Investigation of monoterpenes complexation with hydroxypropyl-β-cyclodextrin

ORIGINAL ARTICLE

Investigation of monoterpenes complexation with hydroxypropyl-b-cyclodextrin

Miriana Kfoury • Lizette Auezova •

Sophie Fourmentin • Helene Greige-Gerges

Received: 12 November 2013 / Accepted: 22 January 2014

� Springer Science+Business Media Dordrecht 2014

Abstract In this study, we investigated the inclusion

complexation of 2-hydroxypropyl-b-cyclodextrin (HP-b-

CD) and eight monoterpenes (eucalyptol, geraniol, limo-

nene, linalool, a-pinene, b-pinene, pulegone, and thymol)

in aqueous solution and solid state. The formation con-

stants (Kf) of inclusion complexes were determined using

fluorescence spectroscopy and static headspace gas chro-

matography. The results indicated the formation of 1:1

inclusion complexes between HP-b-CD and all studied

guests. A linear relationship was found between Kf values

and the hydrophobic character of the monoterpenes

expressed as logP. Solid complexes were prepared by the

freeze-drying method in a 1:1 (HP-b-CD:monoterpene)

molar ratio. Physicochemical characterization of solid

inclusion complexes was carried out using Fourier trans-

form infrared spectroscopy and differential scanning calo-

rimetry. Finally, the encapsulation efficiency (EE%) of HP-

b-CD was determined using HPLC analysis. Noticeable

difference in the EE% was observed between monoterpene

hydrocarbons and oxygenated monoterpenes. These results

suggested that complexation with HP-b-CD could be a

promising strategy to enlarge the application of monoter-

penes in cosmetic, pharmaceutical and food industries.

Keywords Hydroxypropyl-b-cyclodextrin �Monoterpenes � Formation constant � Fluorescence � Static

headspace gas chromatography � Encapsulation efficiency

Introduction

Essential oils (EOs) have been used for centuries in food,

perfumery and traditional medicine [1–3]. They represent

complex mixtures of volatile aroma compounds synthe-

sized in plants to ensure their protection against various

pathogens. Monoterpenes obtained from the condensation

of two isoprene units, are a large group of EOs constitu-

ents. They can be classified into monoterpene hydrocar-

bons and oxygenated monoterpenes such as alcohols,

esters, aldehydes, ketones, ethers, and phenols. Chemical

composition and properties of EOs have been subjects of

numerous studies. Moreover, antibacterial [4], antifungal

[5], antiviral [6], anticancer [7], insecticidal [8], antioxi-

dant and anti-inflammatory [9] activities of EOs and their

constituents were reported. However, their volatility and

low water solubility limit their applications.

Complexation with cyclodextrins (CDs) is an effective

tool to increase aqueous solubility of these molecules and

to protect them against oxidation, thermal degradation, and

evaporation [10]. Moreover, the complexation reversibility

generates controlled release systems of the encapsulated

aroma [11, 12]. CDs are cyclic oligosaccharides, consisting

of six, seven or eight glucopyranose units (named a-, b-

and c-CD, respectively). A basket-shaped structure of CDs

with a hydrophilic outer surface and a relatively hydro-

phobic cavity explain their ability to act as molecular cage

for hydrophobic guests (G). CDs form inclusion complexes

with a wide range of organic compounds, which enter

partly or entirely into their lipophilic cavity. The strength

M. Kfoury � L. Auezova (&) � H. Greige-Gerges

Bioactive Molecules Research Group, Department of Chemistry

and Biochemistry, Faculty of Sciences-2, Doctoral School of

Science and Technology, Lebanese University, Fanar, Lebanon

e-mail: [email protected]; [email protected]

M. Kfoury � S. Fourmentin (&)

Univ Lille Nord de France, 59000 Lille, France

e-mail: [email protected]

M. Kfoury � S. Fourmentin

ULCO, UCEIV EA 4492, 59140 Dunkerque, France

123

J Incl Phenom Macrocycl Chem

DOI 10.1007/s10847-014-0385-7

of host–G complexation depends mainly on the polarity

and geometric accommodation between CD cavity and G

molecule [10]. These parameters play a critical role in

determining the stoichiometry and formation constant (Kf)

of the inclusion complexes [13].

Among different CDs, b-CD is the most used due to its

suitable cavity size for a wide range of G molecules.

However, its application is limited because of its nephro-

toxicity and low aqueous solubility (1.8 % w/v, at 25 �C)

[1, 2]. b-CD derivatives like 2-hydroxypropyl-b-CD (HP-

b-CD) with improved safety [14, 15] and water solubility

(50 % w/v, at 25 �C) were synthesized [2]. HP-b-CD is one

of the most widely used CD derivatives in food, agriculture

and pharmaceutical fields [16].

The encapsulation of monoterpenes in HP-b-CD has

received increasing attention in recent years [12, 17–22].

However, studies concerning the determination of Kf val-

ues of HP-b-CD/monoterpene complexes are still few in

number.

