Chemical composition, antioxidant and antinociceptive properties of L ...

Chemical composition, antioxidant and antinociceptiveproperties of Litchi chinensis leavesRosana C.L. Castellain, Marluci Gesser, Fernanda Tonini, Rafael V. Schulte, Kely Z. Demessiano,Fellippe R. Wolff, Franco Delle-Monache, Daisy J.A. Netz, Valdir Cechinel-Filho,Rilton Alves de Freitas, Márcia M. de Souza and Christiane Meyre-Silva

Centro de Ciências da Saúde, Universidade do Vale do Itajaí (UNIVALI), Itajaí, Santa Catarina, Brazil

Keywordsantinociceptive; antioxidant activity;L. chinensis; lipid peroxidation; phenoliccompounds

CorrespondenceChristiane Meyre-Silva, Programa de Mestradoem Ciências Farmacêuticas, Universidade doVale do Itajaí (UNIVALI), Núcleo deInvestigações Químico-Farmacêuticas(NIQFAR)(UNIVALI), Rua Uruguai, 458, Itajaí,Santa Catarina, 88.302-202, P.O. Box 360,Brazil.E-mail: [email protected]

Received April 2, 2014Accepted July 18, 2014

doi: 10.1111/jphp.12309

Abstract

Objectives Litchi chinensis has been traditionally used in folk medicine to treatseveral ailments. In this study, we investigated the chemical composition, antioxi-dant and antinociceptive activity of L. chinensis leaves.Methods The antioxidant capacity of the extract, fraction and compounds wasevaluated using the 1,1-diphenyl-picrylhydrazyl (DPPH) and 2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) assays, and the liposomemodel with peroxyl radicals generated by 2,2′-azobis (2-amidinopropane)dihydrochloride radical. The pharmacological models of acute nociception used inmice were: writhing test with acetic acid (AA), hotplate (HP), glutamate (GLU),capsaicin (CP) and formalin (FM) tests.Key findings The main compounds isolated were procyanidin A2 (PA2),procyanidin B2 (PB2) and (–)-epicatechin. The biochemical features of the crudeextracts and their ethyl acetate fraction (EtOAcFR) presented high antioxidantactivity, and the antioxidant activity of PA2 and PB2 was remarkably high, withDPPH and ABTS. The crude methanol extract (MeOHEXTR), EtOAcFR and PB2were effective in reducing nociception in FM and HP models. MeOHEXTR andEtOAcFR treatments also reduced pain induced by GLU and AA. In the CP model,only EtOAcFR and PB2 were effective.Conclusions The results demonstrate the antinociceptive and antioxidant ofMeOHEXTR, EtOAcFR and PB2.

Introduction

Litchi chinensis, known as litchi, is a subtropical tree withhigh commercial value because of the worldwide consump-tion of its fruits. Litchi is native to Southern China andSoutheast Asia, but is now cultivated in many countriesaround the world.[1] Litchi pericarp is used in traditionalChinese medicine for its antitussive, analgesic, antipyretic,haemostatic and diuretic properties.[2] Investigations foractive compounds in litchi pericarp and seeds responsiblefor these beneficial effects have identified polyphenolspolysaccharides, triterpenes, phytosterols and polyphenoliccompounds that have been associated with important bio-logical effects, including antioxidant and anticanceractivity.[2–4] Although litchi leaves are extensively used in teainfusions, their biological properties are almost unknown.The antinociceptive effects of this plant have already been

demonstrated previously by Besra et al. that demonstratedthe anti-inflammatory effect of crude ether extract of litchileaves in experimental animals.[5] In the present work, weinvestigate, for the first time, the chemical composition ofL. chinensis leaves, as well as their antioxidant andantinociceptive potential.

Materials and Methods

General

The nuclear magnetic resonance (NMR) spectra were rec-orded on a Bruker DRX-400 instrument (Bruker BioSpinGMbH, Rheinstetten, Germany) in methanol-d4 oracetone-d6 at 300 MHz (1H) and 75.5 MHz (13C).

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And PharmacologyJournal of Pharmacy

Research Paper

© 2014 Royal Pharmaceutical Society, Journal of Pharmacy and Pharmacology, ••, pp. ••–•• 1

mailto:[email protected]

Plant material

Leaves of L. chinensis Sonn. were collected from Itajaí, SantaCatarina, Brazil on May 2009 and were authenticated byProf. Rene Artur Ferreira (Pharmacy Course, Universidadedo Vale do Itajaí (UNIVALI)-Itajaí-SC, Brazil). A voucherspecimen, number 52829, was deposited at the BarbosaRodrigues Herbarium (BRH, Itajaí-SC, Brazil).

