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Publicly Available Published by De Gruyter July 8, 2019

Functionalized polysaccharides with aminoguaiacol: a competition between associative behavior and antibacterial and antioxidant activities

Virginie Dulong, Marie-Carole Kouassi and Luc Picton

Abstract

In a previous study, we presented the development of a series of functionalized carboxymethylpullulan (CMP) grafted with aminoguaiacol (derivative of guaiacol with known antibacterial and antioxidant activities) leading to CMP-G derivatives with various degrees of substitution [DS(Ga)] from 0.16 to 0.58. Our results have shown the efficiency of the grafting both with the evidence of antioxidant and antibacterial activities (Staphylococcus aureus) of the CMP-G derivatives. Nevertheless, an important result has shown surprisingly that such biological activity was not clearly improved with the DS(Ga) unlike the antioxidant activity. These results were probably correlated with a peculiar associative behavior of the derivative (i.e. amphiphilic character) due to the grafted hydrophobic guaiacol groups leading to preferential intramolecular association which was particularly important in the more concentrated regime (polysoap behavior). To complete this study, we propose here two strategies in order to diminish the associative character and notably the polysoap behavior: (i) decrease the DS(Ga) of CMP derivative with a CMP-G0.05 [grafted with a DS(Ga) = 0.05], (ii) conduct the functionalization onto a more rigid polysaccharide backbone as alginate. Our results show a good correlation of the associative physicochemical behaviors with both antioxidant and antibacterial activities. They also confirm the availability of these strategies mainly for the first one (i.e. CMP-G0.05). The main result indicates that the lower is the DS(Ga), the better is the antibacterial activity thanks to a lower associative character. Finally, this study also shows that the grafting of aminoguaiacol is possible onto another anionic polysaccharide (i.e. alginate).

Introduction

Polysaccharides find many applications in food, cosmetic, pharmaceutics or textile industries due to their ability to modify aqueous environments by their extraordinary rheological properties (gelling, thickening, emulsifying… properties). Some polysaccharides also possess intrinsically antibacterial activity such as chitosan [1] or sulfated polysaccharides present in marine algae [2], or antioxidant activity such as polysaccharides issued from plants [3], [4]. Functionalization of polysaccharides (with molecule from natural origin) exhibiting both antioxidant and antibacterial activities together with viscosifying properties seems an interesting approach to develop preservatives for aqueous formulations, in order to replace chemical molecules such as butyl hydroxyl toluene (BHT) or parabens which are suspected to be carcinogenic or endocrine disruptor [5], [6]. Guaiacol or 2-methoxyphenol, is a natural phenolic compound extracted from guaiac tree which presents antioxidant and antibacterial activities [7]. In a previous study we synthesized derivatives of carboxymethylpullulan grafted with aminoguaiacol (CMP-G derivatives) with grafting degrees [DS(Ga)] of 0.16, 0.37 and 0.58 [8]. A physico-chemical study of these derivatives in aqueous solutions showed a tendency to associative behavior increasing with DS(Ga). Indeed, the coupling technique of size exclusion chromatography with multi-angle light scattering, differential refractive index and viscometry (SEC/MALS/DRI/Visco) allowed the determination of molar masses, hydrodynamic radius and intrinsic viscosity demonstrating the high association of CMP-G derivatives. Associations are mainly intramolecular, especially for the most grafted derivative, due to hydrophobic interactions between phenolic moieties leading to hydrophobic clusters (demonstrated by fluorescence spectroscopy with pyrene probe). Low shear viscosity measurements confirmed this tendency: on one hand the critical concentration (corresponding to the transition from diluted to semi-diluted regime) increases when DS(Ga) increases; on the other hand, the apparent viscosity of CMP-G derivatives was lower than that of CMP precursor even for the most grafted sample (CMP-G0.58). This behavior can be assimilated to that of polysoaps [9], [10]. This peculiar behavior can be explained by the predominance of intramolecular hydrophobic associations leading to compact coils which limit the establishment of entanglement even for more concentrated polymer aqueous solution. As a matter of fact the expected viscosifying properties were difficult to reach with these CMP-G derivatives. Nevertheless, antioxidant properties of CMP-G derivatives were studied using the DPPH method and we obtained better antioxidant activity for the most grafted sample. The IC50 (half-inhibition concentration) logically decreased when DS(Ga) increased. The associative behavior of CMP-G derivatives does not limit the antioxidant activity. Concerning the antibacterial properties of CMP-G derivatives versus Staphylococcus aureus, we highlighted a real activity but without reaching the minimal bactericidal or minimal inhibitory concentrations. In summary, hydrophobic clusters composed of grafted aminoguaiacol moieties, were rather favorable to antioxidant properties but not to the antibacterial activity. This is certainly due to the availability of grafted moieties which are enclosed into the hydrophobic clusters.

