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# Harnessing thermal spring water and hydrophobic eutectic solvent for galactoglucomannans, glucans and phenolics recovery from Evernia prunastri and their potential biological applications

Julie Queffelec, Ramón Novoa-Carballal, Nathalie Bourgougnon, Christel

Marty, Filipa Teixeira, Francisca Rodrigues, Paulo C Costa, María D Torres, Herminia Domínguez

► To cite this version:

Julie Queffelec, Ramón Novoa-Carballal, Nathalie Bourgougnon, Christel Marty, Filipa Teixeira, et al.. Harnessing thermal spring water and hydrophobic eutectic solvent for galactoglucomannans, glucans and phenolics recovery from Evernia prunastri and their potential biological applications. International Journal of Biological Macromolecules, 2025, 329 (Part 1), pp.147613. 10.1016/j.ijbiomac.2025.147613. hal-05302991

HAL Id: hal-05302991

https://hal.univ-brest.fr/hal-05302991v1

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# Harnessing thermal spring water and hydrophobic eutectic solvent for galactoglucomannans, glucans and phenolics recovery from *Evernia prunastri* and their potential biological applications

Julie Queffelec<sup>a,d</sup>, Ramón Novoa-Carballal<sup>b,c</sup>, Nathalie Bourgougnon<sup>d</sup>, Christel Marty<sup>d</sup>, Filipa Teixeira<sup>e,f</sup>, Francisca Rodrigues<sup>e,f</sup>, Paulo C. Costa<sup>e,f</sup>, María D. Torres<sup>a,*</sup>, Herminia Domínguez<sup>a</sup>

## ARTICLE INFO

### Keywords:

* Evernia prunastri
* Thermal spring water
* Hydrophobic eutectic solvent
* Antioxidant activity
* Polysaccharides
* Biopolymer film

## ABSTRACT

Lichens produce polysaccharides scarcely studied, in addition to secondary metabolites, that can exhibit high bioactivity. The aim of this work is to evaluate the efficiency of subcritical water extraction using thermal spring water and hydrophobic eutectic solvent for the extraction of these compounds. Subcritical water extraction of *E. prunastri* compounds was performed between 150 and 190 °C with distilled or with thermal spring water, combined or not with eutectic solvent men:lau as modifier. The biochemical composition of the polysaccharide extracts from each process (P-D for distilled water, P-T for thermal spring water, P-DM and P-TM when the eutectic was added) and of the soluble liquid extracts free from the precipitated polysaccharides (E-D, E-T, E-DM and E-TM) was analyzed as well as their antioxidant capacity and biological activity against herpes viruses. After purification through resin of E-T170 and E-TM150, extracts with an antioxidant activity of 1.38 and 1.78 g Trolox eq./ g of extract were obtained. Polysaccharides were identified as galactoglucomannans and glucans by NMR analysis. All polysaccharides were formulated in films, however, those extracted with thermal water presented much better viscoelastic properties than those extracted with distilled water. P-T-170 °C and P-TM-150 °C were non-irritating according to *Skinethic®* test but did not exhibit protective activity against HSV-1 and HSV-2 viruses. E-T170 °C presented a slightly higher antiviral activity (around 40 % protection) with low cytotoxicity. In addition, the films' physical and mechanical properties suggest potential for biomedical applications, especially in topical or mucosal delivery systems.

## 1. Introduction

Lichens are symbiosis between fungus and microalgae that synthesize a large set of compounds, called lichenic substances [1]. They are extracted for the perfume industry and investigated in the pharmaceutical sector for their antibacterial, anti-inflammatory, anti-tumoral, antiviral, and photoprotective properties [2,3]. The cell walls and extracellular matrix of lichens also contain valuable polysaccharides that are less studied than the other compounds and remain in the solid

residue after the extraction of the secondary metabolites. These polysaccharides, mainly glucans and galactomannans, may have immunostimulatory, anti-tumoral and anti-inflammatory activity [4-9]. They have significant potential as biomaterials for skin applications. Indeed, antibacterial biomaterial in film form have also been produced from these polysaccharides [10]. However, their effectiveness is closely linked to the polysaccharide's structure, solubility, and molecular weight.

Among the 20,000 species of lichen, *Evernia prunastri* (L.) Ach

<footnote><sup>a</sup> CINBIO, Universidade de Vigo, Departamento de Ingeniería Química, 32004, Ourense, Spain</footnote>

<footnote><sup>b</sup> CINBIO, Universidade de Vigo, Departamento de Química Orgánica, 36310, Vigo, Pontevedra, Spain</footnote>

<footnote><sup>c</sup> Instituto de Investigación Sanitaria Galicia Sur (IIS Galicia Sur), 36312, Vigo, Pontevedra, Spain</footnote>

<footnote><sup>d</sup> Laboratoire de Biotechnologie et Chimie Marines, Université Bretagne Sud, EMR CNRS 6076, IUEM, 56000, Vannes, France</footnote>

<footnote><sup>e</sup> Associate Laboratory i4HB - Institute for Health and Bioeconomy, Faculty of Pharmacy, University of Porto, 4050-313, Porto, Portugal</footnote>

<footnote><sup>f</sup> UCIBIO - Applied Molecular Biosciences Unit, MedTech-Laboratory of Pharmaceutical Technology, Faculty of Pharmacy, University of Porto, 4050-313, Porto, Portugal</footnote>

<footnote>* Corresponding author.
E-mail address: matorres@uvigo.es (M.D. Torres).</footnote>
(Ascomycota, Parmeliaceae) was selected in this study for its abundance in Spain, its easy identification, and its content in film-forming polysaccharides. Traditional polysaccharide extraction in alkaline or acidic media has the drawback of requiring toxic chemicals and can be both time-consuming and energy intensive. Pressurized hot water extraction, under subcritical conditions, is considered as an eco-friendly technique to extract polysaccharides [11]. Less polar compounds (such as phenolics) can also be extracted by subcritical water as the dielectric constant of water under those pressure and temperature conditions decreases, making it a more apolar solvent [12,13]. Subcritical water extraction also has the advantage of having been successfully scaled up from lab scale to pilot scale.

In this work, thermal spring water was used for the first time to performed subcritical water extraction, as its physicochemical properties may impact the extraction of lichen metabolites. Thermal spring waters have been used since ancient times for both therapeutic and recreational purposes [14–16]. The unique physicochemical composition has demonstrated beneficial effects on the skin, justifying the emergence of thermal spring water therapies. These treatments are said to cure not only skin disorders but also rheumatic diseases or respiratory issues [17]. Thermal spring waters are classified into categories in function of their chemical composition and physical characteristics. For example, they are called oligometalic when their dry residues is lower than 100 mg/L, and highly mineralized when it is higher than 1000 mg/L [18]. In this work, hyperthermal, medium mineralized water from Ourense city spring was used. This water is specifically recommended for different types of dermatoses especially pruritic ones, as sodium and silica content contribute to anti-inflammatory effects.

Among the recently emerging extraction techniques, natural eutectic solvents are also attracting growing interest. They are a mix of at least two compounds, a hydrogen bonds donor and a hydrogen bonds acceptor. The hydrogen bonds they establish make the melting point of the system decrease, forming a homogeneous liquid at a temperature much lower than the melting temperature of each compound. These solvents raised interest for their high solubilization properties [19–21]. Indeed, depending on the compounds selected, their polarity can be tuned to favor the extraction of different target molecules [22].

The novelty of this study lies in the use of thermal spring water and an understudied hydrophobic eutectic solvent to extract polysaccharides and phenolic compounds from *Evernia prunastri* and in their subsequent characterization. The evaluation of some of their potential biological activities, including their antiviral properties against herpes viruses and skin irritation potential, was also carried out. Furthermore, this study includes the formulation of the polysaccharides into films for topical or mucous membrane applications, such as oral mucositis, and their stability study over 3 months.

## 2. Materials and methods

### 2.1. Raw material

Lichen from *Evernia prunastri* (L.) Ach. species has been collected in Ribeira Sacra (Ourense, Spain) (altitude 608 m, 42° 24′ 47″ North, 7° 43′ 34″ West, 31/12/2023). Samples were air dried, then milled and stored in the dark. The lichen was identified using Standard Methods by Prof. María Eugenia López de Silanes (University of Vigo, Spain). A voucher specimen can be found at the Herbarium SANT (University of Santiago de Compostela, Spain).

The lichen was coarsely ground (size <1 cm) and sieved to remove the smallest particles. It was found to have 11 % moisture, and on dry basis, 1.1 ± 0.1 % lipids, 2.3 ± 0.3 % ash, 9.9 ± 0.3 % protein, 14.9 ±

0.1 % acid insoluble residue, and 68.79 ± 2.36 % carbohydrates: mainly glucose 30.9 %, galactose 13.7 %, mannose 12.4 % (Queffelec et al., 2024).

