β-cyclodextrin inclusion compounds

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Spectrochimica Acta Part A 70 (2008) 154–161

Synthesis and spectral investigation of Al(III) catechin/␤-cyclodextrin and Al(III) quercetin/␤-cyclodextrin inclusion compounds Karina Dias a , Sofia Nikolaou b , Wagner F. De Giovani a,∗ a

Departamento de Qu´ımica, Faculdade de Filosofia Ciˆencias e Letras de Ribeir˜ao Preto, Universidade de S˜ao Paulo, Av. Bandeirantes 3900, 14040-901 Ribeir˜ao Preto, SP, Brazil b Departamento de F´ısica e Qu´ımica, Faculdade de Ciˆ encias Farmacˆeuticas de Ribeir˜ao Preto, Universidade de S˜ao Paulo, Av. do Caf´e s/n, 14040-903 Ribeir˜ao Preto, Brazil Received 11 January 2007; accepted 19 July 2007

Abstract Al-catechin/␤-cyclodextrin and Al-quercetin/␤-cyclodextrin (␤-CD) inclusion compounds were synthesized and characterized by IR, UV–vis, H and 13 C NMR and TG and DTA analyses. Because quercetin is sparingly soluble in water, the stability constants of the Al-quercetin/␤-CD and Al-catechin/␤-CD compounds were determined by phase solubility studies. The AL -type diagrams indicated the formation of 1:1 inclusion compounds and allowed calculation of the stability constants. The thermodynamic parameters were obtained from the dependence of the stability constants on temperature and results indicated that the formation of the inclusion compounds is an enthalpically driven process. The thermal decomposition of the solid Al-quercetin/␤-CD and Al-catechin/␤-CD inclusion compounds took place at different stages, compared with the respective precursors, proving that an inclusion complexation process really occurred. © 2007 Published by Elsevier B.V. 1

Keywords: Quercetin; Catechin; Flavonoid complexes; Antioxidants; ␤-Cyclodextrin; Inclusion compounds

1. Introduction Flavonoids are a group of naturally occurring polyphenolic compounds ubiquitous in fruits and vegetables. Their basic structure consists of two aromatic rings (designated as A and B) linked through three carbons that usually form an oxygenated heterocycle (C ring). (Fig. 1 shows the quercetin and catechin structures.) Many of their homeostatic and protective functions have been described [1–3], including their antioxidant properties [4,5], which arise mainly from their ability to scavenge reactive oxygen species and also from their chelating properties. Additionally, complexation may change the antioxidant properties and some biological effects of flavonoids. We have recently reported the synthesis and antioxidant properties of flavonoids complexes with Cu(II), Fe(II), Al(III) and Zn(II) [6,7]. We have shown that the complexed flavonoids were better DPPH (1,1-diphenyl-2-picrylhydrazyl) radical scavengers than the corresponding free flavonoids [8]. However, the ther∗

Corresponding author. Fax: +55 16 6024838. E-mail address: [email protected] (W.F. De Giovani).

1386-1425/$ – see front matter © 2007 Published by Elsevier B.V. doi:10.1016/j.saa.2007.07.022

apeutic application of this potential benefit is limited by the very poor solubility of these complexes in water. Their inclusion into cyclodextrins is a convenient alternative to solve the problems related to the administration of hydrophobic drugs [9–12], once conjugation of flavonoid complexes with cyclodextrins may increase their solubility in water if compared with non-encapsulated ones. The present study was designed to investigate the behavior of the inclusion compounds formed between the Al-quercetin ([Al2 (L1 )(H2 O)8 ]Cl4 ) or Al-catechin ([Al(L2 )(H2 O)4 ]Cl2 ) complexes and ␤-CD, both in the solid state and in aqueous solution, by means of IR, UV–vis, 1 H and 13 C NMR and TG and DTA techniques. 2. Materials and methods 2.1. Chemicals AlCl3 ·6H2 O, quercetin, catechin and ␤-cyclodextrin (␤-CD) were purchased from Aldrich Chemical Co. All solvents were analytical grade.

