Elucidation of the Kinetic Behavior of Quercetin, Isoquercitrin, and

Article pubs.acs.org/IECR

Elucidation of the Kinetic Behavior of Quercetin, Isoquercitrin, and Rutin Solubility by Physicochemical and Thermodynamic Investigations Latifa Chebil,*,† Mohamed Bouroukba,‡ Claire Gaiani,† Céline Charbonel,† Marwa Khaldi,† Jean-Marc Engasser,† and Mohamed Ghoul† †

ENSAIA - LIBio (Laboratoire d’Ingénierie des Biomolécules), 2 avenue de la Forêt de Haye, TSA 40602, 54518 VANDOEUVRE CEDEX, France ‡ Université de Lorraine, ENSIC - LRGP (Laboratoire Réaction et Génie des Procédés), 1 rue Grandville, B.P. 20451, 54001 Nancy, France ABSTRACT: The solubility of flavonoids in organic media is a complex and important process. It is a key factor in extraction, formulation, and biocatalysis processes of flavonoids. In this work, dissolution experiments showed that the solubility of quercetin, isoquercitrin, and rutin in tert-amyl alcohol is higher (20, 30, and 36 g/L) than in acetonitrile (1.7 g/L). Moreover, dissolution kinetic of quercetin in tert-amyl alcohol showed an unusual behavior of the amount dissolved over time. High concentrations of dissolved quercetin were measured at the beginning of the experiment. The maximum concentration was followed by a decrease later on in time until the saturation concentration was obtained. To explain these results, thermodynamic (enthalpy of dissolution and mixing) and physicochemical (microscopy, particle size measurement) experiments were carried out. The enthalpies of dissolution (ΔHdiss) and mixing (ΔHmix), measured in this work, are consistent with the experimental solubility scale for quercetin and rutin, i.e. rutin/tert-amyl alcohol < isoquercitrin/tert-amyl alcohol < quercetin/tert-amyl alcohol < quercetin/acetonitrile, with values ranging from ca. 12 to 180 J/g and −123 to 43 J/g, respectively. Microscopic observations and particle size measurement showed an increase of quercetin particles size, in tert-amyl alcohol, throughout the dissolution process (0.4 and 8 μm at 5 min, 14−60 μm after 68 min of dissolution). This surprising behavior, in contrast to the experience in common dissolution experiments (quercetin, isoquercitrin, and rutin in acetonitrile, isoquercitin and rutin in tert-amyl alcohol), has never been observed for flavonoids and is called kinetic size effect.

1. INTRODUCTION Flavonoids are the largest class of phenolic secondary metabolites in plants. They have a characteristic fifteen-carbon ring structure consisting of two aromatic rings containing phenolic hydroxyl groups.1 In plants, many of them are glycosylated with different sugars. Flavonoids are powerful antioxidants and have been reported to possess a wide range of biological effects: free radical scavenging, inhibition of cellular proliferation, antibiotic, antiallergic, antiulcer, or anti-inflammatory activities.2 For these properties, plant-extracted flavonoids are increasingly used as nutritional, cosmetic, and pharmaceutical active ingredients.3−6 However, the solubility of flavonoids in different media (water, organic solvent, hydrophobic media) is a complex and important process. It is a key factor in extraction, formulation, and biocatalysis processes of flavonoids. In biocatalysis and biotransformation in particular, organic solvents are one of the relevant factors that define the performances of the reaction.7,8 Only few data have been published on the solubility of flavonoids in organic solvents.7,9−11 In acetone and tert-amyl alcohol, the solubilities at 50 °C of quercetin, isoquercitrin, rutin, naringenin, chrysin, and hesperitin were found in the range of 20 to 40 g/L, much higher than their solubilities in water, which do not exceed a few g/L, or in acetonitrile (0.3−1.7 g/L).9 To explain the high solubility values of flavonoids in acetone and tert-amyl alcohol, we investigated, in a previous work, molecular dynamics simulations and free energy perturbation (FEP) methodology to predict © 2012 American Chemical Society

the relative free energies of solvation of quercetin in a variety of solvents. This study shows clearly that the enhanced solvation of quercetin by acetone and tert-amyl-alcohol stems from more pronounced hydrophobic and hydrophilic interactions, respectively, compared to acetonitrile. Moreover, the free energies of solvation (ΔGsolv) computed in this work are consistent with the experimental solubility scale for quercetin, i.e. water < acetonitrile < tert-amyl alcohol ≈ acetone.12 In these works, limited data are available on flavonoid dissolution kinetics, thermodynamic, and physical properties in organic media. The present work aimed to (I) investigate an experimental approach for quantifying the solubility, particle size, and enthalpies of dissolution and mixing of flavonoids in organic media, and (II) establish a relationship between solubility, particle size, and enthalpies.

