Inhibition on cholinesterase and tyrosinase by alkaloids and phenolics from Aristotelia chilensis leaves

Food and Chemical Toxicology xxx (2017) 1e12

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Inhibition on cholinesterase and tyrosinase by alkaloids and phenolics from Aristotelia chilensis leaves Carlos L. Cespedes a, *, Cristian Balbontin c, Jose G. Avila d, Mariana Dominguez f, ~ aloza-Castro e, Julio Alarcon b, Cristian Paz g, Viviana Burgos g, Leandro Ortiz h, Ignacio Pen i j David S. Seigler , Isao Kubo a

Biochemistry and Phytochemical-Ecology Lab, Department of Basic Science, Facultad de Ciencias, Universidad del Bio Bio, Chillan, Chile Synthesis and Biotransformation Lab., Department of Basic Science, Facultad de Ciencias, Universidad del Bio Bio, Chillan, Chile Plant Production Department, Instituto Nacional de Investigaciones Agropecuarias, Quilamapu, Chillan, Chile d Laboratorio de Fitoquimica, Unidad UBIPRO-FES-Iztacala, UNAM, Tlalnepantla de Baz, Mexico, DF, Mexico e Laboratorio de Fisiologia Vegetal, Unidad UBIPRO-FES-Iztacala, UNAM, Tlalnepantla de Baz, Mexico, DF, Mexico f Departamento de Biologia Celular y Desarrollo, Laboratorio 305-Sur, Instituto de Fisiologia Celular, UNAM, Ciudad Universitaria, Coyoacan 04510, Mexico, DF, Mexico g Departamento de Química y Recursos Naturales, Universidad de La Frontera, Av. Francisco Salazar 1011, Temuco, Chile h Instituto de Ciencias Química, Facultad de Ciencias, Universidad Austral de Chile, Valdivia, Chile i Department of Plant Biology, University of Illinois, Urbana-Champaign, Illinois, USA j Natural Products Chemistry Lab., ESPM Department, University of California, Berkeley, USA b c

a r t i c l e i n f o

a b s t r a c t

Article history: Received 30 March 2017 Received in revised form 2 May 2017 Accepted 6 May 2017 Available online xxx

It is reported in this study the effect of isolates from leaves of Aristotelia chilensis as inhibitors of acetylcholinesterase (AChE), butyrylcholinesterase (BChE) and tyrosinase enzymes. The aim of the paper was to evaluate the activity of A. chilensis towards different enzymes. In addition to pure compounds, extracts rich in alkaloids and phenolics were tested. The most active F5 inhibited AChE (79.5% and 89.8% at 10.0 and 20.0 mg/mL) and against BChE (89.5% and 97.8% at 10.0 and 20.0 mg/mL), showing a strong mixed-type inhibition against AChE and BChE. F3 (a mixture of flavonoids and phenolics acids), showed IC50 of 90.7 and 59.6 mg/mL of inhibitory activity against AChE and BChE, inhibiting the acetylcholinesterase competitively. Additionally, F3 showed and high potency as tyrosinase inhibitor with IC50 at 8.4 mg/mL. Sample F4 (anthocyanidins and phenolic composition) presented a complex, mixed-type inhibition of tyrosinase with a IC50 of 39.8 mg/mL. The findings in this investigation show that this natural resource has a strong potential for future research in the search of new phytotherapeutic treatments for cholinergic deterioration ailments avoiding the side effects of synthetic drugs. This is the first report as cholinesterases and tyrosinase inhibitors of alkaloids and phenolics from A. chilensis leaves. © 2017 Elsevier Ltd. All rights reserved.

Keywords: Cholinesterase inhibitors Tyrosinase inhibitors Antioxidants Aristotelia chilensis leaves Aristotelia alkaloids

1. Introduction Neurodegenerative diseases involve that the cholinergic hypothesis is the most widely accepted biochemical theory of these diseases. Declination of cognitive and mental functions ailment known as Alzheimer's disease (AD) are related with the loss of cortical cholinergic neurotransmission (Lai et al., 2013). There acetylcholinesterase (AChE) plays a key role in hydrolysis of acetylcholine (ACh) (Yang et al., 2012). Diverse therapeutic

* Corresponding author. E-mail address: [email protected] (C.L. Cespedes).

approximations for AD treatments are extremely limited to several AChE inhibitors such as tacrine, donepezil, rivastigmine, huperzine or galantamine and the NMDA receptor antagonist memantine (Chen et al., 2017; Vallen-Graham et al., 2017). As a matter of fact, the use of AChE inhibitors is an effective clinical approach to maintain the levels of ACh and enhance cholinergic function. The inhibition of the AChE has become the standard approach in the symptomatic treatment of AD (Howes and Houghton, 2003; Pendota et al., 2013). In similar form to AChE, butyryl cholinesterase (BChE) can also inactive ACh. A decreasing of AChE activity implies a reduction of ACh. If, BChE in AD remains under normal levels. Thus, BChE could be a noticeable

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Please cite this article in press as: Cespedes, C.L., et al., Inhibition on cholinesterase and tyrosinase by alkaloids and phenolics from Aristotelia chilensis leaves, Food and Chemical Toxicology (2017), http://dx.doi.org/10.1016/j.fct.2017.05.009

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C.L. Cespedes et al. / Food and Chemical Toxicology xxx (2017) 1e12

contributor to the loss of ACh in AD (Walsh et al., 2011). On the other hand, BChE inhibition can diminish Ab peptide (Greig et al., 2002; Furukawa-Hibi et al., 2011), additionally, BChE is very important in AD plaque maturation (Darvesh et al., 2012). Thus, selective BChE inhibitors or bivalent ChE inhibitors are important for new treatments for AD (Wang et al., 2012). As long as, Tyrosinase (EC 1.14.18.1), is an enzyme within the group of polyphenol oxidases (PPO) (Mayer, 1987); there is a copper-containing enzyme widely occurring in microorganisms, animals, and plants. This mixed function oxidase catalyzes two distinct reactions of melanin synthesis (Robb, 1984), the hydroxylation of a monophenol (monophenolase activity) and the conversion of an o-diphenol to an o-quinone (diphenolase activity). Whereas, the oxidation of L-tyrosine to melanin catalyzed by tyrosinase is crucial since melanin has many functions in the human systems. Alterations in melanin synthesis occur in many disease states. Melanin plays an important role in the absorption of free radicals that occur within the cytoplasm and in shielding the host from various types of ionizing radiation. The hydroxylation of L-tyrosine, the initial step in melanin synthesis (Holme et al., 2001), is also the initial step in catecholamine synthesis. Melanoma specific anticarcinogenic activity is also being linked with tyrosinase activity (Prezioso et al., 1992). On the other hand, melanin pigments are also found in the mammalian brain. Thus, tyrosinase may play a role in neuromelanin formation in the human brain, particularly in the substantia nigra (Xu et al., 1997). Recently, secondary metabolites from natural plants such as alkaloids, flavonoids and terpenoids have been proven to possess AChE and tyrosinase inhibitory activities (Orhan et al., 2013; Pinho ~ oz et al., 2013). These findings suggest that plants et al., 2013; Mun are excellent and sustainable resources looking for compounds with AChE and tyrosinase inhibitory activities. On the other hand, in the continuation of the general screening program of Chilean flora with bioactivities, there have been reported several bioactivities including antioxidant, cardioprotection, and anti-inflammation among other from fruits of A. chilensis (Cespedes et al., 2008, 2009; 2010a, 2010b; 2010c, 2017). Although it has gained popularity as an ethno-medicine for many years, this plant species is broadly used in Chilean Traditional Medicine for the treatment of diuretic, antimicrobial, neurological diseases (Cespedes, 1996; Silva et al., 1997; Bhakuni et al., 1976), and it is used particularly as an anti-inflammatory agent for kidney pain, stomach ulcers, diverse digestive ailments (tumors and ulcers), fever and healing injuries (Bhakuni et al., 1976; Cespedes et al., 2010b, 2010c). Previously, monoterpene indole alkaloids, flavonoids and phenolics have been reported in the leaves of A. chilensis (Bhakuni et al., 1976; Cespedes et al., 1990, 1993; Cespedes, 1996; Watson et al., 1982, 1989; Silva et al., 1997; Suwalsky et al., 2008). In the present work, extracts, fractions, alkaloids, flavonoids and phenolics from leaves of A. chilensis were evaluated the same as AChE, BChE and Tyrosinase inhibitors. Additionally, the Araucanian people used this species for treating “the nerves”, an ethnomedical disease with symptoms of restlessness, cardiac arrhythmias, and despair, headache and inflammation (these latter are strongly ~ oz associated with AD), (Montes and Wilkomirsky, 1978, 1987; Mun et al., 2001). 2. Materials and methods 2.1. Chemicals and solvents All reagents used were commercially available. Thiamine, sorbate, methyl-paraben, ascorbate, acetic acid, acetaldehyde, tyrosinase (from mushroom, EC 1.14.18.1), AChE, BChE, acetylthiocholine (ATC), 5,5-dithiobis (2-nitrobenzoic acid) (DTNB), choline-chloride,

