Lichens of parmelioid clade as promising multi-target neuroprotective

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Lichens of parmelioid clade as promising multi-target neuroprotective agents Victor Sieteiglesias, Elena González-Burgos, Paloma BermejoBescós, Pradeep Kumar Divakar, and Maria Pilar Gómez-Serranillos Chem. Res. Toxicol., Just Accepted Manuscript • DOI: 10.1021/acs.chemrestox.9b00010 • Publication Date (Web): 24 May 2019 Downloaded from http://pubs.acs.org on May 25, 2019

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Chemical Research in Toxicology

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Lichens of parmelioid clade as promising multi-

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target neuroprotective agents

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Víctor Sieteiglesias, Elena González-Burgos, Paloma Bermejo-Bescós, Pradeep K. Divakar,

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María Pilar Gómez-Serranillos*

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Department of Pharmacology, Pharmacognosy and Botanical, Faculty of Pharmacy, Universidad

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Complutense de Madrid, Plaza Ramon y Cajal s/n, Ciudad Universitaria, 28040, Madrid, Spain

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Keywords: parmelioid lichens; antioxidants; neuroprotection; secondary metabolites.

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“For Table of Contents Only”

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ABSTRACT

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Neurodegenerative diseases such as Alzheimer’s disease and Parkinson’s disease are

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multifactorial disorders which are increasing in incidence and prevalence over world without

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existing effective therapies. The search of new multi-target compounds are the latter

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therapeutically strategy to address these pathological conditions. Lichens have an important and

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unknown therapeutic value attributed to their unique secondary metabolites. The aim of this

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study is to evaluate for first time the in vitro neuroprotective activities and molecular

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mechanisms underlying of methanol extracts of lichens of parmelioid clade and to characterize

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major bioactive secondary metabolites responsible for their pharmacological actions.

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Of the fifteen parmelioid lichen species, our results showed that Parmotrema perlatum and

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Hypotrachyna formosana methanol extracts exhibited the high antioxidant activity as evidenced

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in ORAC, DPPH and FRAP assays. Then, SH-SY5Y cells were pretreated with methanol

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extracts (24 h) followed by Fenton reagent exposure (2 h). Pretreatments with these two more

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antioxidant methanol lichen extracts increased cell viability, reduced intracellular ROS,

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prevented oxidative stress biomarkers accumulation and upregulated antioxidant enzymes (CAT,

ACS Paragon Plus Environment

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SOD, GR and GPx) activity compared to Fenton reagent cells. The neuroprotective activity was

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much higher for H. formosana than for P. perlatum, even equal to or higher than Trolox

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(reference compound). Moreover, H. formosana extracts inhibited both AChE and BuChE

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activities in a concentration dependent manner and P. perlatum only showed concentration

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dependent manner activity against AChE. Finally, chemical composition analysis using TLC and

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HPLC methods revealed that physodic acid, lividic acid and lichexanthone are major secondary

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metabolites in H. formosana are and stictic acid and constictic acid are in P. perlatum.

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These results demonstrated that P. perlatum and specially, H. formosana, are promising multi-

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targeted neuroprotective agents due to their antioxidant and AChE and BuChE inhibition

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activities.

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INTRODUCTION

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Reactive oxygen species (ROS) participate in various biological processes regulating metabolic

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and cellular survival pathways. In physiological situations, these ROS are in equilibrium with the

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endogenous antioxidants system through free radical scavenger, metal chelator and enzymatic

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modulation mechanisms. However, under certain circumstances, ROS may exceed the capacity

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of antioxidants resulting from an oxidative damage to cell structures (membrane lipid

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peroxidation, DNA demethylation and protein oxidation) and even cell death via apoptosis and

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necrosis.1 This situation known as oxidative stress is involved in the pathogenesis and

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progression of many different chronic diseases including age-related neurodegenerative disorders

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such as Alzheimer’s disease (AD), Parkinson’s disease (PD) and amyotrophic lateral sclerosis

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(ALS).2,3 The prevalence and incidence of these central nervous system (CNS) disorders rise

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with increasing age, expecting an exponential growth in the number of cases as average life

