Preparation and Characterization of Inclusion Complexes of Î²

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Ind. Eng. Chem. Res. 2009, 48, 5070–5078

Preparation and Characterization of Inclusion Complexes of β-Cyclodextrin-BITC and β-Cyclodextrin-PEITC Hai-Na Yuan,†,‡ Shan-Jing Yao,*,† Lian-Qing Shen,§ and Jian-Wei Mao‡ Department of Chemical and Biochemical Engineering, Zhejiang UniVersity, 38 Zheda Road, Hangzhou 310027, China, School of Biological and Chemical Engineering, Zhejiang UniVersity of Science and Technology, 318 Liuhe Road, Hangzhou 310023, China, and Zhejiang Gongshang UniVersity, 18 Xuezheng Street, Hangzhou 310018, China

Benzyl isothiocyanate (BITC) and phenylethyl isothiocyanate (PEITC) are two poorly water-soluble plant components that can form inclusion complexes with β-cyclodextrin (β-CD), namely, β-cyclodextrin-BITC and β-cyclodextrin-PEITC, that are two water-soluble complexes. The inclusion complexes were prepared by two independent processes: physical mixing and coprecipitation. The content of guest molecules in the complexes was measured by UV spectrophotometry. Response surface design (RSD) was applied to optimize the preparation conditions of said complexes. The results showed that the embedding ratios for β-CD-BITC and β-CD-PEITC were 94.9% and 94.1%, respectively. Variance analysis revealed that the mass ratio and the inclusion temperature were two important factors in terms of inclusion action. The optimum conditions for the inclusion of β-CD-BITC were a mass ratio of 0.17 and an inclusion temperature of 57.99 °C, and those for β-CD-PEITC were a mass ratio of 0.0057 and an inclusion temperature of 64.87 °C. The inclusion complexes prepared were qualified by thermal methods [thermogravimetry (TG) and differential scanning calorimetry (DSC)], Fourier transform infrared (FTIR) spectroscopy, and X-ray powder diffraction (XRD). The thermal analysis of β-CD and the two complexes indicated that an interaction between the guest and host molecules did occur. BITC and PEITC could be partially embedded in the hydrophobic cavity of β-CD, so the formation of the said complexes was established. The X-ray and FTIR results support this indication of inclusion behavior. Introduction Organic isothiocyanates (ITCs) are enzymatic hydrolysates of glucosinolates occurring in cruciferous vegetables. Watercress is an especially good source of phenylethyl isothiocyanate (PEITC, Figure 1b), the breakdown product of gluconasturtin, and garden cress is an especially good source of benzyl isothiocyanate (BITC, Figure 1a), a breakdown product of glucotropaeolin when the cellular or tissue integrity of the vegetables or plants is disrupted by mechanical influences.1 BITC and PEITC are two poorly water-soluble plant components that have been found to have effective cancer chemopreventive capabilities. It was reported that BITC and PEITC are effective inhibitors of lung tumorigenesis in A/J mice treated with the chemical carcinogens benzo[a]pyrene (BaP) and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), respectively. ITCs can inhibit the metabolic activation of carcinogens in the regulation of phase one and phase two metabolism enzymes.2-4 The cancer chemopreventive function of BITC and PEITC, either commercially available or naturally occurring in brassica vegetables, is linked to bioavailability of the isothiocyanate metabolite.5 Additionally, a direct effect of PEITC on cell viability, induction of apoptosis, and arrest of the cell cycle in various cancer cells including hepatoma, breast, colon, and prostate has been reported.6-8 A pharmaceutical preparation of BITC was used as an antibiotic for the treatment of urinary and respiratory tracts infections in human.9 The aromatic BITC showed biocidal activity to some extent against microbial and * To whom correspondence should be addressed. Fax: 0086-57187951015. E-mail: [email protected]. † Zhejiang University. ‡ Zhejiang University of Science and Technology. § Zhejiang Gongshang University.