To develop this study and to look for solutions in order to improve the antibacterial activity of functionalized polysaccharides with aminoguaiacol, we present here two strategies:

  1. decrease the grafting degree to 5% for the CMP derivatives and compare our results to the previous ones [i.e. DS(Ga) from 0.58 to 0.16]

  2. use a more rigid polysaccharide (such as alginate) to prevent the intramolecular interactions (i.e. the polysoaps behavior) and obtain products with viscosifying properties. This last approach also demonstrates the possibility to extend this functionalization to others polysaccharide.

In this article, we describe these two strategies. The physico-chemical characteristics of these new derivatives are studied by different techniques (SEC/MALS/DRI/Visco and rheology) and compared to results previously obtained, as well as the antioxidant and antibacterial properties. The influence of associative behavior on activities is discussed and notably correlated with chemical and biological activities

Experimental

Materials

Pullulan was purchased from Hayashibara Biochemical Laboratory (Japan); Alginate (ALG) (with a ratio of mannuronate to glucuronate sodium salt of 1.2) were kindly provided by Cargill (France); aminoguaiacol (NH2GA) from Merck KGaA (Germany), 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDCI), α,α-diphenyl-β-picrylhydrazyl (DPPH) and phosphate-buffered saline (PBS) tablets from Sigma-Aldrich (France). Sodium hydroxide (NaOH) and hydrochloric acid (HCl) were purchased from VWR (France). Water was purified with the milli-Q water reagent system (Millipore, USA). All compounds were used without further purification.

Synthesis of carboxymethylpullulan (CMP)

CMP, with a substitution degree in carboxylate groups (DSCOONa) of 0.96, was synthesized by the reaction of pullulan with sodium chloroacetate in the presence of NaOH using a method previously described [11]. DSCOONa, the number of carboxylate groups by anhydroglucose unit (AGU) was determined by conductimetric titration.

Synthesis of functionalized polysaccharides (CMP-G and ALG-G)

CMP-G and ALG-G derivatives were synthesized by reaction of aminoguaiacol with CMP or ALG, in the presence of EDCI at acidic pH as described in our previous article [8]. Briefly, aminoguaiacol was dissolved in HCl at 0.05 mol L−1 for 12 h. Then, the solution of aminoguaiacol was slowly added to a solution of CMP or alginate (1 g) in Milli-Q water (50 mL). 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 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

SEC/MALS/DRI/Visco

The size-exclusion chromatography (SEC) line is composed of a degasser (DGU-20A3 Shimadzu, Japan), a pump (LC10Ai Shimadzu, Japan), an automatic injector (SIL-20A, Shimadzu, Japan), 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 a polyhydroxymethylmetacrylate gel. The chromatographic line is coupled with a multi-angle light scattering detector (MALS, 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), a viscometer (Viscostar II, Wyatt technology Inc., USA) and a differential refractive index detector (DRI, RID-10A shimadzu, Japan) with an uncertainty of 3%.

The eluent, phosphate buffer saline or PBS (0.15 mol L−1, pH 7.4), used as carrier, was filtered through a 0.1 μm filter unit (Millipore, USA). The polymer solutions were prepared from lyophilized CMP-G or ALG-G products at 1 g L−1 in PBS. One hundred microliter of filtered solution (0.45 μm, Millipore, USA), are injected at 0.5 mL min−1 flow rate. The collected data were analyzed using Zimm 1st order from 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, alginate and derivatives). The whole line allows the access to both number and mass average molar masses (respectively Mn and Mw) and their distribution, average hydrodynamic radii (Rh) and intrinsic viscosities [η]. The determination of the average intrinsic viscosity allowed us to obtain the average hydrodynamic volume (Vh) using the Einstein−Simha equation (Eq. 1):

(1) Vh=[η]M/νNA

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 (Eq. 2), we calculated the diffusion coefficient (Dt in m2·s−1):

(2) Rh=kT/6πηDt

where k is the Boltzmann constant, T is the temperature (K), and η is the dynamic viscosity (Pa·s) of the medium.