calcium pantothenate, niacinamide, riboflavin, folic acid, biotin, and vitamin B-12 were purchased from Sigma-Aldrich Chemical Co, Santiago de Chile or in Mexico City. Benzoic acid, 4-hydroxybenzoic acid, t-cinnamic acid, p-coumaric acid, sinapic acid, linarin, luteolin, syringin, kojic acid, gallic acid, chlorogenic acid, quercetin, tannic acid, 1,1-diphenyl-2-p-picrylhydrazyl (DPPH), and L-3,4dihydroxyphenylalanine (L-DOPA) were purchased from SigmaAldrich Chemical Co. (Santiago Chile, Milwaukee, WI, USA). Ltyrosine, and dimethylsulfoxide (DMSO) were obtained from Aldrich Chemical Co. (St. Louis MO, USA). Silica gel 60 F254 precoated aluminum sheets (0.2 mm layer thickness) were purchased from Merck (Darmstadt, Germany). Methanol, ethyl acetate, CuSO4, KCl, NaHCO3, MgCl2, NH4Cl, pyridine, acetic anhydride, Silica gel GF254 analytical chromatoplates, Silica gel grade 60, 70e230, 60 A for column chromatography were purchased from Merck Co., Santiago de Chile. Precoated TLC plates SIL G-100 UV254, 1.0 mm, preparative were purchased from Machery-Nagel (GmbH&Co, KG), Dueren, Germany. Many authentic samples of natural compounds were provided by Dr. J.G. Avila, FES-Iztacala, UNAM, Tlalnepantla, Mexico DF, Mexico. 2.2. Plant material Samples of aerial parts (stems and leaves) of A. chilensis were ~ collected on slope of Andean hills of Araucanian and Nuble regions, southern Chile; the samples were collected during springs of 1985 and 2012. All samples were always from aerial parts (stems and leaves) and were botanically identified by Professor Dr. David S. Seigler (Plant Biology Department, curator of Herbarium (ILLS), University of Illinois, Urbana-Champaign, US), vouchers specimens were deposited at the herbariums of Univ. of Illinois at UrbanaChampaign, US, at the herbarium of Departamento de Ciencias Basicas, Facultad de Ciencias, Universidad del Bio Bio, Chillan, Chile, and at the herbal collection of Department of Chemistry, School of Engineering and Sciences, Universidad de La Frontera, Temuco, Chile. The collected samples were air-dried and frozen at
80  C until use. 2.3. Apparatus For hyphenated analyses were used the following: 1H NMR spectra were recorded at 300, 400 and 500 MHz, 13C NMR at 75 and 125 MHz, on DPX 300 MHz, DRX500 MHz and Avance III (400) NMR Bruker spectrometer, chemical shifts (d) are related to (CH3)4Si as internal reference (d ¼ 0), CDCl3, MeOD and acetone-d6 from Aldrich Chemical Co were used as solvents; coupling constants are quoted in Hz. GC/MS HP5989A, LC/MSD-TOF Agilent. IR spectra were obtained as KBr pellets on Perkin Elmer 283-B and FT-IR Nicolet Magna 750 spectrophotometers. UV spectra of pure compounds were determined on a Shimadzu UV-160 and Spectronic model Genesys 5 spectrophotometers; CHCl3 was used as solvent. Optical rotations were measured on a JASCO DIP-360 Spectropolarimeter; CHCl3 was used as solvent. Melting points were obtained on a Fisher-Johns hot-plate apparatus and they remain uncorrected. Nunc 24-well polystyrene multi-dishes were purchased from Cole-Parmer. LAB-LINE Chamber model CX14601A, with adjustable HieLo protection thermostats safeguard samples. Additionally, a microplate reader Epoch-Biotek UVeVis (200e999 nm) spectrophotometers were used to carry out the spectrophotometric measurements of the cholinesterase activity. The centrifuge used in this study was Thermo-Scientific Heraeus Megafuge 16R. So also, EIMS and TOF data were determined on a Q-TOF Waters and JEOL JMSAX505HA mass spectrometer at 70 eV. FABMS were obtained on a JEOL JMS-SX102A mass spectrometer operated with

Please cite this article in press as: Cespedes, C.L., et al., Inhibition on cholinesterase and tyrosinase by alkaloids and phenolics from Aristotelia chilensis leaves, Food and Chemical Toxicology (2017), http://dx.doi.org/10.1016/j.fct.2017.05.009

