Journal of Microencapsulation, 2010; 27(6): 496505

RESEARCH ARTICLE

Release characteristics of freeze-dried eugenol encapsulated with -cyclodextrin by molecular inclusion method
Eun-Ju Seo1, Sang-Gi Min1 and Mi-Jung Choi2
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1 2

Department of Food Science and Biotechnology of Animal Resources, Konkuk University, Seoul, Korea, and Department of Food Science and Biotechnology, Kyonggi University, Suwon, Korea
Abstract The study investigated the thermo-physical properties of eugenol encapsulated with -cyclodextrin (-CD) by molecular inclusion and eugenol release characteristics at various relative humidities and storage temperatures. Particle size, Zeta-potential, thermal transition and morphology of -CD-Eugenol complex after freeze-drying measured using Nanosizer , differential scanning calorimeter (DSC) and transmission electron microscopy (TEM), respectively. The particle size, Zeta-potential and inclusion efficiency of encapsulated eugenol presented 340 nm, 34.5 mV and 91.7% after freeze-drying, respectively. The relationship between retention rate of eugenol and time during release was described by a mathematical model of Avrami equation. In these events, the parameter of release mechanism and the release rate constant were rapidly elevated with increasing relative humidity and storage temperature. Furthermore, the Arrhenius activation energy for the release of eugenol decreases with increasing relative humidity and storage temperature. Key words: -cyclodextrin; release; molecular inclusion

Introduction
Encapsulation of flavour compounds has become a popular technique in recent decades in the food and cosmetics industries (Karathanos et al. 2007). Most flavour compounds are comparatively highly volatile and chemically labile components as a result of oxidation, chemical interactions or volatilization. For these reasons, the encapsulation of flavour compounds is essential to entrap a volatile substance in a capsule or matrix (Choi et al. 2009). The variety of techniques used to form the carrier for volatile substances include spraying-drying, spray-cooling/chilling, freeze-drying, fluidized bed coating, extrusion and molecular inclusion (Shahidi and Han 1993, Gibbs et al. 1999). Molecular inclusion by cyclodextrin (CD) has been increasing interest as a novel system in functional food delivery. Especially, the encapsulation of volatile flavours as guest molecules in the interior cavity

of CDs can extend the duration of entrapment and enhance the stability of the volatile compound from environmental conditions such as exposure to oxygen, heat or light (Hedges et al. 2004, Li and McGuffin 2007). Furthermore, the CD has been broadly used on account of capability to include insoluble material according to the accessibility of different cavity sizes, potential various sites for chemical modification, low toxicity and protection of the included active substance from the biodegradation (Szente et al. 1998, Nuchuchua et al. 2009). In particular, CD complexation of flavours is an encapsulation process on a molecular scale of single monomer size. The hydrophobic flavour compound can be solubilized in the aqueous phase due to their hydrophilic portions. CDs are a series of cyclic oligosaccharides consisting of six, seven or eight glucose units linked by -1, 4 bonds and called respectively -, - and -CD (Figure 1). They have a truncated cone shape with a hydrophobic cavity and a

Address for correspondence: Mi-Jung Choi, Department of Food Science and Biotechnology, Kyonggi University, 94-6 Yiui-Dong, Yeongtong-gu, Suwon 443-760, Korea. Tel: 82-31-249-9657. Fax: 82-31-249-9650. E-mail: Foodeng301@paran.com (Received 7 Jul 2009; accepted 2 Feb 2010) ISSN 0265-2048 print/ISSN 1464-5246 online 2010 Informa UK Ltd DOI: 10.3109/02652041003681398 http://www.informahealthcare.com/mnc

Eugenol encapulsation by molecular inclusion

(a)

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(b) Journal of Microencapsulation Downloaded from informahealthcare.com by UNICAMP on 08/19/13 For personal use only. Secondary hydroxyl rim2

Hydrophobic cavity Primary hydroxyl rim1

Figure 1. Chemical structure of (a) -, - and -cyclodextrins and (b) their schematic illustration of cyclodextrin. 1, 2 Interaction with hydroxyl group.