In the present study, the complexation of HP-b-CD with

eight monoterpenes (eucalyptol, geraniol, limonene, linal-

ool, a-pinene, b-pinene, pulegone, and thymol) was

investigated in aqueous solution by fluorescence spectros-

copy and static headspace gas chromatography (SH-GC)

and in solid state by Fourier transform infrared spectros-

copy (FTIR) and differential scanning calorimetry (DSC).

Furthermore, the encapsulation efficiency (EE%) of HP-b-

CD towards the studied monoterpenes was evaluated using

HPLC.

Experimental

Materials

HP-b-CD (DS 5.6) was purchased from Sigma-Aldrich

(China). Eucalyptol, a-pinene, b-pinene, limonene, and

thymol were provided by Fluka Chemicals (Germany),

linalool, pulegone, eucalyptol and geraniol were obtained

from Sigma-Aldrich (Germany). Methanol was HPLC

grade (Sigma-Aldrich, Germany). All solutions were pre-

pared with ultrapure water obtained with ‘‘Heal force SNW

Ultra-Pure Water System’’ (China).

Methods

Fluorescence studies

In a final volume of 5 ml, an appropriate volume of HP-b-

CD 0.1 M was added to 0.5 ml of G compound

(1.5 9 10-4 M) to reach a range of concentrations from 1

to 7 9 10-3 M. The solutions were mixed in a thermostatic

shaker at 150 rpm for 24 h at 25 ± 2 �C. After

equilibrium, the suspensions were filtered through 0.45 lm

filters. For the filtrate, the fluorescence spectra were

acquired at maximum excitation and emission wavelengths

of each G compound. Fluorescence measurements were

performed on a F7000 fluorescence spectrophotometer

(Hitachi, High Technologies Corporation, Japan). The

experiments were carried out in duplicate.

Static headspace gas chromatography (SH-GC)

HP-b-CD/G complexation was studied by a SH-GC titra-

tion method developed in UCEIV for volatile organic

compounds [17]. Four concentrations of HP-b-CD were

used at constant G concentration (10 ppm). Measurements

were conducted using an Agilent G 1888 headspace

Autosampler. The sample was analyzed by GC (Perkin

Elmer Autosystem XL) equipped with a flame-ionization

detector using an Agilent J&W DB-5 column. The GC

settings were set as follows: detector temperature, 280 �C;

column temperature, 100 �C for eucalyptol, a-pinene and

b-pinene, 120 �C for limonene and linalool, 140 �C for

geraniol and pulegone, and 180 �C for thymol. An algo-

rithmic treatment was then applied to minimize the dif-

ference between the experimental and theoretical values of

the peak area leading to the adequate Kf value [17].

Preparation of solid HP-b-CD/G inclusion complexes

300 mg of HP-b-CD were dissolved in 20 ml of water and

the required amount of G was added (1:1 molar ratio).

Solutions were then stirred (150 rpm for 24 h at

25 ± 2 �C). The solutions obtained were filtered (0.45 lm

filter), frozen at -80 �C and lyophilized using a freeze

dryer (Martin Christ, Germany). The final dry powders

were stored at 4 �C prior to analysis.

Fourier transform infrared spectroscopy (FTIR)

FTIR spectra were obtained in the frequency range

between 4,000 and 400 cm-1 using Fourier Transform

Infrared spectrometer (FT/IR-6300, JASCO, Japan). FTIR

samples were prepared in the form of potassium bromide

pellets. The IR spectra of the freeze-dried complexes were

analyzed and compared with the spectra of HP-b-CD and G

alone.

DSC analysis

DSC analyses were carried out for monoterpenes, HP-b-

CD, their physical mixtures and inclusion complexes with a

Mettler-Toledo DSC821 differential calorimeter calibrated

with indium (Mettler-Toledo S.P.A., Milan, Italy). Each

sample (3–5 mg) was heated in a crimped aluminium pan


123

at a scanning rate of 10 �C/min between 50 and 300 �C

temperature range under a nitrogen flow of 40 ml/min. An

empty pan sealed in the same way was used as reference.

Reproducibility was checked by running the sample in

triplicate.

Encapsulation efficiency (EE%)

For each monoterpene, three batches of HP-b-CD/

monoterpene inclusion complex were prepared. 10 mg of

the freeze dried inclusion complex were dissolved in

methanol (10 ml). The samples were maintained in an

ultrasonic bath for 15 min and saturated NaCl solution

(0.05 ml) was then added to accelerate the precipitation.

Samples were centrifuged at 18,0009g at 4 �C for

30 min. 0.1 ml of supernatants were taken and added to

0.1 ml of an appropriate internal standard and 0.2 ml of

methanol. Samples were then analyzed by HPLC (Hit-

achi VWR L-2130) using a C18 column 5 lm

(150 9 4.6 mm, Agilent Zorbax Eclipse XDB-C18). The

column temperature was maintained at 25 �C. 20 ll of

the samples were injected into the HPLC column. The

mobile phase consisted of MeOH:H2O and was set as

follows: for linalool and pulegone, 65:35 (v/v), for

eucalyptol, geraniol and thymol, 70:30 (v/v), for limo-

nene, a-pinene and b-pinene 85:15 (v/v). The flow rate

was 1 ml/min. The absorbance was monitored continu-

ously at 204 nm since all the studied monoterpenes

absorb at this wavelength. Monoterpenes were identified

and quantified based on analytical standard curves. The

HPLC method was validated in terms of linearity,

repeatability and limit of detection. The EE% was cal-

culated using the following equation:

EE% ¼ EMC ðmgÞTMC ðmgÞ � 100; ð1Þ

where EMC stands for the experimental monoterpene

content which is the extracted amount of monoterpene

from its solid inclusion complex and TMC is the theoretical

monoterpene content which is the amount of monoterpene

initially used to prepare the solid inclusion complex.