anthelmintic such as Diaporthe phaseolorum, Phytium irregulare,Sclerotiniasclerotiorum,Rhizoctoniasolani,andnematodes.10,11 Resistance to oxidative damage was documented when cells were exposed to low micromolar concentrations of ITCs. BITC at 25 µM and PEITC at 3.5 µM were found to inhibit 12-Otetradecanoylphorbol-13-acetate (TPA)-induced superoxide generation in differentiated HL60 cells by 80% and 50%, respectively.12 However, BITC and PEITC, as one type of significantly functional chemical compound and as important flavor ingredients, have found limited applications because of their poor water solubilities, strong volatilization, and pungent odors. Moreover, owing to the electrophilic behavior of the isothiocyanic group, ITCs can react with some nucleophilic agents such

Figure 1. Chemical structures of (a) BITC, (b) PEITC, and (c) β-CD.

10.1021/ie8015329 CCC: $40.75  2009 American Chemical Society Published on Web 04/10/2009

Ind. Eng. Chem. Res., Vol. 48, No. 10, 2009

as water, resulting in a decrease of their stability. Ohta et al. reported related studies on improving the stability of ITCs and found that cyclodextrin (CD) could be a suitable carrier with ITCs. The inclusion action between ITCs and CD could inhibit the decomposition of ITCs in aqueous solution.13-15 β-Cyclodextrin (β-CD, Figure 1c) consists of seven end-onend, interconnected D-glucose residues. β-CD exhibits the shape of a truncated hollow cone with the characteristic molecular structure of a hydrophilic exterior and a hydrophobic central cavity. Based on molecular interactions, such as the van der Waals force, hydrophobic effect, and dipole-dipole interactions, inclusion complexes can be formed by incorporating various guest compounds into the cavity of β-CD. Such complexation structures would be able to improve the solubility and reduce the volatility of the guests.12,16 Having relatively low molecular weights, BITC and PEITC can be incorporated in β-CD. As an approach similar to the release of allyl isothiocyanate (AITC) from the complexes is available,17 the inclusion complexation of BITC and PEITC is of great benefit in masking their strong odors, meliorating their water solubility problems, and enhancing their functional stability. Therefore, the aims of this work were first to prepare the two complexes of β-CD-BITC and β-CD-PEITC; then to optimize the inclusion conditions; and finally to characterize the inclusion behavior by thermal quality [differential scanning calorimetry (DSC), thermogravimetry (TG)], Fourier transform infrared(FTIR)spectroscopy,andX-raydiffractometry(XRD).18-20 Materials and Methods Materials. BITC (98% purity) and PEITC (96% purity) were purchased from Acros Organics (Morris Plains, NJ). β-CD (g99% purity) was purchased from Shanghai Boao Biotechnology Co. (Shanghai, China). All other reagents used were of analytical grade. Preparation of β-CD-BITC and β-CD-PEITC Complexes. To prepare the complexes, 5.0 g of β-CD was thoroughly dissolved in 200 mL of water at 60 °C under stirring, and then the solution was cooled to 40 °C. Separately, 1 mL of BITC was dissolved in an equal volume of ethanol, and 1 mL of PEITC was dissolved in an equal volume methanol; then, each of these solutions was dribbled into a β-CD solution under agitation for 3 h. The products were filtered under vacuum and dried in a vacuum desiccator to constant mass at 25 °C. The complexes β-CD-BITC and β-CD-PEITC were obtained. Quantitative Determination of BITC and PEITC on the Surfaces of the Complexes. For this analysis, 0.1 g of each complex was deposited in a dry test tube with a cover. Then, 10 mL of hexane was added, and the mixture was oscillated for 30 min at room temperature, after which the top organic phase was collected. The above processes were repeated three times, and the total volume of the organic phase was measured accurately. Analysis of the organic phase by UV spectrophotometer absorption at 251 nm allowed the BITC and PEITC contents on the surface of the complexes to be estimated; the mean values of three repeat tests are reported. Quantitative Determination of BITC and PEITC in the Cavities of the Complexes. For this analysis, 0.1 g of each complex was added hermetically to a mixed solution containing 10 mL of water and 10 mL of hexane in a clean dry test tube. The test tube was held in hot water at 80 °C for 20 min with interval vibration. Then, the organic phase was recovered. The operation was repeated three times, and all organic phases were collected. The contents of BITC and PEITC in the cavities of the complexes were determined by quantitative UV spectro-

