Encapsulation of hydroxycitronellal in β -cyclodextrin and the characteristics of the inclusion complex

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INTRODUCTION
Hydroxycitronellal (with a synonym: 3,7-dimethyl-7-hydroxyoctanal), which has been reported found in pepper, is usually synthesized by hydration of citronellal 1 .Its chemical structural formula is shown in Figure 1.
Hydroxycitronellal has a sweet, fl owery odor reminiscent of lily of valley and linden blossom.As a common fl avor and fragrance material, hydroxycitronellal has been widely used in foods, beverages, perfumery and cosmetics.At 50 ppm, hydroxycitronellal has a taste characteristic of green, waxy, sweet, melon and fl oral notes 2 .Due to its fi ne aroma, hydroxycitronellal is used in large quantities in the fl avor and fragrance industry.As a composition of fl avors, a small quantity of hydroxycitronellal can be adopted in blending fl avors such as cucumber, melon, apple, and mango to impart a fresh feeling.A large quantity of hydroxycitronellal usually is used in many perfume compositions for creating lily of the valley and linden blossom notes due to its fi ne fl owery odor.Hydroxycitronellal can also be used in imitating blossom fragrances such as cyclamen, honeysuckle, and lily 1, 2 .Most importantly, hydroxycitronellal can present an anxiolytic-like effect and can be used to treat anxiety 3 .Furthermore, its anxiolytic-like activity does not present adverse effects such as neuromuscular impairment and sedation commonly caused by benzodiazepines 3 .
Hydroxycitronellal is a slightly viscous, colorless liquid at room temperature.It is soluble in ethyl alcohol, propylene glycol and most fi xed oils.But hydroxycitronellal is insoluble in glycerin and water 2 .In some cases, good water solubility is required.For example, some beverages require fl avors to have good water solubility.Hydroxycitronellal is a volatile liquid and it cannot last long.However, lastingness is one of the most important properties of fl avor and fragrance.The performance of fragrance and fl avor is often measured by its lastingness and pleasantness 4 .
Although the application scope of hydroxycitronellal is wide, it is somewhat unstable.Toward alkali and acid, hydroxycitronellal is relatively unstable.In ethyl alcohol, it tends to form acetal 5 .Furthermore, hydroxycitronellal is prone to polymerization reaction and is easily oxidized when exposed to air 6 .Therefore, there is a need to improve the stability of hydroxycitronellal.
Except for its drawbacks such as water-insolubility, instability and volatility, hydroxycitronellal is often considered as an allergen.It showed a positive patch test 7 .Calnan 8 investigated cases of allergic contact dermatitis to perfumes.Using a patch test, Calnan found that hydroxycitronellal could elicit an allergic contact dermatitis of the face.The potential of hydroxycitronellal to elicit human sensitization reaction or induce hypersensitivity was also investigated by Steltenkamp et al. 9 .They found that, at exposure concentrations greater than 5%, sensitization to hydroxycitronellal appeared to be induced.For its sensitizing potential in human skin, hydroxycitronellal was studied by Ford et al.They found that induction at 12% produced sensitization in 8 of 11 tests.When 100 of the non-reacting panelists were re-exposed in the same way, allergic sensitization reactions appeared during the induction period with concentrations as low as 2.5% 10 .In β-cyclodextrin aqueous solution.2 g of hydroxycitronellal (dissolved in 10 ml ethyl alcohol) was added slowly to the β-cyclodextrin aqueous solution.The mixture was kept at 35 o C in a thermostatic water bath and was stirred for another 3 h.After the complexation reaction fi nished, the mixture was stored in a refrigerator and the temperature of the refrigerator remained constant at 5 o C.After 24 h, the formed precipitate was recovered by vacuum fi ltration, which was then washed with ethyl alcohol 3 times to remove hydroxycitronellal on the surface of β-cyclodextrin.A freezer dryer (FD-1A-50, Shanghai Bilon Instrument Manufacturing Co., Ltd.) was adopted to dry the products.The dry sample was stored in a desiccator for further analysis.

Characterization of the surface shape of hydroxycitronellalβ-cyclodextrin inclusion complex
The surface shape of hydroxycitronellal-β-cyclodextrin inclusion complex was characterized using optical microscopy (LW600LJT, Shanghai Cewei Photoelectric Technology Co., LTD).The images of the samples were obtained by a built-in charge-coupled device (CCD) camera.