2. MATERIALS AND METHODS 2.1. Materials. Quercetin hydrate (≥98%) and rutin hydrate (≥95%) (Figure 1) were purchased from Sigma. Isoquercitrin (≥95%) was purchased from Extrasynthese. 2-Methyl 2-butanol (≥99%), acetone (≥99.9%), and acetonitrile (≥99.9%) were Received: Revised: Accepted: Published: 1464

October 24, 2012 December 3, 2012 December 30, 2012 December 31, 2012 dx.doi.org/10.1021/ie3029202 | Ind. Eng. Chem. Res. 2013, 52, 1464−1470

Industrial & Engineering Chemistry Research

Article

Figure 1. Molecular structures of quercetin, isoquercitrin, and rutin.

purchased from Merck. Molecular sieves (4 Å) were from Grace Davison. Nylon syringe filters (13-mm diameter, 0.22-μm pore size) were from Whatman Co. Dissolution rate experiments were performed in a 27-mL jacketed reactor (Chemspeed ASW 1000). 2.2. Dissolution Experiments. Before each experiment, solvent (15 mL) was dried with 4-Å molecular sieves during 24 h at 50 °C and then transferred to the flavonoid (0.75 g). The solution was stirred at 50 °C and 500 rpm, using a Chemspeed vibration system, until equilibrium was reached. Samples (0.2 mL) were withdrawn with a syringe and filtered through 0.22-μm nylon filters (prewarmed at 50 °C). The filtrate (0.5 or 1 mL) was collected in a vial, and immediately analyzed by HPLC. Measurement of water content in all mixtures was performed using the Karl Fischer titration method. In all these measurements, the water content was about 0.15% (m/m). 2.3. Analysis. Dissolved flavonoids were analyzed by HPLC using LaChrom HPLC system (Merck) with UV detector (Merck) at 254 nm and with an Appolo C18 column (250 × 4.6 mm, Alltech). The eluent used was methanol. The elution flow rate was 1 mL/min. The volume of the injection sample was 10 μL. Elution was performed at 55 °C. 2.4. Particle Size. Particle size distributions during dissolution were performed with a laser light diffusion granulometer (Mastersizer S, Malvern Instruments Ltd., Malvern, UK) with a 5-mW He−Ne laser operating at a wavelength of 632.8 nm with a 300RF lens. The Malvern apparatus is equipped with an isothermal small volume sample cell module which is suited for powders in suspension (stirring was constant at 2000 rpm). For each measure, 0.6 g of the powder was dispersed in 150 mL of

solvent (to obtain a good obscuration and to avoid particle superposition). Particle size was measured during the dissolution process. The results obtained are average diameters calculated using the Mie theory. The criterion selected was d(50), which means that 50% of the particles have a diameter lower than this level. Results are the average of 3 replicate experiments carried out on different days for each powder sample. 2.5. Transmission Electron Microscopy (TEM) and Scanning Electron Microscopy (SEM). Samples were withdrawn at different times from dissolution experiments and examined with a Philips CM-200 transmission electron microscope. The same samples were examined using a Hitachi SEM S2500 instrument operating at 10 kV. 2.6. Dissolution Calorimetry. To quantify the heat of dissolution of powder into the different solvents used, measurements were carried out isothermally (50 °C) in a Calvet calorimeter (Setaram C80, Caluire, France). In short, the liquid (4 mL) and the powder (6 mg) were placed separately in a special cell (reversal mixing), which was then introduced in the calorimeter and thermally equilibrated for at least 4 h. In preliminary studies, several amounts of the solid samples were tested in order to evaluate the influence of the liquid/solid ratio on the enthalpy values measured. It was found that the above quantities did not affect the results; the solid was completely soluble in the amount of liquid utilized. The rotation of the apparatus was started after attaining equilibrium, bringing about contact of the solid and liquid. The heat released or absorbed was measured in relation to a reference cell containing the same amount of solvent. A calorimetric 1465

dx.doi.org/10.1021/ie3029202 | Ind. Eng. Chem. Res. 2013, 52, 1464−1470


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curve was obtained and the data was treated for baseline corrections, and the area under the curve was automatically integrated (SetSoft 2000 Software, Setaram) yielding the heat (J/g) adsorbed or released during the complete dissolution of the solid sample. All the measurements were repeated at least twice and average values are presented. Heat of mixing is equal to heat of dissolution minus heat of fusion. It determined at the limit of flavonoid solubility.

