Ionic liquids derived from vitamin C as multifunctional active

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Ionic liquids derived from vitamin C as multifunctional active ingredients for sustainable stored-product management Micha# Niemczak, Damian Krystian Kaczmarek, Tomasz Klejdysz, Daniela Gwiazdowska, Katarzyna Marchwi#ska, and Juliusz Pernak ACS Sustainable Chem. Eng., Just Accepted Manuscript • DOI: 10.1021/ acssuschemeng.8b04696 • Publication Date (Web): 30 Nov 2018 Downloaded from http://pubs.acs.org on December 3, 2018

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ACS Sustainable Chemistry & Engineering

Ionic liquids derived from vitamin C as multifunctional active ingredients for sustainable stored-product management

Michał Niemczak,*,† Damian Krystian Kaczmarek,† Tomasz Klejdysz,‡ Daniela Gwiazdowska,§ Katarzyna Marchwińska§ and Juliusz Pernak†

†

Department of Chemical Technology, Poznan University of Technology, ul. Berdychowo 4, Poznan 60-965, Poland ‡

Institute of Plant Protection - National Research Institute, ul. Władysława Węgorka 20, Poznan 60-318, Poland

§

Department of Natural Science and Quality Assurance, Poznań University of Economics, ul. Al. Niepodległości 10, Poznan 61-875, Poland, * Corresponding author at: Poznan University of Technology, ul. Berdychowo 4, 60–965 Poznan, Poland; Tel.: +48 616653681. E–mail: [email protected]

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ABSTRACT This study is focused on the utilization of naturally occurring ascorbic acid (vitamin C) in the synthesis of biologically-inspired ionic liquids with attractive application potential. The ionic liquids with the ascorbate anion were obtained in high yield (>90%) via a simple, “green” twostep

procedure

using

alkyltrimethylammonium,

well-known,

cost-effective

dialkyldimethylammonium

and

commercially and

available

alkylmethylbis(2-

hydroxyethyl)ammonium cations. Three positive ions in this group used for syntheses are derived from renewable resources, such as vegetable oil or animal fat. The study confirmed the strong influence of the chemical structure of the cation on the stability of the compounds obtained in the air, as well as in aqueous solutions. In addition, the synthesized ionic forms of vitamin C exhibited very good antibacterial and antifungal properties against different microorganisms, including pathogens. The tests also revealed the excellent or good deterrent activity towards common stored-grain insects (granary weevil, confused flour beetle and khapra beetle), reaching the values determined for azadirachtin, known as the reference antifeedant. The best results were achieved with products containing two long alkyl substituents in the cation (dimethyldioctylammonium and didecyldimethylammonium) that proved to be extremely effective against all the organisms tested. The results of the biological activity indicate that the products synthesized belong to the third generation of ionic liquids. In conclusion, ascorbatebased ionic liquids with multifunctional (antibacterial, antifungal, antifeedant and antioxidant) properties have substantial potential to be used in the storage of crop protection products. KEYWORDS: Ascorbic acid, ionic liquids, stored grain protection, feeding deterrents, fungicides.


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INTRODUCTION With the increasing concerns about the environment, ionic liquids (ILs) have been frequently proposed during the last decade as promising eco-friendly compounds to replace conventional common chemicals. Due to the designability of the ILs, their cations and anions could be selected to obtain the desired physical (1st generation), chemical (2nd generation) and/or biological (3rd generation) properties.1 This fact was used to synthesize modern innovative ionic liquid forms of pharmaceuticals, such as bretylium tosylate, phenazone gentisate, didecyldimethylammonium ibuprofenate, lidocainium docusate and ranitidine docusate.2,3 Ionic liquids are widely described as compounds that exhibit a biological activity, particularly toward microorganisms, including Gram-positive and Gram-negative bacteria.4,5 The combination of quaternary ammonium or phosphonium cations with active ions that possess herbicidal or fungicidal activities recently led to the formation of a new group of ionic liquids designated herbicidal ionic liquids (HILs) or fungicidal ionic liquids.6-8 ILs can also protect harvested grains from insects by acting as feeding deterrents (antifeedants). This class of substances can inhibit pest feeding on stored products, limiting potential pest development without directly killing them. Our previous research indicated that ILs with long alkyl chains in the cation derived from sweeteners or some natural acids, such as abietic acid, could exhibit a deterrent activity comparable to the most effective natural antifeedants, such as azadirachtin. 9,10