Characterization of hydroxycitronellal, β-cyclodextrin, physical mixture and hydroxycitronellal-β-cyclodextrin inclusion complex by FTIR
The FTIR determination method was adopted as described in reference 21 .Hydroxycitronellal, β-cyclodextrin, physical mixture, and the inclusion complex were determined by a Vertex 70 Fourier Transform Spectrometer (Bruker, German).The FTIR spectra were recorded in the frequency range of 400 to 4000 cm -1 .

Characterization of hydroxycitronellal, β-cyclodextrin, physical mixture and hydroxycitronellal-β-cyclodextrin inclusion complex by TGA
The weight loss and the rate of weight loss of hydroxycitronellal, β-cyclodextrin, physical mixture and the inclusion complex were performed with a thermogravimetric analyzer (TGA-Q5000IR, TA Instruments, USA).Approximately 5 mg of the sample was loaded in a ceramic crucible.During the pyrolysis process, N 2 was used and its fl ow was 20 ml/min to avoid oxidation.The heating rate used in the experiment was 10 o C/ min and the pyrolysis temperature was in the range of 30 o C to 600 o C.

Determination of the loading capacity of hydroxycitronellal
The loading capacity of hydroxycitronellal is defi ned as the mass ratio of hydroxycitronellal to hydroxycitronellalβ-cyclodextrin inclusion complex.TGA can provide the mass loss of the samples with the increase of temperature.During the heating process of β-cyclodextrin and hydroxycitronellal-β-cyclodextrin inclusion complex, mass loss occurs because of volatiles.As a volatile, hydroxycitronellal can release from its inclusion complex when the temperature reaches a certain point.The loading capacity of hydroxycitronellal can be obtained from the mass loss difference between hydroxycitronellal-β-cyclodextrin inclusion complex and β-cyclodextrin.
recent years, the safety of the use of hydroxycitronella in consumer products has attracted widespread attention.How to reduce the irritation and improve the safety of the hydroxycitronellal is of great interest.
β-Cyclodextrin can be used in the reduction of skin irritation 11 .β-cyclodextrin is nonallergenic and nontoxic.It is not regarded as irritating to the skin.In some cases, the complexation of a guest molecule with β-cyclodextrin can reduce a possible irritating effect of a guest molecule 12 .It was reported that the skin irritation of retinol could be relieved and the service life of retinol products could be extended 13 .Cyclodextrin molecules have hollow hydrophobic cavities and hydrophilic outer surfaces.Encapsulation of hydrophobic organic molecules in the cavities of cyclodxtrin molecules can protect these hydrophobic organic molecules, improve their water solubility and stability, and prolong their shelf-life activity 14- 16 .Kayaci et al. 17 found that high thermal stability and enhanced surability of geraniol were achieved by encapsulation of geraniol in cyclodextrins.Siva et al. reported that signifi cant enhancement of isoeugenol water solubility was confi rmed after the formation of the inclusion complex with 2-hydroxypropyl-β-cyclodextrin 18 .Real et al. reported that a signifi cant improvement in the aqueous solubility of triclabendazole by complexation with cyclodextrins was achieved 19 .
The major drawbacks regarding the use of hydroxycitronellal are related to water insolubility, volatility, instability, and sensitization.In this work, β-cyclodextrin was adopted as wall material to encapsulate hydroxycitronellal to overcome these concerns.Hydroxycitronellalβ-cyclodextrin inclusion complex was prepared and the product was characterized by optical microscope, Fourier transform infrared spectroscopy (FTIR), and thermogravimetric analysis (TGA).The interaction of hydroxycitronellal and β-cyclodextrin was investigated by molecular simulation (MM).The optimal structure of the inclusion complex and binding energy (BE) were obtained by MM2 calculation.

Materials
Hydroxycitronellal (slightly viscous colorless liquid, molecular weight: 172, chemical pure) was provided by Pu-Jie Aroma Chemicals (Shanghai) Co., Ltd.β-cyclodextrin (molecular weight: 1135, white powder, chemical pure) and ethyl alcohol (analytically pure) were provided by Sinopharm Chemical Reagent Co. Ltd.Without further purifi cation, all these materials were directly used as received.Deionized water was used throughout the experiment.