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Table 1. Factors and Levels of the Designed Experiment in RSD levels -1.414

factors

X1, guest/β-CD mass ratio (mg/g) 0.0645 X2, temperature (°C) 35.86

-1

0

1

1.414

0.11 0.22 0.33 0.3755 40 50 60 64.14

photometric analysis. Furthermore, the embedding ratio was calculated by the equation Y (%) )

Ci × 100% Ci + Cs

(1)

where Y is the embedding ratio, Ci is the content of BITC and PEITC in the cavities of the complexes, and Cs is the content of BITC and PEITC on the surfaces of the complexes. Optimization of the Inclusion Process by Response Surface Design (RSD). To investigate the effects of different mass ratios and temperatures on the embedding ratio, an RSD test with a central composite design was carried out. The mass ratio and temperature were set as the variables X1 and X2, respectively, with the embedding ratio as the response value Y. Table 1 lists the factors and their levels for all of the tests. Determination of the Association Constants of BITC and PEITC with β-CD. For the determination of association constants, 10 mL of a 10 mmol/L solution of BITC in ethanol (or PEITC in methanol) and 10 mL of solutions with a series of concentrations of β-CD were added 50-mL stoppered conical flasks at 40 °C. The final concentrations of β-CD in the reaction solutions were 1, 1.5, 2, and 2.5 mmol/L. All reaction samples were kept for 3 h, and their absorbance values were determined at 251 nm. The association constants were calculated according to the equation

(

CS - CCD CS - CCD ) k [CD]0 C0 CCD CS

)

(2)

where CS and CCD are the concentrations of BITC or PEITC in the absence and presence of β-CD, respectively; [CD]0 is the initial concentration of β-CD; and C0 is the initial concentration of BITC or PEITC. Specifically, each association constant was determined as k, the slope of the straight line obtained by plotting (CS - CCD)/CCD vs {[CD]0 - C0(CS - CCD)/CS}. Analysis Methods. A DSC Q100 V9.5 Build 288 system and an SDT Q600 V8.2 Build 100 system were used. DSC samples in aluminum pans were heated at 10 °C min-1 in nitrogen flowing at a rate of 50.0 mL/min. TG experiments were performed using an alumina pan with samples heated at 10 °C min-1 in nitrogen flowing at a rate of 120 mL/min. XRD experiments were carried out using a Rigaku D/max 2550PC diffractometer system with Cu KR radiation (λ ) 1.54059Å) generated at a voltage of 40 kV and a current of 250 mA. XRD spectra were recorded over the interval 3-70° (2θ) for all samples in angular steps of 0.020°. All samples were prepared by tabletting together with standard potassium bromide pellets. An Avatar 370 Fourier transform infrared (FTIR) spectrometer (Thermo Nicolet, Madison, WI) was used to record all spectra over the range of 7800-375 cm-1. The spectral resolution was 0.5 cm-1, and 32 scans were collected. Results and Discussion Standard Profiles of BITC and PEITC. BITC and PEITC were dissolved in hexane solution with a maximum absorption peak at 251 nm. A set of BITC standard solutions with different concentrations in the range of 0-1.1 mg/mL were prepared,

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Figure 2. Correlation between BITC concentration and absorption value in the concentration range of 0-1.1 mg/mL.

Figure 3. Standard curves for determining the concentrations of BITC and PEITC: (a) BITC, (b) PEITC.

and the absorption values were measured. A plot of BITC sample concentration versus UV absorption at 251 nm is shown in Figure 2. The variation coefficients were compared among different concentration ranges and were consistently found to be less than that in the concentration range from 0.01 to 0.07 mg/mL. Therefore, the concentration range from 0.01 to 0.07 mg/mL was used to construct the standard curves (Figure 3) to quantify BITC and PEITC. The concentrations of the samples Ci and Cs in eq 1 were diluted to this concentration range. Determination of Inclusion Qualifications. Single-factor tests were carried out first to determine the significant factors for the embedding ratio. In Figure 4, the selected mass ratio and inclusion temperature were found to be much more effective in improving the embedding ratio than the agitation speed. The two-factor central composite design experimental scheme and the embedding ratios measured under various test conditions are listed in Table 2. Minitab15 software was used to perform a quadratic regression response surface analysis and a variance analysis for the experimental results. The variance analysis results are reported in Tables 3 and 4 for the β-CD-BITC and β-CD-PEITC complexes, respec-

