Phenolic profile and biological activities of decoctions from Santolina impressa, a Portuguese endemic species

Journal Pre-proof Phenolic profile and biological activities of decoctions from Santolina impressa, a Portuguese endemic species ´ Paulo Madeira, Ana Margarida Rodrigues, Pedro Luis Vieira Fale, ˆ ˜ Maria Luisa Rita Pacheco, Maria Helena Florencio, Lia Ascensao, Marques Serralheiro

PII:

S2210-8033(20)30007-5

DOI:

https://doi.org/10.1016/j.hermed.2020.100335

Reference:

HERMED 100335

To appear in:

Journal of Herbal Medicine

Received Date:

8 June 2018

Revised Date:

5 December 2018

Accepted Date:

20 January 2020

ˆ Please cite this article as: Rodrigues AM, Vieira Fale´ PL, Madeira P, Pacheco R, Florencio ˜ L, Marques Serralheiro ML, Phenolic profile and biological activities of MH, Ascensao decoctions from Santolina impressa, a Portuguese endemic species, Journal of Herbal Medicine (2020), doi: https://doi.org/10.1016/j.hermed.2020.100335

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Phenolic profile and biological activities of decoctions from Santolina impressa, a Portuguese endemic species

Ana Margarida Rodriguesa†,, Pedro Luis Vieira Faléb,c, Paulo Madeirac,d, Rita

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Pachecob,e, Maria Helena Florêncioc,f, Lia Ascensãoa and Maria Luisa Marques Serralheirob,c*

Centro de Estudos do Ambiente e do Mar, Faculdade de Ciências, Universidade de

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a

Lisboa, Campo Grande, 1749-016 Lisboa, Portugal

BioISI - Biosystems & Integrative Sciences Institute, Faculdade de Ciências,

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b

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Universidade de Lisboa, Campo Grande, C8, 1749-016 Lisboa, Portugal Departamento de Química e Bioquímica. Faculdade de Ciências, Universidade de

Lisboa, Campo Grande, 1749-016 Lisboa, Portugal SAPEC-agro. Av.da do Rio Tejo-Herdade das Praias. 2910-440 Setúbal, Portugal

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Área Departamental de Engenharia Química, Instituto Superior de Engenharia de

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d

Centro de Química e Bioquímica. Faculdade de Ciências, Universidade de Lisboa,

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f

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Lisboa, Av. Conselheiro Emídio Navarro, 1959-007 Lisboa, Portugal

Campo Grande, 1749-016 Lisboa, Portugal †Current

address: Plant Metabolomics Laboratory, Instituto de Tecnologia Química e

Biológica António Xavier, Universidade Nova de Lisboa (ITQB NOVA), Av. da República, 2780-157 Oeiras, Portugal

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*Corresponding Author: Maria Luísa Serralheiro, Faculdade de Ciências, Universidade de Lisboa, Edifício C8, Campo Grande, 1749-016 Lisboa, Portugal. Tel.: +351 217500935; Fax: +351 217500088; Email: [email protected]

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Graphical abstract

Highlights  Santolina impressa decoctions have antiacetylcholinesterase activity  S. impressa decoctions pass through the digestive juices without changes in bioactivity  Santolina impressa decoctions can participate in the inhibition of cariogenic bacteria  Decoctions form Santolina impressa have chlorogenic acid, cynarin as main components 2

Abstract Santolina impressa Hoffmanns & Link is an aromatic Asteraceae species endemic to the southwest of Portugal. It is used in folk medicine as an herbal tea for gastrointestinal ailments and mouthwash antiseptic. The present study aimed to relate the chemical composition

of

decoctions

from

the

aerial

parts

S.

impressa

with

their

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antiacetylcholinesterase, antioxidant, antimicrobial and cytotoxic activities, in an attempt to explain the traditional use of this species. LC-MS/MS analysis identified chlorogenic acid and cynarin as the main components. Antimicrobial activity of the extracts was

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investigated against two cariogenic bacteria:- Streptococcus sobrinus and Streptococcus mutans, and these decoctions were not cytotoxic towards Caco-2 and HepG2 cell lines.

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Decoctions from capitula and stems/leaves showed high AChE inhibition, with IC50 values of 328 μg/mL and 579 μg/mL, respectively. The antioxidant activity measured

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using the DPPH radical scavenging assay showed an EC50 of 14.7 μg/mL and 12.9 μg/mL for capitula and stems/leaves extracts, respectively. The biological activities were kept

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constant after in vitro digestive process and the HPLC analysis did not indicate changes in the extract’s chemical composition. The results may explain the traditional use of S.

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impressa decoctions for digestive problems and as a mouthwash antiseptic.