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2.2. Instruments The IR spectra were recorded on a Nicolet FTIR 5ZDX spectrophotometer using KBr pellets. The UV–vis spectra were registered on a Hewlett-Packard 8453 Diode Array UV–vis spectrophotometer using standard 1.0 cm quartz cells. The 1 H and 13 C NMR spectra were recorded from D O solutions, on a NMR 2 Bruker DR X 400 9.4 T spectrometer; the samples were prepared as described below for the phase solubility studies. A TA Instrument DSC q-10 was used to carry out the thermal analyses (TG and DTA); ␣-Al2 O3 was used as the reference substance; experiments were done under nitrogen gas atmosphere; the temperature was increased at a speed of 10 ◦ C min−1 , using a heating ramp from 30 ◦ C to 800 ◦ C. 2.3. Preparation of the metal complexes Both the preparation and characterization of Al(III)-quercetin and Al(III)-catechin complexes have been previously described [6,7]. 2.4. Preparation of the solid inclusion compounds Fig. 1. Structures of

(L1 )

quercetin (A) and

(L2 )

catechin (B).

The Al(III)-quercetin/␤-CD and Al(III)-catechin/␤-CD inclusion compounds were prepared by the co-precipitation method [9], using 1:1 molar ratios. 2.5. Phase solubility studies The phase solubility studies were carried out according to the method reported by Higuchi and Connors [13]. Aliquots of aqueous solutions (10 mL) containing different concentrations of ␤-CD (ranging from 2 × 10−3 mol L−1 to 1 × 10−2 mol L−1 ) were added to excess amounts of quercetin and catechin complexes, and the resulting mixtures were shaken at 300–320 K for 72 h. Aliquots withdrawn from such mixtures were filtered through a Gelman Science Acrodisc® LC PVDF 45 ␮m filter and they were then adequately diluted and analyzed spectrophotometrically, to determine the concentrations of the quercetin and catechin complexes. The experiments were carried out in triplicate. The stability constants, Ka , were calculated according to the Higuchi–Connors equation: Ka =

slope intercept (1 − slope)

and the parameters were obtained from phase solubility diagrams concentration of the compounds vs. ␤-CD concentrations; the intercepts correspond to the solubilities of the compounds in the absence of ␤-CD. The thermodynamic parameters, G, H and S were obtained from graphs of stability constants at different temperatures. 3. Results and discussion Fig. 2. Phase solubility study of Al-quercetin/␤-CD in water at 25 ◦ C (A) and 30 ◦ C (B).

The spectral and electrochemical properties of the catechin and quercetin complexes have been previously reported [8].

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Table 1 Stability constants (Ka ) at different temperatures and thermodynamic parameters for inclusion compounds of Al-quercetin and Al-catechin complexes Inclusion compounds

Ka (L mol−1 )

Al-quercetin/␤-CD 300 K 308 K 315 K 320 K

187.32 163.58 121.63 127.39

Al-catechin/␤-CD 300 K 308 K 315 K 320 K

210.99 196.75 188.14 169.45

G (kJ mol−1 )

H (kJ mol−1 )

S (J (K−1 mol−1 ))

−20.80

−39.61

−63.12

−24.71

−46.34

−72.59

3.1. Phase solubility studies Phase solubility diagrams obtained for the Al-quercetin/␤CD inclusion compound at various temperatures (Fig. 2) are linear for a wide range of antioxidant-␤-CD concentrations and they have profiles corresponding to the AL -type [13]. Taking into account that a slope lower than 1.0 is typical of such phase solubility diagram profile, we assume that the increase in solubility is due to the formation of a 1:1 inclusion compound. Stability constant (Ka ) values (Table 1) for the Alquercetin/␤-CD and Al-catechin/␤-CD inclusion compounds at 300–320 K were estimated from the slopes of the straight lines of the phase solubility diagrams using the Higuchi–Connors equation. The Ka values decrease as the temperature increases. Accordingly, the increase in kinetic energy probably leads to disassembly of the inclusion compound because the in out dynamic equilibrium of the guest from the host cavity is favored. Values of G, H and S related to the formation of the inclusion compounds were obtained from Van’t Hoff plot, as illustrated in Fig. 3 for Al-quercetin/␤-CD. These values are listed in Table 1. The negative values often concern the replacement of inner cavity solvent molecules, which acts as a driving force for the formation of inclusion compounds because of the release of enthalpic-rich water molecules from the ␤-CD cavity [14,15]. The water molecules located inside the cavity cannot

