Physicochemical features and toxicity of some vitamin based ionic liquids

Journal of Molecular Liquids 247 (2017) 411–424

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Journal of Molecular Liquids journal homepage: www.elsevier.com/locate/molliq

Physicochemical features and toxicity of some vitamin based ionic liquids M. Vraneš a,⁎, A. Tot a, S. Papović a, D. Četojević-Simin b, S. Markov c, A. Velićanski c, M. Popsavin a, S. Gadžurić a a b c

University of Novi Sad, Faculty of Sciences, Department of Chemistry, Biochemistry and Environmental Protection, Trg D. Obradovića 3, 21000 Novi Sad, Serbia University of Novi Sad, Faculty of Medicine, Oncology Institute of Vojvodina, Put dr Goldmana 4, 21204 Sremska Kamenica, Serbia University of Novi Sad, Faculty of Technology, Bulevar Cara Lazara 1, 21000 Novi Sad, Serbia

a r t i c l e

i n f o

Article history: Received 1 June 2017 Received in revised form 19 September 2017 Accepted 3 October 2017 Available online 05 October 2017 Keywords: Ionic liquids Vitamins Toxicity Antibacterial activity Physicochemical characterization

a b s t r a c t A three novel vitamin based ionic liquids, cholinium nicotinate, cholinium biotinate and cholinium ascorbate were synthesized and characterized. For the first time the acidity constant for biotin was determined. Physicochemical properties such as density, electrical conductivity and viscosity were measured and nature of interactions was discussed from the obtained experimental results. Also, toxicity study of these ionic liquids has been performed using human non-tumor cell line (normal fetal lung fibroblasts, MRC-5) and rat liver hepatoma cell line (H-4-II-E). Antibacterial activity was determined by disc diffusion method on Gram negative bacteria Pseudomonas aeruginosa and Escherichia coli as well on Gram positive bacteria Staphylococcus aureus and Listeria monocytogenes. © 2017 Elsevier B.V. All rights reserved.

1. Introduction The chemistry of ionic liquids (ILs) has been developed rapidly during the last decade [1,2] founding applications in many classical areas of science. Also, they are important candidates to solve classical problems within several societal challenges, such as more soluble and more bioavailable pharmaceutical compounds, highly efficient carbon capture, clean and efficient energy, through the development of a new energy technologies. The potential of ionic liquids is further emphasised since their physical and chemical properties may be fine tuned by varying the cation and the anion [3]. In order to improve known ILs, or to create novel, so-called risk-conscious design and “thinking in terms of structure-activity relationships” should be applied [4]. According to this strategy, the best solution to reduce toxicity and enhance biodegradability is synthesis of ionic liquids from selected naturally-derived, already nontoxic materials [5,6]. Using this strategy it is possible to expand ILs application in food industry, for example as novel dietary supplements. Synthesis driven from non-toxic starting reagents does not necessarily give a product with lower toxicity [7], since the presence of hydrogen bond acceptors and donor ability significantly affect toxicity [8]. Also, so-called protic ionic liquids attracted attention of scientists recently, but still there is a lack of information about their toxicity in the literature [9]. There are numerous advantages of ionic liquids that can be of interest in food industry, such as good solubility in water, better bioavailability, designed lipophilicity that allows easier transport of the nutrients through the cell membrane and possibility of ILs synthesis with ⁎ Corresponding author. E-mail address: [email protected] (M. Vraneš).

https://doi.org/10.1016/j.molliq.2017.10.015 0167-7322/© 2017 Elsevier B.V. All rights reserved.

synergistic cation and anion performances. One of the most promising cations for this purpose is cholinium, also known as vitamin B4, containing the quaternary ammonium ion with a polar hydroxyl group, which is reason for its low toxicity [7]. Also, choline is biologically widespread micronutrient completely degradable under aerobic conditions [10]. The use of choline in the human diet was officially recognized by the US Institute of Medicine's Food and Nutrition Board in 1998. Choline is essential for brain development of the fetus and improves visomotor performance of healthy humans. Lack of the choline causes serious liver and muscle damages [11–13]. Recently, a numerous cholinium based ILs has been synthesized, showing low toxicity and high biodegradability [14–16]. The anion also contribute to the overall toxicity of ILs, although its effect has often been overlooked, possibly due to the limited anion types reported. Vitamins, as important nutrients for humans, are the promising anion candidates for a new class of edible ionic liquids. Importance of ascorbic acid (vitamin C) in nutrition is well-known: it increases iron absorption, improves collagen synthesis, acts as an antioxidant and biological blocking agent against nitrosamine formation [17,18]. Nicotinic acid, also known as niacin or vitamin B3 is an essential vitamin required for processing fat in the body, lowering cholesterol levels and regulating blood sugar levels. Niacin was primarily used for the treatment of hypercholesterolemia and reduction of cardiovascular risk. A deficiency of niacin causes pellagra, a condition characterized by diarrhea, dermatitis, dementia, inflammation of the mouth, amnesia, delirium and if left untreated, death [19,20]. Even a slight deficiency of niacin can lead to irritability, poor concentration, anxiety, fatigue, restlessness, apathy and depression. Biotin (vitamin H or vitamin B7) is necessary for cell growth, the production of fatty acids, metabolism of fats and amino acids and in