Thermal water, (T = 64–68 °C, dry residue = 649.2 mg/L, pH = 7.56) was collected in Burgas Thermal Spring (Ourense, Spain). Its main compounds are bicarbonate (≈ 462 mg/L), sodium (≈ 102 mg/L), silica (≈ 72 mg/L), chloride (≈ 25 mg/L), fluoride (≈ 14 mg/L), calcium (≈ 11 mg/L) and potassium (≈ 8 mg/L) according to the characterization made by Souto and Vermello in 1990.

The eutectic solvent DL-menthol:lauric acid (2:1) (men:lau) was made by heating and stirring menthol (C₁₀H₂₀O) and lauric acid (C₁₂H₂₄O₂) at 70 °C until a transparent liquid was obtained.

### 2.2. Pressurized hot water extraction

Lichen was mixed with distilled or thermal water, at a liquid-to-solid ratio of 1:30(w/w) in a 0.1 L stainless steel reactor (Parr Company, Illinois, USA), without and with 5 % (w/w) of men:lau. The mixture was heated at 150, 160, 170, 180, 190 °C and immediately cooled down when the temperature was reached. The general scheme of the experiment is displayed on Fig. 1.

### 2.3. Polysaccharides recovery, characterization and film formulation

The soluble extracts were frozen for 2 days. After thawing, insoluble polysaccharides precipitated in the liquid fractions. They were centrifuged at 6000 rpm for 5 min, and subsequently freeze-dried. Their yields (%) were determined gravimetrically. The polysaccharides are coded as P—D if extracted with distilled water, P-T for thermal water, P-DM for distilled water with eutectic and P-TM for thermal water with eutectic. The temperature is added at the end of the code when a specific extract is used (P—D180, for example, corresponds to the polysaccharide extracted with distilled water at 180 °C). The same nomenclature is used for the soluble extracts using E instead of P.

Films were made using the polysaccharides obtained with the highest yield for each solvent: F—D180, F-T170, F-DM160, F-TM150. Polysaccharides (2 % w/w) were dissolved in hot water with glycerol at 40 % (dw polysaccharides). 2 mL of the solution was placed in a circular cup (diameter = 2.4 cm) and dried in an oven at 40 °C.

### 2.4. Structural characterization

#### 2.4.1. Molecular weight distribution

High-performance size exclusion chromatography (HPSEC) was used to determine the molecular weight distribution. Measurements were done on a HPLC (Agilent Technologies, California, USA) equipped with a TSKgel SuperMultiporePW-H column (6 mm × 15 cm) and a TSKgel SuperMP(PW)-H guard column (4.6 mm × 3.5 cm) (Tosoh, Tokio, Japan) at 40 °C. Milli-Q water (0.4 mL min⁻¹) was used as the mobile phase. Standards of polyethylene oxide from 23.6 × 10³ to 786 × 10³ g/mol were used.

#### 2.4.2. NMR spectroscopy

Prior to NMR analysis all samples were suspended at room temperature in excess deuterated water, stirred for 2 h, and then freeze dried. This process was repeated 3 times to diminish HOD signal (NMR in D₂O) and simplify the spectra in DMSO (OH- exchanged into OD). Nuclear Magnetic Resonance was conducted on a Bruker ARX400 spectrometer (Bruker BioSpin GmbH, Germany) at 75 °C in D₂O or DMSO-d₆. Quantitative ¹H NMR was conducted with 8 scans and an inter-scan relaxation delay 10 s (ca. 5xT1) to assure complete relaxation after each scan. One
<figure><img src="image_4.png" /><figcaption>Fig. 1. General scheme of the experiment.</figcaption></figure>

dimensional ¹H diffusion-filtered spectrum was measured at 75°C was measured with 18 scans using the Stimulated-Echo pulse sequence (bruker pulse sequence stebpg1s1d). The diffusion delay Δ was set to 50 ms, and the duration of each PFG pulse to encode diffusion was 1 ms, with a gradient power of 0.5 T/m. Samples for 1H and ¹H diffusion-filtered spectra were prepared at 8 g/L.

Two-dimensional spectra HSQC, COSY and TOCSY and ¹³C NMR spectra were measured at 20 g/L in the polysaccharide obtained after extraction in distilled water at 190 °C. Bruker pulse sequence hsqcetgps was used for the HSQC (112 scans, 512 complex points in the time domain, and an inter-relaxation delay of 1.7 s, D4 was optimized for a typical JCH value of sugar signals: 145 Hz.). Pulse sequence cosygpqf was selected for the COSY (8 scans, 512 complex points in the time domain, and 1.7 s inter-relaxation delay) and pulse sequence mlevetgp for the TOCSY (8 scans, 512 complex points in the time domain, and 1.7 s inter-relaxation delay). 13C NMR was recorded with a zgpg45.anv pulse sequence (16,640 scans, and an inter-relaxation delay of 2.6 s).

For the composition analysis the integration regions selected were 5.50 to 4.70 for galactoglucomannans and 4.60–4.10 ppm for glucans. These regions together with the additional anomeric region 6.20–6.00 were considered as a 100 % of sugars. The acetyl methyl region (2.15–1.95) was integrated versus this 100 %. Given that the sample is a polysaccharide mixture and that the difference in number of H in the acetyl methyl and sugar regions are not considered this cannot be considered as a precise % of acetylation but it is useful to assess changes in acetylation along the extraction conditions.

### 2.4.3. FTIR

The films were analyzed at least in duplicate by Fourier-Transform InfraRed spectroscopy (FTIR) using a Nicolet 6700 spectrometer (Thermo Fisher Scientific, Massachusetts, USA). Samples were blended with KBr and the spectra were monitored from 500 to 4000 nm using a resolution of 4 cm⁻¹ and 32 scans/min using OPUS-2.52 software (Opus Software Limited, UK).

### 2.4.4. Scanning Electron microscopy

The surface morphology of the different films was studied by Scanning Electron Microscopy (SEM), in a JEOL JSM6010LA equipment, after coating them with a 15 nm layer of gold.

## 2.5. Stability study

The weight and the viscoelastic properties of the films made with the polysaccharides having the greatest yield for each solvent were monitored over 3 months at 15, 30, 45, 60, 90 days.

## 2.6. Films characterization

### 2.6.1. Mass uniformity

Buccal films weight was measured using an analytical balance (KERN ALS, Radwag, and Mettler Toledo) to evaluate mass uniformity. Individual 4 cm² sections from various batches were separately weighed, and the average weight along with the standard deviation was calculated.

### 2.6.2. Thickness

Thickness was evaluated using a calibrated electronic digital micrometer (MICROM-DIG, Portugal). The measurements were performed in three different sections of each buccal film portion (4 cm²) from different batches (n = 9).

### 2.6.3. Moisture content

Moisture content was evaluated according to Teixeira et al [23]. Briefly, 4 cm² sections of films from different batches (n = 3) were weighed and placed in an infrared moisture analyzer (AD-4713, A&D Company, Tokyo, Japan), where they were heated at 80 °C for 30min using an infrared lamp. The device automatically determined the moisture content by calculating the difference in weight before and after heating.

### 2.6.4. Mechanical properties

Percent elongation and Young's modulus (Eq. (1) and Eq. (2), respectively) were evaluated in three independent film portions (n = 3) using a texture analyzer (TA.XT plus Texture Analyzer, Stable Micro Systems, Cardiff, UK) equipped with the Texture Exponent 32 software (version 6.1.12.0, Stable Micro Systems; Surrey, UK). Briefly, the buccal films were cut in 4 × 1 cm portions and were vertically held using the tensile grip probe with a separation of 20 mm. Afterwards, films were stretched until rupture by moving the probe at a constant speed of 0.1
mm/s. The force and height calibrations were performed with a weight of 5 kg and Mini Tensile Grips (Stable Micro Systems), respectively.

### 2.7.3. Phenolic compounds

The quantity of phenolic compounds has been estimated with the Folin-Ciocalteau method [25]. In test tubes, 1.875 mL of distilled water,

$$ \text{Percent elongation (\%)} = \frac{\text{distance at the rupture instant} - \text{initial grip distance}}{\text{initial grip distance}} \times 100 \qquad (1) $$

0.125 mL of the Folin reactive diluted at (1:1) with distilled water and

$$ \text{Young's modulus (MPa)} = \frac{\text{force at corresponding strain}}{\text{cross-sectional area of the film} \times \text{corresponding strain}} \qquad (2) $$

0.25 mL of Na₂CO₃ at 20 % were added to 0.25 mL of sample and mixed all together. After one hour in the dark, the absorbance was measured at λ = 765 nm. The standard curve was set up using gallic acid dissolved in water at concentration between 0.1 and 0.00625 mg/mL. Eqs. 4 represents the calibration curve (concentration vs. absorbance).

$$ y = 9.7401 \times + 0.0056 (R^2 = 0.9996) (3). $$

### 2.6.5. Frequency sweep

Viscoelastic properties of the different films were determined in a MCR 302 (Paar Physica, Austria) controlled stress rheometer with a Peltier module, with a parallel sandblasted plate (25 mm) with a gap of 0.2 mm. The elastic (G') and viscous (G") moduli were measured doing a frequency sweep analysis in the linear viscoelastic region and carried out between 1 and 10 Hz.