Bio-ionic liquids are a group of ILs entirely comprising renewable natural resources.11 Their unabated popularity is related to the fact that these compounds are usually less harmful to the environment and are more readily biodegradable than the ILs obtained from synthetic substrates.12,13 Naturally occurring L-carnitine, choline or betaine cations are used to synthesize the majority of the bio-ILs,14,15 while derivatives of fatty acids and carboxylic acids, such as acetic or formic acid, are often utilized as the source of the anion.10,16,17 Bio-ILs derived from amino acids or sugars have also recently been described in the literature.18-20 Another bioinspired approach, based on merging an ion of natural origin with the synthetic counterion, proved to possess many advantages, such as facilitating the implementation of the desired properties while reducing adverse impacts on the environment. The extraordinary application potential of such bio-inspired ILs confirms their usefulness as electrolytes,21 herbicides,22 adjuvants,17 feeding deterrents,9 antibacterial agents,23 plasticizers for biopolymers,24 organocatalysts,25 and extractants of rare-earth elements,26 as well as active pharmaceutical ingredients in drug delivery systems27.



Vitamin C, also known as ascorbic acid, is a lactone of natural origin that is produced by some plants and animals.28 Its structure in the stable ene-diol form and both unstable keto forms is presented in Scheme 1.29 Ascorbic acid is generally non-toxic to living organisms, and the deficiency of this substance has a deleterious impact on the human body. Vitamin C is widely used as an antioxidant, preservative in the food industry and as an ingredient in cosmetics.30,31

Scheme 1. 1,2-Diketone and 1,3-diketone forms of ascorbic acid and their interconversion.31 The majority of most of the recent studies on the ILs derived from vitamin C are focused on exploring their basic physicochemical properties and toxicity. Previous studies have shown that such ILs do not exhibit toxic effects toward human cells.32 In addition, the antioxidative properties of the anion in ascorbate-based ionic liquids are proven to be retained, and the structure of the organic cation seems to only marginally influence them.33 More sophisticated applications of ILs found in the literature that use the ascorbate ion demonstrate their potential to serve as reducing agents during the synthesis of metal nanostructures or ILs-supported copper catalysts in Huisgen’s cycloaddition.34 Other studies also showed that such ILs may exhibit bactericidal activity35 and strong electrical conductivity36. The goal of the study focused on the synthesis of new bio-inspired ILs that contain vitamin C as the anion. A survey of the literature on the biological activity of ILs indicates that the presence of at least one long alkyl chain (longer than octyl) in the cation causes a significant enhancement in both antimicrobial and antifeedant properties.9 Therefore, large, cost effective and commercially available alkyltrimethylammonium, dialkyldimethylammonium and alkylmethylbis(2-hydroxyethyl)ammonium cations have been selected to serve as the counter ions. Since the ascorbate anion decomposes in the presence of water molecules, it was also important to determine the stability of these new ILs. This study also involves an extensive evaluation of the potential of the compounds obtained to protect stored products. Therefore, we provide a thorough analysis on the influence of length of the alkyl chains in the cations of the ILs synthesized on their bactericidal, fungicidal and antifeedant activities. MATERIALS AND METHODS Materials 4 ACS Paragon Plus Environment

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L–(+)–Ascorbic

acid (99+%) were purchased from Alfa Aesar (Kandel, Germany).