Keywords: Acetylcholinesterase inhibition, antioxidant activity, cell toxicity, cariogenic activity, chlorogenic acid, cynarin, Santolina impressa

Keywords: Acetylcholinesterase inhibition, cariogenic activity, chlorogenic acid, cynarin, Santolina impressa

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1. Introduction Herbal teas (infusions and decoctions) are aqueous extracts of herbs containing a diversity of compounds from different classes, mainly flavonoids and phenolic acids that are responsible for important biological activities (Sarwar and Lockwood, 2010; Barroso et al., 2014). It has been shown that polyphenols are important natural antioxidants which

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can also inhibit several key enzyme activities, such as acetylcholinesterase (AChE) (Mata et al., 2007; Hernandez et al., 2010). AChE inhibitors have been used in the treatment of severe gastrointestinal (GI) disorders (Jarvie et al., 2008; Broad et al., 2013) and

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Alzheimer’s disease (Terry and Buccafuscio, 2003). Some GI disorders originate in

inflammation processes due to oxidative cell damage caused by free radicals (Baumgart

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and Carding, 2007). To avoid injuries produced by free radicals, cells have developed protective mechanisms, i.e. antioxidation processes, in which free radicals can be

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captured and oxidative stress reduced (Lü et al., 2010). Natural antioxidants to help prevent cell injury and dysfunction commonly described in inflammatory conditions

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(Conner and Grisham, 1996). Plant extracts with anti-inflammatory action and antioxidant activity are also reported to have a gastro protective effect (Ozbayer et al.,

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2014), but the toxicity of these crude extracts should be assessed before oral

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administration (Mnengi et al., 2014). Santolina L. (Anthemideae, Asteraceae) is a genus endemic to the Mediterranean region, particularly widespread in the western area. Santolina species have a strong characteristic chamomile-like fragrance and are traditionally used in Mediterranean countries to prepare herbal medicinal teas. Decoctions from leaves and inflorescences (capitula) are reported to possess anti-inflammatory, antispasmodic and digestive properties (Giner et al., 1989;

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Silván et al., 1996, Demirci et al., 2000, Da Silva et al., 2005). Decoctions are also used externally as eye and mouth antiseptics (Vallés et al., 1996). Due to their fragrance and extensive ethnobotanical uses, most Santolina species have been investigated for their essential oil composition and biological activities (Demirci et al., 2000; Liu et al., 2007; Khubeiz and Mansour, 2016 and references herein). Only in a few species the composition and biological activities of organic solvent extracts have been reported (Silván et al., 1996; Tavares et al., 2012; Gomes et al., 2015) and to the

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best of the authors knowledge, studies dealing with aqueous extracts, e.g. infusions or decoctions, are still scarce (Giner et al., 1989; Boudoukha et al., 2015).

Phytochemical studies on S. chamaecyparissus, the most studied Santolina species, had

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shown the presence of diverse classes of natural products, such as terpenoids, flavonoids and coumarins with several biological activities (Giner et al., 1989 Demirci et al., 2000;

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Boudoukha et al., 2015; Khubeiz and Mansour, 2016).

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Santolina impressa Hoffmanns. & Link is a Portuguese endemic species that occurs in the dunes throughout the southwestern coast, from the estuary of the Sado River to Cape Sines (Greuter, 2008; Rivero-Guerra, 2010a,b, 2011; Rivero-Guerra and Laurin, 2012).

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Decoctions prepared from leaves and capitula of S. impressa are traditionally used as beverages (herbal teas) for a wide range of gastrointestinal ailments, particularly for

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flatulence, abdominal complaints and gastrointestinal discomforts (Proença-da-Cunha,

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2003), but no phytochemical data relating the decoctions’ composition with the ethnobotanical uses is available. Therefore, the aim of the current study is to investigate the composition of decoctions of Santolina impressa and evaluate the AChE inhibitory activity, antioxidant capacity and cytotoxicity of these aqueous extracts towards Caco-2 and HepG2 human cell lines. Furthermore, the aim was to assess if the active compounds and activities found in these herbal teas were stable under gastrointestinal tract conditions.

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Antiseptic properties of these aqueous extracts were also investigated for the growth of cariogenic bacteria. 2. Materials and Methods 2.1. Reagents All chemicals were of analytical grade. Acetylcholinesterase (AChE) (E.C.3.1.1.7) type VI-S, from electric eel (Electrophorus electricus) 411 U/mg protein, 5,5’-dithiobis[2nitrobenzoic acid] (DTNB), pancreatin, pepsin, acetylthiocholine iodide (AChI),

(DPPH), thiazolyl tetrazolium bromide (MTT),

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tris[hydroxymethyl]aminomethane (Tris–HCl buffer), 1,1-diphenyl-2-picrylhydrazyl HEPES buffer, dimethylsulfoxide

(DMSO), chlorogenic acid, cynarin, were obtained from Sigma Aldrich (Barcelona,

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Spain). Dulbecco’s modified Eagle’s medium (DMEM), FBS (fetal bovine serum), Lglutamine, penicillin and streptomycin were purchase from Lonza (Verviers, Belgium).