Figure 4. Effects of (a) mass ratio, (b) temperature, and (c) agitation on the embedding ratio. For a, at the inclusion temperature is 60 °C, and the agitation rate is 20 rpm. For b, the mass ratio is 0.22, and the agitation rate is 20 rpm. For c, the mass ratio is 0.22, and the inclusion temperature is 60 °C.

tively. For β-CD-BITC inclusion action, the computed F values of 29.15 and 29.44 are much larger than F0.05(2,10), which is only 4.10. The F value of the variance of interaction, 6.92, is larger than F0.05(1,11), which is 4.84. The above results suggest that the linear, square, and interaction items are at high significance levels. Meanwhile, the F value for lack-of-fit, 2.23, is much smaller than F0.05(3,9), 3.86. The F value of the regression item (29.29) is larger than F0.05(5,7) (3.97). These


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Table 2. Central Composite Design and Response Data Y (%) test

X1

X2

β-CD-BITC

β-CD-PEITC

1 2 3 4 5 6 7 8 9 10 11 12 13

-1 -1 1 1 0 0 0 0 0 1.414 -1.414 0 0

-1 1 -1 1 0 0 0 0 0 0 0 1.414 -1.414

70.5 92.1 74.0 81.3 95.8 92.0 92.7 90.0 94.2 78.0 87.3 85.7 69.5

78.2 91.2 68.7 71.1 83.2 82.5 86.5 81.5 84.7 67.4 89.0 82.0 74.0

Table 3. Variance Analysis of the Mode β-CD-BITC source of variance

sum of variance

degrees of freedom

mean square

computed F

regression linear square interaction lack-of-fit pure error total

1082.18 430.72 435.08 51.12 32.37 19.35 1133.90

5 2 2 1 3 4 12

216.437 215.362 217.539 51.123 10.789 4.838

29.29 29.15 29.44 6.92 2.23

Table 4. Variance Analysis of the Mode β-CD-PEITC source of variance

sum of variance

degrees of freedom

mean square

computed F

regression linear square interaction lack-of-fit pure error total

679.582 109.154 110.083 28.090 3.470 15.368 698.420

5 2 2 1 3 4 12

135.916 54.577 55.041 28.090 1.157 3.842

50.51 20.28 20.45 10.44 0.3 Figure 5. (a) Surface plot and (b) contour plot of Y ) f(X1,X2) for β-CD-BITC.

results show that the regression procedure was reliable and that the regression equation fits the experimental results well. For the β-CD-PEITC inclusion results, all regression items were significant at a significance level of 0.05. Additionally, the F value of the lack-of-fit item, 0.3, indicates that the regression equation was reliable. Variance analysis showed that the mass ratio and the inclusion temperature were significant factors for the embedding ratio. The regression equations of the central composite design tests were as follows Y ) -151.614 + 355.127X1 + 7.497X2 - 490.616X12 0.06X22 - 3.25X1X2 (3) Y ) -32.249 + 159.065X1 + 3.904X2 - 243.121X12 0.03X22 - 2.409X1X2 (4) where Y is the embedding ratio, X1 is the mass ratio, and X2 is the inclusion temperature. Equations 3 and 4 depict the relationships between the embedding ratio and the mass ratio and the inclusion temperature for the two complexes. Figures 5 and 6 show surface plots and contour plots of eqs 3 and 4, respectively. The effects of the mass ratio (X1) and inclusion temperature (X2) on the embedding ratio (Y) are statistically remarkable based on the steep curves. The embedding ratio was found to increase with increasing inclusion temperature. However, considering the volatilization qualities of BITC and PEITC samples, the inclusion temperature should be limited. The effect of the mass ratio on the embedding ratio

for the complexes β-CD-BITC and β-CD-PEITC varies in Figures 5 and 6. The complexation action of β-CD-PEITC requires a much higher temperature to achieve a higher embedding ratio than does that of β-CD-BITC. Presumably, some attribute of the R group in the ITC structure RsNdCdS affected the conjugation efficiency between the guest and β-CD. The regression equations were solved as first-order partial derivatives to obtain the optimal inclusion parameters. The solution process and results were as follows

