Carboxymethylpullulan Grafted with Aminoguaiacol: Synthesis

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Carboxymethylpullulan grafted with aminoguaiacol: Synthesis, characterization and assessment of antibacterial and antioxidant properties Marie-Carole Kouassi, Pascal Thébault, Christophe Rihouey, Emmanuelle De, Beatrice Labat, Luc Picton, and Virginie Dulong Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.7b00899 • Publication Date (Web): 26 Jul 2017 Downloaded from http://pubs.acs.org on July 27, 2017

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Polysaccharide grafted with high content of aminoguaiacol (Polysoaps) ( screened by ionic strength)

Flexible anionic polysaccharide ( screened by ionic strength)

RH

Antioxidant properties

( ) + aminoguaiacol

R

.

Antibacterial properties S. aureus

ACS Paragon Plus Environment

dead S.aureus

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Carboxymethylpullulan grafted with aminoguaiacol: Synthesis, characterization and assessment of antibacterial and antioxidant properties Marie-Carole Kouassi, Pascal Thébault, Christophe Rihouey, Emmanuelle Dé, Béatrice Labat, Luc Picton, and Virginie Dulong* Laboratory Polymères, Biopolymères, Surfaces, Normandie Univ, UNIROUEN, INSA Rouen, CNRS, PBS, 76000 Rouen, France

ABSTRACT

Aminoguaiacol, the aminated derivative of guaiacol, a natural phenolic compound, was chemically grafted onto a polysaccharide (Carboxymethylpullulan, CMP) in the presence of the activator agent 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI). The grafted polysaccharides were characterized by FTIR and 1H-NMR spectroscopy to confirm and quantify the grafting. All polysaccharide derivatives (grafting rates of aminoguaiacol between 16% and 58%) were soluble in water. Their physicochemical properties were studied in a dilute regime and a semi-dilute regime by light scattering, fluorescence and rheology, showing associative properties with peculiar polysoap behavior. The antibacterial activities of the synthesized products against Staphyloccocus aureus were assessed using a counting method. The


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antioxidant activities of the derivatives were also highlighted using the α,α-diphenyl-βpicrylhydrazyl (DPPH) method. Finally, the cytotoxicity of the derivatives was studied with fibroblast cells and they showed a very good cytocompatibility. Such polymers could be used to replace chemical preservatives in food and cosmetic aqueous formulations.

KEYWORDS: associative polysaccharide, polysoap, antibacterial, antioxidant. Polysaccharide grafted with high content of aminoguaiacol (Polysoaps) ( screened by ionic strength)

Flexible anionic polysaccharide ( screened by ionic strength)

RH

Antioxidant properties

( ) + aminoguaiacol

R

.

Antibacterial properties S. aureus

dead S.aureus

INTRODUCTION Bacterial contamination or detrimental oxidative effects of materials and aqueous solutions (in several fields such as the medical, food and cosmetic industries), represents a major public health problem and conservation issue in the food and cosmetic fields. Today, preventive measures of hygiene and also antibiotics (medical) and conservators (food and cosmetic) are already used to fight against pathogenic bacteria. Precisely, synthetic preservatives are largely used in food and cosmetics. Thanks to their antibacterial and antioxidant properties, these preservatives protect both the consumer and the formulating product against microbial infections1 and also against diseases caused by oxygen-derived free radicals.2 Even though chemical preservatives are approved for human consumption by government agencies, some of them remain a controversial issue because they are suspected to lead to negative consequences on human health.3,4 Recently, as an alternative, more attention has been given to molecules of natural origin from various


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sources, such as plants, animals and microorganisms,5,6 to replace chemical preservatives. Plantderived compounds are promising candidates. These compounds can be classified according to their chemical structure: phenolic compounds, terpens and steroids or nitrogen compounds (alkaloids). They are active molecules with various antibacterial,7,8,9 antioxidant10 and/or antifungal properties.11 Many comparative studies have found that the family of phenolic compounds is very active compared to others. However, these molecules also possess some drawbacks, such as their possible allergenic issues and often hydrophobic12,13 character, which limit their fields of uses. One alternative is the incorporation of these active compounds within a polymer structure. Most of the time, the selected polymers are polysaccharides because they are natural, biocompatible and/or biodegradable.14,15,16 Moreover, these natural polymers also present interesting viscosifying and/or gelling properties with wide domains of application, including the food and cosmetic industries. Different methods are described in the literature to incorporate active plant-derived compounds within a polymer structure. For example, their incorporation within natural polymer films, such as carboxymethylcellulose or casein,17 their entrapment inside capsules of synthetic polymers like polysulfone,18 their polymerization,19 copolymerization20 and also their chemical grafting onto a polysaccharide such as chitosan have been attempted.21,22 The natural compound guaiacol possesses interesting antibacterial, antifungal23,24 and antioxidant properties.25 Liu et al.19 synthesized a polymer using an acrylamide monomer derived from guaiacol. The authors demonstrated that these new polymers were highly effective against the bio-adhesion of bacteria and also prevented the formation of the Bacillus subtilis biofilm. In this study, we chose to chemically graft an active plant-derived compound onto a polysaccharide. In the literature, most of the time, authors used chitosan as polysaccharide and


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various acid phenolics including ferulic acid,21,26,27 caffeic acid,27,28 or protocatechuic acid29 as active plant-derived compounds. Chitosan, a cationic polysaccharide is known as a bioactive compound with intrinsic antimicrobial and antioxidant properties. However, its poor solubility in neutral pH solutions represents a limit to use it in various applications. The main objectives of the chemical grafting of a natural compound onto chitosan were to improve its solubility and also its antioxidant properties for use as antioxidant agent in food, food packaging, pharmaceuticals or cosmetic industries. In this work, carboxymethylpullulan (CMP) was used as polysaccharide because of its high water solubility and because it does not possess any biological and antioxidant properties. Aminoguaiacol, a phenolic compound was used as the active molecule with antioxidant and antibacterial properties. The main objectives of this work are to attempt to (a) bring antioxidant and also antibacterial properties to a polysaccharide (which present neither antioxidant properties nor antibacterial properties) thanks to the presence of an active plantderived compound. We also attempt to (b) obtain new active systems with viscosifying or gelling properties. These types of systems will be used as natural preservatives for aqueous formulations in cosmetic or food domains. To elaborate this type of product, aminoguaiacol was firstly chemically grafted onto an anionic polysaccharide CMP. Then, the grafting was confirmed by FTIR spectroscopy and quantified by 1

H-NMR spectroscopy. The physicochemical behavior of the derivatives was studied in dilute

and semi-dilute media using various techniques, size-exclusion chromatography (SEC) coupling on-line with a multi-angle light scattering system (MALS), a viscometer, a differential refractive index detector (DRI), fluorescence spectroscopy and also rheological measurements. Then, their antibacterial and antioxidant properties were also evaluated. EXPERIMENTAL SECTION


