Bioactivity of Acanthus mollis – Contribution of benzoxazinoids and phenylpropanoids

Journal of Ethnopharmacology 227 (2018) 198–205

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Bioactivity of Acanthus mollis – Contribution of benzoxazinoids and phenylpropanoids

T

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P. Matosa,b, A. Figueirinhaa,b, , A. Paranhosa,c, F. Nunesd, P. Cruze, C.F.G.C. Geraldese,f, M.T. Cruza,d, M.T. Batistac,g a

Faculty of Pharmacy, University of Coimbra, Azinhaga de Santa Comba, 3000-548 Coimbra, Portugal LAQV, REQUIMTE, Faculty of Pharmacy, University of Coimbra, Azinhaga de Santa Comba, 3000-548 Coimbra, Portugal c Center for Pharmaceutical Studies, Faculty of Pharmacy, University of Coimbra, Azinhaga de Santa Comba, 3000-548 Coimbra, Portugal d Center for Neurosciences and Cell Biology, University of Coimbra, Azinhaga de Santa Comba, 3000-548 Coimbra, Portugal e Coimbra Chemistry Centre (CQC), Department of Chemistry, University of Coimbra, Rua Larga, 3004-535 Coimbra, Portugal f Department of Life Sciences, Faculty of Science and Technology, University of Coimbra, Calçada Martim de Freitas, 3000-393 Coimbra, Portugal g CIEPQPF, Department of Chemical Engineering, Faculty of Science and Technology, University of Coimbra, 3030-790 Coimbra, Portugal b

A R T I C LE I N FO

A B S T R A C T

Keywords: Acanthus mollis DIBOA Verbascoside Antioxidant activity Anti-inflammatory activity

Ethnopharmacological relevance: Acanthus mollis is a plant native to the Mediterranean region, traditionally used as diuretic, anti-inflammatory and soothing of the mucous membranes of the digestive and urinary tract and externally as healing of wounds and burns, also demonstrating analgesic and anti-inflammatory activities. However, studies focused on its phytochemical composition as well as scientific proof of Acanthus mollis efficacy are scarce. Aim of the study: The proposed work aims to perform a phytochemical characterization and evaluation of the therapeutic potential of Acanthus mollis, based on biological properties that support its traditional uses. Material and methods: In this study, an 96% ethanol extract from Acanthus mollis leaves was obtained and its phytochemical composition evaluated using High Performance Liquid Chromatography with Photodiode Array Detector coupled to Electrospray Ionization Mass Spectrometry (HPLC-PDA-ESI/MSn). The chemical structure of the compound isolated was elucidated using 1H and 13C Nuclear Magnetic Resonance (NMR), 1H-correlation spectroscopy (1H–COSY), heteronuclear single quantum correlation (HSQC) and heteronuclear multiple-bond correlation (HMBC). The quantification of the constituents was performed using two external standards (2,4dihydroxy-1,4-benzoxazin-3-one and verbascoside). The antioxidant activity was determined by the 2,2-diphenyl-1-pycrylhydrazyl (DPPH) assay. Anti-inflammatory activity was determined measuring the inhibition of nitric oxide production by RAW 264.7 macrophages stimulated with the TLR4 agonist lipopolysaccharide (LPS) and through lipoxygenase (LOX) inhibition assay. The cytotoxicity was screened on two lines (RAW 264.7 and HaCaT) using the resazurin assay. Results: Compounds such as verbascoside and its derivatives, as well as benzoxazinoids were found as the main constituents. A percentage of 5.58% was verified for the 2,4-dihydroxy-1,4-benzoxazin-3-one (DIBOA) derivatives. DIBOA was the main compound of the extract. Significant concentrations were also found for phenylpropanoids, which constitute about 4.39% of the total compounds identified. This extract showed antioxidant capacity against DPPH (IC50 = 40.00 ± 1.59 μg/mL) and superoxide anion (IC50 = 29.42 ± 1.99 μg/mL). It also evidenced anti-inflammatory potential in RAW 264.7 macrophages, presenting capacity for nitric oxide reduction (IC50 = 28.01 μg/mL). Moreover, in vitro studies have shown that this extract was able to inhibit the lipoxygenase, with an IC50 of 104.39 ± 4.95 µg/mL. Importantly, all effective concentrations were devoid of cytotoxicity in keratinocytes, thus highlighting the safety of the extract for the treatment of skin inflammatory related diseases. Concerning macrophages it was also possible to disclose concentrations showing anti-inflammatory activity and without cytotoxicity (up to 30 µg/mL). The benzoxazinoid DIBOA demonstrated a considerable anti-inflammatory activity suggesting its important contribution to this activity. Conclusions: These results corroborate the anti-inflammatory properties traditionally attributed to this plant. Among the compounds identified in this study, benzoxazinoids exhibited a significant anti-inflammatory activity

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Corresponding author at: Faculty of Pharmacy, University of Coimbra. Pólo das Ciências da Saúde, Azinhaga de Santa Comba, 3000-548 Coimbra, Portugal. E-mail address: amfigueirinha@ff.uc.pt (A. Figueirinha).

https://doi.org/10.1016/j.jep.2018.09.013 Received 30 May 2018; Received in revised form 3 September 2018; Accepted 6 September 2018 Available online 07 September 2018 0378-8741/ © 2018 Elsevier B.V. All rights reserved.