Hydroxycitronellal-β-cyclodextrin inclusion complex preparation
The precipitation method, with some modifi cations, as elucidated in literature 20 was adopted to prepare hydroxycitronellal-β-cyclodextrin inclusion complex.A digital display electric stirrer (JB200-S, Shanghai Suoying Instrument Equipment Co., Ltd.) was turned on.Then, 11 g of β-cyclodextrin was weighed and added slowly to the deionized water (600 ml) to form of β-cyclodextrin and hydroxycitronellal-β-cyclodextrin inclusion complex were shown in Figure 3.

Investigation of the interaction of hydroxycitronellal and β-cyclodextrin by molecular simulation calculations
With some modifi cations, the molecular simulation method described in references 22, 23 was used to study the interaction of hydroxycitronellal and β-cyclodextrin.Hydroxycitronellal-β-cyclodextrin inclusion complex structure optimization and binding energy calculation, defi ned as Eq. ( 1), were performed by Chem 3D Ultra (CambridgeSoft Corporation, MA, USA). ( where BE is binding energy, E guest is the minimum total energy of hydroxycitronellal, E host is the minimum total energy of β-cyclodextrin, and E complex is the minimum total energy of hydroxycitronellal-β-cyclodextrin inclusion complex.
The total energy is formed from stretch energy, bend energy, stretch-bend energy, torsion energy, non-1,4-Van der Walls energy, 1,4-Van der Walls energy, and dipole/ dipole energy.
The docking strategy was to push hydroxycitronellal molecule stepwise through the β-cyclodextrin orifi ce along the Z axis as shown in Figure 2. The minimum total energy of the complex was calculated at each step and MM2 calculation was used.The Z coordinate of C6 atom in hydroxycitronellal molecule was used to mark the relative position between hydroxycitronellal and β-cyclodextrin.

The surface shapes of β-cyclodextrin and hydroxycitronellal-β-cyclodextrin inclusion complex
Hydroxycitronellal-β-cyclodextrin inclusion complex, and cyclodextrin alone, processed for the preparation of the inclusion complexes with hydroxycitronellal, were investigated with an optical microscope.The surface shapes As shown in Figure 3, the size of most β-cyclodextrin and hydroxycitronellal-β-cyclodextrin inclusion complexes is in the range of 2 to 10 μm.Although relatively large aggregates can be observed from Figure 3(a), the shape β-cyclodextrin is similar to that of hydroxycitronellal-βcyclodextrin inclusion complex.During the process of preparation of hydroxycitronellal-β-cyclodextrin inclusion complexes, or cyclodextrin alone processed as for the preparation of the inclusion complexes with hydroxycitronellal, the products formed aggregates which took on different shapes.Some products have irregular shapes, while some of the products are quadrangular or rhombic in appearance as shown in Figure 3. Cyclodextrin is not a cylindrical molecule but rather a slightly conical circular molecule.β-cyclodextrin has a truncated coneshaped molecular structure.In aqueous suspension, hydroxycitronellal-β-cyclodextrin inclusion complex molecules may line up in staggered parallel arrangement.The schematic diagram of hydroxycitronellal-βcyclodextrin inclusion complex molecules in staggered parallel arrangement is shown in Figure 4.
In this way, some products with the shapes of parallelogram and rhombus can be formed.This structure has been reported in the literature 24 .It was reported that β-cyclodextrin inclusion complexes to line up in staggered parallel arrangement were energetically favorable 25 .Therefore, the appearance of some aggregates of hydroxycitronellal-β-cyclodextrin inclusion complex is parallelogram or rhombus as shown in Figure 3.
In the FTIR spectrum of β-cyclodextrin, the broad peak due to the O-H stretching vibration of hydroxyl group absorption appears at 3334 cm -1 .The peaks appearing at 1154 cm -1 and 1028 cm -1 can be assigned to the C-O stretching vibration.The FTIR spectrum of hydroxycitronellal-β-cyclodextrin inclusion complex shows resemblance to that of β-cyclodextrin.In the FTIR spectrum of hydroxycitronellal-β-cyclodextrin inclusion complex, the broad peak due to the O-H stretching vibration of hydroxyl group absorption also appears at 3334 cm -1 , and the peaks due to the C-O stretching vibration appear at 1155 cm -1 and 1130 cm -1 .However, compared with the FTIR spectrum of β-cyclodextrin, two new peaks appear at 2978 cm -1 and 2905 cm -1 respectively in the FTIR spectrum of hydroxycitronellal-βcyclodextrin inclusion complex.It may be caused by the asymmetrical and symmetrical C-H stretching absorption of CH 3 in hydroxycitronellal molecule, which resulted in the peaks at 2970 cm -1 and 2876 cm -1 respectively in the FTIR spectrum of hydroxycitronellal.In the FTIR spectrum of physical mixture of hydroxycitronellal and β-cyclodextrin, two small peaks still occur at 2970 cm -1 and 2876 cm -1 respectively.Furthermore, the peak at 1724 cm -1 due to aldehyde C=O stretching vibration in the FTIR spectrum of hydroxycitronellal disappears in the FTIR spectrum of hydroxycitronellal-β-cyclodextrin inclusion complex 27 .In the FTIR spectrum of physical mixture, the peak at 1724 cm -1 still appeared.The peaks occurring at 1458 cm -1 and 1374 cm -1 in the FTIR spectrum of hydroxycitronellal still appear in the FTIR spectrum of the physical mixture, but disappear in the FTIR spectrum of hydroxycitronellal-β-cyclodextrin inclusion complex.These changes in FTIR spectrum suggest the successful encapsulation of hydroxycitronellal in β-cyclodextrin.