3. RESULTS AND DISCUSSION 3.1. Flavonoid Dissolution. In a first study9 we reported the solubility values of quercetin, isoquercitrin, and rutin in tert-amyl alcohol and in acetonitrile, at the equilibrium. For a more indepth analysis of these values, the experimental dissolution kinetics of the three flavonoids were carried out, at 50 °C, and are presented in Figure 2. It is clear that tert-amyl alcohol is the best solvent to dissolve quercetin (20 g/L), isoquercitrin (30 g/L), and rutin (36 g/L). However, an unusual behavior of the amount dissolved over time was observed for quercetin (Figure 2a). High concentrations of dissolved quercetin were measured at the beginning of the experiment. The maximum concentration was followed by a decrease later on in time until the saturation concentration was obtained. This surprising behavior, in contrast to the case in common dissolution experiments (Figure 2b and 2c), has never been observed for flavonoids but has already reported for several oxidic and barium sulfate nanoparticles.13−15 Through these studies, it was shown that this behavior, known as kinetic size effect, is a consequence of a strong increase of Gibbs free energy of the nanoparticles compared to the bulk material.14 It is a characteristic of the very small particles in the nanometric size range. A decrease of particle size and an increase of surface area available increase the kinetic size effect. The reason for the existence of kinetic size effect only in the case of quercetin dissolution will be explained in the following paragraphs (Particle Size Kinetics). 3.2. Particle Size Kinetics. Particle size kinetics was monitored, during the dissolution of flavonoids, by static light scattering and transmission electron microscopy. Results are shown in Figures 3 and 4. Considering the used solvent, tert-amyl alcohol or acetonitrile, the quercetin particle size kinetics were completely different (Figure 3). In the case of quercetin dissolution in tert-amyl alcohol (Figure 3a), two populations of particles were observed (0.4 and 8 μm) at 5 min. After 68 min of dissolution, only one population was measured (14−60 μm). TEM pictures show that the size of the primary particles became larger at 68 min compared to the size at 5 min. Moreover, Figure 5 shows clearly the aggregation phenomena. Smaller particles aggregated to form a sunflower pattern (Figure 5a). When particles aggregated, micropores were observed (Figure 5b). The existence of micropores in this sample could be due to the presence of a small fraction of fines (nanoparticles measured and observed in Figure 3a) in the quercetin powder. The quercetin particle size is an important factor in the dissolution process. It is well-known that the solubility of small particles increases with decreasing particle size (Kelvin effect). Particles with a size smaller than that of initial nanoparticles, dissolve completely and particles having a size larger that of initial nanoparticles start to grow. Ostwald ripening takes place. The concentration in the system decreases since some of the particles vanish and the remaining particles increase in size. An initial increase of the concentration of the particle-forming species is followed by a decrease of the concentration. This was the kinetic behavior observed experimentally (Figure 2a). Changes of particle size and

Figure 2. Dissolution kinetics of quercetin (a), isoquercitrin (b), and rutin (c) in tert-amyl alcohol (⧫) and acetonitrile (□) at 50 °C.

particle concentration occur simultaneously in a real system nanoparticle-solution. In the case of quercetin dissolution in acetonitrile (Figure 3 b), particle size decreased over time. Similar results were observed for rutin dissolution in tert-amyl alcohol and acetonitrile (Figures 3b, 4a and b). These results are in accordance with those reported the evolution of flavonoid concentration over time (Figure 2). 3.3. Thermodynamics. Dissolution calorimetry was carried out to quantify the thermodynamic effects occurring during the process and potentially provide information on the kinetics and the mechanism of dissolution. Quercetin, isoquercitrin, and rutin were characterized by their enthalpy of dissolution and mixing with solvent. An example of calorimetric curves is shown in 1466



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Figure 3. Quercetin dissolution kinetics in tert-amyl alcohol (a) and acetonitrile (b) observed by static light scattering and transmission electron microscopy.

Table 1. Enthalpy of Fusion (ΔHfus) of Quercetin, Isoquercitrin, and Rutin, Enthalpy of Dissolution (ΔHdiss), Enthalpy of Mixing (ΔHmix), and Solubility (S) of Quercetin, Isoquercitrin, and Rutin in tert-Amyl Alcohol and Acetonitrile ΔHfus (J/g)9 ΔHdiss (J/g) ΔHmix (J/g) S (g/L)9 a

quercetin

isoquercitrin

rutin

137 ± 2

107 ± 1.9

135 ± 0.11

tert-amyl alcohol

acetonitrile

tert-amyl alcohol

tert-amyl alcohol

acetonitrile

85 ± 1.3 −52 ± 0.7 20 ± 0.17

180 ± 4.5 43 ± 2.5 1.7 ± 0.23

45 ± 3 −62 ± 1.1 30 ± 0.9

12 ± 0.03 −123 ± 0.08 36 ± 0.24

n.d.a n.d. 0.3 ± 0.006

n.d.: Nondetermined due to the very low solubility of rutin in acetonitrile.

