Phytochemical study and anti-inflammatory effect of Psychotria stenocalyx ( Rubiaceae )

Article history: Received on: 31/01/2017 Accepted on: 23/03/2017 Available online: 30/04/2017 In this study, an in vivo anti-inflammatory assay was conducted on Psychotria stenocalyx ethanol extract of leaves, evaluating this effect on cell migration and exudate formation in a murine carrageenan-induced model of pleurisy. The extract showed the optimum dose, at 400 mg/kg, inhibiting cellular and vascular parameters: total leucocytes due to polymorphonuclear migration and exudate formation (p < 0.01). A phytochemical study was conducted using the hyphenated techniques HPLC-DAD-SPE/NMR and HPLC-DAD-ESI-TOF-HRMS, in which seven monoterpene indole alkaloids were identified: lyaloside 1, (E)-O-(6′)-cinnamoyl-4”-hydroxy-3”,5”dimethoxy-lyaloside 2, strictosamide 3, pauridianthoside 4, vallesiachotamine lactone 5, E-vallesiachotamine 6 and Z-vallesiachotamine 7, in the alkaloid fraction, obtained by an acid-base extraction on P. stenocalyx crude extract. This is the first study developed with this species.


INTRODUCTION
Psychotria L. is the largest genus in the tribe Psychotrieae (Rubiaceae) and is comprised of around 2000 species worldwide, distributed in tropical regions (Nepokroeff et al., 1999).
Psychotria stenocalyx Müll.Arg. is a shrub that grows up to two meters, and occurs in coastal tropical forests of South and Southeast Brazil, ranging from Rio de Janeiro to Santa Catarina (Taylor et al., 2015).It is popularly known as "gandiúva-d 'anta" and "café-d'anta" (Dillenburg et al., 1985).
In view of the many reports that cite the important biological activity of Psychotria, the aim of this study was to carry out an in vivo anti-inflammatory assay on the leaf extract, and to isolate and structurally elucidate monoterpene indole alkaloids on Psychotria stenocalyx.

Plant Material
The aerial parts of Psychotria stenocalyx were collected in June 2012 in Florianópolis, state of Santa Catarina, Brazil.The plant was identified by botanist Ademir Reis and a voucher was deposited in the "Barbosa Rodrigues" Herbarium in Itajaí, state of Santa Catarina, Brazil, under number HBR55428.

Extraction of plant material
Air-dried and powdered leaves of P. stenocalyx (449 g) were extracted with ethanol 96% (3 x 72h) at room temperature.After filtration, the extract was concentrated to obtain the crude extract, CE (5.54 g/100g dried leaves) for use in the inflammation study.The CE was dissolved in 0.01 mol/L HCl H 2 O/EtOH (80:20 v/v) and then stored in a refrigerator.After 24 h, the extract was filtered and exhaustively extracted with ethyl acetate to remove the nonpolar constituents.The acid extract was alkalinized with 10% NH 4 OH in water up to pH 11 and extracted again with ethyl acetate yielding the purified alkaloid fraction (3.24 g/100g CE) which was used for the isolation and characterization of alkaloids.

Animals
In the present protocol, one month old female Swiss mice weighing 20 to 25 g were housed under standardized conditions (20 ± 2 ºC, 12 h light/dark cycles) with free access to chow and water.The experiments were designed to minimize the animal suffering and the 3R recommendations were followed (replacement, reduction and refinement) (Flecknell 2002).The experiments were carried out on the ethanolic crude extract of P. stenocalyx according to the regulations of the Brazilian College of Animal Experimentation (COBEA) and are in accordance with the rules of the Committee for Ethics in Animal Research of the Federal University of Santa Catarina (CEUA -PP00965).

