Lichens of Parmelioid Clade as Promising Multitarget Neuroprotective

Article pubs.acs.org/crt

Cite This: Chem. Res. Toxicol. 2019, 32, 1165−1177

Lichens of Parmelioid Clade as Promising Multitarget Neuroprotective Agents Víctor Sieteiglesias, Elena Gonzaĺ ez-Burgos, Paloma Bermejo-Bescoś , Pradeep K. Divakar, ́ ez-Serranillos* and María Pilar Gom

Downloaded via BUFFALO STATE on July 23, 2019 at 08:46:51 (UTC). See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.

Department of Pharmacology, Pharmacognosy and Botanical, Faculty of Pharmacy, Universidad Complutense de Madrid, Plaza Ramon y Cajal s/n, Ciudad Universitaria, 28040, Madrid, Spain ABSTRACT: Neurodegenerative diseases such as Alzheimer’s disease and Parkinson’s disease are multifactorial disorders which are increasing in incidence and prevalence over the world without existing effective therapies. The search for new multitarget compounds is the latter therapeutic strategy to address these pathological conditions. Lichens have an important and unknown therapeutic value attributed to their unique secondary metabolites. The aim of this study is to evaluate for the first time the in vitro neuroprotective activities and molecular mechanisms underlying methanol extracts of lichens of the parmelioid clade and to characterize major bioactive secondary metabolites responsible for their pharmacological actions. Of the 15 parmelioid lichen species, our results showed that Parmotrema perlatum and Hypotrachyna formosana methanol extracts exhibited high antioxidant activity as evidenced in ORAC, DPPH, and FRAP assays. Then, SH-SY5Y cells were pretreated with methanol extracts (24 h) followed by Fenton reagent exposure (2 h). Pretreatments with these two more antioxidant methanol lichen extracts increased cell viability, reduced intracellular ROS, prevented oxidative stress biomarkers accumulation, and upregulated antioxidant enzyme (CAT, SOD, GR, and GPx) activity compared to Fenton reagent cells. The neuroprotective activity was much higher for H. formosana than for P. perlatum, even equal to or higher than Trolox (reference compound). Moreover, H. formosana extracts inhibited both AChE and BuChE activities in a concentration dependent manner, and P. perlatum only showed concentration dependent activity against AChE. Finally, chemical composition analysis using TLC and HPLC methods revealed that physodic acid, lividic acid, and lichexanthone are major secondary metabolites in H. formosana and stictic acid and constictic acid are in P. perlatum. These results demonstrated that P. perlatum and, specially, H. formosana are promising multitargeted neuroprotective agents due to their antioxidant and AChE and BuChE inhibition activities.

■

INTRODUCTION Reactive oxygen species (ROS) participate in various biological processes regulating metabolic and cellular survival pathways. In physiological situations, these ROS are in equilibrium with the endogenous antioxidants system through a free radical scavenger, metal chelator, and enzymatic modulation mechanisms. However, under certain circumstances, ROS may exceed the capacity of antioxidants resulting from an oxidative damage to cell structures (membrane lipid peroxidation, DNA demethylation, and protein oxidation) and even cell death via apoptosis and necrosis.1 This situation known as oxidative stress is involved in the pathogenesis and progression of many different chronic diseases including age-related neurodegenerative disorders such as Alzheimer’s disease (AD), Parkinson’s disease (PD), and amyotrophic lateral sclerosis (ALS).2,3 The prevalence and incidence of these central nervous system (CNS) disorders rise with increasing age, expecting exponential growth in the number of cases as average life expectancy increases.4 Therefore, oxidative stress prevention and treatment using exogenous antioxidants has become an emerging © 2019 American Chemical Society

and promising therapeutic strategy to cope with ROS overproduction and, consequently, to improve antioxidant status. Consistent studies have identified numerous compounds of natural origin with antioxidant activity, highlighting in particular molecules with phenolic structure.5 Moreover, targeting AChE and BuChE enzymes is considered a promising strategy for the prevention and treatment of different CNS disorders including senile dementia, AD, myasthenia gravis, and ataxia.6 Natural products are considered as a source of potential compounds with therapeutic activity. The knowledge of traditional medicine, its pharmacological validation, and the discovery of new activities has led to the development of many “lead molecules” for prevention and treatment of different diseases. It is estimated that approximately 25% of the therapeutic arsenal available on the market comes from molecules of natural origin, especially from medicinal plants. Received: January 14, 2019 Published: May 24, 2019 1165

DOI: 10.1021/acs.chemrestox.9b00010 Chem. Res. Toxicol. 2019, 32, 1165−1177

Article

Chemical Research in Toxicology

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

composition includes depsides, depsidones, and molecules derived from pulvinic acid and dibenzofurane structure. Most of the selected species such as Hypotrachyna cirrhata, Parmelinella wallichiana, Parmotrema austrosinense, Parmotrema nilgherrense, Parmotrema perlatum, and Parmelia sulcata have traditionally been used for various purposes including antimicrobial (antibacterial and antifungal) activities.17,18 Therefore, the aim of this work is (1) to investigate for the first time the neuroprotective activities of lichen species belonging to the parmelioid clade, studying their potential antioxidant action and their ability to inhibit AChE and BuChE enzymes, and (2) to identify the secondary metabolites of the most active lichen species which are responsible for these effects.

However, there are natural products whose pharmacological activity has not been investigated, underlining especially these natural products from lichens.7 Lichens are attractive organisms formed from the symbiotic association of a fungus (mycobiont) and an algae and/or cyanobacteria (photobiont). The parmelioid clade, within the Parmeliaceae family, is the largest group of lichens forming fungi with more than 1500 species widely distributed from dry climates to tropical conditions.8 Lichens synthesize a great diversity of secondary metabolites (more than 1000 identified compounds), most of them present exclusively in these organisms and with a phenol structure, such as depsidones, depsides, depsones, dibenzofurans, and xanthones. In recent years, there has been a growing interest in lichens due to their potential pharmacological action such as antioxidant, antitumor, antimicrobial, and anti-inflammatory agents.9,10 Furthermore, the neuroprotective potential of lichen species has been scarcely investigated.11,12 In the present work, the 15 selected species representing different genera belonged to different clades of parmelioid lichens.13 These include Canoparmelia, Hypotrachyna, Myelochroa, Parmelia, Parmelina, Parmelinella, Parmotrema, Pleurosticta, and Xanthoparmelia. The Canoparmelia texana, Hypotrachyna cirrhata, Hypotrachyna formosana, Myelochroa aurulenta, Parmelinella wallichiana, and Parmotrema austrosinense are widely distributed in tropical and temperate regions of the world. Parmotrema nilgherrense is widespread in the Indian subcontinent and Africa. Parmotrema gardneri, Parmotrema pseudocrinitum, and Xanthoparmelia phaeophana are widespread in Africa. Hypotrachyna densirrhizinata is distributed in Africa and Central and South America. Whereas Parmelia sulcata is widespread in the temperate regions of the world, Parmelina tiliacea, Parmotrema perlatum, and Pleurostica acetabulum are widely distributed in Europe.14−16 Their

