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International Journal of Pharmaceutics 369 (2009) 5–11

Contents lists available at ScienceDirect

International Journal of Pharmaceutics

journa l homepage: www.e lsev ier .com/ locate / i jpharm

tudies on coumestrol/�-cyclodextrin association: Inclusion complexharacterization

amila Francoa, Liege Schwingela, Ivana Lulab, Rubén D. Sinisterrab,etícia Scherer Koestera, Valquiria Linck Bassania,∗

Programa de Pós-Graduacão em Ciências Farmacêuticas, Faculdade de Farmácia, Universidade Federal do Rio Grande do Sul. Av. Ipiranga, 2752,orto Alegre 90.610-000, RS, BrazilDepartamento de Química, Instituto de Ciências Exatas, Universidade Federal de Minas Gerais, Belo Horizonte, MG, Brazil

r t i c l e i n f o

rticle history:eceived 2 July 2008eceived in revised form 10 October 2008

a b s t r a c t

Coumestrol is an estrogenic and antioxidant agent, characterized by its low solubility in aqueous andlipophilic media, once in the aglicone form. In order to improve its solubility in water, coumestrol wasassociated with �-cyclodextrin in aqueous media followed by freeze-drying and characterized by SEM,

ccepted 13 October 2008vailable online 5 November 2008

eywords:oumestrolyclodextrin

1H NMR and molecular modeling. The analysis proved the existence of an inclusion complex, with higherprobability of inclusion of the coumestrol B-ring into the wider rim of the �-cyclodextrin molecule.

© 2008 Elsevier B.V. All rights reserved.

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. Introduction

Coumestrol belongs to the group of coumestans, included in theeguminosae family. It is structurally similar to isoflavones and con-titutes a fully oxidized version of pterocarpans (Al-Maharik andotting, 2004; Ganry, 2005). Its molecular structure is presented inig. 1.

This compound is found in clovers and alfalfa sprouts (Al-aharik and Botting, 2004; Ganry, 2005) and has been receiving

ttention due to its estrogenic and antioxidant properties. Coume-trol acts on ER� and ER�, estrogen receptors, having seven timesore affinity for ER� than for ER� (Benassayag et al., 2002; Garey

t al., 2001). These receptors are present in the epidermis (quer-tinocites, Langerhans cells and melanocites), blood vessels andermis (fibroblasts), among other places of the human body (Birtt al., 2001; Krazeisen et al., 2001; Pocock et al., 2002; Lapcik etl., 2003; Thornton et al., 2003; Sator et al., 2004). Coumestrols the most potent phytoestrogen and competes with zearalenol

nd genistein for the 17�-estradiol receptors binding, dependingn which receptor it acts (ER� or ER�) (Benassayag et al., 2002).

Besides its estrogenic activity, coumestrol acts as an antioxidantue to its capacity of donating electrons from the hydroxyl groups

∗ Corresponding author. Tel.: +55 51 33083602; fax: +55 51 33085437.E-mail address: valquiria@pq.cnpq.br (V.L. Bassani).

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378-5173/$ – see front matter © 2008 Elsevier B.V. All rights reserved.oi:10.1016/j.ijpharm.2008.10.026

resent in the A and B-rings; removing free radical and prevent-ng oxidative damages (Mitchell et al., 1998). Mitchell et al. (1998)eported that coumestans can inhibit peroxidation reactions six-een times more than �-tocoferol, a natural antioxidant present inhe membranes. More recently, Georgetti et al. (2003) and Lee et al.2006) demonstrated that the red clover extract (Trifolium pratense.), which contains coumestrol, presents a high antioxidant activity,nd that the pterocarpans from roots of Glycine max L. have potentow-density lipoprotein (LDL) oxidation inhibitory activity, show-ng that coumestrol is 20 times more antioxidant than genisteinnd daidzein.