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maintaining a steady blood sugar level. Insufficient level of biotin can cause growth retardation, neurological disorders and dermatological abnormalities [21,22]. Therefore, in this paper a new vitamin based ionic liquids were synthesized and appropriate spectroscopy and thermal characterization was conducted. For the newly synthesized cholinium based ionic liquids: cholinium biotinate, [Chol][Biot], cholinium nicotinate, [Chol][Nicot] and cholinium ascorbate, [Chol][Asc], density, electrical conductivity and viscosity measurements were performed, together with their toxicity and antibacterial tests. The antimicrobial activities were evaluated against a range of Gram positive and Gram negative bacteria, as well-established method for antibacterial activity determination of newly synthesized compounds [23–25]. In order to evaluate cytotoxicity of obtained ionic liquids, the starting compounds in the synthesis and applied standards (ascorbic acid, nicotinic acid and biotin), determination of cell growth effect in human fetal lung cell line MRC-5 derived from the healthy tissue and the rat hepatoma cell line H-4-II-E, were performed. Multi-endpoint bioassays that are based on whole-cell response in mammalian cell lines are powerful indicators of metabolic, biochemical, and genetic alterations that arise under the influence of evaluated compounds [26]. MRC-5 is a well characterized human diploid fibroblast cell line that is remarkably stable and retains the predominantly diploid normal karyotype of the original tissue cells [27]. The H-4-II-E cell line possess excellent growth characteristics and low basal, but highly inducible CYP1A activity. Mammalian studies have revealed the utility of the H-4-II-E cells to predict toxic effects [28]. The differences that are obtained under the influence of evaluated samples can point out a selective action and give bioassays in mammalian cell lines predictive power in terms of the risk to higher organisms [29]. 2. Experimental section 2.1. General procedure for synthesis of vitamin based ILs All applied chemicals were used without purification, the summary of the provenance and purity is given in Table A1 in Appendix. All cholinium based ILs (structures are presented in Fig. A1) were synthesized by an acid-base titration, measuring the pH change. The reaction was conducted by slow addition of aqueous solution of choline hydroxide (concentration c = 1.7524 mol·dm−3) to aqueous vitamin solution with stirring, until obtaining inflection point (pH values for [Chol][Biot] = 8.80; [Chol][Nicot] = 7.95; [Chol][Asc] = 7.66). From [Chol][Biot] and [Chol][Nicot] water was removed using a rotational evaporator under temperature of 343.15 K. After evaporation, obtained ILs were stored together with P2O5 and heated under the vacuum for the next two weeks. Knowing that [Chol][Asc] is unstable at higher temperatures [30], water was removed under room temperature in inert atmosphere using rotational evaporator and obtained [Chol][Asc] was stored in desiccator under P2O5. After drying, water content in the ILs were determined by the Karl Fisher titration (using Metrohm 831 Karl Fischer coulometer). The water content was found to be b200 ppm in all prepared ILs. The purities of these ILs were estimated from 1H NMR spectra to be higher than 0.96 (mass fraction, Table A1). Two yellow compounds liquid at room temperatures with no tendency for crystallization during the work were obtained ([Chol][Biot] and [Chol][Asc]). [Chol][Nicot] was obtained as brown solid compound, with melting point below 100 °C (Thermal analysis section) and could be classified as ionic liquid, too. 2.2. Material and methods For additional characterization, the IR and NMR spectra of newly synthesized cholinium based ILs were recorded. NMR spectra were recorded in D2O at T = 298.15 K on a Bruker Advance III 400 MHz spectrometer. Tetramethylsilane was used as accepted internal standard for calibrating chemical shift for 1H and 13C. 1H homodecoupling and 2D COSY method

were used routinely for the assignation of obtained NMR spectra. 13C NMR spectra were assigned by selective decoupling technique. Infrared spectra were recorded as neat samples from (4000–650) cm−1 on a Thermo-Nicolet Nexus 670 spectrometer fitted with a Universal ATR Sampling Accessory. The measurements were performed with a total of 60 scans, at T = 298.15 K, and a spectrum resolution of 2 cm−1 in a range of υ from (750 to 4000) cm−1. The software package Omnic version 6.2 was used in the data acquisition and spectral analysis. The thermal stability of ionic liquids is checked applying thermogravimetric (TG) and differential scanning calorimetry (DSC) analysis. The thermal characterization of the sample was performed by thermogravimetric analysis using simultaneous TG/DSC thermal analyzer SDT Q600 (TA Instruments, USA). Sample of about 2.5 mg was placed in an open platinum pan. Measurements were carried out in nitrogen atmosphere (flow rate was 100 cm3·min−1) to 600 °C with a heating rate of 20 °C·min−1. DSC measurements were performed by differential thermal analyzer DSC Q20 (TA Instruments, USA) with a heating rate of 20 cm3·min−1 in nitrogen atmosphere. Firstly, samples were heated up to 150 °C (in order to remove residual water), cooled to −60 °C and re-heated to 150 °C. The density measurements were performed using vibrating tube Rudolph Research Analytical DDM 2911 densimeter equipped with Peltier-type thermostat within and automatically viscosity correction. Before each series of measurements calibration of the instrument was performed at the atmospheric pressure. Each experimental density value is the average of at least three measurements at temperatures ranged from (293.15 to 313.15) K. Repeated experimental measurements showed reproducibility within 0.01%, and an average value is presented in this work. Standard uncertainty of determining the density is b7.5 ∙ 10−4 g∙cm−3. Viscosity of ionic liquids was measured using a Brookfield Viscosimeter DV II+ Pro which is thermostated with an accuracy of ±0.01 K and filled with about 8 cm3 of pure liquid. The spindle type (SC4-18) was immersed and rate per minute (RPM) was set in order to obtain a suitable torque. A viscometer cell protected from moisture with the compartment made by the manufacturer was calibrated using the liquids of different viscosities purchased from the manufacturer. Viscosities of pure ILs were measured in the temperature range from (293.15 to 313.15) K with the rotation speed of 0.5 RPM. Presented experimental values are the mean of three measurements and the measurement uncertainty was found to be about 1%. The electrical conductivity measurements were carried out in a Pyrexcell with platinum electrodes on a conductivity meter Jenco 3107 using DC signal in the temperature range from (293.15 to 313.15) K. The cell constant of 1.0353 cm−1 was checked from time to time to control any possible evolution. The relative standard uncertainty for electrical conductivity was b1.5%. All obtained experimental values represent the mean of three measurements. 2.3. Cell lines and cytotoxicity tests Sulforhodamine B (SRB) and antibiotic/antimycotic (amphotericin B) solution were purchased from Sigma Aldrich, fetal bovine serum (FBS) and Dulbecco's Modified Essential Medium (DМEМ) was from PAA Laboratories GmbH, trypsin was from Serva and EDTA from Laphoma. All substances were diluted in 9 mg·cm−3 NaCl and sterilized using 0.22 μm syringe filters. Ionic liquid and standards of ascorbic acid, biotin and nicotinic acid were investigated in the concentration range from (125 to 2000) μg·cm−3. Cell growth activity was evaluated in vitro in human fetal lung cell line MRC-5 (ECACC 05090501) and rat hepatoma cell H-4-II-E (ATCC CRL-1548). Cells were grown in Dulbecco's Modified Essential Medium supplemented with 10% heat inactivated FCS, 100 μg·cm−3 of penicillin, 100 μg·cm− 3 of streptomycin and 0.25 μg·cm− 3 of amphotericin B. Cells were cultured in 25 cm3 flasks at 37 °C in the atmosphere of 5% CO2 and high humidity, and sub-cultured twice a week. A single cell suspension was obtained using 0.1% trypsin with 0.04% EDTA.

