Food Chemistry 124 (2011) 1069–1075

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Food Chemistry

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Characterisation of inclusion complex of trans-ferulic acid andhydroxypropyl-b-cyclodextrin

Jing Wang *, Yanping Cao **, Baoguo Sun, Chengtao WangCollege of Chemistry and Environment Engineering, Beijing Technology and Business University, 11 Fucheng Road, Beijing 100048, PR ChinaBeijing Higher Institution Engineering Research Center of Food Additives and Ingredients, Beijing 100048, PR China

a r t i c l e i n f o

Article history:Received 26 April 2010Received in revised form 4 June 2010Accepted 26 July 2010

Keywords:Ferulic acidHydroxypropyl-b-cyclodextrinInclusion complexStabilityWater solubility

0308-8146/$ - see front matter � 2010 Elsevier Ltd. Adoi:10.1016/j.foodchem.2010.07.080

* Corresponding author at: College of Chemistry anBeijing Technology and Business University, 11 FuchChina. Tel.: +86 10 68985378; fax: +86 10 68985456.** Corresponding author at: College of Chemistry an

Beijing Technology and Business University, 11 FuchChina.

E-mail addresses: jingw810@yahoo.com (J. Wang), c

a b s t r a c t

The inclusion complex of trans-ferulic acid (FA) with hydroxypropyl-b-cyclodextrin (HP-b-CD) was pre-pared by the freeze-drying method and its characterisation was investigated by different analytical tech-niques including UV–visible spectroscopy, Fourier transform infrared spectroscopy, differential scanningcalorimetry, X-ray diffractometry, and scanning electron microscopy. All these approaches indicated thatFA was able to form an inclusion complex with HP-b-CD, and the FA/HP-b-CD inclusion compoundsexhibited different spectroscopic features and properties from FA. The stoichiometry of the complexwas 1:1. The calculated apparent stability constant of the FA/HP-b-CD complex was 166.3 M�1, andthe water solubility of FA was significantly improved by phase solubility studies. Moreover, the irradia-tion-induced decomposition of FA in aqueous solution was markedly reduced by complexation with HP-b-CD. The results showed that HP-b-CD was a proper excipient for increasing solubility and stability of FA.

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1. Introduction

Ferulic acid (4-hydroxy-3-methoxy-cinnamic acid, FA) is an ex-tremely abundant hydroxycinnamic acid of low toxicity in theplant kingdom, which is synthesised from the shikimate pathwayfrom phenylalanine or L-tyrosine and occurs mainly as a trans iso-mer. It plays important roles in plant cell walls by cross-linkingwith polysaccharides, including protection against pathogen inva-sion, and control of extensibility of cell walls and cell growth. FA isone of the effective components of Chinese medicinal herbs such asAngelica sinensis, Cimicifuga heracleifolia and Lignstcum chuangxiong,which has been reported to have physiological functions, includingantioxidant, anti-carcinogenic, antimicrobial, anti-thrombosis,anti-inflammatory, and anti-cancer properties (Ou & Kwok,2004). It also protects against coronary disease, lowers cholesteroland increases sperm viability. Despite the applicable qualities andbiological activities, the therapeutic usefulness of ferulic acid arelimited because of its unfavourable physicochemical properties,especially very poor water solubility and low oxidative stability(Piber & Koehler, 2005; Strazišar, Andrenšek, & Šmidovnik, 2008).

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d Environment Engineering,eng Road, Beijing 100048, PR

d Environment Engineering,eng Road, Beijing 100048, PR

aoyp@th.btbu.edu.cn (Y. Cao).

It has been recently reported that FA can break down into inactiveproducts under physical and thermal stresses (Graf, 1992). Inter-estingly, these problems can be addressed by complexation withcyclodextrins (CDs) in aqueous solutions.