Taken together, coumestrol seems to be a promising agent forkin aging prevention, especially for post-menopausal women.owever, its activities are conditioned to the aglicone form, whichresents reduced solubility in organic solvents and in hydrophilicehicles (Budavari, 2001; Silva et al., 2001; Havsteen, 2002). Thisact impairs the development of a topical pharmaceutical dosageorm. In order to improve the solubility of the aglicone form inater, and therefore to facilitate its delivery to the skin as well

s its incorporation into a hydrophilic vehicle, the association ofoumestrol with cyclodextrins seems to be a promising strategy.

n fact, the association of coumestrol with a cyclodextrin was forhe first time investigated by Cannavà et al. (2008), who performedhase-solubility studies and employed FTIR-ATR analysis to pointut the implication of particular functional groups of coumestrol inhe inclusion complexes with �-cyclodextrin and hydroxypropyl-

6 C. Franco et al. / International Journal of

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Fig. 1. Chemical structure of coumestrol.

-cyclodextrin prepared by the co-precipitation method. In theresent study, a solid coumestrol:�-cyclodextrin complex was pre-ared by freeze-drying method. The morphology of the complexas characterized by scanning electron microscopy (SEM) and the

patial configuration of this association was firstly proposed byeans of 1H Nuclear Magnetic Resonance (1H NMR) and molec-

lar mechanics calculation. �-Cyclodextrin was chosen due to itsdequate cavity size, which enables the formation of inclusion com-lexes with many substances, as well as to its accessible cost andelated advantages concerning the industrial feasibility of a topicalosage form containing coumestrol (Loftsson and Brewster, 1996;ingh et al., 2002; Del Valle, 2004; Yan et al., 2006).

. Materials and methods

.1. Materials

Coumestrol, code 27885 (95% of purity), was purchased fromigma–Aldrich (São Paulo, Brazil) and �-cyclodextrin was kindlyonated by Roquette et Frères (France). Dimethyl sulfoxide d6 wasurchased from Tedia (Rio de Janeiro, Brazil) and potassium bro-

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Fig. 2. SEM of (a) coumestrol, (b) �-cyclodextrin and (c) coumestrol:�-cyclodex

Pharmaceutics 369 (2009) 5–11

ide from Synth (Porto Alegre, Brazil). All other reagents andolvents used were of analytical grade.

.2. Preparation of coumestrol associations

The complex preparation followed the procedure reported byiguchi and Connors (1965). An excess amount of coumestrol

1.5 mg) was added to a vial containing 2.5 ml of either water or a-cyclodextrin solution (considering a 1:1 molar ratio). These dis-ersions were stirred in a water bath (IKA®-Werke EH4 Basic) at7 ◦C, during 48 h. After this period, the dispersions were cooledown to room temperature, filtered through a 0.45 �m pore diam-ter membrane to volumetric flasks of 5 ml, and the volume wasade up with water. An aliquot (1 ml) of this solution was trans-

erred to other 5 ml volumetric flasks and diluted with methanolor drug assay by ultraviolet spectrophotometry at 343 nm (Hewlettackard 8452A—Diode Array Spectrophotometer) over the concen-ration range of 1–5 �g/ml of coumestrol (R2 > 0.999), following

previously validated method based on ICH guidelines (2005).he solution (with theoretical 1:1 drug:cyclodextrin content),as freeze-dried (Edwards Modulyo 4K, −60 ◦C, under lightrotection) and stored for further analysis and drug contentvaluation.

.3. Characterization of coumestrol associations

The scanning electron microscopy (SEM) was performed to the

aw materials and to coumestrol: �-cyclodextrin freeze-dried prod-ct at three magnifications (2000×, 1000× and 500×), using the

eol 6060 apparatus, after samples had been gold sputtered. Thearameters used were: SEI mode, samples height less than 5 mm;oltage of 20 kV; WD of 11 mm, spotsize of 40 and LC of 60 �A.

trin complex (magnifications of 2000×, 1000× and 500×, respectively).

rnal of Pharmaceutics 369 (2009) 5–11 7

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Table 11H/NMR attributions to coumestrol and �-cyclodextrin.

Coumestrol atoms ı 1H (ppm) �-Cyclodextrin atoms ı 1H (ppm)

H5 7.84 H1 4.83H6 6.93 H2 3.28–3.30H8 6.90 H3 3.61–3.67HHH

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C. Franco et al. / International Jou

pecific adjustments of magnitude, focus, brightness, contrast andstigmatism were performed.

