PHYTOTHERAPY RESEARCH Phytother. Res. 29: 1509–1515 (2015) Published online 22 June 2015 in Wiley Online Library (wileyonlinelibrary.com) DOI: 10.1002/ptr.5401
Anti-TNF-α Activity of Brazilian Medicinal Plants and Compounds from Ouratea semiserrata Priscilla R. V. Campana,1,2 Daniel S. Mansur,3 Grasielle S. Gusman,1 Daneel Ferreira,4 Mauro M. Teixeira3 and Fernão C. Braga1* 1
Faculdade de Farmácia, Universidade Federal de Minas Gerais, Av. Antônio Carlos 6627, campus Pampulha, Belo Horizonte CEP 31.270-901, Brazil 2 Divisão de Ciências Farmacêuticas, Fundação Ezequiel Dias, R. Conde Pereira Carneiro 80, Belo Horizonte CEP 30.510-010, Brazil 3 Departamento de Bioquímica, ICB, Universidade Federal de Minas Gerais, Av. Antônio Carlos 6627, campus Pampulha, Belo Horizonte CEP 31.270-901, Brazil 4 Department of Pharmacognosy and Research Institute of Pharmaceutical Sciences, University of Mississippi, MS 38677, USA
Several plant species are used in Brazil to treat inflammatory diseases and associated conditions. TNF-α plays a pivotal role on inflammation, and several plant extracts have been assayed against this target, both in vitro and in vivo. The effect of 11 Brazilian medicinal plants on TNF-α release by LPS-activated THP-1 cells was evaluated. The plant materials were percolated with different solvents to afford 15 crude extracts, whose effect on TNF-α release was determined by ELISA. Among the evaluated extracts, only Jacaranda caroba (Bignoniaceae) presented strong toxicity to THP-1 cells. Considering the 14 non-toxic extracts, TNF-α release was significantly reduced by seven of them (inhibition > 80%), originating from six plants, namely Cuphea carthagenensis (Lythraceae), Echinodorus grandiflorus (Alismataceae), Mansoa hirsuta (Bignoniaceae), Ouratea semiserrata (Ochnaceae), Ouratea spectabilis and Remijia ferruginea (Rubiaceae). The ethanol extract from O. semiserrata leaves was fractionated over Sephadex LH-20 and RP-HPLC to give three compounds previously reported for the species, along with agathisflavone and epicatechin, here described for the first time in the plant. Epicatechin and lanceoloside A elicited significant inhibition of TNF-α release, indicating that they may account for the effect produced by O. semiserrata crude extract. Copyright © 2015 John Wiley & Sons, Ltd. Keywords: TNF-α inhibition; Brazilian medicinal plants; Ouratea semiserrata; epicatechin; lanceoloside A.
INTRODUCTION Inflammation is a physiological response to infection or sterile injury of tissues that aims to restore tissue functions to their original state. However, excessive or uncontrolled inflammation contributes to the pathogenesis of various diseases including rheumatoid arthritis and inflammatory bowel diseases (Medzhitov, 2008; Alessandri et al., 2013; Souza et al., 2013). TNF-α is a crucial mediator of inflammatory responses. It is rapidly released after inflammatory stimuli, including infection or tissue injury, and triggers a series of intracellular events that culminate in the activation of the transcription nuclear factor kappa B (NF-κB), leading to the production of other proinflammatory cytokines, chemokines and proteases (Choy and Panayi, 2001; Scott and Kingsley, 2006). Consistent with this relevant role of TNF-α in the context of inflammation, injection of TNF-α causes significant influx of leukocytes in vivo, whereas blockade of TNF-α ameliorates inflammatory responses in several preclinical models of inflammatory diseases (Croft et al., 2013). The inhibition of TNF-α is an emerging and promising strategy to overcome chronic inflammatory processes in human diseases like rheumatoid arthritis and inflammatory bowel diseases (Laveti et al., 2013). Indeed, several * Correspondence to: Fernão C. Braga, Faculdade de Farmácia, Universidade Federal de Minas Gerais, Av. Antônio Carlos 6627, campus Pampulha, Belo Horizonte CEP 31.270-901, Brazil. E-mail: [email protected]
Copyright © 2015 John Wiley & Sons, Ltd.
TNF-α inhibitors have been proven to be clinically effective (Croft et al., 2013). Presently, commercially available anti-TNF-α drugs are immunobiological which require intra-articular administration and are costly (Khanna et al., 2007). Therefore, there is demand for new TNF-α inhibitors that can be further developed as antiinflammatory drugs to treat chronic diseases like arthritis and psoriasis. Several plant species are traditionally used in Brazil to treat a plethora of inflammatory conditions (Falcão et al., 2005), which may represent a source of bioactive compounds. Hence, the goal of the present work was to investigate some selected medicinal plants as potential TNF-α inhibitors.
MATERIALS AND METHODS Plant materials and preparation of the crude extracts. Eleven plant species were selected based on their traditional uses in Brazil to treat different inflammatory process, including Cuphea carthagenensis (Jacq.) J.F. Macbr. (Lythraceae), Echinodorus grandiflorus (Cham. & Schltdl.) Micheli (Alismataceae), Erythroxylum gonocladum (Mart.) O.E. Schulz (Erythroxylaceae), Erythroxylum suberosum A. St.-Hil., Erythroxylum tortuosum Mart, Hancornia speciosa Gomes (Apocynaceae), Jacaranda caroba (Vell.) A. DC. (Bignoniaceae), Mansoa hirsuta DC. (Bignoniaceae), Ouratea semiserrata (Mart. & Nees) Engl. (Ochnaceae), Received 11 November 2014 Revised 19 May 2015 Accepted 26 May 2015
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Jacaranda caroba Mansoa hirsuta Ouratea semisserrata Ouratea spectabilis Remijia ferruginea
Aerial parts Leaves Leaves Leaves Leaves Leaves, bark, roots Leaves Aerial parts Leaves, bark Leaves, bark Leaves, stems
Telêmaco Borba Telêmaco Borba Caeté Lagoa Santa Lagoa Santa São Gonçalo do Rio Preto Belo Horizonte Caratinga Serra do Cipó Perdizes São Gonçalo do Rio Preto
51.460 23.862 49.269 48.94 3.833
Vendruscolo, 2004; Lima et al., 2007 Souza and Felfili, 2006; Brandão et al., 2009; Corrêa, 1974 Gonzales-Guevara et al., 2006; Cano and Volpato, 2004 Gonzales-Guevara et al., 2006; Cano and Volpato, 2004 Gonzales-Guevara et al., 2006; Cano and Volpato, 2004 Lima and Martins, 1996; Grandi et al., 1982; Britto and Britto, 1982 Siqueira, 1981; Corrêa, 1974; Di Stasi and Hiruma-Lima, 2002 Chaves and Reinhard, 2003 Corrêa, 1974 Felicio et al., 1995 Corrêa, 1974 Cardiovascular diseases, atherosclerosis Anti-rheumatic, antiinflammatory Antiinflammatory, to treat bronchitis and asthma Antiinflammatory, to treat bronchitis and asthma Antiinflammatory, to treat bronchitis and asthma Antiinflammatory, anti-rheumatic, to treat gout and dermatosis Wound healing, to treat ulcers To treat sore throats Antiinflammatory Antiinflammatory, to treat ulcers To treat ulcers and fever
Bioassay method. The potential antiinflammatory activity of the crude extracts and compounds isolated from O. semiserrata leaves was evaluated by measuring TNF-α produced by LPS-stimulated THP-1 cells, employing an immunoassay as described by Weiss et al. (2004). THP-1 cells (ATCC TIB-202) were cultivated in RPMI 1640 medium (Sigma, USA) supplemented with 0.05 mM 2-mercaptoethanol, 10% FBS, 100 U/mL of penicillin and 100 μg/mL of gentamicin at 37 °C, in an atmosphere containing 5% CO2. The medium was renovated twice a week, when cell concentrations reached 1.0 × 106 cells/mL. The cells were transferred to a 96-well microplate at a density of 100 000 cells per well, incubated for 18 h and pre-treated with the samples for 3 h. LPS (from Escherichia coli 0111: B4, Sigma) was administrated at 1.0 μg/mL as inflammatory stimulus. After incubating the plate at 37 °C overnight, it was centrifuged (1.800 ×g, 5 min, 16 °C), the supernatant was collected and TNF-α was measured using the cytokine-specific sandwich quantitative enzyme-linked immune-sorbent assay (ELISA) according to the manufacturer’s instructions (TNF-α duo set, DY210, R&D Systems, USA). Cell viability was evaluated for all tested samples by the MTT method using untreated cells as reference for viability. Samples that gave cell viability higher than 90% were considered non-toxic for the THP-1 cell line. The percentage of TNF-α inhibition was calculated by the ratio between the TNF-α amount secreted by treated cells (pg/mL) and the baseline level of this cytokine (pg/mL) observed for solvent control (0.1% DMSO). The statistical significance of differences was calculated employing the software GraphPad Prism, version 5.0 (GraphPad Software Inc., USA) using one-way ANOVA followed by Dunnett post-test for multiple comparisons. Results were considered different when p < 0.05. The samples were tested in triplicate at different concentrations (125–500 μg/mL for extracts and 62.5–450 μM for pure compounds). Dexamethasone was employed as positive control (0.1 μM).