■

MATERIALS AND METHODS

Reagents. All reagents were purchased from Sigma-Aldrich (St. Louis, MO, USA). HPLC grade methanol and ferrous sulfate (FeSO4) were acquired from Panreac (Barcelona, Spain). 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): • C. texana (MAF-Lich 21531) and P. nilgherrense (MAF-Lich 21528) were collected from Uttarakhand (India), October 2013. • H. cirrhata (MAF-Lich 21530) was collected from Sikkim (India), April 2014. • H. densirrhizinata (MAF-Lich 4342), H. formosana (MAF-Lich 4481), P. wallichiana (MAF-Lich 46009), P. austrosinense (MAF-Lich 4597), P. gardneri (MAF-Lich 4543), P. pseudocrinitum (MAF-Lich 4623), and X. phaeophana (MAF-Lich 4625) were collected from Kenya, May 2014. • M. aurulenta (MAF-Lich 16565) was collected from New Jersey (USA), May 2010. • Parmelia sulcata (MAF-Lich 21535) and Parmelia tiliacea (MAFLICH 21534) were collected from Á vila (Spain), September 2015. 1166


772.8 302.3 1557.8 49.5 640.8 634 1343.3 819.9 1452.9 503.9 787.1 105.5 1091.1 1700.2 1364.6

± 12.3b,d,l ± 5.2 ± 181.8a,b,d,e,f,h,j,k,l,m ± 0.8 ± 32.3b,d,l ± 23.1b,d,l ± 104.8a,b,d,e,f,h,j,k,l ± 109.7b,d,j,l ± 87.3a,b,d,e,f,h,j,k,l,m ± 49.5d,l ± 11.6b,d,j,l ± 7.9 ± 8.0a,b,d,e,f,j,k,l ± 127.0a,b,d,e,f,g,h,j,k,l,m,ñ ± 21.9a,b,d,e,f,h,j,k,l

DPPH EC50(μg/mL) 3.9 5.9 4.1 7.8 2.9 3.6 11.5 3.7 17.1 7.3 7 22.1 2.3 3.1 2.49

± 0.6e,m,ñ ± 0.2a,c,e,f,h,j,m,n,ñ ± 0.05e,m,n,ñ ± 0.2a,b,c,e,f,h,m,n,ñ ± 0.06 ± 0.3ñ ± 0.08a,b,c,d,e,f,h,j,m,n,ñ ± 0.2m,ñ ± 0.4a,b,c,d,e,f,g,h,j,m,n,ñ ± 0.3a,b,c,e,f,h,m,n,ñ ± 0.2a,b,c,e,f,g,h,m,n,ñ ± 0.5a,b,c,d,e,f,h,i,j,m,n,ñ ± 0.2f ± 0.1 ± 0.29

ORAC value (μmol TE/mg dry extract) 3.3 4.1 2.4 10.9 3.4 3.5 2.0 3.3 2.9 4.5 2.6 24.89 2.0 1.1 1.2

± 0.01n,ñ ± 0.5c,g,m,n,ñ ± 0.2n ± 0.9a,b,c,e,f,g,h,i,j,k,m,n,ñ ± 0.6g,m,n,ñ ± 0.3g,m,n,ñ ± 0.1 ± 0.08n,ñ ± 0.2n,ñ ± 0.1c,g,i,m,n,ñ ± 0.1n ± 1.14a,b,c,d,e,f,g,h,i,j,k,m,n,ñ ± 0.1 ± 0.2 ± 0.05

FRAP (μmol of Fe2+eq/g sample) 36.5 50.8 29.05 77.7 29.7 44.8 103.8 27.4 171.0 49.8 28.5 170.5 29.7 20 18.46

± 3.3n,ñ ± 2.2e,h,k,m,n,ñ ± 1.3c ± 3.5a,b,c,e,f,h,j,k,m,n,ñ ± 3.8 ± 2.0h,n,ñ ± 4.7a,b,c,e,f,h,j,k,m,n,ñ ± 0.2 ± 10.9a,b,c,d,e,f,g,h,j,k,m,n,ñ ± 4.3c,e,h,k,m,n,ñ ± 2.5 ± 9.1a,b,c,d,e,f,g,h,j,k,m,n,ñ ± 1.9 ± 0.5 ± 0.95

total phenolic contents (μg GA/mg) 28.44 41.83 12.22 58.68 29.72 31.03 27.12 27.27 34.54 40.43 31.84 97.93 18.17 6.05 11.98

EAP index

9 3 13 2 8 7 11 10 5 4 6 1 12 15 14

rank

a Multiple comparisons for antioxidant assays and total phenolic content were assessed by Tukey’s test. Statistical significance (ρ < 0.05) is presented in letter 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.

2.0 0.7 0.2 3.7 0.6 4.2 3.6 1.9 0.2 1.1 2.5 6.4 0.4 2.0 1.2

± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

Canoparmelia texana Hypotrachyna cirrhata Hypotrachyna densirrhizinata Hypotrachyna formosana Myelochroa aurulenta Parmelia sulcata Parmelia tiliacea Parmelinella wallichiana Parmotrema austrosinense Parmotrema gardneri Parmotrema nilgherrense Parmotrema perlatum Parmotrema pseudocrinitum Pleurosticta acetabulum Xanthoparmelia phaeophana

18.5 4.9 17.9 16.0 13.4 13.2 23.2 12.3 30.8 10.6 14.1 19.5 11.4 11.2 11.3

yields (% w/w)

lichen species

Table 1. Yields of Extraction, Antioxidant Activities (ORAC, DPPH and FRAP Assays), Total Phenolic Contents, Extract Antioxidant Potency (EAP) Index and Rank of 15 Methanol Extracts of Lichens of the Clade Parmelioida