NMR spectra were recorded in a Bruker DRX400—AVANCE spec-rometer operating at 400 MHz to the hydrogen nucleus, equippedith direct detection 5 mm 1H/13C dual probe and inverse detec-

ion 5 mm probe with z-gradient coil, having dimethyl sulfoxideDMSO-d6) as solvent and tetramethilsilane (TMS) as internal stan-ard (ı 0.0). The 1H/NMR analyses were carried out at 27 ◦C300 K) for coumestrol, �-cyclodextrin and the (1:1) coumestrol/�-yclodextrin complex.

The molecular modeling was obtained with the Software Chem-io Office ChemBio 3D/ChemBio Draw Ultra, v. 9.0. Cambridge Soft2005), using the molecular mechanics method (MM2).

. Results and discussions

The mean aqueous solubility of coumestrol with and without-cyclodextrin was found to be 12.90 (RSD < 1.1%) and 3.23 �g/ml

RSD < 1.8%), respectively. In this way, coumestrol solubility in wateras enhanced up to ∼4 times at the presence of �-cyclodextrin

n a 1:1 molar ratio. After freeze-drying, the solid product (com-lex) was analyzed with respect to coumestrol content and thectual stoichiometry ratio of the complex, after all steps, was ∼1:1.3

oumestrol: �CD.

The SEM images presented in Fig. 2 showed that coumestrolarticles (Fig. 2a) present acicular form, irregular surface, a littlerinkled and no porous while �-cyclodextrin particles (Fig. 2b) hadarallelogram form, smooth surface, with some irregularities. The

Nptbu

Fig. 3. 1H/NMR analysis of (a) �-cyclodextrin, (b) coume

2′ 7.69 H4 3.32–3.383′ 6.95 H5 3.55–3.615′ 7.16 H6 3.61–3.67

omplex was found to be a slightly homogeneous crystal mass withmooth surface, little wrinkled and no porous. Particles of around0 �m prevail in this new arrangement (Fig. 2c), not being possibleo infer, by this technique, about higher interaction.

Modern NMR techniques based on gradient-pulsed field weresed in this study in order to make the assignment and the deter-ination of the coumestrol and its inclusion compound structures

Derome, 1987; Claridge, 1999). 1H NMR resonance assignments ofhe coumestrol molecule were carried out by 2D shift-correlatedMR techniques. The chemical shifts of hydrogens atoms are sum-arized in Table 1.The attributions were made by comparison with the studies of

oravcová and Kleinová (2001), Schneider et al. (1998) and (2D)1

MR analysis. H NMR spectra for coumestrol and its freeze-dried

roduct are presented in Fig. 3, (the �-cyclodextrin 1H NMR spec-ra is also presented only for comparison, Fig. 3a). The comparisonetween 1H NMR spectra for coumestrol and its freeze-dried prod-ct shows that the preparation of the inclusion compound did not

strol and (c) coumestrol:�-cyclodextrin complex.

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egrade the coumestrol molecule. Also, the comparison betweenH NMR spectrum of coumestrol (Fig. 3b), and 1H NMR spectrumf the coumestrol/�-cyclodextrin freeze-dried product in DMSO-6 solution (Fig. 3c) reveals some loss of resolution in the spectralines of the NMR spectra and the coalescence of the OH signalsf the coumestrol, due to the complexation effects with the hostolecule.In order to prove the host:guest interaction and to get informa-

ion about the cyclodextrin geometry complex, Nuclear Overhauserffect measurements (NOESY spectrum) of the coumestrol/�-yclodextrin freeze-dried product in DMSO-d6 solution werebtained (Fig. 4).

The NOEs observed between the hydrogens H5′ (ıH 7.16), H3′ (ıH.95), H6 (ıH 6.93) and H8 (ıH 6.90) of coumestrol molecule andhe hydrogens H-3 (ıH 3.89), H-5 (ıH 3.69–3.80) and OH2 (ıH 5.70),H3 (ıH 5.66) and OH6 (ıH 4.44) of �-cyclodextrin, as detected

n the 2D-NOESY experiments, could only arise if a coumestrol/�-yclodextrin complex has been formed (Fig. 4).