49.269 107.791 118.812 111.068 111.065 49.895
Ouratea spectabilis (Mart. ex Engl.) Engl. and Remijia ferruginea (A. St.-Hil.) DC. (Rubiaceae). The ethnopharmacological uses of these plants are collated in Table 1. The plant materials were collected, and the species were identified by botanists from the Botanical Department, Instituto de Ciências Biológicas (ICB), UFMG, Belo Horizonte, Brazil, where voucher specimens are deposited (see Table 1 for voucher numbers). After drying at 45 °C, during 72 h, the plant materials were powdered and extracted by exhaustive percolation with ethanol or acetone at room temperature (Table 2). The solvent was vacuum removed in a rotatory evaporator at 50 °C, to furnish the crude extracts (see Table 2 for extraction yields). The inhibitory effects on TNF-α production in LPS-activated THP-1 monocytic cells of the crude extracts were evaluated at a concentration of 500 μg/mL. Selected extracts were further assayed at 125, 250 and 500 μg/mL.
Cuphea carthagenensis Echinodorus grandiflorus Erythroxylum gonocladum Erythroxylum suberosum Erythroxylum tortuosum Hancornia speciosa
Traditional uses Voucher number Sample location Part used Plant species
Table 1. Selected Brazilian medicinal plants with their botanical names, part used, ethnopharmacological uses and other information
Literature
P. R. V. CAMPANA ET AL.
Phytochemical investigation of O. semiserrata. A portion (10 g) of the ethanolic extract from O. semiserrata leaves was fractionated over a Sephadex LH-20 column Phytother. Res. 29: 1509–1515 (2015)
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Table 2. Inhibition of TNF-α production by LPS-activated THP-1 monocytic cells elicited by the extracts of Brazilian medicinal plants Species
Plant part
Control
Extract
Yield (% w/w)
RPMI DMSO 0.1%
C. carthagenensis
Aerial parts
Acetone 50%
25.7
Acetone 70%
23.3
E. grandiflorus
Leaves
Ethanol 70%
27.0
E. gonocladum
Leaves
Ethanol 96 GL
23.6
E. suberosum
Leaves
Ethanol 96 GL
18.6
E. tortuosum
Leaves
Ethanol 96 GL
27.2
H. speciosa
Leaves
Acetone 50%
28.4
Acetone 70%
33.9
J. caroba
Leaves
Ethanol 96 GL
23.3
M. hirsuta
Fruits
Ethanol 96 GL
18.6
O. semisserrata
Leaves
Ethanol 96 GL
30.1
O. spectabilis
Bark
Ethanol 96 GL
36.3
R. ferruginea
Leaves
Ethanol 96 GL
ND
Stem
Ethanol 96 GL
21.4
Ethanol 50%
ND
Dexamethasone
TNF-α (pg/mL ± S.D.) 10.9 ± 1.1* 1055.0 ± 63.9 14.2 ± 0.5* 1107.1 ± 93.7 9.7 ± 1.1* 40.0 ± 1.9 4.0 ± 1.0* 1.6 ± 0.8 4.3 ± 0.2* 5.16 ± 0.5 8.9 ± 0.5* 319.7 ± 10.6 9.2 ± 1.1* 1360 ± 43.7 9.4 ± 0.3* 899.1 ± 44.7 18.8 ± 1.2* 315.9 ± 11.0 10.3 ± 1.6* 281.8 ± 16.43* NDa NDa 6.0 ± 0.3* 89.6 ± 21.6 18.7 ± 3.2* 106.4 ± 8.1 15.6 ± 2.7* 217.3 ± 11.2 8.4 ± 1.9* 157.8 ± 49.9 12.2 ± 1.9* 1159 ± 12.59 13.0 ± 0.3* 1076 ± 67.6 147.5 ± 5.3
Inhibition (% ± S.D.) Medium Solvent
96.4 ± 0.2 99.9 ± 0.1 99.5 ± 0.1 71.1 ± 1.0 22.8 ± 3.9 18.8 ± 4.0 71.5 ± 1.0 74.6 ± 1.5
91.9 ± 2.0 90.4 ± 0.7 80.4 ± 1.0 85.7 ± 4.5 4.7 ± 1.1 2.8 ± 6.1 86.7 ± 1.1
*Not stimulated with LPS 1 μg/mL. ND: not determined. a Cell viability < 90%.
(36.0 × 7.0 cm i.d.; bed volume of 1400 mL). The elution was carried out with absolute ethanol (200 proof), followed by mixtures of ethanol/acetone from 90:10 to 20:80. The eluates were combined according to their chromatographic profiles on silica gel TLC plates, affording 22 fractions. Fractions 9, 10 and 11, obtained by elution with pure ethanol, were further purified by preparative HPLC, using a Waters 2996 system composed of a quaternary pump and a Waters 996 PDA detector. An ODS column (250 × 21.2 mm i.d., 5 μm, Luna, Phenomenex, USA) was submitted to a gradient elution of acetonitrile (5 to 95%) and water in 60 min, at a flow rate of 5.0 mL/min and detection at 280 nm. Fraction 9 afforded compound OSF1 (RT = 12.14 min, 50 mg, white amorphous powder). Compounds OSF2 (RT = 14.46 min, 35 mg, yellow amorphous powder) and OSF3 (RT = 44.00 min, 6 mg, yellowish amorphous powder) were obtained from fraction 10, whereas OSF4 (RT = 51.31 min, 22 mg) and OSF5 (RT = 53.26 min, 27 mg) were obtained as yellow amorphous solids from fraction 11. Structure elucidation of compounds OSF1 to OSF5 was conducted by spectroscopic analysis, including UV Copyright © 2015 John Wiley & Sons, Ltd.
spectra obtained on line using a DAD detector and MS data, 1D and 2D NMR data (DEPT-135, HSQC and HMBC), and also by comparison with literature data (Moreira et al., 1999; Velandia et al., 2002; Daniel et al., 2005; Carvalho et al., 2008). NMR spectra were recorded on a Bruker Avance AVIII400 instrument (Germany) operating at 100 MHz for 13C and 400 MHz for 1H. Methanol-d4 and DMSO-d6 were employed as solvents and TMS was used as internal reference for both nuclei. Based on the obtained spectroscopic and spectrometric data and by comparison with literature records, compounds OSF1 to OSF5 were respectively identified as 1-O-(4-hydroxyphenyl)-6-O-(4-hydroxybenzoyl)-β-Dglucopyranoside (1, lanceoloside A), rutin (2), epicatechin (3), amentoflavone (4) and agathisflavone (5) (see Fig. 1 for chemical structures). HPLC analyses of the crude extract and isolated compounds were carried out on a Waters alliance 2695 HPLC system composed of a quaternary pump, an auto sampler, a photodiode array detector (DAD) 2996 and a Waters Empower pro data handling system (Waters Corporation, USA). An ODS column (250 × 4.6 mm i.d., 5 μm; Merck, Phytother. Res. 29: 1509–1515 (2015)
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Figure 1. Chemical structures of compounds isolated from the ethanol extract of Ouratea semiserrata leaves. 1, lanceoloside A; 2, rutin; 3, epicatechin; 4, amentoflavone; 5, agathisflavone.
Germany) in combination with a LiChrospher 100 RP-18 guard column (4 mm × 4 mm i.d., 5 μm; Merck, Germany) was employed for the analysis, eluted with a gradient of acetonitrile and water (5 to 45% ACN) in 35 min, at a flow rate of 1.0 mL/min and detection at 210 nm.
RESULTS AND DISCUSSION The selection of concentrations for testing extracts on cell-based bioassays is not straightforward. If a low concentration is employed, there is always the risk of rejecting potentially active minor constituents, whereas extracts tested at high concentrations may lead to unspecific positive response. The extract concentration employed in the present work was defined based on literature records for similar screenings. A wide assortment of concentrations is reported, ranging from 10 to 1000 μg/mL (Chou et al., 2013; Moon et al., 2013; Kim et al., 2013; Zhong et al., 2013). Some crude extracts elicit marginal TNF-α inhibition when tested at low concentrations, but the maximum effect does not reach high values (Wangchuk et al., 2013). Therefore, we employed the 500 μg/mL concentration for initial screening and established a minimum of 80% TNF-α inhibition to consider a crude extract as positive. The potential toxicity of the test samples was initially assessed against THP-1 monocytic cells to guarantee that the effects observed on TNF-α release were not because of low cell viability. From the 15 crude extracts evaluated, only J. caroba was toxic to the cell line, and, therefore, its effect on TNF-α release was not measured. All other plant extracts showed cell viability higher than 90% and were tested at the established concentration (500 μg/mL). A total of 11 extracts of the 14 non-toxic samples significantly reduced TNF-α release by LPSstimulated THP-1 cells in comparison to the control cells (Table 2). Among the active extracts, eight induced inhibition higher than 80%. It is noteworthy that none of the plant extracts induced the release of TNF-α by unstimulated THP-1 cells, indicating absence of proinflammatory properties (Table 2). Copyright © 2015 John Wiley & Sons, Ltd.