hydrophilic exterior (Yoshii et al. 2001). Correspondingly, CDs represent a good wall material to prolong the shelf-life of the products controlling the release of active substances during storage (Szente et al. 1998). CD complexes are amenable to freeze-drying because of their stability at low temperature. Freeze-drying is also termed lyophilization, which has proven to be one of the most useful processes for thermally sensitive substances, with a high potential of volatile compound entrapment within the void pore structure owing to sublimation of ice crystals (Madene et al. 2006). Moreover, freeze-dried powder can maintain the original colour and can be quickly reconstituted after addition of water (Choi et al. 2008). As two examples, freeze-dried strawberries maintain their colour and flavour with rehydration capacity (Shishehgarha et al. 2002), and freeze-dried production of microencapsulated fish oil generates a product with good resistance to oxidation (Heinzelmann and Franke 1999). As well, microcapsule shape is maintained upon freeze-drying (Toshio et al. 1990). The release characteristics of flavour compounds from the freeze-dried powder are important for estimating storage period and flavour shelf-life, as well as the appropriate application of controlled release rates in food (Reineccius 1995, Pszczola 1998). For the food packaging system, controlling antimicrobial agents releasing rate with food grade preservative is a very important issue to develop the novel packaging system (Han 2005). Related to our main purpose for the food packaging system, we tried to apply into the strawberry surface coated by liquid form Eug-CD to inhibit the mould growth during

preservation at the different preserved temperature or casing system including powder from Eug-CD system for meat product, for example emulsified sausages. According to Han reviews, the releasing kinetics of volatile antimicrobial compounds are dependent on their volatility and chemical interaction with outer environmental condition such as the concentration on the food surface or temperature and so on. The release rate of encapsulated flavour compounds depends on several mutual processes such as diffusion of volatile compound through the matrix type as a continuous phase, shape and geometry of the carriers, diffusion of active substance from the matrix to the outer environment and degradation/dissolution of the carriers for matrix systems encapsulating volatile compounds (Rosenberg et al. 1990, Pothakamury and Barbosa-Canovas 1995, Yuliani et al. 2006). Presently, eugenol (4-allyl-2-methoxyphenol) was selected as a volatile model substance to include inside the cavity of -cyclodextrin (-CD). Eugenol is an allyl chain-substituted guaiacol and a member of the phenylpropanoid class of chemical compounds that are widely used as flavouring agents and essential oils in the food and cosmetic industries (Jadhav et al. 2004). However, eugenol is comparatively light sensitive and chemically unstable. The aim of the present study was to investigate the encapsulation of eugenol as a model volatile active substance by molecular inclusion with -CD and clarify the rate of eugenol release from -CD microcapsules at various conditions of relative humidity (RH) and storage temperature (20, 30 and 40 C). The retention content of eugenol and this activation energy were fitted by Avrami and Arrhenius equations, respectively.

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Each sample was scanned from 25300 C with a heating rate of 10 C min1 under nitrogen gas injection.

Materials and methods

Materials B-CD as a coating material was purchased from Pung-Rim Trading (Seoul, Korea). Eugenol was purchased from Sigma-Aldrich (St. Louis, MO). Sodium bromide (NaBr), sodium chloride (NaCl) and barium chloride (BaCl2) were obtained from Sam-Chun (Seoul, Korea). Phosphorus pentoxide (P2O5) as a strong dehydrating agent was purchased from Duksan Pure Chemical (Kyungkido, Korea). All reagents were of suitable chemical grade and were used without further purification.