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that was never previously described. Ethanol seems to be a good option for the extraction of these bioactive compounds, since relevant antioxidant/anti-radical and anti-inflammatory activities were found for this extract.

1. Introduction

ethanol extract from Acanthus mollis leaves, as well as to disclose its antioxidant and anti-inflammatory activities at concentrations devoid of cytotoxicity. Concomitantly it is intended to glimpse a correlation between these bioactivities and the chemical nature of the extract constituents under study.

Acanthus mollis L. is a Mediterranean plant that belongs to the Acanthaceae family. It was one of the first to be cultivated in gardens because of the beauty of its leaves and believed to have been the source of inspiration for the decoration of Corinthian columns (Sharma et al., 2015). It has been used in traditional medicine in various parts of the world. In southern Europe it is used in cataplasms for intestinal problems, gargles to relieve toothache and inflammation of the mouth, baths and compresses (Ferrão and Liberato, 2015; Rivera and Obón, 1995). The crushed leaves are applied externally to treat wounds, sprains, fractures, bruises, burns and nipples problems due to its soothing and emollient properties (Attard and Pacioni, 2012; FioriniPuybaret, 2011; Rivera and Obón, 1995; Sequeira et al., 2006). Its leaves are often used directly on the skin to alleviate swollen legs, as well as headaches when applied in the forehead (Freitas and Mateus, 2013). Its infusion is also employed internally as diuretic and in the treatment of irritation of the digestive and urinary mucosa, tumors, ulcers and cough (Fiorini-Puybaret, 2011; Rivera and Obón, 1995). Despite its extensive use in folk medicine, scientific studies to validate these traditional uses are scarce. Rezanka and co-workers (2009) isolated lignans with antitumor activity and Attard and Pacioni (2012) verified that an extract from A. mollis containing erpenoids, flavonoids and proteins exhibited similar properties. In 2015, it was found that a methanol extract of the A. mollis leaves increased the biosynthesis of 15hydroxyeicosatetraenoic acid, an anti-inflammatory eicosanoid (Bader et al., 2015). Most recently, various parts of Acanthus mollis were studied, ethanol extracts showing higher concentration in phenolic compounds. Antifungal activity against several strains of Candida was also verified, as well as a correlation with the antioxidant activity (Jara et al., 2017). Plants with anti-inflammatory and/or antioxidant properties are potential sources of bioactive constituents with relevant interest for the treatment of different pathological processes. Inflammatory diseases include a wide variety of disorders and conditions characterized by inflammation, such as allergy, asthma, autoimmune diseases, celiac disease, glomerulonephritis, hepatitis, inflammatory bowel disease, reperfusion injury and transplant rejection (Barmeyer et al., 2017; Kim et al., 2017; Oz, 2017). Other pathologies are also frequently associated with chronic inflammatory processes, such as diabetes, cancer, some cardiovascular, neurological or even pulmonary diseases (Reuter et al., 2010). On the other hand, the so-called oxidative stress, occurs as a result of the imbalance between the production of free radicals and its elimination through the various endogenous antioxidant mechanisms (Khansari et al., 2009), and if persistent sets in and may predispose the host to chronic inflammation, causing chronic diseases. Indeed, inflammation has been highlighted as one of the hallmarks of cancer, and different types of inflammation can lead to different types of cancer (Khansari et al., 2009; Reuter et al., 2010). For the prevention or even cure of some of these diseases, the discovery and identification of compounds with anti-inflammatory properties is of utmost importance to benefit patients. Previous studies have suggested that the phenylpropanoid compounds are mainly responsible for the antioxidant activity, rather than the benzoxazinoid compounds that will be responsible for the anti-inflammatory activity (Matos et al., 2018). Currently, there are several in vitro studies emphasizing their anti-inflammatory, anticancer, antimicrobial and anti-allergic activities, and also describing effects on the central nervous system (Adhikari et al., 2015, 2013). This work aims to characterize these phytoconstituents of an