M. Vraneš et al. / Journal of Molecular Liquids 247 (2017) 411–424

The cell lines were harvested and plated into 96-well microtiter plates at seeding density of 4 × 103 cells/well in a volume of 180 cm3, and preincubated in complete medium supplemented with 5% FBS at 37 °C for 24 h. Serial two-fold dilutions of tested substances were added to achieve final concentrations. Equal volume of solvent was added in control wells. After the addition of dilutions microplates were incubated at 37 °C for 48 h. The cell growth was evaluated by colorimetric SRB assay of Skehan et al. [31] Colour development was measured using Multiscan Ascent photometer at 540 nm against 620 nm as background. The effect on cell growth was calculated as 100 × (AT/AC) (%), where AT is the absorbance of the test sample and AC of the control. Dose effect (concentration-cell growth) curves were drawn for each treatment and IC50 values (concentration that inhibit cell growth by 50%) were determined using OriginPro 8. The results of cell growth activity were obtained in two independent experiments, each performed in quadruplicate (n = 8). 2.4. Test microorganisms Test microoganisms for the determination of antibacterial activity were reference cultures of Gram negative bacteria: Pseudomonas aeruginosa (ATCC 27853) and Escherichia coli (ATCC 25922) and Gram positive bacteria: Staphylococcus aureus (ATCC 25923) and Listeria monocytogenes (ATCC 19111). 2.5. Antibacterial activity

where c(H3O+) is total ionized (from HCl) and potentially ionizable hydronium ions (from HBiot and H2O), [H3O+] is concentration of free hydronium ions (measurable with pH meter) and c(HBiot) is total concentration of biotin. Calculated amount of biotin is dissolved in water, measured amount of HCl (c = 0.09561 mol·dm−3) was added and ionic strength was adjusted to 0.01 by adding the appropriate amount of KCl. Obtained solution is titrated, under thermostatic conditions (T = 298.15 ± 0.01 K), by slow addition of aqueous solution of NaOH (c = 0.09403 mol·dm−3), measuring pH change. On the basis of obtained data, the values were calculated using Eq. (3): nH ¼

  cðHBiotÞ−cðNaOHÞ− H3 Oþ þ ½OH−  þ cðHClÞ cðHBiotÞ

 ð1−nH Þ  H3 Oþ : nH

3.1. Determination of biotin acidity constant

The results are given in Table A3, and average value for pKca calculated by Albert method is 4.77. Based on activity coefficient calculated from expanded Debye-Hückel theory of constant ionic strength (values of activity coefficient for single charged ions is 0.914 for I = 0.01) it is possible to calculate thermodynamic acidic constant (pKaa) of biotin and its value is 4.85 (Appendix of this manuscript). 3.2. Spectroscopy analysis Obtained 1H NMR, 13C NMR and FTIR spectra of [Chol][Asc], [Chol][Biot] and [Chol][Nicot] are presented in Figs. A2–A7 and corresponding assignation is given bellow. 3.3. [Chol][Asc]

Due to the lack of literature data for acidity constant (Kaa) of biotin, determination of Kaa is significant, because synthesis of cholinium biotinate ionic liquid is based on acid-base neutralization reaction. In aqueous solution, the equilibrium of dissociation of biotin can be written:

nH

1.0

HBiot þ H2 O⇄Biot− þ H3 0þ ;

ð1Þ

0.0 c

pKa

Potentiometric determination of Kca is based on calculation of average number of protons attached per biotin molecule:
  c H3 Oþ − H3 Oþ ; nH ¼ cðHBiotÞ

nH

0.5

and an equilibrium constant which describes this process (stoichiometric acid dissociation constant), Kca, can be calculated (Eq. (1)):   ½Biot−  H3 Oþ ½HBiot