Rheological measurements

The viscosity measurements of polysaccharides and their derivatives were performed at a low shear rate (1 s−1) in the Newtonian regime using a Couette-type viscometer (LS400, Lamy Rheology, France) at 25°C. Polymer solutions were prepared in PBS (0.15 mol L−1, pH 7.4) at various concentrations.

Intrinsic viscosity [η] and Huggins coefficient (KH) were determined by plotting reduced viscosity as a function of polymer concentration (Eq. 3).

(3) ηred=ηspeC=[η]+KH[η]2C

Critical overlapping concentration is determined as the slope failure of specific viscosity versus concentration plots (data not shown).

Flow curves were determined using a Discovery HR2 Rheometer from TA Instrument (UK) with a standard-size double concentric cylinder as geometry (aluminum – gap 500 μm) for CMP-G concentrations of 50 and 100 g L−1 and with a cone-plate geometry (diameter 4 cm; angle 2°; gap 57 μm) at 150 g L−1. For ALG-G derivatives a cone-plate geometry was used for all the studied solutions.

Antioxidant properties

The antioxidant properties of the CMP-G and ALG-G derivatives were evaluated using the adapted DPPH method [12] according to Kouassi et al. [8]. Briefly, solutions of the CMP-G derivatives were prepared in NaCl at 0.15 M at various concentrations. 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 were 0.002–2.5 g L−1. For alginate derivatives, the final concentrations were 1, 2 and 3 g L−1 in NaCl 0.15 M. The percentage of the DPPH radical-scavenging activity was calculated according to Eq. 4 at 543 nm [the maximum absorption of DPPH in NaCl/ethanol (3:1)] (spectrophotometer CARY 100 UV-Vis, Agilent).

Due to the brown coloration of derivatives and to overcome the color of aminoguaiacol and facilitate the reading of the absorbance in the UV-visible region, we also prepared a similar range of dilutions for each tested product without DPPH as a blank [Absorbance (blank)].

(4) DPPHscavengingactivity(%)=A0A1A0×100

where A0 corresponds to the absorbance of a standard solution of DPPH and A1 corresponds to the absorbance of the sample solutions.

For the grafted polymers, A1=Absorbance (sample with DPPH)–Absorbance (blank).

The antioxidant concentrations corresponding to 50% inhibition of the DPPH radical [half-inhibition concentration (IC50)] for aminoguaiacol and for derivatives were also determined. Measurements of the scavenging effect was carried out between 0.0488 and 3.125 μg·mL−1 for aminoguaiacol and between 0.002 and 2.5 mg·mL−1 for CMP-G derivatives.

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

(5) [NH2GA]eq=DS(Ga)M0(CMPG)C(CMPG)

where, M0(CMP-G) is the molar mass of the repetition unit of CMP-G product [M0(CMP-G)=162+80×DSCOONa+99.15×DS(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. For alginate derivatives, the same equation can be used with M(ALG-G)=198+99.15×DS(GA).

Antibacterial activity

Antibacterial tests were carried out using a CFU (colony forming unit) counting method according to Lequeux et al. [13] with some modifications. Staphylococcus aureus ATCC 29213 was chosen as the bacterium, and BHI medium (Brain Heart Infusion, Sigma-Aldrich, France) was used as the broth for culture. The tests were conducted according to Kouassi et al. [8]. The antibacterial activity of each sample was calculated using the following equation (Eq. 6):

(6) antibacterialactivity(%)=CFU/mL(reference)CFU/mL(sample)CFU/mL(reference)100

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 and ALG-G derivatives, the reference was the precursors CMP or ALG prepared using the same conditions as derivatives.