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Materials. Pullulan was purchased from Hayashibara Biochemical Laboratory (Japan). Aminoguaiacol (NH2GA) was purchased from Merck KGaA (Germany). 1-Ethyl-3-(3dimethylaminopropyl) carbodiimide (EDCI), α,α-diphenyl-β-picrylhydrazyl (DPPH) and phosphate-buffered saline (PBS) tablets were purchased from Sigma-Aldrich (France). Sodium hydroxide (NaOH) and hydrochloric acid (HCl) were purchased from VWR (France). Water was purified with a Milli-Q water reagent system (Millipore, USA). All compounds were used without further purification. Synthesis of carboxymethylpullulan (CMP). The sodium salt of carboxymethylpullulan (CMP, Na+) was synthesized according to the method previously described.30 The degree of substitution of CMP (DSCOONa), which is the number of carboxylate groups per anhydroglucose unit (AGU), was determined by conductimetric titration and was 0.96 (DSCOONa = 0.96). Synthesis of carboxymethylpullulan grafted with aminoguaiacol (CMP-G). Three theoretical degrees of substitution of aminoguaiacol DS(Ga) have been targeted: 0.25, 0.5 and 1. For example, for theoretical DS(Ga) of 0.25, aminoguaiacol (13 mM) was dissolved in HCl at 0.05 mol L-1 for 12 h. Then, the solution of aminoguaiacol (80 mL) was slowly added to a solution of CMP (1 g)

in

Milli-Q water (50 mL) (Mn (CMP) = 143,000 g.mol-1, Ð = 1.6 and

DSCOONa = 0.96). The coupling reaction of aminoguaiacol with the carboxylate groups of CMP was activated by EDCI (at a molar ratio of EDCI over AGU of CMP equal to 0.3), and the pH of the mixture was adjusted to 4.5 (with HCl at 0.1 mol L-1). The reaction was conducted at ambient temperature for 24 h. At the end of the reaction, the pH was checked and adjusted to 7.2 with NaOH at 1 mol.L-1. Then, CMP-G was first dialyzed against NaOH (0.1 mol L-1) for 24 h to eliminate the unreacted aminoguaiacol and EDCI urea and then against Milli-Q water until a low conductivity of the


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dialysis water was obtained (equivalent to the conductivity of Milli-Q water) (dialysis membrane Spectra-Por 12-14 kDa purchased from Spectrum Europe). The CMP-G was then lyophilized and stored at 4°C. The product obtained was brown. Methods. FTIR spectroscopy. The Fourier transform infrared spectroscopy was performed on a Nicolet IS50 FT-IR spectrometer (Thermo Scientific, USA). The samples were analyzed by transmission from 500 to 4,000 cm-1 (128 scans resolution 4) using the OMNIC software. All the products were deposited in solid form (without prior preparation) on the crystal of the device for analyses. NMR spectroscopy. The 1H-NMR spectra of the CMP-G derivatives were determined with a Bruker Advance AC-P 300 MHz spectrometer (USA) with an internal standard, sodium acetate. The spectra were analyzed with the Topsin software. The spectra provided chemical shifts in parts per million (ppm) and the integrations of the hydrogen peaks of the macromolecules. The degree of substitution of aminoguaiacol (DS(Ga)) was calculated via Equation 1 using the integration of the aromatic protons of aminoguaiacol (3 protons) and the integration of the protons of sodium acetate (3 protons). The DS(Ga) is the number of aminoguaiacol molecules per anhydroglucose unit. The solutions of CMP-G derivatives were prepared for analyses in deuterium oxide (D2O; 99.9 atom % D, Sigma-Aldrich, France) at 10 g.L-1 with sodium acetate at 0.5 g L-1. DS(Ga) =

m (acetate) ∗ I (NH2GA) ∗ M (CMP − G) M (acetate) ∗ I (acetate) ∗ m ( CMP − G)

Equation 1

I(acetate) indicates the peak area of sodium acetate protons; I(NH2GA) is the peak area of aromatic protons of aminoguaiacol. m(acetate) indicates the exact mass of sodium acetate; m(CMP-G) is the exact mass of CMP-G. M(acetate) indicates the molar mass of sodium acetate;


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M(CMP-G) is the molar mass of the CMP-G product (M (CMP-G) = 162 + 80 x DSCOONa + 99.15 x DS(Ga)). SEC/MALS/DRI/Viscometer. The equipment used was a size-exclusion chromatography (SEC) coupling on-line with a multi-angle light scattering detector (MALS), a viscometer and a differential refractive index detector (DRI) with a maximum uncertainty of 3%. This coupling allowed for us to obtain the physicochemical characteristics of the polymers, including their average mass and number molar masses (Mw and Mn, respectively), their intrinsic viscosities [ɳ] and their hydrodynamic radii (Rh) in diluted medium at 25°C. The determination of the average intrinsic viscosity allowed us to obtain the average hydrodynamic volume (Vh) using the Einstein−Simha equation (Equation 2): Vh = [η]M/νNA (Equation 2), where NA is Avogadro’s number, M is the molar mass, [η] is the intrinsic viscosity (g mL−1), and ν is a conformational parameter equal to 2.5 in the case of a spherical conformation, which was expected in our study. Based on the Stokes−Einstein equation (Equation 3), we calculated the diffusion coefficient (Dt in m2.s−1): Rh = kT/6ηDt (Equation 3), where k is the Boltzmann constant, T is the temperature (K), and η is the dynamic viscosity (Pa.s) of the medium. The eluent used was phosphate-buffered saline (0.15 mol L-1, pH 7.4) previously filtered through a 0.1 µm filter unit (Millipore, USA), on-line degassed (DGU-20A3 Shimadzu, Japan), and eluted at a 0.5 mL min-1 flow rate (LC10Ai Shimadzu, Japan). The polymer solutions were prepared with stirring for 24 h at 25°C from lyophilized CMP-G products at 1 g L-1 in filtered PBS (0.45 µm, Millipore, USA), and 100 µL was injected with an automatic injector (SIL -20A,


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Shimadzu, Japan). The SEC line contained an OHPAK SB-G guard column and two OHPAK SB 804 and 806 HQ columns (Shodex Showa Denko K.K., Japan) in series packed with polyhydroxymethylmetacrylate gel. The MALS detector was a DAWN Heleos-II (Wyatt Technology, Inc., USA) fitted with a K5 cell of 50 μL and 18 photodiodes (normalized relative to the 90° detector using bovine serum albumin). The viscometer used was a ViscoStar II (Wyatt Technology, Inc., USA). The collected data were analyzed using the Zimm 1st order fit in the Astra 6.1.1.17 software package. The concentration of each eluted fraction was determined with DRI (RID-10A Shimadzu, Japan) according to the known values of dn/dC (0.140 mL g-1 for CMP and derivatives).31 All the samples were analyzed between 13 and 22 mL of elution volume. Rheological measurements. Solutions were prepared at various concentrations (2.5 - 60 g L-1) in PBS (0.15 mol L-1, pH 7.4). The viscosity measurements of the CMP precursor and CMP-G derivatives were performed at a low shear rate (1 s-1) in the Newtonian regime using a Couettetype viscometer (LS400, Lamy Rheology, France) at 25°C. Flow curves were determined using a Discovery RH2 Rheometer from TA Instrument (U.K.) with a standard-size double concentric cylinder as geometry (aluminum – gap 500 m) for polymer concentrations of 50 and 100 g L-1 and with a cone-plate geometry (diameter 4 cm; angle 2°; gap 57 m) at 150 g L-1. All the measurements were performed with a solvent trap to prevent any evaporation. The analyses were performed with the TA Instrument Trios v4.1.1 software. Fluorescence measurements. Pyrene was used as a hydrophobic fluorescence probe to measure the self-aggregation behavior of the CMP-G products.32 Fluorescence measurements were performed with a Fluoromax-4 spectrophotometer (Horiba Jobin Yvon, Japan) equipped with a


