O A d E A a B b A c B d a A R A A K C C C A G I u “ c m r t a 0 c Revista Brasileira de Farmacognosia 27 (2017) 495–501 ww w.elsev ier .com/ locate /b jp riginal Article nti-inflammatory action of ethanolic extract and clerodane iterpenes from Casearia sylvestris laise G. Pierri a, Rogério C. Castroa, Ednir O. Viziolib, Carla M.R. Ferreirab, Alberto J. Cavalheiroc, risteu G. Tininisd, Chung M. Chinb, André G. Santosa,∗ Laboratório de Farmacognosia, Departamento de Princípios Ativos Naturais e Toxicologia, Faculdade de Ciências Farmacêuticas, Universidade Estadual Paulista, Araraquara, SP, razil Laboratório de Pesquisa e Desenvolvimento de Fármacos, Departamento de Fármacos e Medicamentos, Faculdade de Ciências Farmacêuticas, Universidade Estadual Paulista, raraquara, SP, Brazil Núcleo de Bioensaios, Biossíntese e Ecofisiologia de Produtos Naturais, Departamento de Química Orgânica, Instituto de Química, Universidade Estadual Paulista, Araraquara, SP, razil Instituto Federal de São Paulo, Campus Avanç ado de Matão, Matão, SP, Brazil r t i c l e i n f o rticle history: eceived 1 September 2016 ccepted 12 December 2016 vailable online 4 May 2017 eywords: lerodane diterpene asearin B aseargrewiin F nti-inflammatory activity astric side effects a b s t r a c t The present study aimed to investigate the anti-inflammatory activity of ethanolic extract from Casearia sylvestris Sw., Salicaceae, leaves and to identify the compounds responsible for this activity. The ethanolic extract from C. sylvestris leaves was fractionated by solid phase extraction and the chemical composition of extract and fractions were assessed by chromatographic techniques. Casearin-like clerodane diterpenes were quantified in ethanolic extract (27.4%, w/w) and in fraction 2 of solid phase extraction (50.6%, w/w). Carrageenan-induced paw edema and carrageenan-induced pleurisy assays (rats) were used to evaluate anti-inflammatory activity of ethanolic extract, its fractions and clerodane diterpenes from C. sylvestris – caseargrewiin F and casearin B. The ethanolic extract was tested in the rat paw edema model and the doses tested (10 and 100 mg/kg) had no effect. In the pleurisy model, the extract doses of 300 and 500 mg/kg showed inhibitory effect. The fraction 2 of solid phase extraction (10 mg/kg), caseargrewiin F and casearin B (0.5 mg/kg) showed a significant reduction (p < 0.05) of the carrageenan-induced paw edema in rats compared to indomethacin. Gastric ulcers were not observed in animals treated with samples from C. sylvestris. In conclusion, the ethanolic extract from C. sylvestris, its enriched fraction of clerodane diterpenes, casearin B and caseargrewiin F exhibited anti-inflammatory activity on in vivo models in rats. Casearin-like clerodane diterpenes may be considered active chemical markers for C. sylvestris leaves. On the other hand, these diterpenes are promising compounds in the development of new drugs with anti-inflammatory action without gastric side effects. © 2017 Sociedade Brasileira de Farmacognosia. Published by Elsevier Editora Ltda. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). ntroduction Casearia sylvestris Sw., Salicaceae, is a tree or shrub widely sed in Brazilian folk medicine in which is primarily known as guaç atonga”, a Tupi-Guarani name. This species is geographi- ally distributed throughout Latin America and in Brazil occurs in any biomes as Atlantic Forest, Cerrado and Amazon Forest. In a eview of the plant, Ferreira et al. (2011) described many tradi- ional uses and pharmacological properties emphasizing anti-ulcer, nti-inflammatory, anti-tumor and anti-venom serum activities ∗ Corresponding author. E-mail: santosag@fcfar.unesp.br (A.G. Santos). http://dx.doi.org/10.1016/j.bjp.2016.12.008 102-695X/© 2017 Sociedade Brasileira de Farmacognosia. Published by Elsevier Edit reativecommons.org/licenses/by-nc-nd/4.0/). of C. sylvestris. The Brazilian Health Surveillance Agency (Anvisa) included C. sylvestris in herbal form of the Brazilian Pharma- copoeia (Formulário de Fitoterápicos, 2011) – an infusion of dried leaves indicated as anti-dyspeptic. The hydroethanolic or aqueous extracts of its fresh leaves have been widely used in Brazil as topical wound healing and anti-inflammatory agent (Silva, 1926; Hoehne, 1939; Correa, 1975). Regarding the anti-inflammatory activity, the aqueous extract (Ruppelt et al., 1991), the hydroethanolic extract (Silva et al., 2004; Albano et al., 2013) and the essential oil (Esteves et al., 2005) from