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Methods Preparation of inclusion complex (molecular inclusion). Host (-CD; 0.5 g) was dissolved in 30 ml of distilled water at 55 C for 30 min and guest (eugenol; 68 ml) was added to produce a host:guest molar ratio of 1 : 1. The mixture was incubated at 250 rpm, 55 C for 4 h. The product was frozen at 20 C and lyophilized using laboratory made freeze-dryer equipment under 0.4 mbar vacuums for 24 h and a condenser temperature of 50 C to form a powder. Preparation of the physical mixture. A physical -CD eugenol mixture with the same molar ratio as the inclusion complex was prepared to compare the differences of their thermal properties. -CD and eugenol were homogenized together using an Ultra-Turrax high-speed homogenizer (IKA Labotechnik, Staufen, Germany) at 11 000 rpm for 5 min. Each mixture was frozen at 20 C and freeze-dried to form a powder as described above. Particle size and zeta-potential analysis. Particle size and Zeta-potential were analysed by the dynamic light scattering method using a Zetasizer apparatus (Malvern Instruments, Worcestershire, UK). To measure the size and Zeta-potential of the samples, the samples were diluted with distilled water for dynamic lighter scattering, so that the mean count rate was over 300 kcps. Measurements were made at 25 C in triplicate. Analysis of thermal properties. Differential scanning calorimeter (DSC) studies were done utilizing a DSC 200F3 apparatus (Netzsch-Geraetebau GmbH, Selb, Germany) to confirm the formation of inclusion complex. -CD alone, eugenol alone, -CD-eugenol inclusion complex and -CD-eugenol physical mixture were analysed. Temperature calibration was performed using indium. The samples were weight at an accuracy of 3 0.01 mg and were hermetically sealed in an aluminum pan.

Free eugenol determination (washing method). The amount of free eugenol was determined by a slightly modified version of a previously described washing method by Padukka et al. (2000). Briefly, 0.1 g of freeze-dried powder was added to 9 ml of ethyl acetate and the mixture was gently shaken for 20 min at room temperature. The free eugenol extracted solvent phase was centrifuged for 10 min at 2000 rpm and the supernatant was collected for ultraviolet (UV) analysis. Absorbance at 284 nm was recorded with an UV-visible spectrophotometer (Optizen, Mecasys, Daejeon, Korea). The free eugenol content in the extract was calculated from a standard curve of eugenol with ethyl acetate. The determination was carried out in triplicate.

Total eugenol determination (solvent extraction method). The total content of eugenol in freeze-dried powder was measured according to a previously solvent extraction method described by Padukka et al. (2000) with some modifications. Freeze-dried powder (0.1 g) was weighed and mixed with 7 ml of distilled water and 9 ml of ethyl acetate. Then the mixture was heated at 55 C for 20 min in a shaking incubator. The upper ethyl acetate layer containing the total eugenol was separated by pipette. The ethyl acetate extract was measured at 284 nm as described above. The encapsulation efficiency of CD and loading content were calculated according to equations (1) and (2), respectively: Encapsulation efficiency % Measured total eugenol content g measured free eugenol contentg =Measured total eugenol content g 100 1 Eugenol loading content % Measured total eugenol content g measured free eugenol content g =Total mass of particles g 100

In addition, we measured the percentage of eugenol leakage after freeze-drying by the following calculation: Eugenol leakagecontent % Total extract eugenol content g measured freeeugenol content after freezedrying g=Total extracteugenol content g 100 3

Eugenol encapulsation by molecular inclusion

Transmission electron microscopy (TEM) analysis. We observed the morphology of encapsulated eugenol by a JEOL-1010 microscope (JEOL, Tokyo, Japan) at an accelerating voltage of 60 kV. For TEM, the sample was stained by negative fixation. A drop of sample was directly added to a 400 mesh copper grid covered by a carbon film followed by a drop of 2% sodium phosphotungstic acid (PTA) solution. The PTA was left for 15 min to stain the sample prior to drying at room temperature. The eugenol included in -CD was fixed to the carbon film of the grid by the surfactant polymer filled with heavy W atoms, producing a high TEM absorption contrast. In the resulting negative staining, the particles appear white and the background appears dark ` re et al. 2002). (Guinebretie

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increment of freeze-dried powder was calculated according to the following: Weight increment of -CD-eugenol powder % Measured powder weight at certain time g measured initial weight of powder g = measure initial weight of powder g 100 4

