1. Introduction

Cyclodextrins (CDs) are a common ingredient used by food manufacturers and the pharmaceutical industry. CDs have multifunctional properties and can act as a carrier and stabilizer for flavors and sweeteners (Bibby et al. 1998, Kalogeropoulos et al. 2009, SzenteSzejtli 2004). Specially, the application of CD-assisted molecular encapsulation of neutraceutical ingredients, functional food in food products, hydrophobic drugs, and volatile substances helps protect the active ingredients against oxidation, loss of volatile compounds and light-induced decomposition and increases the solubility of hydrophobic medicines (Choi et al. 2009, Gèze et al. 2004). CDs self-assemble through spontaneous molecular associations under hydrodynamic equilibrium conditions into stable, regular and structurally well-defined aggregates joined by non-covalent bonds. Using molecular self-assembly to build agglomeration systems may give rise to many unique structural characteristics and novel physical/chemical properties. Self-assembly aggregation of CDs has been extensively investigated from the aggregation of native CDs to high-order complex aggregates. The increasing complex order of the aggregates is dependent on parameters such as pH and concentration (He et al. 2008, Memisoglu-Bilensoy et al. 2006). He et al. divided the cyclodextrin aggregates into five types: aggregates of native and modified CDs, inclusion complexes and their aggregates of CDs, CD rotaxanes (a kind of molecular species consisting of linear components threaded into cyclic components) and polyrotaxanes (rotaxanes has more than two cyclic components), CD nanotubes and their secondary assembly, and other high-order aggregates such as nanosphere and network aggregates. There are many parameters that affect the aggregation properties of CDs and their inclusion complexes such as temperature, concentration, guest material, water, and period of storage time in aqueous solution (Daoud-Mahammed et al. 2007, PricePatchan 1993). Bonini et al. confirmed that aggregation of β-CD monomers was dependent on the concentration of β-CD and temperature conditions in the aqueous solution (Bonini et al. 2006).

In this study, eugenol (4-allyl-2-methoxyphenol) was selected as a volatile model substance to examine the encapsulation of an aroma component during CD self-assembly aggregation. Eugenol (Eug) is an allyl chain-substituted guaiacol and a member of the phenylpropanoids class of chemical compounds, which are widely used as flavoring agents, essential oils and antimicrobials by the food and pharmaceutical industries (Jadhav et al. 2004). However, the application of eugenol in food products, pharmaceutics and cosmetics has been limited due to its unstable high volatile behavior and oxidation after light exposure (Choi et al. 2009). For this reason, the use of eugenol by these industries requires that it be encapsulated within the walls of a different material such as cyclodextrin.

In this study, eugenol particles were fabricated using two different methods; inclusion into the cavity of cyclodextrin by molecular inclusion or encapsulation by self-assembling aggregates of cyclodextrin around eugenol droplet such as the micelle type. The release properties of these complex aggregates are important for eventual use of eugenol in different applications. These release properties involve the presence of a trigger for eugenol release or for its efficient entrapment in the particles. We investigated the physical properties, such the size, thermal transition properties, and stability of dispersion, of the eugenol particles. With these two different types of carriers, the releases of volatile compounds were found to be dependent on environmental conditions such as temperature and relative humidity (RH) (Soottitantawat et al. 2005).

2. Materials and Methods

2.1 Materials

β-Cyclodextrin (β–CD), which was used as a coating material, was purchased from Pung-Rim Trading Company (Seoul, Korea). Eugenol was purchased from Sigma-Aldrich (Missouri, USA). Sodium bromide (NaBr), sodium chloride (NaCl) and barium chloride (BaCl2) was obtained from Sam-Chun (Seoul, Korea). Phosphorus pentoxide (P2O5), which was used as a strong dehydrating agent, was purchased from Duksan Pure Chemical (Kyunggido, Korea). All reagents were chemically graded and used without further purification.

2.2.1. Encapsulation of eugenol with β-cyclodextrin by self-aggregation and by molecular inclusion

Two percent β-CD (w/v), which has a maximum solubility of 1.85 % (w/v) at room temperature in water (Astray et al. 2009) was dissolved in distilled water at 50˚C for 30 min, and then, eugenol (CD:eugenol = 1:1 (M:M)) was added into this β-CD solution. These mixtures were put into a shaking-incubator for inclusion at 180 rpm and 50˚C. Different shaking times were applied to induce molecular inclusion (2 h and 6 h) and self-aggregation inclusion (8 h and 24 h). The obtained samples were then frozen at -40°C and freeze-dried to form a powder.