2. Material and methods 2.1. Material of study Leaves of Acanthus mollis L. were collected in the Coimbra city (40°12'28.9"N, 8°25'20.2"W) in November 2015. A voucher specimen (A. Figueirinha 01015) was deposited at the Herbarium of Medicinal Plants, Faculty of Pharmacy, University of Coimbra. 2.2. Extraction, fractionation and isolation procedures The plant material was kept refrigerated at −20 °C, away from light and moisture, until used. Subsequently, the leaves were lyophilized, milled and sieved (2500 meshes/cm2). Powdered plant (42.00 g) was extracted with 2.1 L of 96% ethanol for two hours using an electromagnetic stirrer. After filtration, water was added and the extract placed overnight in the cold and centrifuged to remove chlorophyll. This extract was concentrated on a rotatory evaporator under vacuum and freeze-dried (EEt). The extraction yield was determined gravimetrically in three aliquots (1 mL), subjecting them to heating at 40 °C in a vacuum oven to constant weight, presenting a yield of 13%. Flash chromatography was used to fractionate 4.35 g of EEt extract. Aliquots of 250 mg were chromatographed on a reversed phase C18 column Buchi® (40 × 150 mm, with particle diameter between 40 and 63 µm) (Flawil, Switzerland). The mobile phase was propelled by two pumps Buchi® Pump Module C-605 (Flawil, Switzerland), taking up the water and methanol used in step gradient: 5% (0–12 min), 50% (12–20 min), 80% (20–23 min) and 100% (23–30 min) at a constant flow rate of 30 mL/min. After the control by TLC, five fractions Fr.1–5 were obtained. Subsequently, the fraction Fr.2 (2.25 g) was concentrated and the residue solubilized in water. This solution was re-chromatographed on a Sephadex LH-20 column (40 × 1.6 cm; Sigma-Aldrich, Sweden), equilibrated and eluted with water milliQ to obtain thirty-six fractions (Fr.2 1–36). The fractions were controlled by TLC, with 5% methanol FeCl3, to detect benzoxazinoids. Fr.2 23–28 revealed the same composition and were joint, concentrated and taken to dry residue, yielding a mass of 224.3 mg (ca. 10%). To this residue n-BuOH-toluene (1:1) has been added, however only one portion was solubilized. This soluble portion (192.3 mg) was collected and used for further studies. 2.3. Analytical chromatography 2.3.1. High performance liquid chromatography with photodiode array detector (HPLC-PDA) To separate and quantify the phytoconstituents, HPLC-PDA profiles from the extract were obtained on a HPLC Gilson, equipped with two pumps (models 305 and 306), mixer (Model 811 B), manometric module (model 805) and an auto sampler (Gilson 234 autoinjector), hyphenated to a PDA (Gilson model 170) and a control and processing station Unipoint System data (Unipoint® 2.10). The analyses were carried out on a Waters® RP18 Spherisorb ODS-2 column (250 × 4.6 mm; 5 µm particle size), maintained at 24 °C and protected 199

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quartet (q) and multiplet (m).

by a guard column KS 30/4 Nucleosil 120–5 C-18, Macherey-Nagel (Duren, Germany). The mobile phase consisted of 5% aqueous formic acid solution (A) and methanol (B) used at a flow rate of 1 mL/min. The gradient was of 5–15% B (0–10 min), 15–25% B (10–15 min), 25–50% B (15–40 min), 15–25% B (10–15 min), 25–50% B (15–40 min) and 50–80% B (40–50 min). The UV-V profiles were acquired in the range 200–600 nm and chromatograms were recorded at 280 and 320 nm. Quantification of benzoxazinoids and phenolic acids was obtained by the absorbance recorded on the chromatograms against two external standards: 2,4-dihydroxy-1,4-benzoxazin-3-one (DIBOA) and verbascoside. The detection and quantification limits (LOD and LOQ, respectively) were determined from the parameters of the calibration curves represented in Table 1. Benzoxazinoids were quantified as DIBOA (at 280 nm) and phenolic acids as verbascoside (at 320 nm). Two independent injections were performed for each sample, injecting 100 μL of extract and standards dissolved in water and microfiltered. The identification of extract compounds was performed by comparing retention times and their UV spectra with the verbascoside standard (Extrasynthese S.A., Lyon Nord, Genay, France) and with DIBOA which was isolated in this work.

2.3.3.1. 2,4-dihydroxy-1,4-benzoxazin-3-one (DIBOA). UV/Vis λmáx (water) nm: 212, 255, and 280. 1H NMR (400 MHz, MeOD-d6) δ: 7.38 (1H, dd, J = 7.9, 0.9 Hz, H-5), 7.05 [m, 3H (H-6, H-7, H-8)] 7.12 (1H, m, H-6), 7.09 (1H, m, H-8), 7.02 (1H, m, H-7), 5.72 (1H, s, H-2). 13C NMR (100 MHz, MeOD-d6) δ: 158.74 (C, C-3), 141.11 (C, C-10), 128.24 (C, C-9), 124.20 (C, C-7), 122.39 (C, C-6), 117.09 (C, C-8), 113.02 (C, C5), 92.29 (CH, C-2). 2.4. Antioxidant activity 2.4.1. 2,2-diphenyl-1-pycrylhydrazyl (DPPH) assay The antioxidant activity was evaluated using the method described by Blois (1958). The samples (100 μL) were tested in methanol solution of 500 μM DPPH (500 μL) in the presence of 100 mM acetate buffer at pH 6.0 (1 mL). The reaction was developed for 30 min in the dark at room temperature. The absorbance value of the solutions was measured at 517 nm, in a Cintra 101 (GBC, Australia) spectrophotometer, against a blank. The results were expressed in IC50 values, indicating the concentrations of samples required for 50% reduction of the absorbance of DPPH solution. Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid) was used as a standard to express the results as Trolox equivalent antioxidant capacity (TEAC) values, defined as concentration of extract with antioxidant activity equivalent to a solution of 1 mM of Trolox.