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an acceleration voltage of 10 kV. Samples were desorbed from a nitrobenzyl alcohol matrix using 6 keV Xenon atoms. Column chromatography was carried out on Kiesel-gel G (Merck, Darmstadt, Germany); TLC was performed on Si gel 60 F254. In addition to LC/MSD-TOF (6500 Series Agilent), and GC/MS with GC/ MSD5977A Agilent systems a HPLC system consisted of a Hewlett Packard Series 1100HPLC instrument with DAD and UV detectors set at 320 nm. The column was obtained from Supelco Technologies C18 (150 mm  4.6 mm, 5 mm). Flavonoids and phenolics acids were detected as well as 280 nm and 360 nm. Solvent gradients were made with dual pumping system varying proportions of solvents. The eluent 1 was a mixture of 4% tetrahydrofuran in acetonitrile and water (35:65, v/v) and contained 0.04% phosphoric acid; eluent 2: a mixture of (A: water-acetic acid 97:3 v/v; B: methanol (100%)). The flow rates were 0.85, 1.0 and 1.7 mL/min and the column temperature and pressure were 30  C and 149 bar, respectively, and the injection volume was 20 mL. Verbascoside (a gift from Prof. J. Guillermo Avila, FES-Iztacala. UNAM, Mexico DF), linarin, quercetin, luteolin and syringin (from Sigma-Aldrich S.A. de C.V., Mexico DF) served as standards for phenolics and aristoteline (a gift from Prof. Ralph Bick, Tasmania, Australia), galantamine, huperzine and ajmalicine (Purchased from Sigma-Aldrich SA de CV, Mexico DF) for alkaloids controls. Flavonoids and phenolics acids were detected as well as 280 nm and 360 nm. Solvent gradients were made with dual pumping system varying proportions of solvents. The eluent 1 was a mixture of 4% tetrahydrofuran in acetonitrile and water (35:65, v/v) and contained 0.04% phosphoric acid; eluent 2: a mixture of (A: wateracetic acid 97:3 v/v; B: methanol (100%)). From HPLC-MS fractionation of the F-3 flavonoids and phenolic acids were identified by matching the retention time and their spectral features against standards. Quantitation was made according to the linear calibration curves of standard compounds. Quercetin and 3,4,5trimethoxycinnamic acid were chosen as internal standards due to that their retention times are close to peaks of interest and there is no large light absorbance near their retention times in the chromatograms. 2.4. Isolation of alkaloids and phenolics from leaves of Aristotelia chilensis Dried and milled plant material - aerial parts (leaves and stems, 32.0 kg) collected during spring of 1985 was macerated with methanol during one week. The solvent was evaporated under reduced pressure until a syrup concentrate (10.2 Kg). A portion of this concentrate (3900 g), was dissolved with a mixture H2O/MeOH (6:4) and filtered completing with distilled water until 500 mL, and this solution was extracted with n-hexane yielding fraction F1 (2900 g), after with dichloromethane yielding fraction F2 (750 g), finally with ethyl acetate yielding fraction F3 (830 g), remaining an aqueous residue (F4, 340 g). The four fractions were evaluated as total phenolics content with Folin-Ciocalteu reagent, F3 showed highest levels of phenolics. In a first time F3 and F4 were worked under a conventional open column chromatography and after both fractions were analyzed by hyphenated analytical techniques HPLC-MS-NMR. In total from F3 (630 g) quercetin 9 (Yellow crystals, 5.4 g 0.86%), myricetin 10 (178.0 mg 0.028%), luteolin 15 (95.9 mg 0.015%), scopoletin (88.0 mg 0.014%), anthraquinone (15.0 mg 0.024%), in minor concentrations diosmetin 17, apigenin 14, rhamnetin 11, acacetin 16, a number of phenolic acids and other phenolics that remains unquantified were determined. From F4 was determined quercitrin 12 (5.95 g 0.94%) and rutin 13 (5.33 g 0.846%), together with several phenolic acids and other phenolics that remains undetermined (for details see Scheme 1a). It is will providing a complete metabolomic analyses of leaves, stems and

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roots, for to be published in a next paper. On the other hand, another portion of the total concentrate (1270 g) was dissolved with MeOH/HCl 1 M (6:4) and following a routine alkaloid procedure at basic solutions were obtained with CH2Cl2/EtOAc (6:4) two fractions: FA1 and FA2, at pH ¼ 10, and pH ¼ 12, respectively, after both fractions were collected in an alkaloid concentrate F5 (15.0 g, 0.98%), FA1 and FA2 were controlled with Drangendorff's reagent used as an alkaloids visualization reagent (see Scheme 1b). From F5 by CC, HPLC-DAD-MS/ MS, GC/MS and NMR were identified the following Aristotelia type alkaloids: aristoteline 1 (200 mg), aristotelinine (10 mg), aristotelone (15 mg), aristone (15 mg), makonine 6 (45 mg), aristotelinone 5 (85 mg), 8-oxo-9-dehidrohobartine (20 mg), 8-oxo-9dehidromakomakine 2 (135 mg), hobartine 3 (60 mg), aristoquinoline 4 (15 mg), serratoline (40 mg), makomakine (20 mg), sorreline (15 mg) and other alkaloids that remain unidentified (Cespedes, 1996; Cespedes et al., 1990, 1993; Silva et al., 1997; Watson et al., 1989). The hyphenated conditions for HPLC of alkaloids and phenolics determination from F3 and F5: For hyphenated analyses were used the following: 1H NMR and 13C NMR spectra were recorded at 400 MHz, and 125 MHz, respectively on Avance 400 and 600 MHz on NMR Bruker spectrometers, chemical shifts (ppm) are related to (CH3)4Si as internal reference (d 0), CDCl3, MeOD and acetone-d6 from Aldrich Chemical Co were used as solvents, coupling constants are quoted in Hz. GC/MS HP5989A, LC/MSD-TOF Agilent. Additionally, EIMS and TOF data were determined on a Q-TOF Waters and JEOL JMSAX505HA mass spectrometer at 70 eV. FABMS were obtained on a JEOL JMS-SX102A mass spectrometer operated with an acceleration voltage of 10 kV. Samples were desorbed from a nitrobenzyl alcohol matrix using 6 keV Xenon atoms. In addition to LC/MSD-TOF (6500 Series Agilent), and GC/MS with GC/MSD 5977A Agilent systems a HPLC system consisted of a Hewlett Packard Series 1100 HPLC instrument with DAD and UV detectors set at 320 nm. The column was obtained from Supelco Technologies C18 (150 mm  4.6 mm, 5 mm). For phenolics the eluent was a mixture of 4% tetrahydrofuran in acetonitrile and water (35:65, v/v) and contained 0.04% phosphoric acid; the flow rates were 0.85, 1.0 and 1.7 mL/min and the column temperature and pressure were 30  C and 149 bar, respectively, and the injection volume was 20 mL. Verbascoside, linarin and syringing served as standards. For alkaloids the elution consisted of a linear gradient program from 4% to 25% acetonitrile in 1% formic acid aqueous solution over 60 min. The flow rate was 1 mL/min and 10 mL of samples was injected. A 15 min re-equilibration time was used between HPLC runs. The DAD acquisition wavelength was set in the range of 200e700 nm. After passing through the flow cell of the DAD, the column eluate was split and 0.3 mL/min was directed to a triple-quadrupole tandem mass spectrometer with an electrospray interface (ESI), operating in full scan MS mode from m/z 50 to 1500. Mass spectra were acquired in both negative and positive modes with ion spray voltage at 3.5 kV, capillary temperature at 350 1C, capillary voltage at 35 V, sheath gas pressure at 35 Arb, auxiliary gas pressure at 11 Arb. The MS spectral data are outlined in Table 3. To verify and do correct stereospecific assignments of chemical structures, and for increase the amount of alkaloids available for biological tests, in another independent experiment, dried and milled plant material (stems and leaves) from A. chilensis (20 kg), collected during spring 2012, was extracted in acid water (30 L, pH 3, HCl 1 M) during three days at room temperature (RT) and filtered. The aqueous layer was extracted with ethyl acetate (EtOAc) (3  10 L) and the organic layer was evaporated under reduced pressure at 45  C to afford a gummy residue (extract Hþ: 52 g). The acid water was alkalinized (pH 11, NaHCO3-NaOH diluted) and subsequently extracted with EtOAc (3  15 L). The organic layer was

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Scheme 1. a. Method of obtaining extracts, partitions (fractions), and compounds. For isolation and purification please see Material and Methods. For alkaloids extraction, please see scheme 1b. b. Obtaining procedure for alkaloid. For isolation and purification please see Material and Methods.

evaporated under reduced pressure at 45  C to afford a gummy residue (extract OH
: 87 g). The crude OH
extract (total alkaloid fraction, 87 g) was chromatographed using a silica gel column (200e300 mesh) and increased solvent polarity (from hexane 100% to EtOAc 100%), giving fractions B1-B9. Fractions B1-B4 contained no alkaloids, only fatty acids and chlorophylls. The fraction B6 (5.2 g) was separated by silica gel chromatography using hexane:EtOAc (1:1 v/v), giving 8-oxo-9-dehidromakomakine 2, (100 mg, yellow crystals, 0.0012% yield). The fraction B7 (6.6 g) was applied to a Sephadex LH-20 column and eluted with EtOAc, giving aristoteline 1, (210 mg, colorless crystals, 0.0025% yield). The fraction B8 (2.1 g) was also applied to a Sephadex LH-20 column an eluted with EtOAc, but further separated by silica gel chromatography (200e300 mesh) using EtOAc, giving aristoquinoline 4, (130 mg, yellow oil, 0.0015% yield) and Hobartine 3, (10 mg, colorless crystals, 0.00012% yield). These alkaloids were isolated primarily by Cespedes as part of his doctoral thesis work (Cespedes, 1996) and later confirmed by Paz-Robles et al. (2013, 2014, 2016).