Extraction and isolation

Litchi chinensis leaves were dried at room temperature.The powder was submitted to methanol (MeOH) extrac-tion at room temperature for 7 days. The MeOH extract(MeOHEXTR) was then decanted, and a crudeMeOHEXTR was obtained. Liquid–liquid partitioning wasperformed with the MeOHEXTR using n-hexane, CH2Cl2

and ethyl acetate (EtOAc). The EtOAc fraction (EtOAcFR)was submitted to column chromatography using silica gel60 as stationary phase, and mixtures of dichloromethane-methanol of increasing polarity as mobile phase. The pres-ence of aromatic compounds in the ∼300 collectedfractions (10 ml each) was evaluated by silica gel 60 F254thin layer chromatography (TLC) using dichloromethaneand methanol (CH2Cl2 : MeOH) 7 : 3 as mobile phase.TLC column chromatography yielded three main ranges offractions with aromatic compounds: fractions 197–203,212–221 and 229–240. The combined fractions of thesethree ranges were evaporated to dryness, yielding 0.755,2.77 and 1.40 g of solid material, respectively, and washedusing a mixture of CH2Cl2 : MeOH (7 : 3) to obtain a solidthat was submitted to NMR analysis. Fractions 197–203yielded 480 mg solid, which was shown to be (1) (–)-epicatechin by NMR spectroscopy, based on comparisonwith the literature data.[6] The solids obtained from frac-tions 212–221 and 229–240 were identified as (2)procyanidin A2 (PA2, 60.8 mg) (3) and procyanidin B2(PB2, 400 mg), respectively, based on comparison withNMR spectral data.[1,7]

To assess the influence of the type of solvent on antioxi-dant content (see below), a smaller quantity of dried leaves(223.7 g) was extracted using maceration with 70% ethanol(EtOH) for 10 days at room temperature. Similar to themethanolic extract, the crude extract (15.0 g) was parti-tioned (70% EtOH in 30% water) into fractions usingn-hexane, CH2Cl2 and EtOAc. The residues of the organicsolvents were dissolved in EtOH to produce 1% (w/v) stocksolutions.

Analysis of total phenolic content

The amount of total phenolic compounds in crude extractsor EtOAc partionates was determined with Folin–Ciocalteu(FC) reagent, using the method of Singleton et al.[8] with

modifications. Diluted sample solution (25 μl of 0.01–0.5 mg/ml) was shaken for 1 min with 125 μl of FC reagentand 1.5 ml of purified water. Then 0.5 ml of sodium car-bonate solution was added, the mixture was shaken for1 min and the solution was completed up to 2.5 ml byadding distilled water. The solutions were incubated at 40°Cand after 30 min, the absorbance at 750 nm was evaluatedusing a spectrophotometer (Bel Photonics, SP 2000 UV,Osasco, SP, Brazil). The phenolic content was determinedfrom a calibration curve prepared with gallic acid standard(0.01, 0.05, 0.1, 0.25 and 0.5 mg/ml). The results wereexpressed as mg of gallic acid equivalent (GAE)/gram of drysample. All the samples were prepared in triplicate.

Antioxidant evaluation

DPPH assay

The 1,1-diphenyl-picrylhydrazyl (DPPH) assay was per-formed according Brand-Williams et al.[9] Samples(1–10 μg/ml of stock solution) were mixed with 1 ml of0.1 mM DPPH solution and adjusted with EtOH to avolume of 3 ml. The absorbance were recorded after 30 minat room temperature at 517 nm. The half maximal inhibi-tory concentration (IC50) value was estimated using a plotof the radical scavenging capacity (RSC % = 100 × (A

blank − A sample)/A blank) as a function of sample concentration.All tests were performed in triplicate.

ABTS assay

The 2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonic acid)(ABTS) assay was carried out according Arnao et al.[10]

Samples of crude extract or isolated compound (1–10 μg/ml) were dissolved in 3 ml EtOH. To these solutions, 1 mlstandardized ABTS solution was added, followed by thor-ough mixing. The mixture was incubated at 25°C for30 min, and the absorbance was recorded at 734 nm. Theresults were analysed as for the DPPH assay, using Trolox(6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid)as reference. All the samples were analysed in triplicate.

Determination of inhibition of lipid peroxidationin liposomes

To evaluate lipid peroxidation (LP), a solution of soybeanphosphatidylcholine (50 mg/ml) in chloroform was pre-pared, and the solvent was evaporated under nitrogenflow.[11] The lipid film was dispersed in 0.1 M Tris-HCl, pH7.6, maintained for 10 min in a water bath (50°C), andsonicated in an ultrasonic bath for 3 min. Liposomes werefiltered through a cellulose ester cartridge (0.22 μm).MeOHEXTR, EtOAcFR, PA2 and PB2 (0.05 to 5 mg/ml)were diluted in EtOH, and the evaluation was performedusing the thiobarbituric acid (TBA) reactive substance

Rosana C.L. Castellain et al.Investigation of Litchi chinensis leaves

© 2014 Royal Pharmaceutical Society, Journal of Pharmacy and Pharmacology, ••, pp. ••–••2

(TBARS) method. In each replicate, 200 μl of the liposomesuspension, 100 μl (40 mg/ml) of the 2,2′-azobis(2-amidinopropane) dihydrochloride radical (AAPH) and200 μl of blank (EtOH), sample or positive control (Trolox)were used. The mixture was incubated for 1 h in a waterbath (37°C). For the quantification of malondialdehydegenerated from lipids by AAPH, 400 μl of each sample wasremoved, and 150 μl of 0.375% TBA and 2 μl of 2%butylated hydroxytoluene were added. The materialwas then stirred using a vortex, incubated at 95°C for10 min and placed in an ice bath for 5 min. Next, themixture was centrifuged at 2.29 g for 3 min, extracted withn-butanol, and the absorbance of the extract at 530 nm wasquantified. Inhibition of TBARS formation was calculatedby the following equation: % inhibition = ((absorbanceof blank − absorbance of test sample)/absorbance ofblank) × 100.