{ {

∂Y ) 355.127 - 981.232X1 - 3.25X2 ) 0 ∂X1 ∂Y ) 7.497 - 0.12X2 - 3.25X1 ) 0 ∂X2

(5)

with X1 ) 0.17, X2 ) 57.8, and Y ) 95.52% for β-CD-BITC ∂Y ) 159.065 - 486.242X1 - 2.409X2 ) 0 ∂X1 ∂Y ) 3.904 - 0.06X2 - 2.409X1 ) 0 ∂X2

(6)

with X1 ) 0.0057, X2 ) 64.9, and Y ) 94.77% for β-CD-PEITC. The complexes β-CD-BITC and β-CD-PEITC were prepared with mass ratios of 0.17 and 0.0057 and inclusion temperatures of 57.8 and 64.9 °C respectively. The embedding ratios were 94.9% for β-CD-BITC and 94.1% for β-CD-PEITC, which were in good agreement with the predicted values. Therefore, RSD proved to be a valuable method for optimizing the inclusion conditions.

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Figure 6. (a) Surface plot and (b) contour plot of Y ) f(X1,X2) for β-CD-PEITC.

Figure 7. Plot of (CS - CCD)/CCD versus {[CD]0 - C0(CS - CCD)/CS} for calculating association constants of BITC and PEITC with β-CD.

Association Constants of BITC and PEITC with β-CD. When BITC and PEITC were added to β-CD solution, BITC and PEITC were combined according to the reaction k

BITC ⁄ PEITC + β-CD a β-CD-BITC ⁄ PEITC This complex reaction could reach equilibrium. The association constant k is a parameter that depends only on temperature. CS and CCD were calculated by determining the absorbance values of reaction samples at different concentrations of β-CD. The straight lines obtained by plotting (CS - CCD)/CCD versus {[CD]0 - C0(CS - CCD)/CS} (Figure 7) were used to evaluate k. The experiment results showed that the association constants of β-CD-BITC and β-CD-PEITC were 107.6 and 122.81 L/mol, respectively.

Figure 8. DSC curves for (a) β-CD and complexes of (b) β-CD-BITC and (c) β-CD-PEITC.

Analysis of the Thermal Behaviors of BITC, PEITC, and β-CD and β-CD-BITC and β-CD-PEITC Complexes. The DSC curve for β-CD is shown in Figure 8a. A prominent dehydration endothermic peak was found at 114.28 °C with ∆H ) 747.3 J/g between 75 and 120 °C. Meanwhile, the TG curve of β-CD in Figure 9a shows a 13.56% water mass loss. This is different than the results of 10.9% and 12.5% determined by Orgova´nyi20 and Ndlebe et al.,19 respectively. Accordingly, it


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Figure 9. TG curves for (a) β-CD, (b) BITC, and (c) PEITC standard samples and complexes of (d) β-CD-BITC and (e) β-CD-PEITC.

can be concluded that the thermoanalytical outcome depends on the experimental conditions (exoteric or hermetic) and that the stability of water in β-CD varied in the different environments. The DSC curves of BITC and PEITC standard samples could not be acquired because of the strong volatilization of these compounds. However, the TG curve of BITC in Figure 9b shows an initial mass loss of 18.65% at 63.69 °C and a second-stage mass loss of 33.47%. The TG curve of PEITC in Figure 9c shows an initial mass loss of 18.27% at 76.04 °C, followed by further mass losses of 27.31% and 17.99%. The total mass loss was 52.12% for BITC and 63.57% for PEITC. Consequently, BITC is distinct in its stability to temperature from PEITC.