Results and discussion

In the present study, inclusion complexes of HP-b-CD and

eight monoterpenes were prepared and characterized both

in aqueous solution and in solid state.

Investigation of inclusion complexes in aqueous

solution

Effect of HP-b-CD on the fluorescence behavior

of monoterpenes

Figure 1 shows the fluorescence emission spectra of pule-

gone and b-pinene (as example) in aqueous solution for

different HP-b-CD concentrations. All studied monoter-

penes, with the exception of thymol, were found to exhibit

no or low fluorescence yield in the absence of HP-b-CD.

Thymol showed noticeable emission intensity, due to its

aromatic ring moiety. Upon addition of HP-b-CD, the

fluorescence intensity of monoterpenes was enhanced and

increased gradually with its concentration. This intensity

increment can be obviously seen from the plots (F - F0)

against HP-b-CD concentration (Fig. 1), where F and F0

are the fluorescence intensities of inclusion complex and of

free G, respectively. The best fluorescence enhancement,

F0/F0 (F0, fluorescence intensity in the presence of the

highest HP-b-CD concentration), was obtained for the HP-

b-CD/linalool inclusion complex (Table 1). It might be due

to the better fit of linalool inside the HP-b-CD cavity in

0

500

1000

1500

2000

2500

290 310 330 350

a

F-F

(nm)

[HP- -CD] M

0

500

1000

1500

2000

2500

3000

280 300 320 340

b

[HP- -CD] M

F-F

(nm)

Fig. 1 Fluorescence spectra of a pulegone (1.5 9 10-5 M) and b b-

pinene (1.5 9 10-5 M) in absence and presence of HP-b-CD.

Concentration of HP-b-CD: curves (1 ? 8); (0, 1, 2, 3, 4, 5, 6 and

7 9 10-3 M). Inset plots of fluorescence intensity enhancement

(F - F0), as a function of HP-b-CD concentration


123

comparison to other monoterpenes [23]. The same

enhancement of fluorescence intensity upon complexation

with CDs has been previously demonstrated for a number

of hydrophobic compounds such as eugenol, anethole [24],

aflatoxins [25] and acyclovir [26].

The observed enhancement of fluorescence intensity

suggested that the studied monoterpenes had been suc-

cessfully incorporated inside the CD cavity, forming non-

covalent inclusion complexes. The fluorescence

enhancement might be due to several interactions

between CD and the encapsulated G. For instance, the

spatial restrictions inside the cavity might lead to an

increase of the G molecule rigidity [27] and the inhibi-

tion of its ‘‘free rotor’’ effect [28]. In addition, the

hydrophobic cavity of CD possesses high electron den-

sity produced by the non-bonding electron pairs of the

glycosidic oxygen bonds localized inside the cavity [27,

29]. Furthermore, encapsulation protected the fluorophore

from external quenchers and reduced its interactions with

water [28, 30]. The fluorescence intensity enhancement

was found to be associated with a bathochromic shift

(1–3 nm) of maximum emission wavelength (Table 1). A

hypsochromic shift of maximum excitation wavelength

was also observed (data not shown). These observations

provided an evidence of the G translocation from water

into the hydrophobic cavity of HP-b-CD, a less polar

medium [31]. Zhan et al. [32] reported similar wave-

length shift for eugenol encapsulated in different CDs.

Thus, for all studied monoterpenes, enhanced fluores-

cence and maximum wavelengths shifts were observed

upon HP-b-CD addition, proving their encapsulation in

HP-b-CD cavity.

Determination of formation constants (Kf)

Fluorescence studies

The formation constants (Kf) were estimated assuming a

1:1 stoichiometry for the inclusion complexes between HP-

b-CD and the monoterpenes. The following expression can

be written:

HP-b-CDþ G� HP-b-CD=G; ð2Þ

with overall Kf:

Kf ¼½HP-b-CD/G�½HP-b-CD�½G� : ð3Þ

The Kf values of the inclusion complexes were deter-

mined based on fluorescence intensity. Two different

approaches, the modified Benesi–Hildebrand’s [33] and

Scatchard’s [34] methods were applied.