## 2.7. Biochemical composition analysis

Biochemical composition analysis was done on the soluble extracts after the precipitation of the polysaccharides. These extracts are coded E-D, E-T, E-DM, E-TM in function of the solvent used. Like for the polysaccharides, the extraction temperature is sometimes added to the code when a specific extract is used.

### 2.7.1. Oligosaccharide content

Measurements were conducted in HPLC (1100 series Agilent Technologies, California, USA) to evaluate the oligosaccharide content present in the liquid phase after precipitating the polysaccharides. The HPLC was equipped with refraction index and ultraviolet diode array detectors. An Aminex HPX87H column (300 × 7.8 mm, BioRad, USA) operating with 0.003 M sulfuric acid (Sigma-Aldrich, USA) as mobile phase (0.6 mL/min) at 60 °C. A posthydrolysis was carried out on the liquid phases to determine the oligosaccharide content, employing sulfuric acid (4 %). Liquid phases were autoclaved for 20 min at 121 °C, then, the Pyrex canisters were cooled at room temperature before filtering the samples (0.45 µm). Galactose, mannose and xylose were quantified in a CARBOSep CHO-682 column (Transgenomic Inc., Omaha, NE 68164, USA) operated at 80 °C with deionized water as mobile phase (flow rate 0.6 mL/min) after neutralizing the acid with barium carbonate. The difference between the monosaccharide content in the extract after and before the post hydrolysis was considered as the oligosaccharide content.

### 2.7.2. Soluble protein content

The Bradford micro assay protocol was followed to determine the soluble protein content [24]. Briefly, 0.5 mL of Bovine Serum Albumin from 1 µg/mL to 9 µg/mL were mixed with 0.5 mL of Bradford reagent to set the standard curve. The absorbance at λ = 595 nm was measured after 16 min. The same protocol was followed with the samples.

### 2.7.4. Purification of the phenolic compounds

SP700 resin was previously regenerated shaking 5 mL of methanol for each g of resin, during 15 min at 175 rpm, 25 °C and then filtrated, washed with deionized water and dried in an oven at 50 °C. The resin was then activated with ethanol 96 % following the protocol previously described.

Phenolic compounds from the soluble extracts were recovered shaking 15 g of liquid phase (T170 and TM150, the liquid phase corresponding to the best films) with 3 g of dry SP700 resin during 1 h at 25 °C and 175 rpm. The mixture was then filtrated and washed with distilled water.

The desorption of phenolic compounds was performed by contacting ethanol 96 % with the moist resin (weight ratio 9:2) during 1 h at 175 rpm and 25 °C. The mixture was then filtered and the dry content of the recovered phase in ethanol was calculated. A previous study comparing the TEAC and Folin values of the desorbed product in ethanol at 70, 80 and 96 % was done.

### 2.7.5. HPLC analysis of phenolic compounds

The ethanolic fractions recovered after the resin and the men:lau eutectic phase recovered directly after the extraction, diluted 40 times in ethanol absolute, were analyzed by HPLC (1100 series Agilent Technologies, California, USA) in a ROC C18 column 250 × 4.6 mm (Restek Corporation, Bellefonte, Pennsylvania USA), at 30 °C, with two mobile phases (flow rate of 1 mL/min); Phase A and B consisted in acetonitrile: ultrapure water:formic acid at (10:85:5) and (90:5:5) respectively and with the gradient: 0 min, 100 % A, 40 min 85 % B, 15 % A, 45 min 100 % B and 60 min 100 % A for a total time of 65 min. The equipment was connected to a DAD detector measuring at 250, 260, 270, 280, 360, 520 nm. The spectra at 280 nm were used for peaks identification. Standards used were 4-hydroxybenzoic acid, gallic acid, chloroatranol, ellagic acid, 4-hydroxycinnamic acid, p-coumaric acid, quercetin, caffeic acid, vanillin, benzoic acid, vanillic acid, salicylic acid, evernic acid, myricetin, atranol, orsellinic acid, and usnic acid.

## 2.8. Evaluation of biological activity

### 2.8.1. SkinEthic irritation test

SkinEthic™ irritation test was performed on a selected system based
on the best viscoelastic features to predict and categorize their skin irritation potential by using the SkinEthic™ Reconstructed human Epidermis (RHE) model. This method has been revealed to distinguish skin irritant chemicals and mixtures (Category 2) from chemicals not-classified for skin irritation (no category) following the United Nations Globally Harmonized System of Classification and Labelling of Chemicals (UN GHS). The tests have been conducted according to the guidelines provided by the commercial supplier (EPISKINTM) and the SkinEthic™ (DB-ALM Protocol n° 135). This protocol is compliant with the OECD Test Guideline No. 439 - In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method OECD, Test No. 439: In vitro skin irritation: Reconstructed human epidermis test method, OECD guidelines for the testing of chemicals, section 4, OECD publishing, Paris 2032 (Cho et al., 2014).

### 2.8.2. Cytotoxicity and antiviral activity evaluation by cell viability

Vero cell lines (line no. ATCC CCL81) were cultivated in Eagle's MEM supplemented with 8% fetal calf serum (FCS, Eurobio, France), 1% L-glutamine (200 mM), and 1% PCS (penicillin (10,000 U), colimycin (25,000 U), and streptomycin (10 mg). The culture was maintained at 37 °C under a 5% CO₂ atmosphere, and the medium was renewed daily. Herpes simplex virus type 1 and two acyclovir resistant strain (HSV-1 & HSV-2; family Herpesviridae) was provided by Prof. David Boutolleau Virology Department, Hospital Pitié-Salpétrière, France.

Samples were diluted at 5 different concentrations (100, 250, 500, 750, 1000 µg/mL). Acyclovir (2-amino-1,9-dihydro-9-[(2-hydroxyethoxy)methyl]-6H-purin-6-one) (0.05–5 µg/mL) and heparin (an heterogenous glycosaminoglycan made of repeated units of sulfated iduronic acid or glucuronic acid with glucosamine, N-sulfated and 6-O-sulfated) (1–200 µg/mL) were used as positive control. 2 samples at the 5 concentrations were tested per plate. 100 µL of cell suspension (3.5 × 10⁵ cells/mL) was added in all wells of a 96-well flat-bottom microplates TC Flacon (Corning, France). 100 µL of MEM supplemented was added to the cells control column and 50 µL to the virus control column and to the fourth upper column of the plate, from raw 1 to 10, for the cytotoxicity test (n = 4). Then, 50 µL of sample was added, each concentration in one column. Finally, 50 µL of viral suspension (MOI 0.001 ID₅₀/cells) was added to the virus control column and to the fourth bottom raw, column 1 to 10 for the anti-viral analysis (n = 4). Following the same protocol, another microplate was prepared with heparin (1–200 µg/mL) and Acyclovir (0.05–5 µg/mL) instead of the sample. This plate was prepared each time the antiviral activity of samples was evaluated. A representation of the microplate is displayed in Supplementary material 1. The plates were incubated 72 h, at 37 °C in an oven with 5% CO₂. After incubation, 50 µL of neutral red was added to all wells, and the plate was incubated in the same conditions for 45 min. The wells were washed 2 times with 100 µL of PBS, then 100 µL of ethanol citrate was added and homogenized with a pipette to destroy the living cells and release the neutral red. The optical density at 540 nm was measured in a spectrophotometer as it is related to the quantity of neutral red that enter viable cells and thus directly related to the cytotoxic effect. The EC₅₀ is the effective antiviral concentration, the concentration at which 50% of protection of virus-infected cells from virus-induced destruction is observed. The percentage of protection P is determined by Eq. 3:

$$P(\%) = \frac{(OD_s)_{\text{virus}} - (OD_c)_{\text{virus}}}{(OD_c)_{\text{sample}} - (OD_c)_{\text{virus}}} \times 100 \quad (3)$$

With $(OD_s)_{virus}$ the average optical density (4 wells) of the cells with the tested sample infected by the virus, $(OD_c)_{virus}$ the average optical density (8 wells) of the infested cells, $(OD_c)_{sample}$ the average optical density (4 wells) of the cells with the sample, without the virus.

The cytotoxicity of the samples on cells was characterized by the CC₅₀, the concentration that reduce the OD of the cells treated with the sample by 50% with respect to untreated cells, expressed in destruction

percentage D (Eq. 4):

$$D(\%) = \frac{(OD_c)_{\text{sample}} - (OD_c)_{\text{cells}}}{(OD_c)_{\text{cells}}} \times 100 \quad (4)$$

With $(OD_c)_{cells}$ the average optical density (8 wells) of the control cells without sample neither virus [26–28].

### 2.8.3. Antioxidant activity

The antiradical capacity of the samples was evaluated using the ABTS (2, 2'-Azinobis (3-ethylBenzoThiazoline-6-Sulfonic acid) radical scavenging spectrophotometric technique, following the procedure described by Re [29]. The Trolox Equivalent Antioxidant Capacity (TEAC) reagent was diluted in a Phosphate-Buffered Saline solution (PBS) to reach an absorbance of 0.7 ± 0.05 nm. Then, 1 mL of this reactive was added to 10 µL of sample and incubated for 6 min at 30 °C. The blank was the PBS, the absorbance was measured at λ = 734 nm. A control with water was placed each 15 measures. The standard curve was set using Trolox in water from 0.2 to 2 mM. Eqs. 4 represents the calibration curve (concentration vs. absorbance).

$$y = 1.0463 \times -0.0188 (R^2 = 0.9997) \quad (5)$$

## 2.9. Statistical analysis

Statistical analysis was performed on R studio.