C. sylvestris leaves showed inhibitory effect in inflammation models. However, none of these studies used the ethanolic extract from the leaves that presented antiulcerogenic effect in models of acute and chronic ulcers in rats (Sertié, 1990). Thus, it became relevant ora Ltda. This is an open access article under the CC BY-NC-ND license (http:// dx.doi.org/10.1016/j.bjp.2016.12.008 www.elsevier.com/locate/bjp http://crossmark.crossref.org/dialog/?doi=10.1016/j.bjp.2016.12.008&domain=pdf http://creativecommons.org/licenses/by-nc-nd/4.0/ http://creativecommons.org/licenses/by-nc-nd/4.0/ http://creativecommons.org/licenses/by-nc-nd/4.0/ http://creativecommons.org/licenses/by-nc-nd/4.0/ http://creativecommons.org/licenses/by-nc-nd/4.0/ http://creativecommons.org/licenses/by-nc-nd/4.0/ http://creativecommons.org/licenses/by-nc-nd/4.0/ http://creativecommons.org/licenses/by-nc-nd/4.0/ http://creativecommons.org/licenses/by-nc-nd/4.0/ http://creativecommons.org/licenses/by-nc-nd/4.0/ mailto:santosag@fcfar.unesp.br dx.doi.org/10.1016/j.bjp.2016.12.008 http://creativecommons.org/licenses/by-nc-nd/4.0/ http://creativecommons.org/licenses/by-nc-nd/4.0/ http://creativecommons.org/licenses/by-nc-nd/4.0/ http://creativecommons.org/licenses/by-nc-nd/4.0/ http://creativecommons.org/licenses/by-nc-nd/4.0/ http://creativecommons.org/licenses/by-nc-nd/4.0/ http://creativecommons.org/licenses/by-nc-nd/4.0/ http://creativecommons.org/licenses/by-nc-nd/4.0/ http://creativecommons.org/licenses/by-nc-nd/4.0/ http://creativecommons.org/licenses/by-nc-nd/4.0/ 4 de Fa t t n 2 c m s W 1 g n ( o t t T s p d b l u i a i h M P P t S ( t o A b o t ( f F ( h t r P f i 96 E.G. Pierri et al. / Revista Brasileira o evaluate the anti-inflammatory activity of ethanolic extract from he leaves because one of the main constraints of anti-inflammatory on-steroidal drugs is the appearance of peptic ulcers (Wallace, 008). Furthermore, these studies did not investigate the possible ompounds involved in anti-inflammatory activity. In scientific literature there are a diversity of secondary etabolites described for C. sylvestris leaves: monoterpenes, esquiterpenes (Sousa et al., 2007), nor-isoprenoids (Santos, 2008; ang et al., 2009), diterpenes (Itokawa et al., 1990; Morita et al., 991; Santos et al., 2007), triterpenes, lapachol, caffeic acid, chloro- enic acid and vanillic acid, flavonoids (Raslan et al., 2002), eolignans (Wang et al., 2010), ellagic and gallic acid derivatives Silva et al., 2008) among other compounds. In terms of chemotax- nomy Casearia genus has been characterized by the presence of ypical highly oxygenated tricyclic cis-clerodane diterpenes with etrahydrofuran ring with two acyloxy groups (Xia et al., 2015). wenty eight clerodane diterpenes of this type were isolated from C. ylvestris including casearins A-X and caseargrewiin F that showed ronounced antitumor activity (Ferreira et al., 2011). Clerodane iterpenes also showed antiulcerogenic activity as demonstrated y Santos (2008). In the same study, quantification of total casearin- ike diterpenes in ethanolic extract was determined as 18.1% (w/w) sing HPLC-PDA. Therefore, the present study aimed to investigate the anti- nflammatory activity of ethanolic extract from C. sylvestris leaves nd to identify the compounds responsible for this activity. This nformation is fundamental to the development of traditional erbal medicine from C. sylvestris. aterials and methods hytochemical procedures lant material and extraction Leaves of Casearia sylvestris Sw., Salicaceae, were collected at he Medicinal Garden of the School of Pharmaceutical Sciences of ão Paulo State University (Araraquara, SP, Brazil) in March 2010 S 21.81466, W 48.20215). Voucher specimen is deposited with he herbarium “Maria Eneida Kaufmann” of the Botanical Institute f São Paulo State (São Paulo, Brazil) with the reference number GS102. Dried and powdered leaves of C. sylvestris (2 kg) were extracted y maceration with ethanol (24 l) at 40 ◦C for seven days under ccasional stirring. The pooled extractive solutions were concen- rated under reduced pressure at 40 ◦C and dried in a desiccator silica gel) to give a residue (290.5 g) named dried ethanolic extract rom C. sylvestris leaves (EEC). ractionation of EEC by solid-phase extraction (SPE) EEC (20 g) was separated by SPE from silica gel 60–200 �m)/activated charcoal (1:1, w/w) by elution with exane:ethyl acetate (95:5, v/v), ethyl acetate and methanol o afford three fractions named SPECs1, SPECs2 and SPECs3, espectively (Santos et al., 2010). urification and structural determination of clerodane diterpenes rom SPECs2 Caseargrewiin F (1), casearins B (2) and X were purified and dentified by Santos et al. (2010). rmacognosia 27 (2017) 495–501 TLC and HPLC-PDA analyzes of EEC, SPECs1, SPECs2 and SPECs3 TLC analysis was developed in silica gel plate aluminum backed (20 cm × 20 cm; 200 �m) from Sorbent ® Technologies using hexane:ethyl acetate:isopropanol 70:28:02 (v/v) as elu- ent and sulfuric anisaldehyde as spray reagent (110 ◦C; 10 min). Sample concentration (EEC and SPECs1-3) was 10 mg/ml (ethyl acetate). Caseargrewiin F (1), casearin B (2) and X were used as standards for identification purposes (1 mg/ml; ethyl acetate). HPLC analyzes and sample pretreatment were developed with HPLC grade solvents and ultrapure water (18.2 M� cm). Sample pretreatment included a solid phase extraction on reversed-phase silica cartridge (Phenomenex ® StrataTM C18-E; 15 mm × 10 mm; 55 �m). Samples (EEC: 7.3 mg; SPECs1: 8.2 mg; SPECs2: 8.9 mg) were dissolved in 1 ml of methanol:water 98:2 (v/v) and applied in SPE cartridge. Elution was developed with 4 ml of methanol:water 98:2 (v/v). The obtained solutions were dried in a desiccator (sil- ica gel under reduced pressure) and the residues were dissolved with 1 ml of methanol, and submitted to membrane filtration (0.22 �m, PVDF Millipore ® ). Caseargrewiin F, casearins B and X (1 mg/ml; methanol; membrane filtration) were used as standards for identification purposes. Analytical reversed-phase HPLC-PDA was performed using a Shimadzu ® system (Kyoto, Japan) compris- ing a model Prominence ® LC-20AT pump, SIL-20A autosampler, DGU-20A5 degasser, CTO-20A column oven, SPD-M20A photo- diode array detector and CBM-20A communication bus module, fitted with Hypersil Gold ® C18 (Thermo ® Scientific, USA) column (250 × 4.6 mm, 5 �m), with control and data handling managed by LCsolution ® multi-PDA software. Samples were eluted with methanol:acetonitrile:water 22:44:34 (v/v), changing by linear gradient to 47:53:00 over 42 min, and isocratically with 47:53:00 for 15 min. The solvent flow rate was 0.8 ml/min and detection was at 200–700 nm for all samples. Aliquots of 20 �l were injected (Claudino et al., 2013). The analytical curve for quantification of total casearin-like clerodane diterpenes was obtained by using solutions of caseargrewiin F at concentrations of 0.035, 0.070, 0.140, 0.280 and 0.560 mg/ml (methanol) that were injected in triplicate; both regression equation and linearity factor were deter- mined. Pharmacological experiments Animals and bioassays to evaluate anti-inflammatory activity Male rats Wistar weighing 250–280 g were obtained from the Central Biotery of São Paulo State University (Botucatu, SP, Brazil) and were maintained in a room with controlled temperature (24 ± 1 ◦C) at 12 h light/dark cycle. The animals received food and water ad libitum, except during the 12 h before the experiments in which there was restriction of food. Two bioassays were per- formed to evaluate the anti-inflammatory activity, the paw edema de Fa a A A t o s c E C i w E F t � s a c 5 e e t e c b t C w w 5 a a m t k � c c 4 c i i n b F o p l a a S m o w s E.G. Pierri et al. / Revista Brasileira ccording to the protocol approved by the Ethics Committee on nimal Use of the School of Pharmaceutical Sciences, UNESP, raraquara-SP (CEUA/FCF/CAr no 10/2012) and pleurisy according o the protocol approved by the Ethics Committee on Animal Use f the University Center of Rio Preto (CEUA/UNIRP no 16/2013). The tructure of the proposal was conducted according to ethical prin- iples of animal experimentation of the Brazilian College of Animal xperimentation. arrageenan-induced paw edema in rat A carrageenan-induced paw edema assay was evaluated accord- ng to the method described by Winter et al. (1962). The animals ere treated by oral route with vehicle, indomethacin (10 mg/kg), EC (10 and 100 mg/kg), SPE1 and SPE2 (10 mg/kg), caseargrewiin and casearin B (0.5 and 2.5 mg/kg). One hour after administra- ion of the agents, edema was induced by injection of 100 �l of -carrageenan 1% (w/v, Sigma-Aldrich ® , St. Louis, USA) into the ub-plantar tissue of the right hind paw. The left paw was used s control, which was injected with the same volume of the vehi- le (saline). Paw thickness were measured before and 1, 2, 3, 4, and 6 h after carrageenan injection. Inflammatory swelling was xpressed as thickness variation. Indomethacin was used as refer- nce drug while