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expectancy increases.4 Therefore, oxidative stress prevention and treatment using exogenous

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antioxidants has become an emerging and promising therapeutic strategy to cope with ROS

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overproduction and consequently, to improve antioxidant status. Consistent studies have

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identified numerous compounds of natural origin with antioxidant activity, highlighting in

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particular molecules with phenolic structure.5

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Moreover, targeting AChE and BuChE enzymes is considered a promising strategy for

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the prevention and treatment of different CNS disorders including senil dementia, AD,

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myasthenia gravis and ataxia.6


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Natural products are considered as a source of potential compounds with therapeutic

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activity. The knowledge of traditional medicine, its pharmacological validation and the discovery

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of new activities has led to the development of many “lead molecules” for prevention and

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treatment of different diseases. It is estimated that approximately 25% of the therapeutic arsenal

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available in market comes from molecules of natural origin, especially from medicinal plants.

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However there are natural products whose pharmacological activity has not been investigated,

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underlining especially these natural products from lichens.7

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Lichens are attractive organisms formed from the symbiotic association of a fungi

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(mycobiont) and an algae and/or cyanobacteria (photobiont). Parmelioid clade, within

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Parmeliaceae family, is the largest group of lichens forming fungi with more than 1500 species

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widely distributed from dry climates to tropical conditions.8 Lichens synthesize a great diversity

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of secondary metabolites (more than 1000 identified compounds), most of them present

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exclusively in these organisms and with phenol structure such as depsidones, depsides, depsones,

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dibenzofurans and xanthones. In recent years, there have been a growing interest in lichens due

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to their potential pharmacological action such as antioxidant, antitumor, antimicrobial, and anti-

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inflammatory agents.9,10 Furthermore the neuroprotective potential of lichen species has been

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scarcely investigated.11,12

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In the present work, the fifteen selected species representing different genera were

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belonged to the different clades of parmelioid lichens.13 These include Canoparmelia,

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Hypotrachyna, Myelochroa, Parmelia, Parmelina, Parmelinella, Parmotrema, Pleurosticta and

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Xanthoparmelia. The Canoparmelia texana, Hypotrachyna cirrhata, Hypotrachyna formosana,

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Myelochroa aurulenta, Parmelinella wallichiana and Parmotrema austrosinense are widely

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distributed in tropical and temperate regions of the world; Parmotrema nilgherrense are

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widespread in Indian subcontinent and Africa; Parmotrema gardneri, Parmotrema

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pseudocrinitum and Xanthoparmelia phaeophana are wide spread in Africa. Hypotrachyna

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densirrhizinata is distributed in Africa, Central and South America. Whereas, Parmelia sulcata

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is wide spread in the temperate regions of the world, and Parmelina tiliacea, Parmotrema

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perlatum and Pleurostica acetabulum are widely distributed in Europe.14,15,16 Their composition

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includes depsides, depsidones, and molecules derivated from pulvinic acid and dibenzofurane

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structure. Most of the selected species as Hypotrachyna cirrhata, Parmelinella wallichiana,

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Parmotrema austrosinense, Parmotrema nilgherrense, Parmotrema perlatum, and Parmelia


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sulcata have traditionally been used for various purposes including antimicrobial (antibacterial

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and antifungal) activities.17,18

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Therefore, the aim of this work is 1) to investigate for first time the neuroprotective

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activities of lichen species belonging to parmelioid clade, studying their potential antioxidant

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action and their ability to inhibit AChE and BuChE enzymes 2) to identify the secondary

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metabolites of the most active lichen species which are responsible for these effects.

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MATERIALS AND METHODS

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Reagents. All reagents were purchased from Sigma-Aldrich (St. Louis, MO, USA).

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HPLC grade methanol and ferrous sulphate (FeSO4) were acquired from Panreac (Barcelona,

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Spain).

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Lichen collection and extracts preparation. All the following lichens were collected, identified and authenticated by the experts Dr. P.K. Divakar and Dr. A. Crespo (Figure 1): 

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C. texana (MAF-Lich 21531) and P. nilgherrense (MAF-Lich 21528) were collected from Uttarakhand (India), October 2013.