The data suggest that the coumestrol A-ring is situated in theorus cavity of the cyclodextrin due to the interaction between6 and H8 of coumestrol with H-3 of �-cyclodextrin, being thisydrogen (H3) situated inside the cyclodextrin cavity.

In addition, the cross peak correlation between the coume-trol hydrogens with the �-cyclodextrin OH hydrogens (OH2,H3 and OH6) might be explained by the formation of a large

upramolecular structure, with a high molecular weight, as theelf-assembly of the supramolecular complex besides of an inter-

ction between the coumestrol molecule and the outer face of-cyclodextrin in a projection of the A and B-rings of coume-trol onto the OH2/OH3/OH6 face of �-cyclodextrin (Sousa et al.,008).

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Fig. 4. NOESY contours map (1H/1H) of the coumestrol:�-cyclodextrin complex and

Pharmaceutics 369 (2009) 5–11

The NOESY spectrum also indicated the insertion of the coume-trol B-ring into the cyclodextrin cavity by the interaction between3′ from coumestrol molecule and H3 from the �-cyclodextrin.

The chemical shift of H3 of �-cyclodextrin was confirmed byhe molecular modeling. These chemicals shifts may indicate theormation of four types of complexation: (i) insertion of A-ringf coumestrol in the �-cyclodextrin cavity; (ii) insertion of B-ringf coumestrol in the �-cyclodextrin cavity; (iii) insertion of twoolecules of coumestrol in both sides of a �-cyclodextrin cavity or

iv) the insertion of one molecule of coumestrol in two moleculesf �-cyclodextrins, by both sides.

The simulations were performed by manual insertion of theoumestrol molecule in the vertical position into the cavity in aerpendicular way of its diameter. The dynamic calculations wereerformed at 300 K by molecular mechanics method (MM2). The

nclusion complex models and their total conformational energiesre presented in Figs. 5 and 6. The carbon atoms are represented byray spheres, the oxygen atoms by red spheres and hydrogen atomsy the white ones.

By these preliminary tests, it is possible to infer that allimulated inclusion complex models, which were energeticallyavorable, could be occurring in solution since this can be consid-red a dynamic process in which different parts of the moleculeould be alternatively included in the CD (Loftsson and Brewster,996; Piel et al., 2001; Del Valle, 2004).

The total energy values obtained were very close to each otherhen the complexation occurred with one molecule of coumestrol

nd one of �-cyclodextrin, no matter which ring was introduced orhich side of the cyclodextrin was tested. The low difference in the

nergy values is dependent on the coordinates and was consideredcceptable for comparison in other models cited in the literature,

expansion region of the aromatic resonance hydrogen (DMSO-d6, 400 MHz).

C. Franco et al. / International Journal of Pharmaceutics 369 (2009) 5–11 9

Fig. 5. Complexation models of the insertion of (a) A and (b) B rings of one molecule of coumestrol by the narrower and wider rims of one molecule of �-cyclodextrin, withits total energy value calculated by MM2.

Fig. 6. Complexation models of the insertion of (a) A and (b) B rings of two molecules of coumestrol in the wider and narrower rims of one molecule of �-cyclodextrin, withits total energy value calculated by MM2.

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.g. for rutin (difference of 2.89 kJ/mol) (Haiyun et al., 2003), 3-ydroxyflavone and fisetin (differences of 7.77 and 5.60 kcal/mol,espectively) (Banerjee and Sengupta, 2006) and robinetin (differ-nce of 2.3 kcal/mol) (Banerjee et al., 2007).

The coumestrol B-ring inserted by the wider rim of �-yclodextrin demonstrated the lower energy (105.810 kJ/mol) inhe 1:1 simulations. This can be confirmed by the NOESY spectrumhere it is possible to observe the interaction between H3′ of the

oumestrol molecule and the H3 from the cyclodextrin, which areositioned inside the cavity.

The complexation of two molecules of coumestrol with one of-cyclodextrin was energetically more favorable than the complex-tion of one molecule of each. However, the complex model thatbtained the smaller total energy (74.071 kJ/mol) was that obtainedy the insertion of the B-rings of two molecules of coumestrol inoth sides of the �-cyclodextrin molecule, being this the most prob-ble complexation pathway according to the molecular modelingFig. 6a).