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Xenon lamp. One milliliter of pyrene (4 ×10−7 M in water) was added to the solutions of CMP or CMP-G derivatives (1 mL) to obtain final concentrations in the range of 0.05 – 12 g L-1 in 0.15 M NaCl. The solutions were placed with stirring in darkness for 24 h. Then, each solution was excited at 332 nm, and the emission spectra of pyrene was recorded from 350 to 450 nm. The temperature was 25°C and controlled with a circulating bath. Generally, five vibrational peaks can be observed in the pyrene emission spectrum. The ratio I1/I3 of the intensity of the highest energy vibrational band (I1, 373 nm) to the third highest energy vibrational band (I3, 382 nm) was used to measure the local environment polarity of pyrene in presence of CMP or CMP-G derivatives at the various tested concentrations. A decrease in the I1/I3 ratio indicates the presence of a nonpolar environment, such as hydrophobic clusters. Antibacterial assays. Antibacterial tests were carried out by a CFU (colony forming unit) counting method according to Lequeux et al.33 with some modifications. Staphylococcus aureus ATCC 29213 was chosen as the bacterium, and BHI medium (Brain Heart Infusion, SigmaAldrich, France) was used as the broth for culture. Stock solutions of the tested compounds in ethanol or PBS were made according to the solubility of each compound (Table 1). Serial dilutions of the compounds were then made in BHI broth with a final volume of 2 mL, and bacterial inoculum was added (100 µL) to reach a final density of 1 x 106 CFU/mL. Incubations of these bacterial solutions were made for 24 h at 37°C, allowing bacteria to grow with or without the antibacterial derivatives (aminoguaiacol, CMP-G derivatives or solvents used for solubilization as references). The optical density (OD) values at 595 nm were then determined, and the samples presenting decreased OD values compared to normal growth were plated to assess the CFU/mL. For counting, 10-fold serial dilutions in sterilized PBS of the chosen


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samples were used. Two drops (20 µL) of each dilution were deposited onto BHI agar petri dishes. After the incubation of the plates for 24 h at 37°C, the colonies of bacteria were counted and expressed as CFU/mL. The antibacterial activity of each sample was calculated using the following equation: antibacterial activity (%) =

CFU/mL(reference) − CFU/mL (sample) ∗ 100 Equation 4 CFU/mL (référence)

For aminoguaiacol, the reference was made with BHI/ethanol using the same volume as the one used for the dilution of aminoguaiacol. For CMP-G derivatives, the reference was the precursor CMP prepared using the same conditions as CMP-G derivatives. Table 1: The concentrations and conditions of solubilization of the different compounds used in the antibacterial assays. Aminoguaiacol (NH2GA) and CMP-G derivatives with various substitution degrees of aminoguaiacol DS(Ga): (0.16; 0.37 and 0.58) were tested. Compounds Solvent [stock solution] (g L-1)

[compound] (g L-1)

[NH2GA]eq a (mM)

NH2GA

Ethanol

69.5

[2.8 – 0.35]

[20 – 2.5]

CMP

PBS

50 ou 80

[50 – 3.125] / [60 – 3.75]

-

80

[60 – 3.75]

[38 – 2.4]

80

[60 – 3.75]

[81 – 5]

50

[50 – 3.125]

[98 – 6.1]

CMP-G0.16 b CMP-G0.37 b CMP-G0.58 b

PBS

a

Equivalent concentration of NH2GA (expressed in mM) that is present in each tested product.

b

Average experimental DS(Ga) (see Table 2).

The equivalent concentration of NH2GA in CMP-G derivatives ([NH2GA]eq, expressed in mM) for assays with the CMP-G derivatives were calculated using Equation 5. [NH2GA]eq =

𝐷𝑆(𝐺𝑎) ∗ 𝐶(𝐶𝑀𝑃 − 𝐺) 𝐸𝑞𝑢𝑎𝑡𝑖𝑜𝑛 5 𝑀(𝐶𝑀𝑃 − 𝐺)


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In this equation, M(CMP-G) is the molar mass of the CMP-G product (M(CMP-G) = 162 + 80xDSCOONa + 99.15xDS(Ga)), DS(Ga) is the experimental degree of substitution of NH2GA calculated using the 1H-NMR data and C(CMP-G) is the tested concentration of the CMP-G product. Antioxidant assays. The antioxidant activity of aminoguaiacol grafted onto CMP was evaluated using the DPPH radical according to the method of Woranuch et al.21 with some modifications. The DPPH radical is stable and is a purple color in ethanolic solution. In the visible domain (400-800 nm), this radical has a maximum absorbance at 517 nm. Upon exposure to an antioxidant, the DPPH radical reacts, and its color decreases (i.e., its absorbance at 517 nm decreases). For aminoguaiacol, various solutions in ethanol (1 mL) were prepared at different concentrations (from 0.045 to 1.4 mM). Then, 1 mL of DPPH (100 µM) in ethanol was added, and each solution was incubated with stirring in darkness for 5, 10, 20, 30 min and 24 h at room temperature. After stirring, the absorbance of solutions was measured between 400-800 nm. The percentage of DPPH radical-scavenging activity was calculated according to Equation 6 at 517 nm (the maximum absorption of DPPH in ethanol). For grafted polysaccharides, this protocol was slightly modified as follows: solutions of the CMP-G derivatives were prepared in NaCl at 0.15 M at various concentrations (3.33 – 10 g L1

).Then, 0.5 mL of DPPH (200 µM) in ethanol was added to 1.5 mL of the CMP-G solution. The

final CMP-G concentrations equaled to 2.5; 5 and 7.5 g L-1. The percentage of the DPPH radicalscavenging activity was calculated according to Equation 6 at 543 nm (the maximum absorption of DPPH in NaCl/ ethanol (3:1)).


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All tested products were colored (brown). Their color increased along with the DS(Ga). To overcome the color of aminoguaiacol and to facilitate the reading of the absorbance in the UVvisible region, we also prepared a similar range of dilutions for each tested product without DPPH as a blank. The half-inhibition concentration (IC50) for aminoguaiacol and for CMP-G derivatives which correspond to the concentrations of active compounds causing 50% of inhibition of the DPPH radical were also determined. Measurements of the scavenging effect was carried out between (0.0488 – 3.125) µg.mL-1 for aminoguaiacol and between (0.002 – 0.5) mg.mL-1 for CMP-G derivatives. DPPH scavenging activity (%) =

Absorbance (DPPH) − Absorbance (sample) ∗ 100 Absorbance (DPPH)

Equation 6

For the grafted polymer, Absorbance (sample) = Absorbance (sample with DPPH) – Absorbance (blank). The equivalent concentration of NH2GA (expressed in mM) for the assays with the CMP-G derivatives were calculated using Equation 5. Cytotoxicity assays. Cell culture. We used mouse fibroblasts cells (L929 - ATCC® CCL-1) to check that our CMP-G derivatives were not cytotoxic. Cells were cultured with -MEM culture medium supplemented with 10% foetal bovine serum (FBS, Invitrogen), 100 mg/ml streptomycin (Sigma), 100 mM penicillin (Sigma), 2 mM L-glutamine (Sigma), grown in a 75 cm2 tissue culture flask, and incubated in a humidified atmosphere with 5% CO2 at 37°C. Cells were routinely examined using a Motic inverted microscope (phase-contrast). When subconfluent, cells were enzymatically detached using 0.25% trypsin/1mM EDTA (Sigma). We prepared (7.5g.L-1) solutions of CMP and CMP-G derivatives (CMP-G0.16 and CMP-G0.58) in non-supplemented -MEM, the highest concentration used for antioxydant assays.