According to Benesi–Hildebrand’s [33] approach, the

equation can be written as follows:

1

DF¼ 1

Kf � k � Q � ½G� �1

HP -b- CD½ � þ1

k � Q � ½G]: ð4Þ

[HP-b-CD] and [G] are, respectively, the initial con-

centrations of HP-b-CD and G. DF denotes the variation in

the fluorescence yield of G upon HP-b-CD addition. Q is

the quantum yield for the complex, and k stands for an

instrumental constant. The initial HP-b-CD concentration

was chosen at least ten times higher than that of the G. In

these conditions, the concentration of free HP-b-CD, at the

equilibrium, was presumed to be approximately equal to its

initial concentration [35]. Kf values were calculated by

Table 1 Emission wavelengths of monoterpenes in absence (k1) and

presence (k2) of HP-b-CD, Kf values of HP-b-CD/monoterpene

complexes calculated from Benesi–Hildebrand (Kf1), Scatchard (Kf2)

approaches and SH-GC (Kf3), fluorescence enhancement ratios (F0/F0), logP values, and encapsulation efficiency (EE%)

Guests k1 (nm) k2 (nm) Kf1 (M-1) Kf2 (M-1) Kf3 (M-1) F0/F0 LogP EE%

Eucalyptol 311 309 1,200 1,112 334a 5.24 3.13c 88.0 ± 2.8

Geraniol 311 309 1,320 1,064 712b 5.88 3.47c 85.1 ± 2.8

Limonene 305 304 1,667 1,700 2,787a 5.86 4.38d 15.4 ± 1.5

Linalool 307 304 1,500 1,260 596a 9.54 3.50d 90.3 ± 0.9

a-Pinene 307 305 2,000 1,842 1,637a 7.37 4.48d 24.9 ± 0.6

b-Pinene 308 305 1,667 1,671 3,151a 6.63 4.16d 20.9 ± 4.1

Pulegone 311 309 867 798 676a 5.79 2.76d 82.1 ± 1.9

Thymol 309 306 1,400 1,313 806 3.27 3.30d 90.6 ± 3.0

F0/F0 enhancement of the guest fluorescence in the presence of 7 9 10-3 M of HP-b-CDa See [18]b See [17]c See [37]d See [38]


123

dividing the intercept over the slope of the straight line

obtained in the double-reciprocal plot (Eq. (4)). The major

advantage of the Benesi–Hildebrand approach is that the

variables remain independent on the abscissa and ordinate

axis, whereas other graphical representations allow the

variables to become mixed.

According to Scatchard’s approach, the relationship

between fluorescence intensity enhancement (F - F0) and

the HP-b-CD concentration is given by the following

equation [34]:

ðF � F0ÞHP -b- CD½ � ¼ F1 � F0ð Þ � Kf � F � F0ð Þ � Kf ; ð5Þ

where F0 is the fluorescence intensity of G in the absence

of HP-b-CD, F? is the fluorescence intensity when the G is

essentially complexed with HP-b-CD, F is the fluorescence

at each HP-b-CD concentration, Kf is the formation con-

stant of the inclusion complex and [HP-b-CD] stands for

the initial concentration of HP-b-CD. For a 1:1 complex, a

plot of (F - F0)/[HP-b-CD] versus (F - F0) gives a

straight line, with a slope equal to -Kf.

For all studied monoterpenes, a good linearity was

observed by both treatments indicating 1:1 stoichiometric

ratio of the obtained inclusion complexes. The plots

obtained for pulegone were shown as example in Fig. 2.

Kf calculated by Benesi–Hildebrand and Scatchard

approaches (Table 1) showed similar values ranging from

867 to 2,000 M-1 and from 798 to 1,842 M-1, respectively

(Table 1). A good correlation was observed between Kf

values determined by the two approaches (r2 = 0.9169).

The obtained values of Kf fell between 200 and 2,000 M-1,

a characteristic range of 1:1 inclusion complexes [36]. It

should be noted that Kf values ranging from 200 to

10,000 M-1 were found to be appropriate for drug encap-

sulation in CDs, allowing its controlled release over time

[26].

Static headspace gas chromatography (SH-GC)

HP-b-CD/monoterpenes complexation was also investi-

gated using SH-GC. For all the studied monoterpenes,

experimental variations of their peak areas with HP-b-CD

concentration fit well with the 1:1 complex theoretical

curve (Fig. 3). This indicated that the stoichiometry of the

obtained inclusion complexes was 1:1, confirming the

R = 0.9845

0

0.002

0.004

0.006

0.008

0.01

0.012

0 200 400 600 800 1000 1200

1/F

-F

1/[HP- -CD] (M-1)

R = 0.9573

0

25000

50000

75000

100000

0 50 100 150 200

(F-F

0)/

HP

--C

D]

(F-F0 )

a

b

Fig. 2 a Benesi–Hildebrand and b Scatchard plots for HP-b-CD/

pulegone inclusion complex

0

400000

800000

1200000

1600000

2000000

0.000 0.002 0.004 0.006 0.008 0.010 0.012

Are

a (µ

V.s)

[HP- -CD] M

Fig. 3 Representation of the experimental points (filled diamonds)

obtained for HP-b-CD/thymol compared with theoretical titration

curve (dashed lines) for a 1:1 complex


123

results obtained by fluorescence spectroscopy. Kf were

calculated using an algorithmic treatment developed pre-

viously (Table 1) [17]. These values were in agreement

with those obtained from fluorescence spectroscopy, with

the three monoterpene hydrocarbons showing the highest

Kf values.

To the best of our knowledge, few are the studies con-

cerning the determination of Kf values of HP-b-CD com-

plexes with the studied monoterpenes. Saito et al. [19]

determined the Kf for linalool, limonene, a-pinene, b-

pinene, and geraniol inclusion complexes with HP-b-CD.