control group received the vehicles that were used o dissolve the samples. The group treated with vehicle was consid- red as maximum of inflammation and all others treatments were ompared to this group. Gastric ulcerogenesis was also evaluated y macroscopic visualization of the rat stomachs after the end of he experiments. arrageenan-induced pleurisy in rat Pleurisy was performed as described by Vinegar et al. (1973) ith minor modifications. Initially, the animals (n = 6 per group) ere orally administered by gavage to EEC (100, 300 and 00 mg/kg), SPECs1 (30 mg/kg), SPECs2 (30 mg/kg), dexameth- sone (0.5 mg/kg, positive control) and vehicle (8% Tween 80 nd 0.1% carboxymethylcellulose in water, negative control and igration control). One hour later, the animals were submit- ed to anesthesia/analgesia by intraperitoneal administration of etamine (80 mg/kg) associated with xylazine (7 mg/kg) and then -carrageenan (4 mg/ml in saline) was injected into the pleural avity in a volume of 0.1 ml (400 �g/pleural cavity). The negative ontrol received intrapleural saline (0.1 ml/pleural cavity). After h, the animals were sacrificed in a CO2 chamber and the pleural avity rinsed with 2 ml of phosphate buffered saline (PBS) contain- ng heparin. An aliquot of 50 �l of the pleural fluid was diluted n Turk solution to lyse the erythrocytes and used to count the umber of total leukocytes (LT) in a Neubauer chamber. The num- er of LT by pleura was calculated by means of correction factors. or differential count, another aliquot of lavage fluid was used for btaining smear stained with May–Grunwald–Giemsa in which the ercentage of mononuclear (MN) and polymorphonuclear (PMN) eukocytes were determined. Applying the percentages obtained t the respective numbers of LT, it has reached the number of MN nd PMN by pleura. tatistical analyses The results were presented as mean ± standard error of the ean. Statistical analyzes of data were performed using analysis f variance (ANOVA), Tukey test. For this we used the InStat3 soft- are (GraphPad ® , San Diego, CA, USA) assuming minimal levels of ignificance of p < 0.05. rmacognosia 27 (2017) 495–501 497 Results Chemical profile of EEC and its fractions and purification of clerodane diterpenes EEC, SPECs1, SPECs2 and SPECs3 yields were 11.8, 3.9, 29.6 and 15.6% (w/w), respectively. TLC analysis showed the presence of casearin X (Rf = 0.23) only in EEC and SPECs2. Caseargrewiin F and casearin B showed similar Rf values (0.17 and 0.18). Thus, a spot in EEC and SPECs2 with Rf = 0.18 could be attributed to one of these diterpenes or both of them. Clerodane diterpenes were not iden- tified in SPECs1 and SPECs3. HPLC-PDA identification of clerodane diterpenes was based on peak UV spectra (�max 232–238 nm) as described by Claudino et al. (2013) and a typical UV spectra of casearin-like clerodane diterpenes was presented in Fig. 2. Casear- grewiin F (1) and casearins B (2) and X were identified both in EEC and SPECs2 by tR and UV spectra comparison (Figs. 1 and 2). The quantification of total casearin-like clerodane diterpenes equiva- lents to caseargrewiin F was performed using analytical curve of caseargrewiin F (y = 53,760,775.275 x + 557,587.117; R2 = 0.9987) and considering the sum of the areas of the peaks identified as casearin-like clerodane diterpenes. The results obtained in quantitative analysis of total casearin-like clerodane diterpenes equivalents to caseargrewiin F were 27.4 and 50.6% (w/w) for EEC and SPECs2, respectively. Caseargrewiin F was also quantified in EEC and SPECs2 and the obtained values were 1.1 and 1.4% (w/w), respectively. Bioassays to evaluate anti-inflammatory activity Carrageenan-induced paw edema in rat Edema was measured as the difference between the initial (before the treatment, 0 h) and final hind paw thickness obtained after different periods of time (1, 2, 3, 4, 5 and 6 h) after administra- tion of the agent in the right hind paw of the animals (100 �g/paw, intraplantar). The extract at doses of 10 and 100 mg/kg did not present signif- icant anti-inflammatory activity in the model of paw edema in rat (Fig. 3A and B). SPECs2 (10 mg/kg), caseargrewiin F (1) and casearin B (2) (0.5 and 2.5 mg/kg) showed a significant reduction of the carrageenan- induced paw edema in rats compared to control group. The effect of SPECs2 (Fig. 4) began in the second hour after carrageenan administration and persisted throughout the experiment in a dose dependent manner similarly or higher than indomethacin that also reduced