Fig. 3. Van’t Hoff plot for the formation of the inclusion compound between Al-quercetin and ␤-CD.

satisfy their hydrogen bonding potentials and therefore have higher enthalpy. The energy of the system is lowered when these enthalpic-rich water molecules are replaced by suitable guest molecules, which are less polar than water [16]. Therefore, with respect to thermodynamics, the data summarized in Table 1 is in agreement with the statements above, and they show that the formation of the inclusion compounds is an enthalpically driven process in the case of the quercetin and catechin complexes. 3.2. IR spectra Fig. 4 shows the IR spectrum of the Al-quercetin/␤-CD inclusion compound (1:1) compared with the physical mixture, the Al-quercetin complex alone and ␤-CD alone. • The IR spectrum of ␤-cyclodextrin (␤-CD) (Fig. 4A) displays the ␤-CD characteristic bands and is in agreement with literature data [17]. The intense band at 1652 cm−1 corresponds to the HOH bending of water molecules attached to ␤-CD; the 1419 cm−1 band corresponds to the CCH and OCH bending; the 1330 cm−1 band to the CCH, COH and HCH bending; the 1250 cm−1 band to the OCH, COH and CCH bending; the 1162 cm−1 band to the CO, CC stretching and to the COH bending; the 1028 cm−1 band to the CC stretching and to the CO and COH bending; the 947 cm−1 band corresponds to the skeletal vibration involving the ␣1, 4 linkage; the 857 cm−1 band to CCH bending, CO and CC stretching; the 574 cm−1 band corresponds to the skeletal vibration. • The Al-quercetin complex (Fig. 4B) has a characteristic band at 1634 cm−1 corresponding to the C O conjugated stretching. Specifically, for the free quercetin, this band appears at 1668 cm−1 ; for Al-quercetin complex it was shifted to the lower 1634 cm−1 frequency. This 34 cm−1 displacement suggests that the coordination of quercetin to Al(III) involves the oxygen atom of the carbonyl group. The 1598 cm−1 band corresponds to the stretching of the C C double-bond of the ring conjugated with the carbonyl group (C O); the 1518–1438 cm−1 bands are due to the C C stretching of the ring; the 1272 cm−1 band is attributed to the C–O stretching due to conjugation of the oxygen with the ring. The 1328 cm−1 band corresponds to the O–H in plane bending; the 637 cm−1 band is attributed to the M–O (M = Al(III)) stretching. The IR

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quercetin it appears at 1598 cm−1 . This shift may be due to many factors, including the effect of placing this aromatic ring into the constrained environment of ␤-CD. There may also be a hydrogen-bonding interaction of quercetin with ␤-CD hydroxyl groups. In the case of characteristic C O stretching band of the quercetin complex in the inclusion compound, it appears practically in the same frequency (1638 cm−1 ) and its intensity is very little reduced if compared with the Al-quercetin complex alone Table 2 Chemical shifts (ppm) for free ␤-CD, Al-catechin complex and Al-catechin/␤CD inclusion compound Position

δ (ppm); J (Hz); Integral values

␤-CD (free) H-1 H-2 H-3 H-4 H-5 H-6a,b

5.15;d, J1–2 3.5; 6H 3.73;dd, J1–2 3.3; J2–3 9.7; 6H 4.05; t, J3–4 9.4; 6H 3.66; t, J4–5 9.3; 6H 3.93; bs; 18Ha 3.96; bs; 3.99; br; 18Ha