2.3.2. HPLC-PDA-ESI/MSn analysis Structural elucidation of EEt phytoconstituents was carried out on a Surveyor liquid chromatograph equipped with a photodiode array detector (Surveyor) and interfaced with a Finnigan LCQ Advantage Ion Max tandem mass spectrometer (Thermo Fisher Scientific, Waltham, MA, USA) equipped with an ESI ionization chamber. Separation was performed on a Spherisorb ODS-2 column (150 × 2.1 mm; particle size 3 μm; Waters Corp., Milford, MA, USA) with a Spherisorb ODS-2 guard cartridge (10 × 4.6 mm; particle size 5 μm; Waters Corp., Milford, MA, USA) at 25 °C. A mobile phase consisting of 2% aqueous formic acid (v/ v) (A) and methanol (B) was used with a discontinuous gradient of 5–15% B (0–10 min), 15–20% B (10–15 min), 25–50% B (15–40 min), 50–80% B (40–50 min) at a flow rate of 200 μL min−1. The detection was done with a PDA detector in a wavelength range of 200–500 nm, followed by a second detection in the mass spectrometer. Mass analyses were obtained in the negative mode. Three consecutive scans were performed: full mass (m/z 200–1000), MS2 of the most abundant ion in the full mass, and MS3 of the most abundant ion in the MS2. Source voltage was 4.7 kV and the capillary temperature and voltage were 275 °C and −7 V respectively. Nitrogen was used as the sheath and auxiliary gas at 20 Finnigan arbitrary units. The normalized collision energy was 45%, using helium as the collision gas. Data treatment was carried out with XCALIBUR software (Thermo Scientific, Waltham, MA, USA).

2.4.2. Superoxide anion-scavenging assay Scavenging capacity for superoxide radical was evaluated using a superoxide anion-scavenging assay, based on the reduction of NBT by superoxide radical generated from the photoreduction of riboflavin (Kostyuk et al., 2000). The mixtures (3 mL) contained phosphate buffer (16 mM, pH 7.8), EDTA (0.1 mM), TEMED (0.8 mM), NBT (85 μM), riboflavin (6 μM) and the appropriate volume of the samples. The reaction was carried out under fluorescent light (20 W, 20 cm) and at room temperature (22 °C). The assay was stopped by switching off the light and addition of 50 μL SOD (1 mg/mL). The measurement of absorbance was performed at 560 nm. 2.5. Anti-inflammatory activity 2.5.1. Measurement of nitrite production by Griess reagent A mouse macrophage cell line (Raw 264.7) obtained from the American Type Culture Collection (TIB-71) was kindly supplied by Dr. Otília Vieira (Center for Neurosciences and Cell Biology, University of Coimbra, Portugal). The cells were cultured and grown in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% non-inactivated fetal bovine serum, 3.02 g/L sodium bicarbonate, 100 U/mL penicillin and 100 μg/mL streptomycin at 37 °C, in a humidified atmosphere of 95% air and 5% CO2. During the experiments, the cells were monitored under optical microscope to detect any morphological change. The macrophage cells (RAW 264.7) (0.6 × 106 cell/well) were cultured in 96-well microplates, allowed to stabilize for 12 h, and then incubated with culture medium (control), or stimulated with 1 µg/mL LPS in the absence or in the presence of different concentrations of the extracts, during 24 h.

2.3.3. NMR spectroscopy NMR spectra were obtained on a Bruker Avance II 400 spectrometer operating at 400.13 (1H) and 100.610 (13C) MHz. The chemical shifts are reported in ppm, relative to the CH3 1H (δ = 3.33 ppm) and 13 C (δ = 47.64 ppm) resonances of the methanol solvent used as external reference. The 1H and 13C NMR spectra were assigned using twodimensional COSY (correlated spectroscopy), 1H–13C HSQC (heteronuclear single quantum correlation spectroscopy) and 1H–13C HMBC (heteronuclear multiple-bond correlation spectroscopy) techniques. The spectra were obtained at the temperature of 25 °C and pH 7.5. The following abbreviations are used: singlet (s), doublet (d), triplet (t),

Table 1 Linearity, limit of detection (LOD) and limit of quantification (LOQ) of the two standard compounds used as reference. Standard compound DIBOA Verbascoside (1)

Range concentration (µg/mL) 0.27–500.00

n(1) 4

Slope

Intercept 6

2.02 × 10 1.78 × 106

8.54 × 10 − 7.60 × 106

Number of points used for the regression of standard solutions. Injections were done in duplicate. 200

5

R2

LOD (µg/mL)

LOQ (µg/mL)

0.9987 0.9965

7.92 ± 4.35 17.97 ± 7.08

27.38 ± 4.28 49.93 ± 6.92

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The nitric oxide (NO) production was measured colorimetrically by the accumulation of nitrite in the culture supernatants using the Griess reagent (Green et al., 1982). Briefly, culture supernatants (170 μL) were diluted with equal volumes of the Griess reagent [0.1% (w/v) N-(1naphthyl)-ethylenediamine dihydrochloride and 1% (w/v) sulphanilamide containing 5% (w/v) H3PO4] and maintained in the dark during 30 min. Quantification of nitrites was performed on a Biotek Synergy HT plate reader at 550 nm, using culture medium as blank. Three independent experiments were performed with the extract. The results were expressed as a percentage of the nitrite production by cells cultured with LPS.

fluorescent) into resorufin (fluorescent pink dye) (Rampersad, 2012). Therefore, the magnitude of dye reduction is correlated with the number of viable cells. So, after the treatment described above, macrophages and keratinocytes were incubated with a resazurin solution (50 µM in culture medium) for 2 h at 37 °C in a humidified atmosphere of 5% CO2 and 95% air. Quantification of resorufin was performed in an ELISA microplate reader (Biotek Synergy HT) at 570 nm, using a 620 nm reference filter. A cell-free control was performed to exclude nonspecific effects of the extracts on resazurin.