ð4Þ

1 H NMR (D2O): 3.19 (bs, 9H, N(CH3)3); 3.51 (t, 2H, CH2N(CH3)3); 3.70–3.38 (m, 2H, H-6); 3.98–4.08 (m, 3H, H-5 i CH2OH); 4.50 (bs,1H, H-4). 13 C NMR (D2O): 56.66, 56.70 and 56.74 (N(CH3)3,); 58.45 (HOCH2CH2N(CH3)3); 65.39 (C-6); 70.22, 70.25 and 70.28

3. Results and discussion

K ca ¼

ð3Þ

All calculated results are presented in Table A2. Plot of versus nH pH (protonation curve) is presented in Fig. 1. When nH = 0.5 then [HBiot] = [Biot−] and pKca = pH (note: pKca = −logKca), so acid dissociation constant for biotin was determined using graphical method from Fig. 1, and the obtained value of pKca is 4.81. In order to determine Kca values more precisely, Albert method was applied. In the range nH between 0.4 and 0.6 adequate [H3O+] values were taken and Kca values were calculated according to the Eq. (4). K ca ¼

Antibacterial activity was determined by disc diffusion method [32]. Bacterial strains were grown on Müeller-Hinton agar (Himedia, Mumbai, India) 24 h at 37 °C. Cells were then suspended in a sterile 0.9% NaCl solution. Afterwards, 2 ml of the suspensions for inoculation (1 × 106 cells/ml, estimated by Densichek, Biomérieux, France) were homogenized with 18 ml of melted (45 °C) Müeller-Hinton agar and poured into Petri dishes. After the solidification, sterile 6 mm discs were placed on the inoculated agar plates and impregnated with 15 μl of tested samples. The antibiotic (cefotaxime 30 μg/clavulanic acid 10 μg discs, Bioanalyse, Ankara, Turkey) was used as control. The test plates were refrigerated at 8 °C for 1 h to allow the samples to diffuse into the medium, and then were incubated at 37 °C for 24 h. After the incubation period, the diameters of the inhibition zones were measured using HiAntibiotic Zone Scale™ (HiMedia, Mumbai, India). The evaluation of antibacterial activity was carried out in three repetitions, and the results were recorded as inhibition zone ± standard deviation.

413

2

3

4

5

6

7

8

pH ð2Þ

Fig. 1. nH variation with pH at 298.15 K.

9

10

11

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(CH2N(CH3)3); 72.39 (C-5); 81.22 (C-4); 115.94 (C-2); 178.57 (C-3) and 180.38 (C-1). IR (neat): 3369 (stretching OH); 2925 (sym. stretching CH); 1722 (stretching C_O); 1585 (asym. stretching NH); 1478 (CH3 scissor); 1417 (CH2 waging); 1138 (stretching C\\O); 1110 (stretching C\\O\\C); 1041 (twisted CH2); 957 (asym. stretching C\\C\\O); 827 (C\\C stretching). 3.4. [Chol][Biot] 1 H NMR (D2O): 1.38–1.82 (m, 6H, CH2CH2CH2CH2CO2); 2.22 (t, 2H, J = 7.4 Hz, CH2 CH2 CH 2CH2 CO2 ); 2.81 (d, 1H, J 5a,5b = 13.0 Hz, H-5a); 3.04 (dd, 1H, J5a,5b = 13.0 Hz, J4,5b = 5.0 Hz, H-5b); 3.23 (s,

9H, N(CH3)3); 3.40 (ddd, 1H, J2,3 = 4.5 Hz, J2,CH2 = 6.0 Hz, J2,CH2 = 8.7 Hz, H-2); 3.54 (m, 2H, CH2N(CH3)3); 4.09 (m, 2H, CH2OH); 4.47 (dd, 1H, J2,3 = 4.5 Hz, J3,4 = 7.9 Hz, H-3); 4.63 (dd, 1H, J 4,5 = 4.7 Hz, J3,4 = 7.9 Hz, H-4). 13 C NMR (D2O): 28.60, 30.61 and 31.22 (CH2CH2CH2CH2CO2); 40.25 (CH2CH2CH2CH2CO2); 42.64 (C-5); 56.73, 56.77 and 56.81 (N(CH3)3); 58.28 (C-2); 58.49 (OHCH2CH2N(CH3)3); 63.13 (C-4); 64.88 (C-3); 70.29, 70.32 and 70.35 (OHCH2CH2N(CH3)3). IR (neat): 3221 (stretching OH); 2921 (sym. stretching CH); 1681 (stretching C_O)); 1561 (asym. stretching NH); 1454 and 1390 (CH2 waging); 1263 (stretching C\\C); 1084 (stretching C-C-N); 1008 (asym. stretching C\\COO); 954 (asym. stretching C\\C\\O); 864 (stretching C\\S).

a) 217.32 C 1.898% 199.92 C 2.873%

Weight (%)

205.37 C

Temperature (oC)

Heat Flow (W g-1)

b)

14.45 C 56.44 J g-1

55.97 C

Temperature (oC) Fig. 2. TG analysis (a) of ionic liquids: [Chol][Nicot] (red line), [Chol][Asc] (green line), [Chol][Biot] (black line) and DSC analysis (b) for [Chol][Nicot]. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

M. Vraneš et al. / Journal of Molecular Liquids 247 (2017) 411–424

415

0.0

3.5. [Chol][Nicot]

-1.0

-1.5

2

-1

(S cm mol )

