Synthesis and antibacterial properties of thymol and carvacrol grafted onto lignocellulosic kraft fibers

Abstract

Bacterial infections and surface contaminations are worrying public health issues. It becomes urgent to find solutions. One of the ways to limit bacterial proliferation is to develop new antimicrobial materials. The phenolic compounds of essential oils like thymol and carvacrol, are attractive antibacterial candidates, which have gained great popularity in the food, cosmetic, and pharmaceutical industries. This work describes the elaboration of bioinspired antibacterial materials. Thymol and carvacrol are linked to kraft pulp fibers, via triazine link. This novel material has been investigated for its antibacterial properties against Escherichia coli and Staphylococcus aureus. The developed materials show very interesting antibacterial activity. The grafting of thymol and carvacrol by covalent bond allows to avoid the problem of their release and, thus, could maintain the antibacterial properties of support.

Keywords

Essential oils compounds thymol carvacrol cellulosic fibers kraft pulp functionalization of polymers antibacterial material

Introduction

The control of bacterial proliferation is a major issue in various fields such as in hospitals¹ and in the food² and pharmaceutical industries.³ It is important to reduce or eliminate bacterial contamination on surfaces, avoiding the formation of biofilms. The development of antimicrobial materials appears to be an effective strategy for inhibiting microbial growth. ^4–7 In this context, the use of natural products such as essential oils (EOs), which have antimicrobial properties, appears to be an interesting option.⁸ EOs play a significant role in the protection of plants, notably as an antibacterial. Plants contain a wide variety of secondary metabolites that are capable of inhibiting or slowing the growth of bacteria.⁹ The main advantage in the use of EOs is their high activity, probably due to the synergistic effects of their various components contained in EOs.¹⁰ The main bacterial targets of EOs are the bacterial cell walls and membranes resulting in disturbing adenosine triphosphate (ATP) production and pH homeostasis.¹¹ So, EOs represents good alternatives to synthetic chemical preservatives. Many reports have focused on the development of antibacterial surfaces to prevent attachment and proliferation of bacteria, by essential oil deposits on materials such as chitosan^12–14 or polyester.^15,16 Despite this described antimicrobial behavior, the direct application of EOs has several limitations ^17–20 such as strong sensory properties, high volatility, poor water solubility, degradability, and potential toxicity. To avoid EO release from materials and the leaching into skin resulting in epidermal toxicity, it is important to move toward the immobilization of EO compounds by a covalent link. It has been reported that EOs containing aldehydes or phenols, such as cinnamaldehyde, citral, eugenol, carvacrol, or thymol, as major components showed the highest antibacterial activity.^21–24 The combination of phenolic compounds produced synergistic effects on several microorganisms, in particular, the combination of thymol and carvacrol, which have a synergistic effect against Escherichia coli strains²⁵ and an additive activity against Staphylococcus aureus and Pseudomonas aeruginosa.²⁶

The focus of this work is the development of antibacterial materials by grafting thymol and carvacrol onto kraft pulp (KP) fibers using triazine as a covalent link (Figure 1). S-triazine, which is a symmetrical molecule, is a very useful platform for multifunctional molecular arrangements and stepwise substitution reactions.²⁷

The grafting of thymol and carvacrol by covalent bond avoids the problem of release and thus could maintain the antibacterial properties of support.

The modified KP sheets were tested for their antibacterial properties against two pathogenic bacterial strains frequently found on surfaces, Staphylococcus aureus and Escherichia coli.²⁸ The synergistic effect between the KP containing the thymol and that containing the carvacrol was also studied.

Experimental

Materials

All solvents and reagents were commercially available and, unless otherwise stated, were used as received. Thymol (98%), cyanuric chloride (98%), and N-ethyldiisopropylamine (99%) were purchased from Alfa Aesar (Karlsruhe, Germany). Carvacrol was purchased from Sigma Aldrich (Lyon, France). Dichloromethane anhydrous (99.7%) was purchased from Acros Organics (Geel, Belgium). Reactions were monitored by thin-layer chromatography (TLC) on 0.2 mm silica gel precoated 60 F254 (Merck Chimie, Fontenay-sous-bois, France) plates and revealed with an ultraviolet light source at 254 nm. The bleached hardwood KP was received in wet-laps from a Northeastern Canada mill.

Instrumentation

Fourier transform infrared spectroscopy

A Perkin Elmer 1000 Fourier transform infrared spectroscopy (FTIR) spectrometer equipped with the Spectrum software was used to perform FTIR analysis. The spectra were obtained by preparing dried KBr powder pellets containing 5% w/w of the investigated sample.