The effect of some selected extracts on TNF-α release was further evaluated at different concentrations. The extracts of C. carthagenensis, E. grandiflorus, M. hirsuta, O. semiserrata, O. spectabilis and R. ferruginea inhibited TNF-α release in a concentration-dependent way (Fig. 2). Most of the screenings for TNF-α inhibitors reported in literature have been carried out with one single concentration, in the range from 10 to 1000 μg/mL (Chou et al., 2013; Moon et al., 2013; Kim et al., 2013; Zhong et al., 2013). Therefore, the data here reported demonstrate unequivocally that the active extracts promote a concentration-dependent inhibition of this important pro-inflammatory cytokine. The chemical composition of these active extracts was preliminarily assessed by TLC and RP-HPLC fingerprints, and based on the chemical complexity and effect on TNF-α release, the extracts of M. hirsuta and O. semiserrata were selected for phytochemical studies. The phytochemical investigation of M. hirsuta afforded two new heterotrimeric glucosylated flavonoids named mansoins A and B, with potent TNF-α inhibitory activity (IC50 values of 48.1 ± 1.8 μM and 20.0 ± 1.4 μM, respectively), as recently reported by us (Campana et al., 2014). On its turn, the fractionation of the crude extract from O. semiserrata leaves by column chromatography over Sephadex LH 20 and further purification by RP-HPLC afforded five compounds identified as lanceoloside A (1), rutin (2), epicatechin (3), amentoflavone (4) and agathisflavone (5). The structure elucidation was accomplished by spectroscopic data and comparison with literature records (Moreira et al., 1999; Velandia et al., 2002; Daniel et al., 2005; Carvalho et al., 2008). Compounds 1, 2 and 4 have been previously reported for O. semiserrata leaves (Velandia et al., 2002), whereas the presence of 3 and 5 is described here for the first time. However, their occurrence has been registered for other Ouratea spp. (Moreira et al., 1999; Daniel et al., 2005; Carvalho et al., 2008). The chromatographic profile registered for the crude extract (Fig. 3) allowed us to ascribe the peaks corresponding to the isolated compounds, rutin being identified as the major peak (RT of 15.34 min). Even though the selected plant species are traditionally used to treat a variety of inflammatory disorders, Phytother. Res. 29: 1509–1515 (2015)
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Figure 2. Effect of crude extracts from Brazilian medicinal plants on TNF-α release in LPS-activated THP-1 cells, assayed at different concentrations (125, 250 and 500 μg/mL). Data represent the mean ± S.D. from three separate experiments. ***p < 0.001, statistically significant when compared with vehicle-treated control (0.1% DMSO). LPS only: cells treated with LPS without DMSO as vehicle.
only a few have had their antiinflammatory effects previously evaluated in vitro or in vivo. The aqueous extract of C. carthagenensis reduced the acetic acid-induced writhing in mice by 50% at 100 mg/kg, indicating a potential antinociceptive effect. On the other hand, the same extract only had minor effects on carrageenan-induced paw edema in mice (Fernandes et al., 2002). The antidematogenic activity of hydroethanolic extracts from E. grandiflorus leaves (1000 mg/kg, p.o.) and derived fractions enriched in diterpenes (70– 420 mg/kg, p.o.) and flavonoids (7.2–36 mg/kg, p.o.) was demonstrated by us using the carrageenan-induced paw edema model in mice (Garcia et al., 2010). rans-Aconitic acid obtained from E. grandiflorus also exhibited a significant antiedematogenic effect (270 mg/kg, p.o.). The ethanolic extract of O. semiserrata leaves elicited around 30% inhibition of 5-lipoxigenase in vitro, assayed at 19 μg/mL. Under the same conditions, the extracts of C. carthagenensis and M. hirsuta were inactive (Braga et al., 2000). On its turn, the active species O. spectabilis and R. ferruginea have not been previously investigated for their antiinflammatory potential using in vitro or in vivo assays. Therefore, this is the first report on the inhibition of TNF-α release in a human cell line elicited by the extracts of C. carthagenensis, E. grandiflorus, O. semiserrata, O. spectabilis and R. ferruginea. Targeting pro-inflammatory cytokines, especially TNF-α, constitutes a valid strategy to treat inflammatory disorders. Hence, the results described here for the six active species may be regarded as a first step to corroborate their traditional uses.
The compounds isolated from O. semiserrata extract were tested for their effects on TNF-α release by LPSstimulated THP-1 cells (Fig. 4). The biflavonoids 4 and 5 showed strong toxicity to the cell line and were not assayed: amentoflavone (4) was toxic at all evaluated concentrations while agathisflavone (5) showed toxicity at concentrations higher than 62.5 μM. Epicatechin, lanceoloside A and rutin elicited significant TNF-α inhibition, with inhibition values of 67.1 ± 2.6, 64.8 ± 2.4 and 42.4 ± 4.15% when tested at 250, 400 and 250 μM respectively, corresponding to 72.5, 156.8 and 152.5 μg/mL
Figure 4. Effect of compounds isolated from the ethanol extract of O. semiserrata leaves on TNF-α release in LPS-activated THP-1 cells, assayed at different concentrations (62.5–400 μM). Data represent the mean ± S.D. from three separate experiments. ***p < 0.001, statistically significant when compared with vehicle-treated control (0.1% DMSO). LPS only: cells treated with LPS without DMSO as vehicle.
Figure 3. RP-HPLC profile obtained for the ethanolic extract of O. semiserrata leaves. 1, epicatechin; 2, lanceoloside A; 3, rutin; 4, agathisflavone; 5, amentoflavone. Chromatographic conditions: see Materials and methods. Copyright © 2015 John Wiley & Sons, Ltd.
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(Fig. 4), indicating that these compounds may contribute to the overall activity observed for the crude extract. Epicatechin is a plant polyphenol found in different plant species and foods like tea, wine and cocoa. It possesses several biological activities, including antiinflammatory properties. The effect of catechins on the release of pro-inflammatory cytokines by whole blood cells has been previously evaluated (Crouvezier et al., 2001). Epicatechin showed no significant effect on the production of IL-1β, IL-6 or TNF-α, when assayed at concentrations up to 20 μM. However, its antiinflammatory activity has been evidenced in other models, such as the attenuation of neuroinflammation caused by antineoplasic treatment: it reduced the increase in TNF-α and the expression of iNOs and NF-κ B induced by doxorubicin (Mohamed et al., 2011). Epicatechin was shown to prevent TNF-α-induced activation of cell signals involved in inflammation and insulin resistance, such as NF-κB, mitogen-activated protein kinases (MAPKs), AP-1 and peroxisome proliferator activated receptor c (PPARc) in differentiated white adipocytes (3T3-L1) (Vazquez-Prieto et al., 2012). Rutin is a widespread flavonoid, whose biological activities have been extensively investigated, including its antiinflammatory properties (Kwon et al., 2005). Rutin inhibited the expression of iNOS and COX-2 and decreased the levels of IL-6 and TNF-α in LPS-stimulated RAW 264.7 cells at a concentration of 100 μg/mL (Karki et al., 2013). Additionally, it reduced the expression of NF-κB p65 subunit in the nuclear fraction (Karki et al., 2013). Previously, we have reported the inhibition of TPA-mediated NF-κB activation by rutin, with an IC50 value of 26.8 ± 6.3 μM in HEK293 cell line (Endringer et al., 2009). The antiinflammatory potential of lanceoloside A, an arbutin derivative, has not been investigated so far, even though the antiinflammatory effect of arbutin has been demonstrated using in vitro and in vivo models. Arbutin reduced the ulcer area and associated edema, inflammation and leucocyte infiltration in models of ulcer induced by aspirin or ethanol (Taha et al., 2012). Pre-treatment
with arbutin in the same models (ethanol and aspirin induced ulcers) reduced the levels of the proinflammatory cytokines TNF-α and IL-6 and increased the release of the antiinflammatory cytokine IL-10 (Taha et al., 2012). Arbutin was reported to suppress the production of NO and expression of iNOS and COX-2 by LPS-stimulated microglial cells. It also reduced the production of IL-1β and TNF-α and inhibited nuclear translocation of NF-κB (Lee and Kim, 2012). In summary, our findings are the first evidence to corroborate the traditional use of six medicinal plants to treat inflammatory conditions. Moreover, the activity of the isolated phenolic compounds, especially lanceoloside A, will be further investigated aiming the elucidation of its mechanism of action.
CONCLUSION TNF-α release by LPS-stimulated THP-1 cells was significantly reduced by seven of the 14 evaluated extracts, prepared from six Brazilian medicinal plants. The fractionation of O. semiserrata extract afforded five compounds of which lanceoloside A, epicatechin and rutin induced considerable inhibition of TNF-α release and may account for the overall activity of the crude extract.
Acknowledgements This work received financial support from CNPq, FAPEMIG and the European Community’s Seventh Framework Programme [Fp7-2007– 2013] under grant agreement N. Health-F4-2011-281608. CNPq is also acknowledged for the research fellowships (F.C.B. and M.M.T.).
Conflict of Interest The authors declare no conflict of interest connected to this paper.