-0.5

log

1 H NMR (D2O): 3.05 (s, 9H, N(CH3)3); 3.37 (m, 2H, CH2N(CH3)3); 3.91 (m, 2H, CH2OH); 7.34 (dd, 1H, J4,5 = 7.7 Hz, J5,6 = 5.0 Hz, H-5); 8.09 (bd, 1H, J4,5 = 7.9 Hz, H-4); 8.43 (bs, 1H, H-6); 8.80 (bs, 1H, H-2). 13 C NMR (D2O): 56.57, 56.61 and 56.65 (N(CH3)3); 58.34 (OHCH2CH2N(CH3)3); 70.17, 70.20 and 70.23 (OHCH2CH2N(CH3)3); 126.80 (C-5); 135.32 (C-3); 140.46 (C-4); 151.77 (C-2); 153.05 (C-6); 175.23 (C = O). IR (neat): 3201 (stretching OH); 1598 (stretching C_C); 1558 (asym. stretching NH); 1478 (CH3 scissor); 1361 (sym. stretching C_O from COO−); 1192 (stretching C\\OH, from Ph-OH); 1088 (stretching C\\C\\N); 1025; 953 (asym. stretching C\\C\\O); 832 (C\\C stretching); 759, 708 and 696 (C\\H out plane bending).

-2.0 -2.0

3.6. Thermal analysis In order to determine the thermal stability of newly synthesized ionic liquids [Chol][Asc], [Chol][Biot] and [Chol][Nicot] the thermogravimetric measurements were performed. The results of thermal stability are important, since they indicate which temperature range is suitable for thermal processing of food which contains these ILs as an additives. Obtained thermogravimetric curves are presented in Fig. 2a. As can be seen from Fig. 2a, [Chol][Asc] and [Chol][Nicot] were found to decompose in one step, and the thermal decomposition of [Chol][Biot] is a two stages process. The Tonset values increased in the following order [Chol][Biot] b [Chol][Asc] b [Chol][Nicot] and these values were in the range from (199 to 218) °C, which were comparable to those of cholinium based ILs with amino acid anions [33,34]. In addition, thermal stability of cholinium based ionic liquids is lower in comparison with choline chloride, [Chol]Cl, (Tonset = 320 °C) [35] which is a consequence of weaker interactions between ions in ionic liquid originate from more voluminous and better steric hindrance of vitamin anion compared to chloride. High thermal stability of ionic liquid does not require special temperature conditions during warehousing and preservation. In addition, presented thermal stability enables high temperature technological processes. On the basis of DSC curve in Fig. 2b, melting point is observed for ionic liquid [Chol][Nicot] at T = 55.97 °C. No melting points were observed in the scanned temperature range from − 60 to 130 °C for [Chol][Biot] and [Chol][Asc]. 3.7. Physicochemical properties Density, viscosity and electrical conductivity of synthesized roomtemperature ionic liquids [Chol][Asc] and [Chol][Biot] were measured at temperature range from (293.15 to 313.15) K. The results are tabulated in Table 1 and plotted in Figs. A8–A10. The measurements indicated that [Chol][Asc] have higher values of viscosity comparing to [Chol][Biot]. The experimental results at Table 1 Experimental values of density (d), viscosity (η) and conductivity (κ) and calculated values of thermal expansivity (αp) for [Chol][Biot] and [Chol][Asc] in temperature range from (293.15 K to 313.15) K at atmospheric pressure. T/K

η/MPa·s

κ/mS·cm−1

d/g·cm−3

αp · 104/K−1

293.15 298.15 303.15 308.15 313.15

[Chol][Biot] 1565.67 981.39 619.07 381.11 220.16

0.181 0.293 0.418 0.593 0.791

1.16545 1.16247 1.15939 1.15621 1.15299

4.87 4.88 4.90 4.91 4.92

293.15 298.15 303.15 308.15 313.15

[Chol][Asc] 2887.13 1546.41 801.41 431.55 231.97

0.076 0.164 0.306 0.499 0.766

1.25732 1.25441 1.25136 1.24826 1.24508

5.26 5.27 5.28 5.30 5.31

-1.5

-1.0

log (

-1

) / (mPa s)

-0.5

0.0

-1

Fig. 3. Walden plot for: (■) [Chol][Biot] and (○) [Chol][Asc].

298.15 K are compared with the results of Liu et al. [35]. (Fig. A11) for cholinium amino acids based ionic liquids (ChAAILs). It is shown that stronger intermolecular forces such as van der Waals forces, hydrogen bonding and π-stacking interactions might contribute to the higher viscosity [35] of cholinium based IL presented in this work in comparison to ChAAILs, as well as the occurrence of the additional non-covalent interactions. Higher values of viscosity were obtained for [Chol][Asc] ionic liquid, due to formation of cross-linked hydrogen bond network between OH-group in the cholinium cation and OH-groups of ascorbate anion, which is stronger than such bonds between cholinium cation and cyclourea group from biotin. Variation of viscosity, η, with temperature was calculated using Arrhenius equation: η ¼ ηo expðEA =ðRT ÞÞ

ð5Þ

where η is the viscosity, T is the temperature, ηo is the coefficient of equation and EA activation energy. Obtained values for activation energies are EA([Chol][Asc]) = 1395 kJ·mol− 1 and EA([Chol][Biot]) = 1074 kJ·mol− 1. The higher value E A is, the more difficult ions will move, which might be due to either physical size or the occurrence of more stronger interactions [36]. On the basis of density measurements, the isobaric thermal expansivity, αp, can be calculated using the relation: αp ¼ −

  1 ∂d d ∂T p

ð6Þ

Table 2 Cell growth activity of ionic liquids and standards in MRC-5 and H-4-II-E cell lines after 48 h. Compound

