4. On the methods to follow the interactions between cyclodextrins and phytochemicals and sugar-based surfactants

As it must have already been understood from the previous subsections, an accurate choice of the technique to follow the host-guest association is a key issue for a reliable thermodynamic and kinetic characterization of the association process. In general, the experimental techniques can be subdivided into two different categories, labeled as I and II [68]. Methods from group I (e.g., surface tension) are measuring changes in physical properties that are proportional, in some ways, to the extent of binding, while those from group II (e.g., <sup>1</sup> H NMR spectroscopy) rely on direct measurements of the free and bound ligand in a solution containing a known amount of the CD and guest molecule. Comments on such a division can be found in a couple of reviews (see, e.g., [69]), and it is outside of the scope of this chapter. The same is valid for computational techniques as relevant tools to infer on the structure of the supramolecular compounds [70–72].

In this section, the most relevant techniques used to study the interactions between CDs and sugar-based surfactants (e.g., APGs) and phytochemicals (e.g., polyphenols, EOs, and their components) will be highlighted (Table 2).

value is obtained. This clearly suggests a 1:1 APG/CD complexation [4], and actually it finds support by the Job's plot [61] reported by Bernat et al. [60]. Another important issue is to understand the reliability of the binding constants reported in Table 1. Rymdén et al. found that an increase of a methylene group for a series of alcohols decreases the standard free energy of the alcohol: β-CD binding for ca. 3.0 kJ/mol [62]. This variation is similar to that observed for K1,1 values obtained by self-diffusion coefficients (Table 1). On the other hand, comparing K1,1 values for C8G1 with those from other monoalkyl surfactants, one can conclude that the APG/CD complex is more stable [63, 64]. This has been justified by the occurrence of hydrogen bonds between the sugar structure and the hydroxyl groups located at the rim of the CD. However, studying the effect of the number of sugar moieties in the surfactant head on the free energy of binding, an algebraic increase in the Gibbs free energy is observed. Indeed, comparing the binding constants for the interactions between β-C12G1 and β-C12G2 with β-CD, it is possible to conclude that the addition of an extra sugar moiety in the surfactant head decreases the K values for the supramolecular association. Thus, it can be hypothesized that no significant sugar-sugar interactions are involved in the interaction with CD, as it was previously discussed. Another hypothesis arises from the effect of carbohydrates on the water structure, for example, Ribeiro et al. have found that the presence of carbohydrates leads to an increase of the entropy in water [65], also called a structure "breaking effect" [66]. Consequently, an increase of the concentration of the sugar molecules in solution may contributes for a decrease in the binding entropy change

Up to now, we have been discussing the binding process assuming a 1:1 APG/CD (α- and β-) binding stoichiometry. However, it should be stressed that from the study of the interactions between C12G1, and C18G1, and CDs, there are strong evidences for the occurrence of other species consistent with 1:2 complexes [56]. More recently, Haller and Kaatze, studying the interaction between C8G1 and α-CD by ultrasonic attenuation spectroscopy, concluded that besides 1:1 (APG/CD) complexes, the formation of 1:2 and 2:1 complexes (although in very low

4. On the methods to follow the interactions between cyclodextrins and

As it must have already been understood from the previous subsections, an accurate choice of the technique to follow the host-guest association is a key issue for a reliable thermodynamic and kinetic characterization of the association process. In general, the experimental techniques can be subdivided into two different categories, labeled as I and II [68]. Methods from group I (e.g., surface tension) are measuring changes in physical properties that are proportional, in

rely on direct measurements of the free and bound ligand in a solution containing a known amount of the CD and guest molecule. Comments on such a division can be found in a couple of reviews (see, e.g., [69]), and it is outside of the scope of this chapter. The same is valid for computational techniques as relevant tools to infer on the structure of the supramolecular

H NMR spectroscopy)

and, consequently, to an increase in the binding Gibbs free energy.

concentration) should not be ruled out [67].

76 Cyclodextrin - A Versatile Ingredient

compounds [70–72].

phytochemicals and sugar-based surfactants

some ways, to the extent of binding, while those from group II (e.g., <sup>1</sup>

NMR spectrometry falls in the group II techniques, and it is used to determine association constants through the chemical shift changes noticed either by the guest or by the CD [73–75]. Focusing on EOs, and as previously discussed, they may present undesired features, such as volatility, poor aqueous solubility, and stability. Therefore, host-guest supramolecular complexes are often obtained by using solid-state-based methods [14] such as freeze-drying [76, 77], coprecipitation [78], and the saturated approach [27], improving the solubility of the EO and thus allowing the use of NMR techniques for the quantitative and qualitative assessment of the complexation process [74]. For example, the complexation of eugenol with β-CD was obtained by using the saturation method, and the obtained complex, in the solid state, was characterized by either <sup>1</sup> H, 13C, or 2D NMR techniques, confirming the thread of CD's cavity by the aromatic ring of the eugenol [27]. Other techniques will be mentioned later since they fall on the so-called group II.