2.2.2. Particle size and -potential analysis

The particle size and -potential were analyzed using the dynamic lighter scattering method (Zetasizer®, Nano-ZS90, Malvern Instuments Ltd., Worcestershire, UK). The rehydrated β-CD-Eug complexes were diluted with

distilled water for dynamic lighter scattering. Measurements were made at 25oC in triplicate.

2.2.3. Surface tension analysis

The surface tension was measured by Du Nouy ring method using a Tensiometer®

(Sigma 703D KSV, Monroe, U.S.A.). This method measures the maximum weight of the liquid lifted by a ring that is pulled out of the liquid interface. The force required to lift the ring is related to the surface tension. The rehydrated β-CD-Eug complexes were placed into a sample holder connected to a water bath circulator. The equilibrium time to reach steady state for each measurement was determined to be 10 min. All measurements were carried out at 30oC in triplicate.

2.2.4. Stability behavior of CD-Eug complex in aqueous solution

The stability behavior was monitored by multiple light scattering methods measured by a TurbiScan® (MA2000, Formulaction, Toulouse, France). The detection head is composed of a pulsed near infrared light source (λ=850 nm) and two synchronous detectors. The transmission detector (at 180°) receives the light, which goes through the sample, while the backscattering detector (at 45°) receives the light scattered backward by the sample. The detection head scans the entire height of the sample, acquiring transmission and backscattering data every 40 μm.The aqueous solution of pure β-CD and β-CD-Eug complexes were prepared as previously described prior to analysis by the Turbiscan®. The samples were subsequently placed into cylindrical glass tubes. The backscattering of light from the aqueous solution of pure β-CD and β-CD-Eug complexes was then measured every 2 h for 24 h at 50°C and along sample height from 0 mm to 40 mm. The results are presented as the backscattering profile (Delta Backscattering (DBS) /100%) vs. time.

2.2.5. Encapsulation efficiency determination of freeze-dried powder

The total eugenol content in freeze-dried β-CD-Eug complexes was measured according to a solvent extraction method described by Padukka et al. with some modifications (Padukka et al. 2000). 0.1 g of freeze-dried powder was added to a 20 ml glass tube. 7 ml of distilled water and 9 ml of ethyl acetate was then added to the 20 ml glass tube. The mixture was warmed up for 10 min in a water bath to dissociate the eugenol by dissolving β-CD into the water. The glass tube was cooled to room temperature after heating. The upper ethyl acetate was removed with a pipette, and then the amount of eugenol was detected at 284 nm using a UV-VIS spectrophotometer (UV-VIS spectrophotometer, Optizen, Mecasys,

Korea). Free eugenol from the freeze-dried β-CD-Eug complexes was obtained by a washing method according to Padukka et al. (Padukka et al. 2000). Ethyl-acetate was selected as an organic solvent for extraction of free eugenol. 0.1 g of freeze-dried powder was placed into a 20 ml glass tube and 9 ml of ethyl acetate was added. The solution was then mixed with a vortex mixer for 5 min at room temperature. This mixture was separated by centrifugation for 10 min at 2000 rpm. The absorbance at 284 nm of the upper ethyl acetate layer containing free eugenol was measured using a UV-visible spectrophotometer. The encapsulation efficiency was calculated using the following equation:

Encapsulation efficiency (%) = [(A - B) / A] ⅹ 100 eq. (1)

Where A (g) is the weight of the total amount of eugenol, and B (g) is the weight of free eugenol.