Results and discussion

Characterization of eugenol encapsulated by b-CD Table 1 shows the mean size, mean count rate, Zeta-potential, encapsulation efficiency, loading content and leakage content of the -CD-eugenol inclusion complex after freeze-drying. The -CD-eugenol complex was 340 nm in diameter after rehydration of freeze-dried sample. -CD-eugenol complexes displayed a monodispersed suspension and homogenous particle sizes, with a low PdI value of 0.3. Zeta-potential averaged 34.5 1.2 mV, indicating the complex was highly stable. In general, when the Zeta-potential is relatively lower, attractive force exceeds repulsive force between particles resulting in coalescence and aggregation of particles (Simovic and Prestidge 2003). Thus, particles with a high Zeta-potential (negative or positive) are electrically stabilized as compared with a low Zeta-potential. Encapsulation efficiency and loading content of -CDeugenol complexes after freeze-drying were 91.7% and 58.4%, respectively. Encapsulation efficiency of -CD-eugenol inclusion complexes was previously determined to be 90.9% (Choi et al. 2008). The included flavour content and encapsulation efficiency in the powder are the highest value when -CD is used as an encapsulant compared with maltodextrin/gum arabic or soy matrices (Liu et al. 1999). This suggests that a small molecular weight compound such as eugenol is suitable for inclusion into -CD. Besides, the leakage of the encapsulated eugenol after freeze-drying was 11.0% in total encapsulated eugenol amount before freeze-drying. The -CD-eugenol complex contained higher residual eugenol contents after freeze-drying, which means that the -CD-complex presently displayed low temperature stability as compared to

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Release of eugenol from freeze-dried powder at constant humidity and temperature. Freeze-dried -CD-eugenol complex was placed in a chamber containing a phosphorus pentoxide (P2O5) for 6 h to eliminate residual water remaining after freeze-drying. During the storage, the samples became anhydrous (Lievonen et al. 1998) and their release was analysed at various RHs and three selected storage temperatures. The retention of the -CD-eugenol complex was investigated at a constant humidity and temperature (Soottitantawat et al. 2004, Ko et al. 2008). Freeze-dried powder (0.1 g) was weighed into a glass bottle and 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 20, 30 and 40 C, respectively (Min et al. 1998). The chambers were sealed and maintained at the desired temperature. At designated times, samples were acquired and the amount of eugenol was measured spectrophotmetrically as described above. The retention of eugenol in the freeze-dried powder was expressed as the percentage of the initial total amount of eugenol.

Water content measurement. The water content of the -CD-eugenol complex was investigated at a constant humidity and temperature as described above. The weight

Table 1. Mean size, Zeta-potential, encapsulation efficiency and loading content of eugenol included in -CD after freeze-drying. Mean size (nm SD) -CD-eugenol complexes
a

PdIa

Mean count rate (Kcps SD) 318.2 1.43

Zeta-potential (mV SD) 34.5 1.26

Inclusion efficiency (%) 91.74 0.54

Loading content (w/w %) 58.42 0.11

Eugenol leakage content (%) 10.08 0.64

343.6 1.21b

0.3 0.003

Polydispersity index; bstandard deviation (n 3).

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Release characteristic of eugenol from freeze-dried powder It has been reported that the presence of water is essential for the formation of inclusion complexes between CD and hydrophobic guests (Yoshii et al. 1994). In fact, the inclusion reaction is a substitution process of guest for water in the cavity of CD and vice versa (Rehmann et al. 2003). The release behaviour of eugenol from the freeze-dried powder is shown in Figure 3. The retention of eugenol gradually decreased at the given RH and its releasing rate was greatly accelerated with an increase of RH and storage temperature. Overall, the content of eugenol in the freeze-dried complex was quickly reduced during the initial stage of storage. The releasing rate became very slow after 8 h and thereafter nearly ceased altogether. This trend has been reported previously
Retention of eugenol oil (%) (a) 100 90 80 70 60 50 40 30 20 10 0 02 4 6 8 Retention of eugenol oil (%) (b) 100 90 80 70 60 50 40 30 20 10 0 02 4 (c) exo 0 -CD Eugenol Physical mixture 100 90 80 70 60 50 40 30 20 10 0 02 4 8 12 24 Time (h) 48 Retention of eugenol oil (%) 8 12 24 Time (h) 48 12 24 Time (h) 48

the eugenol-loaded nanocapsules by polymer (Choi et al. 2005). Correspondingly, the interaction strength between eugenol and -CD was strong enough to preserve the complex against low freezing temperature as compared to generally low temperature fragility of the oil.