Didecyldimethylammonium chloride (40% solution in water) octadecyltrimethylammonium chloride (95%) were purchased from KCI Limited (Seoul, South Korea). Oleylmethylbis(2– hydroxyethyl)ammonium chloride (Ethoquad O/12, 80% solution in isopropanol), di(hydrogenated tallow)dimethylammonium chloride (Arquad 2HT–75, 75% solution in isopropanol), dicocodimethylammonium chloride (Arquad 2C–75, 75% solution in isopropanol)

were

purchased

from

AkzoNobel

(Amsterdam,

Netherlands).

Dioctyldimethylammonium chloride (95%) were purchased in Chemos GmbH & Co. KG (Regenstauf, Germany). Tetradecyltrimethylammonium chloride (98%), hexadecyltrimethyl– ammonium chloride (98%), docosyltrimethylammonium chloride (90%) were purchased Stockmeier chemie eilenburg GMBH & CO. KG (Berlin, Germany). Benzalkonium chloride (min. 95%) was purchased from Sigma–Aldrich (Poznan, Poland) and used as obtained. Potassium (min. 99%) hydroxides and solvents (methanol (99,5%), ethanol (96%), DMSO (99%), acetonitrile (99,5%), acetone (99,5%), ethyl acetate (99%), chloroform (98%), toluene (99,5%), hexane (99%)) were delivered by Avantor (Gliwice, Poland) and used without further purification. Synthesis The appropriate quaternary ammonium chloride (0.01 mol) was dissolved with 50 mL of ethanol in a 100 mL reaction glass equipped with a mechanical stirrer. Forty millilitres of the anionic resin Dowex Monosphere 550A in the form of an ethanolic suspension were added, and the mixture was stirred for 1 h at 25 °C. After the anion exchange reaction, the resin was filtered and rinsed three times using small amounts of ethanol. The solutions of quaternary ammonium hydroxides obtained were slowly neutralized using stoichiometric amounts of ascorbic acid. All the neutralization reactions were conducted at 25 °C in a Mettler Toledo semi-automated reactor system EasyMax™ equipped with a glass electrode. The solvent was evaporated under vacuum in a rotary evaporator, and the product was dried under reduced pressure (5 mbar) at 25 °C for 4 h. Analysis The UV spectra were recorded at 25 °C using a UV-1601 spectrophotometer (Rayleigh) in the range of 200-400 nm using 1 cm path length quartz cuvettes and a 1.5 nm bandwidth. Methanolic solutions of the products at concentrations of 1.14 mmol L-1 were loaded into individual cuvettes and analysed (pure methanol was used as the reference). The IR spectra 5 ACS Paragon Plus Environment


were collected using a semi-automated system EasyMax 102 (Mettler Toledo) connected with a ReactIR iC15 (Mettler Toledo) probe equipped with an MCT detector and a 9.5 mm AgX probe with a diamond tip. The data were sampled from 3000 to 650 cm-1 at 8 cm-1 resolution and processed using iCIR 4.3 software. The 1H NMR spectra were recorded on Mercury Gemini 300 and Varian VNMR-S 400 MHz spectrometers operating at 300 MHz and 400 MHz using TMS as the internal standard. The 13C NMR spectra were obtained using the same instruments at 75 and 100 MHz. The elemental analyses (CHN) were performed at the Adam Mickiewicz University, Poznan (Poland). The elemental analysis results, as well as the UV, FT-IR and NMR spectra, are provided in the electronic Supporting Information. The melting points were measured using a MP90 Melting Point System, Mettler Toledo apparatus. Refractive index The refractive index was determined using an Automatic Refractometer J357 with an electronic temperature control. Approximately 1 mL of the samples were analysed at 20 °C. The uncertainty of the measurement was less than 0.00005. Solubilities The solubility of the ILs prepared was determined as described in Vogel’s Textbook of Practical Organic Chemistry.37 Popular representative solvents were chosen and ranked in descending order of their Snyder polarity index value (water, 9.0; methanol, 6.6; DMSO, 6.5; acetonitrile, 6.2; acetone, 5.1; ethyl acetate, 4.4; chloroform, 4.1; toluene, 2.3, and hexane, 0.0). The term “good solubility” refers to the ILs that were dissolved (0.10 g of IL) in 1 mL of the solvent, while the term “limited solubility” indicates that 0.10 g of IL was dissolved in 3 mL of the solvent. The term “poor solubility” indicates that the ILs could not be dissolved in 3 mL of the solvent. All the analyses were conducted at 25 °C under ambient pressure. Stability in aqueous solution The stability of the selected ILs (4, 6, 9 and 10), ascorbic acid and potassium ascorbate was estimated for 0.0572 mol L-1 aqueous solutions (which correspond to a concentration of 1.00% m/v of the ascorbate anion). Measurements were conducted in sealed glass vials containing a magnetic stir bar. A total of 0.572 mmol of the IL selected was introduced into the vial and dissolved in 10 cm3 of distilled water. All the vials had been shaken in a Heidolph MR Hei-End stirrer equipped with a Heat-On anodized block at a constant temperature of 25.0 °C (with accuracy ± 0.1 °C) in the dark. After a period of time, a 0.02 mL sample was collected from 6 ACS Paragon Plus Environment