2.2.6. Release study of freeze-dried -CD-eug complexes at constant humidity and temperature

The release characteristics of the eugenol encapsulated in β-CD at different storage temperatures and RH were evaluated to compare the release rate and release mechanism of eugenol as a function of the aggregation behavior of the β-CD-Eug complexes. Freeze-dried β-CD-Eug complexes induced by using different shaking times were placed into a glass bottle. 0.8 g of the powders was divided into two glass bottles (one to calculate the free eugenol and the other to calculate the total eugenol retention). The glass bottles were placed in a desiccator containing a phosphorus pentoxide (P2O5) for 6 h to remove any the residual water in the freeze-dried β-CD-Eug complexes after the freeze-drying process was complete and to accelerate eugenol release within 24 hours for a comparison among the different treatment conditions. Saturated salt solutions were preparedas shown in Table 1 (Min et al. 1998). The glass bottles was then placed in a chamber containing a saturated salt solution of NaBr (RH = 56.0 ± 3.2%), NaCl (RH = 75.1 ± 0.4%) and BaCl2 (RH = 90.6 ± 0.8%) at storage temperature of 20°C, 30°C and 40°C, respectively. The amount of free and total eugenol was determined after storage times of 2 h, 6 h, 12 h and 24 h using a UV-VIS spectrophotometer.

Table 1 Composition and relative humidity of saturated salt solutions at various

temperatures

SaltsRelative humidity (%) Amount

20°C 30°C 40°C Salts(g)

Water(ml)

NaBr 59.14 56.03 52.83 200 80NaCl 75.47 75.09 74.68 200 60BaCl2 91.20 90.80 89.70 200 70

2.2.7. TEM analysis

The morphology of β-CD-Eug complexes was observed by using a Jeol-1010 microscope (JEOL, Japan) under an accelerating voltage of 60 kV. In order to acquire TEM images, the rehydrated β-CD-Eug complexes were first diluted in distilled water. A drop of diluted rehydrated β-CD-Eug complexes was directly deposited onto a 400 mesh copper grid covered by a holy carbon film. The grid was subsequently stained with an aqueous solution containing 2 % phosphotungstic acid (PTA) for negative staining. The sample was then dried at room temperature before imaging.

2.2.8. Statistical analysis

The data were analyzed by ANOVA using the SAS statistical program 9.1 (SAS Institute, Cary, North Carolina). Differences among the means were compared using Duncan’s multiple range tests, and the correlations between independent variables and measured values were calculated as Pearson’s correlation coefficients. Each treatment had three replicate determinations.

3. Results and Discussions

3.1. Particle size, -potential, surface tension and encapsulation efficiency

Table 2 shows the mean size, mean count rate, polydispersion index (PdI) and encapsulation efficiency of the β-CD-Eug complexes at different shaking times. The mean size of β-CD-Eug complexes was approximately 650 nm and 1118 nm when the mixture was shaken for 8 h and 24 h, respectively. However, the mean size of β-CD-Eug complexes was not determined after 2 h and 6 h of shaking. Seo et al. explained that inclusion complexes between CD and eugenol through the molecular inclusion method could be successfully formed by shaking the mixture up to a certain time (Seo et al. 2010). The β-CD-Eug complexes formed by molecular inclusion started to significantly aggregate after shaking for 8 h. The β-

CD-Eug complexes produced by shaking for 2 h had the lowest encapsulation efficiency (81 %) among all the treatments. This result suggests that the 2 h shaking time was not enough to completely encapsulate eugenol into the cavity of the β-CD complexes when the molecular inclusion method was used. In fact, You et al. reported that a shaking time of at least 4 hours was required to completely encapsulate eugenol inside the -CD cavity (You et al. 2008). In the other word, the hydrophobic interaction between the cavity of β-CD and eugenol might reach an equilibrium state only after 6 h, which is necessary to achieve molecular inclusion. After 8 h of continuous shaking, the β-CD-Eug molecular inclusion complex started to precipitate leading to self-aggregation. As the shaking time was extended, the transparent samples started to appear opaque and turbid. Their particles sizes were agglomerated up to 1 micron after 24 hours of shaking. According to these results, a shaking time of 6 hours may be the minimal shaking time needed for the crystallization of -CD or β-CD-Eug complex molecules. However, the encapsulation efficiency was not significantly different (p>0.05) at shaking times greater than 6 hours.

Table 2 Mean size, polydispersity index (PdI), and encapsulation efficiency of CD-Eug complexes after freeze-drying

ST1

(h)Mean size ± SD

(nm)Count rate

(kcps) Pdl2 EE3

(%)

2 ND ND ND 81.8±0.89a

6 ND ND ND 92.6±0.26b

8 652.1 ± 13.46a 279.50±6.10a 0.50±0.06a 89.7±0.73b

24 1118.0 ± 127.05b 275.10±1.34a 0.17±0.05b 92.2±0.57b

1 Shaking time, 2 Polydispersity index, 3 Encapsulation efficiencyND: not determined.a-d Means with different superscripts within a column differ, P < 0.05.