REFERENCES Alessandri AL, Souza PL, Lucas CD, Rossi AG, Pinho V, Teixeira MM, 2013. Resolution of inflammation: mechanisms and opportunity for drug development. Pharmacol Ther 139: 189–212. Braga FC, Wagner H, Lombardi JA, Oliveira AB. 2000. Screening Brazilian plant species for in vitro inhibition of 5-lipoxygenase. Phytomedicine 6: 447–452. Brandão MGL, Cosenza GP, Grael CF, Netto NL Jr, Montemor R. 2009. Traditional uses of American plant species from the 1st edition of Brazilian official Pharmacopoeia. Rev Bras Farmacogn 19: 478–484. Britto K, Britto IC. 1981/1982. Plantas com atributos medicinais do herbário da Universidade de Feira de Santana. Oréades 8: 152–163. Campana PRV, Coleman CM, Teixeira MM, Ferreira D, Braga FC. 2014. TNF-α inhibition elicited by mansoins A and B, heterotrimeric flavonoids isolated from Mansoa hirsuta. J Nat Prod 77: 824–830. Cano JH, Volpato G. 2004. Herbal mixtures in the traditional medicine in eastern Cuba. J Ethnopharmacol 30: 293–316. Carvalho MG, Albuquerque LRM, Mendes LS, Guilhon GMSP, Rodrigues ST. 2008. Biflavonoids and terpenoids isolated from the leaves of Ouratea microdonta Engl. (Ochnaceae). Rev Latinoam Quim 36: 71–75. Chaves SAM, Reinhard KJ. 2003. Paleopharmacology and pollen: theory, method, and application. Mem Inst Oswaldo Cruz 98: 207–211. Copyright © 2015 John Wiley & Sons, Ltd.
Chou ST, Peng HY, Hsu JC, Lin CC, Shih Y. 2013. Achillea millefolium L. essential oil inhibits LPS-induced oxidative stress and nitric oxide production in RAW 264.7 macrophages. Int J Mol Sci 14: 12,978–12,993. Choy ES, Panayi GS. 2001. Cytokine pathways and joint inflammation in rheumatoid arthritis. N Engl J Med 344: 907–910. Corrêa MP. 1974. Dicionário de Plantas úteis do Brasil e das Exóticas Cultivadas. Di Giorgio: Rio de Janeiro. Croft M, Benedict CA, Ware CF. 2013. Clinical targeting of the TNF and TNFR superfamilies. Nat Rev 12: 147–168. Crouvezier S, Powell B, Keir D, Yaqoob P. 2001. The effects of phenolic components of tea on the production of pro- and anti-inflammatory cytokines by human leukocytes in vitro. Cytokine 13: 280–286. Daniel JFS, Carvalho MG, Cardoso RS, Agra MF, Ebeli MN. 2005. Other flavonoids from Ouratea hexasperma (Ochnaceae). J Braz Chem Soc 16: 634–638. Di Stasi LC, Hiruma-Lima CA. 2002. Plantas Medicinais na Amazônia e na Mata Atlântica (2nd edn). Unesp: São Paulo. Endringer DC, Pezzuto JM, Braga FC. 2009. NF-κB inhibitory activity of cyclitols isolated from Hancornia speciosa. Phytomedicine 16: 1064–1069. Falcão HS, Lima IO, Santos VL, et al. 2005. Review of the plants with anti-inflammatory activity studied in Brazil. Rev Bras Farmacogn 15: 381–391. Phytother. Res. 29: 1509–1515 (2015)
ANTI-TNF-α ACTIVITY OF BRAZILIAN PLANTS AND ISOLATED COMPOUNDS Felicio JD, Gonçalez E, Braggio MM, Constantino L, Albasini A, Lins AP. 1995. Inhibition of lens aldose reductase by biflavones from Ouratea spectabilis. Planta Med 61: 217–220. Fernandes FR, Santos AL, Arruda AMS, et al. 2002. Antinociceptive and anti-inflammatory activities of the aqueous extract and isolated Cuphea carthagenensis (Jacq.) J. F. Macbr. Rev Bras Farmacogn 12: 55–56. Garcia EF, Oliveira MA, Godin AM, et al. 2010. Antiedematogenic activity and phytochemical composition of preparations from Echinodorus grandiflorus leaves. Phytomedicine 18: 80–86. Gonzales-Guevara JL, Velez-Castro H, Gonzalez-Garcia KL, et al. 2006. Flavonoid glycosides from Cuban Erythroxylum species. Biochem Sys Ecol 34: 539–542. Grandi TMS, Lima-Filho FM, Ferreira SMA. 1981/1982. Levantamento das plantas medicinais de Grão Mogol. Oréades 8: 116–125. Karki R, Park CH, Kim DW. 2013. Extract of buckwheat sprouts scavenges oxidation and inhibits pro-inflammatory mediators in lipopolysaccharide-stimulated macrophages (RAW264.7). Chin J Integr Med 11: 246–252. Khanna D, Sethi G, Ahn KS, et al. 2007. Natural products as a gold mine for arthritis treatment. Curr Opin Pharmacol 7: 344–351. Kim MJ, Yang KW, Kim SS, et al. 2013. Chemical composition and anti-inflammatory effects of essential oil from Hallabong flower. EXCLI J 12: 933–942. Kwon KH, Murakami A, Tanaka T, Ohigashi H. 2005. Dietary rutin, but not its aglycone quercetin, ameliorates dextran sulfate sodium-induced experimental colitis in mice: attenuation of pro-inflammatory gene expression. Biochem Pharmacol 69: 395–406. Laveti D, Kumar M, Hemalatha R, et al. 2013. Anti-inflammatory treatments for chronic diseases: a review. Inflamm Allergy Drug Targets 12: 349–361. Lee HJ, Kim KW. 2012. Anti-inflammatory effects of arbutin in lipopolysaccharide-stimulated BV2 microglial cells. Inflamm Res 61: 817–825. Lima JCS, Martins DT. 1996. Screening farmacológico de plantas medicinais utilizadas popularmente com anti-inflamatória, in Resumos do Simpósio de Plantas Medicinais do Brasil, 89 p., Universidade Federal de Santa Catarina, Florianópolis. Lima CB, Bellettini NMT, Silva AS, et al. 2007. Uso de plantas medicinais pela população da zona urbana de bandeirantes, PR. Rev Bras Biociênc 5: 600–602. Medzhitov R. 2008. Origin and physiological roles of inflammation. Nature 454: 428–435.
Copyright © 2015 John Wiley & Sons, Ltd.
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Mohamed RH, Karam RA, Amer MG. 2011. Epicatechin attenuates doxorubicin-induced brain toxicity: critical role of TNF-α, iNOS and NF-κB. Brain Res Bull 86: 22–26. Moon PD, Choi IS, Go JH, et al. 2013. Inhibitory effects of BiRyuCheBang on mast cell-mediated allergic reactions and inflammatory cytokines production. Am J Chin Med 41: 1267–1282. Moreira IC, Carvalho MG, Bastos ABFO, Braz-Filho R. 1999. A flavone dimer from Ouratea hexasperma. Phytochemistry 51: 833–838. Scott DL, Kingsley GH. 2006. Tumor necrosis factor inhibitors for rheumatoid arthritis. N Engl J Med 355: 704–712. Siqueira JC. 1981. Utilização Popular das Plantas do Cerrado. Loyola: São Paulo. Souza CD, Felfili JM. 2006. Uso de plantas medicinais na região de alto paraíso de Goiás, GO, Brasil. Acta Bot Bras 20: 135–142. Souza LP, Alessandri AL, Pinho V, Teixeira MM. 2013. Pharmacological strategies to resolve acute inflammation. Curr Opin Pharmacol 13: 1–7. Taha MME, Salga MS, Ali HM, Abdulla MA, Abdelwahab SI, Hadi AHA. 2012. Gastroprotective activities of Turnera diffusa Willd. ex Schult. revisited: role of arbutin. J Ethnopharmacol 141: 273–281. Vazquez-Prieto MA, Bettaieb A, Haj FG, Fraga CG, Oteiza PI. 2012. ( )-Epicatechin prevents TNFα-induced activation of signaling cascades involved in inflammation and insulin sensitivity in 3T3-L1 adipocytes. Arch Biochem Biophys 527: 113–118. Velandia JR, de Carvalho MG, Braz-Filho R, Werle AA. 2002. Biflavonoids and a glucopyranoside derivative from Ouratea semiserrata. Phytochem Anal 13: 283–292. Vendruscolo GS. 2004. Estudo etnobotânico das plantas utilizadas como medicinais por moradores do bairro Ponta Grossa, Porto Alegre, Rio Grande do Sul, Dissertation, Universidade Federal do Rio Grande do Sul, Porto Alegre. Wangchuk P, Keller PA, Pyne SG, Taweechotipatr M. 2013. Inhibition of TNF-α production in LPS-activated THP-1 monocytic cells by the crude extracts of seven Bhutanese medicinal plants. J Ethnopharmacol 148: 1013–1017. Weiss T, Shalit I, Blau H, et al. 2004. Anti-inflammatory effects of moxifloxacin on activated human monocytic cells: inhibition of NF-κB and mitogen-activated protein kinase activation and of synthesis of proinflammatory cytokines. Antimicrob Agents Chemother 48: 1974–1982. Zhong W, Chi G, Jiang L, et al. 2013. p-Cymene modulates in vitro and in vivo cytokine production by inhibiting MAPK and NF-κB activation. Inflammation 36: 529–537.