DOSY <sup>1</sup> H NMR has been used to study inclusion complexes between CD and different sugarbased substrates [79]. Kfoury et al. report a comprehensive study on the complexation between two phenol isomers (thymol and carvacrol) and CDs by using different NMR techniques, including DOSY [80]. The data allowed concluding that those isomers have a binding constant of 1344 M<sup>1</sup> and 1336 M<sup>1</sup> , respectively.

The self-diffusion measurements are, in principle, applicable to any systems as long as the free and complexed guests are soluble to an extent that allows for a good signal-to-noise ratio. It is important to note that on account of the rapid exchange on the NMR time scale, average diffusion coefficients for both the guest and for the CD are typically obtained. This method, as well as that involving chemical shift changes analysis, is also limited to systems where no overlapping of welldefined resonances is observed. The method relies on the fact that the self-diffusion coefficients of the uncomplexed guest are higher than the self-diffusion of the host-guest complex, as defined by the Stokes-Einstein equation. The change in the self-diffusion coefficient of the CD upon complexation is often small since the complex is often of the same size as the CD molecule, and thus the information from the CD self-diffusion is rather limited [58].

Ultrasonic relaxation technique falls into group II techniques and is based on the application of ultrasound to a given solution, with a frequency ranging from 20 kHz to several GHz, and subsequently measuring the molecular structural relaxation. The relaxation is sensitive to molecular volume changes [81], and thus, it may convey information on the stability constants of the host-guest complexes [82]. Furthermore, the use of a large frequency range allows to follow processes with relaxation times in the range from 20 ps to 20 μs [83], and thus the kinetics of the CD-surfactant association can be investigated. Haller and Kaatze, by using ultrasonic attenuation spectroscopy, were able to quantify the dynamics of unimer-micelle exchange of a sugarbased surfactant (i.e., octyl-β-D-glucopyranoside (C8G1)) in the presence of α-CD [67].

Also from group II, surface tension has also been used to follow the effect of CDs on the aggregation and interfacial properties of surfactants in CD-surfactant-containing solutions


[59, 60]. Surface tension is a measure of cohesive forces between liquid molecules present at the surface, and it represents the quantification of force per unit length of free energy per unit area [84]. In general, the presence of CDs will increase the surface tension of an APG solution. Knowing that natural CDs are not surface-active, they cannot replace APG at the air interface [85]. Therefore, CD molecules contribute for the depletion of APG unimers from the interface due to the great interaction between these unimers and CDs. There are several examples where surface tension measurements have been used to assess the stoichiometry and stability con-

Isothermal titration calorimetry (ITC) is a sensitive and powerful technique to study host-guest interactions by measuring the enthalpy and the free energy of binding [86, 87]. There are also some cases where the kinetic constants of the binding process can be obtained by ITC (see, e.g., [88]). For example, the heat produced by a stepwise addition of HP-β- and β-CD solution to a nootkatone allowed to characterize the complexation process with a binding enthalpy and

tively [71]. Unfortunately, the strict conditions required by this technique do not allow its

As has been pointed out before, some phytochemicals (EOs, in particular) are, in general, poorly soluble in aqueous solutions; therefore, the formation of complexes with CD in solid state is a strategy for further applications. Consequently, there are several available techniques used to evaluate the complexation. Thermal techniques, such as thermal degradation analysis and differential scanning calorimetry, are classical examples of methods used to assess complexation. Moreover, thermal degradation also allows evaluating the thermal stability of the

Other spectroscopic techniques, such as FTIR and XRD, which can be included in group II, have also been used, but the information is, in our opinion, rather qualitative [30, 72, 78, 91]. Another interesting approach to learn about the formation of host-guest complexes, in solid state, is to study the release kinetics of the EO. These studies, although do not allow to quantify the total amount of EO incorporated into the CDs, are of utmost importance to evaluate the presence of the EO in the complex as well as to provide hints on the release mechanism; the

