (n = 6) of the time that the animal spent licking the formalin-injected paw.Statistical significance was calculated by ANOVA followed by Bonferroni's test.*p < 0.05 when compared to vehicle-treated mice; # p < 0.05 when compared to morphine-treated mice.
Gardner were collected in November (2012) at Serra dos Órgãos (Rio de Janeiro State, Southeast of Brazil, −22.464042, −43.011746).The access authorization number is 27035-4 emitted by Ministério do Meio Ambiente.A voucher specimen (Trovó 389 RB) was deposited at the herbarium of the Jardim Botânico do Rio de Janeiro and identified by Marcelo Trovó Lopes de Oliveira.
with their characteristic fragment ions reinforce identification.In our analyzes, butin (m/z 273.1108, [M + H] + ) was detected in positive mode showing that loss of H 2 O (m/z 255.09 [M + H -H 2 O] + ) is characteristic of two OH groups in ortho.Ions at m/z 245.05 [M + H -CO] + and m/z 229.08 [M + H -CO 2 ] + were observed, consistent with the literature.
Ions at m/z 179.06 and m/z 111.92 confirmed the loss of H 2 O [M + H -H 2 O] + , and also the loss of the ring adjacent to the lactone [M + H -H 2 O -C 5 H 8 ] + (Table

Table 2
) was consistent with fragments at m/z 357.20 and m/z 226.95 in the MS/MS spectrum, due to the loss of methoxy group and glucose moiety from the precursor ion, respectively.Elimination of methyl ester from m/z 226.95 [M -H -C 2 H 4 O 2 ] -with subsequent dehydration showed the major product ion at m/z 151.11 [M -H -C 2 H 4 O 2 -H 2 O] -. - 714.19and at m/z 369.17, due to loss of the sugar moiety [M + H -Glc] + .71Thealkaloid N-methyl-1,2,3,4-tetrahydro-β-carboline [M + H] + at m/z 187.1226 yielded ions at m/z 158.09 and m/z 144.16 due to loss of CH 3 N [M + H -CH 3 N] + and C 2 H 5 N [M + H -C 2 H 5 N] + fragments, respectively.

Table 1 .
Characterization of the chemical composition of EtOAc fraction of P. nemorosa leaves by UFLC-DAD-ESI-MS 2 in positive mode

Table 3 .
Characterization of the chemical composition of MeOH fraction of P. nemorosa leaves by UFLC-DAD-ESI-MS 2 in positive mode

Table 4 .
Characterization of the chemical composition of MeOH fraction of P. nemorosa leaves by UFLC-DAD-ESI-MS 2 in negative mode Structures of the identified compounds from P. nemorosa leaves.Vol.28,No. 5, 2017 Figure 2. Structures of the identified compounds from P. nemorosa leaves.(cont.)