A test was also performed with two molecules of �-cyclodextrinnd one molecule of coumestrol, and the four other inclusion mod-ls developed presented higher energies compared with the modelsf one molecule of each compound (data not shown).

The inclusion complexes of other flavonoids with �-cyclodextrinreviously discussed in the literature may help to understandoumestrol interactions with �-cyclodextrin.

Zheng et al. (2005) developed a molecular modeling study ofuercetin:�-cyclodextrin at a 1:1 molar ratio, showing the projec-ion of the B-ring onto the OH2/OH3 face of �-cyclodextrin and therojection of the A-ring from the OH6 face, such relation was con-rmed by NMR results for the coumestrol molecule suggesting theame way of interaction as observed in the quercetin complex. Inhe quercetin complex orientation, four quercetin hydroxyl groupsere outside the hydrophobic �-cyclodextrin cavity. The conforma-

ional arrangement of the quercetin:�-cyclodextrin complex withhe aromatic B-ring positioned inside the cavity and the formationf one hydrogen bound between OH7 of the quercetin A-ring withH6 of �-cyclodextrin molecule helps in the protection from theolecule degradation (Yan et al., 2006).The same orientation (B-ring positioned in the wider side of

he cyclodextrin) was reported to naringin/�-cyclodextrin and 3-O-ethylquercetin/�-cyclodextrin complexes by Fronza et al. (2002)

nd Schwingel et al. (2008), respectively.Bergonzi et al. (2007) confirmed that the inclusion of the B-ring

f flavonoids galangin, kaempferol and quercetin in the wider rimf �-cyclodextrin was the most probable way of interaction amonghe molecules. In addition, freeze-drying was considered the best

ethod to prepare inclusion complexes.Cannavà et al. (2008) suggested that a single coumestrol

olecule could be inserted more or less deeply in the hydropho-ic cavity of the �-cyclodextrin and that new hydrogen bonds areupposed to be formed, turning weaker the C–O–C and C–O bondsuring the inclusion phenomena.

Recently, Daruházi et al. (2008) evaluated the inclusion com-lex of genistein/�-cyclodextrin at a 1:2 molar ratio prepared byneading method. The 1H NMR was performed at 600 MHz/30 ◦Cith ROESY analysis and molecular modeling was made by the

M3 semi-empirical method. It is worth emphasizing that bothenistein and coumestrol molecules present very similar chemicaltructures. This research confirmed the inclusion of the genisteinolecule through the insertion of the B-ring in the wider rim of the

-cyclodextrin. This interaction was detected between the OH4 of

he genistein with H3 and, specially, with H5 of the cyclodextrin.The formation of an inclusion complex between the coume-

trol and �-cyclodextrin is postulated, as demonstrated by NOESYnd molecular modeling experiments. This inclusion may occur by

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Pharmaceutics 369 (2009) 5–11

eans of the insertion of coumestrol A or B-rings in the cyclodex-rin cavity, which will interact with the H3 of the �-cyclodextrin,s demonstrated by NOESY. According to our results, this inclusions likely to occur by means of the insertion of coumestrol B-ring inhe wider side of the cyclodextrin, considering the interaction withne molecule of each substance. However, it was also demonstratedhat a more energetically favorable interaction occurs by the inser-ion of two molecules of coumestrol (B-rings) into both sides of one

olecule of �-cyclodextrin.

. Conclusions

Coumestrol association with �-cyclodextrin improved its solu-ility in water up to 4 times at the 1:1 molar ratio. According tohe NMR data and molecular modeling, the formation of an inclu-ion complex is possible through the insertion of the A or B-rings ofoumestrol into the �-cyclodextrin cavity. However, the insertionf the B-ring of one molecule of coumestrol by the wider side of the-cyclodextrin is supposed to be the most probable complexationathway in a complex 1:1.

cknowledgements

The authors would like to thank the colleagues Cabral Pavei,reice Borghetti and Roberta Hansel de Moraes for their help

n the experimental part, Roquette et Frères for donation of �-yclodextrin, LAREMAR/UFMG that supported the NMR facilitiest 400 MHz, and for the grant and scholarship of CNPQ and CAPES.

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