significantly the edema compared to control group. The effect of caseargrewiin F (1) started at the second hour after carrageenan administration at doses of 0.5 and 2.5 mg/kg (Fig. 5A and B). Caseargrewiin F was able to reduce significantly the edema in both doses evaluated from the second hour and remained throughout the experiment. The effect of casearin B (2) (0.5 and 2.5 mg/kg) (Fig. 6A and B) showed similar activity profile or significantly higher than indomethacin. Animals treated with C. sylvestris samples (EEC, SPECs2, cas B and casgw F) did not present gastric lesions whereas indomethacin group showed evident lesions in the rat stomachs. Carrageenan-induced pleurisy in rat In the evaluation of EEC on pleurisy model, the intrapleural injection of carrageenan (migration control) caused scores of LT, MN and PMN significantly higher than those found for the negative control. Comparing the groups receiving the EEC with the migration control, the dose of 100 mg/kg had no effect, but the doses of 300 and 500 mg/kg significantly reduced the number of LT (36 and 42%, respectively) and PMN (41 and 49%, respectively). Dexamethasone 498 E.G. Pierri et al. / Revista Brasileira de Farmacognosia 27 (2017) 495–501 mAU 2250 2000 1750 1500 1250 1000 750 500 250 0 5.0 10.0 15.0 20.0 25.0 30.0 35.0 40.0 45.0 50.0 55.0 min casB casgF casX Fig. 1. HPLC-PDA chromatogram of EEC and UV spectra of caseargrewiin F peak (tR = 16.58 min). Chromatographic conditions: C18 column (250 mm × 4.6 mm, 5 �m); methanol:acetonitrile:water 22:44:34 (v/v) to 47:53:00 over 42 min (linear gradient) and 47:53:00 for 15 min (isocratic); flow rate 0.8 ml/min; 235 nm; injection volume 20 �l. mAU 2250 2000 1750 1500 1250 1000 750 500 250 0 5.00.0 10.0 15.0 20.0 25.0 30.0 35.0 40.0 45.0 50.0 55.0 min casB casgF casX Fig. 2. HPLC-PDA chromatogram of SPECs2 and UV spectra of caseargrewiin F peak (tR = 16.62 min). Chromatographic conditions: C18 column (250 mm × 4.6 mm, 5 �m); methanol:acetonitrile:water 22:44:34 (v/v) to 47:53:00 over 42 min (linear gradient) and 47:53:00 for 15 min (isocratic); flow rate 0.8 ml/min; 235 nm; injection volume 20 �l. 7.00 6.50 6.00 5.50 5.00 4.50 4.00 3.50 3.00 0 60 120 180 Time (min) P aw th ic kn es s (m m ) 240 300 360 6.50 6.00 5.50 5.00 4.50 4.00 3.50 3.00 0 60 120 180 Time (min) P aw th ic kn es s (m m ) 240 300 360 Control A B Indomethacin (10mg/kg) EECs (10mg/kg) Control Indomethacin (10mg/kg) EECs (100mg/kg) ∗ ∗ ∗ ∗ ∗ ∗ ∗ ∗ ∗ ∗ ∗ ∗∗ ∗ Fig. 3. Influence of EEC (10 mg/kg) (A) and (100 mg/kg) (B) on carrageenan-induced hind paw edema in rats. Values expressed as mean ± SEM (n = 6). Data were analyzed by two-way Tuckey’s test for comparisons between groups. *Statistical significance: p < 0.05 when compared with the control. E.G. Pierri et al. / Revista Brasileira de Fa 6.00 5.50 5.00 4.50 4.00 3.50 3.00 2.50 0 60 120 180 Time (min) P aw th ic kn es s (m m ) 240 300 360 Control (carrageenan) Indomethacin (10mg/kg) SPECs2 (10mg/kg) ∗ ∗ ∗ ∗ ∗ ∗ ∗ ∗ ∗ ∗ ∗ ∗ ∗ ∗ Fig. 4. Influence of SPECs2 (10 mg/kg) on carrageenan-induced hind paw edema in rats. Values expressed as mean ± SEM (n = 6). Data were analyzed by two-way T w ( a i M t c r D n c D d In the present study, in addition to using the EEC, its fractions F b F t uckey’s test for comparisons between groups. *Statistical significance: p < 0.05 hen compared with the control. positive control) reduced by 57, 45 and 60% the number of LT, PMN nd MN, respectively, compared to control migration (Table 1). Also in the evaluation of SPECs1 and SPECs2, the intrapleural njection of carrageenan (migration control) caused scores of LT, N and PMN significantly higher than those found for the nega- ive control. Comparing the groups receiving the fractions with the ontrol of migration, SPECs1 had no effect, but SPECs2 significantly educed the number of PMN and LT (47 and 48%, respectively). examethasone (positive control) reduced by 61, 51 and 65% of the umber of LT, PMN and MN respectively, compared with migration ontrol (Table 2). iscussion The present study supports that ethanolic extract, its enriched iterpene fraction and isolated clerodane diterpenes from C. 6.00 5.50 5.00 4.50 4.00 3.50 3.00 0 60 120 180 Time (min) P aw th ic kn es s (m m ) A B 240 300 360 Control (carrageenan) Indomethacin (10mg/kg) casgwf (0.5mg/kg)∗ ∗ ∗ ∗ ∗ ∗ ∗ ∗ ∗ ∗ ∗ ∗ ∗ ∗ ig. 5. Influence of casgwF (0.5 mg/kg) (A) and (2.5 mg/kg) (B) on carrageenan-induced hi y two-way Tuckey’s test for comparisons between groups. *Statistical significance: p < 0 6.00A B 5.50 5.00 4.50 4.00 3.50 3.00 P aw th ic kn es s (m m ) 0 60 120 180 Time (min) 240 300 360 Control (carrageenan) Indomethacin (10mg/kg) casB (0.5mg/kg)∗ ∗ ∗ ∗ ∗ ∗ ∗ ∗ ∗ ∗ ∗ ∗ ∗ ∗ ig. 6. Influence of casB (0.5 mg/kg) (A) and (2.5 mg/kg) (B) on carrageenan-induced hind wo-way Tuckey’s test for comparisons between groups. *Statistical significance: p < 0.05 rmacognosia 27 (2017) 495–501 499 sylvestris leaves exhibit anti-inflammatory activity reinforcing pre- vious results obtained by other authors as discussed below. Ruppelt et al. (1991) demonstrated that the aqueous extract from C. sylvestris leaves presents analgesic and anti-inflammatory actions in mice that received intraperitoneal injection of acetic acid. The analgesic action was confirmed by the reduction in the number of writhes and anti-inflammatory action by reducing of the Evans blue extravasation into the peritoneal cavity. Silva et al. (2004) showed that the hydroethanolic extract from C. sylvestris leaves exhibits anti-inflammatory effects in skin edema model in rats. Esteves et al. (2005) demonstrated that the essential oil from C. sylvestris leaves (EOCS) exhibits anti-inflammatory activity on carrageenan-induced paw edema model and granulomatous tissue test, both in rats. These authors also demonstrated that the EOCS exhibits analgesic action by reducing the number of writhes after intraperitoneal injection of acetic acid in mice. Mattos et al. (2007) evaluated the effect of hydroethanolic extract from C. sylvestris leaves on three models of nociception in mice, one model of pain not associated with inflammation (hot-plate test) and two models of pain associated with inflammation (acetic acid-induced abdom- inal writhes and ovalbumin-induced licking behavior in sensitized animals). About the last two tests, this extract showed inhibitory effect with all doses tested whereas in the hot plate test, showed an inhibitory effect only at the highest dose. More recently, Albano et al. (2013) demonstrated that the hydroethanolic extract from C. sylvestris leaves reduced paw edema in mice and leukocyte migra- tion in pleurisy in rats, both induced by carrageenan. However, none of these studies used the ethanolic extract from C. sylvestris leaves (EEC) which presented antiulcerogenic effect in models of acute and chronic ulcers in rats (Sertié, 1990). Also, these studies did not investigate the possible compounds involved. and isolated clerodane diterpenes were also tested to investi- gate the possible compounds involved with the anti-inflammatory activity of EEC in two models, one to evaluate the formation of 6.00 5.50 5.00 4.50 4.00 3.50 3.00 P aw th ic kn es s (m m ) 0 60 120 180 Time (min) 240 300 360 Control (carrageenan) Indomethacin (10mg/kg) casgwF (2.5mg/kg) ∗ ∗ ∗ ∗ ∗ ∗ ∗ ∗ ∗ ∗ ∗ ∗ ∗∗ nd paw edema in rats. Values expressed as mean ± SEM (n = 6). Data were analyzed .05 when compared with the control. 6.00 5.50 5.00 4.50 4.00 3.50 3.00 P aw th ic kn es s (m m ) 0 60 120 180 Time (min) 240 300 360 Control (carrageenan) Indomethacin (10mg/kg) casB (2.5mg/kg)∗ ∗ ∗ ∗ ∗ ∗ ∗ ∗ ∗ ∗ ∗ ∗ ∗ ∗ paw edema in rats. Values expressed as mean ± SEM (n = 6). Data were analyzed by when compared with control. 500 E.G. Pierri et al. / Revista Brasileira de Farmacognosia 27 (2017) 495–501 Table 1 Effect of EEC orally administrated on the number of leukocytes present in the pleural cavity 4 h after intrapleural injection of carrageenan (400 �g/pleural cavity) in rats. Group (Cells/pleural cavity) × 106 Total leukocytes Mononuclear leukocytes Polymorphonuclear leukocytes Negative control 8.29 ± 1.27 5.78 ± 0.94 2.51 ± 0.50 Migration control 47.47 ± 4.17a 10.85 ± 1.58a 36.62 ± 5.01a Positive control 20.58 ± 1.09a,b 5.96 ± 0.82b 14.62 ± 1.44a,b EEC 100 mg/kg 43.33 ± 2.84a 7.76 ± 2.03 35.57 ± 2.87a 300 mg/kg 30.37 ± 3.84a,b 8.74 ± 2.01 21.63 ± 1.87a,b 500 mg/kg 27.40 ± 4.47a,b 8.68 ± 2.27 18.72 ± 2.45a,b Results are expressed as mean ± standard error of the mean (n = 6 animals per group). Negative control, vehicle orally + intrapleural saline (0.1 ml/pleural cavity); migration control, vehicle orally; positive control, dexamethasone orally (0.5 mg/kg). a p ≤ 0.05 compared to the negative control. b p ≤ 0.05 compared with migration control (ANOVA, Tukey’s test). Table 2 Effect of SPECs1 and SPECs2 orally administrated on the number of leukocytes present in the pleural cavity 4 h after intrapleural injection of carrageenan (400 �g/pleural cavity) in rats. Group (Cells/pleural cavity) × 106 Total