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H. cirrhata (MAF-Lich 21530) was collected from Sikkim (India), April 2014.

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H. densirrhizinata (MAF-Lich 4342), H. formosana (MAF-Lich 4481), P. wallichiana

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(MAF-Lich 46009), P. austrosinense (MAF-Lich 4597), P. gardneri (MAF-Lich 4543), P.

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pseudocrinitum (MAF-Lich 4623) and X. phaeophana (MAF-Lich 4625) were collected

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from Kenya, May 2014.

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M. aurulenta (MAF-Lich 16565) was collected from New Jersey (USA), May 2010.

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Parmelia sulcata (MAF-Lich 21535) and Parmelia tiliacea (MAF-LICH 21534) were

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collected from Ávila (Spain), September 2015. 

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Parmotrema perlatum (MAF-Lich 19077) was collected from Tenerife (Spain), March 2014.

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Pleurosticta acetabulum (MAF-Lich 21533) was collected from Soria (Spain), July 2015. Lichen samples are preserved in MAF herbarium of Faculty of Pharmacy, UCM (Spain).

Images of parmelioid lichens are shown in Figure 1.

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2 ml of methanol was added to 50 mg of dry thalli of lichen (2 ml), shaking with vortex

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(20 sec, every 15 min, 2 h) and macerated for 24 h. Afterwards extracts were filtered using nylon

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filters (0.45 μm pore). Methanol extracts were evaporated at room temperature until dry residue.


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Antioxidant activity. 1,1-diphenyl-2-picrylhydrazyl (DPPH) method: DPPH method was

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carried out as Amarowicz et al. (2004) with some modifications.19 Dilutions of lichens methanol

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extracts were incubated with DPPH solution (50 µM) for 30 min. Absorbance was measured in a

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FLUOstar Optima fluorimeter (BMG Labtech, Ortenberg, Germany) at 517 nm.

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Oxygen Radical Absorbance Capacity (ORAC) method: ORAC method was measured as

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described by Dávalos et al. (2004).20 Dry lichen extracts were dissolved in methanol (1 mg/ml

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stock solution) and serial dilutions in PBS were obtained (concentrations from 10 to 500

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μg/mL). Then, samples were incubated with fluorescein (70 nM) for 10 minutes darkness. After

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incubation, AAPH was added (12 mM) and fluorescence was measured for 98 min in a

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FLUOstar Optima fluorimeter (BMG Labtech, Ortenberg, Germany) at 485 nm excitation

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wavelength and at 520 nm emission wavelength.

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Ferric Reducing Antioxidant Power (FRAP) method: FRAP assay was performed as described

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Avan et al. (2016) with some modifications.21 Lichen methanol extracts dilutions were mixed

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with FRAP reagent and incubated for 30 min at 37 °C. Absorbance was determined at 595 nm

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using a Spectrostar Nanomicroplate reader (BMG Labtech Inc., Ortenberg, Germany).


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Canoparmelia texana (Tuck.) Elix & Hale

Hypotrachyna cirrhata (Fr.) DivakarA. Crespo, Sipman, Elix & Lumbsch

Hypotrachyna densirrhizinata (Kurok.) Hale

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Hypotrachyna formosana (Zahlbr.) Hale

Myelochroa aurulenta (Tuck.) Elix & Hale

Parmelinella wallichiana (Taylor) Elix & Hale

Parmelia sulcata Taylor

Parmotrema nilgherrense (Nyl.) Hale

Parmelia tiliacea (Hoffm.) Hale

Parmotrema perlatum (Huds.) M. Choisy

Parmelinella wallichiana (Taylor) Elix & Hale

Parmotrema austrosinense (Zahlbr.) Hale

Parmotrema gardneri (C.W. Dodge) Serus

Parmotrema pseudocrinitum Pleurosticta acetabulum Xanthoparmelia phaeophana (Stirt.) Hale (Abbayes) Hale (Neck.) Elix & Lumbsch

Figure 1. Thallus of the studied lichens of Parmelioid clade.