Results and discussion

Synthesis of polysaccharides derivatives with low DS(Ga) or with alginate

The two strategies consist on the one hand to reduce the grafting rate of aminoguaiacol onto the CMP derivatives and on the other hand to synthesize such derivatives from the more rigid alginate. These strategies have led to the functionalized derivatives listed in Table 1. Let us notice that the CMP-G derivatives with DS(Ga) of 0.16, 0.37 and 0.58 have already been presented in our previous article [8] and are given here to follow the whole influence of the DS(Ga). The grafting is evidenced by 1H-NMR and infrared spectroscopy proving the presence of aminoguaiacol onto CMP and alginate (data not shown). The experimental DS(Ga) were determined by 1H-NMR spectroscopy by the method described by Kouassi et al. [8].

Table 1:

Functionalized polysaccharides with aminoguaiacol.

Sample DStheo(GA)a DSexp(GA)b Solubility in water
CMP-G0.05 0.15 0.05±0.01 YES
CMP-G0.16c 0.25 0.16±0.02 YES
CMP-G0.37c 0.50 0.37±0.03 YES
CMP-G0.58c 1 0.58±0.08 YES
ALG-G0.06 0.15 0.06±0.01 YES
ALG-G0.10 0.25 0.10 YES
ALG-G0.50theo 0.50 ND NO
ALG-G1theo 1 ND NO

  1. aMolar ratio NH2GA/CMP.

  2. bExperimental DS(Ga) calculated using 1H-NMR (average of seven separate syntheses except for ALG-G0.10).

  3. c[8].

  4. ND means not determined.

The first difference between CMP and alginate derivatives concerns their water solubility. Indeed, syntheses of alginate derivatives with theoretical DS(Ga) of 0.50 and 1 lead to insoluble products in water while those obtained with CMP at the same theoretical DS(Ga) are water soluble. This let us suppose that for high grafting degree, phenolic moieties are less associated, notably in an intramolecular way, in alginate derivatives than in CMP derivatives. The higher rigidity of alginate chains can explain this difference. Alginate is considered as a semi-flexible polysaccharide [α(1,4) and β(1,4) links] with a persistence length of 15 nm in salted medium [14], [15] compared to 1.3 nm for pullulan (perhaps a little bit more for CMP) due to more flexible backbone [α(1,4) and α(1,6) links] [16]. It can be expected that for more flexible chains (i.e. CMP derivatives), intramolecular associations prevail and lead to quite a polysoaps behavior more favorable to water solubility. On the contrary, for semi-flexible backbone of alginate, likely intermolecular associations can lead to its insolubility in water for the highest DS(Ga). In order to complete these first results, the associative character is studied in the following by different physicochemical approaches thanks to the techniques described above.

Physico-chemical properties

SEC/MALS/DRI/Visco

The impact of the hydrophobic grafted groups on the macromolecular chains molar masses and sizes (Mn; Mw; Rh; [ɳ]) was studied by SEC/MALS/DRI/Visco in dilute regime of concentration. Table 2 summarizes all the results obtained and elution profiles together with molar masses distributions are given in Fig. 1. Before analyzing the results, it has to be noted that the steps of purification of the grafted derivatives (dialysis against various solvents including 0.1 M NaOH) have led to a slight degradation of the alginate chains. Therefore, in the following study, we compared the behavior of the ALG-G derivatives with the control alginate (Alginate Ctrl, having undergone the same purification treatment).

Table 2:

Results of SEC/MALS/DRI/Visco analyses of CMP-G and ALG-G derivatives and precursors (at 25°C, in PBS 0.15 mol·L−1 and at 1.0 g·L−1 for polymer concentration).

Mn (g mol−1) Mw (g mol−1) Mo a (g mol−1) DPn Apparent (Mn/M0) Rh(n) (nm) [ɳ]n (mL g−1)
CMP 143 000 (±1.6%) 229 000 (±0.4%) 239 598 12.6 (±0.5%) 100 (±1.0%)
CMP-G0.05 150 000 (±4.3%) 241 000 (±1.2%) 244 615 12.3 (±1.0%) 90 (±0.9%)
CMP-G0.16 166 000 (±1.6%) 262 000 (±0.6%) 255 651 12.3 (±0.5%) 77 (±1.1%)
CMP-G0.37 181 000 (±1.4%) 294 000 (±0.6%) 276 656 10.9 (±0.6%) 52 (±1.3%)
CMP-G0.58 279 000 (±2.1%) 307 000 (±1.7%) 297 « 939 » 8.9 (±1.9%) 16 (±5.2%)
Alginate ctrl 119 000 (±0.9%) 163 000 (±0.4%) 198 601 20.0 (±0.4%) 453 (±0.2%)
Alg-G0.06 144 000 (±2.0%) 193 000 (±1.1%) 204 706 20.0 (±0.7%) 383 (±0.3%)
Alg-G0.10 147 000 (±1.6%) 195 000 (±1.1%) 208 707 18.7 (±0.7%) 301 (±0.2%)