2.5.2. Lipoxygenase (LOX) inhibition assay Lipoxygenase (E.C. 1.13.11.12) inhibiting activity of plant extracts was carried out according to the method described by Malterud and Rydland (2000), adapted, using sodium phosphate buffer (100 mM, pH 8.0). Assay mixture (1 mL) contained buffer with or without test sample, soybean lipoxygenase type 1-B (167 units, Sigma-Aldrich) and linoleic acid (134 μM, Sigma-Aldrich) as substrate. The mixture was preincubated at 25 °C for 10 min before adding substrate to start the reaction. The increase in absorbance at 234 nm was monitored for at least 3 min, with readings recorded every 10 s. The percentage of the lipoxygenase activity inhibition was calculated as follows: inhibition (%) = [(A–B)/A] × 100, where A and B represent the absorbance of the control and test samples, respectively, between incubation times of 30 and 90 s.

Statistical analysis was made using GraphPad Prism program, version 5.02 (GraphPad Software, San Diego, CA, USA). The results from the anti-inflammatory activity and cytotoxicity were expressed as mean ± standard error media (SEM) of the indicated number of experiments (n = 3). Comparison between treatment conditions and control was performed using two-sided unpaired t-test. One-way ANOVA followed by Bonferroni's test was used to evaluate the effect of different treatments to LPS-stimulated cells. IC50 values for the anti-inflammatory activity was calculated from the calibration line (median effect plot) obtained after linearization of the dose-response curve, as described by Chou (2006).

2.7. Statistical analysis

3. Results and discussion 3.1. Structural elucidation

2.6. In vitro cytotoxicity evaluation 3.1.1. HPLC-PDA and HPLC-PDA-ESI/MSn EEt extract profile obtained by HPLC-PDA is illustrated in Fig. 1. It can be observed that this extract consists of benzoxazinoids, phenolic acids and flavonoids. The phenolic acids present in this extract identified by the characteristic shape of the on-line UV spectra represent only phenylpropanoids (Plazonić et al., 2009), presenting also apigenin derivatives, with maxima at 272 nm (band II) and 335 nm (band I) (Waksmundzka-Hajnos et al., 2010). The extract contained benzoxazinoid compounds, identified by its characteristic spectrum with a maximum at 254 nm and 280 nm and shoulder at 286 (Hanhineva et al., 2011). A phytochemical characterization of the compounds present in the extract was tentatively performed by HPLC-PDA-ESI/MSn (Table 2).

2.6.1. Cell culture Two different cell lines were used to assess the potential cytotoxicity of the extract: the macrophage cell line Raw 264.7, cultured as described previously, and the human keratinocytes cell line (HaCaT) obtained from DKFZ (Heidelberg) and kindly supplied by Dr. Eugenia Carvalho (Center for Neurosciences and Cell Biology, University of Coimbra, Portugal). Keratinocytes were cultured in Dulbecco's Modified Eagle Medium (high glucose) supplemented with 10% inactivated fetal bovine serum, 3.7 g/L sodium bicarbonate, 25 mM glucose, 100 U/mL penicillin and 100 μg/mL streptomycin at 37 °C in a humidified atmosphere of 95% air and 5% CO2. During the experiments, the cells were monitored under optical microscope to detect any morphological change.

3.1.1.1. Benzoxazinoids. The compounds eluted at 15.68 and 16.86 min exhibited UV spectral profile similar to benzoxazinoids spectra. The presence of a λmáx at 254 nm and a shoulder near to 280 nm suggest the presence of DIBOA derivatives (Pihlava and Kurtelius, 2016). The

2.6.2. Assessment of cell viability by resazurin assay Resazurin assay was used to evaluate the cell viability, which is based on the ability of living cells to convert resazurin (blue non-

Fig. 1. HPLC-PDA profile of EEt extract from Acanthus mollis leaves, recorded at 280 and 320 nm. 201

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Table 2 Benzoxazinoids and phenolic compounds identified in EEt by HPLC-PDA-ESI/MSn. Peak

Rt (min)

λmáx (nm)

Precursor Ion [M-H]-

HPLC-ESI-MSna m/z

1 2 3 4 5 6 7 8 9 10

15.68 16.86 25.39 29.20 29.96 31.16 31.88 33.03 33.71 39.21

254; 254; 247; 249; 248; 248; 247; 246; 247; 273;

– – 639 623 623 667 667 623 667 –

– – MS2: MS2: MS2: MS2: MS2: MS2: MS2: –

279sh 280sh 286sh; 286sh; 286sh; 288sh; 289sh; 290sh; 287sh; 334

300sh; 299sh; 300sh; 300sh; 300sh; 328 327

331 330 331 331 330

Attempt to identify

3

459; 529; 621MS : 459 461MS3: 315 461MS3: 279; 297; 315 621MS3: 179; 459 621MS3: 179; 459; 469 461MS3: 315 621MS3: 179; 251; 441; 459