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Quantification of total polyphenols: Total polyphenols were quantified by Folin-Ciocalteu method.22 Briefly, methanol lichen extracts were incubated with Folin-Ciocalteu reagent for 5 min in dark. Then, Na2CO3 solution (10%) and milliQ water were added to the mixture and incubated for 40 min. Absorbance was read at 752 nm in a Spectrostar Nanomicroplate reader (BMG Labtech Inc., Ortenberg, Germany). Neuroprotective activity Cell line and treatments. SH-SY5Y cells (human neuroblastoma cell line) were grown in DMEM with 10% FBS and 0.5% gentamicin at 37 °C and 5% CO2/95% air. Cells were pretreated with P. perlatum and H. formosana methanol extracts (from 0.5 μg/mL to 250 μg/mL, 24 h), previous Fenton's reagent (300 µM H2O2 + 300 µM FeSO4, 2 h). Lichen extracts were dissolve in DMSO and PBS as stock solutions (1 mg/ml) and then serial dilutions were made in PBS. DMSO final concentration in cells was less than 0.1%. MTT assay. Cytotoxicity and cytoprotection was assessed by MTT assay.23 After treatments, MTT solution (2 mg/mL) was added and plates incubated for 1 h at 37◦C. Finally, formazan crystals were solubilized in DMSO and absorbance was read at 550 nm using a microplate reader Digiscan 340 (Asys Hitech, Eugendorf, Austria). Dichlorodihydrofluorescein diacetate (DCFH-DA) assay. Intracellular ROS level was determined using DCFH-DA assay.24 After treatments, DCFH-DA solution in PBS (20 µM) was added and plates were incubated for 30 min at 37◦C. Then, plates were washed with glucosePBS. Fluorescence was determined for emission (λ = 510 nm) and for excitation (λ = 480 nm) in a Microplate Fluorescence Reader (BioTek Instrumentation, Inc., Winooski, Vermont, USA). TBARS assay. Lipid peroxidation products level was determined according to TBARS assay.25 Total lysates were mixed with TCA 40%, HCl 5N, TBA and millliQ water and then, they were heated (100◦C, 10 min) and centrifuged (3000 rpm, 10 min). Absorbance was read at 535 nm. Glutathione assay. GSH and GSSG levels was performed with the fluorometric method previously described.26 Total lysates were incubated with OPT and sodium phosphate buffer for 15 min. For GSSG assay, was also necessary to previously incubate samples with NEM for 5 min in dark. Fluorescence was measured at emission wavelength of 485 nm, and at excitation wavelength of 528 nm.


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Antioxidant enzymes activity. CAT activity. Total lysates were mixed with 15 mM H2O2 in 50 mM of phosphate buffer (pH 7.4). Absorbance was measured at 240 nm for 1 min.27 SOD activity. Total lysates were mixed with pyrogallol (0.15 mM) dissolved in HCl (10 mM) and Tris–DTPA (pH 8.2). Absorbance was measured at 420 nm for 1 min.28 GPx activity. Total lysates were incubated with phosphate buffer (pH 7.4) (50 mM), EDTA (1 mM), GSH (4 mM), glutathione reductase (27 U), NADPH (0.2 mM) and sodium azide (4 mM) for 4 min. After incubation, cumene solution was added. Absorbance was measured at 340 nm for 3 min.29 GR activity. Total lysates were mixed with EDTA (6.3 mM, pH 7.4), GSSG (80 mM), NADPH (6 mM) and phosphate buffer (50 mM). Absorbance was measured at 340 nm for 4 min with 1 min of delay time.30 AChE and BuChE enzymatic activities. AChE and BuChE activities were determined according to Ellman's colorimetric method.31 Reaction mixture consisted on DTNB (1.2 mM final concentration), AChE or BuChE (0.0008 U/well final concentration), 50 mM Tris–HCl buffer (pH 8.0), 0.1% (w/v) BSA, lichen extract at different concentrations and, the substrates acetylcholine iodide for AChE and butyrilthiocholine for BuChE (3 mM final concentration). Galanthamine was used as positive control. Absorbance was measured at 412 nm for 3 min each 30 sec in a Digiscan Microplate Reader (Assys Hitech, Korneuburg, Austria). Chemical analysis. High performance liquid chromatography method. HPLC analysis was performed according to the method described previously in detaile.32 The identification of lichen substances was determined by comparing retention times and UV-absorption spectrums with commercial compounds, individual compounds isolated in our lab and other lichen species. Thin-layer chromatography. TLC was performed on pre-coated aluminium sheets ALUGRAM® SIL G (0.20 mm of layer thickness) in the system solvent toluene-acetic acid (170:30). Spots were visualized under UV light (254 and 350 nm). Then, TLC plates were sprayed with sulfuric acid (10%) and heated at 100 ºC. Finally, Rf values were calculated for each detected components. Statistical analysis All assays were performed in at least in triplicate and media and standard deviation are shown. Data were analyzed by one-way ANOVA followed by Tukey’s test using Graph Pad