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Trifluoracetic acid, NaCl, MgCl2, formic acid, methanol (HPLC grade) were acquired

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from Merck (Darmstadt, Germany). Brain Heart Infusion (BHI) medium supplied by Fluka (Steinheim, Germany). 2.2. Plant Material

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Branches of Santolina impressa Hoffmanns. & Link were collected in full bloom (June/July) from natural populations occurring in the dunes throughout southwestern

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Portugal from the estuary of the Sado River to Cape Sines (about 100 km south of Lisbon).

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A voucher specimen was deposited in the Herbarium of Botanical Garden of the University of Lisbon, Portugal, under the accession number LISU: 233493. 2.3. Preparation of plant aqueous-extracts (decoctions) Plant aqueous-extracts from stems/leaves and inflorescences (capitula) of S. impressa were prepared as decoctions, using 20 g of fresh plant material boiled in 200 mL of distilled water for 10 minutes. Decoctions were filtered through a grade 1 Whatman paper

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and lyophilized. Extracts were stored at -20 ºC in the dark prior to HPLC analysis and biological activities evaluations. 2.4. HPLC-DAD analysis The HPLC-DAD analysis of the aqueous plant extracts was carried using a Liquid Chromatograph Finnigan™ Surveyor® Modular LC System, Thermo-Finnigan equipped with a RP-8 (5μm) Lichrospher® column and Xcalibur ® software. Aqueous plant extracts (1 mg/mL) were analyzed by HPLC-DAD, injecting 25 µL with an auto injector

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and using a linear gradient composed of solution A [0.05% (v/v) trifluoroacetic acid] and solution B (methanol) as follows: 0 min: 70% A, 30% B; 40 min: 30% A, 70% B; 45 min: 30% A, 70% B; 47 min: 70% A, 30% B; 50 min: 70% A, 30% B; with a 0.8 mL/min flow

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rate. The detection was carried out between 200 and 600 nm with a diode array detector Finnigan Surveyor PDA Plus Detector. Standards of chlorogenic acid and cynarin were

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analyzed under the same conditions and used for quantifying these compounds in the

2.5. LC-MS analysis

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decoctions.

The LC–MS and LC–MS/MS analysis were carried out with a liquid chromatograph

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Surveyor Plus Modular LC system connected to a LCQ Duo ion trap mass spectrometer equipped with an electrospray ionisation (ESI) source, from Thermo Scientific (Bremen,

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Germany). The column and gradient used were the same as in the HPLC-DAD analysis.

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25 µL of each extract was injected at a concentration of 10 mg/mL, and the solution A was 1.0 % (v/v) formic acid. The mass spectrometer was operated in both positive and negative ion modes in the range m/z 120–1000 and the parameters were adjusted in order to optimize the signal-to-noise ratios (S/N) for the ions of interest. Briefly, the nebulizing and auxiliary gas (nitrogen) flow rates were 40 and 20 (arbitrary units) and the capillary temperature was set to 250 oC. Collision induced dissociation (CID) experiments were

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performed by isolating the ions within the ion trap and accelerating them to suffer multiple collisions with the background gas present in the ion trap (helium) using a data dependent acquisition mode. The ions of interest were activated by applying a percentage of a supplementary a.c. potential in the range of 0.75–1.75 Vp–p (peak-to-peak) to the end cap electrodes of the ion trap at the resonance frequency of the selected ion (referred to as the Normalized Collision Energy, NCE). The injection times were 50 ms in a full scan and 200 ms in an MS/MS scan Xcalibur ® software from Thermo Scientific was

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used to acquire and process the data. 2.6. Acetylcholinesterase inhibition

The AChE enzymatic activity was measured using an adaptation of the Ellman’s

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colorimetric method (Porfirio et al., 2010). Briefly, 325 μL of 50 mM Tris–HCl buffer (pH 8), 100 μL of the lyophilized extract solutions at different concentrations in water,

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25 μL of AChE (0.1 U/mL) solution in HEPES buffer pH 8 were incubated for 15 min.