Figure 1 shows the correlation between the -potential and surface tension as a function of the applied shaking time (2 h, 6 h, 8 h and 24 h). Up to 6 hours, the shaking time did not affect the surface tension and -potential, which was around 45 mN/m and -21 mV. However, above 6 hours, the surface tension increased and the value of the zeta potential decreased at increasing shaking time. In terms of the particles size, aggregates of the -CD eugenol complex were observed after a shaking time of 6 hours, where the -potential value started to decrease even though the composition was the same. In general, the eugenol oil droplet had a high negative charged -potential that was around -34.1 mV (Choi et

al. 2009). As the shaking time was increased over 6 hours, the solid particles may precipitate more and accelerating the aggregation of -CD or -CD-Eug complexes resulting in a decrease in the -potential value due to the embedding of the oil region. Using TEM, Choi et al. reported that a high amount of fish oil can induce and accelerate the precipitation of -CD causing a thicker membrane to form around the oil droplet (Choi et al. 2009). He found that this process resulted in a reduction in the -potential value. Furthermore, after 6 hours, the point where aggregation was first observed and the -potential started to decrease, the shaking time influenced the surface tension. Some researchers have found that surface tension and -potential are not strictly related. According to Riddick et al., a surfactant can decrease surface tension but also decrease the -potential to a lower electro-negative number (Riddick 2008). In addition, they explained that the decrease in the direction of the electro-negative charge can be related to an increase in surface tension for many cases in aqueous solutions. This tendency is in good agreement with our results. Similarly, Acevedo et al. found that an increase in surface tension was caused by a low negative charge in the oil-alkaline systems in the aqueous phase (Acevedo et al. 1999). From these results, it can be assumed that the reduction of -potential and increase in surface tension may be related to the aggregation process of β-CD-Eug complexes induced by shaking.

Figure 1. Surface tension and zeta-potential of β-CD-Eug complexes aggregated by shaking time 2 h, 6 h, 8 h and 24 h, A-D Means with different superscripts within a surface tension values differ, P < 0.05, a-c Means with different superscripts within a zeta-potential values differ, P < 0.05. 3.2. Stability behavior of CD-Eug complex in aqueous solution

To determine the stability behavior of eugenol encapsulated -CD complexes,

pure β-CD in aqueous solution and -CD-Eug complex including molecular inclusion and self-aggregates were scanned by the optical characteristic distribution of Turbiscan® (λ = 850 nm) through the Rayleigh and Mie theory (Graff 2006). Turbiscan optical analyzer was further used to study the breaking emulsion system such as creaming or sedimentation during the storage time. Figure 2 shows the delta backscattering (DBS) intensity (%) of β-CD-Eug complexes (a) and pure β-CD (b) as a function of sample height and scanning time. The DBS intensity of β-CD-Eug complexes increased by approximately 0.8% as the scanning time was increased from 0 h to 8 h at a height from 0 mm to 40 mm. The DBS intensity of β-CD-Eug complexes started to decrease as the scanning time was increased from 8 h to 24 h. The optical characteristics of small homogeneous spherical light-scattering particles can be approximated using calculations that are based on the Rayleigh and Mie theories. The propagation of light in a random dispersed medium may be considered independent or incoherent when the photon mean path length λ(, d) is larger than the wavelength of the incident radiation (Cunha-SilvaTeixeira-Dias 2005).

λ(, d) = 1 / n(d2 / 4) Qe = 2d / 3 Qe and = d 3 / 6 n eq.(2)