Animals

Male Swiss albino mice (25–30 g), bred and maintained ina vivarium at the Universidade do Vale do Itajaí, were usedin the present investigation. The animals were housed intemperature-controlled rooms (20–22°C), under a 12-hlight/dark cycle (light on at 0700 h) and 55% ± 1% relativehumidity. Standard rodent diet and tap water were pro-vided ad libitum. The experiments were performed aftergaining the protocol approved by the Committee on theEthical Use of Animals of UNIVALI (CEUA/UNIVALIprotocol number 291/2010) and were carried out accord-ing the ethical guidelines for investigations of experimentalpain in conscious animals, similar to those described pre-viously.[12] The numbers of animals (6–8 per group) andintensities of noxious stimuli used were the minimum nec-essary to demonstrate the consistent effects of the drugtreatments. Each animal was used only once (except in thehotplate (HP) test). The treatments were administeredorally 60 min before the test in all cases. The standarddrugs used as positive control were administered by theintraperitoneal or subcutaneous routes, 30 min before thetests. In-vivo assays were performed after the in-vitroassays.

Drugs

Glutamate (GLU), capsaicin (CP), indomethacin, morphineand naloxone were obtained from Sigma Chemical Co. (St.Louis, MO, USA), and Trolox were purchased from Sigma-Aldrich (St. Louis, MO, USA). The stock solutions of alldrugs used were prepared in phosphate buffered saline(PBS) (1–10 mM) in silicone plastic tubes, kept at −20°Cand diluted to the appropriate concentration at the time ofuse. The drugs were prepared daily in 0.9% w/v NaCl.

Pharmacological assays

Rotarod test

This test was carried out according to previous descriptionsby Holzmann et al.,[13] with adaptations. The animals wereselected 24 h previously by eliminating those mice that didnot remain on the bar for three consecutive periods of 60 s.In the test, the animals were treated 60 min before beingsubmitted to the apparatus. The results were expressed asthe time(s) the animals remained on the rotarod (rotatingat 12 revolutions per minute).

Formalin-induced pain in mice

The procedure used was similar to that described previ-ously, with minors alterations.[14] Sixty minutes after treat-ments, the animals received 20 μl 2.5% formalin (FM;0.92% formaldehyde) made up of phosphate-buffered solu-tion, which was injected under the surface of the right hindpaw. After injection of the phlogistic agent, the mice wereobserved for two periods: 0–5 min (the early phase, inwhich neurogenic pain is caused by direct stimulation ofthe nociceptors) and 15–30 min (the late phase, in whichinflammatory pain is caused by the release of inflammatorymediators).

Acetic acid-induced writhing test in mice

This test was conducted using the method described byCollier et al., with minor modifications.[15] After the treat-ments, mice in all the groups received an injection of aceticacid (AA; 0.6%, 0.45 ml/mouse, i.p.), and the number ofabdominal constrictions were counted cumulatively over aperiod of 20 min.

Capsaicin-induced nociception

The method used was similar to that described previ-ously.[16] Sixty minutes after treatments, 20 μl of CP (1.6 μg/paw prepared in a phosphate-buffered solution) wasinjected under the skin of the dorsal surface on the righthind paw. The amount of time spent licking the injectedpaw was timed with a chronometer for 5 min and was con-sidered indicative of nociception.

Glutamate-induced nociception

The procedure used in this test was similar to that describedby Beirith et al.[17] After the treatments, the animals wereobserved individually for 15 min following GLU injection(20 μmol/paw). The amount of time spent licking theinjected paw was timed using a chronometer and was con-sidered indicative of nociception.

Rosana C.L. Castellain et al. Investigation of Litchi chinensis leaves


Hotplate test

The HP test was used to measure response latency accord-ing to the method described previously by Eddy andLeimbach,[18] with minor modifications. Sixty minutes afterthe treatments, mice were placed individually on an HPmaintained at 55 ± 1°C. The time (seconds) that elapseduntil the animal jumped or licked one of its hind paws wasrecorded as the reaction time. To avoid damage to theanimals’ paws, a cut-off time of 30 s was established.

Statistical analysis

All the in-vitro experiments were conducted in triplicate.The statistical significance was determined by analysis ofvariance (ANOVA) with the Kruskal–Wallis test and subse-quently, Dunn’s method (P < 0.05). The results of thein-vivo experiments were present as mean ± standard errorof the mean (SEM), except for ID50 (corresponds to doses ofextracts or compound that reduced the response by 50% inrelation the control group), which were presented with geo-metric means accompanied by their respective confidencelimits (95%). The percentages of inhibition were cited asmean ± SEM difference (in percentage) obtained for eachindividual experiment relative to the corresponding controlgroup. All the analyses mentioned above were performedusing GraphPad Instat or GraphPad Prism 4.0 (SanDiego, CA, USA). The results were compared usingone-way ANOVA, followed by the Dunnett’s pairwise orStudent–Newman–Keuls tests and the Bonferroni test;P values < 0.05 were considered significant.