Thermal properties Figure 2 presents the DSC results of -CD, eugenol, -CD-eugenol inclusion complex and -CD-eugenol physical mixture. Pure eugenol exhibited a endothermic peak at 249.3 C, which corresponds to the boiling point. The thermal transition enthalpy was calculated as 113 J g1. On the other hand, the -CD thermal transition graphs ranged between 100200 C, with onset and maximal peak temperatures appearing at 187.3 and 188.7 C, respectively. Thermal transition of -CD-eugenol complex appeared at 196.8 C with the endothermic peak. The melting temperature of -CD alone was somewhat lower than that of the -CD-eugenol inclusion complex. Interaction of the guest with CD provides a higher energy barrier, so that an increase in phase transition temperature of 10 C is observed (Hedges 2004, Li and McGuffin 2007). On the other hand, two endothermic peaks were evident in the thermogram of the physical mixture: one was nearly identical to that of pure eugenol at 231.7 C, and the other corresponded to that of -CD, showing that the partial inclusion reaction occurred by physical mixture due to the similar transition temperature of -CD-eugenol complexes. Interestingly, the inclusion complex did not show the boiling peak of eugenol in the temperature range investigated. The disappearance of the boiling peak of eugenol was the obvious evidence of the inclusion complex formation between eugenol and -CD.

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Heat Flow (mW)

2 4

Inclusion complex 6 8 50 100 150 200 Temperature (C) 250

Figure 2. DSC analysis of thermal properties on each component.

Figure 3. Release behaviour of eugenol from -CD-eugenol complex under various RH conditions and storage temperatures of (a) 20 C, (b) 30 C and (c) 40 C. (^) is RH of 56.0%; (#) is RH of 75.1% and (m) is RH of 90.6%.

Eugenol encapulsation by molecular inclusion

(Yoshii et al. 2001, Li et al. 2007). However, the releasing rate was quickly decreased in all RHs and storage temperatures, in which eugenol was almost completely released in 24 h using P2O5 as a dehydrating agent. The dehydrated samples quickly absorbed water from the atmosphere at the various humidities and storage temperatures early during storage. Furthermore, release was complete within 24 h. The explanation is likely that the high RH induced high moisture absorption into freeze-dried powder during the storage time resulting in the fast release of eugenol as a consequence of marked dissolution of -CD. In particular, over 48 h at 20 C, eugenol retention decreased to almost 51, 42 and 30% at a RH of 56.0, 75.1 and 90.6%, respectively. At 40 C, retention decreased to 44, 30 and 19% at the same respective RHs. Similar results have been reported for increasing the release of encapsulated guest from wall matrices with an increase of relative humidity (Yoshii et al. 2001). These authors explained that the water uptake caused a change of matrix structure to increase the CD dissolution.

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retention time curve of flavour in -CD complex powder (Li et al. 2007). In this study, the Avrami equation was used to evaluate the release rate constant of eugenol, as follows (Avrami 1939): R expkt n 5

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where R is the retention of flavours during release, t is time, k is the release rate constant and n is a parameter representing the release mechanism. Theoretically, the parameter n 1 corresponds to the first-order release kinetics and a linear equation. The parameter n 0.54 represents the diffusion-limited system and the exponential function (Shiga et al. 2001). Taking the double logarithm of both sides, equation (5) yields equation (6): ln ln R n ln k n ln t
(a)

Absorbed water content (%)

12 10 8 6 4 2 0 02 4 8 12 24 Time (h) 48

Water content measurement during release study Figure 4 shows the change in weight of the freeze-dried -CD-eugenol complex at various RHs under the different storage temperatures. The slower releasing rate at the low RH was most likely due to the lower mobility of water molecules. In all cases, the water content in the -CD-eugenol complex gradually increased according to the elevating RH and the same storage temperature. The water content increment of complexes at a RH of 90.6% at all three storage temperatures was faster than the other RHs at the storage temperature. On the contrary, the water content increment tended to be slow at a low RH and storage temperature. Particularly, the water content of the -CD-eugenol complex was dramatically increased to 9.11, 9.29 and 11.26% at RH of 90.6% under storage temperatures of 20, 30 and 40 C, respectively. Otherwise, the water content indicated that the -CD-eugenol complex was slowly augmented and had water content of 3.49, 5.38 and 5.84% at RH of 56.0% under storage temperatures of 20, 30 and 40 C, respectively. These results demonstrate that RH and storage temperature have a strong influence on the water increment of -CD-eugenol powder owing to the rapid dehydration of the complex. Analysis of release characteristics by Avrami equation The Avrami equation (equation 5), which has been recently applied to represent solidgas and solidliquid reaction schemes (Shiga et al. 2001), well simulated the