The TG and DTG curves of hydroxycitronellal, β-cyclodextrin, physical mixture, and hydroxycitronellal-βcyclodextrin inclusion complex
TGA can be used to investigate the thermal stability and lastingness of the guest molecule encapsulated in β-cyclodextrin 28 .The TG and DTG curves of hydroxycitronellal, β-cyclodextrin, physical mixture, and hydroxycitronellal-β-cyclodextrin inclusion complex obtained were shown in Figure 6.
As shown in Figure 6, from 32 o C to 68 o C, a slight mass loss of hydroxycitronellal can be observed in the TG curve hydroxycitronellal.In this stage, hydroxycitronellal vaporized slowly.With the increase of temperature, hydroxycitronellal vaporized quickly, and the rate of mass loss attained its maximum value at 133.6 o C. A strong peak appeared at this temperature in the DTG curve of hydroxycitronellal.Although the atmospheric boiling point of hydroxycitronellal is 241 o C, hydroxycitronellal almost vaporized completely at the temperature of 180 o C.
Compared with the TG curve of hydroxycitronellal, from 32 o C to 68 o C relatively high mass losses can be observed in the TG curves of β-cyclodextrin, physical mixture and hydroxycitronellal-β-cyclodextrin inclusion complex.These mass losses were mainly due to the desorption of bound water on the surface of β-cyclodextrin.From 68 o C to 150 o C, relatively high mass loss can be observed again in the TG curve of the physical mixture.The temperature of the initial decomposition of β-cyclodextrin is about 269.5 o C. A more interesting thing was that the mass loss of hydroxycitronellal-βcyclodextrin inclusion complex was higher than that of β-cyclodextrin during the initial period of decomposition of β-cyclodextrin.From 269.5 o C to 286.6 o C, the mass losses of hydroxycitronellal-β-cyclodextrin inclusion complex and β-cyclodextrin were 9.2% and 3.1% respectively.The difference, 6.1%, can also be attributed to the release of hydroxycitronellal.When β-cyclodextrin decomposed, the release of hydroxycitronellal from its inclusion complex still occurred.Free hydroxycitronellal almost vaporized completely when the temperature reached 180 o C under the experimental conditions.Hydroxycitronellal-β-cyclodextrin inclusion complex still gave off hydroxycitronellal at 286.6 o C. It indicated that hydroxycitronellal was successfully encapsulated in β-cyclodextrin.Compared with free hydroxycitronellal, the release of hydroxycitronellal from its inclusion complex at a relatively higher temperature supported the formation of an inclusion complex.It also revealed that the thermal stability and lastingness of hydroxycitronellal were improved by the formation of the inclusion complex.From 180 o C to 286.6 o C, the mass loss difference between hydroxycitronellal-β-cyclodextrin inclusion complex and β-cyclodextrin was about 8.5%.The loading capacity of hydroxycitronellal, which is defi ned as the mass ratio of hydroxycitronellal to hydroxycitronellal-β-cyclodextrin inclusion complex, was 8.5%.