IC50/(μg·cm−3) MRC-5

H-4-II-E

[Chol][Asc]

1967.86 ± 15.42

[Chol][Biot]

N2000 (IC13.71 = 2 mg cm−3) N2000 (IC16.66 = 2 mg cm−3) 398.60 ± 24.96 N2000 (IC6.02 = 2 mg cm−3) N2000 (IC33.2 = 2 mg cm−3)

N2000 (IC36.52 = 2 mg cm−3) N2000 (IC11.23 = 2 mg cm−3) N2000 (IC11.67 = 2 mg cm−3) / N2000 (IC1.14 = 2 mg cm−3) 1060.38 ± 5.50

[Chol][Nicot] Ascorbic acid Biotin Nicotinic acid

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Table 3 Results of antibacterial tests on four different bacteria in mass concentration 1%, 5% and 10% of ionic liquids. Sample

[Chol][Asc]

[Chol][Biot]

[Chol][Nicot]

Test microorganism

1% 5% 10% 1% 5% 10% 1% 5% 10%

Cefotaxime/clavulanic acid

Escherichia coli

Pseudomonas aeruginosa

Staphylococcus aureus

Listeria monocytogenes

nd nd nd nd nd nd nd nd nd 35.5a ± 0.7

nd nd nd nd nd nd nd nd nd 15.33a ± 0.58

nd nd nd nd nd nd nd nd nd 37.0a ± 2.0

Nd Nd Nd Nd Nd Nd Nd Nd Nd 11.0a ± 0.0

nd – not detected inhibition zone. a Clear zone around the disc.

Calculated values are presented in Table 1. It can be seen that these values do not significantly differ at different temperatures in the range from (293.15 to 313.15) K. The value of relative deviation in αp was found to be b1%. Thus, it was demonstrated that the thermal expansion coefficient of [Chol][Asc] and [Chol][Biot] is temperature independent which is also observed by Tao et al. [34] Based on experimental values of viscosity and conductivity, Walden plot was applied, in order to examine ionicity of [Chol][Asc] and [Chol][Biot] [37]. The relation between molar conductivity and viscosity can be demonstrated by Eq. (7): logΛ m ¼ logC þ α logη−1

ð7Þ

where, Λm is molar conductivity, η−1 is fluidity, α is slope of the line in the Walden plot which reflects the decoupling of the ions and C is temperature dependent constant. The Walden plot is presented in Fig. 3, showing that [Chol][Biot] is more close to ideal KCl line comparing to [Chol][Asc]. In order to quantify ionicity, Angell method was applied by measuring the vertical distance between ionic liquids line and the KCl line [37–40]. Obtained ionicity results at 298.15 K are 85.96% for [Chol][Biot] and 56.47% for [Chol][Asc]. Lower value of ionicity in the case of [Chol][Asc] is indicating stronger interactions between cation and anion, causing higher level of association for this ionic liquid which is in agreement with obtained high values of viscosity and density. 3.8. Toxicity of vitamin based ILs In order to consider potential application of vitamin based ILs [Chol][Asc], [Chol][Biot] and [Chol][Nicot] in food industry, their cell growth activity was evaluated in human non-tumor cell line (normal fetal lung fibroblasts MRC-5) and H-4-II-E (rat liver hepatoma). Obtained

results are shown in Table 2. Besides that, influence of commercial substances ascorbic acid, nicotinic acid and biotin on the cell growth were examined and obtained results are presented in Table 2. Antiproliferative activity was expressed as IC50 value. The highest overall cell growth inhibition was demonstrated by ascorbic acid and biotin with IC50 values obtained at 400 and 1000 μg cm−3, respectively (Table 2). Cell growth inhibition and obtained IC50 values at these concentrations are considered as mild [25]. Ascorbic acid shows much higher cytotoxicity in comparison with [Chol][Asc]. Ionic liquid [Chol][Biot] has slightly higher cytotoxicity toward both cell lines in comparison to biotin. In order to compare newly synthesized ionic liquids, inhibitory activity at highest concentration (2 mg·cm−3) were compared and presented in Table 2 (in parenthesis). For all ILs IC50 was achieved only for [Chol][Asc] at highest investigated concentration (2 mg∙cm−3). At these highest concentrations percentage of cell growth inhibition induced by ILs was from 11–36% (Table 2) and proved them to be non-toxic. Results of antibacterial activity of newly synthesized [Chol][Asc], [Chol][Biot], [Chol][Nicot] and control sample (cefotaxime/clavulanic acid) are presented in Table 3 and in Fig. 4. Samples were dissolved in distilled water to a mass concentration of 1%, 5% and 10% of ionic liquid. As can be seen from Table 3, all tested bacteria showed susceptibility to control sample (cefotaxime/clavulanic acid). Tested samples in all applied concentrations did not show any antibacterial activity against Gram positive and Gram negative bacteria, regardless of the different cell wall structures of the tested strains. Cholinium based ILs possess high biodegradability nature and nontoxic profile [40]. Additionally, B-group vitamins are often necessary for microbial growth, that means microorganisms are auxotrofic according to some of them. On the other hand, several nicotinic acid and biotin analogues have shown good antimicrobial activity [41]. As for ascorbic acid, it is known that it possess antimicrobial activity

Fig. 4. Antibacterial activity of vitamin based IL (H1 = [Chol][Asc]; H2 = [Chol][Biot], H3 = [Chol][Nicot]) at concentration 5% and 10% on different bacteria: a) Escherichia coli, b) Pseudomonas aeruginosa, c) Listeria monocytogenes and d) Staphylococcus aureus.