The enthalpies of dissolution (ΔHdiss) measured in this work are consistent with the experimental solubility scale for quercetin, isoquercitrin, and rutin, i.e. rutin/tert-amyl alcohol < isoquercitrin/tert-amyl alcohol, quercetin/tert-amyl alcohol < quercetin/acetonitrile, with values ranging from ca. 12 to 180 J/g. Similarly, the calculated enthalpies of mixing (ΔHmix), at the limit of flavonoid solubility, are in accordance with the solubility scale for quercetin, isoquercitrin, and rutin, i.e. rutin/tert-amyl alcohol < isoquercitrin/tert-amyl

Figure 6. The dissolution was found to be endothermic whatever the flavonoid and the solvent used. In general, endothermic values were measured for crystalline forms of samples. However, only the calorimetric cure of quercetin dissolution in tert-amyl alcohol showed a double peak. This behavior can be related to the decrease of solubility observed, over a shorter time, in Figure 2 which characterizes an aggregation of quercetin particles in tert-amyl alcohol. Table 1 summarizes the values obtained by integration of curves. 1467



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Figure 4. Rutin dissolution kinetics in tert-amyl alcohol (a) and acetonitrile (b) observed by static light scattering and transmission electron microscopy.

Figure 5. TEM (a) and SEM (b) pictures of quercetin particles in tert-amyl alcohol of the initial sample (dissolution time 5 min).

alcohol (−123, −62, and −52 J/g). This was attributed to the strong attractive interactions between rutin and tert-amyl alcohol molecules due to the formation of hydrogen bonding between the molecules. The lesser enthalpy of mixing of quercetin with tert-amyl alcohol can be the result of both repulsive and attractive interactions. These results are confirmed by the high measured solubilities of rutin, isoquercitrin, and quercetin in tert-amyl alcohol (20, 30, and 36 g/L) and the kinetic profiles reported in Figure 2.

alcohol < quercetin/tert-amyl alcohol < quercetin/acetonitrile, with values ranging from ca. −123 to 43 J/g. The enthalpy of mixing of quercetin with acetonitrile was show to have a positive value (43 J/g), thus indicating repulsive interactions between the flavonoid and the solvent and consequently a low solubility value (1.7 g/L). Rutin and tert-amyl alcohol had, however, a higher negative value for the enthalpy of mixing than quercetin and tert-amyl alcohol and isoquercitrin and tert-amyl 1468



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The calorimetric cure of quercetin dissolution in tert-amyl alcohol showed a double peak endothermic for dissolution and exothermic for aggregation. The enthalpies of dissolution (ΔHdiss) and mixing (ΔHmix), measured in this work, are consistent with the experimental solubility scale for quercetin, isoquercitrin, and rutin, i.e. rutin/ tert-amyl alcohol < isoquercitrin/tert-amyl alcohol < quercetin/ tert-amyl alcohol < quercetin/acetonitrile, with values ranging from ca. 12 to 180 J/g and −123 to 43 J/g, respectively. The results obtained are of importance when these solvents are used as a medium in biocatalysis reactions or as solvent in flavonoid extraction from plants.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Tel.: +33383586195. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We acknowledge the technical assistance of Dr. Jaafar Ghanbaja for help to accomplish scanning and electron microscopy at SCMEM Nancy, France.

Figure 6. Typical calorimetric curves for the dissolution of quercetin, rutin, and isoquercitrin in tert-amyl alcohol.