Chemical Research in Toxicology Article

1167


Article

Chemical Research in Toxicology • Parmotrema perlatum (MAF-Lich 19077) was collected from Tenerife (Spain), March 2014. • Pleurosticta acetabulum (MAF-Lich 21533) was collected from Soria (Spain), July 2015. Lichen samples were preserved in the MAF herbarium of the Faculty of Pharmacy, UCM (Spain). Images of parmelioid lichens are shown in Figure 1. A total of 2 mL of methanol was added to 50 mg of dry thalli of lichen (2 mL), shaken with a vortex (20 s, every 15 min, 2 h), and macerated for 24 h. Afterward extracts were filtered using nylon filters (0.45 μm pore). Methanol extracts were evaporated at room temperature until they became a dry residue. Antioxidant Activity. 1,1-Diphenyl-2-picrylhydrazyl (DPPH) Method. The DPPH method was carried out as in Amarowicz et al. with some modifications.19 Dilutions of lichens’ methanol extracts were incubated with a DPPH solution (50 μM) for 30 min. Absorbance was measured in a FLUOstar Optima fluorimeter (BMG Labtech, Ortenberg, Germany) at 517 nm. Oxygen Radical Absorbance Capacity (ORAC) Method. The ORAC method was measured as described by Dávalos et al.20 Dry lichen extracts were dissolved in methanol (1 mg/mL stock solution), and serial dilutions in PBS were obtained (concentrations from 10 to 500 μg/mL). Then, samples were incubated with fluorescein (70 nM) for 10 min of darkness. After incubation, AAPH was added (12 mM), and fluorescence was measured for 98 min in a FLUOstar Optima fluorimeter (BMG Labtech, Ortenberg, Germany) at a 485 nm excitation wavelength and at a 520 nm emission wavelength. Ferric Reducing Antioxidant Power (FRAP) Method. FRAP assay was performed as described Avan et al. (2016) with some modifications.21 Lichen methanol extracts dilutions were mixed with FRAP reagent and incubated for 30 min at 37 °C. Absorbance was determined at 595 nm using a Spectrostar Nanomicroplate reader (BMG Labtech Inc., Ortenberg, Germany). Quantification of Total Polyphenols. Total polyphenols were quantified by the Folin-Ciocalteu method.22 Briefly, methanol lichen extracts were incubated with Folin-Ciocalteu reagent for 5 min in the dark. Then, Na2CO3 solution (10%) and Milli-Q 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) and the previous Fenton’s reagent (300 μM H2O2 + 300 μM FeSO4, 2 h). Lichen extracts were dissolved 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 were 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 Digiscan 340 microplate reader (Asys Hitech, Eugendorf, Austria). Dichlorodihydrofluorescein Diacetate (DCFH-DA) Assay. The intracellular ROS level was determined using a 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 glucose-PBS. 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. The lipid peroxidation product level was determined according to a 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 the GSSG assay, it was also necessary to previously incubate samples

with NEM for 5 min in the dark. Fluorescence was measured at an emission wavelength of 485 nm and at an excitation wavelength of 528 nm. Antioxidant Enzymes Activity. CAT activity: Total lysates were mixed with 15 mM H2O2 in 50 mM 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 The reaction mixture consisted of 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 a positive control. Absorbance was measured at 412 nm for 3 min, every 30 s 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 detail.32 The identification of lichen substances was determined by comparing retention times and UVabsorption spectra with commercial compounds, individual compounds isolated in our lab, and other lichen species. Thin-Layer Chromatography. TLC was performed on ALUGRAM SIL G precoated aluminum sheets (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 °C. Finally, Rf values were calculated for each detected component. Statistical Analysis. All assays were performed 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 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 × 100). Parmotrema austrosinense (30.8%), Parmotrema tiliacea (23.2%), and Parmotrema perlatum (19.5%) presented the highest yields, whereas Table 2. Pearson’s Correlation Coefficients of Total Phenolic Compounds and Antioxidant Activity (ORAC, DPPH, FRAP, and EAP)a bioactive capacities phenolic content ORAC DPPH FRAP EAP

phenolic content

ORAC

DPPH

FRAP

EAP index

1 0.654** 0.713** 0.758**

1 0.781** 0.970**

1 0.938**

1

1 0.733** 0.83** 0.933** 0.908**

Statistical differences are represented as follows: *p < 0.05 and **p < 0.01. a

1168


Article


Figure 2. Neuroprotective effect of H. formosana and P. perlatum against Fenton reagent-induced neuroblastoma cell death. (A) Effect of methanol extract of the lichen species P. perlatum and H. formosana (range of concentrations from 0.5 to 250 μg/mL for 24 h) on neuroblastoma SH-SY5Y cell viability using MTT assay. (B) Potential neuroprotective effect of noncytotoxic concentrations of methanol extract of the lichen species P. perlatum and H. formosana for 24 h against Fenton reagent (H2O2 300 μM, FeSO4 300 μM for 2 h) on SH-SY5Y cell viability using MTT assay. (C) Effect of the most protective concentrations of P. perlatum and H. formosana on cell morphology. Trolox (0.1 mM, 24 h) was used as a positive control. Values are the mean ± SD from at least three independent experiments. #p < 0.05 vs control; *p < 0.05 vs Fenton reagent.

radicals so that the antioxidant becomes a radical (R• + ArOH → RH + ArO•).33 The 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 μg/mL) and Hypotrachyna densirrhizinata (1557.8 μg/mL). Regarding the ORAC assay, Parmotrema perlatum (22.1 μmol 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 a FRAP assay. Parmotrema perlatum and Hypotrachyna formosana

Hypotrachyna cirrhata (4.9%) was the lichen species with the lowest extraction yield. The free-radical-scavenging and iron-reducing properties of methanol extracts of 15 lichen species of the 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 will inhibit the formation of OS byproducts. The differences of these methods lie in the mechanism of action. While DPPH and FRAP methods measure SET capacity of antioxidants, the ORAC method is based on HAT. In the SET method, antioxidant compounds donate an electron to free radicals so that the antioxidants become a radical cation (R• + ArOH → R− + ArOH•+), whereas in the HAT method, antioxidants donate hydrogen atoms to free 1169


Article


Figure 3. Hypotrachyna formosana and Parmotrema perlatum extract prevention of the intracellular Fenton reagent-induced ROS production. Effect of P. perlatum (25 μg/mL) and H. formosana (1 μg/mL) for 24 h, previous to the exposure of Fenton reagent (FR; 300 μM H2O2 + 300 μM FeSO4) for 2 h, on intracellular ROS generation in SH-SY5Y cells. Trolox (0.1 mM, 24 h) was used as a positive control. Values are the mean ± SD from at least three independent experiments. #p < 0.05 vs control; **p < 0.01 vs FR.

Figure 5. Hypotrachyna formosana and Parmotrema perlatum methanol extract prevention of Fenton reagent-induced lipid peroxidation in neuroblastoma cells. Effect of P. perlatum (25 μg/ mL) and H. formosana (1 μg/mL) for 24 h, previous to the exposure of Fenton reagent (FR; 300 μM H2O2 + 300 μM FeSO4) for 2 h, on TBARS levels in SH-SY5Y cells. Trolox (0.1 mM, 24 h) was used as a positive control. Values are the mean ± SD from at least three independent experiments. #p < 0.05 vs control, **p < 0.01 and *p < 0.05 vs FR, and ··p < 0.05 vs P. perlatum extract.

exhibited the highest ferric reducing antioxidant potential (24.89 and 10.9 μmol of Fe2+ eq/g sample, respectively). However, methanol lichen extracts which were 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). On the basis of all these antioxidant assays, the EAP index was calculated as follows: EAP = [(sample score/best score) ×