Phytother. Res. 29: 1509–1515 (2015)
Anti-TNF-α Activity of Brazilian Medicinal Plants and Compounds from Ouratea semiserrata Priscilla R. V. Campana,1,2 Daniel S. Mansur,3 Grasielle S. Gusman,1 Daneel Ferreira,4 Mauro M. Teixeira3 and Fernão C. Braga1* 1
Faculdade de Farmácia, Universidade Federal de Minas Gerais, Av. Antônio Carlos 6627, campus Pampulha, Belo Horizonte CEP 31.270-901, Brazil 2 Divisão de Ciências Farmacêuticas, Fundação Ezequiel Dias, R. Conde Pereira Carneiro 80, Belo Horizonte CEP 30.510-010, Brazil 3 Departamento de Bioquímica, ICB, Universidade Federal de Minas Gerais, Av. Antônio Carlos 6627, campus Pampulha, Belo Horizonte CEP 31.270-901, Brazil 4 Department of Pharmacognosy and Research Institute of Pharmaceutical Sciences, University of Mississippi, MS 38677, USA
Several plant species are used in Brazil to treat inflammatory diseases and associated conditions. TNF-α plays a pivotal role on inflammation, and several plant extracts have been assayed against this target, both in vitro and in vivo. The effect of 11 Brazilian medicinal plants on TNF-α release by LPS-activated THP-1 cells was evaluated. The plant materials were percolated with different solvents to afford 15 crude extracts, whose effect on TNF-α release was determined by ELISA. Among the evaluated extracts, only Jacaranda caroba (Bignoniaceae) presented strong toxicity to THP-1 cells. Considering the 14 non-toxic extracts, TNF-α release was significantly reduced by seven of them (inhibition > 80%), originating from six plants, namely Cuphea carthagenensis (Lythraceae), Echinodorus grandiflorus (Alismataceae), Mansoa hirsuta (Bignoniaceae), Ouratea semiserrata (Ochnaceae), Ouratea spectabilis and Remijia ferruginea (Rubiaceae). The ethanol extract from O. semiserrata leaves was fractionated over Sephadex LH-20 and RP-HPLC to give three compounds previously reported for the species, along with agathisflavone and epicatechin, here described for the first time in the plant. Epicatechin and lanceoloside A elicited significant inhibition of TNF-α release, indicating that they may account for the effect produced by O. semiserrata crude extract. Copyright © 2015 John Wiley & Sons, Ltd. Keywords: TNF-α inhibition; Brazilian medicinal plants; Ouratea semiserrata; epicatechin; lanceoloside A.
INTRODUCTION Inflammation is a physiological response to infection or sterile injury of tissues that aims to restore tissue functions to their original state. However, excessive or uncontrolled inflammation contributes to the pathogenesis of various diseases including rheumatoid arthritis and inflammatory bowel diseases (Medzhitov, 2008; Alessandri et al., 2013; Souza et al., 2013). TNF-α is a crucial mediator of inflammatory responses. It is rapidly released after inflammatory stimuli, including infection or tissue injury, and triggers a series of intracellular events that culminate in the activation of the transcription nuclear factor kappa B (NF-κB), leading to the production of other proinflammatory cytokines, chemokines and proteases (Choy and Panayi, 2001; Scott and Kingsley, 2006). Consistent with this relevant role of TNF-α in the context of inflammation, injection of TNF-α causes significant influx of leukocytes in vivo, whereas blockade of TNF-α ameliorates inflammatory responses in several preclinical models of inflammatory diseases (Croft et al., 2013). The inhibition of TNF-α is an emerging and promising strategy to overcome chronic inflammatory processes in human diseases like rheumatoid arthritis and inflammatory bowel diseases (Laveti et al., 2013). Indeed, several * Correspondence to: Fernão C. Braga, Faculdade de Farmácia, Universidade Federal de Minas Gerais, Av. Antônio Carlos 6627, campus Pampulha, Belo Horizonte CEP 31.270-901, Brazil. E-mail: [email protected]
Copyright © 2015 John Wiley & Sons, Ltd.
TNF-α inhibitors have been proven to be clinically effective (Croft et al., 2013). Presently, commercially available anti-TNF-α drugs are immunobiological which require intra-articular administration and are costly (Khanna et al., 2007). Therefore, there is demand for new TNF-α inhibitors that can be further developed as antiinflammatory drugs to treat chronic diseases like arthritis and psoriasis. Several plant species are traditionally used in Brazil to treat a plethora of inflammatory conditions (Falcão et al., 2005), which may represent a source of bioactive compounds. Hence, the goal of the present work was to investigate some selected medicinal plants as potential TNF-α inhibitors.
MATERIALS AND METHODS Plant materials and preparation of the crude extracts. Eleven plant species were selected based on their traditional uses in Brazil to treat different inflammatory process, including Cuphea carthagenensis (Jacq.) J.F. Macbr. (Lythraceae), Echinodorus grandiflorus (Cham. & Schltdl.) Micheli (Alismataceae), Erythroxylum gonocladum (Mart.) O.E. Schulz (Erythroxylaceae), Erythroxylum suberosum A. St.-Hil., Erythroxylum tortuosum Mart, Hancornia speciosa Gomes (Apocynaceae), Jacaranda caroba (Vell.) A. DC. (Bignoniaceae), Mansoa hirsuta DC. (Bignoniaceae), Ouratea semiserrata (Mart. & Nees) Engl. (Ochnaceae), Received 11 November 2014 Revised 19 May 2015 Accepted 26 May 2015
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Jacaranda caroba Mansoa hirsuta Ouratea semisserrata Ouratea spectabilis Remijia ferruginea
Aerial parts Leaves Leaves Leaves Leaves Leaves, bark, roots Leaves Aerial parts Leaves, bark Leaves, bark Leaves, stems
Telêmaco Borba Telêmaco Borba Caeté Lagoa Santa Lagoa Santa São Gonçalo do Rio Preto Belo Horizonte Caratinga Serra do Cipó Perdizes São Gonçalo do Rio Preto
51.460 23.862 49.269 48.94 3.833
Vendruscolo, 2004; Lima et al., 2007 Souza and Felfili, 2006; Brandão et al., 2009; Corrêa, 1974 Gonzales-Guevara et al., 2006; Cano and Volpato, 2004 Gonzales-Guevara et al., 2006; Cano and Volpato, 2004 Gonzales-Guevara et al., 2006; Cano and Volpato, 2004 Lima and Martins, 1996; Grandi et al., 1982; Britto and Britto, 1982 Siqueira, 1981; Corrêa, 1974; Di Stasi and Hiruma-Lima, 2002 Chaves and Reinhard, 2003 Corrêa, 1974 Felicio et al., 1995 Corrêa, 1974 Cardiovascular diseases, atherosclerosis Anti-rheumatic, antiinflammatory Antiinflammatory, to treat bronchitis and asthma Antiinflammatory, to treat bronchitis and asthma Antiinflammatory, to treat bronchitis and asthma Antiinflammatory, anti-rheumatic, to treat gout and dermatosis Wound healing, to treat ulcers To treat sore throats Antiinflammatory Antiinflammatory, to treat ulcers To treat ulcers and fever
Bioassay method. The potential antiinflammatory activity of the crude extracts and compounds isolated from O. semiserrata leaves was evaluated by measuring TNF-α produced by LPS-stimulated THP-1 cells, employing an immunoassay as described by Weiss et al. (2004). THP-1 cells (ATCC TIB-202) were cultivated in RPMI 1640 medium (Sigma, USA) supplemented with 0.05 mM 2-mercaptoethanol, 10% FBS, 100 U/mL of penicillin and 100 μg/mL of gentamicin at 37 °C, in an atmosphere containing 5% CO2. The medium was renovated twice a week, when cell concentrations reached 1.0 × 106 cells/mL. The cells were transferred to a 96-well microplate at a density of 100 000 cells per well, incubated for 18 h and pre-treated with the samples for 3 h. LPS (from Escherichia coli 0111: B4, Sigma) was administrated at 1.0 μg/mL as inflammatory stimulus. After incubating the plate at 37 °C overnight, it was centrifuged (1.800 ×g, 5 min, 16 °C), the supernatant was collected and TNF-α was measured using the cytokine-specific sandwich quantitative enzyme-linked immune-sorbent assay (ELISA) according to the manufacturer’s instructions (TNF-α duo set, DY210, R&D Systems, USA). Cell viability was evaluated for all tested samples by the MTT method using untreated cells as reference for viability. Samples that gave cell viability higher than 90% were considered non-toxic for the THP-1 cell line. The percentage of TNF-α inhibition was calculated by the ratio between the TNF-α amount secreted by treated cells (pg/mL) and the baseline level of this cytokine (pg/mL) observed for solvent control (0.1% DMSO). The statistical significance of differences was calculated employing the software GraphPad Prism, version 5.0 (GraphPad Software Inc., USA) using one-way ANOVA followed by Dunnett post-test for multiple comparisons. Results were considered different when p < 0.05. The samples were tested in triplicate at different concentrations (125–500 μg/mL for extracts and 62.5–450 μM for pure compounds). Dexamethasone was employed as positive control (0.1 μM).