Where n is the particle density, d the particle mean diameter, f the particle volume fraction and Qe the extinction efficiency factor for the scattering and absorption phenomena (ratio of the extinction cross-section to the geometrical cross-section). In this work, the particles were considered non-absorbing with an extinction efficiency factor Qe equal to the scattering efficiency factor Qs. The anisotropic scattering of light by a particle can be characterized by the asymmetry factor, g, which is the average cosine <cos > of the scattering angles weighted by the phase function or scattering diagram P( ) of the scatterers (g = 0 for isotropic Rayleigh scatterers and 0<g<1 for Mie scatterers of size larger than the wavelength). Therefore, in the turbiscan application, the characteristic of backscattering is modeled using the Rayleigh theory when the particle size appears below 600 nm. In contrast, when the particle size appears greater than 600 nm (Table 2), the backscattering is modeled using the Mie theory (Cunha-SilvaTeixeira-Dias 2005). The increase in the DBS intensity means β-CD-Eug complexes aggregated around 600 nm in aqueous solution after 8 h. However, after 8 h the β-CD-Eug complexes continued to aggregate with time. This increase in particle size was demonstrated by an increase in backscattering for particles smaller than the incident light in the Rayleigh range. For particles bigger than the incident light, an increase in size leads to a decrease in backscattering in the Mie range (Cunha-SilvaTeixeira-Dias 2005). Furthermore, the precipitation of particles was increased at the bottom of the sample (around 3 mm and 8 mm) resulting in a sharp increase in the DBS intensity at high concentration. However,

there was no migration of particles throughout the whole height when creaming or sedimentation did not occur during the scanning time period. Therefore, these results indicate that the aggregation of β-CD-Eug complexes was accelerated by the increase in scanning time throughout the sample length without causing any breaking emulsion conditions such as sedimentation and creaming. As shown in Figure 2(b), the DBS intensity of pure β-CD increased by about 0.1 % at a height from 0 mm to 40 mm during a 24 h scanning period. This means that pure β-CD was slightly more aggregated throughout the sample height during the scanning time as compared with the aggregation process of the β-CD-Eug complex. In general, pure CDs can form aggregates in aqueous solution that have a size of about 200-300 nm (Bonini et al. 2006). Moreover, this cyclodextrin aggregation process was related to water content when present with the guest material (Cunha-SilvaTeixeira-Dias 2005). In contrast, inclusion complexes of CDs formed larger aggregates than pure CDs in aqueous solution. Furthermore, CDs can solubilize themselves or lipophilic water-insoluble drugs through non-inclusion complexation or micelle-like structures (Ayala-Zavala et al. 2008).

In summary, Figure 3 indicates that variations in the mean values of DBS intensity from 0 mm to 40 mm of sample height vs. time for β-CD-Eug complexes and pure β-CD occurred. The variation in DBS of β-CD-Eug complexes was significantly higher than pure β-CD in aqueous solution. This result proved that the agglomeration process of β-CD-Eug complexes was remarkably stronger than pure β-CDs and aggregation depended on the period of time the complexes were in aqueous solution. From these experiments, it was shown that the agglomeration of β-CD (host) could be accelerated in the presence of eugenol (guest) for the formation of β-CD-Eug complexes in aqueous solution. Moreover, an increase of storage time appears to affect the stability of β-CD-Eug complexes leading to aggregation, which was governed by forming association complexes. This phenomena can be explained on the basis of our previous work. A partial inclusion complex is formed between eugenol and β-CD at the eugenol/water interface or just simple adsorption between β-CD and eugenol. Secondly, when energy is supplied by shaking, the interface curves and surrounds the oily globules to form an eugenol–water emulsion. Then, due to the interaction between β-CD and eugenol, CD molecules crystallize around the oily globules. At the same time, eugenol may exist randomly among the precipitated β-CD to promote the formation of supramolecules, especially depending on shaking time.

Figure 2. Delta backscattering (%) of β-CD-Eug complexes (a) and pure β-CD (b) as a function of tube height (0-40mm), Data are given for different period of time of 4 h ( ), 8 h ( ), 12 h ( ), and 24 h ( ).

0 5 10 15 20 25 30 35 40

Height (mm)

-0.6-0.4

-0.2

0

0.2

0.4

0.6

0.8

1

1.2

1.4

Delta backscattering (%)

(a)

0 5 10 15 20 25 30 35 40

Height (mm)

-0.6

-0.4

-0.2

0

0.2

0.4

0.6

0.8

1

1.2

1.4

Delta backscattering (%)

(b)

Figure 3. Variation of mean values of DBS intensity from 0 to 40 mm of sample height vs. time for β-CD-eugenol complexes: solid line (—); pure β-CD : dotted line (---).