Figure 4. Hypotrachyna formosana and Parmotrema perlatum methanol extract prevention of changes in Fenton reagent-induced glutathione levels in neuroblastoma cells. Effect of P. perlatum (25 μg/mL) and H. formosana (1 μg/mL) for 24 h, previous to the exposure of Fenton reagent (FR; 300 μM H2O2 + 300 μM FeSO4) for 2 h, on (A) GSH levels, (B) GSSG levels, and (C) GSH/GSSG ratio in SH-SY5Y cells. Trolox (0.1 mM, 24 h) was used as a positive control. Values are the mean ± SD from at least three independent experiments. #p < 0.05 vs control, **p < 0.01 vs FR, and ··p < 0.05 vs H. formosana extract. 1170


Article


Figure 6. Hypotrachyna formosana and Parmotrema perlatum methanol extracts upregulated antioxidant enzymes activity in neuroblastoma cells. Effect of P. perlatum (25 μg/mL) and H. formosana (1 μg/mL) for 24 h, previous to the exposure of Fenton reagent (FR; 300 μM H2O2 + 300 μM FeSO4) for 2 h, on CAT, SOD, GR, and GPx enzymatic activities in SH-SY5Y cells. Trolox (0.1 mM, 24 h) was used as a positive control. Values are the mean ± SD from at least three independent experiments. #p < 0.05 vs control, **p < 0.01 and *p < 0.05 vs Fenton reagent, and ··p < 0.01 and ·p < 0.05 vs lichen extracts.

100] where the best score is assigned a value of 100 for the best value of each test.34 The mean value for all three 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 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 the capacity to reduce Fe3+ , this suggests that its methanol extracts contain bioactive secondary metabolites with antioxidant molecules involved in HAT and SET mechanistic pathways. On the other hand, Hypotrachyna formosana methanol extract has resulted to be significantly active in a DPPH free-radical scavenging test and in FRAP assay, so that it contains secondary lichen compounds with antioxidant properties in which the SET mechanism predominates. 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 oxidative damage to biological macromolecules. Previous works have demonstrated the antioxidant potency of other lichen extracts (i.e., Cladonia f urcata, Lecanora atra, and Lecanora muralis)

and isolated compounds (i.e., physodic acid, salazinic acid).35,36 Therefore, we measured total phenolic content using the Folin−Ciocalteu method. Parmotrema 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 the 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 to be moderate (0.7 < r < 0.85). Regarding the 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 terminate 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 1171


Article


byproducts (4-hydroxyl-2-nonenal), and protein oxidation products (protein carbonyls).3 We therefore studied the potential neuroprotective effect of Parmotrema perlatum and Hypotrachyna formosana methanol extracts, which possess the highest in vitro antioxidant activity. Initially, we evaluated whether a range of concentrations from 0.5 to 250 μg/mL for 24 h could affect SHSY5Y cell viability using an MTT assay. As shown in Figure 2A, Parmotrema perlatum methanol extract was not cytotoxic at any assayed concentration except for 50 μg/mL, 100 μg/mL, and 250 μg/mL (cell viability was reduced by 90.0%, 90.2%, and 90.8%, respectively, compared with control cells). On the other hand, Hypotrachyna formosana was significantly cytotoxic at concentrations above 25 μg/mL. However, from 0.5 μg/mL to 10 μg/mL, SHSY5Y cell viability was not affected by the Hypotrachyna formosana treatments. Hydrogen peroxide, the major ROS in the human body, can react with a ferrous salt mixture leading to hydroxyl radical production through a Fenton reaction. The Fenton reagent is commonly used in in vitro systems to mimic a pathophysiological situation of oxidative stress. Both H2O2 and •OH are related to aging-related human diseases such as PD and AD.2,38 Until now, no studies have been performed with the Fenton reagent as an oxidative stress inductor in neuroprotective studies with lichen extracts. In previous works, some lichenic molecules such as usnic acid and salazinic acid have been demonstrated to protect astrocytes against oxidative stress induced by hydrogen peroxide.39 Moreover, lichen extracts such as Ramalina terebrata, Cladonia kalbii, and Xanthoparmelia conspersa have shown antioxidant properties diminishing hydrogen peroxide levels.40,41 Then, we evaluated whether noncytotoxic concentrations of Parmotrema perlatum and Hypotrachyna formosana methanol extracts could protect against oxidative stress induced by a Fenton reagent. SHSY5Y cells were pretreated with noncytotoxic concentrations of Parmotrema perlatum (from 0.5 to 25 μg/mL) and Hypotrachyna formosana (from 0.5 to 10 μg/mL) methanol extracts, and then they were exposed to the Fenton reagent. As shown in Figure 2B, when cells were treated with the Fenton reagent, cell viability was significantly reduced by 57% compared with control cells. Pretreatments with all noncytotoxic Hypotrachyna formosana concentrations significantly increased cell viability, 1 μg/mL being the most active concentration (cell viability was increased around 20% more compared with the Fenton reagent cells). On the other hand, Parmotrema perlatum significantly protected neuroblastoma cells at the highest assayed concentrations of 10 and 25 μg/mL (14.27% and 22.38% of protection compared to Fenton reagent cells). Therefore, we used a 1 μg/mL concentration for Hypotrachyna formosana and a 25 μg/mL concentration for Parmotrema perlatum for further experiments. Finally, the effect of lichen extracts on cell morphology was evaluated. The Fenton reagent caused significant morphological changes on SHSY5Y cells as evidenced by a loss of shape, being less bound to the surface, and there being fewer viable cells. However, pretreatments with lichen extracts avoid these morphological changes as well as Trolox did (Figure 2C). Intracellular ROS Production. It is well-known that Fenton reagent leads to the formation of the highly toxic •OH. When there is ROS overproduction such as that of H2O2 and • OH, cells fail to keep normal physiological redox-regulated processes.1 ROS overproduction is able to damage cell

Figure 7. Hypotrachyna formosana and Parmotrema perlatum extracts’ inhibition of acetylcholinesterase and butyrylcholinesterase enzymatic activities. Galanthamine was used as a positive control. Values are the mean ± SD from at least three independent experiments. ap < 0.05 vs 25 μg/mL; bp < 0.05 vs 50 μg/mL.

the lichen extracts. The higher is the phenol content, the higher is the antioxidant effect. The antioxidant power can be attributed to different factors such 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 MTT Viability and Neuroprotective Effect. Consistent evidence of oxidative stress in brain tissues of patients with neurodegenerative diseases support its contribution in the pathogenic development of these diseases. Hence, samples of post-mortem brains of patients with AD revealed a decline in SOD, GPx, and GST antioxidant enzymes and in GSH content as well as an increase in protein carbonyls and lipid peroxidation product levels.2 Moreover, oxidative stress is also responsible for dopaminergic neuronal degeneration as evidenced by DNA and RNA oxidation products (8-hydroxyguanosine and 8-hydroxy-deoxyguanosine), lipid peroxidation 1172


Article


Figure 8. HPLC chromatograms of methanol extracts of (A) Hypotrachyna formosana and (B) Parmotrema perlatum. Detection was at 254 nm.