M. Vraneš et al. / Journal of Molecular Liquids 247 (2017) 411–424

in concentration 0.4% [42]. It can be assumed that sample [Chol][Asc] acts by reducing or eliminating antimicrobial activity of ascorbic acid. 4. Conclusions In this paper synthesis of novel vitamin based ionic liquids has been reported, with adequate spectroscopic and thermal characterization. The measurements of density, conductivity and viscosity of [Chol][Asc] and [Chol][Biot] were also performed. Based on the obtained results, it was concluded that interactions in [Chol][Asc] are stronger due to hydrogen bonds formation. Using Walden plot, lower value of ionicity for [Chol][Asc] was obtained comparing to those calculated for [Chol][Biot]. Toxicity of cholinium based ionic liquids has been investigated using human non-tumor cell line and rat liver hepatoma cell line. Obtained results are compared with toxicity results of their vitamin analogue, ascorbic acid, biotin and nicotinic acid. Also antibacterial activity was determined by

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disc diffusion method on Gram negative bacteria: Pseudomonas aeruginosa and Escherichia coli and Gram positive bacteria: Staphylococcus aureus and Listeria monocytogenes. According to obtained results it was shown that these ILs can be considered as non-toxic. It can be concluded that using simple and green chemical route a non-toxic ionic liquids based on vitamins can be prepared, in which both cation and anion are biologically active components. Therefore, through their synthesis is completely neglected the problem of their low solubility in water, low absorbability, polymorphic forms and on the other side, reached the possibility to deliver two or more active ingredients at once as the novel food supplements. Acknowledgments This work was financially supported by the Ministry of Education, Science and Technological Development of Republic of Serbia under project contract ON172012.

Appendix A Table A1 Provenance and purity of the chemicals. Chemical name

Provenance

CAS number

Purification method

Final mass fraction, ω

Choline hydroxide (water solution) D (+) – Biotin L - ascorbic acid Nicotinic acid [Chol][Biot] [Chol][Asc] [Chol][Nicot]

Sigma-Aldrich Merck Sigma Aldrich Sigma Aldrich Synthesis Synthesis Synthesis

123-41-1 58-85-5 50-81-7 59-67-6

None None None None Rotary evaporation followed by vacuum Rotary evaporation followed by vacuum Rotary evaporation followed by vacuum

ω ≥ 0.46 ω ≥ 0.99 ω ≥ 0.99 ω ≥ 0.98 ω ≥ 0.96a ω ≥ 0.96a ω ≥ 0.96a

a

Determined by NMR measurement.

Table A2 Results of biotin acid-base titration. V(NaOH) cm3

c(biotin) mol·dm−3

c(NaOH) mol·dm−3

pH

[H3O+] mol·dm−3

[OH−] mol·dm−3

nH

0.00 0.20 0.35 0.50 0.60 0.65 0.70 0.75 0.80 0.85 0.90 0.93 0.95 1.00 1.05 1.07 1.10 1.12 1.15 1.17 1.20 1.23 1.25 1.27 1.30 1.33 1.35 1.40 1.42 1.45 1.47 1.50 1.53 1.55 1.57

5.576 · 10−4 5.554 · 10−4 5.538 · 10−4 5.523 · 10−4 5.512 · 10−4 5.507 · 10−4 5.502 · 10−4 5.496 · 10−4 5.491 · 10−4 5.486 · 10−4 5.481 · 10−4 5.478 · 10−4 5.476 · 10−4 5.470 · 10−4 5.465 · 10−4 5.463 · 10−4 5.460 · 10−4 5.458 · 10−4 5.455 · 10−4 5.452 · 10−4 5.450 · 10−4 5.447 · 10−4 5.445 · 10−4 5.442 · 10−4 5.440 · 10−4 5.437 · 10−4 5.435 · 10−4 5.429 · 10−4 5.427 · 10−4 5.424 · 10−4 5.422 · 10−4 5.419 · 10−4 5.417 · 10−4 5.414 · 10−4 5.412 · 10−4

0.00 1.84 · 10−4 3.22 · 10−4 4.59 · 10−4 5.50 · 10−4 5.95 · 10−4 6.41 · 10−4 6.86 · 10−4 7.32 · 10−4 7.77 · 10−4 8.22 · 10−4 8.45 · 10−4 8.68 · 10−4 9.13 · 10−4 9.58 · 10−4 9.81 · 10−4 1.00 · 10−3 1.03 · 10−3 1.05 · 10−3 1.07 · 10−3 1.09 · 10−3 1.12 · 10−3 1.14 · 10−3 1.16 · 10−3 1.18 · 10−3 1.21 · 10−3 1.23 · 10−3 1.27 · 10−3 1.30 · 10−3 1.32 · 10−3 1.34 · 10−3 1.36 · 10−3 1.39 · 10−3 1.41 · 10−3 1.43 · 10−3

2.98 3.07 3.15 3.25 3.32 3.37 3.42 3.48 3.54 3.62 3.69 3.72 3.77 3.86 3.95 4.01 4.09 4.16 4.23 4.28 4.36 4.42 4.48 4.57 4.65 4.71 4.76 4.89 4.96 5.04 5.14 5.26 5.4 5.55 5.78