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Aminoguaiacol was first solubilized in ethanol (200 mM) and then diluted down to 1 mM with culture medium so that the final concentration of ethanol was 0.5% (v/v). In parallel, we tested a solvent control (EtOH 0.5% - v/v). We also used 10% DMSO and complete culture medium as positive and negative controls of cytotoxicity, respectively. 24h before any cytotoxicity assay, we plated L929 cells at a density of 30 000 cells.cm-2 either on top of glass slides or in 48-well plates, for viability and metabolic activity, respectively. First, we assessed the cell viability by using Live/Dead kit (Molecular Probes) 24h after contacting with the compounds. Live cells were stained by calcein-AM (green), a highly lypophilic dye that only stains viable cells after enzymatic conversion of non-fluorescent calcein-AM into fluorescent calcein, whereas dying or dead cells were stained by ethidium heterodimer (red), a nuclear staining dye that can only pass through damaged membranes. For fluorescence examination, we used an epifluorescence microscope (Zeiss Axio Scope A1) equipped with a Motic CCD camera for image acquisition. Second, 48h after contacting, we tested the metabolic activity of L929 cells by using Alamar Blue® (AB) assay (AbD Serotec) which is an indicator of oxidation−reduction reactions during cell proliferation and is based on the metabolic activity of viable cells. Pre-warmed 10% AB-containing culture medium was prepared and added to cells for a 2h30 period. Finally, the absorbance was read at 570 and 600 nm on UV-Vis spectrophotometer (Spectronic Unicam – UV 300). All tests were performed in triplicate. RESULTS AND DISCUSSION Synthesis of CMP-G. A hydrophobic compound, aminoguaiacol (NH2GA) which is a phenolic compound (colored in dark brown) derived from guaiacol, was chemically grafted onto CMP, a hydrosoluble polysaccharide, using an EDCI coupling reagent in aqueous acid medium (pH 4.5) for 24 h. EDCI activates the carboxylate groups of CMP.34 An amide covalent bond was


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created between the amine functional group of NH2GA and the carboxylate group of CMP. Amphiphilic CMP-G derivatives were obtained. The grafting reaction is described in Scheme 1.

Scheme 1. The grafting reaction of aminoguaiacol onto CMP activated by EDCI in acid aqueous medium for 24 h, at room temperature To achieve the grafting reaction, various theoretical degrees of substitution of aminoguaiacol DS(Ga) were fixed at 1, 0.5 and 0.25. All reaction conditions are summarized in Table 2. These conditions allowed us to control the solubility of CMP-G derivatives and to obtain a wide range of products for biological and antioxidant tests. All synthesized CMP-G derivatives were soluble in water and changed colors from brown to a dark brown color according to the DS(Ga). The mass and substitution yields were approximately 60%. The precision of the reaction was also studied, and the experimental DS(Ga) values showed that the reproducibility was good (Table 2). Table 2: The general conditions for the synthesis of CMP grafted with aminoguaiacol in acid aqueous medium for 24 h at ambient temperature.


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Theoretical DS(Ga) a

EDCI/CMP b

Experimental DS(Ga) c

0.25

0.30

0.50 1

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Mass yield (%)

0.16 ± 0.02

% (g/100 g) aminoguaiacol content in product 8.7

0.60

0.37 ± 0.03

18.7

66 ± 0.22

1.20

0.58 ± 0.08

27.2

62 ± 0.21

a

Molar ratio NH2GA/CMP.

b

Molar ratio EDCI/CMP.

58 ± 0.31

c

Experimental DS(Ga) calculated using 1H-NMR (Equation 1) (average of seven separate syntheses). Characterization of CMP-G. FTIR spectroscopy. FTIR spectroscopy was used to confirm the covalent grafting of aminoguaiacol onto CMP by the means of amide bond detection. The FTIR spectrum of aminoguaiacol possessed characteristic peaks at 3390 cm-1 (N-H stretching), 3310 cm-1 (O-H stretching), 1600 cm-1 (Ar-NH2), 1390 cm-1 (Ar-OH), 1530 cm-1 and 1450 (C=C stretching), 1250 cm-1 (C-N stretching of Ar-NH2 band) and 1220 cm-1 (C-O stretching of Ar-OH band). The spectrum of CMP exhibited characteristic peaks at 3660-3000 cm-1 (O-H stretching), 1590 cm-1 (C=O stretching of carboxylate groups) and 1010 cm-1 (C-O-C stretching of ether function). For CMP-G spectra, the new peaks appeared at 1240 cm-1 and 1510 cm-1 corresponding to the C-O stretching of the Ar-OH band and to the C=C stretching of the aromatic ring, respectively. Moreover, shoulders at 1650 cm-1 were also visible, and they corresponded to the amide bonds, demonstrating the grafting of NH2GA onto CMP. As qualitative evidence, the intensities of the shoulders at 1650 cm-1 increased with the DS(Ga) (Supporting information Figure S1). 1H-NMR

spectroscopy.

1

H-NMR spectroscopy was used to detect the presence of

aminoguaiacol in CMP-G derivatives and to quantify the grafting by determining the DS(Ga). Figure 1 shows the spectra of CMP, aminoguaiacol and the CMP-G derivative with the highest


15

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Biomacromolecules

DS(Ga). The spectrum of CMP (Figure 1a) exhibited the characteristic signals for D2O between 3.1-4.5 ppm corresponding to CH and CH2 (except anomeric protons) and between 5-6 ppm corresponding to anomeric protons (Ha). The spectrum of aminoguaiacol (Figure 1b) exhibited peaks in DMSO-d6 at 8.6 ppm (1H: OH); between 5.9-6.7 ppm corresponding to aromatic protons (3H), at 4.5 ppm (2H: NH2) and at 3.6 ppm (3H: O-CH3). In comparison with the spectrum of CMP (Figure 1a), the spectrum of the CMP-G derivative (Figure 1c) showed a new large peak between 6.2-7 ppm. This new peak corresponded to the aromatic protons (3H) of aminoguaiacol and allowed the confirmation of the presence of aminoguaiacol in CMP-G products. In the literature, the 1H-NMR spectrum of a grafted polysaccharide (chitosan) with aromatic compounds also exhibited peaks between 6-8 ppm.22,27 In comparison with the spectrum of free aminoguaiacol, the resolution of the aromatic peaks in the CMP-G spectrum was low, and it was not possible to distinguish each aromatic proton peak. It was probably due to the use of different solvents (D2O for CMP-G and DMSO-d6 for free aminoguaiacol) but also because of the reduced mobility of the aromatic protons in the CMP-G derivatives. Moreover, we noticed that the aromatic protons peaks of grafted aminoguaiacol in the CMP-G products were shifted (ppm) towards higher values. This could be attributed to the environment of the aromatic protons, which was different because of the grafting (amide bond instead of amine bond). To quantify the grafting rate, the DS(Ga) values were calculated by means of the ratio between the integrated peaks of sodium acetate and the aromatic aminoguaiacol protons (Equation 1). Table 2 summarizes the experimental DS(Ga) that were obtained for the CMP-G derivatives. These DS(Ga) values corresponded to the contents of aminoguaiacol in the products, which was important. For example, for an experimental DS(Ga) of 0.58, the amount of NH2GA was 27.2 g for 100 g of CMP-G. The efficiency of the reaction was good, between 58% for the theoretical


16

Biomacromolecules

DS(Ga) = 1 and 74% for the theoretical DS(Ga) = 0.5. We obtained substitution degrees higher than those found in the literature. For example, caffeic acid was grafted onto chitosan, and the maximum content of caffeic acid obtained was 13.6 g/100 g of product.28 Moreover, ferulic acid was grafted onto carboxymethylpullulan with an efficiency of 14% (for a maximum experimental DS equal to 0.11).31 The authors also used EDCI as coupling agent. a

OCH2COONa CH2

Pullulan : R = H

O

CMP: R = H ou CH2COONa

O --OR

OR

Ha

OR n

b

f OCH 3 b d

Ha

b b a OH

c

f

NH2 e

a

b

c

d

e

c

OH CH2 O

b

OH

O--O C

b

n

H2C O

Sodium acetate

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 18 of 46

NH

c OH

c

OCH3

Figure 1. The 1H-NMR spectra of (a) CMP, (b) aminoguaiacol and (c) CMP-G derivatives with DS(Ga) of 0.58 at 10 g L-1 in D2O in the presence of sodium acetate at 0.5 g L-1. SEC/MALS/DRI/Viscometer. The size exclusion chromatography coupled with multi-angle light scattering, differential refractive index and viscometer detectors was used to characterize the physico-chemical behavior of the amphiphilic CMP-G products in diluted medium by the


17

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Biomacromolecules

determining the intrinsic characteristic variables (Mn; Mw; Rh; [ɳ]). Samples (CMP precursor and CMP-G derivatives) were analyzed at 1 g L-1 in PBS at 0.15 mol L-1. Figure 2 illustrates the fractionation profiles with both light scattering (LS at 90°) and DRI responses, together with the molar mass distribution of the CMP precursor (DSCOONa = 0.96) and the CMP-G derivatives with various experimental DS(Ga) values (0.16, 0.37 and 0.58). Table 3 summarizes the intrinsic characteristic variables obtained from the fractionation profiles for all analyzed samples. Table 3. The results obtained with SEC/MALS/DRI/Visco testing for the analyses of the CMP precursor and the CMP-G derivatives (at 25°C, in PBS at 0.15 mol L-1 and at 1.0 g L-1).