biological structures and, consequently, cell biological functions, leading to cell damage and even cell death. There are recent studies that demonstrate that the increase of ROS production leads to cell apoptosis, and this mechanism has been implicated in neurodegenerative diseases. The hydroxyl radical is the most potent of all known radicals, so it is able to do more damage to biomolecules than the other ROS.42TherTherefore, we evaluated whether Hypotrachyna formosana and Parmotrema perlatum extracts could inhibit Fenton-reagentinduced ROS production in SHSY5Y cells using a DCFH-DA assay (Figure 3). The antioxidant Trolox (water-soluble vitamin E analogue) was employed as a positive control. The Fenton reagent significantly increased ROS overproduction in SHSY5Y cells as evidenced in the rise in fluorescence intensity (an increase of 32% of ROS generation compared to control cells). However, pretreatments with Hypotrachyna formosana (1 μg/mL) and Parmotrema perlatum (25 μg/mL) for 24 h reduced intracellular ROS accumulation (51.98% and 47.63% of ROS reduction, respectively). The effect on intracellular ROS generation of both lichen extracts was similar to that of Trolox, which reduced ROS accumulation 43.7%.43 Other lichen extracts such as Cetraria islandica and Vulpicida canadensis extracts have demonstrated neuroprotective effects by reducing ROS production in the SH-SY5Y cell line.44

Moreover, ramalina, a compound isolated from the lichen Ramalina terbrata, was able to regulate ROS in murine macrophage Raw264.7 cells. Our results are in accordance with previous studies that show that lichen extracts and molecules isolated from lichens could be effective in the reduction of oxidative stress. Gluthation Levels. Glutathione in its reduced form is involved in the detoxification of radical species. It is the main nonenzymatic antioxidant present in our cells. In some diseases, such as Lou Gehrig’s disease, Parkinson’s disease, and HIV, a deficiency of GSH has been seen in the brain. In AD, oxidized glutathione is increased, and the glutathione Stransferase activity is decreased. GSH is a tripeptide crucial in the free radical scavenging of singlet oxygen and hydroxyl radicals. It also binds to heavy metals that can increase oxidative stress. It also binds strongly to electrophiles because of the sulfhydryl group of cysteine. Recent therapies have tried to increase GSH levels to restore them.45 Exposure of neuroblastoma cells with the Fenton reagent resulted in a significant reduction of GSH and of the ratio GSH/GSSG by 0.64 and 0.52 fold versus control cells, respectively (Figure 4). On the other hand, pretreatment with both lichens’ extracts significantly increased GSH levels. Particularly, Hypotrachyna formosana increased GSH content 2.91 fold and Parmotrema 1173


Article


cells for Hypotrachyna formosana), highlighting especially Hypotrachyna formosana that reduced the levels of lipid peroxidation to Trolox levels. Antioxidant Enzymes Activities. To further study the mechanism involved in the neuroprotective effect of Parmotrema perlatum and Hypotrachyna formosana extracts against Fenton reagent-induced oxidative stress cell damage, we then examined its effect on the antioxidant enzymes CAT, SOD, GR, and GPx activities (Figure 6). Human cells have developed mechanisms to protect them against ROS and oxidative stress; these mechanisms include nonenzymatic (gluthatione) and enzymatic pathways, including the enzymes CAT, SOD, GR, and GPX.52 CAT is the enzyme responsible for eliminating H2O2 and transforming it into H2O and O2. SOD catalyzes the reaction of superoxide anion into H2O2. GPx has the same action as catalase but uses GSH for the reaction. GR is responsible for the conversion of GSSG into GSH.53 Exposure to the Fenton reagent reduced significantly CAT, SOD, GR and GPx activities in SHSY5Y cells by 0.64, 0.75, 0.80, and 0.61 folds versus control. However, pretreatments with Hypotrachyna formosana extracts significantly increased the activity of all antioxidant enzymes, this effect being notably superior to the effect of Parmotrema perlatum (for SOD, GR, and GPx enzymatic activities) and even for the reference compound Trolox (for GR enzymatic activity). Previous studies with Usnea ghattensis extract showed an increase in the activity of SOD, CAT, and GPX in a mice liver slice culture model.54 This indicates that the lichen extract could be a more effective antioxidant so that it reduced antioxidant enzyme activities in a significant quantity.55 These results suggest that the two lichen extracts, but especially Hypotrachyna formosana, are capable to induce or at least maintain the antioxidant mechanisms of the cell and protect it against oxidative stress induced by Fenton reagent. AChE and BuChE Enzymatic Inhibition. Acetylcholine plays a key role in neuronal transmission and function. A deficiency in this neurotransmitter in the brain has been related to AD and other CNS diseases. Acetylcholine is degraded by AChE and BuChE. Therefore, compounds that competitively inhibit the action of these enzymes and maintain acetylcholine levels are clinically effective for symptomatic treatment of these CNS diseases.6 AChE inhibitors are the first line treatment for mild to moderate AD. Nowadays, new AD treatments are needed, and multitarget anti-AD agents seem to be good alternatives to actual treatments. Some lichenic compounds have already shown AChE inhibition activity in a dosedependent manner, as is the case of biruloquinone, extracted from the lichen Cladonia macilenta.56 Therefore, we evaluated Hypotrachyna formosana and Parmotrema perlatum methanol extract potential capacity to inhibit AChE and BuChE enzymatic activities. Hypotrachyna formosana extracts inhibited both AChE and BuChE activities in a concentration dependent manner. Particularly, the AChE inhibition potential of Hypotrachyna formosana extracts was 31.8% for 25 μg/mL, 39.5% for 50 μg/mL, and 45.7% for 100 μg/mL, whereas BuChE inhibition potential was 24.4% for 25 μg/mL, 35.2% for 50 μg/mL, and 41.2% for 100 μg/mL. However, Parmotrema perlatum only showed concentration dependent activity against AChE (11.4% for 25 μg/mL, 22.1% for 50 μg/ mL, and 31.4% for 100 μg/mL). None of both lichen extracts inhibited the activity of enzymes as much as galanthamine (positive control; Figure 7). The two lichens were less efficient than biruloquinone at inhibiting the AChE activity (IC50 was

Figure 9. TLC fingerprints of (1) Hypotrachyna formosana (a, lichexantone; b, lividic acid; c, physodic acid) and (2) Parmotrema perlatum (d, stictic acid; e, constictic acid).