Levene and Shapiro-Wilk tests were performed to assess the homogeneity of variances and the normal distribution of the residues, respectively. Among all the response variables tested (overall extraction yield, protein content, phenolic content, antioxidant activity, and polysaccharide yield), the phenolic content and antioxidant activity met both assumptions, (p-values >0.05). For these two response variables, two-way ANOVA followed by Tukey Post-hoc tests were performed at 5% level of significance to analyze the impact of the solvent and the temperature. For the overall extraction yield, the protein content, and the polysaccharide yield, at least one of the assumptions was not met. It was thus chosen to perform Sheirer-Ray-Hare non-parametric test followed by Dunn Post-hoc tests using Bonferroni correction (each significant factor one by one, level of significant 5 %). In this preliminary study, each condition was tested in triplicate; however, this low number of replicates may limit the statistical robustness of the findings. To reduce this uncertainty, estimated marginal means were also calculated using “emmeans” package on RStudio. 95% Confidence intervals were thus obtained.

# 3. Results and discussion

## 3.1. Extraction yields and biochemical characterization of the soluble extracts

### 3.1.1. Overall extraction yield

Fig. 2.A illustrated the influence of the solvent and temperature on total extraction yield. It is observed that the total extraction yield increases with the temperature, as the severity of the extraction increases, and with the addition of eutectic solvent, varying from 34.6% for distilled water at 150 °C to 92% for thermal water with eutectic as modifier at 190 °C. However, the type of solvent used (distilled or thermal water, with or without eutectic) has not a statistically significant effect on this variable (Sheirer-Ray-Hare test).

### 3.1.2. Protein extraction

Fig. 2.B showed the protein content of the different liquid phases. The highest protein content, 10.6 mg BSA/g is reached at 160 °C with thermal water and eutectic. The temperature and the interaction between solvent and temperature didn't have a significant effect on the protein content, in contrast to the solvent, which showed a significant
<figure><img src="image_5.png" /><figcaption>**Fig. 2.** Influence of the solvent and temperature on total extraction yield (A), protein content (B), total phenolic content (C), and ABTS antiradical capacity determined by the TEAC method (D) of the *Evernia prunastri* lichen extracted with pressurized hot distilled water, thermal spring water alone or modified by men:lau at 5 %. Results with the same letters are not significantly different (Two-way ANOVA followed by Tukey Test, 5 % level of significance).</figcaption></figure>

impact (Sheirer-Ray-Hare and Bunn Post-hoc tests). Indeed, the use of thermal water has a significant positive impact on the extraction of protein multiplying by almost 10 the extraction protein compared to distilled water. This may be due to the higher ionic strength in thermal water (estimated around $7 \times 10^{-3}$ mol/L), which might disrupt the protein-matrix interactions within the biomass and increase the solubility of proteins that are amphiphilic. Arodundade et al. [30], observed that, at slightly acidic to neutral pH, (which is the case here as the pH of the liquid phases with distilled water are around 4.3–4.4 and with thermal water are between 5.3 and 5.8), the solubility of broad bean proteins increased at low ionic strengths (maximum observed at a ionic strength of 0.4 mol/L) before decreasing at higher ionic strengths. Too high ionic strength can impact negatively proteins’ solubility as it may affect their secondary structure, making them more hydrophobic [31]. The decrease in protein yield from 170 °C is probably due to their thermal degradation.

<figure><img src="image_6.png" /><figcaption>**Fig. 3.** Yield of the recovered compounds after passing the soluble extracts from the process with thermal spring water at 170 °C (E-T170) and with thermal spring water and eutectic at 150 °C (E-TM150) through SP700 resin and desorption with ethanol at 70, 80 and 96 % and the phenolic content and Trolox Equivalent Antioxidant Capacity (TEAC) of each fraction. The ethanol concentration (for all concentrations) has a significant impact on the phenolic content and TEAC results at a 5 % level of significance.</figcaption></figure>

### 3.1.3. Phenolic compounds extraction and antioxidant capacity

The solvent, the temperature and the interaction had a significant impact on the extraction of phenolics and on the antioxidant activity according to Two-way ANOVA test (Estimated marginal means are displayed in Supplementary materials 2–7). The total phenolic content (Fig. 2.C) and the Trolox Equivalent Antioxidant Capacity (TEAC) (Fig. 2.D) exhibit similar trends with a maximum reached at 150 °C for all solvents. However, the maximum values were obtained for thermal water, with a phenolic content of 19.58 g/100 g of extract, a TEAC value of 96.9 g Trolox eq./100 g of extract against a phenolic content of 18.08 g/100 g of extract and a TEAC value of 77.4 g Trolox eq./100 g of extract for distilled water. Thermal water seems to enhance the extraction of phenolics and the antioxidant activity of the extracts. In that case, the addition of the eutectic solvent significantly lowered the phenolic content in the aqueous phase (14.6 g phenolic compounds/100 g of extract with thermal water) and its antioxidant activity (63.3 g Trolox eq./100 g of extract with thermal water). As the overall extraction yields are globally similar or higher with the addition of the eutectic solvent, these lower yields seem to indicate that the most hydrophobic of these compounds are concentrated in the men:lau phase which is immiscible with water. Queffelec et al. [10] found usnic acid in the men:lau phase after hydrothermal microwave assisted extraction with this eutectic solvent as modifier.

### 3.1.4. Phenolic compounds purification and identification

Phenolic compounds from E-T170 and E-TM150 (the best extraction yields for precipitated polysaccharides, described further below) were concentrated using SP700 resin. Three different ethanol concentration were used for the desorption. Fig. 3 shows the phenolic content, the antioxidant activity using TEAC method and recovery yield of the extracts in function of the ethanol concentration used for the desorption. It can be first observed that more compounds are recovered from E-T170 than E-TM150 with maximum value of 0.56 and 0.43 g/L, obtained with ethanol at 70 %. This can be explained by the fact that some apolar compounds concentrated in the eutectic phase for E-TM150. The decrease in the recovery yield with the increase in ethanol content is probably due to a higher purification of the phenolics, as some other
more polar compounds probably desorbed at the lowest ethanol con-
centration. The ethanol concentration had a significant impact on the
total phenolic content and the antioxidant activity, that followed the
same trend with increasing results with the increase in ethanol con-
centration reaching phenolic content values of 46.7 and 72.6 g GAE/
100 g and 138.1 and 177.7 g Trolox eq./100 g respectively for E-T170
and E-TM150. These results, significantly higher than those of the ex-
tracts before the resin step indicates that the purification was success-
fully performed. It is interesting to observed that, at the highest degree
of purification, the highest phenolic content and antioxidant activity are
observed in E-TM150. The highest phenolic content of E-T170 before
purification could thus be related to other non-phenolic compounds
reacting with the Folin reagent. This may indicate that the addition of
the eutectic solvent could purifies the aqueous extracts from the less
water-soluble compounds, leading to less compounds but with higher
antioxidant activity than in E-T170. The lower temperature that can be
used thank to the addition of ment: lau might also potentially prevent
some thermal degradation of the phenolic compounds. The particularly
high ABTS+ radical scavenging activity of the purified extracts should
be emphasized. Such high antioxidant activities of natural compounds
have been observed only in a few studies such as for clove oil or Cen-
teurea damascena extracts [32,33]. This suggests the potential benefits
of lichen metabolites for health. Compounds recovered using ethanol at
96 % were then used for HPLC analysis.

The chromatograms of the phenolic extracts from the aqueous phase of the extraction using thermal spring water at 170 °C and using thermal spring water with eutectic at 150 °C (the conditions that lead to the best viscoelastic properties of the films formulated with the polysaccharides) after adsorption and desorption from SP700 resin are presented in Supplementary material 8.A. It can be observed that the same peaks are present in both chromatograms. However, their height is much lower for TM150 extracts, which seems to confirm that some compounds are concentrated in the men:lau phase. Caffeic acid was identified in low quantity while sharper peaks of atranol and chloroatranol are observed. These compounds are degradation product of atranorin and chloroatranorin. Their presence suggests that the high extraction temperature or the acidification of the medium (caused by the release of acids during

the hydrolysis process) degraded the initially present atranorin and
chloroatranorin. They are severe allergens, prohibited in cosmetics that
are not rinsed off since 2017 by the Scientific committee for the con-
sumers and European commission. Even though no other health risks
have been reported. These compounds have been rarely investigated for
other uses such as pharmaceutical application or surface disinfectants,
despite their potential antioxidant or antibacterial activity [34,35]. The
use of the hydrophobic eutectic solvent offered the advantage of
significantly reducing the presence of these compounds in the aqueous
phase. The high antioxidant activity of E-TM150 might potentially be
due to other compounds, whose chemical characterization should be
carefully determined and whose safety should be assessed before any
use. A better characterization of these compounds would be also needed.
HPLC chromatograms of the compounds extracted at 150 °C with ther-
mal water, recovered in the eutectic phase, diluted 40 times, are dis-
played in Supplementary material 8.B. As it was hypothesized, atranol
and chloroatranol are also observed in the eutectic phase as well as usnic
acid, an interesting anti-bacterial, anti-viral, antioxidant, anti-
inflammatory compound [36]. However, its safety must still be further
investigated.