3.3. Release characteristic of freeze-dried CD-Eug complex

To examine the effect of shaking time on the release rate of eugenol from the -cyclodextrin, each freeze-dried sample was maintained at a different relative humidity for 24 hours (Figure 4 and 5). Overall, the content of eugenol in the freeze-dried complex was quickly reduced during the initial stage of storage. The release rate became very slow after 6 h and at longer times almost no eugenol release was observed. This trend has been reported previously (Ayala-Zavala et al. 2008, Berthod et al. 2005, Seo et al. 2010). However, the release rate quickly decreased at all RH and storage temperatures, in which eugenol had almost all been completely released within 24 h. The dehydrated samples quickly absorbed water from the atmosphere at the various humidity and storage temperatures early during storage. In regards to the effect of shaking time on the eugenol release, the amount of eugenol released remarkably decreased as the shaking time was increased from 6 h to 24 h at all RH storage. More specifically, the amount of

Time (h)

0 4 10 20

-0.2

-0.1 0

0.1

0.2 0.3

0.4

0.5

0.6

0.7

6 8 15 24

Del

ta b

acks

catt

erin

g (%

)

eugenol released after 24 hours of shaking was the slowest at a relative humidity of 56%, 75%, and 90 % and 50 % of the eugenol was still retained within the particles. In particular, eugenol did not remain inside of the cavity of the -cyclodextrin complexes for particles assembled using the molecular inclusion method, in which the samples were prepared by shaking for 2 and 6 hours, when moisture had absorbed and migrated into the freeze-dried -CD-Eug complexes as compared to particles assembled using the self-aggregate method. Based on these results, it appears that the aggregation of β-CD through self-assembly may result in the formation of a thick β-CD membrane, which would effectively retard dissolution. Similarly, Soottitantawat et al. explained the release of flavor was further retarded as the thickness of the wall material increased, which could be achieved by increase the cyclodextrin concentration. The higher wall material content increased the rate of formation of a semi-permeable membrane resulting in the reduction of flavor release. In addition, volatile release from the wall material increased as the relative humidity and temperature was increased up to the point where the wall material collapsed (AgbeninVan Raij 2001, Péter et al. 2001, Soottitantawat et al. 2005).

Based on the previous findings, we also examined the effect of relative humidity on the release rate. As shown in figure 4 (a) and (c), the release rate of eugenol at high relative humidity was faster than at low relative humidity. We observed this same trend at high storage temperature (Fig. 5). These results were explained by the fact that β-CD-Eug complex powders have high water uptake at high relative humidity, which can then rapidly dissolve the β-CD resulting in accelerated release of eugenol. As water penetrates the encapsulating matrix, continued inclusion of the hydrophobic molecule becomes energetically unfavorable (AgbeninVan Raij 2001, Choi et al. 2010).

The most significant environmental stress parameter that speeds up the release of eugenol is the storage temperature. When the release rate was examined at storage temperatures between 20°C and 40°C (Fig. 4(c) and 5(c)), despite the fact that its relative humidity was around 90 % (Table 1), eugenol at a storage temperature of 40°C was shown to migrate more quickly than when the sample was preserved at 20°C in all treatments. Similar trends were observed under other experimental conditions. We hypothesized that a high storage temperature might enhance the solubility of -CD due to increased absorbance of water molecules from the external environmental as compared to low storage temperature. Similar results have also been reported in other studies. In these studies, the release of an encapsulated guest from the wall matrices increased with an increase in relative humidity and temperature (Boyd et al. 1947, Seo et al. 2010, Sparks 1985). These authors explained that the high water uptake of the inclusion complex powders caused a change in the matrix structure, which increased CD dissolution.

Figure 4. Eugenol release behaviour from β-CD-Eug complex at relative humidity at 59.14% (a), 75,47% (b) and 91.20% (c) storage temperature at 20°C after shaking for 2 h ( ), 6 h ( ), 8 h, ( ) and 24 h ( ).

(a)

(b)

(c)

Figure 5. Eugenol release behaviour from β-CD-Eug complex at relative humidity at 52.83% (a), and 74.68% (b) and 89.70% (c) storage temperature at 40°C after shaking for 2 h ( ), 6 h ( ), 8 h, ( ) and 24 h ( ).