1.16 · 10−3 9.46 · 10−4 7.87 · 10−4 6.25 · 10−4 5.32 · 10−4 4.74 · 10−4 4.22 · 10−4 3.68 · 10−4 3.20 · 10−4 2.67 · 10−4 2.27 · 10−4 2.12 · 10−4 1.89 · 10−4 1.53 · 10−4 1.25 · 10−4 1.09 · 10−4 9.03 · 10−5 7.69 · 10−5 6.54 · 10−5 5.83 · 10−5 4.85 · 10−5 4.22 · 10−5 3.68 · 10−5 2.99 · 10−5 2.49 · 10−5 2.17 · 10−5 1.9 · 10−5 1.43 · 10−5 1.22 · 10−5 1.01 · 10−5 8.05 · 10−6 6.11 · 10−6 4.42 · 10−6 3.13 · 10−6 1.84 · 10−6

1.06 · 10−11 1.30 · 10−11 1.57 · 10−11 1.97 · 10−11 2.32 · 10−11 2.60 · 10−11 2.92 · 10−11 3.35 · 10−11 3.85 · 10−11 4.63 · 10−11 5.44 · 10−11 5.83 · 10−11 6.54 · 10−11 8.05 · 10−11 9.90 · 10−11 1.14 · 10−10 1.37 · 10−10 1.61 · 10−10 1.89 · 10−10 2.12 · 10−10 2.54 · 10−10 2.92 · 10−10 3.35 · 10−10 4.13 · 10−10 4.96 · 10−10 5.70 · 10−10 6.39 · 10−10 8.62 · 10−10 1.01 · 10−9 1.22 · 10−9 1.53 · 10−9 2.02 · 10−9 2.79 · 10−9 3.94 · 10−9 6.69 · 10−9

0.7375 0.7902 0.8233 0.8622 0.8618 0.8823 0.8914 0.9061 0.9080 0.9218 0.9097 0.8951 0.8950 0.8750 0.8430 0.8302 0.8214 0.8038 0.7825 0.7533 0.7290 0.6982 0.6659 0.6362 0.6031 0.5666 0.5286 0.4530 0.4145 0.3758 0.3372 0.2983 0.2589 0.2188 0.1787 (continued on next page)

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Table A2 (continued) V(NaOH) cm3

c(biotin) mol·dm−3 −4

1.60 1.65 1.70 1.80 1.90 2.00

c(NaOH) mol·dm−3 −3

5.409 · 10 5.404 · 10−4 5.399 · 10−4 5.389 · 10−4 5.379 · 10−4 5.369 · 10−4

1.45 · 10 1.50 · 10−3 1.54 · 10−3 1.63 · 10−3 1.72 · 10−3 1.81 · 10−3

pH 6.05 6.77 9.05 9.71 9.96 10.15

[H3O+] mol·dm−3 −7

9.90 · 10 1.89 · 10−7 9.90 · 10−10 2.17 · 10−10 1.22 · 10−10 7.87 · 10−11

[OH−] mol·dm−3

nH

1.25 · 10−8 6.54 · 10−8 1.25 · 10−5 5.70 · 10−5 1.01 · 10−4 1.57 · 10−4

0.1377 0.0542 −0.0078 −0.0958 −0.1841 −0.2515

In order to calculate Kc of acid it is necessary to obtain values of nH in range from 0.4 to 0.6. From this range, adequate [H3O+] values were taken and were calculated values of Kca according to the Eq. (1). K ca ¼

 ð1−nH Þ  H3 Oþ nH

ð1Þ

Using appropriate activity coefficient, [H3O+] and [OH−] values were calculated:   H3 Oþ ¼ 10−pH =f 

ð2Þ

  ½OH−  ¼ K cw = H3 Oþ

ð3Þ

For ionic strength I = 0.01 at T = 298.15 K, values of Kcw used for calculation was 1.23·10−14 mol2·dm−6 and activity coefficient is f± = 0.914. For solution with constant ionic strength it is possible to calculate thermodynamic values of acidity constant from: K aa ¼


  a H3 Oþ aðBiot− Þ H3 Oþ f  ½Biot− f  2 ¼ ¼ K ca ð f  Þ aðHBiotÞ ½HBiot

ð4Þ

pK aa ¼ pK ca −2 logf  Table A3 Results for Albert method. c mol·dm−3

pH

[H3O+] mol·dm−3

Kca

pKca

0.60309 0.56663 0.52859 0.45299 0.41449 0.37583

4.65 4.71 4.76 4.89 4.96 5.04

2.49 · 10−5 2.17 · 10−5 1.93 · 10−5 1.43 · 10−5 1.22 · 10−5 1.01 · 10−5

1.64 · 10−5 1.66 · 10−5 1.72 · 10−5 1.73 · 10−5 1.72 · 10−5 1.68 · 10−5

4.79 4.78 4.76 4.76 4.76 4.77

Fig. A1. Structures of vitamin based ionic liquids: a) [Chol][Asc], b) [Chol][Biot], c) [Chol][Nicot].

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Fig. A2. 1H and 13C NMR spectra of [Chol][Asc].

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Fig. A3. FTIR spectrum of [Chol][Asc].

Fig. A4. 1H and 13C NMR spectra of [Chol][Biot].

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Fig. A5. FTIR spectrum of [Chol][Biot].

Fig. A6. 1H and 13C NMR spectra of [Chol][Nicot].

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Fig. A7. FTIR spectrum of [Chol][Nicot].

Fig. A8. Variation of density with temperature for: (■) [Chol][Biot] and (○) [Chol][Asc].

Fig. A9. Variation of lnη with T−1for: (■) [Chol][Biot] and (○) [Chol][Asc].

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Fig. A10. Variation of electrical conductivity with temperature for: (■) [Chol][Biot] and (○) [Chol][Asc].

Fig. A11. Comparison of viscosity for [Chol][Asc] and [Chol][Biot] with cholinium based amino acids ionic liquids.

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