49.269 107.791 118.812 111.068 111.065 49.895
Ouratea spectabilis (Mart. ex Engl.) Engl. and Remijia ferruginea (A. St.-Hil.) DC. (Rubiaceae). The ethnopharmacological uses of these plants are collated in Table 1. The plant materials were collected, and the species were identified by botanists from the Botanical Department, Instituto de Ciências Biológicas (ICB), UFMG, Belo Horizonte, Brazil, where voucher specimens are deposited (see Table 1 for voucher numbers). After drying at 45 °C, during 72 h, the plant materials were powdered and extracted by exhaustive percolation with ethanol or acetone at room temperature (Table 2). The solvent was vacuum removed in a rotatory evaporator at 50 °C, to furnish the crude extracts (see Table 2 for extraction yields). The inhibitory effects on TNF-α production in LPS-activated THP-1 monocytic cells of the crude extracts were evaluated at a concentration of 500 μg/mL. Selected extracts were further assayed at 125, 250 and 500 μg/mL.
Cuphea carthagenensis Echinodorus grandiflorus Erythroxylum gonocladum Erythroxylum suberosum Erythroxylum tortuosum Hancornia speciosa
Traditional uses Voucher number Sample location Part used Plant species
Table 1. Selected Brazilian medicinal plants with their botanical names, part used, ethnopharmacological uses and other information
Literature
P. R. V. CAMPANA ET AL.
Phytochemical investigation of O. semiserrata. A portion (10 g) of the ethanolic extract from O. semiserrata leaves was fractionated over a Sephadex LH-20 column Phytother. Res. 29: 1509–1515 (2015)
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Table 2. Inhibition of TNF-α production by LPS-activated THP-1 monocytic cells elicited by the extracts of Brazilian medicinal plants Species
Plant part
Control
Extract
Yield (% w/w)
RPMI DMSO 0.1%
C. carthagenensis
Aerial parts
Acetone 50%
25.7
Acetone 70%
23.3
E. grandiflorus
Leaves
Ethanol 70%
27.0
E. gonocladum
Leaves
Ethanol 96 GL
23.6
E. suberosum
Leaves
Ethanol 96 GL
18.6
E. tortuosum
Leaves
Ethanol 96 GL
27.2
H. speciosa
Leaves
Acetone 50%
28.4
Acetone 70%
33.9
J. caroba
Leaves
Ethanol 96 GL
23.3
M. hirsuta
Fruits
Ethanol 96 GL
18.6
O. semisserrata
Leaves
Ethanol 96 GL
30.1
O. spectabilis
Bark
Ethanol 96 GL
36.3
R. ferruginea
Leaves
Ethanol 96 GL
ND
Stem
Ethanol 96 GL
21.4
Ethanol 50%
ND
Dexamethasone
TNF-α (pg/mL ± S.D.) 10.9 ± 1.1* 1055.0 ± 63.9 14.2 ± 0.5* 1107.1 ± 93.7 9.7 ± 1.1* 40.0 ± 1.9 4.0 ± 1.0* 1.6 ± 0.8 4.3 ± 0.2* 5.16 ± 0.5 8.9 ± 0.5* 319.7 ± 10.6 9.2 ± 1.1* 1360 ± 43.7 9.4 ± 0.3* 899.1 ± 44.7 18.8 ± 1.2* 315.9 ± 11.0 10.3 ± 1.6* 281.8 ± 16.43* NDa NDa 6.0 ± 0.3* 89.6 ± 21.6 18.7 ± 3.2* 106.4 ± 8.1 15.6 ± 2.7* 217.3 ± 11.2 8.4 ± 1.9* 157.8 ± 49.9 12.2 ± 1.9* 1159 ± 12.59 13.0 ± 0.3* 1076 ± 67.6 147.5 ± 5.3
Inhibition (% ± S.D.) Medium Solvent
96.4 ± 0.2 99.9 ± 0.1 99.5 ± 0.1 71.1 ± 1.0 22.8 ± 3.9 18.8 ± 4.0 71.5 ± 1.0 74.6 ± 1.5
91.9 ± 2.0 90.4 ± 0.7 80.4 ± 1.0 85.7 ± 4.5 4.7 ± 1.1 2.8 ± 6.1 86.7 ± 1.1
*Not stimulated with LPS 1 μg/mL. ND: not determined. a Cell viability < 90%.
(36.0 × 7.0 cm i.d.; bed volume of 1400 mL). The elution was carried out with absolute ethanol (200 proof), followed by mixtures of ethanol/acetone from 90:10 to 20:80. The eluates were combined according to their chromatographic profiles on silica gel TLC plates, affording 22 fractions. Fractions 9, 10 and 11, obtained by elution with pure ethanol, were further purified by preparative HPLC, using a Waters 2996 system composed of a quaternary pump and a Waters 996 PDA detector. An ODS column (250 × 21.2 mm i.d., 5 μm, Luna, Phenomenex, USA) was submitted to a gradient elution of acetonitrile (5 to 95%) and water in 60 min, at a flow rate of 5.0 mL/min and detection at 280 nm. Fraction 9 afforded compound OSF1 (RT = 12.14 min, 50 mg, white amorphous powder). Compounds OSF2 (RT = 14.46 min, 35 mg, yellow amorphous powder) and OSF3 (RT = 44.00 min, 6 mg, yellowish amorphous powder) were obtained from fraction 10, whereas OSF4 (RT = 51.31 min, 22 mg) and OSF5 (RT = 53.26 min, 27 mg) were obtained as yellow amorphous solids from fraction 11. Structure elucidation of compounds OSF1 to OSF5 was conducted by spectroscopic analysis, including UV Copyright © 2015 John Wiley & Sons, Ltd.
spectra obtained on line using a DAD detector and MS data, 1D and 2D NMR data (DEPT-135, HSQC and HMBC), and also by comparison with literature data (Moreira et al., 1999; Velandia et al., 2002; Daniel et al., 2005; Carvalho et al., 2008). NMR spectra were recorded on a Bruker Avance AVIII400 instrument (Germany) operating at 100 MHz for 13C and 400 MHz for 1H. Methanol-d4 and DMSO-d6 were employed as solvents and TMS was used as internal reference for both nuclei. Based on the obtained spectroscopic and spectrometric data and by comparison with literature records, compounds OSF1 to OSF5 were respectively identified as 1-O-(4-hydroxyphenyl)-6-O-(4-hydroxybenzoyl)-β-Dglucopyranoside (1, lanceoloside A), rutin (2), epicatechin (3), amentoflavone (4) and agathisflavone (5) (see Fig. 1 for chemical structures). HPLC analyses of the crude extract and isolated compounds were carried out on a Waters alliance 2695 HPLC system composed of a quaternary pump, an auto sampler, a photodiode array detector (DAD) 2996 and a Waters Empower pro data handling system (Waters Corporation, USA). An ODS column (250 × 4.6 mm i.d., 5 μm; Merck, Phytother. Res. 29: 1509–1515 (2015)
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Figure 1. Chemical structures of compounds isolated from the ethanol extract of Ouratea semiserrata leaves. 1, lanceoloside A; 2, rutin; 3, epicatechin; 4, amentoflavone; 5, agathisflavone.
Germany) in combination with a LiChrospher 100 RP-18 guard column (4 mm × 4 mm i.d., 5 μm; Merck, Germany) was employed for the analysis, eluted with a gradient of acetonitrile and water (5 to 45% ACN) in 35 min, at a flow rate of 1.0 mL/min and detection at 210 nm.
RESULTS AND DISCUSSION The selection of concentrations for testing extracts on cell-based bioassays is not straightforward. If a low concentration is employed, there is always the risk of rejecting potentially active minor constituents, whereas extracts tested at high concentrations may lead to unspecific positive response. The extract concentration employed in the present work was defined based on literature records for similar screenings. A wide assortment of concentrations is reported, ranging from 10 to 1000 μg/mL (Chou et al., 2013; Moon et al., 2013; Kim et al., 2013; Zhong et al., 2013). Some crude extracts elicit marginal TNF-α inhibition when tested at low concentrations, but the maximum effect does not reach high values (Wangchuk et al., 2013). Therefore, we employed the 500 μg/mL concentration for initial screening and established a minimum of 80% TNF-α inhibition to consider a crude extract as positive. The potential toxicity of the test samples was initially assessed against THP-1 monocytic cells to guarantee that the effects observed on TNF-α release were not because of low cell viability. From the 15 crude extracts evaluated, only J. caroba was toxic to the cell line, and, therefore, its effect on TNF-α release was not measured. All other plant extracts showed cell viability higher than 90% and were tested at the established concentration (500 μg/mL). A total of 11 extracts of the 14 non-toxic samples significantly reduced TNF-α release by LPSstimulated THP-1 cells in comparison to the control cells (Table 2). Among the active extracts, eight induced inhibition higher than 80%. It is noteworthy that none of the plant extracts induced the release of TNF-α by unstimulated THP-1 cells, indicating absence of proinflammatory properties (Table 2). Copyright © 2015 John Wiley & Sons, Ltd.