Results

Extraction yields and total phenolic contentin L. chinensis leaves

The amounts of extractable compounds in the methanolic(c) and ethanolic crude extracts, expressed relative to the

dry weight of powdered leaves, were 8.0% and 6.7%, respec-tively. As shown in our experiments (Table 1), the crudeextracts presented high levels of phenolic components, irre-spective of the solvent used (413 and 397 mg GAE/g formethanol or EtOH, respectively). The total amount of phe-nolic compounds almost doubled in the EtOAc partitionedfractions, reaching 810 and 774 mg GAE/g for themethanolic and ethanolic extracts, respectively. There wasno statistically significant difference between methanolicand ethanolic extracts (P = 0.1266), but statistically signifi-cant difference was observed between the EtOAc partitionedfractions (P = 0.0495).

Analysis of the NMR spectra and comparison with litera-ture data identified three phenolic compounds: (–)-epicatechin in pool 1, PA2 in pool 2 and PB2 in pool 3,respectively (Figure 1). All the substances were isolated withhigh grade purity (>95%).

As shown in our experiments (Table 1), the methanoland the EtOH crude extracts have radical scavenging prop-erties because their DPPH IC50 values were comparable(4.45 and 5.60 μg/ml). Statistically significant difference wasobserved between the extracts (P = 0.0495), the methanolicextract being more active. The antioxidant activity in theEtOAcFR of both crude extracts presented similar values tothose detected for the crude extracts (4.76 and 4.79 μg/ml)(P = 0.2752). Analysis of the isolated components showedthat PA2 presented the highest antioxidant activity com-pared with PB2 (IC50 of 2.29 and 3.14 μg/ml, respectively).Statistically significant difference was observed betweenvitamin C and PA2 (P < 0.05).

The ABTS radical scavenging activity of the crude metha-nol extract was similar to that of the EtOH extract (IC50

varying from 3.36 to 3.43 μg/ml) without statistically sig-nificant difference (P = 0.1767). At the EtOAcFR evaluated,the antioxidant activity of the methanolic extract also wasslightly higher than that of the EtOH, with IC50 values of2.88 and 3.02 μg/ml, respectively. The PBA and PB2 were

Table 1 The extraction yield, total phenolic content of methanolic extract and acetyl acetate fraction, the evaluation of the IC50 values in the DPPHand ABTS free radical scavenging of L. chinensis leaves

Sample Yield (%)Total phenoliccontenta (mg GAE/g)b DPPH IC50 (μg/ml) ABTS IC50 (μg/ml)

Inhibition ofLP IC50 (μg/ml)

Solv. 1 (MeOH) MeOHEXTR 8.0 413 ± 12 4.45 ± 0.14 3.36 ± 0.36 0.046 ± 0.005EtOAcFR 1.4 810 ± 40 4.76 ± 0.08 2.88 ± 0.06 0.037 ± 0.060

Solv. 2 (EtOH) EtOHEXTR 6.7 397 ± 8 5.60 ± 0.15 3.43 ± 0.18 0.058 ± 0.038EtOAcFR 0.92 774 ± 12 4.79 ± 0.09 3.02 ± 0.15 0.038 ± 0.086

PA2 0.002 – 2.29 ± 0.20 0.96 ± 0.15 0.032 ± 0.006PB2 0.017 – 3.14 ± 0.02 1.51 ± 0.86 0.042 ± 0.008Ascorbic acid – – 5.00 ± 0.13 1.11 ± 0.24 –Trolox – – – – 0.040 ± 0.036

aEach value represents the mean ± standard deviation (SD) of three experiments. bmg GAE/g: mg of gallic acid equivalent per gram of dry plantextract or fraction. ABTS, 2,2′-azinobis (3-ethylbenzothiazoline-6-sulfonic acid; DPPH, 1,1-diphenyl-picrylhydrazyl; EtOAcFR, ethyl acetate fraction;EtOH, ethanol; EtOAcFR, ethyl acetate fraction; EtOHEXTR, ethanolic crude extracts; MeOH, methanol; MeOHEXTR, methanol extract; PA2,procyanidin A2; PB2, procyanidin B2; Trolox, 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid.



more active than crude extracts, with IC50 0.96 and 1.51 µg/ml, respectively. Both extracts and fractions exhibited statis-tically significant difference when compared with vitamin C(P = 0.039) revealed that both EtOAcFR were more activethan vitamin C (P < 0.05).

PB2 (IC50 of 0.042 μg/ml) also presented an equally excel-lent antioxidant capacity in this assay. In comparisonwith compounds, the crude methanolic and ethanolicextracts were less efficient (IC50 of 0.046 and 0.058 μg/ml,respectively).