Mn

Mw

Mo a

(g mol-1)

(g mol-1)

(g mol-1)

Apparent DPn

Rh (n) (nm)

(Mn/M0)

a exponent from

[ɳ]n

[ɳ]LSr c

(mL g-1)

(mL g-1)

116

0.4

30

60

1.0

55

31

2.4

67

8

18

-

KH d

Ccr e (g L-1)

Rg vs Mb CMP

CMP-G0.16

CMP-G0.37

CMP-G0.58

143,000

229,000

(± 1.6 %)

(± 0.4 %)

166,000

262,000

(± 1.6 %)

(± 0.6 %)

181,000

294,000

(± 1.4 %)

(± 0.6 %)

279,000

307,000

(± 2.1 %)

(± 1.7 %)

239

255

276

297

598

651

656

939

12.6

0.69

100

(± 0.5 %)

(± 0.2%)

(± 1.0 %)

12.3

0.61

77

(± 0.5 %)

(± 0.3%)

(± 1.1 %)

10.9

0.57

52

(± 0.6 %)

(± 0.5%)

(± 1.3 %)

8.9

0.49

16

(± 1.9 %)

(± 4.5 %)

(± 5.2 %)

a

Mo is the molar mass of the repeating unit calculated as 162 + 80DSCOONa + 99.15 DS(Ga) with DSCOONa=0.96. b

determined on valuable Rg values (above 10nm related to anisotropic scattering, see text)

c

Intrinsic viscosities obtained at a low shear rate (1 s-1) using a LS400 viscometer.

d

Huggin’s constant determined by Huggin’s equation35 from low-shear measurements.


18

Biomacromolecules

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Page 20 of 46

e

Critical concentration of a polymer that delimited the transition between the diluted and semidiluted medium determined by the Utracki and Simha representation36 (Supporting Information Figure S2). The chromatograms (Figure 2) show that both the DRI and LS responses shifted toward a higher elution volume (i.e., a lower size) when the DS(Ga) increased. This seemed consistent with a decrease in coil size when the amount of grafted NH2GA increased. This could be explained either by the degradation of the polymer during chemical modification (i.e., the decrease of molar mass) or by the establishment of a more compact conformation because of strong intra- or inter-hydrophobic associations of NH2GA groups onto the polymer backbone. No degradation was observed according to the molar masses values (Table 3). It was also noticed that the molar masses were larger when the DS(Ga) increased for a given eluted volume (i.e., for the same size), which traduced the dense conformation of the eluted grafted fractions. The results showed an increase in the CMP-G derivatives molar masses (Mn and Mw) together with the NH2GA grafting amount (DS(Ga)). This was not only due to the increase in the monomer unit molar mass (M0) since a clear increase in the apparent DPn was also observed. This clearly indicated an associative behavior, even with the dilute regime, with the presence of aggregate structures in solution because of hydrophobic associations between the grafted NH2GA onto the polymer backbones. In the same time, an important decrease in both the intrinsic viscosities [ɳ] and Rh was observed when increasing the DS(Ga) value. This result was consistent with a very compact and dense structure of single coil or aggregates in dilute solution. This was also a consequence of both the intra- and intermolecular hydrophobic associations between the NH2GA-grafted groups. Slight differences between the intrinsic viscosities obtained on-line ([]n) and by a low shear Couette viscometer ([]LSr) were observed. In the last case, the values were determined by the


19

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Biomacromolecules

extrapolation of Huggin’s law (Supporting Information Figure S3) from macroscopic and batch solution (i.e., coexistence of isolated compact coil and aggregates). Thus, the on-line values seemed to appear more relevant. Nevertheless, Hugging’s constant (KH) had been found to be largely higher when the DS(Ga) increased as a confirmation of the great aggregation tendency of grafted polymers.37,38,39 Let us notice that no average gyration radii (Rg) have been given in Table 3 because a part of them could not be determined by light scattering. This was due to isotropic scattering (no angular dependency) of the smallest coils (below about 10nm) that not allowed the determination of Rg. This concerned the smallest fractions in size of CMP and of their derivatives (particularly with compact structures) eluted at the end of the profiles. Nevertheless for Rg values available (above 10nm), it was possible to plot logarithmic relation of Rg vs. molar mass (Rg~Ma) giving exponent ‘a’ related to conformational information. Such plots are reported in the supporting information (Figure S4) and the ‘a’ values are reported in Table 3. Our results clearly indicated a decrease of ‘a’ exponent with increasing DS(Ga) in the CMP derivatives. This was well correlated with more compact conformation due to hydrophobic association between Ga groups that reinforced our previous conclusions. Such hydrophobic interactions between polymers in aqueous media is supported by associative behavior because of the amphiphilic character of the CMP-G derivatives. This was often observed in previous studies with other hydrophobic grafted pendant groups.40,41,38 It has also been largely demonstrated that the associative behavior depends on various parameters, including the nature of a polysaccharide (neutral or polyelectrolyte), the nature and amount of grafted compound, and the solvent (aqueous or salt media).30,31,38,41,42,43,44,45,46,47,48 However, although the compact structures seemed to be preferentially established by intramolecular associations in the dilute regime, the occurrence of intermolecular associations, as observed by


20

Biomacromolecules

increases in the average Mn and Mw, could not be neglected.38,46,49 On the contrary, with the behavior of the polymer in solution without specific polymer/polymer associations, an entangled critical concentration (C*) could be shown between the dilute and semi-dilute regime, and the transition of concentration domains in the case of CMP-G derivatives was not only governed by steric consideration because of associative behavior. A thermodynamic competition occurred between compact low size objects because of intramolecular associations (increasing the critical concentration, Ccr) and intermolecular associative connections (decreasing the critical concentration, Ccr).38 For the CMP-G samples, the Ccr was largely shifted toward higher values when the DS(Ga) increased (Table 3) in relation with the strong compact conformation of polymers in aqueous media. This behavior seemed similar to polysoaps.50 LS

10000000

DRI

1

0,8 1000000

CMP 0,6

Reltive scale

Molecular weigth (g. mol -1)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 22 of 46

CMP-G0.16 CMP-G0.37 CMP-G0.58

0,4

100000

0,2

10000

0 12

14

16

18

20

22

Volume (mL)

Figure 2. The elution profiles of CMP-G derivatives (PBS at 0.15 M). Light scattering signal (LS): full line curves; Differential refractive index (DRI): dash line curves and Molecular weight distributions: full line curves in bold for CMP DS 0.96 (red), CMP-G0.16 (blue), CMP-G0.37 (orange), and CMP-G0.58 (green). Fluorescence measurements. The associative behavior of the CMP-G derivatives in aqueous media was also shown using a pyrene fluorescence probe that shows different characteristics of fluorescence depending on the polarity of the solution in which it is solubilized. More precisely, the ratio between the first and the third fluorescence peaks (I1/I3) is approximately 0.6 in