  1. aMo is the molar mass of the repeating unit calculated as 162+80DSCOONa+99.15 DS(Ga) with DSCOONa=0.96 for CMP-G and as 198+99.15 DS(Ga) for ALG-G.

Fig. 1: Chromatograms of derivatives (PBS at 0.15 M). Light scattering signal (LS): full line curves; differential refractive index (DRI): dash line curves; molar masses distributions on right axe: dash line curves. (a) CMP DS 0.96 (red), CMP-G0.05 (black), CMP-G0.16 (blue), CMP-G0.37 (green), and CMP-G0.58 (orange). (b) Alginate ctrl (red), ALG-G0.06 (blue), ALG-G0.10 (green).

Fig. 1:

Chromatograms of derivatives (PBS at 0.15 M). Light scattering signal (LS): full line curves; differential refractive index (DRI): dash line curves; molar masses distributions on right axe: dash line curves. (a) CMP DS 0.96 (red), CMP-G0.05 (black), CMP-G0.16 (blue), CMP-G0.37 (green), and CMP-G0.58 (orange). (b) Alginate ctrl (red), ALG-G0.06 (blue), ALG-G0.10 (green).

Concerning the CMP derivatives (Fig. 1a), it clearly appears that elution profiles are shifted towards lower elution volumes when DS(Ga) increases but in the same time the range of molar masses seems quite unchanged whatever the derivatives. These results indicate the presence of more compact coils when DS(Ga) increases. This is largely in favor of predominance of intramolecular hydrophobic interactions between grafted guaiacol moieties. This observation is fully confirmed by the evolution of size parameters (i.e. Rh and [η]) which decrease in a sensitive way as DS(Ga) increases (Table 2). In the same time, it appears that molar masses are not strongly modified by the grafting. We can just notice a slight increase of Mw indicating the presence of a very small amount of aggregated structures. Nevertheless, the main behavior of modified CMP consists in very compact and dense structure of single coil due to strong intramolecular associations of grafted guaiacol. As already seen [8], this behavior can be compared to that of polysoaps. Nevertheless, this study shows that the new CMP-G0.05 derivative with the lower grafting degree shows a different behavior. Its elution profiles together with molar masses and size (i.e. Rh and [η]) are quite similar to that of the CMP precursor. This indicates a great decrease of intramolecular association tendency for the lowest grafted derivative (i.e. CMP-G0.05). This is a nice result because we have shown that intramolecular associations seem to affect the antibacterial activity for accessibility reasons [8]. We will evaluate below the specific biological activity of the CMP-G0.05 compared to other CMP-G derivatives.

Concerning ALG and ALG-G derivatives, the higher rigidity of alginate when compared to CMP, is fully confirmed by higher Rh and [η] for equivalent molar masses. The random coil volume of alginate and modified alginate are higher than that of CMP, suggesting better rheological properties. The elution profiles of ALG-G derivatives (Fig. 1b) show only a slight shift towards low elution volume together with a very slight increase of molar masses (presence of low amount of aggregated structure). This effect is similar to that of CMP-G derivatives but is largely diminished. To sum up, it appears that a more rigid backbone of the polysaccharide effectively leads to a decrease of the polysoap behavior.