As seen in Table 1, PA2, PB2 and the EtOAcFR frommethanolic extract presented IC50 of 0.032, 0.042 and0.037 μg/ml, respectively, with excellent antioxidant activitywhen compared with the reference antioxidant Trolox (IC50

of 0.040 μg/ml). The crude methanolic and ethanolicextracts were less efficient (IC50 of 0.046 and 0.058 μg/ml,respectively), but compared with the pure phenolic com-pounds, still had very high activity, counteracting LP. Nosignificant differences were found in LP activity of bothextracts, fractions and compounds.

Pharmacological assays

Rotarod test

Oral treatment with the MeOHEXTR and EtOAcFR frommethanolic extract (100 mg/kg, p.o) and pure substancePB2 (6 mg/kg, p.o.), vehicle or indomethacin (10 mg/kg,p.o.), failed to significantly affect the motor performance(time spent moving the cylinder) of animals when assessed1 h after treatment in the rotarod test (results not shown).

Formalin-induced pain in mice

The MeOHEXTR (Figure 3a and 3b) and EtOAcFR(Figure 2c and 2d) at doses (25 mg/kg; 50 mg/kg; 100 mg/kg, p.o) caused a dose-dependent reduction in pain inducedby the two irritant two phases of FM and neurogenicinflammation (P < 0.01). The ID50 calculated for the experi-ment in the first and second phase with MeOHEXTR were48.06 (37.19–61.62) mg/kg and 57.94 (41.99–79.93) mg/kg,and EtOAcFR, were 57.64 (49.76–66.77) mg/kg and 42.03(35.40–49.91) mg/kg, respectively. In both phases of the FMtest, there was also an antinociceptive response with thegroup of animals pretreated with PB2 (6 mg/kg, p.o.,P < 0.05).

Acetic acid-induced writhing test in mice

In this model, the MeOHEXTR and the EtOAcFR reducedthe writhing induced by AA at all doses, in a dose-dependent manner (P < 0.05, P < 0.01, P < 0.001), whencompared with the control group (Figure 3a and 3b). TheID50 calculated were 43.41 (29.94–62.95) mg/kg forMeOHEXTR and 25 (20.13–31.84) mg/kg to EtOAcFRrespectively. Treatment of animals with PB2 did notproduce statistically significant results (Figure 3c). In thismodel, antinociceptive effect was only observed with indo-methacin (P < 0.01).

Capsaicin-induced nociception

There was no antinociceptive action with the MeOHEXTR(Figure 4a), and the EtOAcFR showed antinociceptive effect(Figure 4b) only at a dose of 100 mg/kg (P < 0.01).However, the group treated with PB2 (6 mg/kg) (Figure 4c)reduced the CP-induced pain response at a dose of 6 mg/kg.

Glutamate-induced nociception

In the model of pain induced by GLU (Figure 5a), all dosesof MeOHEXTR produced an antinociceptive effect with apharmacological profile of dose dependency (P < 0.01). TheID50 calculated in this experiment was 48.85 (31.89–74.83)mg/kg. Reduction of pain was only seen at doses of 50and 100 mg/kg (Figure 5b) (P < 0.01), with ID50 of 85.11(80.67–89.79) mg/kg. PB2 was inactive until the maximaldose tested (Figure 5c).

Hotplate test

In the HP model (Figure 6a), the MeOHEXTR producedincreased nociceptive threshold at doses of 50 (P < 0.05)and 100 mg/kg (P < 0.01). The EtOAcFR (Figure 6b) wasonly effective at the dose of 100 mg/kg (P < 0.05). In bothexperiments, naloxone, a non-selective opioid antagonist,did not alter the antinociceptive effects obtained with the

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Figure 1 Compounds isolated from L. chinensis Sonn. leaves.



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Figure 2 Effect of (a, b) MeOH extract (MeOHEXTR) (6, 12.5, 25, 50 and 100 mg/kg, p.o), (c, d) ethyl acetate fraction (EtOAcFR) (12.5, 25, 50 and100 mg/kg, p.o) and (e, f) procyanidin B2 (PB2) (3, 6, 10 mg/kg, p.o) obtained from the leaves of L. chinensis and indomethacin (10 mg/kg, p.o) onthe neurogenic phase (0–5′) (a, c, e/in the left) and on the chronic phase (15–30′) (b, d, f/in the right) of pain induced by formalin. Mice were pre-treated 1 h before formalin injection. Each bar represents the mean of the experiments followed by standard error of the mean (SEM). Reaction timeis the time at which the animals were kept licking the injected paw formalin (2.5%). Asterisks denote statistical significance (*P < 0.05, **P < 0.01)compared with control. Analysis of variance (ANOVA), Dunnett. The values shown on the bars represent the percentage of inhibition painful processinduced by formalin (IM) obtained for each individual experiment relative to the corresponding control group.



treatments, but altered the antinociceptive effect of mor-phine. In Figure 6c, animals that received PB2 (6 mg/kg)and morphine treatments showed an increase in nociceptivethreshold calculated at 43 ± 9% and 74 ± 4%, respectively.But these responses were only reversed by naloxone inanimals treated with morphine.