21

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Biomacromolecules

hydrocarbon solvents, close to 1.1 in ethanol and approximately 1.6 in water.48 The I1/I3 ratio provided an indication of the local environment polarity of pyrene in the presence of CMP or CMP-G derivatives at various tested concentrations. It can also be used to determine the critical aggregation concentration (CAC).51,52 As expected, no variation in the I1/I3 ratio had been observed with the increase in the CMP precursor concentration. Indeed, at all tested concentrations, the value of the I1/I3 ratio was constant and close to the corresponding solvent ratio of 1.6 (NaCl 0.15 M/water) (Figure 3). For CMP-G derivatives, a decrease in the I1/I3 ratio was clearly observed with an increase in the concentrations (Figure 3). For example, for CMP-G0.16, the I1/I3 ratio moved from 1.6 (polar environment) at the lowest concentration to approximately 1.2 at the highest concentration, indicating a more nonpolar environment.51 This was explained by the formation of hydrophobic clusters mainly due to the intramolecular associations between the NH2GA grafted pendant groups in which pyrene could penetrate. As a confirmation, the decrease was more significant and occurred at lower polymer concentrations when the DS(Ga) increased. This evolution fully confirmed the behavior previously described according to the SEC/MALS/DRI/Viscometer analysis. Therefore, the CAC of the CMP-G derivatives were determined and equal to 1.2, 0.6 and 0.15 g L-1, respectively, for CMP-G0.16, CMP-G0.37 and CMP-G0.58. I1 / I3 1,7 1,6 1,5

CMP

1,4

CMP-G0.16 1,3

CMP-G0.37 CMP-G0.58

1,2 1,1 1

C (polymer) g/L

0,9 0,03

0,3

3

30


22

Biomacromolecules

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Page 24 of 46

Figure 3. The I1/I3 ratio of the vibronic band intensities of pyrene as a function of the polymer concentration at 25°C for CMP and CMP-G derivatives in NaCl at 0.15 M. CMP DS 0.96 (red); CMP-G0.16 (blue); CMP-G0.37 (orange); CMP-G0.58 (green). Rheological measurements. Flow curves of CMP, CMP-G0.16, CMP-G0.37 and CMP-G0.58 (in the range 1-1000 s-1 in PBS at 0.15 M were determined (Figure 4) in the semi-dilute regime above Ccr using a standard-size double concentric cylinder as the geometry at 50 and 100 g L-1. A cone-plate geometry was used at 150 g L-1 because the solution of CMP-G0.58 appeared to be a viscoelastic solution. CMP and CMP-G had Newtonian behavior in the studied range of shear rates and for the whole studied concentrations, except for CMP-G0.58 at 150 g L-1. It was also remarkable that the grafted samples always had lower viscosities than the CMP precursor at a given concentration, with the exception of CMP-G0.58 at 150 g L-1. This rheological behavior is closer to polysoap behavior. Polysoaps are defined as amphiphilic polymers showing a very low viscosity, even for higher concentrations, when compared to their precursor. This is explained by very compact coils (small hydrodynamic radius) because of the intramolecular aggregation of hydrophobic moieties 53,54,55,56. Polysoaps usually possess a high hydrophobic moiety content with a random distribution and a flexible backbone of the polymer. These characteristics are not far from that of the CMP-G derivatives, since the pullulan is considered a flexible polysaccharide and the amount of NH2GA is consequent. The behavior of the CMP-G0.58 at 150 g L-1 is strongly different and atypical. Here, the modified CMP showed a higher viscosity than its precursor for the low shear rates. This indicated that some intermolecular associations took place because both the amount of grafted


23

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Biomacromolecules

NH2GA and the polymer concentration were sufficient. More surprisingly, the flow curve of CMP-G0.58 at 150 g L-1 gave rise to a shear thickening behavior for intermediate shear rates (between 5 - 30 s-1) with increases in the viscosity by 2 orders of magnitude. For the higher shear rates, a more classical shear thinning behavior could be observed. Such a shear thickening behavior has already been shown for polyelectrolytes.57 This peculiar behavior was also observed for neutral associative systems58 and loaded systems.59,60 It could be explained by the intra or intermolecular association shifts induced by the shear rate. In our case, no gelling occurred, and when the shear rate become too high (above 30 s-1), the intermolecular associations were disrupted, leading to shear thinning behavior. ɳapp (Pa.s) 0,1

CMP CMP-G0.16

0,01

CMP-G0.37 CMP-G0.58

shear rate (1/s)

0,001 1

10

100

1000

ɳapp (Pa.s) 0,1

CMP CMP-G0.16 CMP-G0.37 CMP-G0.58

shear rate (1/s)

0,01 1

10

100

1000

ɳapp (Pa.s) 100

10

CMP CMP-G0.16 CMP-G0.37

CMP-G0.58 1

shear rate (1/s)

0,1 1

10

100

1000


24

Biomacromolecules

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Page 26 of 46

Figure 4. The plot of the apparent viscosity of aqueous solutions for CMP and CMP-G derivatives as a function of shear rate. A) 50 g L-1 double concentric geometry; B) 100 g L-1 double concentric geometry; C) 150 g.L-1 cone-plate geometry with CMP (red); CMP-G0.16 (blue); CMP-G0.37 (orange); and CMP-G0.58 (green). At 50 g L-1, the measurements were disturbed at a low shear rate (< 10 s-1) because of the very low viscosities of the solutions and also the sensitivity of the device. Antibacterial assays. In the literature, the antibacterial activity of polysaccharides grafted with active compounds is usually evaluated by qualitative methods.20,43,61 However, because the color of the active compound (aminoguaiacol) disturbs the OD determination, the antibacterial activity of free aminoguaiacol and grafted aminoguaiacol (CMP-G derivatives) was evaluated by the more precise quantitative method of CFU counting. This activity was tested against the Gram-positive bacterium S. aureus, which is commonly found in cosmetics and foodstuffs.62,63 The results are summarized in Figure 5 and Table 4. First, we determined the biocidal activity of the free aminoguaiacol. However, to prevent the killing effect of ethanol for concentrations > 10% (v/v), the counting method was performed for solutions of aminoguaiacol at 15, 10, 5 and 2.5 mM with ethanol concentrations from 7.5 to 1.5%. Figure 5 shows that more than 99.999% of the S. aureus population was killed with 5 and 10 mM NH2GA, and a total eradication was reached for 15 mM, as no bacterial growth was observed on seeded plates after 24 h at 37°C. (This result was also checked without any dilution (dilution 0), see Supporting information Figure S5). The MBC (minimal bactericidal concentration) of aminoguaiacol which corresponded to the lowest concentration of aminoguaiacol for which 99.99% of the initial inoculum was killed was found to be equal to 10 mM.