X-ray photoelectron spectroscopy

A Kratos Axis Ultra spectrometer was used for XPS analysis. Composition information (atomic percentage) was provided within a depth of a few nanometers from the material surface. Survey scans were taken with 1.0 eV step and 160 eV analyzer pass energy, while the high-resolution regional spectra were recorded with 0.1 eV step and 40 eV pass energy. A 225 W monochromatic aluminum source (Al Kα) was used. The pressure was 10⁻⁹ Torr. The analysis was carried out on three different spots (zone 2 mm²) before averaging over the sample, to avoid any bias due to any eventual heterogeneity. The detector was positioned at an angle of 90° to the sample surface. A SUN Sparc Station IPX computer was used to perform deconvolution analysis. The spectrum analysis was done with Casa XPS 2.3.9.

Nuclear magnetic resonance spectroscopy

¹H nuclear magnetic resonance (NMR) spectra were recorded at 500.14 MHz with a Bruker DPX-500 spectrometer using DMSO-d6 as solvent at room temperature. The chemical shifts (δ) are expressed in ppm with Me₄Si as the internal standard (δ0).

Environmental scanning electron microscopy

An environmental scanning electron microscope (ESEM; Quanta FEG-250 SEM) was used to observe the KP sample in the presence of bacteria after 24 h of contact. This microscope permits to analyze samples with controlled humidity and temperature, which are crucial parameters in life-science samples. Samples were observed without prior treatment.

Methods

Example of substitution degree (DS) calculation for triazine-CEO grafting from (XPS)²⁹ data:

In a triazine-CEO unit, there are three nitrogen atoms and the percentage of triazine-CEO is

% [T r i a z i n e - C E O] = \frac{[%N]}{3}

When the percentage of triazine-CEO is obtained, the percentage of oxygen atoms of AGU is the total percentage oxygen atoms subtracting the percentage of oxygen supplied by the triazine-CEO as

% [AGU] = \frac{% [O] - % [T r i a z i n e - C E O]}{5}

Finally, the DS is obtained with this

D S = \frac{% [T r i a z i n e - C E O]}{% [AGU]}

Synthesis

Monosubstitution of triazine with thymol or carvacrol

To a solution of thymol or carvacrol (1 mmol) and N-ethyldiisopropylamine DIPEA (1 eq, 170 µL) in anhydrous dichloromethane (4 mL) was added 1.05 mmol of cyanuric chloride at −40°C. (Figure 2) The reaction was monitored by TLC. Once the reaction was complete, the mixture was diluted with dichloromethane (20 mL) and the organic layer was washed with water (10 mL) and dried over MgSO₄. Removal of solvent under vacuum resulted in a crude product, which was purified by preparative TLC, using chloroform/petroleum ether (1/1) as mobile phase.

Triazine-thymol

Yellow oil; yield 79%; Rf: 0.59 (chloroform/petroleum ether, 1/1).

¹H NMR/DMSO-d6: δppm = 1.14 (d, J = 7.4 Hz, H8 and H9); 2.29 (s, H10); 3.01 (sept, J = 7.3 Hz; H7); 6.99 (brs, H6); 7.12 (dd, J = 2.5 Hz, J = 8.1 Hz, H4); 7.29 (d, J = 8.2 Hz, H3).MS (MALDI) m/z for C₁₃H₁₃ON₃Cl₂ calcd. 297.9, found 298.05, 300.05 and 302.05[M+H]⁺.

Triazine-carvacrol

White crystals; yield 68%; Rf: 0.65 (chloroform/petroleum ether, 1/1); mp: 81°C.

¹H NMR/DMSO-d6: δppm = 1.19 (d, J = 6.9 Hz, H8’ and H9’); 2.11 (s, H10’); 2.88 (sept, J = 6.95 Hz, H7’); 7.08 (dd, J = 1.65 Hz and J = 9.05 Hz, H4’); 7.23 (brs, H6’); 7,25 (d, J = 9.10 Hz, H3’). MS (MALDI) m/z for, C₁₃H₁₃ON₃Cl₂ calcd. 297.9, found 298.05, 300.05, and 302.05 [M+H]⁺.

Grafting of triazine-thymol or triazine-carvacrol on KP

KP fibers (1 g) were suspended in 40 mL of NaOH solution (0.5 M) and the mixture was stirred during 24 h, at room temperature, resulting in a dispersion of the fibers in NaOH solution.³⁰ A total of 9.25 mmol of triazine-thymol or triazine-carvacrol in dichloromethane (30 mL) was then added. After 48 h at room temperature while stirring, the mixture was filtered and washed with dichloromethane (2 × 50 mL), water (2 × 50 mL) and ethanol (2 × 50 mL), and then dried at 60°C.

KP-triazine-thymol

Mass yield: 101%; IR (cm⁻¹): 823(C-Cl), 1577 (C = N).

XPS (eV): 533.15 (O1s: 35.9%), 286.74 (C1s, 62.8%), 399.20 (N1s, 1.4%), DS = 0.07.