2.5. Cholinesterases inhibition An enzyme extract containing acetylcholinesterase (AChE) was obtained per the method of Grundy and Still (1985). About 100 adults of the insect S. frugiperda were frozen at
20  C for 7 days. The heads of frozen adults were detached, then milled and homogenized in 20 mL of 0.1 M phosphate buffer at pH 8.0. The crude homogenate was centrifuged at 15.000 g for 15 min at 5  C, and the supernatant was used for the enzyme activity. ATC (cholinesterase substrate) was dissolved in 0.1 M phosphate buffer (pH 8.0). DTNB (3-carboxy-4-nitrophenyldisulfide), Ellman's reagent and a sensitive sulfhydryl reagent 39.6 mg of this compound were dissolved in 10 mL of 0.1 M phosphate buffer at pH 7.0, and 15.0 mg of NaHCO3 was added (Miyazawa and Yamafuji, 2006). Inhibition of AChE was determined according to a modified spedes et al., 2001) Ellman's procedure (colorimetric method) (Ce using both the control (MeOH) and test solutions. The reaction mixture contained 0.2 mL of the enzyme solution and 0.1 mL of DTNB added to 2.4 mL of 0.1 M phosphate buffer (pH 8.0). Aliquots

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of the reaction mixtures were added to each of the test compounds dissolved in 50 mL of EtOH. The control solution was similarly prepared by the addition of 50 mL of EtOH. Both control and each of the test solutions were preincubated at 25  C for 10 min. After preincubation, the enzyme reaction was started by the addition of 40 mL of ATC followed by incubation at 25  C for 20 min. Once that an enzyme is added to the reaction mixtures and after 20 min, the absorbance was measured spectrophotometrically at 420 nm and compared with the control. Reading was repeated for 5 min at 30 s intervals to verify that the reaction occurs linearly. Blank reaction was measured by substituting saline for the enzyme. AChE activity was calculated with the absorption coefficient 1.56 mmoL/min. The same procedure was conducted with AChE and BChE purchased from Sigmae Aldrich Chem. Co. Identical procedures were performed with electric eel AChE (Type VI-S, EC 3.1.1.7, Sigma) and with horse serum BChE (EC 3.1.1.8, Sigma) as enzyme sources, while acetylthiocholine iodide and butyrylthiocholine chloride (Sigma, Mexico) were employed as substrates for the reaction, and DTNB, was used for the measurements of the cholinesterase activity. In brief, 140 mL of 0.1 mM sodium phosphate buffer (pH 8.0), 20 mL of DTNB, 20 mL of sample solutions and 20 mL of AChE/BChE solution were added by multichannel automatic pipette in a 96-well microplate and incubated for 15 min at 25  C. The reaction was then initiated with the addition of 10 mL of acetylthiocholine iodide/ butyrylthiocholine chloride. The hydrolysis of acetylthiocholine iodide/butyrylthiocholine chloride was monitored by the formation of the yellow 5-thio-2- nitrobenzoate anion because of the reaction of DTNB with thiocholines, catalyzed by enzymes at a wavelength of 412 nm using a BIOTECH/EPOCH reader. All experiments were repeated three times and the results were analyzed by SAS ANOVA and GLM procedures and graph by Microcal Origin version 8.1. Percentage of inhibition of AChE/BChE was determined by comparison of the rates of reaction of samples relative to control (Ethanol in phosphate buffer, pH 8) using the formula (EeS)/E x 100, where E is the activity of the enzyme without test sample and S is the activity of enzyme with test sample. Additionally, the TLC autobiographic preliminary evaluation was used according to procedures reported by Marston (2011). Galantamine (Sigma) an AChE alkaloid drug, quercetin, and huperzine were used as positive control and reference compounds, respectively. 2.6. Tyrosinase assay In order to obtain a high specific activity of mushroom tyrosinase (EC 1.14.18.1) used for this bioassay, the tyrosinase (Sigma Chemical Co. Santiago de Chile, Chile) was repurified according to a previously reported procedure (Espin and Wichers, 1999). Although mushroom tyrosinase differs somewhat from that from other sources (Van Gelder et al., 1997), this fungal source was used for all experiments because of its ready availability. The preliminary assay was tested at 167 mg/mL, unless otherwise specified. All samples were first dissolved in DMSO and diluted 30 times for use in the experiments. Tyrosinase catalyzes a reaction between two substrates, a phenolic compound and oxygen, but the assay was carried out in air-saturated aqueous solutions. The enzyme activity was monitored by dopachrome formation at 475 nm up to the appropriate time (not exceeding 10 min, unless otherwise specified.). The extent of inhibition by the addition of samples is expressed as the percentage necessary for 50% inhibition (IC50). This assay was performed as previously described (Kubo et al., 2003a, 2003b). First, 1 mL of a 2.5 mM L-DOPA solution was mixed with 1.8 mL of 0.1 M phosphate buffer (pH 6.8) and incubated at 25  C for 10 min. Then, 0.1 mL of the sample solution and 0.1 mL of the aqueous solution of the mushroom tyrosinase (138 units) were added in this order to the mixture. This solution was

5

immediately monitored for the formation of dopachrome by measuring the linear increase in optical density at 475 nm. The preincubation mixture consisted of 1.8 mL of 0.1 M phosphate buffer (pH 6.8), 0.6 mL of water, 0.1 mL of the sample solution (equivalent amount of IC50), and 0.1 mL of the aqueous solution of mushroom tyrosinase (138 units). The mixture was preincubated at 25  C for 5 min. Then, 0.4 mL of 6.3 mM L-DOPA solution was added, and the reaction monitored at 475 nm for 2 min. The percentage of inhibition of tyrosinase reaction was calculated as follows:

% inhibition ¼ f½ðA
BÞ
ðC
DÞ=ðA
BÞg  100 where A is optical density at 475 nm without test sample, B is optical density at 475 nm without test sample and enzyme, C is optical density at 475 nm with test sample, and D is optical density at 475 nm with test sample, but without enzyme. Gallic acid (10), kojic acid (11), quercetin (12), and tannic acid (13) were used as positive controls. All measurements were carried out in triplicate. The bathochromic shift of phenolic samples was monitored by adding 0.05 mM CuSO4. 2.7. Statistical analyses Data shown in figures and tables are average results obtained by means of three or 5 replicates and are presented as average ± standard errors of the mean. Data were subjected to analysis of variance (ANOVA) with significant differences between means identified by General Linear Procedures (GLM). Results are given in the text as probability values, with p < 0.05 adopted as the criterion of significance. Differences between treatment were established with a Student
Newman
Keuls test. The IC50 values for each activity were calculated by probit analysis based on percentage of inhibition obtained at each concentration of the samples. IC50 is the concentration producing 50% inhibitory activity. Complete statistical analysis was performed by means of the MicroCal Origin 8.1 statistical and graphs PC program. 3. Results and discussion In our screening program looking for biological activities of native plants from Latin-American regions was found that A. chilensis showed interesting ethno-medicinal activities, being used as anti-inflammatory agent for kidney pain, stomach ulcers, diverse digestive ailments (tumors and ulcers), diverse neurodegenerative ailments, fever and healing injuries (Bhakuni et al., 1976; Cespedes et al., 2010b, 2010c). Based on this information it has been carried out a study of enzyme inhibition by extracts, fractions and compounds obtained from aerial parts (leaves and stems) of A. chilensis. 3.1. Alkaloids, flavonoids, and phenolics Acacetin, apigenin, diosmetin, luteolin, myricetin, quercetin, quercitrin, rutin, scopoletin, anthraquinone, cinnamic acid, p-coumaric acid, gentisic acid, ferulic acid, sinapic acid, gallic acid, 4hydroxy-benzoic acid, among many other compounds that remain unidentified and have been ascertained according to their retention times and spectral data (Fig. 2). The presence of quercetin, myricetin and other phenolics (such as caffeoylquinic acids and ellagic acid for instance) together with hydroxycinnamic acids have been reported previously in fruits (Cespedes et al., 2010a; GironesVilaplana et al., 2012, 2014; Brauch et al., 2016; Ruiz et al., 2016). The chemical structures were determined by 1H - 13C NMR and MS data (Table 3).

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C.L. Cespedes et al. / Food and Chemical Toxicology xxx (2017) 1e12

The polar part of the methanol extractable fraction of the ethyl acetate extract F3 consists almost exclusively of quercetin, myricetin, luteolin, and there are in minor amounts other flavonoids (apigenin, rhamnetin, acacetin, diosmetin, quercitrin and rutin), together with phenolic acids and other unidentified compounds. It is important to indicate that in F1 (n-hexane) and F2 (CH2Cl2) there are a complex mixture of many fatty acids/chlorophylls, triterpenes/sterols (F1) and diterpenoids (F2), respectively, whose chemical structures remain unidentified until now. From a conventional procedure for alkaloids isolation was obtained fraction F5 (see Scheme 1b), from which by phytochemical analyses through column chromatography several Aristotelia alkaloids were obtained. By HPLC-MS-NMR hyphenated procedures the alkaloids aristoteline, aristotelinine, aristotelone, aristone, makonine, aristotelinone, 8-oxo-9-dehydrohobartine, 8-oxo-9dehydromakomakine, hobartine, serratoline, makomakine, and sorreline were identified (Fig. 1), (Cespedes, 1996). On the other hand, from an independent experiment the following alkaloids were purified: Aristoteline 1, (210 mg, colorless crystals, 0.0025% yield). This compound is formed by five fused rings, with a planar region composed by an indole moiety, linked to three aliphatic rings one of them is a N- heterocyclic with lipophilic properties (Fig. 1). The

O

H H N

N H

H N H

H

N H

H

1

2 H

H H N

3.2. Cholinesterases inhibition

HN

H

H

H

H

N H N

3 O

4 H H N

O

N

H N H

H

H

N H

5

H

6 OH

Me

H O

H N

MeO

Me N

7

chromatographic and spectroscopic data results are in excellent agreement with previously published data (Watson et al., 1982, 1989; Silva et al., 1997; Cespedes et al., 1990, 1993). 8-oxo-9-dehydromakomakine 2, (100 mg, yellow crystals, 0.0012% yield). The molecule consists of an indolyl ketone fragment and a nested three-ring system, with both groups linked by a CdC bridge, which was crystallized as two polymorphs with a remarkable color difference, one of them was a deep red crystal and the other was a yellow crystal, the main difference between them was to stablish by a torsion angle in the system indolyl ketone and the planar portion of the heterocyclic six-membered ring, with differences in the dihedral angle indole-ketone system, [130.7 in the yellowish and 161.6 in the deep-red](Cespedes, 1996; Paz-Robles et al., 2013). Hobartine 3, (10 mg, colorless crystals, 0.00012% yield), C20H26N2, is a tetracycle indole alkaloid related to the compound 3 which has an endo double bond in despite to the exo reported in 3, also the crystal structure is colorless probably because the indoyl portion is not conjugated as in 3 which has a ketone at C-8. (Cespedes, 1996; Paz-Robles et al., 2014). Aristoquinoline 4, (130 mg, yellow oil, 0.0015% yield). The structure of 4 is formed by a planar region composed by a quinoline moiety linked to the aliphatic system by a single bond which confer rotation around C4 and C9, this is the main difference between tested compounds which are formed by an indoyl moiety (Cespedes et al., 1993; Cespedes, 1996; Paz-Robles et al., 2014). To obtain more details about sites and mechanisms of action of the enzyme inhibition activity of F3, F4 and alkaloid extract F5, and the isolated compounds were assayed as cholinesterases and tyrosinase inhibitors, verbascoside, quercetin, and galantamine were used as well as pattern compounds and positive control of inhibitory activity of AChE, BChE and tyrosinase bioassays were carried out.

H2N

O

Me

8

Fig. 1. Molecular structures of alkaloids from A. chilensis, and positive control assayed against AChE, BChE and Tyrosinase. Aristoteline 1, 8-oxo-9-dehydromakomakine 2, hobartine 3, aristoquinoline 4, aristotelinone 5, makonine 6, galanthamine 7, huperzine 8.

Inhibition of AChE was carried out per the colorimetric method of Ellman et al. (1961), to investigate the mode of action of enzyme inhibition. Inhibition of AChE by F3, F4, F5, aristoteline 1, 8-oxo-9dehidromakomakine 2 (8-oxo-DHMMK), hobartine 3, aristoquinoline 4, aristotelinone 5, makonine 6, and compounds 7e17, are outlined in Table 1. F3, F5, 1, 2, 3, 4, galanthamine 7, huperzine 8, quercetin 9, myricetin 10, apigenin 14 and luteolin 15 showed the greatest inhibitory effects with IC50 between 0.32 and 38.79 mg/mL against AChE and between 0.23 and 59.6 mg/mL against BChE, respectively (Table 1). F5, 1, 2, 3, 4, galantamine, huperzine, apigenin, and luteolin showed stronger activity levels against AChE with IC50 of 38.79, 31.21, 11.02, 19.67, 20.67, 1.32, 0.32, 19.7, and 15.9 mg/mL, respectively, and against to BChE the most active were F5, 1, 2, 4, 7, 8, 11, and 17 with IC50 of 20.43, 24.9, 14.7, 16.4, 7.3, 0.23, 7.8, and 12.9 mg/ mL, respectively (Table 1). Thus, against cholinesterases F5 was more active than F3, a fraction rich in flavonoids and phenolics acids such as quercetin and myricetin (Table 1). The inhibitory activity of AChE showed by fractions F-3, F-5, alkaloids 1, 2, 3, 4, flavonoids 9, 10, 14, and 15 occurred in a dose-dependent manner. Thus, it is possible to infer that alkaloid compounds together a mix of flavonoids and phenolic acids in these samples could be the active inhibitors of acetylcholinesterase in leaves of Aristotelia chilensis. Additionally, the presence or absence of a carboxyl group in the assayed alkaloids increases or decreases respectively the strength of these compounds on inhibition of AChE and BChE, as in the case of 8-oxo-9-dehidromakomakine (8-oxo-DHMMK) compared with aristoteline and hobartine, it is interesting the behavior of seco-

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C.L. Cespedes et al. / Food and Chemical Toxicology xxx (2017) 1e12

7

OH

OH OH

OH

O

HO

O

HO

OH OH

OH OH

O

O

OH

9

10

OH

OH

OH

OH MeO

O

HO

O

O-rhamnosyl

OH O

OH

O

OH

11

12

OH OH O

HO

O-rhamnoglucosyl OH

O

13 OH OH O

HO

OH

OH HO

O

O

O

OH

14

15

OH OMe O

HO

OH

O

16

OMe O

HO

OH

O

17

Fig. 2. Chemical structures of flavonoids: quercetin 9, myricetin 10, rhamnetin 11, quercitrin 12, rutin 13, apigenin 14, luteolin 15, acacetin 16, diosmetin 17.