3.1.5. *Soluble oligosaccharides characterization*

Fig. 4 represents the yield of polysaccharides that precipitated and the yield of oligo and polysaccharides still in the aqueous phase after the precipitation, as a function of the solvent used (meaning that the sum of these two yields represents the total polysaccharides/oligosaccharides extraction yield). Low concentrations (< 1 %) of glucuronic acid, arabitol, formic acid and acetic acid were found in the samples but are not represented in this figure. Regardless of the solvent, the three main monosaccharides found after hydrolysis of the aqueous phases are glucose, galactose and mannose, which aligns with results obtained with hydrothermal microwave assisted extraction [37]. For all solvents, the content of glucose decreases with the increase in temperature whereas the galactose and mannose content increases. Although the precise location of lichen polysaccharides remains unclear, Honnegger and Haisch [38] identified the lichenin (linear (1 → 3), (1→4)-β-glucan presents in *E. prunastri*) in the extracellular matrix of the peripheral

<figure><img src="image_7.png" /><figcaption>**Fig. 4.** Influence of the solvent and temperature on the yield of polysaccharides precipitated (right scale, line) and the yield of poly and oligosaccharides in the aqueous phase after the polysaccharide's precipitation (left scale, histograms).</figcaption></figure>
cortex and in a large outer wall layer of the hyphae from the medulla. Roth et al. [39] also described the cortical hyphae of lichens as embedded in extracellular matrices mainly made of β-glucan-based polysaccharides. This might explain why glucans are more easily extracted.

The highest overall extraction yield (92 %) was, thus, obtained at 190 °C for thermal water combined with eutectic. Soluble extracts after polysaccharide precipitation were mainly composed of mannose, galactose and glucose oligosaccharides, phenolic compounds (that concentrated in the eutectic phase when added) and proteins. The use of thermal spring water strongly enhanced the extraction of protein and also increased the phenolic content and antioxidant capacity of the extracts.

## 3.2. Extraction and characterization of polysaccharides

### 3.2.1. Yield as a function of solvent and temperature

It can be also noticed on Fig. 4 that the use of thermal spring water and then, of eutectic, shifted to lower temperatures the best extraction yield of polysaccharides that precipitated: for distilled water, 27.4 % at 180 °C, for thermal water, 18.1 % at 170 °C, for distilled water with men:lau, 33.1 % at 160 °C and for thermal water with men:lau, 25.2 % at 150 °C. The increase in the polysaccharide yield with the addition of 5 % men:lau eutectic may be due to the hydrogen bonding formed between the solvent and the biomass, favoring its disruption and leading to a

higher solubilization of polysaccharides. Wang et al. [40] studied the efficiency of different eutectic solvents on the extraction of cellulose, hemicellulose and lignin from *R. palmatum*. They concluded that eutectics containing carboxylic acids (the case with lauric acid) had a better extraction yield as they reacted with the hydroxyl groups of the saccharides, disrupting the cell-wall matrices of the biomass. The main differences in the chemical properties of distilled and thermal water that may explain the lower recovery of precipitated polysaccharides using thermal water, are the pH and the ionic strength. Indeed, the pH of the soluble phase after extraction with distilled water (with and without eutectic) was close to 4.3–4.4 for all temperatures, that of the extraction with thermal water (with and without eutectic) was between 5.3 and 5.8. As a more neutral to slightly alkaline pH usually enhances the solubility of β-glucans [41,42] the decrease in the yields using thermal water seems to be more related with the higher ionic strength of the thermal water. Indeed, the ionic strength might reduce the solubility of neutral polysaccharides such as β-glucans and galactomannans [43].

### 3.2.2. Structural features from NMR and FTIR

NMR spectroscopy was used to investigate the composition of the polysaccharide fraction precipitated after freeze-thaw. ¹H NMR spectra of precipitated were recorded at 75 °C in D₂O and DMSO (data not shown). The samples exhibited higher solubility and a more complex composition in DMSO, leading to the selection of this solvent (and temperature) for subsequent analyses of polysaccharides extracted using

<figure><img src="image_8.png" /><figcaption>**Fig. 5.** NMR spectra recorded for the precipitated polysaccharide (P—D for distilled water, P-T for thermal water, P-DM for distilled water with eutectic, P-TM for thermal water with eutectic) fraction in DMSO-d₆ (400 MHz, 75 °C): A ¹H NMR with diffusion filter (50 ms), B HSQC spectra; C COSY, and D 13C spectra.</figcaption></figure>

<figure><img src="image_9.png" /></figure>
<caption>**Table 1**</caption>

Experimental ¹H and ¹³C NMR chemical shifts (δ, ppm) of the main components in extracted mixture of lichen polysaccharide. The data was measured at 75 °C in DMSO-d. Residues A-D are not part of a unique polysaccharide chain. The polysaccharide mixture extracted from in *Evernia Prunastri* is composed of glucans and galactoglucomannans.

<table><thead><tr><th>Residue</th><th>H1</th><th>H2</th><th>H3</th><th>H4</th></tr></thead><tbody><tr><td></td><td>C1</td><td>C2</td><td>C3</td><td>C4</td></tr><tr><td>A, α-Gal/Glc<br>(1 → 3)</td><td>5.13<br>100.9</td><td>3.43<br>69.8</td><td>3.61<br>83.2/73.2</td><td>–</td></tr><tr><td>B, α-Gal<br>→)1</td><td>5.04<br>100.2</td><td>3.35<br>73.6</td><td>3.80<br>70.04</td><td>3.35<br>73.6</td></tr><tr><td>C, β-Man<br>(1 → 2)</td><td>4.93<br>99.2</td><td>3.45<br>79.8</td><td>3.93<br>71.2</td><td>–</td></tr><tr><td>D, β-Glc<br>(1 → 3)</td><td>4.54<br>103.3</td><td>3.30<br>73.4</td><td>3.49<br>86.7</td><td>3.73<br>61.4</td></tr></tbody></table>

eutectic solvent. It should be noted that as the samples were not fully soluble, the reported data pertain only to the DMSO-soluble fraction. ¹H NMR were recorded with and without diffusion filter (D-filter) to remove the solvent signal and diminish the intensity of low molecular weight molecules. Thus, maximizing the intensity of the polysaccharide fraction. An overview of the ¹H NMR spectra (Fig. 5A) indicate a similar overall composition among the samples, with notable variations in the relative intensity of specific characteristic signals (see below).

¹³C NMR and bidimensional experiments (HSQC, COSY, TOCSY) were recorded at 75 °C on the sample extracted in distilled water at 190 °C. This sample was chosen because it exhibited sufficient signal intensity for all characteristic NMR peaks. Examining HSQC highlights the mixture's complexity, with at least eleven anomeric signals visible (Fig. 5A). Among them, the more intense correspond to 4 main sugar residues (1H/13C cross peaks: residue A, 5.13/100.9; residue B, 5.04/100.2; residue C, 4.93/99.0; and residue D, 4.53/103.3 ppm). The analysis was performed on the polysaccharide mixture, not on isolated fractions, and therefore the identification of the complete spin system (H1-H6) of each residue was not possible, due to signal overlap. Nevertheless, analysis of the COSY and TOCSY allowed to identify key signals of each spin system and combination of this information with the HSQC rendered relevant information. Moreover, a detailed comparison of the data obtained with the information available from the NMR of lichen and fungal glucans, galactomanans and glucogalactomanans allowed to identify characteristic signals of the residues A-D as described in Table 1.

### 3.2.2.1. β-Glucans.
The anomeric signals of the β-glucan fraction are observed in the region between 4.6 and 4.25 ppm of the ¹H NMR spectrum. Bidimensional NMR analysis indicates that the β-glucan is primarily (1 → 3) linked, as evidenced by the characteristic signals of residue D (H3/C3 at 3.49/86.1 ppm) [44]. Additionally, less intense signals corresponding to β-glucans with different substitution patterns are detected in the HSQC spectrum including ¹H/¹³C, 4.38/104.1 and ¹H/¹³C 4.27/103.7 ppm. The peak at 4.45 ppm, although lacking a clear correlation in the HSQC spectrum, is identified as part of the β-glucan structure based on the COSY (4.45/3.29) and TOCSY (4.45/3.50, and 4.45/3.30) correlations. The predominance of 1,3-substitution in *E. prunastri* β-glucans was previously reported through methylation analysis [45] and is now confirmed for the second time through our NMR spectroscopy.