Al-catechin/␤-CD inclusion compound H-1 4.94;d, J1–2 3.5; 6H H-2 3.52; dd, J1–2 3.5; J2–3 9.8; 6H H-3 3.78; t, J3–4 9.3; 6H H-4 3.47; t, J4–5 9.4; 6H H-5 3.63; d, J5–6 9.8; 6H H-6a,b 3.71; s; 12Ha Al-catechin complex H-2 H-4␣; H-4␤b

Fig. 4. IR spectra of ␤-CD (A), Al-quercetin (B), physical mixture of Alquercetin + ␤-CD (C) and Al-quercetin/␤-CD inclusion compound (D).

H-6 H-8 H-2 H-5 H-6 13 C C-2 C-3 C-4 C-5 C-7 C-9 C-10 C-2 C-3 C-4 C-5 C-6

3.95; dd, J 5; J 10; 2H H-4␤b , 2.25; d, J 7.8; H-4␤b , 2.3; d, J 8; H-4␣, 2.58; d, J 5.3; 2.62; d, J 5.3; 4H 5.83; s; 1H 5.75; s; 1H 6.71; m; 5H 6.64; s; 2H 6.58; d, J5 −6 2; 6.6; d, J5 −6 1.8; 4H 81.2 66.9 26.7 155.1 164 155.5 101 115 144.4 144.6 116 120

spectra of Al-quercetin and Al-catechin complexes have been previously reported [6,7]. • The bands observed for the physical mixture (Fig. 4C) are the well defined vibrational bands, and the spectrum of this mixture is practically the superposition of the main bands of the Al-quercetin complex with those of ␤-CD.

Al-catechin/␤-CD inclusion compound H-2 4.13; dd, J 6.8; J 12.3; 2H H-4␤b 2.46; d, J 7.3; H-4␤b 2.5; d, J 7.4; H-4␣; H-4␤b H-4␣ 2.72; d, J 4.5; H-4␣ 2.67; d, J 5; 1H H-6 5.99; s; 1H H-8 5.9; s; 1H H-2 6.81; m; 1H H-5 6.79; s; 5H 6.71; d, J5 −6 2.2; 6.74; d; J5 −6 2; 3H H-6

In the IR spectrum of the Al-quercetin/␤-CD inclusion compound (Fig. 4D), the stretching vibration of the quercetin aromatic ring appears at 1616 cm−1 , while for the “free”

s, singlet; d, doublet; t, triplet; dd, double doublet; m, multiplet; bs, broad singlet; br, broadened signal. a The superimposed signal of protons H5 and H6 integrate to 18H. b For the notation H-4␣ and H-4␤, see Ref. [18].

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(1634 cm−1 ). The C C aromatic stretching vibration appears in the same position as that observed for the non-included guest (1438 cm−1 ), which is an indication that the structural integrity of the guest molecule is maintained. Fig. 4D shows that there is also an overlapping effect in the region centered at 637 cm−1 . It probably corresponds to an overlap of the band characteristic of Al-quercetin M–O stretching with the intense band corresponding to the ␤-CD skeletal vibration, located in the same region. 3.3. 1 H and 13 C NMR spectra 1 H, 13 C

and 1 D NOE NMR experiments were carried out to obtain structural information. Table 2 summarizes the NMR results obtained for the Al-catechin complex and its inclusion compound. A tentative assignment was carried out by comparison with literature data. Fig. 5A shows the 1 H NMR spectrum of the Al-catechin complex dissolved in D2 O. A comparison between 1 H spectrum of free catechin [18] with that of the Al(III) complex allowed the assignment of the proton signals of the complex, although significant variations in the chemical shift values were not observed, on going from the free ligand to

Fig. 5. 1 H NMR spectra of Al-catechin complex: 1 H (A) and 1.0 × 10−3 mol L−1 in D2 O at 25 ◦ C.