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Table 1 AChE and BChE inhibitory activity of compounds, extracts and reference compounds IC50 [mg/mL].a AChE

BChE

Inhibition type

F-1 F-2 F-3 F-4 Chlorogenic acidb F-5 Aristoteline (1) 8-oxo-DHMMK (2) Hobartine (3) Aristoquinoline (4) Aristotelinone (5) Makonine (6) Galantamine (7)b Huperzine (8)b Quercetin (9) Myricetin (10) Rhamnetin (11) Quercitrin (12) Rutin (13) Apigenin (14) Luteolin (15) Acacetin (16) Diosmetin (17) p-coumaric acid 4-OH-benzoic acid Ajmalicineb Verbascosideb

n.d. 189.9 90.7 220.6. 195.6 38.79 31.21 11.02 19.67 20.67 166.1 144.2 1.32 0.32 47.8 37.2 89.9 66.9 169.8 19.7 15.9 112.3 45.6 n.d. n.d. 22.5 190.1

n.d. 202.4 59.6 n.d. n.d. 20.43 24.9 14.7 35.9 16.4 55.1 88.8 7.3 0.23 38.8 70.7 7.8 78.8 95.1 24.5 49.8 177.8 12.9 n.d. n.d. 45.9 105.9

e Mixed Competitive Mixed Mix/competitive Mix/competitive Competitive Competitive Competitive Competitive Competitive Competitive Mixed/non-competitive Competitive Competitive Mixed Mixed Mix/competitive Mixed Mixed Mixed Mixed Mix/noncompetitive e e Mix/competitive Competitive

a Values correspond to average of three experiments; units of concentrations are expressed as [mg/mL]. n.d. - Not determined (due to concentrations were higher than 200 mg/mL). b Compounds used as positive controls.

indole alkaloids v/s non-seco, as in hobartine/aristoteline and 8oxo-DHMMK 2, particularly this last alkaloid has a carboxyl group ab conjugated to a double bond vicinal to nitrogen, this alkaloid showed to be more active. On the other hand, it was suggested that cholinesterases inhibitory activity of F3, and F5 are not caused by any strong inhibitor, but rather by a synergistic effect (Table 1). At current knowledge, the inhibition of AChE activity by phenolics and alkaloids has been reported on many related references (Gulcan et al., 2015; Orhan et al., 2013; Orhan, 2013, 2015, 2016; Pang et al., 2012; Pinho et al., 2013). Therefore, alkaloids and plant phenolics may be considered AChE antagonists (Pinho et al., 2013). Similarly, naphthoquinones (Changwong et al., 2012) and phenylethanoids (Georgiev et al., 2011) have been shown to act as AChE inhibitors and these types of compounds are occurring in leaves of A. chilensis. Like AChE inhibitory activity (Table 1), quercetin and myricetin showed butyrylcholinesterase inhibitory activity; in contrast, phenolic acids appeared to have low active (Table 1). It is obvious that the nature of the carbonyl substituent at C-8 vicinal to indole moiety and to the nitrogen trough a double bond, respectively, play an important role for the cholinesterase activities of the assayed alkaloids, and fraction F5. In anticholinesterase assays the most active compounds (galantamine and huperzine) contained a small and relatively hydrophilic hydroxyl group (Galantamine) and additionally a carbonyl vicinal to Nitrogen (huperzine) as in compound 2 (a 3-carboxy-indole), whereas compounds 1, 3 and 4 with some bulky and more lipophilic double bonds exhibited moderated activity level (Table 1, Fig. 3). Interestingly, compounds 5 and 6 showed better activity against BChE than AChE (Table 1, Fig. 3) showing the possibility that the bonding between these compounds and the active site of enzyme have best performance in BChE than AChE. These results confirm previous findings on the quantitative structureeactivity relationship of carbonyl

80

% inhibition

samples Tested

100

60

40

20

0 0

50

100

150

200

Concentration [mg/ml] Fig. 3. Inhibition [%] of AChE by Aristotelia alkaloids, aristoteline 1 (-), 8-oxo-9dehydromakomakine 2 (C), hobartine 3 (:), and aristoquinoline 4 (;).

derivatives, namely that the inhibitory activity of the respective natural products depends on the position of the carboxyl and on the position of methyl substituents as in huperzine and galantamine, respectively (Kitisripanya et al., 2011; Radwan et al., 2007; Uriarte-Pueyo and Calvo, 2011; Pang et al., 2012; Pinho et al., 2013; Georgiev et al., 2011; Changwong et al., 2012; Park, 2010; Orhan et al., 2015). With respect to the flavonoids our findings indicate that the presence of a methoxy substituent in the aromatic rings seems to be the cause of increasing or decreasing inhibitory activities of BChE as shown by acacetin, diosmetin and rhamnetin with IC50 at 17.8, 12.9, mg/ml, respectively, of inhibition (Table 1). It is important to mention that the innocuous effect of several flavonoids was reported showing that it was not genotoxic for Drosophila wing spot test crosses (Santos-Cruz et al., 2012). Those effects are in accordance with other results in D. melanogaster assays (Heres-Pulido et al., 2005) and show that is possible to use flavonoids on live organisms without secondary effects. In relation to phenolic acids our results are moderate, but are somewhat consistent with those reported by Changwong et al., 2012. The high activity against AChE and BChE observed with F3 and F5 also could be attributed to synergistic effects by the components. Finally, fraction F3 showed a good activity level in this assay, we have determined the presence of flavonoids and phenolic acids (unpublished results). The inhibitory effects of flavonoids on cholinesterases have been reported by many authors (Cao et al., 2013a, 2013b; Orhan et al., 2007; Shen et al., 2009; Zhao et al., 2014).