CDs are untoxic macrocyclic oligosaccharides, consisting of (a-1,4)-linked a-L-glucopyranose units, with a hydrophilic outer sur-face and hollow hydrophobic interior. CDs are widely used in thefood industry as food additives, for stabilisation of flavours, forelimination of undesired tastes or other undesired compoundssuch as cholesterol and to avoid microbiological contaminationsand browning reactions (Astray, Gonzalea-Barreiro, Mejuto, Riao-Otero, & Simal-Gándara, 2009). They have the ability to form inclu-sion complexes with a wide variety of organic compounds, whichenter partly or entirely into the relatively hydrophobic cavity ofCDs simultaneously expelling the few high-energy water mole-cules from inside (Karathanos, Mourtzinos, Yannakopoulou, &Andrikopoulos, 2007). The most common CDs used as formulationvehicles are a-, b- and c-CDs containing six, seven and eight gluco-pyranose units, respectively. There are few studies that have beenperformed on the inclusion complex of FA with CDs. Anselmi et al.(2008) prepared the inclusion complex of FA with a-CD bycoprecipitation from an aqueous solution, which improves itslight-stability and slow-down the delivery in cosmetic sunscreenpreparations. Górnas, Neunert, Baczynski, and Polewski (2009)reported that no inclusion complex was detected for b-CD withFA in aqueous solution by fluorescence spectroscopy. Casolaro,Anselmi, and Picciocchi (2005) investigated the physicochemicalproperties of FA and its inclusion compounds with c-CD in aqueous

1070 J. Wang et al. / Food Chemistry 124 (2011) 1069–1075

solution by potentiometry and solution calorimetry. FA can beefficiently complexed with c-CD in a relatively high proportionforming an inclusion complex (Anselmi et al., 2006).

a-, b- and c-CDs are all used successfully to incorporate drugsinto aqueous vehicles and their toxicity profile has been studiedextensively. The toxicity profile of CDs can differ depending onthe route of administration. In addition, the application of CDs inthe pharmaceutical field is limited by its rather low aqueous solu-bility. Thus numerous chemical modified CDs have been developedto counter the solubility limits and safety concerns of the parentCD. 2-Hydroxylpropyl-b-cyclodextrin (HP-b-CD), a hydroxyalkylderivative, is an alternative to a-, b- and c-CDs, with improvedwater solubility and may be more toxicologically benign (Gould& Scott, 2005). The natamycin/HP-b-CD complex has allowed ahomogeneous delivery system of natamycin to the shreddedcheese surface without the clogging of spray nozzles during cheeseproduction (Koontz & Marcy, 2003). Zhang, Li, Jia, Chao, and Zhang(2009) reported that HP-b-CD could form stable inclusion com-plexes with FA and therefore could be used for the encapsula-tion/release of this potent antioxidant. However, to the best ofour knowledge, there are, so far, few reports on the FA/HP-b-CDcomplex. The object of this paper was to prepare the inclusioncomplex of FA/HP-b-CD, and investigate its characterisation by dif-ferent analytical techniques including UV–visible spectroscopy,Fourier transform infrared spectroscopy (FT-IR), differential scan-ning calorimetry (DSC), X-ray diffractometry (XRD), and scanningelectron microscopy (SEM).

2. Materials and methods

2.1. Materials

Ferulic acid (trans-4-hydroxy-3-methoxycinnamic acid, FA)was purchased from Aldrich. Hydroxypropyl-b-cyclodextrin (HP-b-CD) was obtained from Zibo Qianhui Fine Chemical Co. Ltd., Shan-dong, China. All other chemicals and solvents were of analyticalgrade.

2.2. Preparation of FA and HP-b-CD complex

The complex was prepared by mixing (at 1:1 M ratio) FA andHP-b-CD according to the freeze-drying method described by Pral-had and Rajendrakumar (2004). Briefly, a mixture of HP-b-CD(0.003 mol) and FA (0.003 mol) was diluted in 50 ml of water.The mixture was stirred for 5 h in the dark at room temperature,and left in the dark for 12 h, and then filtered to remove the excessdrug. The resulting solution was frozen at �80 �C and then subjectto lyophilisation in a freeze-drier (FD-1E-50, Beijing BoyikangInstrument Co. Ltd., Beijing, China) for 24 h to obtain a powder.The inclusion ratio of FA was calculated as follows:

Inclusion ratio ð%Þ¼ ½FA content of inclusion complexðmolÞ=0:003ðmolÞ� � 100

The total recovery was calculated according to the followingequation:

Total recovery ð%Þ ¼ Recovered powder=InitialðCDþ FAÞ � 100:

2.3. Preparation of FA and HP-b-CD physical mixture

FA and HP-b-CD were separately pulverised in ceramic mortars.The calculated amounts of both compounds were weighted out at amolar ratio of 1:1 and mixed together by a spatula until a homoge-neous mixture was obtained.