### 3.2.2.2. Galactoglucomannans.
The residue C (H1/C1 at 4.93/99.0) is identified as mannose in base of the low intensity observed in the COSY H1/H2 correlation, that arises from small [3]J coupling due to eq-eq geometry in the ⁴C₁ chair conformation. The chemical shift of the H2 in this mannose is compatible with a β configuration where the H1 is downfield (4.93 ppm) as a consequence of the observed substitution (H2/C2 at 3.45/79.0 ppm). The observed resonances of residues A and B

are compatible with α-glucoses and α-galactoses which are difficult to distinguish, given the lack of information on H4-H6 (due to overlap). Residue B shows no substitution in the H1-H4 region. Due to the similarity between the chemical shifts of residue B and those found in the galactose branches of galactomannans containing β-mannose, we propose that residue B is a galactose, likely branched to Mannose C [46]. The residue A presents HSQC correlations of the H3 at 73.2 (non-substituted) and 83.2 ppm (substituted and commonly described in α-(1 → 3)glucans) [47,48]. Altogether the NMR of residues A-C are in agreement with the structure proposed for a galactomannan found in *Evernia prunastri* by Miljovic-Stojanovic: a main chain of mannose with numerous galactose branches [49]. This mannose is substituted in position 2 (HSQC 3.45/79.8) and very likely substituted in another position, commonly 6 according to the literature [49]. But this substitution is not clearly observed due to the overlap in the spectra of the mixture of polysaccharides. It should anyway be mentioned that our data are also compatible with the presence of α-(1 → 3) glucan overlapping with residue A.

Another characteristic of the polysaccharide is the presence of acetyl methyl substituents, as indicated by signals at 2.05 ppm in ¹H NMR and 2.05/21.1 ppm (¹H / ¹³C) in HSQC. Furthermore, acetylation occurs at multiple hydroxyl positions, albeit in a small proportion of the residues. This conclusion is supported by the detection of minor yet distinct signals in the HSQC spectrum, exhibiting cross-peaks in the region of 6.00–6.20/103–120 ppm (five signals), which are consistent with previously reported chemical shifts for peracetylated galactoglucomannans [50].

This is further supported by the HSQC cross-peaks at 4.94/72.4 ppm, where the non-anomeric resonance at 4.93 ppm corresponds to the H2 of peracetylated galactoglucomannans [50]. Interestingly, the anomeric signal at 6.00–6.20 ppm, which was predominant in polysaccharides from *E. prunastri* extracted via microwave-assisted extraction in our previous study, represents only ~3% of the sample extracted in distilled water at 190 °C and varies within the extraction conditions from 1 to ca. 8%.

Once the main composition is discussed in a representative sample it is appropriate to investigate the changes in composition along the extraction conditions. ¹H spectra were recorded at each condition with and without a diffusion filter (D-filter ¹H). In D-filtered ¹H NMR spectra, signals from low-Mw impurities are attenuated thanks to the application of a pulsed filed gradient [51], due to their higher diffusion coefficients, allowing better observation of polymeric components. A careful integration of the spectra was added with and without diffusion filter renders information of the relative movement of the NMR signals (and hence Mw of the corresponding molecules). The regions of the alpha and beta sugars (galactoglucomannanas and beta glucans) and the regions of the acetyl methyl group were integrated. The detailed information is included in the supplementary material (Table S9 and Table S10). Only the conclusions of such analysis are mentioned here. It can be observed in Fig. 6 that, in distilled water, a step decrease of the proportion of galactoglucomannan as the extraction temperature raises, from 65 % until 47 %. The contrary happens in thermal water where the proportion raises from a 63 until a 74 %. When the extraction is performed in the presence of eutectic solvent again a decrease of the proportion of galactoglucomannan from 68 % (150 °C) until 45 % (190 °C) is observed for distilled water while the % of galactomannans is close to a 70 % independently of the temperature of extraction.

It is also observed (at all extraction conditions) that the proportion of galactoglucomannan raises with the application of the diffusion filter (Table S9 and S10), meaning that this polysaccharide fraction has a higher Mw than the β-glucan fraction. Moreover, a consistent increase in the acetyl methyl signal at 2.05 ppm in the D-filter ¹H strongly suggest that the acetylation occurs at the galactoglucomannan.

Fig. 7. A shows the FTIR spectra of the films made with the polysaccharides. All films presented the peaks related to polysaccharides. Peaks at 850 and 925 cm⁻¹ may be respectively due to the presence of
<figure><img src="image_10.png" /><figcaption>Fig. 6. Evolution of the galactoglucomannans and glucans content as a function of the solvent used and the extraction temperature.</figcaption></figure>

<figure><img src="image_11.png" /><figcaption>Fig. 7. A) FT-IR spectra of the films formulated with these polysaccharides and B) HPSEC chromatograms of the precipitated polysaccharides extracted with distilled water without (D) and with (DM) men:lau and thermal water without (T) and with men:lau (TM).</figcaption></figure>

α-D-galactopyranose or α-glucans and β-glycosidic linkages in β-glucans, peaks at 1035 cm⁻¹ to C—O vibration in primary alcohol, 1070 cm⁻¹ to stretching vibration of C—C bonds, at 1110 cm⁻¹, to the stretching of C—O, C=C, and C—C—O in sugar rings and at 1150 cm⁻¹ probably related to the stretching vibration C-O-C in hemicellulosic materials [10,56–58]. Although no discrepancy is observed between the films in the polysaccharide’s region, differences in region between 1410 and 1700 cm⁻¹ are observed between those formulated with and without the eutectic. On F-DM160 and F-TM150 spectra only, a much sharper peak is observed at 1410 cm⁻¹, and then two peaks at 1560 and 1650 cm⁻¹ when only one broad peak is observed at 1650 cm⁻¹ for F—D180 and F-T170. In this zone, signals from proteins are usually observed, for example at 1650 cm⁻¹ for amide I and at 1550 and 1400 cm⁻¹ [56,59]. However, some authors reported signals from aromatic rings vibration of phenolics from lignin residue, for example around 1410 cm⁻¹ for C—O stretching and aromatic skeletal vibration in lignin or hemicellulosic material [60], around 1547–1599 cm⁻¹ for aromatic vibration, and around 1580–1615 cm⁻¹ for the stretching of the C—H and

C=C-C aromatic bond [61–63]. As the extraction of protein was increased using thermal spring water, peaks for proteins should be observed not only in F-TM150 but also in F-T170 which is not the case, and shouldn’t be observed in F-DM160. The fact that these peaks are only observed for the polysaccharides extracted with the eutectic seems to orientate to phenolics that would have been extracted due to the highest solubilizing properties of the eutectic. The sharp peaks at 2850 and 2920 cm⁻¹ in the film with the eutectic is probably due to a residue of solvent, as it was also observed on the NMR spectra.

### 3.2.3. Molecular weight distribution via HPSEC

The HPSEC chromatograms displayed on Fig. 7.B show that all polysaccharides, regardless of the solvent used, exhibited similar molecular weights with a main peak around 786 kDa. Similar molecular weights were found for polysaccharides extracted from *Evernia prunastri* by hydrothermal microwave assisted extraction at 180 °C [52]. These molecular weights are also in range with galactomannans and glucans molecular weight from other biomass [53–55]. The broad distribution observed in the chromatogram starting at 5 min and showing considerable intensity until 10 min, corresponds to a high Mw polysaccharide with a maximum around 6 min accompanied by lower Mw oligopolysaccharide with a maximum near 8 min. This broad distribution is explained by presence of the different polysaccharides identified by NMR.

<table><caption>**Table 2**

Weight, thickness, moisture content.</caption><thead><tr><th>Sample</th><th>Weight (mg)</th><th>Thickness (mm)</th><th>Moisture content (%)</th></tr></thead><tbody><tr><td>F-TM150</td><td>60.17 ± 9.65</td><td>0.075 ± 0.013</td><td>69.23 ± 1.36 *</td></tr><tr><td>F-T170</td><td>60.17 ± 1.15</td><td>0.088 ± 0.029</td><td>48.85 ± 2.46</td></tr></tbody></table>

<footnote>* in the same column means significant differences (p < 0.05) between the films.</footnote>
<table><caption>**Table 3**

Mechanical properties of the formulated buccal films: resistance to extension, percent of elongation and Young's modulus.</caption><thead><tr><th>Sample</th><th>Resistance to extension (N)</th><th>Percent of elongation (%)</th><th>Young's modulus (MPa)</th></tr></thead><tbody><tr><td>F-TM150</td><td>3.20 ± 0.01 <sup>a</sup></td><td>36.59 ± 1.01 <sup>a</sup></td><td>138.37 ± 15.22 <sup>b</sup></td></tr><tr><td>F-T170</td><td>2.41 ± 0.01 <sup>b</sup></td><td>25.41 ± 0.58 <sup>b</sup></td><td>126.25 ± 7.65 <sup>b</sup></td></tr></tbody></table>

Different letters (a, b) in the same column mean significant differences (p < 0.05).