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Subsequently, 75 μL of 79 mM AChI (0.023 mg/mL) and 475 μL of 3 mM DTNB in Tris–HCl buffer (pH 8) containing 0.05 M NaCl and 0.021 M MgCl2, were added to initiate the reaction. The initial rate of the enzymatic reaction was quantified by

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measuring the absorbance at 405 nm during 5 min. A control reaction was carried out using water instead of the extract solution and it was considered 100 % activity. The

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percentage of AChE inhibition by each concentration of extract was calculated by using: 𝑣

𝐼 (%) = 100 − 100 × (𝑣𝑠𝑎𝑚𝑝𝑙𝑒 ) , where I is the percent inhibition of AChE, vsample is

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𝑐𝑜𝑛𝑡𝑟𝑜𝑙

the initial rate of enzymatic reaction for the extract and vcontrol is the initial rate for the control AChE reaction in the absence of extract. Tests were carried out in triplicate. The IC50 value for each extract was calculated and represents the concentration of the extract that inhibited 50 % of the AChE activity.

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2.7. Antioxidant activity The antioxidant activity of decoctions was determined using the DPPH• radical scavenging method slightly modified (Porfirio et al., 2010). To a 2.5 ml solution of DPPH (0.002% (w/v) in methanol), 25 μL of lyophilized extract solutions were added at various concentrations in water. The mixture was incubated for 30 min at room temperature and the absorbance was measured at 517 nm against a corresponding blank. The antioxidant activity of each extract concentration was calculated using the equation: 𝐴

𝐴𝐴(%) = 100— 100 × ( 𝐴𝑠𝑎𝑚𝑝𝑙𝑒 ),

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𝐷𝑃𝑃𝐻

where AA is the antioxidant activity, Asample is the absorbance of the aqueous extract and ADPPH is the absorbance of the DPPH• solution. The assays were carried out in triplicate.

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The EC50 value was calculated as the concentration of the extract providing 50% of

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antioxidant activity.

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2.8. In vitro gastrointestinal metabolism

Assays were carried out by incubating the decoctions with artificial gastric and pancreatic

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juices, as described by Porfirio et al. (2010). All the assays were carried out in triplicate using blank assays with water. The in vitro gastric metabolism was performed by adding

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to gastric juice (3.2 mg/mL of pepsin and 2 mg/mL NaCl at pH 1.2) the same volume of a 20 mg/mL lyophilized extract solution in water. For the in vitro pancreatic metabolism

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assay, the pancreatic juice, consisting of 25 mg pancreatin/mL of potassium phosphate buffer 50 mM at pH 8, was added to the same volume of a 20 mg/mL lyophilized extract solution in water. Both mixtures were incubated at 37oC during 4 h. Samples (100 µL) were taken hourly and added to 900 µL of ice-cold methanol to stop the reaction. These aliquots were centrifuged 5 min at 5000 g and the supernatants were analyzed by HPLCDAD, for AChE inhibition and antioxidant activity as above described. 9

2.9. In vitro assay for cytotoxicity activity Caco-2 cells (ATCC® HTB37, human colorectal adenocarcinoma epithelial cell line), Hep G2 (ATCC® HB-8065, human hepatocellular carcinoma cell line), supplied were cultured in DMEM supplemented with 10% FBS, 100 U/mL penicillin, 100 U/mL streptomycin and 2 mM L-glutamine. Cells were maintained in CO2 incubator (5 % CO2) at 37 °C.

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The cytotoxicity effect of the decoction was assessed using the MTT viability test, adapted to the aqueous extracts under evaluation (Mosman, 1983). The cells were seeded into sterile 96-well plates at 5 x 103 cells per well in growth medium and incubated for

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48 h at 37°C in an atmosphere with 5% CO2 to assure attachment and 80% confluency.

The medium was discarded and replaced by lyophilized extract solutions in DMEM

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medium at various concentrations. A control assay was performed by adding DMEM

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medium to the wells. Two times 8 replicates were performed for each concentration and control. The plates were incubated for 24h at the same conditions. After incubation, the extract solutions were removed and replaced by 0.05 mg MTT per well in DMEM

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medium. Subsequently, the plates were incubated for 2-4h at 37 ºC and the MTT solution was removed and replaced with DMSO to mix the formazan crystals until dissolved. The

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absorbance at 570 nm was registered. For each extract the half-maximal inhibitory

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concentration (IC50) of the extracts was calculated. 2.10. Determination of antibacterial activity against two cariogenic bacteria The bacteriostatic activity of S. impressa stems/leaves aqueous extract decoction against cariogenic bacteria was determined by calculating MIC values, according to the procedure described by Figueiredo et al. (2010). Two cariogenic species of mutans streptococci, Streptococcus sobrinus (CETC 4010) and Streptococcus mutans (CETC