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prism 6 software. In antioxidant assays, Pearsons correlation coefficient (R) was calculated to measure the linear correlation between the different columns of the table. RESULTS AND DISCUSSION Antioxidant activities and total phenolic content. Table 1 summarizes results for extraction yields (dry extract weight/ lichen thallus weight x 100). Parmotrema austrosinense (30.8%), Parmotrema tiliacea (23.2%) and Parmotrema perlatum (19.5%) presented the highest yields whereas Hypotrachyna cirrhata (4.9%) was the lichen specie with the lowest extraction yield. The free-radical-scavenging and iron-reducing properties of methanol extracts of fifteen lichen species of parmelioid clade were evaluated using different in vitro free radical generating systems along with the determination of total phenolic content (Table 1). The ability of lichen extracts to scavenge DPPH and ORAC radicals and to reduce ferric (III) iron to ferrous (II) iron, there will inhibit the formation of OS by-products. The differences of these methods lie in the mechanism of action. While DPPH and FRAP methods measure SET capacity of antioxidants, ORAC method is based on HAT. In SET method, antioxidant compounds donate an electron to free radicals so that the own antioxidants become a radical cation (R• + ArOH → R- + ArOH•+), whereas in HAT method, antioxidants donate hydrogen atoms to free radicals so that the own antioxidant becomes a radical (R• + ArOH → RH + ArO•).33 DPPH assay revealed that Hypotrachyna formosana exhibited the highest free radical DPPH scavenging activity (EC50 value of 49.5 µg/ml) followed by Parmotrema perlatum and Hypotrachyna cirrhata (105.5 and 302.3 µg/ml, respectively). The lowest DPPH scavenging potential values were found for Parmotrema acetabulum (1700.2 μ g/mL) and Hypotrachyna densirrhizinata (1557.8 μg/mL). Regarding ORAC assay, Parmotrema perlatum (22.1 μ mol TE/mg dry extract), Parmotrema austrosinense (17.1 μmol TE/mg dry extract) and Parmotrema tiliacea (11.5 μmol TE/mg dry extract) showed the highest ORAC values. Parmotrema pseudocrinitum had the lowest ORAC value (2.3 μmol TE/mg dry extract). Finally, lichen methanol extracts were subjected to FRAP assay. Parmotrema perlatum and Hypotrachyna formosana exhibited the highest ferric reducing antioxidant potential (24.89 and 10.9 μmol of Fe2+ eq/g sample, respectively). However, methanol lichen extracts which


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demonstrated to possess the lowest capacity to reduce Fe3+ to Fe2+ were Parmotrema acetabulum (1.1 μmol of Fe2+ eq/g sample) and Xanthoparmelia phaeophana (1.2 μmol of Fe2+ eq/g sample).