### 3.3. Films characterization

#### 3.3.1. Films physical characterization

The physical characteristics of the films (with a view to potential use in pharmacy, such as buccal films) play a critical role in determining dosage precision, release profile of active compounds, ease of handling, patient comfort, and most importantly, therapeutic efficacy. To confirm that films meet their intended use, it is necessary to assess parameters such as uniformity in weight and thickness or mechanical flexibility. Additionally, hydration-related properties, such as moisture content, are key aspects that must be evaluated. Tables 2 and 3 summarize the results obtained for the formulated buccal films.

According to the results, the formulated films demonstrated favorable physical and mechanical characteristics relevant to mucosal delivery. As shown in Table 2, the weights were equal for both, with this value falling within the acceptable range for buccal dosage forms, as other studies have reported weights between 40 and 80 mg for 4 cm² films to ensure ease of handling and dosage control [64,65]. Teixeira et al. developed mucoadhesive buccal films containing *Actinidia arguta* extract and reported slight high weight values [23]. In what concerns to films thickness, the values ranged from 0.065 to 0.091 mm for F-TM150 and F-T170, respectively, without significant differences among them. According to the results, the films have uniform thickness and weight,

which ensured homogeneity.

In what concerns to moisture, F-TM150 has the highest content (69.23 %), while F-T170 achieved the lowest (48.85 %). This difference may be attributed to the higher content in highly branched galactomannans and lower amount of β-glucan in P-TM150 compared to P-T170 (70 % and 27 % respectively for TM150 and 56 % and 39 % respectively for T170), which may influence the water retention. Nonetheless, it should be highlighted that the moisture content plays a crucial role in flexibility and disintegration behavior, depending on the polymer matrix and drying methods [66].

Table 3 presents the mechanical properties of the formulated films.

The film's integrity and mechanical performance are strongly influenced by its composition, particularly the type and amount of polymer, plasticizer, and active compounds. According to Table 3, the resistance to extension, also known as tensile strength when the cross-sectional area is taken into account, varied from 2.41 N (F-T170) to 3.20 N (F-TM150). This parameter indicates the maximum force or load that a film can withstand before breaking or undergoing permanent deformation.

The percent elongation evaluates the films elasticity. The highest results were achieved for F-TM150 (~36 %), while the lowest ones were obtained for the F-T170 sample (25.41 %), interestingly the one with lowest moisture. Comparable flexibility was observed by Teixeira et al. for the films prepared by ultrasound assisted extraction. These results

<caption>**Table 4**</caption>

Visual appearance and SEM images of the different films formulated with the polysaccharides extracted with distilled water at 180 °C (F-D180), thermal water at 170 °C (F-T170), distilled water with eutectic at 160 °C (F-DM160) and thermal water with eutectic at 150 °C (F-TM150).

<table><thead><tr><th></th><th>Films for the stability study</th><th>Visual appearance of the films</th><th>SEM images</th></tr></thead><tbody><tr><td>F-D180</td><td></td><td></td><td></td></tr><tr><td>F-T170</td><td></td><td></td><td></td></tr><tr><td>F-DM160</td><td></td><td></td><td></td></tr><tr><td>F-TM150</td><td></td><td></td><td></td></tr></tbody></table>
<figure><img src="image_12.png" /><figcaption>**Fig. 8.** Viscoelastic moduli G4 and G" of the films formulated with the polysaccharides extracted with distilled water at 180 °C (F—D180), thermal spring water at 170 °C (F-T170), distilled water with eutectic at 160 °C (F-DM160) and thermal spring water with eutectic at 150 °C (F-TM150).</figcaption></figure>

emphasize the comfort and retention advantages of elastic materials.

Finally, the Young's modulus, also known as elastic modulus or stiffness, represents the resistance of a film to deformation under applied stress and measures the film ability to return to its original shape after the stress removal. The values were similar for both films type, namely 138.37 MPa for F-TM150 and 126.25 MPa for F-T170, suggesting stiffness across formulations. Young's modulus generally rises with increased film thickness or weight due to a denser polymer matrix and stronger molecular interactions, while hydration can enhance elasticity by increasing polymer chain mobility [67]. These values fall within the 50–200 MPa range typical of polymeric films and align with those reported by Teixeira et al., who highlighted the balance between film strength and user comfort. Therefore, a higher modulus, indicates a more rigid film, potentially improving durability at the expense of comfort.

### 3.3.2. Film surface appearance and stability study

Table 4 displays the visual appearance of the films as well as SEM images of their surface. The visual appearance of the films differs in function of the solvent used. Those made with the polysaccharides extracted with distilled water were more homogeneous and transparent than those made with the polysaccharides extracted with thermal water. However, they were also much stickier and weaker. It is also interesting to note that those made with the polysaccharides extracted with the eutectic are whiter. SEM images give an idea of the surface morphology of the films. Polysaccharide clumps, possibly due to steric hindrance and hydrogen bonds between the polysaccharide chains [68,69]. This phenomena is relatively common with polysaccharide-based films [70,71]. F-T170 seems however to have less aggregates than the three other films. On the other hand, more granules are observed on the films extracted with the eutectic solvent. This might be due to some residues of the eutectic solvent sticking to the surface of the film during drying.

Fig. 8 shows the elastic (G') and viscous (G") moduli of the films formulated with polysaccharides from the different extractions. All films exhibit viscoelastic properties of a gel-like system with the elastic modulus much higher than the viscous one and both invariant with the

frequency. However, at $T = 0$ day, the films with the polysaccharides extracted with thermal spring water (both without and with men:lau) have a much higher moduli than those from distilled water treatment. G' of the films from thermal spring water extraction is around 6 times higher than that of distilled water treatment. Similarly, G' of the films from thermal spring water with eutectic treatment is almost 3 times higher than that of distilled water with eutectic. The NMR data on Fig. 6 gave us the galactoglucomannans and glucans content of the precipitates at each temperature. It is thus possible to calculate the galactoglucomannans/glucans ratio of each fraction used to make the films. This ratio is 1.28 for P—D180, 1.44 for P-T170, 1.74 for P-DM160 and 2.59 for P-TM150. The particularly high weakness of DM180 films may be due to the low galactoglucomannans content with respect to the glucan content. Indeed, as galactomannans are better film-forming polysaccharides than glucans that are more gel-forming, a higher galactoglucomannans/glucans ratio may mean higher film resistance. Polysaccharides extracted using thermal spring water presented a higher number of acetyl methyl groups compared to all other extracts (6% for P—D180, 10% for P-T170, 5% for P-DM160 and 4% for P-TM150) (S9 and S10). It has been observed that the presence of a reasonable amount of acetyl methyl groups, that substitute OH groups in the polysaccharides, can improve film formation, as well as making them more hydrophobic [72,73]. Egués et al., Schmidt et al., and Zhang et al., [73–75] observed an increase in the mechanical properties of acetylated-starch and xylans with respect to non-acetylated ones. This might be due to higher flexibility of the polysaccharide chains, given by the plasticizing effect of the acetyl methyl groups, slightly lowering the strength of intermolecular hydrogen bonding [74,75]. However, too high degree of acetylation decreases these properties as too many OH groups are substituted, excessively lowering the hydrogen bonding capacity of the polysaccharides [75]. The good properties of the F-T170 and F-TM150 might be then influenced by the ratio galactoglucomannans/glucans and the degree of acetylation.

While all films maintain their gel-like properties, a decrease in the value of their moduli is observed until 2 months (Fig. 8). Then, a recovery of the viscoelastic properties is observed for all films the 3rd
month. The variation of the elastic and viscous moduli might be related to the water loss observed at $t = 30$ and $t = 60$ and the decrease in the water loss ($t = 90$) of the films when stored (Supplementary material 11). This might explain while a recovery of the viscoelastic properties occurred at $t = 90$ days. The stability of the films in terms of their water uptake/loss and viscoelastic properties is crucial before exploring their potential biomedical and cosmetical application. Since this initial stability study is quite conclusive, it may be beneficial to conduct similar studies under varying environmental conditions (high moisture, dry environment) and to evaluate additional properties to achieve a full understanding of the films' behavior, beyond just their viscoelastic properties over time. Schnabl et al. [76] recently assessed the chemical stability of chitosan films, over 400 days, reporting high stability depending on the additive used. All of these findings support the use of natural polysaccharides for the development of bio-based materials. These films appear indeed promising for functionalization (such as incorporation of antioxidant compounds) or for assessing their biological activities.

## 3.4. Antiviral activity and cytotoxicity

### 3.4.1. Irritation test

Skinethic irritation test was performed on the polysaccharides T170 and TM150 as their respective film's properties were much better. According to the UN GHS classification, a compound can be classified into two different categories: Category 2 which means skin irritating and non-classified meaning non skin irritating. If the cell tissue viability is lower than 50 %, the compound is Category 2, higher, it is non-classified. Both samples were non-classified, with a viability of 93.9 ± 4.6 % and 91.5 ± 10.5 % for P-T170 and P-TM150 respectively. These high viability values are encouraging for potential skin application of the films made with these polysaccharides. To go further, the in vivo performance of the films could be evaluated.