However, the values obtained were higher than those

obtained in our work, certainly due to the difference in the

applied experimental approaches. Namely, authors used the

same CD concentration for different G concentrations,

while in our study the G concentration was constant and

CD concentration varied [19]. Kf values reported in the

literature for HP-b-CD/linalool were 1610 M-1 [20],

958 M-1 [21], 720 M-1 [12] and for HP-b-CD/limonene

3350 M-1 [20], which were consistent with our results.

Solubility diagram for HP-b-CD/thymol was investigated

by Demian [21]. However, the Kf value could not be

compared to our result since the slope was more than unity,

indicating the formation of inclusion complexes with a

molar ratio other than 1:1. On the other hand, a strong

correlation was found between the logP bibliographic

values of the monoterpenes [37, 38] and Kf values obtained

from fluorescence (Fig. 4) and SH-GC (data not shown)

studies. Thus, the main driving force of the complexation

seemed to be related with the hydrophobic character of G.

Correlations between Kf values of CD/aroma complexes

and logP were also observed in other study [35].

Solid inclusion complexes investigation

FTIR spectroscopy analysis

FTIR spectroscopy had been commonly applied to prove

the formation of CD/G inclusion complex [39]. None of the

complexes studied in this paper was previously character-

ized by FTIR spectroscopy. FTIR spectra of b-CD/geraniol

[40], b-CD/pulegone and c-CD/pulegone complexes were

reported in the literature [41].

The FTIR spectra and prominent peak assignments of

HP-b-CD, limonene, linalool and their subsequent inclu-

sion complexes were shown in Fig. 5 and Table 2,

respectively.

As can be seen in Fig. 5a and Table 2, HP-b-CD spec-

trum showed prominent absorption bands at: 3437 cm-1

(free O–H stretching vibrations), 2930 cm-1 (C–H

stretching vibrations), 1373 cm-1 (C–H deformation),

1156 cm-1 (C–O stretching vibrations) and 1034 cm-1

(C–O–C stretching vibrations) [42, 43]. The OH stretching

peak of HP-b-CD presented an overlapping of primary and

secondary groups [44]. The IR spectrum of limonene

(Fig. 5b) showed the following characteristic bands:

3077 cm-1 (=C–H stretching vibrations), 2964 and

2921 cm-1 (C–H stretching vibrations), 1644 cm-1 (C=C

stretching vibrations), 1442 and 1375 cm-1 (C–H defor-

mation vibrations) and 889 cm-1 (C–H deformation

vibrations in the gem-disubstituted alkene). Whereas the

spectrum of linalool (Fig. 5c) displayed the characteristic

bands at: 3440 cm-1 (O–H stretching vibrations),

3086 cm-1 (=C–H stretching vibrations), 2971 and

2922 cm-1 (C–H stretching vibrations), 1644 cm-1 (C=C

stretching vibrations), 1450 and 1374 cm-1 (C–H defor-

mation vibrations), 1113 cm-1 (C–O stretching vibrations),

919 cm-1 (C–H deformation vibrations in the vinyl) and

834 cm-1 (C–H deformation vibrations in the trisubstituted

alkene). The spectra of inclusion complexes showed small

differences in comparison to that of HP-b-CD. This indi-

cated that no covalent interactions between encapsulated

molecules and C–C, C–O–C, and OH groups of HP-b-CD

existed [45]. Moreover, no new peaks were observed

demonstrating that no new chemical bonds were created.

However, the hydroxyl group absorption band of HP-b-CD

R = 0.8872

0

500

1000

1500

2000

2 2.5 3 3.5 4 4.5 5

a K

(M

-1 )

Log P

R = 0.9232

0

500

1000

1500

2000

2 2.5 3 3.5 4 4.5 5

K (

M-1

)

Log P

b

Fig. 4 Relationship between a Benesi–Hildebrand and b Scatchard

formation constants (Kf) and hydrophobicity parameter (logP) of

monoterpenes


123

was slightly shifted towards lower wavenumbers and

showed a substantial decrease in intensity as well as

broadening, upon complexation. These findings could be

explained by the formation of hydrogen bonds between G

molecule and HP-b-CD as well as by the release of

included water molecules from the cavity [40, 41]. It

should be noted that the bands of free G molecules were

generally covered up by the peaks of HP-b-CD/G complex

because the quantities of G molecules were no more than

10–15 % (w/w) in the inclusion complexes [11]. Never-

theless, for each G molecule, some characteristic bands

were reproduced in its corresponding inclusion complex

spectrum (Table 2). Thus, the bands at 1,652 and

852 cm-1, attributed respectively to C=C stretching

vibrations and C–H deformation vibrations in the gem-

disubstituted alkene of limonene, were also reproduced in

inclusion complex spectrum (Fig. 5d). In the case of lin-

alool, the bands at 1647, 945, and 852 cm-1 attributed to

C=C stretching vibrations, C–H deformation vibrations in

the vinyl and C–H deformation vibrations in the trisubsti-

tuted alkene of linalool were also found in HP-b-CD/lin-

alool inclusion complex spectrum (Fig. 5e). However, they

had a decreased intensity due apparently to low quantity of

G molecule in the complex and its restricted vibrating and

bending inside the HP-b-CD cavity [46]. In addition, these

peaks showed slight shifts relative to those of the

0

50

100

400 1400 2400 3400

% T

Wavenumber (cm )-1

a

3437

30

60

90

400 1400 2400 3400

% T

Wavenumber (cm-1)