leukocytes Mononuclear leukocytes Polymorphonuclear leukocytes Negative control 7.41 ± 0.78 5.18 ± 0.60 2.23 ± 0.40 Migration control 46.82 ± 3.45a 11.15 ± 2.23a 35.66 ± 3.95a Positive control 17.88 ± 1.19a,b 5.41 ± 1.10b 12.47 ± 0.92a,b SPECs1 (30 mg/kg) 44.03 ± 2.47a 8.26 ± 1.29 35.77 ± 2.22a SPECs2 (30 mg/kg) 24.74 ± 3.86a,b 6.23 ± 0.95 18.44 ± 3.32a,b Results are expressed as mean ± standard error of the mean (n = 6 animals per group). Negative control, vehicle orally + intrapleural saline (0.1 ml/pleural cavity); migration c e t h d a w i p e ( s 6 i S S p r d e c t c B i i h t o f w s s ontrol, vehicle orally; positive control, dexamethasone orally (0.5 mg/kg). a p ≤ 0.05 compared to the negative control. b p ≤ 0.05 compared with migration control (ANOVA, Tukey’s test). dema and the other to evaluate the cell migration (pleurisy). Ini- ially, the EEC was tested in the rat paw edema model and the ighest dose tested (100 mg/kg) had no effect (Fig. 3B). Thus, the oses of 100, 300 and 500 mg/kg were used in the pleurisy model nd the two higher doses showed inhibitory effect. In this way, the effects of SPECs1 and SPECs2 fractions ere investigated in these models. Only SPECs2 showed anti- nflammatory effect. Santos (2008) characterized SPECs1 as a low olarity fraction containing volatile compounds from the leaves ssential oil and spathulenol was the main volatile compound 17.5%, w/w). In the fraction SPECs1 obtained in the present study, pathulenol was also the main volatile compound, representing 0.6% (w/w) of volatiles in SPECs1. Spathulenol was also identified n the leaves essential oil from C. sylvestris (Esteves et al., 2005; ousa et al., 2007). Clerodane diterpenes were not identified in PECs1 (TLC and HPLC-PDA). On the other hand, clerodane diterpenes are the main com- ounds in SPECs2. These results suggested that the compounds esponsible for the anti-inflammatory activity of EEC were clero- ane diterpenes. Thus, casearin B and caseargrewiin F were valuated in paw edema model and demonstrated activity ompared to indomethacin. Furthermore, through bioguided frac- ionation, Santos (2008) showed that the concentrated fraction in lerodane diterpenes (SPECs2) and clerodane diterpenes (casearins , D, O and X and caseargrewiin F) showed antiulcerogenic activity n acute ulcer model induced by ethanol in rats. Regarding of probable mechanisms of anti-inflammatory activ- ty of C. sylvestris, the literature is relatively sparse. Some studies ave suggested that they might be associated with reduced produc- ion of prostaglandins (PG) by inhibition of cyclooxygenase (COX) r secreted phospholipase A2 (sPLA2). In the first case, there are ew and weak evidences, one suggested by Sassioto et al. (2004) ho found that the decoction from C. sylvestris leaves showed a imilar effect to non-steroidal anti-inflammatory drugs (COX clas- ical inhibitors) in a model of osteogenesis. Esteves et al. (2005) suggest a similar mechanism since EOCS reduced paw edema induced by carrageenan, but not the increased vascular permeabil- ity induced by substances that degranulate mast cells. Furthermore, this hypothesis raises the following question: how can the same extract or clerodane diterpene from C. sylvestris leaves inhibit COX and at the same time provide antiulcerogenic effect? In addition, SPECs2, cas B and casgw F showed anti-inflammatory action in paw edema model without gastric effects. One possible response to such inquiry would be the highly selective inhibition of COX-2, isoform involved in the production of PG in the site of inflammation. Thus, the production of PG in the stomach for COX-1 was not affected. With respect to the inhibition of sPLA2, Borges et al. (2000) showed that the aqueous extract from C. sylvestris leaves inhibited in vitro enzymatic activity of sPLA2 isolated from the venom of Bothrops pirajai and B. jararacussu. In addition, studies have demon- strated that extracts from C. sylvestris show inhibitory effect on biological activities of sPLA2 isolated from poisons (Raslan et al., 2002; Cavalcante et al., 2007). However, it is important to note that there are ten different groups of sPLA2 and the main sPLA2 involved in the inflammatory reaction in humans belong to the group V while sPLA2 found in poisons belong to other groups (Dennis et al., 2011). Regarding of probable mechanisms, Albano et al. (2013) suggested that anti-inflammatory activity of C. sylvestris may be related to the inhibition of nitric oxide production because the hydroethano- lic