3.3. Tyrosinase inhibition Bioassays with extracts and fractions rich in phenolics showed a concentration-dependent inhibitory effect on the oxidation of LDOPA by mushroom tyrosinase. The IC50 values for F2, F3, and F4 were determined as 191.2, 8.4 and 39.8 mg/mL (F1 and F5 do not show any activity), which show that F3 has better level activity than those of tannic acid (Table 2), a well-documented tyrosinase inhibitor (Kubo et al., 2003a,b). It seems possible that flavonoids such as quercetin, myricetin, luteolin, apigenin, acacetin, diosmetin, rhamnetin and several phenolic acids could be responsible for the original tyrosinase inhibitory activity of these samples (F3,

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C.L. Cespedes et al. / Food and Chemical Toxicology xxx (2017) 1e12 Table 2 Antioxidant (AOX) and Tyrosinaseb inhibitory activity, IC50 of compounds, extracts and reference controls. Samples

IC50 [mg/ml]

Inhibition type

IC50 AOXa [mg/ml]

F2 F3 F4 F-5c Alkaloids 1e8 Gallic acidd Chlorogenic acidd Benzoic acidd Tannic acidd Kojic acidd Quercetin (9) Myricetin (10) Rhamnetin (11) Quercitrin (12) Rutin (13) Apigenin (14) Luteolin (15) Acacetin (16) Diosmetin (17)

191.2 8.4 39.8 n.d. n.d. 4.5 7.9 n.d. 22.8 14.2 70.1 98.4 133.3 n.d. n.d. n.d. 25.6 189.8 177.1

Competitive Competitive Competitive n.d. n.d. N.d. mixed n.d. Competitive Competitive Competitive Competitive Mixed e e e Competitive Mix/competitive Mix/competitive

98.2 3.5 45.3 n.d. n.d. 12.2 15.8 40.2 1.06 2.22 25.0 33.33 99.8 >200 >200 111.0 27.8 73.1 66.2

a

IC50 for inhibition of DPPH radical formation. With respect to L-DOPA. c Value correspond to complete F-5 including the alkaloids, units of concentrations are expressed as [mg/mL]. n.d. - Not determined as the values are higher than 200 mg/mL. d Compounds used as positive controls. b

IC50 ¼ 8.4 mg/mL) and F4 (IC50 ¼ 39.8 mg/mL) observed in the screening. Noticeably, in the extract F3, quercetin, myricetin and apigenin are the main components, so also luteolin, diosmetin, acacetin, rhamnetin, and phenolic acids were also found. Then, we can infer that the combined compounds contribute to the inhibitory activity of the ethyl acetate extract. However, in the F4 sample the activity could be attributable to the presence of catechol moieties in the anthocyanidins and phenolic acid molecules that was determined in this sample. Interestingly, quercetin, myricetin, other flavonoids and phenolic acids can be oxidized as substrates by the tyrosinase

enzyme due to the reductory activity of Cuþ2 to Cuþ1 (Duckworth and Coleman, 1970). Oxidation of quercetin was characterized by a new peak with a maximum at 377 nm (shift of Cu2þ, from 329.5 to 379 nm) in accord with data reported previously (Karioti et al., 2007) and a new peak at 420 nm for myricetin (shift of Cu2þ, from 390 to 420 nm). The shift of quercetin was not observed in the case of acacetin, diosmetin and rhamnetin (methoxylated flavonoids); thus, the inhibitory activity of apigenin, luteolin, myricetin and quercetin could be attributed to the presence of orthohydroxyls on the phenolic ring A vicinal to carbonyl group of this type of flavonoids, which give them the chelating property of metals. These flavonoids which have a hydroxyl group vicinal to carbonyl, and where the carbonyl group has vicinal hydroxyls (one at the A ring and other at the B ring) and two catechol hydroxyls in the C ring, increasing the inhibitory potency compared with assayed methoxylated flavonoids. Noteworthy it should be noted that this oxidation was at a much slower rate but did not have the lag period. Oxidation of L-DOPA is not subject to the lag period because the recruitment step takes place as soon as the substrate is presented with rapid conversion of met-enzyme to the oxygen-binding deoxy form (Naish-Byfield and Riley, 1998). In contrast, there was a progressive increase in the observed rate of this oxidation as soon as catalytic amounts (0.01 mM) of L-DOPA became available as a cofactor. The oxidation products of compounds in F3 and F4 are a complex mixture of polar compounds, and the unstable nature of the intermediates make their characterization difficult. On the other hand, now we are doing a complete metabolomic analysis of these extracts and we are doing additional experiments ongoing to elucidate these facts. Enzymes are known to catalyze the oxidation of flavonoids especially ortho-diphenols to corresponding semiquinones and quinones that are highly reactive species (Handerson et al., 1988; Singh et al., 2001; Eghbaliferiz and Iranshahi, 2016). These chemical species can react with other phenols, amino acids and proteins, the hydroxyl-quinone moieties of the flavonoids may condense with one another through a Michael-type addition, yielding a relatively stable quinol-quinone intermediate (Singh et al., 2001; Sayre and Nadkarni, 1994). Nevertheless, these phenolic components that can form adducts with other nucleophilic groups in the

Table 3 MS data of identified compounds from leaves of A. chilensis. Alkaloids

M.W.

MS-MS ions m/z [fragment ions]þ (Relative abundance, %)

Aristoquinoline Aristoteline Aristotelinone Hobartine Makonine 8-oxo-dehydrohobartine 8-oxo-dehydromakomakine Aristone Serratoline Sorreline Makomakine

306 294 308 294 306 306 306 308 310 292 327

306 294 308 294 306 306 306 308 310 292 327

(10); 292 [M-15]þ (12); 197 (38); 157 (39); 93 (44); 74 (64); 59 (100) (60); 279 [M-15]þ (100); 251 [M-43]þ (5); 237 [M-57]þ (55); 211 [M-83]þ (60); 58 (15) (24); 309 (36); 293 [M-15]þ (78); 294 (100); 251 [M-56]þ (38); 112 [C7H14N]þ, (88); 57 (48) (45); 279 [M-15]þ (8); 210 [M-84]þ (8); 174 [M-120]þ (60); 164 (100); 57 (55) (90); 291 [M-15]þ (20); 251 [M-55]þ (100); 236 [M-70]þ (40); 167; 125; 118; 57 (78) (30); 291 [M-15]þ (8); 144 [acylindol]þ (60); 71 (60); 57 (100) (35); 291 [M-15]þ (12); 144 [acylindol]þ (100); 136 (25); 93 (50) (100); 293 [M-15]þ (30); 237 [M-71]þ (35); 208 [M-100]þ (20); 194 (90); 182 (90), 130, 70, 55 (10); 295 [M-15]þ (100); 277 [M-33]þ (35); 207 [M-103]þ (45); 84 (70) (40); 277 [M-15]þ (25); 235 [M-57]þ (30); 199 [M-93]þ (55); 162 [M-130] (100); 93 (80) (2); 279 (2); 253 (2); 209 (10); 191 (5); 177 (2); 164 (100); 130 (30); 96 (15); 73 (10)

MS-MS ions m/z [fragment ions]- (Relative abundance, %)

Phenolics Quercetin Myricetin Rhamnetin Quercitrin Rutin Apigenin Luteolin Acacetin Diosmetin p-coumaric acid 4-OH-benzoic acid

[M]þ [M]þ [M]þ [M]þ [M]þ [M]þ [M]þ [M]þ [M]þ [M]þ [M]þ

302 318 316 448 610 270 286 284 300 164 138

301 317 315 447 609 269 285 283 299 164 138

9

[M-H]- (60); 151 (100) [M-H]- (60); 302 [M-H-15]- (100), 151 (80) [M-H]- (60); 300 (100); 151 (10) [M-H]- (25); 301 [A-H]- (100) [M-H]- (100); 301 [A-H]- (40) [M-H]- (60); 151 (100) [M-H]- (100); 217 (10); 199 (20); 175 (20); 151 (85); 133 (50); 107 (10) [M-H]- (2); 268 [M-15]- (100); 240 [M-H-CO-15]- (5); 211 [M-H]- (12); 284 [M-H-15]- (100); 256 [M-H-CO-15][M]þ (100); 147 [M-17]þ (60); 119 [M-45]þ (25); 107 (15); 91 (20); 65 (20); 39 (18) [M]þ (60); 121 [M-17]þ (100); 93 [M-45]þ (30); 65 (22); 39 (18)