(b)

Absorbed water content (%)

12 10 8 6 4 2 0 02 4 8 12 24 Time (h) 48

(c)

Absorbed water content (%)

14 12 10 8 6 4 2 0 0 2 4 8 12 24 Time (h) 48

Figure 4. Changes in the weight of freeze-dried -CD-eugenol powder at various RH conditions under different storage temperatures of (a) 20 C, (b) 30 C and (c) 40 C. (^) is RH of 56.0%; (#) is RH of 75.1% and (m) is RH of 90.6%.

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equation (6) in all treatments. The release rate constant k of eugenol in freeze-dried powder at a RH of 90.6% and storage temperature of 40 C was 5.7-times larger than that at RH of 56.0% and 20 C. The increase in k value was followed by high RH and storage temperature. In fact, this is the complex that is not stable and allows the release of eugenol and could be rapidly released from the freeze-dried complexes as an increment of RH and storage temperature. All of the n values estimated were near 0.54 (p 5 0.05), which indicated that the release of eugenol corresponded to the diffusion limited system. The parameter n in Figure 5(a) was 0.42, 0.48 and 0.52 for RH of 56.0, 75.1 and 90.6%, respectively, during storage at 20 C. On the other hand, higher values of n were obtained at 40 C, which appeared 0.48, 0.53 and 0.56 for RH of 56.0, 75.1 and 90.6%, respectively in Figure 5(c). The k-value can be a significant index to indicate the rate constant based on the data for eugenol remaining depending on time or storage temperature. Comparison of n and k value among the different RHs and storage temperatures revealed that the k and n values were high, suggesting that the eugenol can be quickly released according to the dissociation of CD due to the increment of water sorption at high RH and storage temperature. On the contrary, the low k and n values indicate that eugenol can be slowly released from the -CD-eugenol complex. Water uptake at a high RH can dissolve -CD and dissociate the -CD-eugenol complex during storage. The release of entrapped flavouring materials is closely related with the adsorption of water in the wall materials and hydration of the powder (Whorton and Reinecciu 1995). The water begins to penetrate the surface wall of the particles, followed by cracks that appear near the surface of the particle and subsequent release of the flavour. However, the reason for these observations is still not well understood and further research in this area is needed (Shiga et al. 2001).

From equation (6), one can obtain parameter n as a slope by plotting ln(lnR) vs lnt, and the release rate constant k from the interception at ln t 0. The release time-courses of eugenol at different conditions were analysed by equation (6), plotting ln(lnR) against ln t (Figure 5). Their estimated parameters of the equation are listed in Table 2. The release of eugenol fitted well to

(a)

0.5 0

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ln (ln R)

0.5 1 1.5 2 2.5 7 8 9 ln t (s) 10 11

(b)

1 0.5 0

ln (ln R)

0.5 1 1.5 2 2.5 7 8 9 ln t (s) 10 11

(c)

1 0.5 0

ln (ln R)

0.5 1 1.5 2 2.5 7 8 9 ln t (s) 10 11

Analysis of the activation energy by Arrhenius equation The Arrhenius equation (equation 7) defines the quantitative basis of the relationship between the activation

Figure 5. Correlation of release time-course of eugenol oil under storage temperatures of (a) 20 C, (b) 30 C and (c) 40 C by Avrami equation. (^) is RH of 56.0%; (#) is RH of 75.1% and (m) is RH of 90.6%.