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479), obtained from the Colección Española de Cultivos Tipo, were used. These bacterial strains were cultured in Brain Heart Infusion (BHI) medium, in anaerobic conditions. S. mutans and S. sobrinus from overnight cultures were adjusted to 0.5 AU (absorbance units) at 630 nm and diluted 10 fold in broth. The stems/leaves decoction of S. impressa was serially diluted with broth to concentrations ranging from 0 to 5 mg/mL. In sterile 96-well plates, 100 µL of diluted extract samples were added to 100 µL of bacterial suspension. For all the assays a positive control without extract, and a negative control

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without inoculation, were prepared. BHI replaced the plant extract in the positive control and the inoculum was substituted by BHI in the negative control. After an incubation period of 48 h at 37°C under anaerobic conditions, the bacterial growth was estimated

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spectrophotometrically at 630 nm. The MIC was defined as the minimum concentration

of the test extract limiting bacterial growth to 90% and IC50 the concentration required

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for 50% of growth inhibition. All assays were carried out in triplicate and the values

mutans, respectively. 2.11. Statistical analysis

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determined using dose-response curves with r2 of 0.906 and 0.863 for S. sobrinus and S.

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All statistical analysis was performed using Microsoft® Excel 2010. Additional F-test variance analysis (ANOVA) was performed at a 5% significance level (P= 0.05). All

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data were expressed as means ± standard deviation.

3. Results and Discussion 3.1. Chromatographic profile and chemical composition of S. impressa decoctions In contrast with the large amount of phytochemical knowledge available on the essential oils of Santolina species, only a few studies have been conducted in aqueous or organic

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solvent extracts. As far as the authors know this is the first study on S. impressa aqueous extracts, particularly on decoction of its capitula. Two decoctions of Santolina impressa from stems/leaves and capitula were analyzed by HPLC-DAD and the chromatograms are shown in Fig. 1a and 1b, respectively. The compounds present in both extracts were identified by LC–MS and LC–MS/MS, in positive and negative ion modes, and identical results were obtained. Table 1 shows the results in the negative ion mode for the extracts of stems/leaves and capitula. Both extracts

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were mainly composed of caffeoylquinic and dicaffeoylquinic acids, being chlorogenic acid (3-O-caffeoylquinic acid) and cynarin (1,3-dicaffeoylquinic acid) the dominant constituents. The non-identified peaks by mass spectrometry are also caffeic acid

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derivatives according to the DAD UV-Vis spectra.

The concentrations of the main compounds in the extracts of S. impressa were calculated

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using the HPLC-DAD chromatograms. Stems/leaves decoctions contained 127.1 ± 6.2

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µg/mg of chlorogenic acid and 92.1 ± 3.3 µg/mg of cynarin, while the capitula decoction contained 134.3 ± 0.3 µg/mg and 144.7 ± 2.7 µg/mg, respectively. Chlorogenic acid and cynarin represented 24.7% and 43.4% of peak area in stems/leaves decoctions, and 19.4%

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and 56.4% in capitula decoctions, respectively. Two other flavonoid derivatives:myricetin-3-O glucoside and homoorientin were also identified in these decoctions.

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Chlorogenic acid, present in most Asteraceae species, is nowadays considered a

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biochemical marker of this family (Jaiswal et al., 2011; Venditti et al., 2018; Caprioli et al., 2017; Silva et al., 2017) and cynarin, first reported by Panizzi and Scarpati (1953) has also been reported in other Asteraceae decoctions (Falé et al., 2014) Flavonoids and coumarins at different concentrations were detected by HPTLC (High Performance Thin Layer Chromatography) in various solvent extracts from roots, stems and leaves of S. insularis in the vegetative and flowering periods (Sacchetti et al., 1997). Recently, the

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presence of hydroxicinammic acids derivatives, flavones, simple phenolic acids and coumarins were detected by HPLC-DAD-ED in organic extracts from stems/leaves of S. semidentata (Gomes et al., 2015) and from the aerial parts of plants of S. impressa (Tavares et al., 2012). In the hydroethanolic extract of S. impressa studied by Tavares et al. (2012), the main constituents found were ferulic acid and derivatives. The differences in the main constituents of the aqueous extracts now studied and of the hydroethanolic extract of S. impressa reported by Tavares et al. (2012) may be partly attributed to several

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factors, including plant developmental stage, organ studied, yearly climatic parameters, harvest time and extraction method (Vinutha et al., 2007; Carpinella et al., 2010; Henriques et al., 2017).

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3.2. AChE inhibitory activity, antioxidant capacity and cytotoxicity of S. impressa decoctions

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The use of AChE inhibitors to stimulate gastrointestinal motility was indicated in the

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treatment of functional gastrointestinal disorders (Jarvie et al., 2008; Broad et al., 2013). As in traditional medicine, herbal teas of S. impressa are used to treat digestive and intestinal problems, adding importance to the evaluation of AChE inhibitory activity and

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antioxidant capacity of these aqueous extracts, as well as their potential toxicity. All results are summarized in Table 2.