2.4. Physicochemical characterisation

2.4.1. UV–visible spectroscopyThe UV–visible absorption spectra were recorded for FA, HP-b-

CD, their physical mixture and the inclusion complex by using aUV–visible recording spectrophotometer (Rayleigh AnalyticalInstruments, Beijing, China). Each sample (50 mmol) was dissolvedwith water at the room temperature. The aqueous solutions werescanned, respectively, in the range from 200 to 400 nm to obtainthe UV–visible absorption spectra.

2.4.2. Fourier transform infrared spectroscopy (FT-IR)The FT-IR spectra of FA, HP-b-CD, their physical mixture and the

inclusion complex were collected between 4000 and 500 cm�1

(Mid infrared region) on a Nicolet Nexus Avater 370 FT-IR spectro-photometer (Nicolet, USA) with 256 scans at a resolution of 4 cm�1.Each sample was ground with spectroscopic grade potassium bro-mide (KBr) powder and then pressed into 1 mm pellets (2 mg ofsample per 200 mg dry KBr). A blank KBr disk was used as back-ground. FT-IR spectra were smoothed and the baseline was cor-rected automatically using the built-in software of thespectrophotometer (OMNIC 3.2).

2.4.3. Differential scanning calorimetry (DSC)DSC analysis was carried out for FA, HP-b-CD, their physical

mixture and the inclusion complex with a Mettler–ToledoDSC821 differential calorimeter calibrated with indium (Mettler–Toledo S.P.A., Milan, Italy). All samples were previously dried for24 h at 110 �C. Each dried powder (3–5 mg) was heated in acrimped aluminium pan at a scanning rate of 5 �C/min between50 and 230 �C temperature range under a nitrogen flow of 40 ml/min. An empty pan sealed in the same way was used as reference.Reproducibility was checked by running the sample in triplicate.

2.4.4. X-ray diffractometry (XRD)The X-ray powder diffraction patterns were obtained with a

XRD-6000 X-ray diffractometer (Shimadzu, Japan) using a Ni-fil-tered, Cu Ka radiation, a voltage of 40 kV and a 30 mA current.Analyses were performed on the same samples prepared for DSCstudies. All samples were measured in the 2h angle range between10� and 80� with a scan rate of 8�/min and a step size of 0.02�. Allsamples were analysed in triplicate.

2.4.5. Scanning electron microscopy (SEM)The surface morphology of samples was examined by a scan-

ning electronic microscopy (SEM) (TESCAN VEGA II Tescan SROCorp., Czech). Prior to examination, samples were prepared bymounting about 0.5 mg of powder onto a 5 mm � 5 mm siliconwafer affixed via graphite tape to an aluminium stub. The powderwas then sputter-coated for 40 s at beam current of 38–42 mAwith a 200 layer of gold/palladium alloy, and the samples wereexamined using SEM set at 15 kV.

2.5. Phase solubility of FA/HP-b-CD complex

Phase solubility studies were carried out according the methoddescribed by Higuchi and Connors’s methods (1965). Excessamounts of FA were added to 10 ml of water solution of HP-b-CDs at different concentrations ranging from 0 to 8.5 mM. Themixtures were magnetically stirred for 1 h in the dark at roomtemperature and left in the dark for 24 h. After equilibrium wasreached, a small volume of the supernatant was withdrawn and fil-tered through a 0.45 lm hydrophilic membrane filter. All sampleswere prepared in triplicate. The concentration of FA in the filtratewas determined at 313 nm by HPLC. The phase solubility profileswere obtained by plotting the solubility of FA vs. the concentration

Fig. 1. UV–visible absorption spectra of FA (a), FA and HP-b-CD physical mixture (b)and FA/HP-b-CD complex (c).

J. Wang et al. / Food Chemistry 124 (2011) 1069–1075 1071

of HP-b-CDs. The apparent stability constant, Kc, of FA and HP-b-CDcomplex can be calculated from the slope and the intercept of thelinear segment of the phase solubility line, according to the follow-ing equation:

Kc ¼ k=S0ð1� kÞ

where S0 is the intrinsic solubility of FA in deionised water in theabsence of HP-b-CD and k is the slope of the straight line.