25

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Biomacromolecules

For comparison, the guaiacol compound which possesses a very close chemical structure to aminoguaiacol was also tested in similar conditions against S. aureus. No MBC was found in the range of tested concentrations (15 mM -2.5 mM). Indeed, we only observed 99.91% of eradication for 15 mM guaiacol (data not shown). This suggests that guaiacol possesses a MBC which is higher than aminoguaiacol. For comparison, others phenolic compounds as creosol, eugenol and acid ferulic which also exhibit a very close chemical structure to aminoguaiacol possess a bactericidal activity against S.aureus with MBC equal to 6.2mM,64 25 mM,65 25.7mM,66 respectively. The antibacterial activities of the grafted polysaccharides (CMP-G) were then evaluated at various concentrations depending on the DS(Ga) (Table 4). All activities were calculated by comparison with the CFU numbers of reference, which were equal to 3.7 x 109. All CMP-G derivatives demonstrated antibacterial activity against S. aureus at all the tested concentrations. Moreover, this activity increased with both the DS(Ga) and the equivalent concentrations of grafted aminoguaiacol [NH2GA]eq (Table 4). It was found that the [NH2GA]eq was higher than 5 mM for all CMP-G derivatives. The MBC of grafted polysaccharides are higher than 60 g/L for CMP-G0.16 and CMP-G0.37 and higher than 50 g/L for CMP-G0.58. Nevertheless, the antibacterial activities of the CMP-G derivatives were lower than 99.999%, demonstrating a decrease in the aminoguaiacol activity once grafted. Two hypotheses could explain this phenomenon. The first is the possible loss of the mobility of aminoguaiacol after the chemical grafting on CMP. In the literature, the importance of the mobility of phenolic compounds in their mechanism of action has already been underlined.7 The second is the presence of compact aggregate structures in solution for CMP-G derivatives, as demonstrated by the coupling SEC/MALLS/DRI/Visco data, low shear measurements and fluorescence analyses. Indeed, if aminoguaiacol moieties


26

Biomacromolecules

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Page 28 of 46

participate in intra- and/or intermolecular associations, the molecules are no longer available for interactions with bacteria. Ethanol concentrations in

Ethanol 7.5%

Ethanol 5%

Ethanol 2.5%

Ethanol 1.25%

15 mM

10 mM

5 mM

2.5 mM

Numbers of CFU/mL for the reference a

5.8 x 1011

8.4 x 1011

7.3 x 1011

7.3 x 1011

Numbers of CFU/mL for NH2GA sample a

0

5

5.3 x 106

5.3 x 109

Antibacterial activity (%)

100

99.99999

99.9993

99.27

references

Pictures of seeded plates after 24h at 37°C

Concentrations of NH2GA

Pictures of seeded plates after 24h at 37°C

Figure 5. The pictures of the seeded plates after 24 h at 37°C for dilutions 5 (D5) to 8 (D8) and the percentage values of the antibacterial activities of aminoguaiacol and the corresponding references in ethanol. The arrow indicates the way of the decreasing dilutions (D5-8). The uncountable numbers of colonies are in yellow. At dilutions for which colonies were countable; a black felt marker was used to mark and count them. a

Equation 7 was used to calculate the CFU numbers CFU 1 1 = CFU (counted) ∗ ∗ Equation 7 mL f Vdeposited (mL)

With f : dilution factor. As example: f = 10-4 means that the bacteria colonies were countable at the dilution 4; Vdeposited = 20 µL = 20 x 10-3 mL


27

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Biomacromolecules

Table 4: The percentages values of the antibacterial activity for the CMP-G derivatives after 24 h at 37°C. [compound] g.L-1

[NH2GA]eq mM

CFU/mL (compound)

Antibacterial activity (%) a

a

CMP-G0.58

CMP-G0.37

CMP-G0.16 a

50

98

2.0 x 108

94.5

25

49

2.6 x 108

92.9

12.5

24

4.2 x 108

88.6

60

81

3.7 x 108

89.9

30

40

4.9 x 108

86.7

15

20

8.7 x 108

76.3

60

38

7.4 x 108

79.8

16

8

76.0

30

8.8 x 10

15 9.4 2.4 x 109 34.6 determined by Eq.4 with CFU/mL of reference equal to 3.7 x 109 (mean of two values).

Antioxidants assays. The DPPH radical scavenging percentage of the samples was determined by measuring the decrease in the absorbance of DPPH according to Equation 6. The CMP precursor did not possess any antioxidant activity at all tested concentrations (Figure 7A). Indeed, no decrease in the DPPH absorbance was observed in the presence of CMP. Free aminoguaiacol had antioxidant activity against the DPPH radical at all tested concentrations (Figure 7B). At the lowest tested concentration (0.022 mM), the scavenging effect was equal to 81.2 ± 1.8 %. At higher concentrations, the scavenging effect increased and was higher than 90%. Additionally, at 0.18 mM of aminoguaiacol, the scavenging percentage was 93.3 ± 0.20 %. In the literature, similar assays with guaiacol in the presence of DPPH were also performed, and a 100%-scavenging effect was found at 0.20 mM.25 Thus, the antioxidant activity of aminoguaiacol was close to that of guaiacol.


28

Biomacromolecules

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Page 30 of 46

The CMP-G derivatives were also tested, and Figure 7C shows that they possessed antioxidant activities. At a fixed concentration of CMP-G, the antioxidant activity increased with the DS(Ga), proving that the antioxidant activity was only due to the grafted aminoguaiacol. At a fixed DS(Ga), the antioxidant activity slightly increased with the tested concentration, indicating that the maximum of scavenging effect was achieved in this range of concentrations (2.5 – 7.5 g L-1). To compare the scavenging activity of the CMP-G derivatives with aminoguaiacol, the equivalent concentration of grafted aminoguaiacol [NH2GA]eq in the CMP-G derivatives was calculated (Equation 5) and added to Figure 7C on the right vertical axis. Compared to the tested concentrations of free aminoguaiacol, the equivalent concentration of aminoguaiacol in the CMP-G derivatives was always higher. However, the scavenging percentages of CMP-G were lower than 90%. Consequently, free aminoguaiacol was more active than grafted aminoguaiacol. This could be attributed to the greater difficulty of the DPPH radical to diffuse into the polymer solution than into a molecular solution and into hydrophobic cluster because of the alreadymentioned associative behavior of CMP-G derivatives. Moreover, antioxidant tests were carried out for various time points, 5, 10, 20, 30 min and 24 h. The time of the scavenging reaction showed no effect on the activities of the tested samples (data not shown). However, it was important to underline that the antioxidant activities of free and grafted aminoguaiacol reached their maxima within 5 min, and no modification in the percentage of scavenging was observed after 5 min. Furthermore, for a better comparison of the antioxidant activity of the tested samples with that of other scavenging agents in literature, the half-inhibition concentration (IC50) of aminoguaiacol and also grafted derivatives were determined. The curves of scavenging effect (%) versus the


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concentrations of tested compound (aminoguaiacol or grafted derivatives) are presented in supporting information Figure S6. Table 5. Values of the concentrations corresponding to 50% inhibition of the DPPH radical (IC50) of aminoguaiacol and CMP-G derivatives.

IC50 (µg.mL-1) IC50 (µmol NH2GA.L-1)

NH2GA

CMP-G0.16

CMP-G0.37

CMP-G0.58

1.9

179

24

13

13.7

112.5

72.5

25.4

Aminoguaiacol demonstrated a high antioxidant activity against DPPH with a concentration IC50 equal to 1.9 µg.mL-1 (13.7 µmol.L-1). It is almost 2 times more active than other phenolic compounds as guaiacol67 (IC50 = 4.15 µg.mL-1 or 33.5 µmol.L-1) or ferulic acid68 (IC50 = 5.4 µg.mL-1 or 27.8 µmol.L-1) and also almost 6 times more active than eugenol69 (11.7 µg.mL-1 or 71.3 µmol.L-1). For CMP-G0.16, CMP-G0.37 and CMP-G0.58, IC50 was respectively 179 µg.mL-1, 24 µg.mL-1 and 13 µg.mL-1. IC50 logically decreased with increasing DS (Ga). These concentrations correspond to equivalent aminoguaiacol concentration of 112.5 µmol.L-1, 72.5 µmol.L-1 and 25.4 µmol.L-1 respectively (calculated according to Eq. 5). Grafted aminoguaiacol is less active than free aminoguaiacol. However, these derivatives exhibited high antioxidant activities compared to chitosan grafted with phenolic compounds. Indeed, in literature, Liu et al.70 grafted ferulic acid onto N,O-carboxymethyl chitosan. Authors determined the IC50 of their chitosan derivative equal to 590 µg.mL-1. Also, Aytekin et al.28 grafted caffeic acid onto chitosan and authors determined the IC50. For the chitosan derivative containing 13.6g of caffeic acid for 100g of product (this


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derivative can be compared to the CMP-G0.37 looking at the phenolic compound content), the IC50 was equal to 90 µg.mL-1.