Discussion

Free radicals could play an important role in the degenera-tive, biological or pathological processes of various diseases.Reactive oxygen species (ROS) cause oxidative stress, whichinterferes with normal cell functioning, resulting in celldamage. Cellular damage caused by ROS may be associatedwith diseases which may be accompanied by pain, especiallychronic pain.[19] Moreover, ROS is also closely related tocentral sensitization and may also interfere with the inter-pretation of acute nociceptive processes.[20]

Phenolic compounds belong to a group of natural sub-stances found in dietary products, and these compoundshave gained considerable attention due to their potent anti-oxidant activity.[21]

The high polyphenol content of litchi pericarp is welldescribed, and consumption of this fruit may provide ben-eficial antioxidant effects.[22,23] Although the use of litchileaves is widespread, studies comparing the phytochemicalcomposition of leaves of litchi pericarp were lacking.

Here, we demonstrate that the total phenolic content oflitchi leaves is threefold to fivefold higher than that of litchifruit pericarp.[24] The main compounds isolated from leaveswere identified as (–)-epicatechin, and their derivedoligomers PA2 and PB2, polyphenols also encountered inthe fruit pericarp. Enrichment of phenolic compounds inthe leaves, compared with those of the litchi pericarp atcomparable concentrations, was also reflected by the higherradical scavenging activity in the DPPH assay when com-pared with positive control (ascorbic acid) and previousresults with pericarp and seeds of L. chinensis.[4,24] In thisstudy, from the three isolated compounds, PA2 was the

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Figure 3 Effect of (a) MeOH extract (MeOHEXTR), (b) ethyl acetatefraction (EtOAcFR) (12.5. 25, 50 and 100 mg/kg, p.o) and(c) procyanidin B2 (PB2) (3, 6, 10, 30, 60 and 100 mg/kg, p.o) obtainedfrom the leaves of L. chinensis on the number of writhings inducedby acetic acid (0.6%) in mice. Indomethacin (10 mg/kg, p.o) was usedas positive control. The number of stretchings occurred immediatelywithin 60 min after the acetic acid injection was recorded. Eachbar represents the mean of the experiments followed by standard errorof the mean (SEM). Asterisks denote statistical significance (*P < 0.05,**P < 0.01 and ***P < 0.001) compared with control. Analysis of vari-ance (ANOVA), followed by the Dunnett test. Values in parentheses arepercentages of inhibition of pain formalin (IM)-induced pain obtainedfor each individual experiment relative to the corresponding controlgroup.◀



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EtOAcFR(b)

Control PB2

C 6

Capsaicin (1.6 µg/paw)

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ctio

n t

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(s)

300

200

100

0

(c)

Figure 4 Effect of (a) MeOH extract (MeOHEXTR), (b) ethyl acetatefraction (EtOAcFR) (25, 50, 100 mg/kg, p.o) and (c) procyanidin B2 (PB2)(6 mg/kg, p.o) obtained from the leaves of L. chinensis, on thenociceptive response of mice to test the capsaicin. Reaction time is thetime at which the animals were kept licking the injected paw capsaicin(1.6 μg/paw) Each bar represents the mean of the experiments followedby standard error of the mean (SEM). Statistical analysis was determinedby one-way analysis of variance (ANOVA) followed by Dunnett’s test.Asterisks denote statistical significance (*P < 0.05, **P < 0.01) comparedwith control. The values shown on the bars represent the percentage ofinhibition painful process induced by capsaicin (IM) obtained for eachindividual experiment relative to the corresponding control group.

(a) Control

C 25 50 100


32%**

63%**

29%**

54%**

67%**

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(s)

300

200

100

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MeOHEXTR

Control

C 25 50 100



Rea

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n t

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(s)

300

200

100

0

EtOAcFR(b)

Control PB2

C 6

Glutamate (20 µmol/paw)

Rea

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n t

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(s)

300

200

100

0

(c)

Figure 5 Effect of (a) MeOH extract (MeOHEXTR), (b) ethyl acetatefraction (EtOAcFR) (25, 50, 100 mg/kg, p.o) and (c) procyanidin B2 (PB2)(6 mg/kg, p.o) obtained from the leaves of L. chinensis, on the nocicep-tive response of mice to the glutamate test. Reaction time is the time atwhich the animals continued licking the glutamate injected paw(20 μmol/paw). Each bar represents the mean of the experiments fol-lowed by standard error of the mean (SEM), and statistical analysis wasdetermined by one-way analysis of variance (ANOVA) followed byDunnett’s test. Asterisks denote statistical significance (**P < 0.01) com-pared with control.



most active antioxidant species, as judged from the valuesobtained with ABTS and DPPH assays.

To test the ability of L. chinensis leaf extracts or isolatedphenolic compounds to inhibit formation of active speciesin liposomes, we used the well-standardized TBARS assay.Both samples presented the highest anti-LP capacity, reach-ing an antioxidant strength that was slightly better than thereference antioxidant Trolox.