KP-triazine-carvacrol

Mass yield: 101%; IR (cm⁻¹): 826(C-Cl), 1524 (C = N).

XPS (eV): 533.09 (O1s: 35.4%), 286.70 (C1s, 63.1%), 399.26 (N1s, 1.5%), DS = 0.07.

Antibacterial assays

Growth conditions of bacteria

Gram-positive bacteria (S. aureus CIP76.25) and Gram-negative bacteria (E. coli CIP54.8T) were purchased by the Institut Pasteur Paris (Paris, France). The different bacterial strains were inoculated into liquid tryptic soy (pancreatic casein extract 17 g/L, soy flour papaic digest 3 g/L, dextrose 2.5 g/L, NaCl 5 g/L, and K₂HPO₄ 2.5 g/L) and incubated at 37°C overnight under aerobic conditions. The stock solution was further diluted to give a working suspension of approximately 2 × 10⁶ colony-forming units per mL (CFU/mL).

Treatment with KP fiber surfaces

To evaluate the antibacterial effect of the different KP fiber surfaces, a protocol based on AATCC100 standard was implemented. Sterile samples of KP fibers (8 mm diameter) were impregnated with 50 µL of bacterial suspension at a cell density of approximately 2 × 10⁶ CFU/mL. To determine the CFU number initially deposited onto disks (t = 0), each sample was transferred into 1 mL of extraction solution, composed of Triton X-100 0.5% (v/v) and physiological saline water (NaCl 0.9% w/v). After 2 h of gentle stirring at room temperature, serial dilutions of each extraction solution were performed, and each dilution was spread on tryptic soy agar plates using an automatic easySPIRAL^® plater (Interscience, St Nom la Bretêche, France). After incubation at 37°C for 24 h, bacterial colonies were counted to determine the total CFU per mL. Each experiment was performed in triplicate and was conducted along with necessary control: untreated KP and KP grafted with triazine (KP-triazine).

For the experiment, a sample was processed immediately after bacterial impregnation (t = 0) and other samples were also processed in the same conditions after 24 h at 37°C under aerobic conditions.

The determination of CFU number was performed in quadruplicate for each sample. Microbiological tests were repeated thrice.

Results and discussion

Synthesis

In the first step (Figure 3), cyanuric chloride was monosubstituted by thymol or carvacrol, compound of essential oil (CEO). An amount of 1.05 eq. of cyanuric chloride was added to a solution of 1 eq. of CEO and 1 eq. of DIPEA in anhydrous dichloromethane. Typically, monosubstitution of triazine requires low temperature.^31,32 The assays at −10°C, −20°C, and −30°C led to formation of di and tri substituted triazine. The reaction carried out at −40°C leads to the formation of a sole product resulting from the monosubstitution. The synthesized compounds were purified and characterized by ¹H NMR and MS confirming the structures of triazine-thymol and triazine-carvacrol.

After the triazine-thymol and triazine-carvacrol synthesis, we proceeded to their grafting on KP fibers. For this, the KP fibers are pretreated with sodium hydroxide (0.5 M).

The NaOH pretreatment leads to better fiber dispersion, which allows cellulosic fibers swelling and weakening of the hydrogen bonds, thus making hydroxides more accessible^33,34

Grafting of triazine-CEO on KP

KP fibers were suspended in NaOH solution (0.5 M) during 24 h, at room temperature. Then triazine-CEO in dichloromethane was added (1.5 eq/AGU). After 48 h at room temperature, the reaction product was recovered by filtration and washed with dichloromethane to remove the triazine-CEO, which has not reacted, until TLC analysis of the washing solution shows the absence of free triazine-CEO. Then we washed with water until a neutral pH of filtrate. The product was washed with ethanol in the last step before drying at 60°C.

The FTIR spectrum of modified KP fibers (Figure 4) shows the peaks corresponding to the (C-Cl) bond around 826 cm⁻¹and the (C = N) bond around 1574 cm⁻¹.

KP fibers consisted mostly of carbon and oxygen (Table.1). The appearance on the XPS spectrum of nitrogen signal at 399.20 eV (N1s) for KP-triazine-thymol (Figure 5) and at 399.26 eV for KP-triazine-carvacrol confirm the grafting. A DS of 0.07 was obtained for both the materials. Since the DS is less than 1, triazine-CEOs grafting is done surely on position C6, the most accessible and so the most reactive.³⁵

Table 1.

Chemical data of products.

	O1s (%at)	C1s (%at)	N1s (%at)	DS
KP	36.0	64.0
KP-triazine-thymol	35.9	62.8	1.4	0.07
KP-triazine-carvacrol	35.4	63.1	1.5	0.07

KP: kraft pulp.