Benzoxazinoids Benzoxazinoids β-OH-verbascoside* Verbascoside isomer** Verbascoside isomer** β-EtOH- verbascoside isomer*** β-EtOH- verbascoside isomer*** Verbascoside isomer** β-EtOH- verbascoside isomer*** Apigenin derivative

Identification based on the UV–Vis spectra, the molecular weight and the fragmentation patterns, which are according to authors as (*) Cardinali et al. (2012); Sanz et al. (2012); (**) Blazics et al. (2011); Cardinali et al. (2012); Nenadis et al., 2007; Owen et al. (2003); Sanz et al. (2012); Savarese et al., 2007; Ying et al., 2004; (***) Innocenti et al. (2006). (a)The base peaks in MS spectra are in bold.

3.2. Antioxidant activity

compound eluting at 15.68 min was isolated from the EEt extract and characterized by 1H and 13C NMR. The 1H and 13C shift assignments were based on the 1H–COSY, HSQC and HMBC NMR analyses, which were consistent with the structure of the 2,4-hydroxy-1,4-benzoxazin-3one molecule (DIBOA) (Fig. 2). Some of the coupling constants could not be obtain due to the proton signals overlap. In the 96% ethanol extract, compound DIBOA was the major compound present representing about 5.58% of the total compounds present in this extract (Table 3).

Aiming a therapeutic application, the ethanol extract (EEt) was assessed for its antioxidant/antiradical capacity against DPPH and one reactive oxygen species involved in cellular oxidation processes, the superoxide anion. This extract, obtained from the leaves of Acanthus mollis, exhibited antioxidant capacity for the DPPH assay, with a IC50 = 40.00 ± 1.59 μg/mL and TEAC = 23.54 ± 1.10. Moreover, this extract also evidenced activity for the superoxide anion, a radical often generated in cells and involved in genesis and progression of several acute and chronic diseases. An IC50 value of 29.42 ± 1.99 μg/mL was verified for this radical. Verbascoside derivatives, that were identified in the EEt, have been referred to as possessing antiradicalar activity (Blazics et al., 2011) and could contribute for the free radicals scavenging activity of this extract. In several experimental models for antioxidant activity, the verbascoside was tested, revealing a potent antioxidant/ antiradicalar ability in the assays against DPPH (Alipieva et al., 2014). Studies previously performed by us, allowed to verify that an enriched fraction of benzoxazinoid compounds did not demonstrate an antioxidant activity against the DPPH radical so relevant (Matos et al., 2018). Thus, it may be inferred that the phenylpropanoids will be probably the compounds that could contribute most to this activity, since they have already been described as good antioxidants / antiradicals. This property is often one of the mechanisms responsible for the anti-inflammatory effect of medicinal plants and/or its phytoconstituents (Arulselvan et al., 2016; Francisco et al., 2011; Santos et al., 2017).

3.1.1.2. Hydroxycinnamic acids. The compounds, eluted at 25.39, 29.20, 29.96, 31.16, 31.88, 33.03 and 33.71 min exhibited the same type of UV spectra, common to hydroxycinnamic acid derivatives and with a maximum absorption peak at 327–331 nm and a shoulder at 286–300 nm, which is often attributed to caffeic or ferulic acid and to their derivatives according to Achour et al. (2018). A minor compound eluted at 25.39 min was tentatively identified as β-OH-verbascoside (Table 2), because it exhibited a molecular ion at m/z 639 and a fragmentation pattern as that described by Sanz et al. (2012); fragment at m/z 621 originated by a loss of one water molecule followed by the loss of a caffeoyl group with formation of ion at m/z 459 (Cardinali et al., 2012; Sanz et al., 2012). The compounds eluted at 29.20, 29.96 and 33.03 min, present the same molecular ion at m/z 623 and fragments at m/z 461 and 315 from the loss of a caffeoyl and a rhamnosyl groups respectively. This fragmentation behavior was described before for verbascoside (acteoside) (Cardinali et al., 2012; Owen et al., 2003; Sanz et al., 2012) and therefore could be verbascoside isomers. These compounds represent about 3.02% of the identified compounds. Other compounds eluted at 31.16, 31.88, 33.71 min were tentatively identified as β-EtOH-verbascoside isomers, based in Innocenti et al. (2006). It presents a molecular ion at m/z 667 and fragments at m/z 621 and 459 from the loss of ethanol and the caffeoyl group, respectively, and the fragment at m/z 179 corresponding to deprotonated caffeic acid molecule. In turn, these compounds represent 1.07% of total identified compounds. Thus, three distinct types of caffeic acid derivatives, which constitute about 4.39% of the total compounds identified, were detected in this extract (Table 3). The less representative constituent (ca. 0.30%) was the β-OH-verbascoside. Verbascoside, as well as its isomer, had already been identified in a methanol extract of A. ilicifolius (Van Kiem et al., 2008). However, to our knowledge, these compounds were identified for the first time in A. mollis. Several biological properties have been attributed to these compounds, including mainly the antioxidant activity, in vivo and ex vivo, but also the anti-inflammatory, antimicrobial, hepatoprotective, antitumor and photoprotective activities (Alipieva et al., 2014; Cardinali et al., 2012; Funes et al., 2009; Koo et al., 2006; Tripoli et al., 2005; Vertuani et al., 2011).