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REFERENCES

(1) Cao, G.; Sofic, E.; Prior, R. L. Antioxidant and Prooxidant Behavior of Flavonoids: Structure-Activity Relationships. Free Radical Biol. Med. 1997, 22, 749−760. (2) Heim, K. E.; Tagliaferro, A. R.; Bobilya, D. J. Flavonoid antioxidants: Chemistry, metabolism and structure-activity relationships. J. Nutr. Biochem. 2002, 13, 572−584. (3) Boumendjel, A.; Mariotte, A.-M.; Bresson-Rival, D.; Perrier, E. Hesperitin Esters: Highly Stable Flavanones With Both Free Radical Scavenging and Anti-Elastase Activities. Pharm. Biol. 2003, 41, 546−549. (4) Gorou, H. Cosmetic for fair-complexioned face containing fatty acid ester of quercetin. JP 58131911, 1983. (5) Haslam, E. Natural Polyphenols (Vegetable Tannins) as Drugs: Possible Modes of Action. J. Nat. Prod. 1996, 59, 205−215. (6) Havsteen, B. H. The biochemistry and medical significance of the flavonoids. Pharmacol. Therapeut. 2002, 96, 67−202. (7) Chebil, L.; Anthoni, J.; Humeau, C.; Gerardin, C.; Engasser, J. M.; Ghoul, M. Enzymatic acylation of flavonoids: Effect of the nature of substrate, origin of lipase and operating conditions on conversion yield and regioselectivity. J. Agric. Food Chem. 2007, 55, 9496−9502. (8) Chebil, L.; Humeau, C.; Falcimaigne, A.; Engasser, J. M.; Ghoul, M. Enzymatic acylation of flavonoids, review article. Process Biochem. 2006, 41, 2237−2251. (9) Chebil, L.; Humeau, C.; Anthony, J.; Dehez, F.; Engasser, J. M.; Ghoul, M. Solubility of flavonoids in organic solvents. J. Chem. Eng. Data 2007, 52, 1552−1556. (10) Xiao, M.; Shao, Y.; Yan, W.; Zhang, Z. Measurement and correlation of solubilities of apigenin and apigenin 7-O-rhamnosylglucoside in seven solvents at different temperatures. J. Chem. Thermodyn. 2011, 43, 240−243. (11) Xiao, M.; Yan, W.; Zhang, Z. Solubilities of Apigenin in Ethanol plus Water at Different Temperatures. J. Chem. Eng. Data 2010, 55, 3346−3348. (12) Chebil, L.; Chipot, C.; Archambault, F.; Humeau, C.; Engasser, J. M.; Ghoul, M.; Dehez, F. Solubilities Inferred from the Combination of Experiment and Simulation. Case Study of Quercetin in a Variety of Solvents. J. Phys. Chem. B 2010, 114, 12308−12313. (13) Roelofs, F.; Vogelsberger, W. Dissolution kinetics of nanodispersed gamma-alumina in aqueous solution at different pH: Unusual kinetic size effect and formation of a new phase. J. Colloid Interface Sci. 2006, 303, 450−459.

Figure 7. Correlation between solubility and enthalpy of dissolution (⧫) or enthalpy of mixing (□) of flavonoids (rutin, isoquercitrin, and quercetin) in organic solvents (acetonitrile and tert-amyl alcohol).

When reporting solubility values of flavonoids in acetonitrile and tert-amyl alcohol as a function of measured enthalpy of dissolution or enthalpy of mixing (Figure 7), a negative linear correlation was observed. A similar correlation was reported between the enthalpy of dissolution and the rate constant of sucrose spheres dissolved in different solutions.16

4. CONCLUSION With the aim of explaining the difference of flavonoid (quercetin, isoquercitrin, and rutin) solubility in organic solvents (acetonitrile and tert-amyl alcohol), we investigated a particle size and thermodynamic experiments. Dissolution kinetics of quercetin in tert-amyl alcohol showed an unusual behavior of the amount dissolved over time. High concentrations of dissolved quercetin were measured at the beginning of the experiment. The maximum concentration was followed by a decrease later on in time until the saturation concentration was obtained. This surprising behavior, in contrast to the experience in common dissolution experiments, has never been observed for flavonoids and is called kinetic size effect. In fact, particle size increase was observed (0.4 and 8 μm at 5 min, 14−60 μm after 68 min of dissolution). 1469



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(14) Vogelsberger, W.; Schmidt, J. Studies of the Solubility of BaSO(4) Nanoparticles in Water: Kinetic Size Effect, Solubility Product, and Influence of Microporosity. J. Phys. Chem. C 2011, 115, 1388−1397. (15) Vogelsberger, W.; Schmidt, J.; Roelofs, F. Dissolution kinetics of oxidic nanoparticles: The observation of an unusual behaviour. Colloids Surf., A 2008, 324, 51−57. (16) Marabi, A.; Mayor, G.; Burbidge, A.; Wallach, R.; Saguy, I. S. Assessing dissolution kinetics of powders by a single particle approach. Chem. Eng. J. 2008, 139, 118−127.

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Elucidation of the Kinetic Behavior of Quercetin, Isoquercitrin, and

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