Rheological study

A rheological study, at low shear rate in the Newtonian regime (1 s−1) and at variable shear rates (flow experiments between 1 and 1000 s−1) was performed to complete the physicochemical study. Fig. 2 shows the reduced viscosity of samples as a function of polymer concentration and Table 3 gives the viscometric data ([η], KH and Ccr) obtained by low shear rate measurements. We had demonstrated that CMP-G0.16, CMP-G0.37 and CMP-G0.58 present mainly compact structures of single coils due to hydrophobic intramolecular interactions [8] improved by the increase of the DS(Ga). This has been highlighted by the decrease of [η] together with an increase of the Huggins constant (KH), traducing the increase of polymer-polymer interactions. These results were consistent with the observed increase of the critical overlapping concentration (Ccr, Table 3). Ccr was determined by the Utracki and Simha representation and confirms this tendency (data not shown): indeed, Ccr of CMP-G0.05 is almost the same as CMP, while for the other CMP-G derivatives Ccr increases and is even not available for CMP-G0.58. The rheological behavior of CMP-G0.05 compared with the previously studied CMP-G derivatives (Fig. 2a) confirms the SEC/MALS/DRI/Visco results presented above: CMP-G0.05 behaves nearly the CMP precursor, with value of [η], Kh and Ccr quite similar to that of the CMP (Table 3). Let us notice that the slight differences which are observed with [η] values between SEC/MALS/DRI/Visco and low shear measurement, can easily be explained by the presence of very low amount of aggregated structures which are taken into account in batch measurement (i.e. low shear) while they are separated by the SEC fractionation for SEC/MALS/DRI/Visco results. Nevertheless, these results confirm the very low associative character of CMP-G0.05 as expected.

Fig. 2: Reduced viscosity of derivatives as a function of polymer concentration in PBS 0.15 M at 25°C at a shear rate of 1 s−1. (a) CMP (black), CMP-G0.05 (red), CMP-G0.16 (grey), CMP-G0.37 (orange), CMP-G0.58 (green). (b) Alginate (red), Alginate ctrl (blue), ALG-G0.06 (green), ALG-G0.10 (orange).

Fig. 2:

Reduced viscosity of derivatives as a function of polymer concentration in PBS 0.15 M at 25°C at a shear rate of 1 s−1. (a) CMP (black), CMP-G0.05 (red), CMP-G0.16 (grey), CMP-G0.37 (orange), CMP-G0.58 (green). (b) Alginate (red), Alginate ctrl (blue), ALG-G0.06 (green), ALG-G0.10 (orange).

Table 3:

Intrinsic viscosity and Huggins coefficient (determined according to Eq. 3) and critical overlap concentration.

CMP CMP-G0.05 CMP-G0.16 CMP-G0.37 CMP-G0.58 Alg crtl ALG-G0.06 ALG-G0.10
[ɳ] mL g−1 116 100 60 31 8 663 490 351
KH 0.4 0.4 1 2.4 18 0.7 0.8 1
[ɳ]n a mL g1 100 90 77 52 16 453 383 301
Ccr b g L−1 42 40 55 67 NA 4 5 9

  1. aIntrinsic viscosity obtained by SEC/MALS/DRI/Visco.

  2. bCritical concentration of a polymer determined by the Utracki and Simha representation.

  3. NA means not available.

ALG-G derivatives show the same tendency with a decrease of [η] and a slight increase of KH but in a lower extend compared to CMP derivatives (Fig. 2b and Table 3). So ALG-G derivatives still present intramolecular interactions leading to compact coils even in semi-diluted medium. Moreover, the results also confirm that ALG-G derivatives present a more rigid structure than CMP-G derivatives since their critical concentrations are largely lower.

The flow experiments in the semi-diluted medium showed that all derivatives, except CMP-G0.58, present apparent viscosities which are lower than that of precursors (Fig. 3a). These results indicate that the CMP-G derivatives keep their associative character with the predominance of intramolecular hydrophobic interactions even in semi-dilute regime. An exception is noted for CMP-G0.58 showing an atypical behavior discussed in our previous article [8]. One more time, in this context, the behavior of CMP-G0.05 appears close to its precursor confirming its low associative character even at high concentration. ALG-G derivatives also show lower viscosities that their precursor (Fig. 3b). Nevertheless, for equivalent grafting degrees, the rheological behavior of ALG-G derivatives is largely higher than that of CMP-G derivatives, leading to better viscosifying properties. Moreover, ALG-G derivatives solutions appear shear-thinning indicating more entangled solutions while CMP-G solutions behave more as Newtonian fluids according to their polysoaps like properties. This is yet a difference that can be correlated to the more rigid backbone of alginate compared to CMP.