perlatum did 2.14 fold versus Fenton reagent treated cells. Moreover, Hypotrachyna formosana extracts but not Parmotrema perlatum extracts significantly increased the GSH/GSSG ratio versus the Fenton reagent by 1.6 fold. No marked modifications were detected in GSSG levels for any treatments. Particularly, it is noteworthy that the Hypotrachyna formosana effect significantly increased both GSH levels and GSH/GSSG ratio compared with Parmotrema perlatum extract and Trolox. Previous studies have demonstrated that the extracts of some lichens (i.e., Cetraria islandica) are capable of increasing the levels of reduced glutathione.46,47 Lipid Peroxidation. Lipid peroxidation is an early event of oxidative damage in CNS disorders. Unsaturated phospholipids present in the cell membranes are vulnerable to ROS attack. This leads to a chain reaction that finishes with products as MDA and unsaturated aldehydes. This leads to cell membrane disrupture and cell death as a final step.48 Some lichenic compounds, such as atranorin, usnic acid, and fumarprotocetraric acid, have been demonstrated to be effective at reducing lipid peroxidation in the SH-SY5Y cell line.49−51 Therefore, we investigated the effect of Hypotrachyna formosana and Parmotrema perlatum extracts on lipid peroxidation using TBARS assay. As shown in Figure 5, the Fenton reagent caused a significant increase in lipid peroxidation (3.90 fold versus control). However, pretreatments with both lichen extracts significantly reduced lipid peroxidation (0.26 fold versus Fenton reagent cells for Parmotrema perlatum and 0.45 fold versus Fenton reagent 1174


Chemical Research in Toxicology 27.1 μg/mL). However, this inhibition combined with the antioxidant effect could be of interest for the treatment of AD. In addition, inhibition of BuChE is important in AD when the AChE activity decreases and the BuChE increases, especially in cortical regions of the brain.57 So the inhibition of both enzymes by Hypotrachyna formosana makes them a better treatment for the disease. Phytochemical Analysis. Lichens possess many unique metabolites; out of 1000 metabolites studied, 80% are restricted to a lichen state. The most common compounds are aromatic polyketides, more concrete depsides, depsidones, or dibenzofurans. For this reason, lichens have become a wide source of phenolic compounds. Phenols are potent antioxidants, so they are capable of donating protons to free radicals to neutralize them. They become a free radical themselves, but this radical is stabilized by resonance delocalization within the aromatic ring and formation of quinone structures.58 We investigated Parmotrema perlatum and Hypotrachyna formosana extract composition by TLC and HPLC methods. HPLC chromatograms of Parmotrema perlatum and Hypotrachyna formosana methanol extracts are shown in Figure 8. The major metabolite for Parmotrema perlatum was stictic acid (tR = 17.964 min ± 0.01 and UV λmax 213, 313, 237, and 270 nm) followed by constitic acid (tR = 13.845 min ± 0.01 and UV λmax 212, 310 nm). For H. formosana, the major compound was the orcinol depsidone lividic acid (tR = 27.271 min ± 0.02, 254 nm) followed by another orcinol depsidone physodic acid (tR = 24.517 min ± 0.01 and UV λmax 212, 263, 314 nm) and the xanthone lichexanthone (tR = 31.311 min ± 0.02 and UV λmax 209, 244, 308, 341 nm). TLC results are shown in Figure 9. P. perlatum showed stictic acid (orange color and Rf 18) and constictic acid (orange color and Rf 2) as secondary metabolites. Both stictic acid and constictic acid are depsidones. On the other hand, H. formosana contained physodic acid (pale orange color and Rf 11), lividic acid (orange brown color and Rf 33), and lichexanthone (pale yellow color and Rf 70).

■

ABBREVIATIONS

■

REFERENCES

AAPH, 2,2′- azobis(2-methylpropionamidine) dihydrochloride; AChE, acetylcholinesterase; ALS, amyotrophic lateral sclerosis; AD, Alzheimer’s disease; BuChE, butyrylcholinesterase; CAT, catalase; CNS, central nervous system; DCFH-DA, 2,7-dichloro-dihydrofluorescein diacetate; DMEM, Dulbecco’s modified Eagle’s medium; DMSO, dimethyl sulfoxide; DPPH, 1,1-diphenyl-2-picrylhydrazyl; EAP, extract antioxidant potency; FBS, fetal bovine serum; GPx, glutathione peroxidase; GR, glutathione reductase; GSH, reduced glutathione; GSSG, oxidized glutathione; GST, glutathione-S-transferase; HAT, hydrogen atom transfer; HPLC, high-performance liquid chromatography; MTT, 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyltetrazolium bromide; ORAC, oxygen radical antioxidant capacity; OS, oxidative stress; PBS, phosphate saline buffer; PD, Parkinson’s disease; ROS, reactive oxygen species; SET, single electron transfer; SOD, superoxide dismutase; tR, retention time; TBA, thiobarbituric acid; TPTZ, 2,4,6-Tris(2-pyridyl)-1,3,5-triazine

(1) Sies, H., Berndt, C., and Jones, D. P. (2017) Oxidative Stress. Annu. Rev. Biochem. 86, 715−748. (2) Ansari, M. A., and Scheff, S. W. (2010) Oxidative stress in the progression of Alzheimer disease in the frontal cortex. J. Neuropathol. Exp. Neurol. 69, 155−167. (3) Dias, V., Junn, E., and Mouradian, M. M. (2013) The role of oxidative stress in Parkinson’s disease. J. Parkinsons Dis. 3, 461−491. (4) Checkoway, H., Lundin, J. I., and Kelada, S. N. (2011) Neurodegenerative diseases. IARC Scientific Publications. 163, 407− 419. (5) Zhang, Y. J., Gan, R. Y., Li, S., Zhou, Y., Li, A. N., Xu, D. P., and Li, H. B. (2015) Antioxidant Phytochemicals for the Prevention and Treatment of Chronic Diseases. Molecules 20, 21138−21156. (6) Kumar, S. (2015) Dual inhibition of acetylcholinesterase and butyrylcholinesterase enzymes by allicin. Indian J. Pharmacol. 47, 444−446. (7) Krause, J., and Tobin, G. (2013) Discovery, Development and Regulation of Natural Products. In Using Old Solutions to New Problems-Natural Drug Discovery in the 21 Century (Kulka, M., Ed.), pp 3−35, st InTech Publishers. (8) Thell, A., Crespo, A., Divakar, P. K., Kärnefelt, I., Leavitt, S. D., Lumbsch, H. T., and Seaward, M. R. D. (2012) A review of the lichen family Parmeliaceae − history, phylogeny and current taxonomy. Nord. J. Bot. 30, 641−664. (9) Zambare, V. P., and Christopher, L. P. (2012) Biopharmaceutical potential of lichens. Pharm. Biol. 50, 778−798. (10) Fernández-Moriano, C., Gómez-Serranillos, M. P., and Crespo, A. (2016) Antioxidant potential of lichen species and their secondary metabolites. A systematic review. Pharm. Biol. 54, 1−17. (11) Fernández-Moriano, C., Divakar, P. K., Crespo, A., and GómezSerranillos, M. P. (2015) Neuroprotective activity and cytotoxic potential of two Parmeliaceae lichens: Identification of active compounds. Phytomedicine 22, 847−855. (12) Fernández-Moriano, C., Divakar, P. K., Crespo, A., and GómezSerranillos, M. P. (2017) Protective effects of lichen metabolites evernic and usnic acids against redox impairment-mediated cytotoxicity in central nervous system-like cells. Food Chem. Toxicol. 105, 262−277. (13) Divakar, P. K., Crespo, A., Kraichak, E., Leavitt, S. D., Singh, G., Schmitt, I., and Lumbsch, H. T. (2017) Using a temporal phylogenetic method to harmonize family-and genus-level classification in the largest clade of lichen-forming fungi. Fungal Divers. 84 (1), 101−117.