The DSC spectra of the β-CD-BITC and β-CD-PEITC complexes are shown in Figure 8b,c. Series endothermic peaks were detected. For the β-CD-BITC complex, enthalpy changes of ∆H ) 263.2 J/g at 44.22 °C and ∆H ) 24.68 J/g at 127.40 °C were found. An enthalpy change of ∆H ) 361.9 J/g occurred at 51.41 °C for the β-CD-PEITC complex. Compared to BITC and PEITC standard samples, the changes for the complexes in the DSC curves indicate the occurrence of an interaction between BITC or PEITC and β-CD. Moreover, modifications of the endothermic peaks and enthalpy changes were associated with the melting, state transition, and decomposition of BITC and PEITC. Additionally, the characteristic dehydration endo-

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Figure 10. X-ray powder diffraction diagrams of (a) β-CD, (b) physical mixture of BITC and β-CD, (c) physical mixture of PEITC and β-CD, (d) β-CD-BITC complex, and (e) β-CD-PEITC complex. (1, d ) 19.83; 2, d ) 14.11; 3, d ) 9.821; 4, d ) 8.279; 5, d ) 7.583; 6, d ) 7.086; 7, d ) 7.335; 8, d ) 14.71; 9, d ) 12.24; 10, d ) 8.879.)

thermic peak at 114.28 °C for the β-CD sample was weakened significantly in the β-CD-BITC complex and even disappeared in the β-CD-PEITC complex. The association reaction between BITC or PEITC and β-CD, especially the hydrophobic cavity of β-CD, thus altered the thermal behavior of both the wall material and the guests. The TG spectra of the β-CD-BITC and β-CD-PEITC complexes are compared with those of two standard samples in Figure 9. The β-CD-BITC complex showed an initial mass loss of 5.915% and three further mass losses of 1.929%, 1.559%, and 1.784%, respectively. However, the mass loss of the β-CD-PEITC complex was divided into five stages as follows: 6.581%, 1.285%, 0.9622%, 1.047%, and 0.8946%. The total mass losses of the β-CD-BITC and β-CD-PEITC complexes were calculated as 11.187% and 10.77%, respectively, which were much lower than those of BITC, PEITC, and even β-CD. This result further suggests that the inclusion action improved the thermal stability of the BITC and PEITC compounds to some extent. X-ray Diffraction Results. The X-ray powder diffraction patterns (Figure 10) of the inclusion complexes and physical mixtures of BITC and PEITC with β-CD show significant differences from one another. The most intense peak of β-CD (d ) 7.086) is still clear in the patterns of the physical mixtures, where it transferred to the peak at d ) 7.335. Some characteristic peaks of β-CD are evident in the patterns of the physical mixtures patterns, whereas they are absent or obscure in the inclusion patterns, such as the intense peaks at d ) 7.583, d ) 8.279, d ) 9.821, d ) 14.11, and d ) 19.83. Three fresh peaks at d ) 8.879, d ) 12.24, and d ) 14.71 appeared distinctively in the spectra of the complexes. However, a peak at d ) 12.24 is weakly present in the patterns of the mixtures. The results further illustrate that an inclusion reaction did occur between the guests and the wall material. Meanwhile, BITC and PEITC were possibly embedded lightly in the β-CD cavity in the physical mixtures. Infrared Results. For the preparation of IR sample, 44 mg of BITC and 44 mg of PEITC were separately mixed with 2 g of β-CD powder. The mixtures were kneaded manually in an agate mortar with a pestle to obtain homogeneous samples.