For such poor soluble compounds, the complexation can also be evaluated by carrying out phase-solubility studies. These can be performed by using complexes in solid state or by checking the ability of increasing concentrations of CD to solubilize saturated solutions of EO. This allows assessing how much the solubility of the EO is improved upon complexation as well as the corresponding complex binding constant. The details on the quantitative determination of those parameters will be given in the next section. Different techniques can be applied to obtain the phase-solubility profiles. For instance, the solubility of black pepper EO in the presence of hydroxypropyl-beta-CD (HP-β-CD) was evaluated by UV-visible spectroscopy [76]. On the other hand, phase-solubility profiles for the encapsulation of polymethoxyflavones, obtained from mandarin EO, into HP-β-CD were obtained by using HPLC [93]. Kfoury et al. have used gas chromatography to study the ability of sulfobutylether-β- and sulfobutylether-γ-CD to encapsulate EOs components, such as limonene, estragole, and α- and

, and 14.38 kJmol<sup>1</sup>

, and 5801 M<sup>1</sup>

Interactions between Bio-Based Compounds and Cyclodextrins

http://dx.doi.org/10.5772/intechopen.73531

, respec-

79

stants of host-guest complexes [60, 86].

binding constant of 6.99 kJmol<sup>1</sup> and 4838 M<sup>1</sup>

latter is quite relevant for EOs used as fragrances [91, 92].

routine implementation on a large scale [89].

EO upon complexation [90, 78].

1 CDs: alpha-cyclodextrin (α-CD), beta-cyclodextrin (β-CD), gamma-cyclodextrin (γ-CD), 2 hydroxypropyl-β-cyclodextrin (HP-β-CD), randomly methylated-beta-cyclodextrins (RAMEB), low methylated beta-cyclodextrin (CRYSMEB), and sulfobutylether <sup>β</sup>-cyclodextrin (SBE-β-CD) <sup>2</sup>

PPs: trans-anethole, estragole, eugenol, isoeugenol (phenylpropenes), caffeic acid, p-coumaric acid, and ferulic acid (hydroxycinnamic acids)

3 UAS: ultrasonic attenuation spectroscopy

4 APGs: glucopyranosides (octyl G8, decyl G10, dodecyl G12, tetradecyl G14) and two maltosides (decyl M10, dodecyl M12)

5 EOs: essential oils of cedarwood, clove, eucalyptus, and peppermint

6 EOS: essential oils of Artemisia dracunculus, Citrus reticulata Blanco, Citrus aurantifolia, Melaleuca alternifolia, Melaleuca quinquenervia, and Rosmarinus officinalis cineoliferum

Table 2. Compilation of the most relevant techniques used to study the interactions between CDs and bio-based compounds.

[59, 60]. Surface tension is a measure of cohesive forces between liquid molecules present at the surface, and it represents the quantification of force per unit length of free energy per unit area [84]. In general, the presence of CDs will increase the surface tension of an APG solution. Knowing that natural CDs are not surface-active, they cannot replace APG at the air interface [85]. Therefore, CD molecules contribute for the depletion of APG unimers from the interface due to the great interaction between these unimers and CDs. There are several examples where surface tension measurements have been used to assess the stoichiometry and stability constants of host-guest complexes [60, 86].

Experimental methods System Obs. NMR Nerolidol + β-CD [74] UV-vis Nerolidol + CDs<sup>1</sup> [74] Phase solubility studies Cabreuva essential oil + HP-β-CD [74] Phase solubility studies β-caryophyllene + HP-β-CD [76] UV-vis Black pepper essential oil + HP-β-CD [76] Phase solubility studies CDs<sup>1</sup> + PPs2 [77] Phase solubility studies, NMR, TGA, DSC β-CD + estragole [78] NMR β-CD + eugenol [27] NMR β-CD + rosmarinic acid [75] NMR Cyclohexylacetic acid + β-CD [79] NMR Cholic acid + β-CD [79] UV-vis, NMR Thymol and carvacrol + CDs<sup>1</sup> [80]

NMR n-Octyl-β-D-glucoside and n-nonyl

1

3

4

5

6

compounds.