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Table 1. Yields of extraction, antioxidant activities (ORAC, DPPH and FRAP assays), total phenolic contents, extract antioxidant potency (EAP) index and rank of fifteen methanol extracts of lichens of the clade parmelioid. Multiple comparisons for antioxidant assays and total phenolic content were assessed by Tukey’s test. Statistical significance (𝑝 < 0.05) is presented in letters superscripts. a: versus Canoparmelia texana; b: versus Hypotrachyna cirrhata; c: versus Hypotrachyna densirrhizinata; d: versus Hypotrachyna formosana; e: versus Myelochroa aurulenta; f: versus Parmelia sulcata; g: versus Parmelia tiliacea; h: versus Parmelinella wallichiana; i: versus Parmotrema austrosinense; j: versus Parmotrema gardneri; k: versus Parmotrema nilgherrense; l: versus Parmotrema perlatum; m: versus Parmotrema pseudocrinitum; n: versus Pleurosticta acetabulum; ñ: versus Xanthoparmelia phaeophana Lichen species

Canoparmelia texana

Yields (% w/w)

DPPH EC50 (μg/mL)

ORAC value (μmol TE/mg dry extract)

FRAP (μmol of Fe2+ eq/g sample)

Total phenolic contents (μg GA/mg)

EAP Index

Rank

18.5±2.0

772.8±12.3b,d,l

3.9±0.6e,m,ñ

3.3±0.01n,ñ

36.5±3.3n,ñ

28.44

9

4.1±0.5c,g,m,n,ñ

50.8±2.2e,h,k,m,n,ñ

41.83

3

Hypotrachyna cirrhata

4.9±0.7

302.3±5.2

5.9±0.2a,c,e,f,h,j,m,n,ñ

Hypotrachyna densirrhizinata

17.9±0.2

1557.8±181.8a,b,d,e,f,h,j,k,l,m

4.1±0.05e,m,n,ñ

2.4±0.2n

29.05±1.3c

12.22

13

Hypotrachyna formosana

16.0±3.7

49.5±0.8

7.8±0.2a,b,c,e,f,h,m,n,ñ

10.9±0.9a,b,c,e,f,g,h,i,j,k,m,n,ñ

77.7±3.5a,b,c,e,f,h,j,k,m,n,ñ

58.68

2

Myelochroa aurulenta

13.4±0.6

640.8±32.3b,d,l

2.9±0.06

3.4±0.6g,m,n,ñ

29.7±3.8

29.72

8

Parmelia sulcata

13.2±4.2

634±23.1b,d,l

3.6±0.3ñ

3.5±0.3g,m,n,ñ

44.8±2.0h,n,ñ

31.03

7

Parmelia tiliacea

23.2±3.6

1343.3±104.8a,b,d,e,f,h,j,k,l

11.5±0.08a,b,c,d,e,f,h,j,m,n,ñ

2.0±0.1

103.8±4.7a,b,c,e,f,h,j,k,m,n,ñ

27.12

11

Parmelinella wallichiana

12.3±1.9

819.9±109.7b,d,j,l

3.7±0.2m,ñ

3.3±0.08n,ñ

27.4±0.2

27.27

10

Parmotrema austrosinense

30.8±0.2

1452.9±87.3a,b,d,e,f,h,j,k,l,m

17.1±0.4a,b,c,d,e,f,g,h,j,m,n,ñ

2.9±0.2n,ñ

171.0±10.9a,b,c,d,e,f,g,h,j,k,m,n,ñ

34.54

5

Parmotrema gardneri

10.6±1.1

503.9±49.5d,l

7.3±0.3a,b,c,e,f,h,m,n,ñ

4.5±0.1c,g,i,m,n,ñ

49.8±4.3c,e,h,k,m,n,ñ

40.43

4

Parmotrema nilgherrense

14.1±2.5

787.1±11.6b,d,j,l

7±0.2a,b,c,e,f,g,h,m,n,ñ

2.6±0.1n

28.5±2.5

31.84

6

Parmotrema perlatum

19.5±6.4

105.5±7.9

22.1±0.5a,b,c,d,e,f,h,i,j,m,n,ñ

24.89±1.14a,b,c,d,e,f,g,h,i,j,k,m,n,ñ

170.5±9.1a,b,c,d,e,f,g,h,j,k,m,n,ñ

97.93

1

Parmotrema pseudocrinitum

11.4±0.4

1091.1±8.0a,b,d,e,f,j,k,l

2.3±0.2f

2.0±0.1

29.7±1.9

18.17

12

Pleurosticta acetabulum

11.2±2.0

1700.2±127.0a,b,d,e,f,g,h,j,k,l,m,ñ

3.1±0.1

1.1±0.2

20±0.5

6.05

15

Xanthoparmelia phaeophana

11.3±1.2

1364.6±21.9a,b,d,e,f,h,j,k,l

2.49±0.29

1.2±0.05

18.46±0.95

11.98

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Based on all these antioxidant assays, EAP index was calculated as follows: EAP = [(sample score/best score) x 100] where the best score is assigned a value of 100 for the best value of each test.34 Mean value for all 3 tests for each methanol extract was used to classify lichens and establish a rank according to their antioxidant potency. The highest EAP index was found for Parmotrema perlatum (EAP 97.93) and Hypotrachyna formosana (EAP 58.68) whereas Parmotrema acetabulum was the last in the ranking (EAP 6.05). Therefore, Parmotrema perlatum and H. formosana are of interest to continue with further investigations