Table 2. Parameters of Avrami equation describing the release of eugenol at various RH and storage temperature. 20 C RH* (%) 56.0 3.2 75.1 0.4 90.6 0.8 n 0.423 0.481 0.523 k 105 (s1) 1.069 2.180 3.976 r2 0.941 0.942 0.953 n 0.466 0.503 0.519 30 C k 105 (s1) 1.648 2.750 4.689 r2 0.928 0.951 0.959 n 0.477 0.531 0.558 40 C k 105 (s1) 1.935 3.659 6.103 r2 0.930 0.960 0.968

*Relative humidity.

Eugenol encapulsation by molecular inclusion

energy and the rate of chemical reaction (Li et al. 2007). To study the calculation of activation energy Ea for the release of eugenol, the release rate constants k were calculated and plotted against the reciprocal of absolute temperature according to the Arrehenius equation. Activation energy can be expressed as k A expEa =RT 7

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activation energy for the release of eugenol decreased with the increasing of RHs in Figure 6. From the Arrhenius equation, the activation energy values were calculated as 22.75, 19.65 and 16.31 kJ mol1 at RH of 56.0, 75.1 and 90.6%, respectively, during storage at 20, 30 and 40 C, respectively. This means that the release of eugenol occurred easier in the higher RH.

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where k is the release rate constant that depends on the pre-exponential factor A and activation energy Ea. R is the universal gas constant (kJ mol1 K1) and T is the absolute temperature (K ). The activation energy was calculated from the logarithmic form of the Arrhenius equation. From equation (8) the parameter Ea is calculated as a slope by plotting ln k vs 1/T. ln k ln A Ea =R1=T 8

Morphological observation Prior to observing the morphology of encapsulated eugenol, TEM of eugenol as a core material and -CD as a wall material was done following staining with 2% PTA. This staining method is useful for discerning morphological characteristics of -CD encapsulated eugenol. According to Figure 7, the stained -CD-eugenol complex appeared brighter than the surrounding stain. The encapsulated eugenol was evident as small spheres with a diameter of 50120 nm. Large aggregates of the complex were presented as an agglomerated form with a diameter of 250 500 nm. Previous TEM analysis indicated that -CD possesses abnormally low water solubility (18.5 g L1) (Dessoudexi et al. 2001), especially when compared to and -CD, since it can easily aggregate. The above results suggest that the irregular form of the -CD-eugenol complex is a consequence of the self-assembly of CD in water. Inclusion complexes of -CD can form larger aggregates in aqueous solution at room temperature, which precipitate as a consequence of self-assembly, or lipophilic water-insoluble active substances through non-inclusion complexation or micelle-like structures (Loftsson et al. 2004). From our images, we suppose that the -CD-eugenol molecular inclusion was dispersed as a monomer in liquid solution below their solubility and then, as the complex dries on the electron microscope grid, self-assembly of the monomer might occur causing aggregation of complex. This would explain why we

According to Agbenin and Raij (2001), activation energy is a measure of the energy to be overcome, and thus can be interpreted as the relative index of the binding strength of host with guest. The release rate constants k was raised with the increase of temperature. Furthermore, the

10 ln k 11 12 3.15

3.20

3.25

3.30 1/T (X103K1)

3.35

3.40

3.45

Figure 6. Relation between release rate constant and temperature in eugenol using Arrhenius equation. (^) is RH of 56.0%; (#) is RH of 75.1% and (m) is RH of 90.6%.

Figure 7. TEM observation of -CD-eugenol complexes after PTA staining at a magnification of 8000 (left) and 12 000 (right).

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found no correlation between the Nanosizer and TEM observations.

Conclusions
The present study demonstrates the utility of molecular inclusion in the -CD encapsulation of eugenol. The storage stability of eugenol at various RHs and storage temperatures was low, with substantial release of eugenol occurring during the initial stage of storage at all storage conditions. The release rate of eugenol in the freeze-dried powder was also significantly influenced by increasing RH and storage temperature. Furthermore, the activation energy for the release of eugenol increases with increasing RH and storage temperature. For these reasons, high water absorption promotes eugenol release rate, resulting in a dissociation of the -CD as a host from the -CD-eugenol complex.

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Declaration of interest: This work was financially supported by grant no. U2.3 E/UR3G/5G031/5A802 by the r Wehrtechnik und Beschaffung German Bundesamt fu (BWB), Koblenz, Germany.

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