The effect of some selected extracts on TNF-α release was further evaluated at different concentrations. The extracts of C. carthagenensis, E. grandiflorus, M. hirsuta, O. semiserrata, O. spectabilis and R. ferruginea inhibited TNF-α release in a concentration-dependent way (Fig. 2). Most of the screenings for TNF-α inhibitors reported in literature have been carried out with one single concentration, in the range from 10 to 1000 μg/mL (Chou et al., 2013; Moon et al., 2013; Kim et al., 2013; Zhong et al., 2013). Therefore, the data here reported demonstrate unequivocally that the active extracts promote a concentration-dependent inhibition of this important pro-inflammatory cytokine. The chemical composition of these active extracts was preliminarily assessed by TLC and RP-HPLC fingerprints, and based on the chemical complexity and effect on TNF-α release, the extracts of M. hirsuta and O. semiserrata were selected for phytochemical studies. The phytochemical investigation of M. hirsuta afforded two new heterotrimeric glucosylated flavonoids named mansoins A and B, with potent TNF-α inhibitory activity (IC50 values of 48.1 ± 1.8 μM and 20.0 ± 1.4 μM, respectively), as recently reported by us (Campana et al., 2014). On its turn, the fractionation of the crude extract from O. semiserrata leaves by column chromatography over Sephadex LH 20 and further purification by RP-HPLC afforded five compounds identified as lanceoloside A (1), rutin (2), epicatechin (3), amentoflavone (4) and agathisflavone (5). The structure elucidation was accomplished by spectroscopic data and comparison with literature records (Moreira et al., 1999; Velandia et al., 2002; Daniel et al., 2005; Carvalho et al., 2008). Compounds 1, 2 and 4 have been previously reported for O. semiserrata leaves (Velandia et al., 2002), whereas the presence of 3 and 5 is described here for the first time. However, their occurrence has been registered for other Ouratea spp. (Moreira et al., 1999; Daniel et al., 2005; Carvalho et al., 2008). The chromatographic profile registered for the crude extract (Fig. 3) allowed us to ascribe the peaks corresponding to the isolated compounds, rutin being identified as the major peak (RT of 15.34 min). Even though the selected plant species are traditionally used to treat a variety of inflammatory disorders, Phytother. Res. 29: 1509–1515 (2015)
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Figure 2. Effect of crude extracts from Brazilian medicinal plants on TNF-α release in LPS-activated THP-1 cells, assayed at different concentrations (125, 250 and 500 μg/mL). Data represent the mean ± S.D. from three separate experiments. ***p < 0.001, statistically significant when compared with vehicle-treated control (0.1% DMSO). LPS only: cells treated with LPS without DMSO as vehicle.
only a few have had their antiinflammatory effects previously evaluated in vitro or in vivo. The aqueous extract of C. carthagenensis reduced the acetic acid-induced writhing in mice by 50% at 100 mg/kg, indicating a potential antinociceptive effect. On the other hand, the same extract only had minor effects on carrageenan-induced paw edema in mice (Fernandes et al., 2002). The antidematogenic activity of hydroethanolic extracts from E. grandiflorus leaves (1000 mg/kg, p.o.) and derived fractions enriched in diterpenes (70– 420 mg/kg, p.o.) and flavonoids (7.2–36 mg/kg, p.o.) was demonstrated by us using the carrageenan-induced paw edema model in mice (Garcia et al., 2010). rans-Aconitic acid obtained from E. grandiflorus also exhibited a significant antiedematogenic effect (270 mg/kg, p.o.). The ethanolic extract of O. semiserrata leaves elicited around 30% inhibition of 5-lipoxigenase in vitro, assayed at 19 μg/mL. Under the same conditions, the extracts of C. carthagenensis and M. hirsuta were inactive (Braga et al., 2000). On its turn, the active species O. spectabilis and R. ferruginea have not been previously investigated for their antiinflammatory potential using in vitro or in vivo assays. Therefore, this is the first report on the inhibition of TNF-α release in a human cell line elicited by the extracts of C. carthagenensis, E. grandiflorus, O. semiserrata, O. spectabilis and R. ferruginea. Targeting pro-inflammatory cytokines, especially TNF-α, constitutes a valid strategy to treat inflammatory disorders. Hence, the results described here for the six active species may be regarded as a first step to corroborate their traditional uses.
The compounds isolated from O. semiserrata extract were tested for their effects on TNF-α release by LPSstimulated THP-1 cells (Fig. 4). The biflavonoids 4 and 5 showed strong toxicity to the cell line and were not assayed: amentoflavone (4) was toxic at all evaluated concentrations while agathisflavone (5) showed toxicity at concentrations higher than 62.5 μM. Epicatechin, lanceoloside A and rutin elicited significant TNF-α inhibition, with inhibition values of 67.1 ± 2.6, 64.8 ± 2.4 and 42.4 ± 4.15% when tested at 250, 400 and 250 μM respectively, corresponding to 72.5, 156.8 and 152.5 μg/mL
Figure 4. Effect of compounds isolated from the ethanol extract of O. semiserrata leaves on TNF-α release in LPS-activated THP-1 cells, assayed at different concentrations (62.5–400 μM). Data represent the mean ± S.D. from three separate experiments. ***p < 0.001, statistically significant when compared with vehicle-treated control (0.1% DMSO). LPS only: cells treated with LPS without DMSO as vehicle.
Figure 3. RP-HPLC profile obtained for the ethanolic extract of O. semiserrata leaves. 1, epicatechin; 2, lanceoloside A; 3, rutin; 4, agathisflavone; 5, amentoflavone. Chromatographic conditions: see Materials and methods. Copyright © 2015 John Wiley & Sons, Ltd.
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(Fig. 4), indicating that these compounds may contribute to the overall activity observed for the crude extract. Epicatechin is a plant polyphenol found in different plant species and foods like tea, wine and cocoa. It possesses several biological activities, including antiinflammatory properties. The effect of catechins on the release of pro-inflammatory cytokines by whole blood cells has been previously evaluated (Crouvezier et al., 2001). Epicatechin showed no significant effect on the production of IL-1β, IL-6 or TNF-α, when assayed at concentrations up to 20 μM. However, its antiinflammatory activity has been evidenced in other models, such as the attenuation of neuroinflammation caused by antineoplasic treatment: it reduced the increase in TNF-α and the expression of iNOs and NF-κ B induced by doxorubicin (Mohamed et al., 2011). Epicatechin was shown to prevent TNF-α-induced activation of cell signals involved in inflammation and insulin resistance, such as NF-κB, mitogen-activated protein kinases (MAPKs), AP-1 and peroxisome proliferator activated receptor c (PPARc) in differentiated white adipocytes (3T3-L1) (Vazquez-Prieto et al., 2012). Rutin is a widespread flavonoid, whose biological activities have been extensively investigated, including its antiinflammatory properties (Kwon et al., 2005). Rutin inhibited the expression of iNOS and COX-2 and decreased the levels of IL-6 and TNF-α in LPS-stimulated RAW 264.7 cells at a concentration of 100 μg/mL (Karki et al., 2013). Additionally, it reduced the expression of NF-κB p65 subunit in the nuclear fraction (Karki et al., 2013). Previously, we have reported the inhibition of TPA-mediated NF-κB activation by rutin, with an IC50 value of 26.8 ± 6.3 μM in HEK293 cell line (Endringer et al., 2009). The antiinflammatory potential of lanceoloside A, an arbutin derivative, has not been investigated so far, even though the antiinflammatory effect of arbutin has been demonstrated using in vitro and in vivo models. Arbutin reduced the ulcer area and associated edema, inflammation and leucocyte infiltration in models of ulcer induced by aspirin or ethanol (Taha et al., 2012). Pre-treatment
with arbutin in the same models (ethanol and aspirin induced ulcers) reduced the levels of the proinflammatory cytokines TNF-α and IL-6 and increased the release of the antiinflammatory cytokine IL-10 (Taha et al., 2012). Arbutin was reported to suppress the production of NO and expression of iNOS and COX-2 by LPS-stimulated microglial cells. It also reduced the production of IL-1β and TNF-α and inhibited nuclear translocation of NF-κB (Lee and Kim, 2012). In summary, our findings are the first evidence to corroborate the traditional use of six medicinal plants to treat inflammatory conditions. Moreover, the activity of the isolated phenolic compounds, especially lanceoloside A, will be further investigated aiming the elucidation of its mechanism of action.
CONCLUSION TNF-α release by LPS-stimulated THP-1 cells was significantly reduced by seven of the 14 evaluated extracts, prepared from six Brazilian medicinal plants. The fractionation of O. semiserrata extract afforded five compounds of which lanceoloside A, epicatechin and rutin induced considerable inhibition of TNF-α release and may account for the overall activity of the crude extract.
Acknowledgements This work received financial support from CNPq, FAPEMIG and the European Community’s Seventh Framework Programme [Fp7-2007– 2013] under grant agreement N. Health-F4-2011-281608. CNPq is also acknowledged for the research fellowships (F.C.B. and M.M.T.).
Conflict of Interest The authors declare no conflict of interest connected to this paper.