extract from the leaves of this plant reduced levels of nitrite and nitrate in pleural lavage of rats subjects to pleurisy induced by carrageenan. They also suggested a relationship with antioxidant activity, because this study demonstrated the reduction of various parameters of oxidative stress in the lung of these animals. Conclusion The results of the present study demonstrated that ethanolic extract from C. sylvestris leaves (EEC), its enriched fraction of clero- dane diterpenes (SPECs2), casearin B and caseargrewiin F exhibited de Fa a a c m u s b c d g E P t t t l C a R n A i r A h c C A a ( R A B C C E.G. Pierri et al. / Revista Brasileira nti-inflammatory activity on in vivo models in rats. These results re in agreement with Brazilian traditional use of the species. Thus, asearin-like clerodane diterpenes may be considered chemical arkers for C. sylvestris leaves, its derived plant drug or extracts sed for anti-inflammatory therapeutic purposes. Therefore this tudy contributed for standardization of a further herbal medicine ased on Brazilian traditional medicine data. On the other hand, asearin-like clerodane diterpenes are promising compounds in the evelopment of new drugs with anti-inflammatory action without astric side effects. thical disclosures rotection of human and animal subjects. The authors declare hat the procedures followed were in accordance with the regula- ions of the relevant clinical research ethics committee and with hose of the Code of Ethics of the World Medical Association (Dec- aration of Helsinki). onfidentiality of data. The authors declare that no patient data ppear in this article. ight to privacy and informed consent. The authors declare that o patient data appear in this article. uthors’ contributions EGP, RCC, EOV, CMRF participated in the evaluation of anti- nflammatory activity, acquisition and interpretation of data. EGP ealized phytochemical procedures and analysis. AJC, AGT, CMC and GS participated in the design and coordination of the work and elped to draft the manuscript. All the authors have contributed to ritical reading of the final manuscript and approved its submission. onflicts of interest The authors declare that there are no conflict of interest. cknowledgments Authors are grateful to CAPES, CNPq and Scientific Support nd Development Program of School of Pharmaceutical Sciences UNESP). eferences lbano, M.N., Silveira, M.R., Danielski, L.G., Florentino, D., Petronilho, F., Piovezan, A.P., 2013. Anti-inflammatory and antioxidante properties of hydroalcoholic crude extract from Casearia sylvestris Sw. (Salicaceae). J. Ethnopharmacol. 147, 612–617. orges, M.H., Soares, A.M., Rodrigues, V.M., Andrião-Escarso, S.H., Diniz, H., Hamaguchi, A., Quintero, A., Lizano, S., Gutiérrez, J.M., Giglio, J.R., Homsi- Brandeburgo, M.I., 2000. Effects of aqueous extract of Casearia sylvestris (Flacourtiaceae) on actions of snake and bee venoms and on activity of phos- pholipases A2. Comp. Biochem. Physiol., Part B: Biochem. Mol. 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http://refhub.elsevier.com/S0102-695X(16)30299-X/sbref0145 http://refhub.elsevier.com/S0102-695X(16)30299-X/sbref0145 http://refhub.elsevier.com/S0102-695X(16)30299-X/sbref0145 http://refhub.elsevier.com/S0102-695X(16)30299-X/sbref0145 http://refhub.elsevier.com/S0102-695X(16)30299-X/sbref0145 http://refhub.elsevier.com/S0102-695X(16)30299-X/sbref0145 http://refhub.elsevier.com/S0102-695X(16)30299-X/sbref0145 http://refhub.elsevier.com/S0102-695X(16)30299-X/sbref0145 Anti-inflammatory action of ethanolic extract and clerodane diterpenes from Casearia sylvestris Introduction Materials and methods Phytochemical procedures Plant material and extraction Fractionation of EEC by solid-phase extraction (SPE) Purification and structural determination of clerodane diterpenes from SPECs2 TLC and HPLC-PDA analyzes of EEC, SPECs1, SPECs2 and SPECs3 Pharmacological experiments Animals and bioassays to evaluate anti-inflammatory activity Carrageenan-induced paw edema in rat Carrageenan-induced pleurisy in rat Statistical analyses Results Chemical profile of EEC and its fractions and purification of clerodane diterpenes Bioassays to evaluate anti-inflammatory activity Carrageenan-induced paw edema in rat Carrageenan-induced pleurisy in rat Discussion Conclusion Ethical disclosures Protection of human and animal subjects Confidentiality of data Right to privacy and informed consent Authors’ contributions Conflicts of interest Acknowledgments References