The binding energy and the optimized structure of hydroxycitronellal-β-cyclodextrin inclusion complex obtained by molecular simulation
Hydroxycitronellal molecule entered the cavity of β-cyclodextrin molecule along the Z-axis.When hydroxycitronellal molecule was at different position in the cavity of β-cyclodextrin molecule, the obtained binding energies by MM2 calculation were different.The Z coordinate of C6 atom in hydroxycitronellal molecule was used to mark the relative position between hydroxycitronellal and β-cyclodextrin.The binding energy change with the Z coordinate of C6 atom in hydroxycitronellal molecule was shown in Figure 7.
As shown in Figure 7, the values of binding energies are negative.It means that energy was released during the When hydroxycitronellal molecule entered the cavity of β-cyclodextrin molecule, energy was released during this process.The maximum value of energy release was 151.2 kJ/mol when the Z coordinate of C6 is -2.5 × 10 -10 m.The more energy was released, the more stable the inclusion complex was formed.Therefore, the structure of the hydroxycitronellal-β-cyclodextrin inclusion complex with the minimum energy is the most stable one.The optimal structure with the minimum energy obtained by MM2 calculation is shown in Figure 8.
As shown in Figure 8, hydroxycitronellal molecule was encapsulated in the cavity of β-cyclodextrin molecule.Two hydrogen bonds can be observed in the hydroxycitronellalβ-cyclodextrin inclusion complex molecule.One hydrogen bond was formed between the oxygen atom of aldehyde group in hydroxycitronellal molecule and the hydrogen atom of hydroxy group in β-cyclodextrin molecule, and the other hydrogen bond was formed between the hydrogen atom of hydroxy group in hydroxycitronellal molecule and the oxygen atom of hydroxy group in β-cyclodextrin molecule.The formation of hydrogen bond is one reason for the stability of hydroxycitronellal-β-cyclodextrin inclusion complex.It can be seen from Figure 6 that aldehyde group in hydroxycitronellal molecule is encapsulated in the cavity of β-cyclodextrin molecule after the formation of hydroxycitronellal-β-cyclodextrin inclusion complex.Therefore, the FTIR peak at 1724 cm -1 due to aldehyde C=O stretching vibration in the FTIR spectrum of hydroxycitronellal disappeared in the FTIR spectrum of hydroxycitronellal-β-cyclodextrin inclusion complex.It can also be seen from Figure 8 that the methyl group in hydroxycitronellal molecule is not completely encapsulated in the cavity of β-cyclodextrin molecules.The methyl group in hydroxycitronellal molecule is sticking out.Therefore, compared with the FTIR spectrum of β-cyclodextrin, two new peaks at 2978 cm -1 and 2905 cm -1 appeared in the FTIR spectrum of hydroxycitronellalβ-cyclodextrin inclusion complex because of the asymmetrical and symmetrical C-H stretching absorption of the uncovered CH 3 in hydroxycitronellal molecule.The results of molecule simulation are helpful to understand the interaction between hydroxycitronellal and β-cyclodextrin.