Experimental design of the murine model of pleurisy
The pleurisy induction was performed as previously reported (Saleh et al., 1996).Briefly, on the day of the experiments the animals were randomly divided into different groups (n = 6animals/group) and challenged with Evans Blue dye solution (25 mg/kg) administered intravenously (i.v).Next, the groups received the appropriate treatment as follows: (a) blank control (S), animals pre-treated with intraperitoneal (i.p.) saline (vehicle) 0.5 h before pleurisy induction with saline; (b) negative control (Cg), animals treated with i.p. saline (vehicle) 0.5 h before pleurisy induction with carrageenan; (c) positive control (Dex), animals pre-treated with i.p. dexamethasone 0.5 mg/kg before pleurisy induction with carrageenan; and (d) experimental groups, animals pre-treated with i.p.Psychotria stenocalyx crude extract at doses ranging from 100 to 400 mg/kg before pleurisy induction with carrageenan.
After 0.5 h of intraperitneal (i.pl) treatment with saline, each group received a single injection of a λ-carrageenan solution (0.1 mL, 1%) from the right side of the thoracic cavity, to induce pleurisy induction, except for the blank control group (S), which received an i.pl.sterile saline injection (0.1 mL).Four hours after pleurisy induction, the animals were euthanized using an overdose of xylazine and ketamine (50 mg/kg and 250 mg/kg, i.p.), and the pleural cavity was exposed and washed with 1.0 mL of sterile phosphate buffered saline (PBS, pH 7.2) (Laborclin, Pinhais, Paraná, Brazil) containing heparin (20 IU/mL).The pleural fluid was used to measure the inflammatory parameters: total and differential leukocyte count and exudate concentration.

Quantification of the leukocyte content and exudation
To quantify the leukocyte content, pleural fluid samples were submitted to a veterinarian automatic counter (BC-2800 Vet, Mindray, Nanshan, Shenzhen, China).For the differential leukocyte count, cytospin preparations from the exudates were stained with May-Grünwald-Giemsa.The results were expressed as the total number of cells (× 10 6 cells/mL).
The exudate quantification was performed indirectly by measuring the amount of Evans blud dye in the pleural cavity.The fluid samples were centrifuged (300 g for 5 min) (SorvallTMST40, ThermoScientific®, Swedesboro, New Jersey, USA) and 200 µL of the supernatant was transferred to a 96 well-ELISA plate.The amount of dye was estimated by a colorimetric measurement at 620 nm, using an ELISA plate reader (OrganonTeknika, Roseland, New Jersey, USA).The results were expressed in μg/mL by interpolation from an Evans blue dye standard curve ranging from 0.01 to 50μg/mL.

Statistical analysis
All data were expressed as mean ± SEM.Statistical differences between groups were tested by one-way analysis of variance (ANOVA) followed by Dunnett's post-hoc test.The results were analyzed using GraphPad Prism v5.0 software (GraphPad Software Inc., San Diego, California, USA) and values of P < 0.05 were considered significant.

HPLC analyses and isolation of compounds
The alkaloid fraction of P. stenocalyx crude extract (50 mg) was dissolved in 1 mL MeOH.The solution was filtered through a PVDF membrane syringe filter (25 mm, 0.45 μm; Tedia Brazil) before HPLC analysis.A reversed-phase Luna C18 (Phenomenex, 150 × 4.6 mm, 3 μm, 100 Å) was used for the chromatographic separation.Gradient elution was performed using a combination eluent (A) consisting of H 2 O/MeCN (95:5, v/v), and organic eluent (B) consisting of H 2 O/MeCN (5:95, v/v), both acidified with 0.1% formic acid (Tedia Brazil).The gradient method was: 0 min, 15% B; 60 min, 65% B; 65-75 min, 100% B; 76-85 min, 15% B. Elution rate 0.5 mL/min.The injection volume was 10 μL and UV traces were monitored at 254, 280, and 330 nm.HPLC-DAD-SPE/NMR analysis was carried out on an Agilent 1200 HPLC.Compounds 3-7 were adsorbed on solidphase extraction cartridges (HySphere Resin GP, 10 mm × 2 mm, 10 μm spherical polydivinylbenzene stationary phase) using an automatic cartridge exchanger (Bruker Biospin GmbH).Twenty consecutive chromatographic runs were performed, with 10 μL injections and a flow rate of 1.0 mL/min.After the adsorption process, the cartridges were dried with nitrogen for 30 min to remove residual solvent.Deuterated MeOH-d4 (99.8% D) was used to elute the compounds from the SPE cartridges directly into NMR tubes (Bruker,3 mm o.d.).NMR experiments were performed with a Bruker Avance III system ( 1 H operating frequency of 600.13 MHz) equipped with a Bruker SampleJet sample changer and a cryogenically cooled gradient inverse tripleresonance 5.0 mm TCI probe-head (Bruker Biospin) optimized for 1 H and 13 C observation.Icon NMR (ver.4.2, Bruker Biospin) was used to control the automated acquisition of NMR data, which were then processed using Topspin (version 3.0, Bruker Biospin).The HRMS and UV spectra were obtained in positive mode in a HPLC-DAD-ESI-TOF-MS instrument consisting of a Shimadzu Prominence LC-20AD UFLC system (Kyoto, Japan), using a Tpiece splitter to direct 1% of the HPLC eluate to a micrOTOF-Q II mass spectrometer (Bruker Daltonik, Bremen, Germany) equipped with an electrospray ionization interface.Mass spectra were acquired in positive-ion mode, using a drying temperature of 200 °C, capillary voltage of 4500 V, nebulizer pressure of 3.0 bar, and drying gas flow of 12 L/min.