Fig. 3: Apparent viscosity of aqueous solutions as a function of shear rate for (a) CMP and CMP-G derivatives at 150 g L−1. (b) Alginate and ALG-G derivatives at 30 g L−1 in PBS 0.15 M.

Fig. 3:

Apparent viscosity of aqueous solutions as a function of shear rate for (a) CMP and CMP-G derivatives at 150 g L−1. (b) Alginate and ALG-G derivatives at 30 g L−1 in PBS 0.15 M.

To sum up and from a physico-chemical point of view, the strategy that consists to decrease the DS(Ga) of CMP-G derivatives leads to reduce their associative character. Indeed, CMP-G0.05 behaves mostly like its precursor CMP, with only a very low tendency to intramolecular association. Concerning the second strategy with the more rigid alginate derivatives, we have provided evidence that intramolecular interactions are still present and better viscosifying properties are obtained when compared to CMP derivatives. With these two strategies, the improvement of antibacterial activities is expected.

Antioxidant activity

The DPPH radical scavenging percentage of the samples was determined by measuring the decrease in the absorbance of DPPH according to Eq. 4. For CMP-G derivatives, the absorbance measurements were performed after 5 min of reaction [this time is enough because a kinetic study has shown that the same results were obtained after 30 min (data not shown)]. In our previous study, we showed that the antioxidant activity of CMP-G derivatives increased with DS(Ga) and with the polymer concentration but was lower than that of free aminoguaiacol. Indeed, as shown in Fig. 4a, showing the scavenging effect of the CMP-G derivatives as a function of equivalent grafting quantities of aminogaïacol ([NH2GA]eq calculated according to Eq. 5), it clearly appears that the antioxidant activity is higher for the higher grafted derivatives at equivalent amount of grafted Ga. It could have been expected that the scavenging effect remains the same for all samples at an equivalent amount of NH2GA. This is not the case and consequently our results indicate that hydrophobic clusters are favorable to antioxidant activity excepted for the very low amount of [NH2GA]eq (i.e. below 10 μM) since there is probably not enough clusters. IC50 of CMP-G0.05 (corresponding to the concentration in polymer when the percentage of scavenging is 50%) is 1300 μg mL−1. This value is higher than those obtained with the other derivatives. 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. As a confirmation, hydrophobic clusters have a real positive effect on antioxidant activity. From this point of view CMP-G0.05 appears less active than derivatives with higher grafting rates.

Fig. 4: (a) DPPH scavenging effect (%) of CMP-G derivatives versus equivalent aminoguaiacol concentration (in μM) after 5 min. CMP-G0.05 (red), CMP-G0.16 (grey), CMP-G0.37 (orange), CMP-G0.58 (green). (b) DPPH scavenging effect (%) of ALG-G derivatives after 30 min. Alginate (red), ALG-G0.06 (blue), ALG-G0.10 (green). Equivalent concentration of NH2GA in mM (♦) are on the right axis (calculated according to Eq. 5).

Fig. 4:

(a) DPPH scavenging effect (%) of CMP-G derivatives versus equivalent aminoguaiacol concentration (in μM) after 5 min. CMP-G0.05 (red), CMP-G0.16 (grey), CMP-G0.37 (orange), CMP-G0.58 (green). (b) DPPH scavenging effect (%) of ALG-G derivatives after 30 min. Alginate (red), ALG-G0.06 (blue), ALG-G0.10 (green). Equivalent concentration of NH2GA in mM (♦) are on the right axis (calculated according to Eq. 5).

For ALG-G derivatives, the scavenging effect was measured after 30 min instead of 5 min like for CMP-G derivatives but it was always lower than 50%. Surprisingly, ALG precursor seems to have a slight antioxidant activity for the higher concentration (i.e. 3 g L−1, Fig. 4b). Thus, ALG-G derivatives appear less antioxidant than CMP homologues. This could be due to the lower amount of hydrophobic clusters in the case of alginate derivatives as shown above. This could also be correlated to the higher viscosities of alginate solutions leading to greater difficulty of the DPPH radical to diffuse into a more entangled polymer solution. Finally, the two strategies did not allow the improvement of the antioxidant activity of functionalized polysaccharides with aminoguaiacol.