13 C

(B);

the complexed form (Table 2). On the other hand, a significant variation in the C-7 signal was observed in the 13 C NMR spectra of free catechin and its Al(III) complex. This signal shifts from 155.5 ppm in free catechin [18] to 164.4 ppm in the Al(III) complex (Fig. 5B), suggesting that the coordination to the metal ion may occur via the 7-OH group, instead of the usual coordination mode, via the 3 -OH and 4 -OH positions [19]. Inclusion of the Al-catechin complex into the ␤-cyclodextrin cavity leads to shift in the δ values of the catechin ligand ranging from 0.10 ppm to 0.21 ppm, without significant changes in the spectral profile of catechin. However, when comparing Fig. 6A and B, the changes in the chemical shift values of the inner protons in the ␤-cyclodextrin cavity, 3-H and 5-H, are evident and strongly suggest that inclusion took place. In fact, 1 D NOE experiments show that upon selective irradiation of the ␤-CD 3-H and 5-H protons there is a drastic change in the 2 H, 5 H and 6 H signals, which indicates that the inclusion occurs via the B ring of the flavonoid (Fig. 7). It should be mentioned that many attempts were made to acquire NMR data for the quercetin species (inclusion compound quercetin/␤-CD, Al-quercetin complex and inclusion compound Al-quercetin/␤-CD) in aqueous media (neutral pH). Although NMR data have been reported for the quercetin/␤-CD system [20,21], in this work the NMR signals of the quercetin and Al-quercetin compounds were hardly detectable by 1 H

Fig. 6. 1 H NMR spectra in D2 O at 25 ◦ C: free ␤-CD (1 × 10−3 mol L−1 ) (A) and Al-catechin/␤-CD inclusion compound (1 × 10−3 mol L−1 ) (B).

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Fig. 7. 1 D NOE spectrum of the Al-catechin/␤-CD inclusion compound dissolved in D2 O at 25 ◦ C (1.0 × 10−3 mol L−1 ).

NMR spectra due to their very poor water-solubilities and the presence of excess ␤-CD (which is much more soluble), thus preventing an NMR structural characterization. 3.4. TG and DTA Fig. 8A shows the TG/DTA curves of free ␤-CD (for comparison purposes [22]). Fig. 8B and C show the curves obtained for the inclusion compounds quercetin/␤-CD and catechin/␤CD, while Fig. 9A and B shows the curves obtained for the compounds Al-quercetin/␤-CD and Al-catechin/␤-CD. Table 3 summarizes the thermal analytical data. The decomposition of ␤-CD is well documented, and the data obtained in this work are in good agreement with those reported in the literature [22]. Dehydration occurs in a single step, in the case of free ␤-CD, with loss of about eight moles of hydration water molecules (11%, 30–98 ◦ C). Its thermal decomposition also occurs in one stage, with a rapid mass loss of 79.7%. Dehydration process occurs in two steps for quercetin/␤-CD: in the first one there is loss of two moles of water molecule (9%); in the second there is loss of one mole (4%). The split dehydration process in the case of the inclusion compound shows that the water molecules occupy different positions in the structure. By comparison with data previously reported for free quercetin [23], it is possible to assign the first step to water loss from the host, and the second to water loss from the guest. The endothermic peaks observed in the DTA curve of this inclusion compound are consistent with the TG curve, although its thermal stability is slightly decreased in comparison with that of the host molecule (114–240 ◦ C). In the case of the catechin/␤-CD inclusion compound, there is no resolution in the TG curve in the range of temperatures where the dehydration occurs, so this process appears as a single step (30–110 ◦ C) with a mass loss of 6%. For both inclusion compounds, the thermal decomposition of the molecules occurs in two stages: the first one is related to the decomposition of the host fragment and the second one is due to the decomposition of the flavonoids (Fig. 8B, C and

Fig. 8. DTA and TG curves of free ␤-CD (A), quercetin/␤-CD (B) and catechin/␤-CD (C).