The assembly of hydroxycitronellal-β-cyclodextrin inclusion complex molecules obtained by molecular simulation
β-Cyclodextrin can form aggregates in water.For a better understanding of the assembly of hydroxycitronellalβ-cyclodextrin inclusion complex molecules, molecular simulation was also performed.The structure of hydroxycitronellal-β-cyclodextrin inclusion complex molecule was optimized previously by MM2.This optimal structure was used as starting geometry to investigate the assembly of two hydroxycitronellal-β-cyclodextrin inclusion complex molecules.Three types of structures for hydroxycitronellal-β-cyclodextrin inclusion complex were evaluated: perpendicular, staggered parallel and ideally parallel orientation of the inclusion complexes.The energy changes (∆E) with the distances of one hydroxycitronellal-β-cyclodextrin inclusion complex molecule from another were shown in Figure 9.
As shown in Figure 9, when the distance of the two hydroxycitronellal-β-cyclodextrin inclusion complex molecules is in the range of 7 × 10 -10 to 8 × 10 -10 m, the energy changes are approximately equal to 0 kJ/mol.The interaction of the two hydroxycitronellal-β-cyclodextrin inclusion complex molecules is very weak.For the three orientations, as the two hydroxycitronellal-β-cyclodextrin inclusion complex molecules approach each other from 6 × 10 -10 to 2.6 × 10 -10 m, the energy of the system tends to go down.The values of ∆E are negative.The interaction of hydroxycitronellal-β-cyclodextrin inclusion complex molecules to each other is attractive.When the distances of the two molecules are at 2.6 × 10 -10 , 2.5 × 10 -10 , 2.6 × 10 -10 m for perpendicular, staggered parallel and ideally parallel orientation respectively, the energy changes reach their bottoms.These values are -28.6,-69.9, -66.8 kJ/mol respectively.Among them, the minimum value is found for the staggered parallel arrangement.With the decrease of the distances of the two molecules, the values of ∆E increase.When the distances of the two molecules are less than 2.0 × 10 -10 m, the energy changes increase rapidly and the values of ∆E are positive.Highly positive value of ∆E means that the interaction of hydroxycitronellal-β-cyclodextrin inclusion complex molecules to each other is strongly repulsive.
Based on Figure 9, it can be inferred that it is energetically favorable for hydroxycitronellal-β-cyclodextrin inclusion complex molecules to line up in staggered parallel arrangement.Thus, it resulted in the structure shown in Figure 4. Similar structures were also reported for (-)-menthol-β-cyclodextrin inclusion complex and dimethyl sulfi de-β-cyclodextrin inclusion complex 29, 30 .Some of these products were in the shape of parallelograms.

CONCLUSIONS
Hydroxycitronellal was successfully encapsulated in the cavity of β-cyclodextrin and hydroxycitronellal-βcyclodextrin inclusion complex was prepared.The loading capacity of hydroxycitronellal was about 8.5%.The products take on different shapes (parallelogram, rhombus, and irregular shapes).The size of most of the products is in the range of 2 to 10 μm.The peak at 1724 cm -1 due to aldehyde C=O stretching vibration in the FTIR spectrum of hydroxycitronellal disappears in the FTIR spectrum of hydroxycitronellal-β-cyclodextrin inclusion complex.Two new peaks at 2978 cm -1 and 2905 cm -1 appeared in the FTIR spectrum of hydroxycitronellal-β-cyclodextrin inclusion complex because of the asymmetrical and symmetrical C-H stretching absorption of the uncovered CH 3 in hydroxycitronellal molecule.These changes in FTIR spectrum suggest the successful encapsulation of hydroxycitronellal in β-cyclodextrin.Thermal stability and hydroxycitronellal release characteristics were obtained by thermal analysis.Free hydroxycitronellal almost vaporized completely when the temperature reached 180 o C. Hydroxycitronellal-β-cyclodextrin inclusion complex still gave off hydroxycitronellal at 286.6 o C. It also revealed that the thermal stability and lastingness of hydroxycitronellal were improved by the formation of the inclusion complex.The optimal structure of the inclusion complex and binding energy were obtained by molecule simulation.When the Z coordinate of C6 is -2.5 × 10 -10 m, the binding energy is minimum (-151.2kJ/mol).The assembly of hydroxycitronellal-β-cyclodextrin inclusion complex molecules was also investigated by molecule simulation.It revealed that β-cyclodextrin inclusion complexes to line up in staggered parallel arrangement were energetically favorable.These basic data are useful to understand the interaction between hydroxycitronellal and β-cyclodextrin.

Figure 2 .Figure 3 .
Figure 2. The movement direction of hydroxycitronellal to the cavity of β-cyclodextrin along the Z axis

Figure 8 .
Figure 8.The optimal structure with the minimum energy obtained by MM2 calculation ((a) β-cyclodextrin and hydroxycitronellal display mode: ball and stick (b) β-cyclodextrin display mode: stick, hydroxycitronellal display mode: ball and stick)

Figure 9 .
Figure 9.The energy changes with the distances of one hydroxycitronellal-β-cyclodextrin inclusion complex molecule from another.Orientation of the inclusion complexes: perpendicular (a), staggered parallel (b), and ideally parallel (c)