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The decoctions from S. impressa capitula showed the highest AChE inhibitory activity at

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a 95% significance level (P=0.05), with an IC50 value of 329 µg/mL, whereas decoctions from stems/leaves exhibited an IC50 of 579 µg/mL. IC50 values obtained for the AChE inhibitory activity of S. impressa decoctions were of the same magnitude of those found for other plant extracts, namely for other Asteraceae species (Tavares et al. 2012; Falé et al., 2013b; Gomes et al. 2015). In the study performed by Tavares et al. (2012), the hydroethanolic extracts of S. impressa showed a lower AChE inhibitory activity, when

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compared with the values found in the aqueous extracts herein reported. These differences are probably due to the different composition of the extracts, due to different efficacy in the extraction of using water or ethanol-water solvents. According to the literature, chlorogenic acid has inhibitory activity towards AChE with an IC50 value of 196 µg/mL (Hernandez et al., 2010) and cynarin, is also able to inhibit AChE with an IC50 value of 77.2 µg/mL (Falé et al., 2013). Thus, the amount of these two compounds in the S. impressa extracts could explain 53% and 42% of the AChE inhibitory activity displayed

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by the stems/leaves and the capitula decoctions, respectively, i.e the total of the IC50 value obtained. Although these two phenolic compounds are approximately one hundred times

less potent than the commercial AChE inhibitor donepezil, a drug used in the most serious

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cases of gastrointestinal motility (Lepkowsky, 2018) and Alzheimer Disease (Cacabelos, 2007) with a IC50 value of 2.4 ng/mL (Silva et al., 2017), they are responsible for the

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enzymatic inhibition activity detected in these extracts.

The antioxidant activity of S. impressa extracts, measured using the DPPH• free radical

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scavenging assay, gave an effective concentration (EC50) of 14.7 μg/mL and 12.9 μg/mL for the stems/leaves and capitula decoctions, respectively (P=0.05). These EC50 values

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are similar to those determined in extracts from other Asteraceae (Barroso et al., 2014) or even in extracts from other Santolina species (Chibani et al., 2012), however closer to

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that shown by the standard BHT, commonly used in the food industry, which presented

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an EC50 value of 12 ± 0.7 µg/mL (Mata et al., 2007). This strong antiradical activity of S. impressa extracts is probably due to their phenolic components, especially to the dominant constituents, chlorogenic acid and cynarin, two compounds well-known for their antioxidant activity (Hernandez et al., 2010; Fratianni et al., 2014; Pistón et al., 2014; Raudonėa et al., 2015).

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As decoctions from S. impressa capitula presented the best AChE inhibitory activity and antioxidant capacity, only the toxicity of these extracts was tested in Caco-2 and HepG2 cells, using several concentrations of plant extract to calculate the cell viability by the MTT assay. In Caco-2 and HepG2 cells, IC50 toxicity values of 1 mg/mL and 0.53 ± 0.03 mg/mL were obtained, respectively. HepG2 were more sensitive to the decoctions than Caco-2. The toxicity of chlorogenic acid was tested relatively to HepG2 and IC50 of 0.84 mg/mL was determined. These results mean that the extracts from capitula decreased in

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50% the cell viability at concentrations higher than 0.1 mg/mL, a reference value that is considered toxic to human cell lines (Cardenas et al., 2006; Oonsivilai et al., 2007).

Consequently, the decoctions from S. impressa have no relevant toxicity as other

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Asteraceae aqueous extracts with a similar composition (Falé et al., 2013; Arantes et al., 2016).

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3.3. In vitro gastrointestinal metabolism

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Phenolic compounds may be modified during the digestive process (Spencer, 2003) and these changes can affect the biological activities of the extracts which are known to be structure-dependent. To determine the effect of the gastrointestinal digestion on the

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chemical structure of the phenolic compounds present in S. impressa extracts, they were incubated with artificial gastric and pancreatic juices and aliquots were collected at

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several time-points for HPLC-DAD analysis. Comparing the chromatographic profiles of

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the decoctions, before and after in vitro digestion, no significant changes (P=0.05) could be observed (Fig. 2a, 2b). Furthermore, the AChE inhibition activity and the antioxidant capacity of the extracts were kept constant during and after the in vitro gastrointestinal digestion (Table 3). As in the current study, aqueous extracts from leaves of Cynara cardunculus (Asteraceae), Fraxinus angustifolia (Oleaceae) and Pterospartum tridentatum

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(Fabaceae), also containing chlorogenic acid, cynarin and flavonoid derivatives, were not modified by gastric digestive juices (Falé et al., 2013). In fact, chlorogenic acid is stable and not hydrolyzed in the stomach at a low pH (Friedman and Jürgens, 2000; Gumienna et al., 2011) and, it has been reported to be in vitro quickly absorbed in its intact form (Lafay et al., 2006). Although this topic is still a matter of discussion, most polyphenols are quite stable during in vitro gastro digestion, reaching intact the small intestine, the main organ involved in the absorption and metabolism of polyphenols (Correa- Betanzo

3.4. Antibacterial activity against cariogenic bacteria

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et al., 2014; Mihailovic´et al., 2016).