3.3. Anti-inflammatory activity Inflammation is associated with several progressive diseases, such as metabolic disorders, cancer, Alzheimer's disease and cardiovascular diseases (Chen et al., 2018), and the anti-inflammatory drugs normally used, the nonsteroid anti-inflammatory agents (NSAIDs), have adverse effects. So, it is important to find new drugs without toxicity and secondary effects. Since anti-inflammatory properties have been reported

Fig. 2. Structure of the DIBOA compound. 202

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therapeutic target for several inflammatory-related diseases.

Table 3 Quantification of the benzoxazinoids and phenolic compounds in EEt by HPLCPDA.

The safety of the extract (EEt) was assessed by resazurin assay in macrophages (RAW 264.7) and keratinocytes (HaCaT). As shown in Table 4, the extract showed no significant cytotoxicity in macrophages for the concentration effective to inhibit half of NO production (IC50 = 28.01 μg/mL). Moreover, this extract showed no significant cytotoxicity in keratinocytes for concentrations up to 120 µg/mL emphasizing its potential therapeutic effect for skin inflammatory related disease at concentrations showing both anti-inflammatory and anti-lipoxygenase activities. Among the compounds present in the extract, verbascoside has been the most studied as a preventive for skin diseases (Alipieva et al., 2014). On the other hand, the DIBOA compound isolated from de EEt extract also showed no cytotoxicity for the IC50 concentration (5 μg/mL) thus attesting its anti-inflammatory potential at safe conditions (Fig. 4-B).

(1) Values are mean ± standard deviation of two replicates. *LOQ (Limit of quantification).

for this plant by the popular medicine its scientific validation constituted the starting point for the realization of this study. 3.3.1. In vitro inhibition of nitric oxide production by macrophages The EEt capacity to inhibit nitric oxide production on RAW 264.7 macrophages stimulated with LPS was studied and we detected relevant anti-inflammatory activity for the Acanthus mollis leaves, with an IC50 of 28.01 μg/mL value (Fig. 3). Curiously, the isolated benzoxazinoid 2,4hydroxy-1,4-benzoxazin-3-one (DIBOA) showed significant anti-inflammatory activity, presenting a nearly 50% reduction in nitrite production (56.33 ± 7.06%) with only 5 µg/mL (Fig. 4-A). From our knowledge and the literature consulted, DIBOA compounds have not yet been related with anti-inflammatory properties, which reinforces the potential of this plant as a source of new anti-inflammatory drugs. Previous research demonstrated that verbascoside inhibits lipopolysaccharide-inducible nitric oxide synthase expression through blocking AP-1 activation (Lee et al., 2005). These results seem to indicate that several pathways could be involved in the anti-inflammatory activity exhibited by this extract. Recently, Lee et al. (2018) studied the inflammatory effect of a Paulownia tomentosa Steud extract enriched with verbascoside and its isomer, the isoverbascoside, in lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages. This extract has been shown to inhibit the activation of the nuclear factor-kappa B (NFκB) transcription factor, to activate superoxide dismutase 3 (SOD3), as well as to decrease the production of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), reactive oxygen species (ROS) and NO. Based on these assumptions we may suggest that those compounds, in addition to exhibit significant antioxidant activity, also assign anti-inflammatory properties to the extracts. This may explain the significant anti-inflammatory activity of the Acanthus mollis EEt extract, since in the concentration effective to inhibit the NO production by half (IC50 = 28.01 μg/mL) only 1.56 µg of the DIBOA compound is present in the extract. In fact, to obtain the same extract NO inhibition, a DIBOA concentration of 5 µg/mL was required, thus meaning that other compounds present in the extract may contribute to the same activity, including the phenylpropanoids.

4. Conclusion In this work, the phytochemical composition, antioxidant and antiinflammatory activities, as well as the safety of an ethanol extract of Acanthus mollis leaves were evaluated. The extract includes benzoxazinoid compounds (DIBOA and its derivatives) (5.58 ± 0.08 g%) and phenylpropanoids (verbascoside, its isomers and derivatives) (4.39 ± 0.16 g%), which, to the best of our knowledge, was no previously reported in Acanthus mollis. Research on biological activity and safety of Acanthus mollis extracts were performed to validate the traditional use of this plant and to explore its potential for health care. The results obtained suggest that the EEt can be an alternative for preventive and/or therapeutic purposes, because it contains bioactive compounds such as the benzoxazinoid 2,4hydroxy-1,4-benzoxazin-3-one (DIBOA), which demonstrated, for the first time, a safe and excellent capacity to reduce nitrite production in macrophages RAW 264.7, and verbascoside derivatives, that have been referred as good antioxidants and holders of relevant activities as antiinflammatory, antitumor or even neuroprotective. Our results also support the safe topical use of Acanthus mollis leaves in traditional medicine as an anti-inflammatory and antioxidant drug and can be further envisioned and explored as an alternative source for the production of therapeutic agents.