Antibacterial activity against Staphylococcus aureus

The antibacterial activities of free aminoguaiacol and functionalized polysaccharides were evaluated using the quantitative method of CFU counting. This activity was tested against the Gram-positive bacterium S. aureus, which is commonly found in cosmetics and foodstuffs. Results are given in term of antibacterial activity (calculated according to Eq. 6 and Kouassi et al. [8]). They are summarized in Fig. 5. Firstly, the results show that for CMP-G0.16, CMP-G0.37 and CMP-G0.58, the antibacterial activity increases with DS(Ga) but seems to reach a plateau below 100%. Secondly, for approximately the same aminoguaiacol equivalent concentration, the antibacterial activity of CMP-G0.05 is quite the same as free NH2GA. Moreover, the antibacterial activity of CMP-G0.05 appears strongly better than that of more grafted CMP-G derivatives and is near to reach 100%. This is a relevant result which confirms the first strategy and demonstrates that decreasing DS(Ga) of CMP-G derivative leads to the lowering of the associative behavior (notably intramolecular association and clusters) and improves the biological activity.

Fig. 5: Left axis: antibacterial activity of NH2GA and CMP-G derivatives (grey histogram); right axis: [NH2GA]eq (red diamond).

Fig. 5:

Left axis: antibacterial activity of NH2GA and CMP-G derivatives (grey histogram); right axis: [NH2GA]eq (red diamond).

Concerning the alginate derivatives, we could not demonstrate real antibacterial activity. This result is unexpected and not yet understood. Perhaps the higher viscosities of alginate derivatives could limit the antibacterial properties of the grafted guaiacol. Further work will be proceeding on.

Conclusion

In a previous study, we have demonstrated the possibility to elaborate functionalized CMP with various amount of aminoguaiacol grafted groups with DS(Ga) from 0.16 to 0.58. Such CMP derivatives present both antioxidant and antibacterial activities. They also present a clear associative behavior in aqueous solution which increases with the DS(Ga) resulting in mainly intramolecular interactions between the grafted hydrophobic moieties. This behavior has been assimilated to polysoap’s one and appears to limit the viscosifying properties but also the antibacterial activity, while the antioxidant activity seems to be improved by the associative character notably by the cluster formation.

Based on these first results we have developed two strategies in order to decrease the associative tendency while preserving or improving the chemical and biological activities, by (i) decreasing DS(Ga) for CMP-G derivatives and (ii) using a more rigid polysaccharides backbone as alginate.

In this study we have synthetized three new derivatives: CMP-G0.05, Alg-G0.06 and Alg-G0.10. Two other alginate derivatives with higher DS(Ga) have also been elaborated but were found not soluble in aqueous solution while their CMP homologues with same amount of grafting remain soluble. This has been interpreted by the difference of rigidity between the two polysaccharides: CMP (very flexible) and alginate (more rigid).

CMP-G0.05 appears effectively less or not associative compared to the CMP-G with higher DS(Ga). Consequently, and as expected through our starting hypothesis, the biological activity (antibacterial one) is clearly improved compared to higher grafted derivatives. These results well correlate the associative character (notably polysoaps type) with the low biological activity. We suppose that when grafted guaiacol are strongly associated in clusters, bacteria have more difficulties to access to the heart of hydrophobic clusters. Concerning the antioxidant activity, clusters and associative behavior appear more favorable.

Due to its more rigid backbone, alginate derivatives appear effectively less associative than CMP derivatives but still present some intramolecular associations. Nevertheless, the rigidity of alginate leads to largest coils in solution (in the same range of molar masses) and better rheological properties compared to CMP. Alginate derivatives present antioxidant activities but in a lower extent than for CMP derivatives probably due to a lower amount of clusters and also higher viscosities. Surprisingly, no biological activities have been evidenced for alginate derivatives perhaps for viscosity reasons.


Article note

A collection of papers presented at the 17th Polymers and Organic Chemistry (POC-17) conference held 4–7 June 2018 in Le Corum, Montpellier, France.


  1. Funding: Région Normandie, Grant Number: doctoral contract.

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Published Online: 2019-07-08
Published in Print: 2020-02-25

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