■

CONCLUSION The results demonstrated that Parmotrema perlatum and, especially, Hypotrachyna formosana are promising multitargeted neuroprotective agents. This neuroprotection action was mediated by the reduction of ROS and lipid peroxidation. Moreover, they increased the levels of GSH and the activity of the antioxidant enzymes GPx, CAT, SOD, and GR. In addition, the extract inhibited the enzymes AChE and BuChE implicated in the treatment of AD. These activities could be attributed to its unique secondary metabolites with polyphenol structure such as physodic acid. The results suggest that these lichens could be a source of natural antioxidants. However, it would be necessary to study the biological activity of the compounds present in the methanolic extract and study the effect of the extract in vivo.

■

Article

AUTHOR INFORMATION

Corresponding Author

*Tel.: +34 913941717. E-mail: [email protected]. ORCID

María Pilar Gómez-Serranillos: 0000-0003-2119-8768 Notes

The authors declare no competing financial interest. 1175


Article

Chemical Research in Toxicology (14) Divakar, P. K., Misra, S., and Upreti, D. K. (2004) Parmelioid Lichens in India: a Revisionary Study, Bishen Singh Mahendra Pal Singh. (15) Swinscow, T. D. V., and Krog, H. (1988) Macrolichens of East Africa, British Museum (Natural History). (16) Smith, C. W. (2009) Lichens of Great Britain and Ireland, British Lichen Society. (17) Upreti, D. K., Divakar, P. K., and Nayaka, S. (2005) Commercial and ethnic use of lichens in India. Econ. Bot. 59 (3), 269. (18) Gómez-Serranillos, M. P., Fernández-Moriano, C., GonzálezBurgos, E., Divakar, P. K., and Crespo, A. (2014) Parmeliaceae family: phytochemistry, pharmacological potential and phylogenetic features. RSC Adv. 4 (103), 59017−59047. (19) Amarowicz, R., Pegg, R. B., Rahimi-Moghaddam, P., Barl, B., and Weil, J. A. (2004) Free-radical scavenging capacity and antioxidant activity of selected plant species from the Canadian prairies. Food Chem. 84, 551−562. (20) Dávalos, A., Gómez-Cordovés, C., and Bartolomé, B. (2004) Extending applicability of the oxygen radical absorbance capacity (ORAC-fluorescein) assay. J. Agric. Food Chem. 52, 48−54. (21) Avan, A. N., Demirci Ç ekiç, S., Uzunboy, S., and Apak, R. (2016) Spectrophotometric determination of phenolic antioxidants in the presence of thiols and proteins. Int. J. Mol. Sci. 17, 1325. (22) Cásedas, G., Les, F., Gómez-Serranillos, M. P., Smith, C., and López, V. (2016) Bioactive and functional properties of sour cherry juice (Prunus cerasus). Food Funct. 7, 4675−4682. (23) Mosmann, T. (1983) Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J. Immunol. Methods 65, 55−63. (24) LeBel, C. P., Ischiropoulos, H., and Bondy, S. C. (1992) Evaluation of the probe 20,70 - dichlorofluorescin as an indicator of reactive oxygen species formation and oxidative stress. Chem. Res. Toxicol. 5, 227−231. (25) Mihara, M., and Uchiyama, M. (1978) Determination of malonaldehyde precursor in tissues by thiobarbituric acid test. Anal. Biochem. 86, 271−278. (26) Hissin, P. J., and Hilf, R. (1976) A fluorometric method for determination of oxidized and reduced glutathione in tissues. Anal. Biochem. 74, 214−226. (27) Aebi, H. (1984) Catalase in vitro. Methods Enzymol. 105, 121− 126. (28) Marklund, S., and Marklund, G. (1974) Involvement of the superoxide anion radical in the autoxidation of pyrogallol and a convenient assay for superoxide dismutase. Eur. J. Biochem. 47, 469− 474. (29) Paglia, D. E., and Valentine, W. N. (1967) Studies on the quantitative and qualitative characterisation of erythrocyte glutathione peroxidase. J. Lab. Clin. Med. 70, 158−169. (30) Barja de Quiroga, G., Perez-Campo, R., and Lopez Torres, M. (1990) Anti-oxidant defences and peroxidation in liver and brain of aged rats. Biochem. J. 272, 247−250. (31) Ellman, G. L., Courtney, K. D., Andres, V., and Feather-Stone, R. M. (1961) A new and rapid colorimetric determination of acetylcholinesterase activity. Biochem. Pharmacol. 7, 88−95. (32) Craft, B. D., Kerrihard, A. L., Amarowicz, R., and Pegg, R. B. (2012) Phenol-based antioxidants and the in vitro methods used for their assessment. Compr. Rev. Food Sci. Food Saf. 11, 148−173. (33) Seeram, N. P., Aviram, M., Zhang, Y., Henning, S. M., Feng, L., Dreher, M., and Heber, D. (2008) Comparison of antioxidant potency of commonly consumed polyphenol-rich beverages in the United States. J. Agric. Food Chem. 56, 1415−1422. (34) Ranković, B. R., Kosanić, M. M., and Stanojković, T. P. (2011) Antioxidant, antimicrobial and anticancer activity of the lichens Cladonia f urcata, Lecanora atra and Lecanora muralis. BMC Complementary Altern. Med. 11, 97. (35) Kosanić, M., Manojlović, N., Janković, S., Stanojković, T., and Ranković, B. (2013) Evernia prunastri and Pseudoevernia f urf uraceae lichens and their major metabolites as antioxidant, antimicrobial and anticancer agents. Food Chem. Toxicol. 53, 112−118.