Nitrite production (% of LPS)

150

3.3.2. Lipoxygenase assay Acanthus mollis ethanol extract was able to inhibit the lipoxygenase enzyme (LOX), with an IC50 of 104.39 ± 4.95 µg/mL, which corroborates results obtained by previous authors in this same species of Acanthus (Bader et al., 2015; Bonesi et al., 2016). This enzyme has as main function to form leukotrienes, through the metabolization of polyunsaturated fatty acids (Yahaya et al., 2014). Leukotrienes, in turn, function as leukocyte recruiters, and have vasomotor properties in cardiovascular, renal, pulmonary and cutaneous manifestations (Iranshahi et al., 2009). Thus, lipoxygenases in their various isoforms are associated with several inflammatory diseases such as asthma, cancer, osteoporosis, atherosclerosis, skin diseases, thrombosis, obesity and diabetes, as well as neurodegenerative diseases (Lang et al., 2016; Sadeghian and Jabbari, 2016). They have thus become a very attractive

100

*** ***

50

***

***

*** L µg

0 24

0

µg

/m

/m

/m

L

L

L /m

µg 12

15

LP S

0

60

5.58 ± 0.08 0.30 ± 0.05 3.02 ± 0.07 1.07 ± 0.04 < LOQ* < LOQ* Not quantified

L

Benzoxazinoids β-OH-verbascoside Verbascoside isomer β-EtOH-verbascoside isomer Verbascoside isomer β-EtOH-verbascoside isomer Apigenin derivative

µg

1, 2 3 4, 5 6, 7 8 9 10

3.4. Cytotoxicity of the extract on macrophages and keratinocytes

(1)

/m

g of compound / 100 g of extract

30

Compounds

µg

Peak

Fig. 3. Anti-inflammatory effect of the extract EEt in LPS-stimulated RAW 264.7 macrophages. Results are expressed as a percentage of nitrite production by cells stimulated with LPS. Each value represents the mean ± standard error (SEM) in the bars, from three experiments performed in duplicate. (*** P < 0.001, versus LPS). 203

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A

B 150

Resazurin reduction (% of control)

Nitrite production (% of LPS)

150

100

***

***

***

***

***

50

0

100

##

##

###

###

50

m L

C on tr ol 5 µg /m L 7. 5 µg /m L 10 µg /m L 12 .5 µg /m L 15 µg /m L

12 .5

15

µg /

µg /

m L

m L µg /

m L

10

L

µg /

/m

7. 5

µg 5

LP S

0

Fig. 4. Anti-inflammatory effect of the DIBOA compound in LPS-stimulated RAW 264.7 macrophages: (A) NO production and (B) cell viability (resazurin assay). NO release was determined in the supernatants of the cultures using the Griess reagent (A) and cell viability was assessed on adherent cells using the resazurin reagent and expressed as percentage of cell viability by control cells (B). Results are shown as mean ± standard error (SE) in the bars, from three experiments performed in duplicate. (*** P < 0.001, versus LPS) (### P < 0.001; ## P < 0.01, versus control cells). Table 4 Cytotoxicity of the extract EEt on macrophages (RAW 264.7) and keratinocytes (HaCat). Cell viability (% of control) EEt95 Control

RAW 264.7 100.0

15 µg/mL 30 µg/mL 60 µg/mL 120 µg/mL 240 µg/mL

90.33 79.33 69.33 57.00 44.33

± ± ± ± ±

5.70 5.78 2.73 5.29 3.71

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(1)

HaCat 100.0

# ### ### ###

(1)

100.00 98.67 96.33 87.00 78.67

Values are mean ± SEM of three replicates. (### P < 0.001; versus control cells).

#

± ± ± ± ±

2.08 5.21 4.81 5.86 4.10

#

P < 0.05,

Acknowledgments This work received financial support from the European Union (FEDER funds POCI/01/0145/FEDER/007265) and National Funds FCT/MEC-Portugal (Portuguese Foundation for Science and Technology and Portuguese Ministry of Education and Science) under the Partnership Agreement PT2020 UID/QUI/50006/2013, Programa de Cooperación Interreg V-A España–Portugal (POCTEP) 2014–2020 (project 0377_IBERPHENOL_6_E). C.F.G.C.G. and P.C. thank FCTPortugal (Portuguese Foundation for Science and Technology) and FEDER–European Regional Development Fund through the COMPETE Programme (Operational Programme for Competitiveness) for funding (UID/QUI/00313/2013 and PEst-OE/QUI/UI0313/2014). NMR data was collected at the UC NMR facility, supported in part by REEQ/481/ QUI/2006, RECI/QEQ-QFI/0168/2012, CENTRO-07-CT62-FEDER002012 and Portuguese NMR Network (RNRMN). Appendix A. Supporting information Supplementary data associated with this article can be found in the online version at doi:10.1016/j.jep.2018.09.013. References Achour, M., Saguem, S., Sarriá, B., Bravo, L., Mateos, R., 2018. Bioavailability and metabolism of rosemary infusion polyphenols using Caco-2 and HepG2 cell model systems. J. Sci. Food Agric. Adhikari, K.B., Laursen, B.B., Gregersen, P.L., Schnoor, H.J., Witten, M., Poulsen, L.K., Jensen, B.M., Fomsgaard, I.S., 2013. Absorption and metabolic fate of bioactive

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