REFERENCES Alessandri AL, Souza PL, Lucas CD, Rossi AG, Pinho V, Teixeira MM, 2013. Resolution of inflammation: mechanisms and opportunity for drug development. Pharmacol Ther 139: 189–212. Braga FC, Wagner H, Lombardi JA, Oliveira AB. 2000. Screening Brazilian plant species for in vitro inhibition of 5-lipoxygenase. Phytomedicine 6: 447–452. Brandão MGL, Cosenza GP, Grael CF, Netto NL Jr, Montemor R. 2009. Traditional uses of American plant species from the 1st edition of Brazilian official Pharmacopoeia. Rev Bras Farmacogn 19: 478–484. Britto K, Britto IC. 1981/1982. Plantas com atributos medicinais do herbário da Universidade de Feira de Santana. Oréades 8: 152–163. Campana PRV, Coleman CM, Teixeira MM, Ferreira D, Braga FC. 2014. TNF-α inhibition elicited by mansoins A and B, heterotrimeric flavonoids isolated from Mansoa hirsuta. J Nat Prod 77: 824–830. Cano JH, Volpato G. 2004. Herbal mixtures in the traditional medicine in eastern Cuba. J Ethnopharmacol 30: 293–316. Carvalho MG, Albuquerque LRM, Mendes LS, Guilhon GMSP, Rodrigues ST. 2008. Biflavonoids and terpenoids isolated from the leaves of Ouratea microdonta Engl. (Ochnaceae). Rev Latinoam Quim 36: 71–75. Chaves SAM, Reinhard KJ. 2003. Paleopharmacology and pollen: theory, method, and application. Mem Inst Oswaldo Cruz 98: 207–211. Copyright © 2015 John Wiley & Sons, Ltd.
Chou ST, Peng HY, Hsu JC, Lin CC, Shih Y. 2013. Achillea millefolium L. essential oil inhibits LPS-induced oxidative stress and nitric oxide production in RAW 264.7 macrophages. Int J Mol Sci 14: 12,978–12,993. Choy ES, Panayi GS. 2001. Cytokine pathways and joint inflammation in rheumatoid arthritis. N Engl J Med 344: 907–910. Corrêa MP. 1974. Dicionário de Plantas úteis do Brasil e das Exóticas Cultivadas. Di Giorgio: Rio de Janeiro. Croft M, Benedict CA, Ware CF. 2013. Clinical targeting of the TNF and TNFR superfamilies. Nat Rev 12: 147–168. Crouvezier S, Powell B, Keir D, Yaqoob P. 2001. The effects of phenolic components of tea on the production of pro- and anti-inflammatory cytokines by human leukocytes in vitro. Cytokine 13: 280–286. Daniel JFS, Carvalho MG, Cardoso RS, Agra MF, Ebeli MN. 2005. Other flavonoids from Ouratea hexasperma (Ochnaceae). J Braz Chem Soc 16: 634–638. Di Stasi LC, Hiruma-Lima CA. 2002. Plantas Medicinais na Amazônia e na Mata Atlântica (2nd edn). Unesp: São Paulo. Endringer DC, Pezzuto JM, Braga FC. 2009. NF-κB inhibitory activity of cyclitols isolated from Hancornia speciosa. Phytomedicine 16: 1064–1069. Falcão HS, Lima IO, Santos VL, et al. 2005. Review of the plants with anti-inflammatory activity studied in Brazil. Rev Bras Farmacogn 15: 381–391. Phytother. Res. 29: 1509–1515 (2015)
ANTI-TNF-α ACTIVITY OF BRAZILIAN PLANTS AND ISOLATED COMPOUNDS Felicio JD, Gonçalez E, Braggio MM, Constantino L, Albasini A, Lins AP. 1995. Inhibition of lens aldose reductase by biflavones from Ouratea spectabilis. Planta Med 61: 217–220. Fernandes FR, Santos AL, Arruda AMS, et al. 2002. Antinociceptive and anti-inflammatory activities of the aqueous extract and isolated Cuphea carthagenensis (Jacq.) J. F. Macbr. Rev Bras Farmacogn 12: 55–56. Garcia EF, Oliveira MA, Godin AM, et al. 2010. Antiedematogenic activity and phytochemical composition of preparations from Echinodorus grandiflorus leaves. Phytomedicine 18: 80–86. Gonzales-Guevara JL, Velez-Castro H, Gonzalez-Garcia KL, et al. 2006. Flavonoid glycosides from Cuban Erythroxylum species. Biochem Sys Ecol 34: 539–542. Grandi TMS, Lima-Filho FM, Ferreira SMA. 1981/1982. Levantamento das plantas medicinais de Grão Mogol. Oréades 8: 116–125. Karki R, Park CH, Kim DW. 2013. Extract of buckwheat sprouts scavenges oxidation and inhibits pro-inflammatory mediators in lipopolysaccharide-stimulated macrophages (RAW264.7). Chin J Integr Med 11: 246–252. Khanna D, Sethi G, Ahn KS, et al. 2007. Natural products as a gold mine for arthritis treatment. Curr Opin Pharmacol 7: 344–351. Kim MJ, Yang KW, Kim SS, et al. 2013. Chemical composition and anti-inflammatory effects of essential oil from Hallabong flower. EXCLI J 12: 933–942. Kwon KH, Murakami A, Tanaka T, Ohigashi H. 2005. Dietary rutin, but not its aglycone quercetin, ameliorates dextran sulfate sodium-induced experimental colitis in mice: attenuation of pro-inflammatory gene expression. Biochem Pharmacol 69: 395–406. Laveti D, Kumar M, Hemalatha R, et al. 2013. Anti-inflammatory treatments for chronic diseases: a review. Inflamm Allergy Drug Targets 12: 349–361. Lee HJ, Kim KW. 2012. Anti-inflammatory effects of arbutin in lipopolysaccharide-stimulated BV2 microglial cells. Inflamm Res 61: 817–825. Lima JCS, Martins DT. 1996. Screening farmacológico de plantas medicinais utilizadas popularmente com anti-inflamatória, in Resumos do Simpósio de Plantas Medicinais do Brasil, 89 p., Universidade Federal de Santa Catarina, Florianópolis. Lima CB, Bellettini NMT, Silva AS, et al. 2007. Uso de plantas medicinais pela população da zona urbana de bandeirantes, PR. Rev Bras Biociênc 5: 600–602. Medzhitov R. 2008. Origin and physiological roles of inflammation. Nature 454: 428–435.
Copyright © 2015 John Wiley & Sons, Ltd.
1515
Mohamed RH, Karam RA, Amer MG. 2011. Epicatechin attenuates doxorubicin-induced brain toxicity: critical role of TNF-α, iNOS and NF-κB. Brain Res Bull 86: 22–26. Moon PD, Choi IS, Go JH, et al. 2013. Inhibitory effects of BiRyuCheBang on mast cell-mediated allergic reactions and inflammatory cytokines production. Am J Chin Med 41: 1267–1282. Moreira IC, Carvalho MG, Bastos ABFO, Braz-Filho R. 1999. A flavone dimer from Ouratea hexasperma. Phytochemistry 51: 833–838. Scott DL, Kingsley GH. 2006. Tumor necrosis factor inhibitors for rheumatoid arthritis. N Engl J Med 355: 704–712. Siqueira JC. 1981. Utilização Popular das Plantas do Cerrado. Loyola: São Paulo. Souza CD, Felfili JM. 2006. Uso de plantas medicinais na região de alto paraíso de Goiás, GO, Brasil. Acta Bot Bras 20: 135–142. Souza LP, Alessandri AL, Pinho V, Teixeira MM. 2013. Pharmacological strategies to resolve acute inflammation. Curr Opin Pharmacol 13: 1–7. Taha MME, Salga MS, Ali HM, Abdulla MA, Abdelwahab SI, Hadi AHA. 2012. Gastroprotective activities of Turnera diffusa Willd. ex Schult. revisited: role of arbutin. J Ethnopharmacol 141: 273–281. Vazquez-Prieto MA, Bettaieb A, Haj FG, Fraga CG, Oteiza PI. 2012. ( )-Epicatechin prevents TNFα-induced activation of signaling cascades involved in inflammation and insulin sensitivity in 3T3-L1 adipocytes. Arch Biochem Biophys 527: 113–118. Velandia JR, de Carvalho MG, Braz-Filho R, Werle AA. 2002. Biflavonoids and a glucopyranoside derivative from Ouratea semiserrata. Phytochem Anal 13: 283–292. Vendruscolo GS. 2004. Estudo etnobotânico das plantas utilizadas como medicinais por moradores do bairro Ponta Grossa, Porto Alegre, Rio Grande do Sul, Dissertation, Universidade Federal do Rio Grande do Sul, Porto Alegre. Wangchuk P, Keller PA, Pyne SG, Taweechotipatr M. 2013. Inhibition of TNF-α production in LPS-activated THP-1 monocytic cells by the crude extracts of seven Bhutanese medicinal plants. J Ethnopharmacol 148: 1013–1017. Weiss T, Shalit I, Blau H, et al. 2004. Anti-inflammatory effects of moxifloxacin on activated human monocytic cells: inhibition of NF-κB and mitogen-activated protein kinase activation and of synthesis of proinflammatory cytokines. Antimicrob Agents Chemother 48: 1974–1982. Zhong W, Chi G, Jiang L, et al. 2013. p-Cymene modulates in vitro and in vivo cytokine production by inhibiting MAPK and NF-κB activation. Inflammation 36: 529–537.
Phytother. Res. 29: 1509–1515 (2015)