Anti-inflammatory effect
The inflammatory model adopted in the present work uses carrageenan as the phlogistic agent, to trigger an inflammatory reaction in the pleural cavity and the consequent damage associated with this process.In this specific model, it is possible to evaluate the cell migration, mainly polymorphonuclear, and exudate formation that is present in several acute inflammatory conditions (Luz et al., 2016;Saleh et al., 1996;Schmid-Schönbein, 2006).
Based on these results, we consider 400 mg/kg to be the optimum dose, as this dose was able to inhibit cell migration and exudate formation triggered by the phlogistic agent in this pleurisy model.

Phytochemical characterization of the alkaloids
In order to isolate and/or identify the compounds from P. stenocalyx crude extract, an acid-base extraction was performed to obtain a purified alkaloid fraction.The HPLC chromatogram at 280 nm showed the presence of nine major compounds, of which seven were identified ( Fig) .The UV spectra displayed by the peaks for these compounds are consistent with β-carboline (1, 2 and 4), tetrahidroβ-carboline (3) (Passos et al., 2013a) and vallesiachotamine-like nuclei (5-7) (Djerassi et al., 1966).
The alkaloids lyaloside 1 and (E)-O-(6′)-cinnamoyl-4"hydroxy-3",5"-dimethoxy-lyaloside 2 were identified without isolation by their UV and HRMS data compared to the data available in the literature (Valverde et al., 1999;Passos et al., 2013a).The hyphenated technique HPLC-DAD-SPE was used to isolate the peaks 3-7 of the chromatogram, which was characterized by UV, HRMS, and 1D and 2D NMR spectra compared with data available in the literature.
The 1 H NMR spectra of all isolated compounds showed, due to the presence of the benzene ring of the indole system, four aromatic hydrogens: two doublets (δ H9 and δ H12 ) ortho-coupled with two double-doublets (δ H10 and δ H11mutually ortho-coupled).All the couplings were confirmed by the 2D COSY spectrum.For the β-carboline-like alkaloid (4), the 1 H NMR spectra showed two doublets (δ H5 -δ H6 ), while for the tetrahidro-β-carboline-like (3) and vallesiachotaman-like alkaloid (5, 6, and 7) it showed four signals (δ H5a-b -δ H6a-b ) presenting multiplicities coherent with the pattern AA'BB' coupling system.The glycosyl alkaloids 3 and 4 showed the signal of the anomeric hydrogen as a doublet with coupling constant coherent with a β-glucose (J ≈ 8 Hz).The HMBC spectrum showed correlations between C21-H1' and C1'-H21, confirming that the glucoside was linked to the acetal carbon C21.
The isomers E/Z-vallesiachotamine, 6 and 7, were distinguished by the difference between their olefinic H19 signals in the 1 H NMR spectra.The E-isomer showed a quartet at δ H19 6.79 ppm while for the Z-isomer, there was a quartet at δ H19 6.58 ppm.The olefinic hydrogen is relatively more deshielded in the Eisomer due to the anisotropy of the carbonyl C21 of the aldehyde group.On the other hand, in the Z-isomer, the anisotropy of the aldehyde leads to a more deshielded 18-methyl group.Thus, a doublet at δ H18 2.10 ppm was observed for the E-isomer and at δ H18 2.20 ppm for the Z-isomer (Waterman et al., 1982).The 1:1 ratio of these E/Z isomers was calculated by the integration of these correspondent peaks in the HPLC chromatogram at λ max 290 nm.For the vallesiachotamine lactone 5, the NMR data were quite similar to compounds 6 and 7, except for the signals of the α-βunsaturated γ-lactone (δ CH18-21 ), linked to the rest of molecule by the carbons C15-C20.