d

1652 852

3362

0

30

60

90

400 1400 2400 3400

% T

Wavenumber (cm-1)

b

1644

889 30

60

90

400 1400 2400 3400

% T

Wavenumber (cm-1)

c

1644

919

834

20

50

80

400 1400 2400 3400

% T

Wavenumber (cm-1)

e

1647

852

3386 945

Fig. 5 FTIR spectra of a HP-b-

CD, b limonene, c linalool and

their relative inclusion

complexes, d HP-b-CD/

limonene and e HP-b-CD/

linalool

Table 2 Wavenumbers (cm-1) assignment of FTIR spectra of HP-b-CD, limonene, linalool and their relative inclusion complexes

Chemical functional groups Wavenumber (cm-1)

Vibration modes HP-b-CD Limonene HP-b-CD/limonene Linalool HP-b-CD/linalool

t(O–H) 3,437 – 3,362 3,440 3,386

t(=C–H) – 3,077 O/l 3,086 O/l

Methylene/methyl t(C–H) 2,930 2964, 2921 2,931 2971, 2922 2,930

Alkene t(C=C) – 1,644 1,652 1,644 1,647

Methylene/methyl d(C–H) O/l, 1373 1442, 1375 O/l, 1375 1450, 1374 O/l, 1374

t(C–O)/(C–O–C) 1156, 1034 – 1155, 1034 1,113 1155, 1034

Vinyl d(C–H) – – – 919 945

Alkene gem d(C–H) – 889 852 – –

Trisubstituted alkene d(C–H) – – – 834 852

O/l overlapped


123

respective free compounds, providing an evidence of host–

G interactions. Similar results were obtained for other

studied G indicating the formation of inclusion complexes

with HP-b-CD. Thus, the FTIR results clearly indicated

that HP-b-CD was able to form inclusion complexes with

all monoterpenes studied in solid state.

DSC analysis

The thermograms of HP-b-CD, linalool, b-pinene, their

inclusion complexes and physical mixtures were presented

in Fig. 6 as examples.

The thermogram of HP-b-CD showed a very broad

endothermic band, between 100 and 150 �C (Fig. 6b, f),

indicating dehydration process. The DSC curves of linalool

and b-pinene illustrated sharp endothermic peak at about

204 and 167 �C (Fig. 6a, e, respectively). However, the

thermal behavior of the freeze-dried inclusion complexes

(Fig. 6c, g) was different from that found for the individual

components. The endothermic peak corresponding to the

free G disappeared, suggesting a complex formation.

Similar observations were done for b-CD/thymol inclusion

complex [47]. Moreover, the broad endothermic peak,

corresponding to the free HP-b-CD has been shifted,

indicating that G molecules had replaced water molecules

in the cavity of HP-b-CD after inclusion complex forma-

tion. The same effect, with lower intensity, was also found

in their relative physical mixtures (Fig. 6d, h). It might be

explained by the diffusion of these highly volatile G into

the CD cavity during the heating process provided by the

DSC analysis [48]. The DSC results confirmed that mon-

oterpenes were successfully included into the cavity of HP-

b-CD.

Encapsulation efficiency

For each monoterpene, an HPLC method was developed to

determine the EE% of HP-b-CD. EE% is a quantitative

parameter indicating the amount of included monoterpene

in the solid complex. EE% values were calculated using

Eq. (1) and are listed in Table 1.

As can be seen in Table 1, the oxygenated monoter-

penes (eucalyptol, geraniol, linalool, pulegone and thymol)

were entrapped in HP-b-CD cavity much more effectively

(EE% [82.1) than monoterpene hydrocarbons (limonene,

a- and b-pinene; EE% from 15.4 to 24.9 %). The lower

values of EE% obtained for monoterpene hydrocarbons

might be due to their very low water solubility. For such

highly hydrophobic molecules, increasing starting ratios of

HP-b-CD to G may produce higher inclusion of these

molecules and may be more adequate for their complexa-

tion [49]. On the other hand, a small amount of water

miscible solvent could be added to increase the solubility

of G in the medium [50].

Shukla et al. [50] found no statistically significant dif-

ference (at the 5 % level) between EE% of b-CD and that

of HP-b-CD. Indeed, our results concerning geraniol and

thymol were consistent with literature data for EE% of b-

CD [47]. To the best of our knowledge, no other reports on

EE% values for the studied monoterpenes have been

published.