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each vial and diluted 625-fold. Subsequently, the absorbance of the solutions obtained was measured at λmax = 265 nm for the ascorbate anion, as well as for ascorbic acid, using a Rayleigh UV-1800 spectrophotometer. The concentrations of the compounds in water were determined on the basis of calibration curves using plots of absorbance vs. concentration for each substance. The results are presented as the mean of three separate measurements. Stability in air The stability of the selected ILs (4, 6, 9 and 10) in air, ascorbic acid and potassium ascorbate was measured in a MEMMERT UF55 oven equipped with an air ventilation system. First, accurately weighed quantities (approximately 0.50 g) of the ionic liquids selected were placed in a Petri dish. All the Petri dishes were placed in a MEMMERT UF55 oven in which the air turbine allowed for effective forced air circulation. A constant temperature of 25.0 °C (with an accuracy ± 0.1 °C), as well as 65% humidity, were maintained during the experiment. After a period of time, a sample of 0.0080-0.030 g (50 µmol) was collected from each Petri dish and dissolved in 20 mL of distilled water in a volumetric flask. Subsequently, all solutions were diluted 25-fold, and their absorbance was measured at λmax = 265 nm for the ascorbate anion, as well as for ascorbic acid, using a Rayleigh UV-1800 spectrophotometer. The content of the ascorbate anion in the samples was determined using calibration curves for each substance. The experiment was performed in triplicate. Microorganisms and culture media Antimicrobial tests were conducted using the Gram-positive bacteria Clostridium perfringens ATCC 13124, Bacillus subtilis ATCC 11774, Micrococcus luteus ATCC 4698, and Lactobacillus plantarum PCM 493; and the Gram-negative bacteria Escherichia coli ATCC 8739, Pseudomonas aeruginosa ATCC 9027, Salmonella enterica Serovar Enteritidis ATCC 13076, as well as fungi, including the yeast Candida albicans ATCC 10231 and the filamentous fungi Alternaria alternata KZF 13, Botrytis cinerea BPR 187, Fusarium graminearum KZF 1 and Sclerotinia sclerotiorum KZF 23. Indicator strains were obtained from the American Type Culture Collection (ATCC), Polish Collection of Microorganisms (PCM) and the Collection of the Institute of Plant Protection-National Research Institute in Poznan (BPR, KZF). MIC and MBC/MFC determination The minimum inhibitory concentration (MIC) of the ionic liquids derived from vitamin C was determined using the broth microdilution method. An aliquot of 100 μL of two-fold 7 ACS Paragon Plus Environment