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As decoctions of Santolina are used externally as mouthwash antiseptic, it was evaluated whether decoctions from S. impressa stems/leaves had antibacterial activity. Two mutans

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streptococci (Streptococcus mutans and Streptococcus sobrinus), generally considered as

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the major agents of dental caries, were tested (Pires et al., 2018). The extract presented similar antimicrobial activity against the two mutans streptococci assayed, with MIC90 values of 1.63 ± 0.39 mg/mL and 1.41 ± 0.08 mg/mL and IC50 values

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of 0.46 ± 0.06 and 0.44 ± 0.14 for S. mutans and S. sobrinus, respectively (P=0.05). The extracts had moderate antimicrobial activity against cariogenic bacteria, according to the

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classification proposed by Aligiannis et al. (2001) and Duarte et al. (2007), that classified

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MIC values as strong (MIC < 500 μg/mL), moderate (MIC of 600–1500 μg/ mL) or weak (MIC > 1600 μg/ mL). MIC values obtained in this study are in the same range as other plant extracts (Yatsuda et al., 2005; Figueiredo et al., 2010; Palombo, 2011; Pires et al., 2018). The values obtained in the present study indicate less efficiency compared to the standard compound chlorhexidine used in commercial mouth washes but this compound has several harmful side-effects (Santos, 2003). Plant extracts can be good alternatives to

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the standard compound (Vinod et al., 2018). The values obtained are of the same magnitude when using pure natural compounds like eucalyptol or carvacrol where 0.5 mg/mL (Park et al., 2003). Many studies have reported relationships between the anticariogenic activity of plant extracts and their amount in phenolic compounds (Ferrazzano et al., 2011), and the mild antimicrobial activity displayed by S. impressa extracts can be due to their phenolic content, most likely to the dominant components, chlorogenic acid and cynarin, whose antimicrobial activity against different bacteria species has been

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reported (Zhou et al., 2003). 4. Conclusions

The high AChE inhibition activity and antioxidant capacity found in the decoctions of

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stems/leaves and capitula of Santolina impressa have been related to the presence of chlorogenic acid and cynarin. Gastrointestinal digestion, using artificial pancreatic and

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gastric juices did not modify the anti-AChE and antioxidant activities. Extracts have no

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relevant toxicity and stems/leaves decoction showed mild antimicrobial activity against cariogenic bacteria. According to the results, both extracts can be considered safe and nontoxic, and the activities reported here support, at least partially, the traditional use of

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this species for digestive and gastrointestinal problems. The stems/leaves decoctions may be also useful as antiseptic mouthwash for the prevention of oral infectious diseases

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caused by Streptococcus mutans and S. sobrinus, the major agents of dental caries.

AUTHOR DECLARATION We wish to confirm that there are no known conflicts of interest associated with this publication and there has been no significant financial support for this work that could have influenced its outcome. We confirm that the manuscript has been read and approved by all named authors and that there are no other persons who satisfied the criteria for authorship but are not listed. We 17

further confirm that the order of authors listed in the manuscript has been approved by all of us. We confirm that we have given due consideration to the protection of intellectual property associated with this work and that there are no impediments to publication, including the timing of publication, with respect to intellectual property. In so doing we confirm that we have followed the regulations of our institutions concerning intellectual property

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We understand that the Corresponding Author is the sole contact for the Editorial process (including Editorial Manager and direct communications with the office). He/she is responsible for communicating with the other authors about progress, submissions of revisions and final approval of proofs. We confirm that we have provided a current, correct email address which is accessible by the Corresponding Author and which has been configured to accept email from ([email protected])

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Author statement

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Acknowledgements

This work was financially supported by Fundação para a Ciência e Tecnologia (FCT)

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through the projects contract numbers: Pest – OE / QUI / UI0612 / 2013; UID / MULTI /00612 /2013, PEst-UID/AMB/50017/2013, BioISI (UID/MULTI/04046/2013).

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Declarations of interest: none.

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na

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7272-7278.