Conclusion

HP-b-CD inclusion complexes of eucalyptol, geraniol,

limonene, linalool, a-pinene, b-pinene, pulegone, and

thymol were successfully prepared as demonstrated by

fluorescence spectroscopy, SH-GC, FTIR and DSC. The

main driving force of complex formation was hydrophobic

interactions as was evidenced by the strong positive cor-

relation between the Kf values of inclusion complexes and

50 100 150 200 250 300

Hea

t fl

ow

(w

/g)

a

d

c

b

Exo

50 100 150 200 250 300

Hea

t fl

ow

(w

/g)

e

f

h

g

Exo

Fig. 6 DSC thermograms of (a) linalool, (b) HP-b-CD, (c) HP-b-CD/

linalool inclusion complex, (d) HP-b-CD/linalool physical mixture,

(e) b-pinene, (f) HP-b-CD, (g) HP-b-CD/b-pinene inclusion complex

and (h) HP-b-CD/b-pinene physical mixture


123

logP of monoterpenes. FTIR and DSC analyses had also

revealed the interactions between host and G molecules in

solid state. For each monoterpene, the EE% of HP-b-CD

was evaluated using HPLC analysis. The presence of an

oxygenated function in the monoterpene structure appeared

to be crucial for an effective encapsulation. Our findings

suggested that encapsulation with HP-b-CD could be a

very effective vector to design new monoterpenes formu-

lations in cosmetic, pharmaceutical and food industries.

Acknowledgments Authors would like to thank Dr. Samar Eid for

her valuable comments during the preparation of this manuscript and

Benoit Duponchel for DSC analyses. Authors are grateful to Lebanese

National Council for Scientific Research (CNRS-L) for the financial

support of M. Kfoury’s PhD thesis. The study was financially sup-

ported by the Doctoral School of Science and Technology of Leba-

nese University (ER28).

References

1. Astray, G., Gonzalez-Barreiro, C., Mejuto, J.C., Rial-Otero, R.,

Simal-Gandara, J.: A review on the use of cyclodextrins in foods.

Food Hydrocoll. 23, 1631–1640 (2009)

2. Szejtli, J.: Utilization of cyclodextrins in industrial products and

processes. J. Mater. Chem. 7(4), 575–587 (1997)

3. Rajewski, R., Stella, V.J.: Pharmaceutical applications of cyclo-

dextrins. 2. In vivo drug delivery. J. Pharm. Sci. 85, 1142–1168

(1996)

4. Deans, S.G., Ritchie, G.: Antibacterial properties of plant

essential oils. Int. J. Food Microbiol. 5, 165–180 (1987)

5. Tserennadmid, R., Tako, M., Galgoczy, L., Papp, T., Pesti, M.,

Vagvolgyi, C., Almassy, K., Krisch, J.: Antiyeast activities of

some essential oils in growth medium, fruit juices and milk. Int.

J. Food Microbiol. 144, 480–486 (2011)

6. Schnitzler, P., Astani, A., Reichling, J.: Screening for antiviral

activities of isolated compounds from essential oils. Evid. Based

Complement. Altern. Med. 2011, 1–8 (2011)

7. Timsina, B., Shukla, M., Naduman, V.K.: A review of few

essential oils and their anticancer property. Int. J. Shoulder Surg.

6(2), 1–8 (2012)

8. Kim, S.I., Roh, J.Y., Kim, D.H., Lee, H.S., Ahn, Y.J.: Insecticidal

activities of aromatic plant extracts and essential oils against

Sitophilus oryzae and Callosobruchus chinensis. J. Stored Prod.

Res. 39, 293–303 (2003)

9. Tsai, M.L., Lin, C.C., Lin, W.C., Yang, C.H.: Antimicrobial,

antioxidant and anti-inflammatory activities of essential oils from

selected herbs. Biosci. Biotechnol. Biochem. 75(10), 1977–1983

(2011)

10. Marques, H.M.C.: A review on cyclodextrin encapsulation of

essential oils and volatiles. Flavor Fragr. J. 25, 313–326 (2010)

11. Yang, Y., Song, L.X.: Study on the inclusion compounds of

eugenol with a-, b-, c- and Heptakis(2, 6-di-O-methyl)-b-cyclo-

dextrins. J. Incl. Phenom. Macrocycl. Chem. 53, 27–33 (2005)

12. Numanoglu, U., Sen, T., Tarimci, N., Kartal, M., Koo, O.M.Y.,

Onyuksel, H.: Use of cyclodextrins as a cosmetic delivery system

for fragrance materials: linalool and benzyl acetate. AAPS

PharmSciTech. 8(4), 34–42 (2007)

13. Kopecky, F., Kopecka, B., Kaclik, P.: Solubility study of nimo-

dipine inclusion complexation with a- and b-cyclodextrin and

some substituted cyclodextrins. J. Incl. Phenom. Macrocycl.

Chem. 39, 215–217 (2001)

14. Stella, V.J., He, Q.: Cyclodextrins. J. Toxicol. Pathol. 36, 30–42

(2008)

15. Gould, S., Scott, R.C.: 2-Hydroxypropyl-b-cyclodextrin (HP-b-

CD): a toxicology review. Food Chem. Toxicol. 43, 1451–1459

(2005)

16. Yuan, C., Jin, Z., Li, X.: Evaluation of complex forming ability of

hydroxypropyl-b-cyclodextrins. Food Chem. 106, 50–55 (2008)

17. Decock, G., Landy, D., Surpateanu, G., Fourmentin, S.: Study of

the retention of aroma components by cyclodextrins by static

headspace gas chromatography. J. Incl. Phenom. Macrocycl.