dilutions of the ionic liquids were examined at concentrations ranging from 0.5 to 1000 μg mL-1 and prepared in 96-well microtiter plates. The bacterial suspensions in Mueller-Hinton broth (Oxoid, Canada), and MRS broth for L. plantarum (Merc, Germany) or the yeast solution in Sabouraud broth (Oxoid, Canada) from 24 h cultures were standardized to obtain a density of 0.5 McFarland’s standard. One hundred microlitres of strain suspensions were introduced into the wells to achieve a final density of 5 x 105 CFU mL-1. The plates were incubated at 30-37 °C for 24 h, depending on the indicator organism. The negative control was the medium with the addition of ionic liquids, and the positive control was the bacterial or fungal culture without an inhibitor. After incubation, the optical density of the microbial growth was determined at 600 nm using a BioTek Epoch 2 microplate reader. The results are expressed as the average of three replicates. The MIC value was defined as the concentration of the ionic liquid that inhibited the growth of the microorganism by at least 90%. Ten microlitres of cultures from wells with an ionic liquid concentration higher than the MIC were cultured on agar media and incubated at 30 or 37 ºC for 24-48 h. The lowest concentration of the ionic liquids tested that prevented the growth of the microorganisms was defined as the minimum bactericidal/fungicidal concentration (MBC/MFC). Assays of antifungal activity as potential fungicides Antifungal activity was assayed against selected filamentous fungi using the food poison technique. The strains were incubated at 25 °C in Petri dishes (90 mm diameter) on potato dextrose agar (BioShop, Canada) for 5-7 days. The ILs tested were dissolved in water or water:DMSO (1:1) and added to sterile media to achieve concentrations of 10, 100 and 1000 μg mL-1. Six millimetre discs of the fungal mycelia were placed at the centre of each Petri plate (55 mm diameter). The media mixed with an appropriate amount of water or water:DMSO solution served as the control growth plates. The plates were incubated at 25 ± 2 °C until the mycelia in the control reached the edge of the Petri dish. After incubation, the diameter (mm) of the fungal colony was measured. The results are expressed as the average of three replicates, subtracting the initial diameter of the fungal plugs (6 mm). Antifeedant properties The bioassay experiments were conducted using Tribolium confusum Duv. (larvae and adults), Sitophilus granarius L. (adults) and Trogoderma granarium Ev. (larvae). The insects were grown on a wheat grain or whole-wheat meal diet in laboratory colonies, which were maintained at 26 ± 1 °C and 60 ± 5% relative humidity. Choice and no choice tests for insect 8 ACS Paragon Plus Environment

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feeding were conducted as previously described.9 Wheat wafer discs (1 cm in diameter and 1 mm thick) were saturated by dipping in either ethanol only (control) or a 1% solution of the ILs in ethanol. After evaporation of the solvent (30 min of air-drying), the wafers were weighed and offered to the insects in plastic boxes as the sole food source for five days. The feeding of the insects was recorded under three sets of conditions: (1) on two control discs (CC), (2) on a choice between one treated disc (T) and one control disc (C; choice test), and (3) on two treated discs (TT; no-choice test). Each of the three experiments was repeated five times with three adults of S. granarius, 20 adults or 10 larvae of T. confusum and 10 larvae of T. granarium. The number of individual insects depended on the intensity of their food consumption. The adults used for the experiments were unsexed, 7-10 days old, and the larvae were 5-30 days old. After five days, the discs were weighed, and the average weight of the food consumed was calculated. The values of the coefficients, A (absolute coefficient of deterrence) and R (relative coefficient of deterrence) were calculated as follows: 𝐶𝐶 ― 𝑇𝑇

𝑅 = 𝐶𝐶 + 𝑇𝑇 × 100 (1) 𝐶―𝑇

𝐴 = 𝐶 + 𝑇 × 100 (2) where CC is the average weight of the food consumed in the control; TT indicates the average weight of the food consumed in the no-choice test, while C and T express the average weights of the food consumed in the choice test. The sum of these two coefficients explain the deterrent activity using the criteria: 200-151 very good, 150-101 good, 100-51 medium, 50-0 weak, AA (τ1/2 ≈ 5.87 days) > IL 10 (τ1/2 ≈ 3.03 days) > IL 6 (τ1/2 ≈ 2.91 days) > IL 9 (τ1/2 ≈ 2.77 days) > IL 4 (τ1/2 ≈ 1.80 days). The τ1/2 values suggest that the ILs comprised of an ascorbate anion are approximately 2-3-fold less stable than KA. This phenomenon may be associated with the alteration of the electron distribution within the lactone ring due to the presence of the large organic cations. The molecular modelling allowed us to associate the influence of the electron density of the anion on the stability of the ILs (see Table S13 in the Supporting Information). The optimized structures of KA and the analogue of 10 are characterized by charge values distributed within the lactone ring lower than AA and higher than the analogues of 4, 6 and 9. This observation strongly suggests that the increase in electron density in the ascorbate anion facilitates its decomposition.