Although clinical studies demonstrated the in-vivo effi-cacy of procyanidins in reducing cellular oxidation levels, itcan probably provide benefits in pathologies associated withhigh ROS production, especially in inflammatory processes(for a comprehensive review see Fraga et al.).[25]

The relationship between ROS and pain has been exten-sively studied in chronic pain, especially neuropathic andinflammatory pain.[19,26] However, it has recently been

(a) Control MeOHEXTR

Nal/MeOHEXTR Nal/Morp

49%**

**

**

**

##

##

##

Morphine (s.c)Naloxone (i.p) + MeOHEXTR (p.o)Naloxone (i.p) + Morphine (s.c)

Morphine (s.c)Naloxone (i.p) + EtOAcFR (p.o)Naloxone (i.p) + Morphine (s.c)

Morphine (s.c)Naloxone (i.p) + PB2 (p.o)

Naloxone (i.p) + Morphine (s.c)

47%*

48%*

63%*

C 25 50 100 Morp

Nal/EtOAcFR Nal/MorpC

C 6

25 50 100 Morp

Nal/PB2 Nal/MorpMorp

Control EtOAcFR

Control PB2

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(s)

40

30

20

10

0

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40

30

20

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20

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(b)

(c)

Figure 6 Effect of (a) MeOH extract (MeOHEXTR, (b) ethyl acetate fraction (EtOAcFR) and (c) procyanidin B2 (PB2) and morphine in the hotplatetest. In the first experiment, animals were pretreated with vehicle (10 ml/kg, p.o), MeOHEXTR (25, 50 and 100 mg/kg, p.o), EtOAcFR (25, 50 and100 mg/kg, p.o), PB2 (6 mg/kg, p.o) and morphine (5 mg/kg, s.c) and tested. In the second experiment, different groups of animals were pretreatedwith: naloxone (1 mg/kg, i.p) + MeOHEXTR (100 mg, p.o), naloxone (1 mg/kg, i.p) + EtOAcFR (100 mg/kg, p.o), naloxone (1 mg/kg, i.p) + morphine(5 mg/kg, s.c) and naloxone (1 mg/kg, i.p) + PB2 (6 mg/kg, p.o), and the nociceptive response of the mice was evaluated in the hotplate test. Eachbar represents the mean of the experiments followed by standard error of the mean (SEM). Statistical analysis was determined by one-way analysisof variance (ANOVA) followed by Dunnett’s test. Asterisks denote statistical significance (**P < 0.01, and *P < 0.05) when compared with control,## denotes statistical significance (**P < 0.01) when compared with morphine. ANOVA, Dunnett.



reported that ROS may participate in processes of acute pain.Ko et al.[27] demonstrated that intraperitoneal and intrathecaladministration of phenyl N-tert-butylnitrone, a free radicalscavenger, decreases analgesic behaviours, leading us to believethat ROS is mainly responsible for acute pain and centralsensitization. These authors suggest that flavonoids possesssignificant antioxidant potential because of presence of phe-nolic hydroxyl groups. In addition, many studies are available,which relate analgesic, anti-inflammatory and antioxidantactivity with flavonoids by interacting with prostaglandinsand superoxides.[28] Inhibition of these mediators may partlyexplain the mechanism of action of the antinociceptive andanti-inflammatory property of extracts rich in flavonoids, inmodels of pain and inflammation where there is a release ofthese mediators

Despite the evaluation of the antinociceptive effect of theplant under study, four analgesic testing methods were usedwith the objective of identifying possible peripheral andcentral effects of the test substances. In addition, differentnoxious stimuli (thermal or chemical) used in these modelsinduced the release of several mediators, suggesting differ-entiated mechanisms of action for a substance with analge-sic potential.

The acetic writhing test is used to study the peripheralanalgesic effects of the drugs.[15] The model of AA hasperipheral and central mechanisms for inducing responsealgic. Related studies have demonstrated that AA indirectlyinduces the release of endogenous mediators of pain such asprostaglandin E2 and prostaglandin F2 alpha[29] thatstimulate the nociceptive neurons, which are sensitive tonon-steroidal anti-inflammatory drugs (NSAIDs). Pavao-de-Souza et al.[30] demonstrated that spinal MAP kinases areactivated by peripheral stimulus with AA. In addition, pro-longed irritation of the peritoneal cavity has been associatedwith an increase in PGE levels in the peritoneal fluid, whichenhances capillary permeability and the release of GLU andsubstance P from peripheral afferent fibre terminals.[31,32] Inthis study, MeOHEXTR and EtOAcFR (but not PB2)showed dose-dependent and significant inhibition ofAA-induced writhing in mice, which suggests the involve-ment of peripheral mechanisms of analgesia.

FM-induced paw pain produced a distinct biphasicnociception, the first phase (lasting the first 5 min) corre-sponding to acute neurogenic pain and a second phase(lasting from 15 to 30 min) corresponding to inflammatorypain responses[33] mediated by prostaglandins, histamine,serotonin, bradykinin and cytokines such as IL-1b, IL-6,tumour necrosis factor-α, eicosanoids and nitric oxide(NO).[14,34–37] The inhibitory effect of MeOHEXTR,EtOAcFR and PB2 in both phases of FM-induced pain sug-gests that the mechanism related to neurogenic pain andinflammatory pain can also be modulated by compoundsidentified in the plant, especially PB2.