based on their antioxidant activity. Since Parmotrema perlatum has potent ORAC and DPPH scavenging effects and capacity to reduce Fe3+ suggest that its methanol extracts contain bioactive secondary metabolites with antioxidant molecules involved HAT and SET mechanistic pathways. On the other hand, Hypotrachyna formosana methanol extract has resulted to be significant active in DPPH free-radical scavenging test and in FRAP assay, so that it contains secondary lichen compounds with antioxidant properties in which predominate SET mechanism. Lichens contain secondary metabolites, most of them exclusive, which have phenolic structure. Phenols have the ability to quench unstable free radicals by donating hydrogen atoms of the phenolic OH, leading to the formation of a resonance stabilized phenoxy radical which accounts for preventing chain reaction-propagation and thus the oxidative damage to biological macromolecules. Previous works have demonstrated the antioxidant potency of other lichen extracts (i.e. Cladonia furcata, Lecanora atra and Lecanora muralis) and (i.e. and isolated compounds (i.e. physodic acid, salazinic acid).35,36 Therefore, we measured total phenolic content using Folin-Ciocalteu method. PParmotrema austrosinense, Parmotrema perlatum and Parmotrema tiliacea exhibited the highest values (171, 170.5 and 103.8 µg gallic acid/mg, respectively) whereas Parmotrema acetabulum showed the lowest total phenolic content (20 µg gallic acid/mg). In order to investigate whether there is a correlation between phenolic compounds and antioxidant activity of methanol extracts of lichens, we calculated Pearson’s coefficient (r) to quantify the relationship between two quantitative variables. Results (Table 2) showed the highest correlation coefficients for FRAP and total phenolic content, and for EAP index and total phenolic content (0.9 < r < 1). The interactions between DPPH and total phenolic content as well as ORAC and total phenolic content were found moderate (0.7 < r < 0.85). Regarding the


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antioxidant methods, there was a moderate correlation between DPPH and ORAC methods (r = 0.654), FRAP and ORAC assays (r = 0.713) and FRAP and DPPH methods (r = 0.781). It has been demonstrated that phenolic compounds can counteract free radical action and terminating the chain reaction that can potentially cause cell damage. Some of the mechanisms that make phenols good antioxidants include releasing hydrogen atoms and electrons, inducing the release of protective conjugate enzymes, increasing apoptosis, inhibiting lipid peroxidation, inhibiting angiogenesis, and inhibiting DNA oxidation37 These results suggest that the content of phenolic compounds may be responsible for the antioxidant activity of the lichen extracts. The higher is the phenol content, the higher is the antioxidant effect. The antioxidant power can be attributed to different factors as the number and position of hydroxyl groups in the molecule, the degree of hydroxylation (the longer the distance between carbonyl group and aromatic ring of a phenolic acid the better is the antioxidant activity) and the number of hydroxyl aromatic rings (flavonoids have demonstrated better antioxidant activity than phenolic acids).38 Table 2. Pearson’s correlation coefficients of total phenolic compounds and antioxidant activity (ORAC, DPPH, FRAP and EAP). Statistical differences are represented as follows: *p

Lichens of parmelioid clade as promising multi-target neuroprotective

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