The FTIR spectra of the physical mixtures and complexes are shown in Figure 11. The characteristic peaks of the isosulfocyanic group (sNdCdS) in all treated samples shifted. The intense sNdCdS stretching band shifted from 2092 to 2089 cm-1 for the β-CD-BITC complexe and from 2110 to 2088 cm-1 for the β-CD-PEITC complex. For the two physical mixtures, the isosulfocyanic group shifted from 2092 to 2089 cm-1 and from 2110 to 2125 cm-1, respectively, as shown in Figure 11c,d. Low wavenumbers shifting revealed that the strength of double bonds (sNdCdS) decreased due to the occurrence of inclusion action. Aside from the major absorbance range of the isosulfocyanic group (sNdCdS), there were two weak adjacent bands, 2360 and 2341 cm-1. They disappeared in the complexes but became stronger in the physical mixtures. A series of absorbance peaks of BITC and PEITC vanished or weakened in the complexes as follows: stretching vibration of sH at 3030 cm-1 in the benzyl group, bending vibration of sCH3 at 1450 and 1380 cm-1, plane bending vibration of CsH in benzyl at about 700 cm-1. This was possibly due to the action of OsH stretching at about 3350 cm-1 and the CsO stretching region from 1300 to 1000 cm-1 in the β-CD molecule. The results indicate that the embedding reaction impacted the optical behavior of BITC and PEITC. Conclusions The inclusion complexes β-CD-BITC and β-CD-PEITC were effectively constructed by the coprecipitation method. However, the inclusion efficiency was predominantly affected by both the mass ratio and the inclusion temperature. The interaction between the guests and the wall material, β-CD, was studied by applying thermoanalytical methods (DSC, TG), FTIR spectrometry, and X-ray diffractometry. The DSC spectra of BITC and PEITC could not be obtained because of their strong volatilization qualities. However, the DSC-TG spectral analysis of β-CD-BITC and β-CD-PEITC revealed that the thermal behavior of the complexes underwent modification. Moreover, the mass losses of the complexes were lower than those of the guests or wall material. The thermal stability of BITC and PEITC was improved in complexes. This


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Figure 11. FTIR spectra of β-CD, physical mixtures, and complexes: (a) β-CD-BITC complex, (b) β-CD-PEITC complex, (c) physical mixture of BITC and β-CD, (d) physical mixture of PEITC and β-CD, (e) BITC, (f) PEITC, and (g) β-CD.

result is presumed to be related to the association reaction occurring during embedding. BITC and PEITC could be bonded into the hydrophobic cavity of β-CD.

X-ray powder diffraction analysis showed that characteristics of β-CD were significantly altered for the complexes rather than for physical mixtures. Meanwhile, some fresh and distinct

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signals were found, which verified the occurrence of modified crystal structure in the patterns of the complexes. Additionally, the optical behavior of BITC and PEITC exhibited by FTIR spectrometry shifted to some extent. Overall, the results of a series of techniques confirmed basically the presence of supramolecular entities in the inclusion complexes. The hydrophobicity of BITC and PEITC, which is consistent with the structure of β-CD, could be a reasonable factor for the formation of inclusion complexes. For other ITC samples, including extracts from crucifer vegetables, this method is advisible. Acknowledgment This work was supported by the National Natural Science Foundation of China. Literature Cited (1) Kassie, F.; Laky, B.; Gminski, R.; Mersch-Sundermann, V.; Scharf, G.; Lhoste, E.; Kansmu¨ller, S. Effects of garden and water cress juices and their constituents, benzyl and phenethyl isothiocyanates, towards benzo(a)pyrene-induced DNA damage: A model study with the single cell gel electrophoresis/Hep G2 assay. Chem.-Biol. Interact. 2003, 142, 285–296. (2) Hecht, S. S.; Kenney, P. M. J.; Wang, M.; Trushin, N.; Upadhyaya, P. Effects of phenethyl isothiocyanate and benzyl isothiocyanate, individually and in combination, on lung tumorigenesis induced in A/J mice by benzo[a]pyrene and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone. Cancer Lett. 2000, 150, 49–56. (3) Hecht, S. S.; Kenney, P. M. J.; Wang, M.; Upadhyaya, P. Benzyl isothiocyanate: An effective inhibitor of polycyclic aromatic hydrocarbon tumorigenesis in A/J mouse lung. Cancer Lett. 2002, 187, 87–94. (4) Satyan, K. S.; Swamy, N.; Dizon, D. S.; Singh, R.; Granai, C. O.; Brard, L. Phenethyl isothiocyanate (PEITC) inhibits growth of ovarian cancer cells by inducing apoptosis: Role of caspase and MAPK activation. Gynecol. Oncol. 2006, 103, 261–270. (5) Song, L.; Thornalley, P. J. Effect of storage, processing and cooking on glucosinolate content of Brassica vegetables. Food Chem. Toxicol. 2007, 45 (2), 216–224. (6) Hu, R.; Kim, B. R.; Chen, C.; Hebbar, V.; Kong, A. N. The roles of JNK and apoptotic signaling pathways in PEITC-mediated response in human HT-29 colon adenocarcinoma cells. Carcinogenesis 2003, 24, 1361– 1367. (7) Rose, P.; Whiteman, M.; Huang, S. H.; Halliwell, B. Phenylethyl isothiocyanate-mediated apoptosis in hepatoma HepG2 cells. Cell. Mol. Life Sci. 2003, 60, 1489–1503.