DPPH 0,3

CMP + DPPH

0,2

100

20

90

90

18

80

80

16

70

70

14

60

12

scavenging effet (%)

CMP

scavenging effect (%)

0,5 0,4

B

100

60 50 40 30 20 10

0,1

0 400

500

600

700

10

40

8

30

6

20

4

10

C (NH 2GA) mM

0

wavelength (nm)

50

0,022

0,045

0,09

0,18

0,36

0,72

C CMP-G0.16 CMP-G0.37 CMP-G0.58

[NH2GA]eq (mM)

A

0,6

Absorbance

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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2

0

0 2,5

5

7,5

C (CMP-G) g/L

800

Figure 6. A) The absorbance of DPPH (50 µM) (red) and CMP (2.5 g L-1) without DPPH (blue) and in the presence of DPPH (black) after 5 min. B) The free-radical scavenging activity of aminoguaiacol after 5 min. C) The free-radical scavenging activity of CMP-G derivatives with various DS(Ga) after 5 min. CMP-G0.16 (orange), CMP-G0.37 (grey), CMP-G0.58 (green). Equivalent concentration of NH2GA () are on the right axis. Cytotoxicity assays. Cytotoxicity of CMP and its derivatives was assessed in terms of cell viability and metabolic activity. For that, we used L929 cells that were put in contact with the compounds during 24h and we first evaluated the cell viability by using a Live/Dead assay (Figure 7A). We observed that for CMP, CMP-G0.16 and CMP-G0.58, cells exhibited a good viability while cells contacting free aminoguaiacol (NH2GA-1mM) were stained in red, indicating a potent cytotoxic effect. To exclude a possible toxic effect of the solvent, we tested viability of cells with the same solvent concentration (0.5% EtOH) and as showed in Figure 7A, cells were still viable. Noteworthy, for all tested concentrations of grafted derivatives, the equivalent concentrations in aminoguaiacol were higher than 1mM and equaled to 4.7mM and 14.6mM for CMP-G0.16 and CMP-G0.58, respectively. This suggests that the grafting of aminoguaiacol leads to no cytotoxic effect. This is probably due to the reduced mobility of the


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natural compound once grafted and/or the replacement of amine function (usually considered as antibacterial) by an amide link.71,72 We then confirmed these first results by using the Alamar Blue® assay. As indicated in Figure 7B, cells contacting all CMP and derivatives displayed excellent metabolic mitochondrial activity, reflecting a non-toxic effect of these compounds. Besides, compared to the negative control, the percentages of AB reduction obtained for CMP and derivatives were twice more important. This suggested that the metabolic activity of fibroblast cells was enhanced. On the contrary and as anticipated from the previous results, the metabolic activity of cells cultured in the presence of NH2-GA was similar to that of the positive control. Consequently, the free natural compound was undoubtedly toxic for L929 cells. The toxicity of free aminoguaiacol could be a problem considering a possible release of this compound caused by a hydrolysis reaction during use. However, it seems unlikely that a release of aminoguaiacol would occur. Indeed, aminoguaiacol is grafted onto the CMP backbone with an amide covalent bond. This type of bonding is less hydrolysable than ester group and seems less hydrolysable than a peptide bond.73,74 However, some additional analyzes of the possible release of aminoguaiacol will be carried out.

Figure 7. Cytotoxicity evaluation of CMP and CMP derivatives (CMP-G0.16 and CMP-G0.58) solutions (7.5 g.L-1); Controls: positive (10% DMSO), negative (complete culture medium), solvent (0.5% EtOH), and aminoguaiacol (1 mM NH2-GA). All the compounds were put in


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contact with L929 mouse fibroblast cells. A) Cell viability by Live/Dead assay 24h after contact - Live (green)/Dead (red). B) Cell metabolic activity 48h after contact assessed by Alamar Blue® (AB) reduction. Data are expressed as mean values ± SEM. CONCLUSION The main objectives of this work were to synthesize polymer systems based on natural compounds with viscosifying, antibacterial and/or antioxidant properties. In this type of system, the polysaccharide carboxymethylpullulan was chemically grafted with various amounts of aminoguaiacol moieties in the presence of EDCI as activator agent for the formation of an amide covalent bond. Many syntheses were carried out with good reproducibility. The substitution degrees of aminoguaiacol (DS(Ga)) were determined by

1

H-NMR

spectroscopy. The efficiency of grafting was high (between 58 and 74%), and the DS(Ga) values obtained were between 0.16 ± 0.02, 0.37 ± 0.03 and 0.58  0.08. All the CMP-G derivatives were soluble in water, even for the highest grafting amount. The physico-chemical behavior of the derivatives were studied in dilute (CCcr) media using various techniques. No polymer degradation was noticed. The derivative polymers exhibited lower sizes (Rh, intrinsic viscosities) and a compact coil conformation compared to the precursor, indicating strong associative characters in salt media because of the occurrence of mainly intramolecular hydrophobic associations between aminoguaiacol moieties. The formation of hydrophobic micro-domains was confirmed by fluorescence spectroscopy. In the semi-dilute regime, the rheological behavior was similar to a polysoap with lower viscosities than the precursor. Consequently, intramolecular associations (i.e., compact structures) appeared to predominate, even when the concentration increased. This behavior could be of great interest for drug delivery applications but does not have viscosifying


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properties. However, for the most grafted CMP-G derivative, a great enhancement of its viscosity compared to the precursor was observed at a higher concentration (150 g L-1) and an outstanding shear thickening behavior was indicated over a more classical shear thinning one. This probably induced the shift from intramolecular interactions toward intermolecular hydrophobic interactions under shear before their disruption. Finally, the antibacterial and antioxidant tests indicated that CMP-G derivatives were active systems with combined antibacterial and antioxidant properties. Precisely, CMP-G exhibited antibacterial activities against S. aureus higher than 90% at 50 g L-1 for CMP-G0.58 and at 60 g L1

for CMP-G0.37 at a fixed seeding equal to 106 CFU/mL. They also possessed free radical

scavenging activity against the DPPH radical between [60 - 90] % at 7.5 g L-1. However, grafted aminoguaiacol was less active than free aminoguaiacol. The good cytocompatibility of the CMPG derivatives with mouse fibroblasts make them good candidates for food or cosmetic applications. In summary, even if some grafted NH2GA was involved in strong clusters and probably less available, both antibacterial and antioxidant activities still occurred. Future studies will be conducted with lower grafted amounts of NH2GA and/or using of a more rigid polysaccharide with the idea to conserve these activities in hopes to diminish the polysoap behavior.

ASSOCIATED CONTENT Supporting information. The infrared spectra of the CMP precursor and CMP-G products with various experimental substitution degrees. The specific viscosity and reduced viscosity versus polymer concentration for the CMP precursor and CMP-G derivatives in PBS at 0.15 M at


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25°C. A picture of the seeded plate after 24 h at 37°C without dilution for aminoguaiacol at 15 mM and for the corresponding reference in ethanol at 7.5%. AUTHOR INFORMATION Corresponding author *Email: [email protected] Notes The authors declare no competing financial interest. ACKNOWLEGMENTS We thank the region of Normandy for financial support. REFERENCES (1)

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Carboxymethylpullulan Grafted with Aminoguaiacol: Synthesis

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