In an attempt to determine whether the MeOHEXTR,EtOAcFR and PB2 attenuated either the peripheral orcentral levels of nociception, or both, the thermal-inducednociceptive model (e.g., HP test) was performed. Thismodel of nociception, which is predominantly a spinalreflex, is thought to involve supraspinal nociceptive pro-cessing and to be selective for centrally acting (opioids),but not peripherally acting analgesic compounds(NSAIDs).[38] According to Mohd et al.[29] centrally actingdrugs activate the release of endogenous peptides via theperiaqueductal grey matter, which is then carried to thespinal cord to inhibit pain impulse transmission withinthe dorsal horn. Based on the ability of MeOHEXTR andEtOAcFR to prolong the latency to feeling discomfortexperienced by the animals in this test, we suggest that thisplant possesses mild centrally mediated antinociceptiveactivity against thermal-induced nociception. However, wecannot say that these substances are acting directly inopioid receptors because their effects were not reversed bynaloxone.

To study the effect of MeOHEXT, EtOAcFR and PB2against nociceptive transmission via the vanilloid receptors,the extract was assayed against the CP-induced paw-lickingtest. CP, an active ingredient in hot chilli peppers, directlystimulates vanilloid receptor 1 or transient receptor poten-tial cation channel subfamily V member 1 (TRPV1).[39]

These types of receptors, which are involved in the trans-mission and modulation of nociceptive activity, as wellas the integration of diverse painful stimuli, act selectivelyon unmyelinated C-fibres and thinly myelinated Aprimary sensory neurons within the peripheral nervoussystem, as well as tissues within the central nervous system(CNS).[39]

In addition, TRPV1 receptor antagonists have beenreported to exhibit pain-relieving activity[40] and were effec-tive in reducing nociception from inflammatory as well asneuropathic pain models in rats.[41] Based on our finding,the oral administration of EtOAcFR and PB2 (but notMeOHEXT) produced a neurogenic inhibition againstCP-induced nociception, indicating the ability of EtOAcFRand PB2 to inhibit nociceptive transmission initiated byTRPV1 activation. As the TRPV1 receptors are also trig-gered by heat and could be involved in the thermal-inducednociception (e.g., HP test),[42] the present findings seem tosuggest that EtOAcFR may contain other compounds(beyond PB2) with potential antagonist of TRPV1 receptorsat the peripheral and central levels. These componentsare more concentrated in the EtOACFR instead ofMeOHEXTR.

GLU is a major excitatory neurotransmitter in theCNS,[43] and various reports have shown that the GLUand glutamatergic receptors (both ionotropic andmetabotropic GLU receptors) are important in peripheral,



spinal and supraspinal nociceptive neurotransmission.[44]

Thus, in another attempt to determine the role of theglutamatergic system in the MeOHEXT, EtOAcFRand PB2 modulation of antinociception, these sub-stances were administered to mice subjected to theGLU-induced pain model, which is strongly mediatedby both the N-methyl-D-aspartate (NMDA) and non-NMDA receptors, as well as by the release of NO andNO-related substances.[17] In addition, activation ofthe GLU receptors has also been reported as contributingto the maintenance of peripheral nociceptive processesthat are associated with inflammatory but not phy-siological pain.[44,45] Based on our findings, we alsosuggest that glutamatergic system may be involved inthe modulation of MeOHEXT, EtOAcFR and PB2antinociception.

Taken together, our results open prospects for the use ofleaf extracts of L. chinensis for cosmetic and/or therapeuticpurposes. There are several advantages to using leavesinstead of whole fruits: easier seasonal management (inde-pendence from fructification), lower loss due to pestsand predatory animals, less fouling, easier storage/conservation and a higher mass yield. Because of the easeof extracting and manipulating leaves, compared with less-enriched parts of the plant, we will be able, in the future,to develop and test the antioxidant and analgesic effects ofcosmetic or therapeutic formulations based on L. chinensisleaves.

Conclusion

This study demonstrated that L. chinensis possesses bothcentral and peripheral antinociceptive activity that maybe involved in inhibition of COX activity or PGE synthesis,and inactivation of the glutamatergic system. L. chinensisalso exhibited inhibitory effect against TRPV1-receptor-mediated nociceptive transmission. The antinociceptiveactivity of this plant may also be associated with its antioxi-dant effect. Combining the results presented in this study,the use of this plant part is validated in pain processes inwhich ROS may be involved.

Declaration

Conflict of interest

The Author(s) declare(s) that they have no conflicts ofinterest to disclose.

Acknowledgements

The authors are grateful to Conselho Nacional deDesenvolvimento Científico e Tecnológico, Fundação deAmparo a Pesquisa e Inovação do Estado de Santa Catarinaand ProPPEC/UNIVALI for financial support and Prof.Renê A. Ferreira (UNIVALI) for classification and collectionof plant material. Special thanks to Dr A.J. Pierik and DrD.J. Netz, Philipps University, Marburg, Germany, for helpwith the manuscript.

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