(8) Pullar, J. M.; Thomson, S. J.; King, M. J.; Tumbull, C. I.; Midinter, R. G.; Hampton, M. B. The chemopreventive agent phenethyl isothiocyanate sensitizes cells to Fas-mediated apoptosis. Carcinogenesis 2004, 25, 765– 772. (9) Adebowale, A.; Ganesan, A. P.; Prasad, R. N. V. Effect of benzyl isothiocyanate on spontaneous and induced force of rat uterine contraction. Pharmacol. Res. 2004, 49, 415–422. (10) Troncoso, R.; Espinoza, C.; Sanchez-Estrada, A. Analysis of the isothiocyanates present in cabbage leaves extract and their potential application to control Alternaria rot in bell peppers. Food Res. Int. 2005, 38, 701–708. (11) Kermanshai, R.; McCarry, B. E.; Rosenfeld, J. Benzyl isothiocyanate is the chief or sole anthelmintic in papaya seed extracts. Phytochemistry 2001, 57, 427–435. (12) Zhang, Y.; Li, J.; Tang, L. Cancer-preventive isothiocyanates: Dichotomous modulators of oxidative stress. Free Radical Biol. Med. 2005, 38, 70–77. (13) Jiang, Z.; Zhang, Q.; Tian, H. The Reaction of Allyl Isothiocyanate with Hydroxyl/Water and β-Cyclodextrin Using Ultraviolet Spectrometry. Food Technol. Biotechnol. 2006, 44 (3), 423–427. (14) Ohta, Y.; Takatani, K.; Kawakishi, S. Effects of ionized cyclodextrin on decomposition of allyl isothiocyanate in alkaline solutions. Biosci, Biotechnol. Biochem. 2004, 68, 433–435. (15) Ohta, Y.; Matsui, Y.; Takatani, K.; Kawakishi, S. Retarding effects of cyclodextrins on the decomposition of organic isothiocyanates in an aqueous solution. Biosci. Biotechnol. Biochem. 2004, 68, 671–675. (16) Reinsborough, V. C.; Stephenson, V. C. Inclusion complexation involving sugar-containing species: β-cyclodextrin and sugar surfactants. Can. J. Chem. 2004, 82, 45–49. (17) Li, X.; Jin, Z.; Wang, J. Complexation of ally isothiocyanate by R- and β-cyclodextrin and its controlled release characteristics. Food Chem. 2007, 103, 1327–1331. (18) Aigner, Z.; Hassan, H. B.; Berkesi, O.; Kata, M.; Eros, I. Thermoanalytical, FTIR and X-ray studies of gemfibrozil-cyclodextrin complexes. J. Therm. Anal. Calorim. 2005, 81, 267–272. (19) Ndlebe, V. J.; Brown, M. E.; Glass, B. D. The thermal stability of triprolidine hydrochloride and its mixtures with cyclodextrin and glucose. J. Therm. Anal. Calorim. 2004, 77, 445–457. (20) Orgova´nyi, J. Thermaoanalytical method for studying the guest content in cyclodextrin inclusion complexes. J. Therm. Anal. Calorim. 2005, 81, 261–266.

ReceiVed for reView October 9, 2008 ReVised manuscript receiVed March 12, 2009 Accepted March 14, 2009 IE8015329

Preparation and Characterization of Inclusion Complexes of Î²

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