2.6. The stability of FA/HP-b-CD complex

Aqueous solutions of free FA and the FA/HP-b-CD complex wereirradiated by placing them under a UVB lamp with emission wave-length in the range of 290–320 nm and a power of 4.5 W. The solu-tions were positioned 10 cm away from a light source in anincubator with temperature set at room temperature. The solu-tions were sampled at the specified time intervals (0, 0.5, 1, 2, 4,6, 8, 10, 12 h) and the concentration of FA was analysed by HPLC.The results were expressed as percentages of the remaining FA,i.e., the ratio Ct=C0 � 100, where C0 is the initial concentration ofFA alone or in the FA/HP-b-CD complex, Ct the concentration attime t and t the time of irradiation (in hours). The calculated deg-radation rate constant was determined by linear regression analy-sis of the natural logarithm of the percentages of remaining FAversus time. Each test was repeated at least three times.

2.7. HPLC of FA

The contents of FA in aqueous solutions were analysed by HPLCusing a C18 Symetry column (150 mm � 3.9 mm i.d., 5 lm particlesize, Waters, USA). The column was maintained at 30 �C. A samplevolume of 10 ll was injected into the HPLC column, and FA waseluted with methanol/water/acetic acid (50:50:0.5) at a flow rateof 0.8 ml/min in an isocratic programme for 15 min. The absor-bance of the eluate was monitored continuously at 313 nm.

2.8. Statistical analysis

The obtained data were expressed as the mean ± standard devi-ation of triplicate determinations. Data were analysed by an anal-ysis of variance (P < 0.05) and the means separated by Duncan’smultiple range test. Statistical analysis was performed using thesoftware STATISTICA 6.0.

3. Results and discussion

3.1. Preparation of FA/HP-b-CD complex

In recent years, HP-b-CD has gained appreciable acceptanceamong the various types of cyclodextrins. The inclusion complexesof HP-b-CD have been successfully used to improve solubility,chemical stability and bioavailability of a number of poorly solublecompounds. Various known methods used for the formation of theinclusion complexes like coprecipitation, neutralisation, kneading,spray drying, freeze-drying, solvent evaporation, and ball-millingand sealed-heating in the laboratory have been widely reported(Yamada et al., 2000). The freeze-drying method as the most effi-cient technique can protect against chemical decomposition, mini-mise loss of activity due to low processing temperatures, andreduction of the moisture content to very low levels. In this study,the freeze-drying method was selected to prepare the FA/HP-b-CDinclusion complex. The content of FA in the FA/HP-b-CD complexwas 11.05 ± 0.26%, and the inclusion ratio of FA was 60.16 ± 0.19%,and the total recovery was 65.28 ± 0.23%. Anselmi et al. (2008) re-ported that 15.1 ± 0.25% of FA was included in the FA/a-CD complex

prepared by the co-precipitation method. Zhao, Wang, Yang, andTao (2010) reported that the inclusion ratio of chlorogenic acid withb-CD was calculated to be 79.86 ± 1.92%. The driving forces betweenCDs and drugs which have been proposed to justify the complex for-mation are hydrogen bonds, van der Waals forces, hydrophobicinteractions and the release of ‘‘high-energy water” molecules fromthe cavity (Salústio, Feio, Figueirinhas, Pinto, & Marques, 2009). Thequantity of FA in the FA/HP-b-CD complex was lower than that ofthe FA/a-CD complex. Yuan, Jin, Xu, Zhuang, and Shen (2008)reported that the quantity of astaxanthin in the astaxanthin/HP-b-CD complex was lower than that of the astaxanthin/b-CD complex.Probably the hydroxylpropyl substituents concentrated at the edgeof the cavity of the CDs made it more difficult for the FA moleculesto enter.