(36) Kohanski, M. A., Dwyer, D. J., Hayete, B., Lawrence, C. A., and Collins, J. J. (2007) A common mechanism of cellular death induced by bactericidal antibiotics. Cell 130, 797−810. (37) Purnamasari, R., Winarni, D., Permanasari, A. A., Agustina, E., Hayaza, S., and Darmanto, W. (2019) Anticancer Activity of Methanol Extract of Ficus carica Leaves and Fruits Against Proliferation, Apoptosis, and Necrosis in Huh7it Cells. Cancer Inf. 18, 117693511984257. (38) Zhang, H., and Tsao, R. (2016) Dietary polyphenols, oxidative stress and antioxidant and anti-inflammatory effects. Curr. Opin. Food Sci. 8, 33−42. (39) Wang, J., Zhao, Y. M., Zhang, B., and Guo, C. Y. (2015) Protective effect of total phenolic compounds from inula helenium on hydrogen peroxide-induced oxidative stress in sh-sy5y cells. Indian J. Pharm. Sci. 77 (2), 163. (40) Amo de Paz, G., Raggio, J., Gómez-Serranillos, M. P., Palomino, O. M., González-Burgos, E., Carretero, M. E., and Crespo, A. (2010) HPLC isolation of antioxidant constituents from Xanthoparmelia spp. J. Pharm. Biomed. Anal. 53 (2), 165−171. (41) Paudel, B., Bhattarai, H. D., Koh, H. Y., Lee, S. G., Han, S. J., Lee, H. K., Oh, H., Shin, H. W., and Yim, J. H. (2011) Ramalin, a novel nontoxic antioxidant compound from the Antarctic lichen Ramalina terebrata. Phytomedicine 18, 1285−1290. (42) Melo, M. G. D., dos Santos, J. P. A., Serafini, M. R., Caregnato, F. F., de Bittencourt Pasquali, M. A., Rabelo, T. K., Moreira, J. C. F., da Rocha, R. F., Quintans, L., de Souza Araújo, A. A., da Silva, F. A., and Gelain, D. P. (2011) Redox properties and cytoprotective actions of atranorin, a lichen secondary metabolite. Toxicol. In Vitro 25 (2), 462−468. (43) Luo, Y., Li, X., Liu, T., Cao, Y., Zhang, J., Yaseen, A., Hu, W., Sun, F., Zheng, W., Jiang, Y., and Si, C.-L. (2019) Senkyunolide H protects against MPP+-induced apoptosis via the ROS-mediated mitogen-activated protein kinase pathway in PC12 cells. Environ. Toxicol. Pharmacol. 65, 73−81. (44) Fernández-Moriano, C., Divakar, P. K., Crespo, A., and GómezSerranillos, M. P. (2015) Neuroprotective activity and cytotoxic potential of two Parmeliaceae lichens: Identification of active compounds. Phytomedicine 22 (9), 847−855. (45) Pocernich, C. B., Cardin, A. L., Racine, C. L., Lauderback, C. M., and Butterfield, D. A. (2001) Glutathione elevation and its protective role in acrolein-induced protein damage in synaptosomal membranes: relevance to brain lipid peroxidation in neurodegenerative disease. Neurochem. Int. 39 (2), 141−149. (46) Kotan, E., Alpsoy, L., Anar, M., Aslan, A., and Agar, G. (2011) Protective role of methanol extract of Cetraria islandica (L.) against oxidative stress and genotoxic effects of AFB1 in human lymphocytes in vitro. Toxicol. Ind. Health 27 (7), 599−605. (47) Anar, M., Orhan, F., Alpsoy, L., Gulluce, M., Aslan, A., and Agar, G. (2016) The antioxidant and antigenotoxic potential of methanol extract of Cladonia foliacea (Huds.) Willd. Toxicol. Ind. Health 32 (4), 721−729. (48) Luo, Y., Li, X., Liu, T., Cao, Y., Zhang, J., Yaseen, A., Hu, W., Sun, F., Zheng, W., Jiang, Y., and Si, C.-L. (2019) Senkyunolide H protects against MPP+-induced apoptosis via the ROS-mediated mitogen-activated protein kinase pathway in PC12 cells. Environ. Toxicol. Pharmacol. 65, 73−81. (49) Fernández-Moriano, C., Divakar, P. K., Crespo, A., and GómezSerranillos, M. P. (2017) In vitro neuroprotective potential of lichen metabolite fumarprotocetraric acid via intracellular redox modulation. Toxicol. Appl. Pharmacol. 316, 83−94. (50) Rabelo, T. K., Zeidán-Chuliá, F., Vasques, L. M., dos Santos, J. P. A., da Rocha, R. F., de Bittencourt Pasquali, M. A., Gelain, D. P., Rybarczyk-Filho, J. L., Araújo, A. A. S., and Moreira, J. C. F. (2012) Redox characterization of usnic acid and its cytotoxic effect on human neuron-like cells (SH-SY5Y). Toxicol. In Vitro 26 (2), 304−314. (51) Melo, M. G. D., dos Santos, J. P. A., Serafini, M. R., Caregnato, F. F., de Bittencourt Pasquali, M. A., Rabelo, T. K., Moreira, J. C. F., da Rocha, R. F., Quintans, L., de Souza Araújo, A. A., da Silva, F. A., and Gelain, D. P. (2011) Redox properties and cytoprotective actions 1176


Article

Chemical Research in Toxicology of atranorin, a lichen secondary metabolite. Toxicol. In Vitro 25 (2), 462−468. (52) Liu, Z., Dong, L., Jia, K., Zhan, H., Zhang, Z., Shah, N. P., Wei, H., and Tao, X. (2019) Sulfonation of Lactobacillus plantarum WLPL04 exopolysaccharide amplifies its antioxidant activities in vitro and in a Caco-2 cell model. J. Dairy Sci. In press, DOI: 10.3168/ jds.2018-15831. (53) Rodriguez-Rodriguez, A., Egea-Guerrero, J., Murillo-Cabezas, F., and Carrillo-Vico, A. (2014) Oxidative stress in traumatic brain injury. Curr. Med. Chem. 21 (10), 1201−1211. (54) Verma, N., Behera, B. C., and Makhija, U. (2008) Antioxidant and hepatoprotective activity of a lichen Usnea ghattensis in vitro. Appl. Biochem. Biotechnol. 151 (2−3), 167−181. (55) Rabelo, T. K., Zeidán-Chuliá, F., Vasques, L. M., dos Santos, J. P. A., da Rocha, R. F., de Bittencourt Pasquali, M. A., Gelain, D. P., Rybarczyk-Filho, J. L., Araújo, A. A. S., and Moreira, J. C. F. (2012) Redox characterization of usnic acid and its cytotoxic effect on human neuron-like cells (SH-SY5Y). Toxicol. In Vitro 26 (2), 304−314. (56) Luo, H., Li, C., Kim, J. C., Liu, Y., Jung, J. S., Koh, Y. J., and Hur, J. S. (2013) Biruloquinone, an acetylcholinesterase inhibitor produced by lichen-forming fungus Cladonia macilenta. J. Microbiol. Biotechnol. 23 (2), 161−166. (57) Greig, N. H., Utsuki, T., Ingram, D. K., Wang, Y., Pepeu, G., Scali, C., Chen, D., et al. (2005) Selective butyrylcholinesterase inhibition elevates brain acetylcholine, augments learning and lowers Alzheimer β-amyloid peptide in rodent. Proc. Natl. Acad. Sci. U. S. A. 102 (47), 17213−17218. (58) Brewer, M. S. (2011) Natural antioxidants: sources, compounds, mechanisms of action, and potential applications. Compr. Rev. Food Sci. Food Saf. 10 (4), 221−247.

1177


Lichens of Parmelioid Clade as Promising Multitarget Neuroprotective

Recommend Documents