### 3.4.2. Antiviral activity

Some natural cell wall sulfated polysaccharides extracted from algae have been shown to exhibit antiviral activity against a wide spectrum of viruses including HSV. The efficacy of these polysaccharides, primarily due to their ability to inhibit viral attachment to host cell surfaces. They can also prevent viral entry and uncoating as well as its transcription and translation process [77–79]. However, some antiviral properties against HSV-1 [80], Tobacco Mosaic [81], or murine noro viruses [82] of glucans and galactomannans have also been observed. In this study, the cytotoxicity and antiviral activity of P-T170 and P-TM150 and their freeze-dried corresponding soluble extracts, E-T170, E-TM150, (free of the precipitated polysaccharides) have been evaluated in vitro by cell viability on the model Vero cell lines/ herpes simplex 1, 2 (Table 5). P-T170 and E-T170 were not cytotoxic against Vero cells up to the tested concentration of 1000 µg/mL. However, the use of men:lau significantly increased cytotoxicity of the resulting polysaccharide samples. $CC_{50}$ of 220 and 790 µg/mL respectively were observed for P-TM150 and E-TM170. It may be due to the presence of a residue of eutectic solvent that induces a change in pH [83,84]. This hypothesis requires further analysis to be confirmed. As P-T170 and P-TM150 did not exhibit protective activity against HSV-1, they were not further tested against acyclovir-resistant HSV-1 and HSV-2 strains. E-T170, mainly composed of shorter molecular weight glucans, galactoglucomannans and some of the lichenic substances, provide slight protection (42.0 % for HSV-1, 36.8 % for HSV-1 acyclovir resistant and 38.2 % for HSV-2 acyclovir resistant). Although some protective activity was observed, it occurred at significantly higher concentrations compared to standard antivirals such as acyclovir and heparin. Due to the low protection level, it was not possible to perform experiments to investigate the mechanisms responsible for the antiviral effect. This study allows for the exclusion of potential antiviral applications of these lichen-derived compounds, given their low activity even at concentrations much higher than other

<caption>**Table 5**</caption>

Anti-viral and cytotoxicity results of the polysaccharides extracted with thermal water and thermal water with eutectic (P-T170, P-TM150) and their corresponding freeze-dried liquid phases (E-T170, E-TM150).

<table><thead><tr><th>Samples</th><th>HSV 1 Protection (%)</th><th>HSV 1 acyclovir resistant Protection (%)</th><th>HSV 2 acyclovir resistant Protection (%)</th><th>Cytotoxicity $CC_{50}$ (µg/mL)</th></tr></thead><tbody><tr><td>P-T170</td><td>No protection</td><td>Not tested</td><td>Not tested</td><td>&gt; 1000</td></tr><tr><td>P-TM150</td><td>No protection</td><td>Not tested</td><td>Not tested</td><td>220</td></tr><tr><td>E-T170</td><td>42.0 ± 3.3% At 1000 µg/mL</td><td>36.8 ± 14.4% At 750 µg/mL</td><td>44.2 ± 5.3% At 1000 µg/mL</td><td>&gt;1000</td></tr><tr><td>E-TM150</td><td>No protection</td><td>No protection</td><td>No protection</td><td>790</td></tr><tr><td>Controls</td><td>HSV 1 $EC_{50}$ (µg/mL)</td><td>HSV 1 acyclovir resistant $EC_{50}$ (µg/mL)</td><td>HSV 2 acyclovir resistant $EC_{50}$ (µg/mL)</td><td>Cytotoxicity $CC_{50}$ (µg/mL)</td></tr><tr><td>Acyclovir</td><td>$EC_{50}$ = 0.31 µg/mL</td><td>$EC_{50}$ &gt; 100 µg/mL</td><td>$EC_{50}$ = 20.0 µg/mL</td><td>&gt;200</td></tr><tr><td>Heparin</td><td>$EC_{50}$ = 45.1 µg/mL</td><td>$EC_{50}$ = 33.2 µg/mL</td><td>$EC_{50}$ = 14.3 µg/mL</td><td>&gt;200</td></tr></tbody></table>

natural sulfated polysaccharides such as carrageenan. The slightly higher activity for E-T170 seems to be more related to the lichenic substances than to the oligosaccharides or soluble phenolics as the main difference between E-T170 and E-TM150 is that the lichenic substances tend to concentrate in the eutectic phase for E-TM150, reducing their presence in the aqueous extracts. This activity could be possibly related to the presence of atranol and chloroatranol, although their direct antiviral role remains to be verified. Potential limitations may arise due to the restricted use of these compounds.

## 4. Conclusions

This study provides a first insight into the use of subcritical thermal water and hydrophobic eutectic solvent for the extraction of polysaccharides and bio-compounds from *Evernia prunastri*. To date, none of these solvents have been evaluated for this purpose. An increase in the protein and phenolics content and in the antioxidant capacity of the soluble extracts was observed with the use of thermal spring water. After purification through SP700 resin of E-T170 and E-TM150, extracts with an antioxidant activity 1.38 and 1.78 g Trolox eq./ g of extract was obtained. However, E-T170 contains a non-negligible amount of atranol and chloroatranol, which may limit its use due to the regulatory restrictions on these compounds. Since E-TM150 contains only a limited amount of atranol and chloroatranol, likely due to their concentration in the men:lau phase, its outstanding antioxidant activity may be attributed to other, safer phenolic compounds that may have applications in pharmaceutic or cosmetics formulations. However, the identification of these presumed compounds, as well as their safety, still needs to be assessed. The use of a small quantity of men:lau as modifier enable optimal extraction of polysaccharides at lower temperature (150–160 °C). However, a decrease in the polysaccharides yield was observed with the use of thermal spring water. NMR analyses make it possible to identify that the extracted polysaccharides were glucans and highly branched galactoglucomannans. They did not exhibit antiviral activity against HSV-1 or HSV-2. Moreover, the use of men:lau eutectic significantly increased their cytotoxicity.

The films formulated with the polysaccharides extracted with thermal water presented much better viscoelastic properties than those extracted with distilled water. These properties over the first two months but almost recovered their initial value the third month. F-TM150 and F-T170 presented suitable mechanical properties for potential medical application of topical or buccal films. Polysaccharides P-T170 and P-TM150 were non-irritating according to Skinethic test. These films, that exhibit good mechanical properties, could be
promising biomaterial for biomedical application. Further characterization and evaluation of additional biological activities are planned to be carried out in a more in-depth study focusing on their biomedical applications. These analyses would also need to be completed with in vivo assays.

### CRediT authorship contribution statement

**Julie Queffelec:** Writing – original draft, Methodology, Investigation, Funding acquisition, Formal analysis, Data curation. **Ramón Novoa-Carballal:** Writing – original draft, Investigation, Formal analysis, Data curation. **Nathalie Bourgougnon:** Writing – review & editing, Supervision, Formal analysis, Conceptualization. **Christel Marty:** Investigation. **Filipa Teixeira:** Investigation. **Francisca Rodrigues:** Writing – review & editing. **Paulo C. Costa:** Writing – review & editing. **María D. Torres:** Writing – review & editing, Writing – original draft, Supervision, Investigation, Funding acquisition, Formal analysis, Conceptualization. **Herminia Domínguez:** Writing – review & editing, Supervision, Funding acquisition, Formal analysis, Conceptualization.

### Declaration of competing interest

Authors declare there is no conflict of interest.

### Acknowledgements

Authors acknowledge Consellería de Cultura, Educación e Universidad da Xunta de Galicia (GRC-ED431C 2022/08 and Centro Singular CINBIO, ED431G-2023/06) and FTIR, NMR, SEM measurements to the services of analysis of Universidade de Vigo (CACTI). M.D.T. acknowledges to the Ministry of Science, Innovation and Universities of Spain for her postdoctoral grants (RYC2018-024454-I) and to the Consellería de Cultura, Educación e Universidade da Xunta de Galicia (ED431F 2020/01). J.Q. thanks the University of Vigo. This work was supported by ISblue project, Interdisciplinary graduate school for the blue planet (ANR-17-EURE-0015) and co-funded by a grant from the French government under the program "Investissements d'Avenir" embedded in France 2030. Ramon Novoa-Carballal thanks his mother, Regina Carballal, a pioneer in lichenology in Galicia, for fostering his interest in nature and science. Filipa Teixeira is thankful for her Ph.D. grant (2024.01202.BD) financed by FCT/MCTES. Francisca Rodrigues was supported by FCT – Fundação para a Ciência e a Tecnologia, I.P under the TENURE program [2023.14718.TENURE.016]. The authors acknowledge the support from the Aliança para a Investigação e Ensino Superior, and the funding received by FCT – Fundação para a Ciência e a Tecnologia, I.P., in the scope of the project UIDP/04378/2020 (DOI: 10.54499/UIDB/04378/2020) and UIDB/04378/2020 DOI:10.54499/UIDB/04378/2020) of the Research Unit on Applied Molecular Biosciences - UCIBIO and the project LA/P/0140/2020.

## Appendix A. Supplementary data

Supplementary data to this article can be found online at https://doi.org/10.1016/j.ijbiomac.2025.147613.

### Data availability

Data will be made available on request.

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