The results of storage in the air (Fig. 3B) revealed that the reference substances (AA and KA) were highly stable. Only approximately 8% of the ascorbate anion was degraded in both samples after 30 days. Alternatively, all the ILs tested proved to be drastically less stable, and the rate of the degradation of the ascorbate anion depended significantly on the structure of each cation. Thus, the cations used could be arranged in descending order of their τ1/2 values: behenyltrimethylammonium (4, τ1/2 ≈ 28.7 days) > benzalkonium (9, τ1/2 ≈ 22.4 days) > oleylmethylbis(2-hydroxyethyl)ammonium (10, τ1/2 ≈ 7.8 days) > didecyldimethylammonium (6, τ1/2 ≈ 2.6 days).

Fig. 3. Changes in the content of the ascorbate anion in ILs 4, 6, 9 and 10 compared to potassium ascorbate (KA) and ascorbic acid (AA) during storage in water (A) or in air (B). In addition, the comparison of Figs. 3A and 3B provide information that the most stable ILs during in storage in water turned out to be the less stable when stored in air. This leads to the conclusion that some other factor must influence the rate of decomposition of the ascorbate anion in air. The thorough analysis of the data collected allowed us to correlate the degree of decomposition with the hygroscopicity of the cations utilized. The didecyldimethylammonium cation proved to be the most hygroscopic, while the behenyltrimethylammonium cation absorbed water the most slowly. Therefore, the increase in the rate of absorption of the water molecules from the IL surroundings facilitates the degradation of the ascorbate anion using the same mechanism that is shown in Scheme 3. These results prove that additional experiments should be conducted to fully elucidate the relationship between the size and stability of the cations in the ascorbate-based ILs.


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Antimicrobial activity The quality and quantity of the cereal microbiota depend on varied factors, such as the type of cereal, climatic conditions and the region. Microbial contamination comes from several sources, such as dust, water, insects, soil and fertilizers, as well as animal faeces. Microorganisms occurring in grains include bacteria from the families Pseudomonadaceae, Micrococcaceae, Lactobacillaceae and Bacillaceae, as well as fungi from the genera Alternaria, Fusarium, Botrytis, and Cladosporium55,56 In addition, microorganisms pathogenic to mammals, such as Clostridium perfringens, Salmonella enterica Serovar Enteritidis and Escherichia coli may occur, posing a threat to the health of animals and people.57 Therefore, the antimicrobial properties of the ILs tested were assayed against bacteria and fungi representing the saprophytic and pathogenic microbiota of cereals. The MIC, MBC and MFC values determined for the ILs tested (1-10), KA and AA are presented in Table 3. Table 3. MIC (μg mL-1) and MBC/MFC (μg mL-1) values determined for the ionic liquids (110), didecyldimethylammonium chloride (DDACl), potassium ascorbate (KA) and ascorbic acid (AA). Gram-positive bacteria Clostridium perfringens

Bacillus subtilis

Gram-negative bacteria

Lactobacillus plantarum

Micrococcus luteus

Pseudomonas aeruginosa

Salmonella Enteritidis

Yeast

Escherichia Coli

Candida albicans

MIC

MBC

MIC

MBC

MIC

MBC

MIC

MBC

MIC

MBC

MIC

MBC

MIC

MBC

MIC

MFC

1

2

4

1

1

1

1

Ionic liquids derived from vitamin C as multifunctional active

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