Fig. 1 :
Fig.1: Psychotria stenocalyx leaf extract (100-400 mg/kg)effect on (a) leukocyte migration, (b) polymorphonuclear migration, and (c) exudate concentration when administered intraperitoneally 0.5 h prior to carrageenan induced pleurisy in mice.S: blank control group, animals pre-treated with intraperitoneal (i.p.) saline 0.5 h before pleurisy induction with saline; Cg: negative control, animals treated with i.p. saline 0.5 h before pleurisy induction with carrageenan; Dex: positive control, animals pre-treated with i.p. dexamethasone 0.5 mg/kg before pleurisy induction with carrageenan; Psychotria stenocalyx: experimental groups, animals pre-treated with i.p.Psychotria stenocalyx extract at doses ranging from 100 to 400 mg/kg before pleurisy induction with carrageenan.The results are expressed as mean ± SEM.N = 6 animals/group.** p< 0.01 compared to Cg group, *** p< 0.001 compared to Cg group, ### p< 0.001 compared to S group.

Fig.
Fig. The compiled data on retention time, mass and UV spectra for all compounds are given inTable 1.The 1 H NMR spectra of all isolated compounds showed, due to the presence of the benzene ring of the indole system, four aromatic hydrogens: two doublets (δ H9 and δ H12 ) ortho-coupled with two double-doublets (δ H10 and δ H11mutually ortho-coupled).All the couplings were confirmed by the 2D COSY spectrum.For the β-carboline-like alkaloid (4), the 1 H NMR spectra showed two doublets (δ H5 -δ H6 ), while for the tetrahidro-β-carboline-like (3) and vallesiachotaman-like alkaloid (5, 6, and 7) it showed four signals (δ H5a-b -δ H6a-b ) presenting multiplicities coherent with the pattern AA'BB' coupling system.The glycosyl alkaloids 3 and 4 showed the signal of the anomeric hydrogen as a doublet with coupling constant coherent with a β-glucose (J ≈ 8 Hz).The HMBC spectrum showed correlations between C21-H1' and C1'-H21, confirming that the glucoside was linked to the acetal carbon C21.The isomers E/Z-vallesiachotamine, 6 and 7, were distinguished by the difference between their olefinic H19 signals in the 1 H NMR spectra.The E-isomer showed a quartet at δ H19 6.79 ppm while for the Z-isomer, there was a quartet at δ H19 6.58 ppm.The olefinic hydrogen is relatively more deshielded in the Eisomer due to the anisotropy of the carbonyl C21 of the aldehyde group.On the other hand, in the Z-isomer, the anisotropy of the aldehyde leads to a more deshielded 18-methyl group.Thus, a doublet at δ H18 2.10 ppm was observed for the E-isomer and at δ H18 2.20 ppm for the Z-isomer(Waterman et al., 1982).The 1:1 ratio of these E/Z isomers was calculated by the integration of these correspondent peaks in the HPLC chromatogram at λ max 290 nm.For the vallesiachotamine lactone 5, the NMR data were quite similar to compounds 6 and 7, except for the signals of the α-βunsaturated γ-lactone (δ CH18-21 ), linked to the rest of molecule by the carbons C15-C20.

Table 1 :
Description of the main peaks observed in the HPLC-DAD-ESI-TOF-MS analyses performed for the alkaloid fraction of P. stenocalyx leaf extract.