3.2. Physicochemical characterisation of FA/HP-b-CD complex

3.2.1. UV–visible spectroscopy analysisUV–visible spectroscopy is an important tool to study the com-

plexation of FA with HP-b-CD. HP-b-CD had no UV absorption. Theobtained absorption spectra of FA, FA and HP-b-CD physical mix-ture and FA/HP-b-CD complex are presented in Fig. 1. In the spec-trum of FA in water solution (Fig. 1a), three kmax values were foundat 310 nm (p–p* transition of the double bond, hyperchromic),286 nm (p–p* transition of the phenolic group, hypochromic),217 nm (p–p* transition of the phenyl ring, hypochromic). Thespectrum of FA and HP-b-CD physical mixture was identical withthat of FA (Fig. 1b). However, as shown in Fig. 1c, the absorptionmaxima of FA/HP-b-CD complex bathochromically shifted to 313and 291 nm, respectively, compared to that of FA alone. It has beenreported that FA alone in water exhibited two absorption peaks at283 and 310 nm, and the absorption peak of FA/HP-b-CD complexbathochromically shifted to 290 and 315 nm Zhang et al. (2009).Zhao et al. (2010) reported that a blue shift of ca. 2 nm for chloro-genic acid was detected after inclusion with b-CD. However, Gór-nas et al. (2009) reported that a bathochromic shift of theabsorption peaks of chlorogenic and caffeic acids in the presenceof b-CD was observed. These results might suggest that the possi-bility of an interactions between FA and HP-b-CD existed as a re-sult of a partial shielding of the chromophore electrons in theHP-b-CD cavity, indicating that FA is capable of forming inclusioncomplex with HP-b-CD. In addition, all three spectra feature a highpeak around at 217 nm, which prove insensitive to HP-b-CD.

1072 J. Wang et al. / Food Chemistry 124 (2011) 1069–1075

3.2.2. FT-IR analysisFT-IR is a useful technique used to confirm the formation of an

inclusion complex. The FT-IR spectra of FA, HP-b-CD, FA and HP-b-CD physical mixture, FA/HP-b-CD inclusion complex are presentedin Fig. 2. The FT-IR spectrum of FA (Fig. 2a) consisted of the prom-inent absorption bands of hydroxyl group (3436 cm�1), of an aro-matic conjugated carbonyl (1691 cm�1), and of an aromaticnucleus (1620, 1595, 1515, 1431, 1205 cm�1). The occurrence ofthe peak at 2836 cm�1 in the spectrum is attributed to the methylC–H stretching vibration. The peak at 1272 cm�1 is attributed tothe C–O–C asymmetric stretching vibration. Absorption at1464 cm�1 is indicative of C–H deformations and aromatic ringvibrations. The band at 1172 cm�1 is characteristic of the carbonyl.In addition, the sharp bands at 804 and 850 cm�1 are due to thetwo adjacent hydrogen atoms on the phenyl ring of FA (Mathew& Abraham, 2007). The FT-IR spectrum of HP-b-CD (Fig. 2b)showed prominent absorption bands at 3418 cm�1 (for O–Hstretching vibrations), 2930 cm�1 (for C–H stretching vibrations)and 1154, 1085 and 1036 cm�1 (C–H, C–O stretching vibration).The FT-IR spectrum of the physical mixture (Fig. 2c) did not differsignificantly from those of the single components. However, theFT-IR spectrum of the FA/HP-b-CD inclusion complex shows no fea-tures similar to pure FA (Fig. 2d). The bands located at 1620, 1595,1515, 1464, 1272 and 850 cm�1 had been shifted and diminished,whereas the bands at 2836, 1431 and 804 cm�1 totally disap-peared. The broad peak around 3415 cm�1 in the spectrum of FA/HP-b-CD complex was increased in intensity owing to the increasein the number of hydroxyl groups. This can be probably due toinclusion complexation of FA into the HP-b-CD cavity. Also, accord-ing to theses changes we might suggest that the phenyl ring of FAwas involved in the inclusion process. It has been reported thatNMR analysis and molecular modelling demonstrated that the aro-matic ring and the ethylene side chain of FA embedded inside the

Fig. 2. FT-IR spectra of FA (a), HP-b-CD (b), FA and HP-b-CD physical mixture (c)and FA/HP-b-CD complex (d).

cavity of c-CD, leaving the more polar groups exposed outside thecavity (Anselmi et al., 2006).

3.2.3. DSC analysisThe thermal curves of FA, HP-b-CD, FA and HP-b-CD physical

mixture, and FA/HP-b-CD are shown in Fig. 3. FA displayed onesharp thermogram endothermic peak at 178 �C, corresponding tothe melting point of the crystalline form of the drug followed byan exothermic effect due to decomposition phenomena at highertemperatures. In case of HP-b-CD owing to its amorphous nature,a broad endothermic peak was observed at about 96 �C (Fig. 3b).A similar behaviour was observed for FA in the physical mixtureswith HP-b-CD (Fig. 3c). In fact, the thermal profile showed besidesCD unchanged broad bands at 96 �C due to the HP-b-CD dehydra-tion and decomposition, a well distinct melting peak, which ap-peared substantially unaffected in its shape and meltingtemperature. A different pattern was observed in the thermogramof the FA/HP-b-CD complex (Fig. 3d). The disappearance of themelting peak of FA at 178 �C and a new small peak at 163 �C to-gether with the shifting and broadening of one characteristic band(from 96 �C to 87 �C) is indicative of a change in the substancestructure and of a tight interaction between FA and HP-b-CD. Thesefindings show that no association takes place when the two pow-ders are simply mixed together and that conversely freeze-dryinggives rise to an association compound.