3.4. TEM analysis

(a)

(b)

(c)

The time-dependant aggregated structure of β-CD-Eug complexes was observed by TEM (Fig. 6). We could not clearly distinguish between eugenol and β-CD apart from these aggregates. However, their structures were very similar, aspreviously reported in a study of CD with various core materials. Spherical vesicles with diameters ranging from 100-200 nm were observed for the β-CD-Eug complexes after a shaking time of 6 h (Fig. 6a). Meanwhile, the β-CD-Eug complexes adopted polygon shapes with diameters of 200-700 nm after a shaking time of 8 h (Fig. 6b). After a shaking time of 24 h, the β-CD-Eug complexes were more agglomerated with diameters around 1.3 um (Fig. 6c). Similarly, Choi et al. observed aggregated structure of β-CD-Eug complexes that adopted spherical and hexagonal shapes. In general, the low voltage used in this study (60 kV) was not suitable for distinguishing between β-CD and eugenol. Even with PTA staining, the shape and morphology of the individual components were obscure and not clear under the low voltage operating condition of TEM (Choi et al. 2009). From these results, we observed that the-CD-Eug complexes became larger and more aggregated through crystallization of β-CD molecules at longer shaking times, which produced the mechanical force.

Figure 6. TEM observation of β-CD-Eug complexes with PTA staining, β-CD-Eug complexes after shaking for 6 h (a), 8 h (b) and 24 h (c).4. Discussion

In general, the molecular inclusion method is a quite well-known preparation method to capture aroma compounds such as low molecular volatile components. However, the interactions during self-random aggregation of CD molecules have not yet been clearly elucidated. Based on the experiments presented in this study, the cyclodextrin-complexion behavior dictates the particles formation properties such as their morphological shape, CD membrane thickness, particle sizes and so on. There are currently several published theories regarding the effect of different parameters on the specific shape of particles. Choi et al (2009) examined the mechanism of self-random aggregation between long chain fatty acids as a core material and -cyclodextrin as a wall material (Choi et al. 2009). Briefly, a mechanism involving self-assembly of -CD around fish oil was schematically proposed as follows: (i) A partial inclusion complex would be formed between fish oil and -CD at the oil/water interface. That would explain the presence of a film that was observed between the oily and aqueous phases. (ii) When submitted to external shaking, the film would curve and surround oily globules to form O/W emulsions. (iii) Because of the -CD-FO interaction, crystallization of CD molecules would occur around the oily globules. At the same time, fish oil could exist randomly among the precipitated -CD and form supramolecules, which would depend on the fish oil concentration (Choi et al. 2010, Trichard et al. 2008). Eugenol is a model material that can be completely encapsulated in the cavity of -cyclodextrin one by one when mixed at the same molar ratio. Therefore, at the beginning of shaking, only single eugenol molecules were included into the hydrophobic cavity of -cyclodextrin. During continuous shaking, the aggregation of β-CD-Eug complexes can be induced by promoting interactions between β-CD-Eug complexes prepared by molecular inclusion, where mechanical stimulation, such as stirring time, is responsible for initiating these interactions. The aggregation properties of β-CD-Eug complexes, induced by shaking, influenced the release rate of eugenol. This result can be attributed to the increase of particle size and slow eugenol diffusion rate of β-CD-Eug complexes as compared to the single molecular inclusion complex in the dried form (You et al. 2008). The most aggregated β-CD-Eug complexes had the slowest diffusion rate due to the formation of a thick β-CD membrane, which retarded the dissolution of β-CD. Based on our experiments, the main aggregation mechanism of β-CD-Eug complexes can be explained by the interactions between β-CD molecules and eugenol during shaking in aqueous solution. This interaction reached an equilibrium state after 6 h, at which point molecular inclusion started to occur. At longer continuous shaking times, the β-CD-Eug molecular inclusion complex started to precipitate leading to their self-aggregation. In the absence of eugenol,

however, the agglomeration process of β-CD was retarded, which means the interaction between β-CD and water through hydrogen bonds did not significantly influence the aggregation process of β-CD.

5. Conclusions

The time-dependent β-CD self-aggregates and molecular inclusion complexes for encapsulation were characterized and the release behavior of eugenol was examined at different relative humidity and storage temperatures. The aggregation of β-CD due to it’s the self-assembling behavior influenced the release characteristics of eugenol. The agglomeration process of the β-CD-Eug complex followed by molecular inclusion could be promoted by continuous shaking over an extended period of time. This process led to the self-aggregation of molecular inclusion and the formation of a thick β-CD membrane, which retarded the release of eugenol. To retard the diffusion rate of eugenol, the CD might have become aggregated during long term shaking; however, we must pay attention to the particle size for the proper applications related to the aggregation process.

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