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Figure 1. HPLC-DAD chromatograms of Santolina impressa decoctions: (a) stems/leaves extracts; (b) capitula extracts. Numbers correspond to the identified compounds: 1 - chlorogenic acid; 2 - myricetin-3-O-glucoside; 3 - cynarin; 4 homoorientin; 5 -dicaffeoylquinic acid. Figure 2. HPLC-DAD chromatograms of Santolina impressa decoctions before and after 4h digestion with gastric and pancreatic juices: (a) stems/leaves extracts; (b) capitula

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extracts. (–—) before; (------) gastric; (····) pancreatic

28

(a)

Intensity (µAU)

300000

3 200000

1 5

100000

4

0 0

10

20

30

40

50

Retention time (min)

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(b)

3

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1

200000 100000

5

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Intensity (µAU)

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Intensity (uAU)

1000000

1

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3

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600000 400000

5

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10

20

30

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50

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Table 1 – Electrospray ionization negative-ion mass spectra and abundance (relative peak area, %) of the main constituents of the aqueous extracts from stems/leaves and capitula of Santolina impressa. Values expressed in mean ± s.d. (n = 3).

m/z precursor ion [M-H]-

m/z product ions (rel. ab.%)

Constituents

1

353

191 (100), 179 (13), 173 (3)

2

479

3

Peak

Santolina impressa Capitula

Chlorogenic acid

24.7 ± 2.4

19.4 ± 0.6

317 (100)

Myricetin-3-O-glucoside

t

7.2 ± 0.1

515

353 (100), 335 (6), 299 (3), 203 (3)

Cynarin

4

447

285 (100)

Homoorientin

5

515

353 (100), 335 (3), 317 (10), 299 (16), 203 (14)

Dicaffeoylquinic acid

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Stems/leaves

56.4 ± 0.4

6.4 ± 0.9

t

16.8 ± 1.2

12.3 ± 1.2

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43.4 ± 0.8

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t – traces ≤ 1.0 %).

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Table 2: Bioactivity of Santolina impressa decoctions: anti-acetylcholinesterase (antiAChE, µg/mL), antioxidant activity (DPPH scavenging, µg/mL), cytotoxicity in Caco2 and HepG2 cell lines (mg/mL), minimum inhibitory concentration (MIC) and concentration required for 50% inhibition (IC50) against Streptococcus mutans and Streptococcus sobrinus (mg/mL). All values expressed as mean ± standard deviation (n

Capitula

Stems/Leaves

Anti-AChE (µg/mL)

328.3±3.3

578.9±13.7

Antioxidant activity (µg/mL)

12.9±0.1

14.7±0.1

1.48±0.16

-

0.53±0.05

-

-

1.63±0.39

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Bioactivity

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= 3).

Cytotoxicity Caco2 cells (mg/mL) HepG2 cells (mg/mL) Antibacterial activity against cariogenic bacteria MIC (mg/mL) IC50 (mg/mL)

-

0.46±0.06

Streptococcus sobrinus (CETC 479)

MIC (mg/mL)

-

1.41±0.08

-

0.44±0.14

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Streptococcus mutans (CETC 4010)

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IC50 (mg/mL)

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Table 3. Anti-acetylcholinesterase (Anti-AChE) and antioxidant (DPPH scavenging) activities of the aqueous extracts from Santolina impressa stems/leaves (S/L) and capitula (C), after 4 h digestion with gastric and pancreatic artificial juices. Values expressed in % ± standard deviation (n=3).

In vitro gastric digestion Antioxidant

Anti-AChE

Antioxidant

S/L

C

S/L

C

S/L

C

S/L

C

100.0 ± 0.3 99.8 ± 2.0 100.3 ± 2.9 99.5 ± 1.2 100.2 ± 3.2

100.0 ± 0.9 99.7 ± 1.3 97.0 ± 2.5 97.4 ± 3.3 96.6 ± 5.1

100.0 ± 1.3 93.9 ± 3.5 99.5 ± 2.7 99.8 ± 1.3 97.7 ± 2.1

100.0 ± 1.4 98.7 ± 2.1 97.5 ± 1.3 98.8 ± 0.5 100.7 ± 1.2

100.0 ± 1.8 93.0 ± 1.2 88.9 ± 1.9 86.4 ± 1.3 86.6 ± 4.5

100.0 ± 0.2 92.2 ± 2.5 92.3 ± 4.3 90.3 ± 1.1 88.6 ± 2.8

100.0 ± 3.0 100.0 ± 3.2 98.7 ± 2.3 99.2 ± 3.1 101.5 ± 1.2

100.0 ± 2.0 103.0 ± 3.1 101.7 ± 1.3 99.0 ± 2.0 98.5 ± 3.0

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0 1 2 3 4

Anti-AChE

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hours

In vitro pancreatic digestion

33