3.2.4. XRD analysisPowder X-ray diffractometry is a useful method for the detec-

tion of CD complexation in powder or microcrystalline states.The diffraction pattern of the complex is supposed to be clearly dis-tinct from that of the superposition of each of the components if atrue inclusion complex is formed (Veiga, Teixeira-Dias, Ked-zierewicz, Sousa, & Maincent, 1996). As shown in Fig. 4, the XRDpattern of FA showed intense, sharp peaks that prove the crystal-line nature of the compound (Fig. 4a). FA had strong crystallinitypeaks at 2h of 9�, 10.4�, 12.7�, 15.6�, 17.3�, 26.3�, 29.4�, and severalminor peaks at 21�, 24.5�, 31.5�, 34.6�, 36� and 39�. On the otherhand, the XRD pattern of HP-b-CD revealed a broad peaks in therange of 15�–25� (2h), confirming its amorphous character. In thecase of FA and HP-b-CD physical mixture with a molar ratio of1:1, the diffraction pattern (Fig. 4c) was simply the superpositionof the two patterns of the crystalline FA and the amorphous HP-b-CD. The sharp peaks of the pattern indicated the retention ofthe crystalline structure of FA in the physical mixture. However,the inclusion complex of FA with HP-b-CD gave a large, broad

Fig. 3. DSC thermograms of FA (a), HP-b-CD (b), FA and HP-b-CD physical mixture(c) and FA/HP-b-CD complex (d).

Fig. 4. Powder X-ray diffraction patterns of FA (a), HP-b-CD (b), FA and HP-b-CD physical mixture (c) and FA/HP-b-CD complex (d).

J. Wang et al. / Food Chemistry 124 (2011) 1069–1075 1073

background under the crystalline peaks, which was similar to thatof the amorphous HP-b-CD and did not exhibit the characteristicpeaks of FA, indicating the formation of a significant amount ofamorphous material (Fig. 4d).

3.2.5. SEM analysisThe surface morphology of the powders derived from FA, HP-b-

CD, FA and HP-b-CD physical mixture, and FA/HP-b-CD complexwas assessed by SEM. As illustrated in Fig. 5, FA existed in

Fig. 5. SEM micrographs of FA (a), HP-b-CD (b), FA and HP-b

needle-like crystal, whereas HP-b-CD was observed as amorphous,‘‘shrinked” cylindrical spheres. In the FA and HP-b-CD physicalmixture, the characteristic FA crystals, which were mixed withexcipient particles or adhered to their surface, were clearlyobserved. In contrast, the FA/HP-b-CD inclusion complex appearedin the form of irregular particles in which the original morphologyof both components disappeared and tiny aggregates of amor-phous pieces of irregular size were present. The comparison ofthese images revealed that the inclusion complex was structurally

-CD physical mixture (c) and FA/HP-b-CD complex (d).

1074 J. Wang et al. / Food Chemistry 124 (2011) 1069–1075

distinct from the isolated components, and the physical mixture ofFA and HP-b-CD. The sizes and shapes of FA and HP-b-CD particleswere different from those of the inclusion complex, which con-firmed the formation of the inclusion complex of FA and HP-b-CD.

3.3. Phase solubility of FA/HP-b-CD complex

The stoichiometry of the FA/HP-b-CD complex was determinedby the solubility technique. A linear relationship was obtained be-tween the amount of FA solubilised and the concentration of HP-b-CD in solution, which classified as a typical AL-type. The regressionequation was as follows:

Y ¼ 0:0231X þ 0:0151 R2 ¼ 0:9984 ð1Þ

where Y is the concentration (mM) of FA, X is the concentration(mM) of HP-b-CD. According to Higuchi and Connors’s theory (Hig-uchi & Connors, 1965), this may be attributed to the formation of a1:1 inclusion complex between FA and HP-b-CD. Karathanos et al.(2007) reported that the stoichiometry of the complex of vanillinwith b-CD was 1:1. The calculated apparent stability constant ofthe FA/HP-b-CD complex was 166.3 M�1, which indicated that theinteractions between FA and HP-b-CD are very strong. As comparedwith the solubility of FA in deionised water in the absence of HP-b-CD, there is a 15-fold increase in the presence of 8.4 mM HP-b-CD.Zhang et al. (2009) reported a sixfold increase in the solubility of FAwas observed with FA-HP-b-CD complex in the presence of1 � 10�2 M CD. Strazišar et al. (2008) reported that the apparentstability constants in aqueous solution of 0.39 � 103 M�1,2.81 � 103 M�1 and 49 � 103 M�1 for o-, m- and p-coumaric acidcomplexes with b-CD, respectively, were determined by phase sol-ubility tests.

3.4. Stability of free FA and FA/HP-b-CD

UV irradiation is one of the several factors that induce FA deg-radation. FA has been reported to undergo the trans–cis isomerisa-tion induced by UV light exposure (Hartley & Jones, 1975). Anselmiet al. (2008) reported that encapsulation with a-CD improves thechemical stability of FA against UVB stress (10 minimal erythemaldose) since no degradation products are formed after irradiation. Inthis study, the photo-stability of FA alone and FA/HP-b-CD inclu-sion complex was examined in aqueous solution. The decomposi-tion of free FA was found to be very marked upon exposure toUVB lamp. After irradiation of the aqueous solution of FA alonefor 0.5 h, the percentage of the remaining FA in the solution was94.7%. The degradation of FA increased with the increase of irradi-ation time. The percentage of remaining FA reached 51.2% after UVirradiation for 12 h. As shown in Fig. 6a, the plots show an expo-nential asymptotic trend which indicates that the degradation of

Fig. 6. Photodegradation profiles of FA under UVB irradiation of an aqueoussolution containing FA alone (a) and FA/HP-b-CD (b).

FA followed apparent first-order kinetics. The calculated degrada-tion rate constant for free FA was 0.0579 per hour under irradiationby UVB lamp. However, the degradation of FA was retarded whenFA was included in HP-b-CD as shown in Fig. 6b. The percentage ofremaining FA in FA/HP-b-CD complex solution was 87.4% after UVirradiation for 12 h. The calculated degradation rate constant for FAin the inclusion complex was 0.0114 per hour under irradiation byUVB lamp. Free FA underwent more than fivefold faster degrada-tion on exposure to UVB irradiation that FA in the FA/HP-b-CDinclusion complex, while the times for 10% drug degradation werereached after about 1 and 8 h of irradiation by UVB lamp for free FAand FA/HP-b-CD inclusion complex, respectively. This indicatedthat FA/HP-b-CD inclusion complex made FA insensitive to thetested UVB stress. Yuan et al. (2008) reported that the formationof the inclusion complex of astaxanthin with hydroxypropyl-b-cyclodextrin greatly enhanced the stability of astaxanthin againstlight and oxygen. The encapsulation in b–CD improves the thermalstability of nutraceutical antioxidants in Hypericum perforatum ex-tract such as epicatechin, catechin and euercetin (Kalogeropoulos,Yannakopoulou, Gioxari, Chiou, & Makris, 2010). The improvementin the photo-stability of FA by complexing with HP-b-CD is extre-mely important in the food field.

4. Conclusions

The results of this study clearly demonstrated that FA could beefficiently complexed with HP-b-CD form an inclusion complex byfreeze-drying method in a molar ratio of 1:1. The results of UV–vis-ible spectroscopy, FT-IR, DSC, XRD and SEM demonstrated that FA/HP-b-CD complex has different physicochemical characteristicsfrom free FA. The aqueous solubility and stability of FA were signif-icantly increased by inclusion in HP-b-CD. The results showed thatHP-b-CD complexation technology might be a promising strategyto improve the food application of FA.

Acknowledgement

This work was supported by a grant from the National HighTechnology Research and Development Program of China (863 Pro-gram) (No. 2007AA10Z306) and Funding Project for Academic Hu-man Resources Development in Institutions of Higher Learning, theJurisdiction of Beijing Municipality and General Project of BeijingMunicipal Education Commission (No. KM200910011002) and Bei-jing Nova Program (No. 2008B07).

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