M10, dodecyl M12)

ferulic acid (hydroxycinnamic acids)

78 Cyclodextrin - A Versatile Ingredient

UAS: ultrasonic attenuation spectroscopy

UAS<sup>3</sup> Octyl-β-D-glucopyranoside + α-CD [67] Surface tension, NMR APGs<sup>4</sup> + β-CD [59] Surface tension Octyl-β-D-glucopyranoside + α-CD [60] Phase solubility studies, ITC, NMR Nootkatone + β-CD and HP-β-CD [71] TGA Cinnamon essential oil + β-CD [90] NMR, FTIR, release kinetics Monochlorotriazinyl β-CD + EOs<sup>5</sup> [91] XRD, NMR, TGA Thymol + γ-CD [30] DSC, TGA, FTIR, XRD, GC/MS, NMR Isopulegol + α-CD and β-CD [72] Phase solubility studies, NMR, HPLC Polymethoxyflavones + HP-β-CD [93] GC, total organic carbon, phase-solubility studies SBE-β-CD, SBE-γ-CD and HP-β-CD + EOS<sup>6</sup> [94, 95]

CDs: alpha-cyclodextrin (α-CD), beta-cyclodextrin (β-CD), gamma-cyclodextrin (γ-CD), 2 hydroxypropyl-β-cyclodextrin (HP-β-CD), randomly methylated-beta-cyclodextrins (RAMEB), low

PPs: trans-anethole, estragole, eugenol, isoeugenol (phenylpropenes), caffeic acid, p-coumaric acid, and

APGs: glucopyranosides (octyl G8, decyl G10, dodecyl G12, tetradecyl G14) and two maltosides (decyl

EOS: essential oils of Artemisia dracunculus, Citrus reticulata Blanco, Citrus aurantifolia, Melaleuca

Table 2. Compilation of the most relevant techniques used to study the interactions between CDs and bio-based

methylated beta-cyclodextrin (CRYSMEB), and sulfobutylether <sup>β</sup>-cyclodextrin (SBE-β-CD) <sup>2</sup>

EOs: essential oils of cedarwood, clove, eucalyptus, and peppermint

alternifolia, Melaleuca quinquenervia, and Rosmarinus officinalis cineoliferum


[58]

Isothermal titration calorimetry (ITC) is a sensitive and powerful technique to study host-guest interactions by measuring the enthalpy and the free energy of binding [86, 87]. There are also some cases where the kinetic constants of the binding process can be obtained by ITC (see, e.g., [88]). For example, the heat produced by a stepwise addition of HP-β- and β-CD solution to a nootkatone allowed to characterize the complexation process with a binding enthalpy and binding constant of 6.99 kJmol<sup>1</sup> and 4838 M<sup>1</sup> , and 14.38 kJmol<sup>1</sup> , and 5801 M<sup>1</sup> , respectively [71]. Unfortunately, the strict conditions required by this technique do not allow its routine implementation on a large scale [89].

As has been pointed out before, some phytochemicals (EOs, in particular) are, in general, poorly soluble in aqueous solutions; therefore, the formation of complexes with CD in solid state is a strategy for further applications. Consequently, there are several available techniques used to evaluate the complexation. Thermal techniques, such as thermal degradation analysis and differential scanning calorimetry, are classical examples of methods used to assess complexation. Moreover, thermal degradation also allows evaluating the thermal stability of the EO upon complexation [90, 78].

Other spectroscopic techniques, such as FTIR and XRD, which can be included in group II, have also been used, but the information is, in our opinion, rather qualitative [30, 72, 78, 91]. Another interesting approach to learn about the formation of host-guest complexes, in solid state, is to study the release kinetics of the EO. These studies, although do not allow to quantify the total amount of EO incorporated into the CDs, are of utmost importance to evaluate the presence of the EO in the complex as well as to provide hints on the release mechanism; the latter is quite relevant for EOs used as fragrances [91, 92].

For such poor soluble compounds, the complexation can also be evaluated by carrying out phase-solubility studies. These can be performed by using complexes in solid state or by checking the ability of increasing concentrations of CD to solubilize saturated solutions of EO. This allows assessing how much the solubility of the EO is improved upon complexation as well as the corresponding complex binding constant. The details on the quantitative determination of those parameters will be given in the next section. Different techniques can be applied to obtain the phase-solubility profiles. For instance, the solubility of black pepper EO in the presence of hydroxypropyl-beta-CD (HP-β-CD) was evaluated by UV-visible spectroscopy [76]. On the other hand, phase-solubility profiles for the encapsulation of polymethoxyflavones, obtained from mandarin EO, into HP-β-CD were obtained by using HPLC [93]. Kfoury et al. have used gas chromatography to study the ability of sulfobutylether-β- and sulfobutylether-γ-CD to encapsulate EOs components, such as limonene, estragole, and α- and β-pinene; their solubility in water was improved more than one order of magnitude [94]. It is worth noticing that, recently, a technique based on the total organic carbon determination has been reported and validated to follow the solubility improvement of EOs when increasing the concentration of CD [95].