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enzyme and inactivate cannot be entirely ruled out. This analysis and result indicate that the enzyme was not inactivated by a Kcattype inhibition (inactivation of the enzyme by products of the reaction) (Golan-Goldhirsh and Whitaker, 1985) as is current in experiments with this type of compounds. Additionally, tannic acid and quercetin exhibit an excellent tyrosinase inhibitory activity level, with an IC50 of 22.8 mM and 70.1 mg/mL, respectively; and kojic acid has an IC50 of 14.2 mg/mL (Table 2). The assayed compounds in this work are in the middle of this range. The inhibition kinetics of flavonoids quercetin 9, myricetin 10, and luteolin 15 for the oxidation of L-DOPA by mushroom tyrosinase were analyzed and found to be those characteristics for competitive inhibitors. Because phenolics are known to react with proteins by crosslinks, they react of course in a process known as tanning (Ryan, 1990). Thus, the flavonoids 9, 10 and 15 should irreversibly inactivate the tyrosinase (protein); however, in this case does not occur, some of which competitively inhibits tyrosinase. It appears that tannic acid exhibits much more potent tyrosinase inhibitory activity compared to that of flavonoids 9, 10 and 15 (Passi and Nazzaro-Porro, 1981). 3.4. Antioxidant activity The antioxidant activity shown by the extracts and compounds from aerial parts of A. chilensis seems to correlate with the tyrosinase inhibitory activity, but it should be borne in mind that plant secondary metabolites can act according to many mechanisms in biological systems. Nevertheless, it important to emphasize that phenolic acids such as gallic acid, chlorogenic acid, tannic acid, Kojic acid, p-coumaric acid, gentisic acid, ferulic acid, sinapic acid and other phenolic components of this fraction inhibited tyrosinase (Table 2, and data not show), but did not inhibit both AChE and BChE (Orhan et al., 2016). Taking account, the antioxidant and prooxidant action of phenolics (Eghbaliferiz and Iranshahi, 2016;  et al., 2011) and with the results obtained we hyProch azkova pothesized that the oxidized products also are responsible for the tyrosinase inhibitory activity showed by phenolic acids (Miyazawa et al., 2003). Radical scavenging activity, which can be measured as decolorizing activity following trapping of the unpaired electron of DPPH, was examined. In fact, F3 (IC50 ¼ 3.5 mg/mL) and F4 (IC50 ¼ 45.3 mg/mL) exhibited radical scavenging activity, as shown in Table 2. The possibility that their adverse effects are a consequence of their potential to act as a prooxidant should also be considered (Carocho and Ferreira, 2013). In fact, gallic acid, under certain conditions, can produce superoxide anion (Serrano et al., 1998) and by esterases can be decarboxylated to pyrogallol (Jana et al., 2014). On the other hand, different studies report that extracts rich in flavonoids can act as antioxidants and as functional perturbation of cell membranes (Arora et al., 1998, 2000; Nakagawa et al., 2000; Suwalsky et al., 2008). The total phenolic and flavonoid content of the F3 and F4 samples was as high as 15,654.6 (mmol of catechin equiv/g of sample) that is indicative of great amount of phenolics in these samples from leaves of A. chilensis, explaining their high potency as antioxidants. 4. Concluding remarks Our findings show that alkaloid extracts and its components possess inhibition of acetylcholinesterase or butyrylcholinesterase, and this target has also been demonstrated for similar compounds (Alout et al., 2012; Cao et al., 2013a, 2013b; Changwong et al., 2012; Georgiev et al., 2011; Orhan et al., 2007, 2013; Orhan, 2013; 2015,

Orhan and Senol, 2016; Orhan et al., 2016). Our findings show the strengthen action of the extracts and fractions of leaves from A. chilensis as inhibitors of cholinesterases and tyrosinase. We have ongoing studies for determination of sites, mechanisms and mode of action of the more active samples from A. chilensis aerial parts and these could be due to specific inhibition or a combination of different mechanisms of the samples, as well as, with assayed enzymes and other PPO binding with the samples, since these targets were also found for other natural products spedes et al., 2001; Casida, 2009; Casida and Durkin, 2013; (Ce ~ oz et al., 2013; Orhan et al., 2013, Orhan, Green et al., 2013; Mun 2013, 2015; Orhan and Senol, 2016; Orhan et al., 2016). In summary, the enzyme inhibitory activity of F3, F4, and F5 from aerial parts of A. chilensis may be due to synergistic effects shown by the components of the mixtures in the test system used in this investigation. F2 showed greater inhibition against BChE than against AChE. In contrast, F3 showed greater inhibition power against AChE than against BChE. This can be due to the presence of diterpenes in F2, since the greatest effect of this type of compounds against BChE is known (Orhan, 2013; Changwong et al., 2012), as well as the inhibitory power of flavonoids against AChE (Orhan et al., 2007; Shen et al., 2009). In the same sense the aristoquinoline alkaloid showed a greater inhibitory effect against BChE than on AChE, correlating very well with the reports of effects of quinolines and isoquinolines against BChE (Orhan and Senol, 2016). Finally, here we demonstrate that leaves of A. chilensis is an excellent resource as cholinesterase and tyrosinase inhibitors. Additionally, the added value of the findings is to expand the range of biological activities of this natural resource towards the inhibition of essential enzymes in neurodegenerative diseases and in the generation of melanogenesis, such as cholinesterases and tyrosinase, respectively. Competing financial interest The authors declare no competing financial interest. Conflict of interest The authors declare that there are no conflicts of interest. Acknowledgments This work was supported in part by internal grant from Direccion de Investigacion, Universidad del Bio Bio, Chillan, Chile. This paper is based on work supported by partial grants from: DGAPAPAAPIT-UNAM: IN-243802-1, IN-211105-3, and in part by grants from UC-MEXUS-CONACYT (#2008-02). The authors thanks to Prof David S. Seigler, curator Herbarium of University of Illinois at Urbana-Champaign, USA, for botanical identification of the plant. We thank Ma. Teresa Ramirez-Apan and Antonio Nieto for technical assistance: Chemistry Institute, Ana Ma. Garcia-Bores: UBIPRO FESIztacala, UNAM, Mexico D.F., Mexico. Anne Murray (ESPM, UC, Berkeley); M.D. thanks to TIES-ENLACES USAID Program for a research fellowship. CLC and IK acknowledge to Seed Funds Program of Conicyt-Chile and UC-Berkeley “2013 UC Berkeley-Chile Seed Grants”, grant (# 2013-02): A New Connection: Potential Cancer Treatment Agents. Transparency document Transparency document related to this article can be found online at http://dx.doi.org/10.1016/j.fct.2017.05.009.

Please cite this article in press as: Cespedes, C.L., et al., Inhibition on cholinesterase and tyrosinase by alkaloids and phenolics from Aristotelia chilensis leaves, Food and Chemical Toxicology (2017), http://dx.doi.org/10.1016/j.fct.2017.05.009

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Please cite this article in press as: Cespedes, C.L., et al., Inhibition on cholinesterase and tyrosinase by alkaloids and phenolics from Aristotelia chilensis leaves, Food and Chemical Toxicology (2017), http://dx.doi.org/10.1016/j.fct.2017.05.009