Table 3). Nevertheless, it is worthy to mention that the thermal decomposition of free quercetin begins around 140 ◦ C [23], so it probably contributes to the endothermic peak at 321 ◦ C assigned to the decomposition of ␤-CD. It seems that the encapsulation of the free flavonoid into the ␤-CD cavity causes little effect on the thermal behavior of both quercetin and catechin. On the other hand, coordination to the Al(III) metal at center leads to

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Table 3 Thermal data for ␤-CD, quercetin/␤-CD, catechin/␤-CD, Al-quercetin/␤-CD and Al-catechin/␤-CD Compounds

Dehydration stage (◦ C) First

␤-CD (weight loss, %) Quercetin/␤-CD (weight loss, %) Catechin/␤-CD (weight loss, %) Al-quercetin/␤-CD (weight loss, %) Al-catechin/␤-CD (weight loss, %)

30–98 (11) 30–75 (9) 30–110 (6) 30–118 (6) 30–114 (6)

DTA peak (◦ C)

Second 75–120 (4) 118–184 (33) 114–220 (6)

important changes in the case of the quercetin complex, and it has a less pronounced effect in the case of the catechin complex. In the case of the Al-quercetin/␤-CD inclusion compound (Fig. 9A), there is an initial mass loss of 6% due to the dehydration process. In the next step, there is a large mass loss (33%), which possibly involves loss of coordinated water molecules from the two Al(III) ions. This event may lead to the colapse of the structure, with a significant decrease in its thermal stability, e.g., from the mass loss percentage, it can be inferred that the decomposition of the inclusion compound begins around 120 ◦ C (Table 3) (endothermic peak at 152 ◦ C). The profile of

Decomposition stage (◦ C) First

91 75; 98; 320 110; 317 152 123; 304

247–360 (79.7) 240–330 (44) 240–338 (55) 220–328 (33)

Second 330–618 (19) 338–676 (18) 184–657 (29) 328–677 (22)

the TG curve suggests that the quercetin further decomposes (184–657 ◦ C) with a mass loss of 29%, and this process also begins in a lower temperature range. In the case of the Al-cathechin/␤-CD inclusion compound (Fig. 9B), the profile of the TG curve looks like a superposition of the processes observed for the isolated fragments, although the second water loss step occurs at higher temperatures (114–220 ◦ C). This is because this second process involves coordinated water molecules, which are more strongly bound to the inclusion compound than the hydration water molecules. 4. Conclusions Formation of the Al-quercetin/␤-CD and Al-catechin/␤-CD inclusion compounds was observed by means of phase solubility studies, which pointed to the 1:1 stoichiometry. The IR spectrum of the Al-quercetin complex indicates that the flavonoid coordination to the metal also involves the oxygen of the carbonyl group. The IR spectrum of the Al-quercetin/␤CD inclusion compound indicates that the aromatic ring of the flavonoid is located in the constrained environment of the host. The IR, TG and DTA data give evidence for the formation of the Al-quercetin/␤-CD and Al-catechin/␤-CD inclusion compounds in the solid state. The 13 C NMR of the Al-catechin complex also suggests that the Al(III) coordination also occurs via the 7-OH group. NMR, including 1 D NOE experiments, gives evidence for the formation of the Al-catechin/␤-CD inclusion compound and these techniques show that the inclusion occurs via the B ring of the flavonoid. Acknowledgments Acknowledgments are made to Fundac¸a˜ o de Amparo a Pesquisa do Estado de S˜ao Paulo (FAPESP) and Conselho Nacional de Desenvolvimento Cient´ıfico e Tecnol´ogico (IMINOFAR CNPq 420015/05-1) for financial support. K.D. thanks the PhD fellowship from Conselho Nacional de Desenvolvimento Cient´ıfico e Tecnol´ogico (CNPq). References

Fig. 9. DTA and TG curves of the inclusion compounds Al-quercetin/␤-CD (A) and Al-catechin/␤-CD (B).

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