Antibacterial assay against S. aureus and E.coli

The antibacterial activity of modified KP sheets was evaluated against the Gram-positive bacteria S.aureus CIP76.25 and the Gram-negative bacteria E.coli CIP54.8T. KP and KP grafted with triazine (KP-triazine) were used as controls.

We began by carrying out a qualitative test to confirm the absence of free thymol and free carvacrol in the elaborated materials. For this purpose, we evaluated the formation of a zone of inhibition around the materials elaborated in our study. As supplementary control, a solution of thymol and carvacrol was deposited on unmodified KP.

A total of 1 mL of a bacterial suspension at 10⁵ CFU/mL (S.aureus or E.coli) was seeded on tryptic soy agar plates. After that, the disks of different materials (KP, KP-triazine, KP-triazine-thymol, and KP-triazine-carvacrol) were placed upon agar and plates were incubated at 37°C for 24 h. In parallel, we deposited 50 µL of CEO solution at 8.4 g/L on KP disks, which corresponds to the amount of 0.42 mg grafted onto a disk of 7 mg. The results are shown in Figure 6.

For free EO compounds deposited on KP, the results show the presence of a zone of inhibition due to the diffusion of EO compounds relative to their antibacterial activity. S.aureus showed less sensitive to free carvacrol at 8.4 g/L. In the case of the grafted materials elaborated in our study, we observed the absence of zone of inhibition, indicating the absence of free EO compounds, confirming the effective covalent grafting of thymol and carvacrol on KP.

Subsequently, we performed quantitative tests to evaluate the antibacterial activity of the grafted materials. Bacterial suspensions were deposited on disks and then incubated at 37°C. After 24 h, the total CFU per mL were determined. The two controls (KP and KP-triazine) showed no antibacterial activity against the two bacterial strains used in this study. Indeed, after 24 h of contact, an increase of bacterial growth of 4 log was observed, in comparison to reference t₀ (Figure 7).

For S.aureus, a bacteriostatic effect was obtained for KP-triazine-thymol. However, S.aureus appears to be less sensitive to KP-triazine-carvacrol material. This result is in accordance with previous studies, indicating that S.aureus strains are less sensitive to carvacrol.³⁶

For E.coli, after 24 h of contact with KP-triazine-thymol and KP-triazine-carvacrol, a bacteriostatic effect was obtained with a 2.5 log reduction for each material in comparison to control KP-triazine. The bacterial growth is equivalent to the growth observed at t₀.

Antibacterial activities of some EOs may result from disruption of bacterial cell envelopes. ESEM analysis of materials in the presence of bacteria after 24 h of contact was carried out. In the case of E. coli, after 24 h of contact with PK-triazine-thymol, a modification of the structure of bacterial cell wall was observed in comparison with bacteria in contact with untreated KP (Figure 8).

As indicated in the literature,¹⁰ the main bacterial targets of EOs are the bacterial cell walls and membranes; grafted thymol on KP seems to have an effect on the cell wall of E.coli. This disruption of the structure of cell wall could induce a change in the potential membrane, leading to the release of ions, ATP molecules, and to cell death.

To evaluate the synergistic effect of thymol and carvacrol, we prepared a material containing both products. For this, we have made a homogeneous mixture of KP-triazine-thymol and KP-triazine-carvacrol in various proportions (Table 2).

Table 2.

Different proportions of the mixtures.

	KP-triazine-thymol	KP-triazine-carvacrol
Mixture 1	30%	70%
Mixture 2	50%	50%
Mixture 3	70%	30%

KP: kraft pulp.

The antibacterial activity of the different mixtures was evaluated as previously. For S.aureus, all mixtures induce a bacteriostatic effect. For E.coli, mixtures 1 and 2 showed a bacteriostatic activity (Figure 9).

Interestingly, a stronger antibacterial effect was observed after the contact of E.coli with mixture 3, with a level of inhibition of 40% compared to the reference t₀. Indeed, a 6 log reduction in comparison to KP fibers, was obtained with this mixture 3.

Conclusion

In this study, we developed new bacteriostatic materials based on KP fibers. We covalently grafted two EO compounds, thymol and carvacrol, known for their antibacterial activity. The grafting of these molecules on KP fibers made it possible to develop a paper having a bacteriostatic activity against two pathogenic bacterial strains, involved in microbial infections. Even with a very low DS, less than 0.1, these new materials block the growth of bacteria, limiting bacterial proliferation and biofilm formation. Furthermore, the method of manufacturing this material is simple and can be easily scaled-up.

The developed materials can be used to limit the growth of bacteria on surfaces. They could be used in the protection of items that are handled by several people like newspapers, magazines, and so on.

Footnotes

Acknowledgements

The authors are thankful to Mr Yann Launay (European Center for Ceramics—ESTER TECHNOPOLE—Limoges) for ESEM analysis.

Declaration of conflicting interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) received no financial support for the research, authorship, and/or publication of this article.

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