Chem. 62, 297–302 (2008)

18. Ciobanu, A., Landy, D., Fourmentin, S.: Complexation efficiency

of cyclodextrins for volatile flavor compounds. Food Res. Int. 53,

110–114 (2013)

19. Saito, Y., Tanemura, I., Sato, T.: Interaction of fragrance mate-

rials with 2-hydroxypropyl-b-cyclodextrin by static and dynamic

headspace methods. Int. J. Cosmet. Sci. 21, 189–198 (1999)

20. Matsuda, H., Ito, K., Fujiwara, Y., Tanaka, M., Taki, A., Uejima,

O., Sumiyoshi, H.: Complexation of various fragrance materials

with 2-hydroxypropyl-b-cyclodextrin. Chem. Pharm. Bull. 39(4),

827–830 (1991)

21. Demian, B.A.: Correlation of the solubility of several aromatics

and terpenes in aqueous hydroxypropyl-b-cyclodextrin with ste-

ric and hydrophobicity parameters. Carbohydr. Res. 328,

635–639 (2000)

22. Ciobanu, A., Mallard, I., Landy, D., Brabie, G., Nistor, D.,

Fourmentin, S.: Inclusion interactions of cyclodextrins and

crosslinked cyclodextrin polymers with linalool and camphor in

Lavandula angustifolia essential oil. Carbohydr. Polym. 87(3),

1963–1970 (2012)

23. Al Kindy, S.M.Z., Suliman, F.D.O., Al Hamadi, A.A.: Fluores-

cence enhancement of coumarin-6-sulfonyl chloride amino acid

derivatives in cyclodextrin media. Anal. Sci. 17, 539–543 (2001)

24. Ochocka, R.J., Marzec, A., Lamparczyk, H.: Fluorescence

enhancement of two terpenes commonly present in essential oils.

J. Pharm. Biomed. Anal. 10, 809–812 (1992)

25. Dallasta, C., Ingletto, G., Corradini, I., Galaverna, G., Marchelli,

R.: Fluorescence enhancement of aflatoxins using native and

substituted cyclodextrins. J. Incl. Phenom. Macrocycl. Chem. 45,

257–263 (2003)

26. Aicart, E., Junquera, E.: Complex formation between purine

derivatives and cyclodextrins: a fluorescence spectroscopy study.

J. Incl. Phenom. Macrocycl. Chem. 47, 161–165 (2003)

27. Jiang, H., Zhang, S., Cui, Y., Xie, Y., Shi, Q.: NMR and fluo-

rescence enhancement effect studies of ternary inclusion com-

plexes among naphthalenediamines–triethylene tetramine

modified b-cyclodextrin–lanthanide metal ions. Afr. J. Pure Appl.

Chem. 5(6), 136–144 (2011)

28. Liu, Y., Yang, Y.W., Zhao, Y., Li, L., Zhang, H.Y., Kang, S.Z.:

Molecular recognition and cooperative binding ability of fluo-

rescent dyes by bridged bis(b-cyclodextrin)s tethered with aro-

matic diamine. J. Incl. Phenom. Macrocycl. Chem. 47, 155–160

(2003)

29. Szejtli, J.: Introduction and general overview of cyclodextrins

chemistry. Chem. Rev. 98, 1743–1753 (1998)

30. Easton, C.J., Lincoln, S.F.: Modified Cyclodextrins. Scaffolds

and Templates for Supramolecular Chemistry, pp. 1–293. Impe-

rial College Press, London (1999)

31. Gazpio, C., Sanchez, M., Zornoza, A., Martin, C., Martinez-

Oharriz, C., Velaz, I.: A fluorimetric study of pindolol and its

complexes with cyclodextrins. Talanta 60, 477–482 (2003)

32. Zhan, H., Jiang, Z.T., Wang, Y., Li, R., Dong, T.S.: Molecular

microcapsules and inclusion interactions of eugenol with b-

cyclodextrin and its derivatives. Eur. Food Res. Technol. 227,

1507–1513 (2008)


123

33. Benesi, H.A., Hildebrand, J.H.: A spectrophotometric investiga-

tion of the interaction of iodine with aromatic hydrocarbons.

J. Am. Chem. Soc. 71, 2703–2707 (1949)

34. Connors, K.A.: Binding Constants. The Measurements of

Molecular Complex Stability. Wiley, New York (1987)

35. Astray, G., Mejuto, J.C., Morales, J., Rial-Otero, R., Simal-

Gandara, J.: Factors controlling flavors binding constants to

cyclodextrins and their applications in foods. Food Res. Int. 43,

1212–1218 (2010)

36. Challa, R., Ahuja, A., Ali, J., Khar, R.K.: Cyclodextrins in drug

delivery: an updated review. AAPS PharmaSciTech 6(2),

329–357 (2005)

37. Goodner, K.L.: Practical retention index models of OV-101, DB-

1, DB-5, and DB-wax for flavor and fragrance compounds

practical retention index models of OV-101, DB-1, DB-5, and

DB-wax for flavor and fragrance compounds. LWT Food Sci.

Technol. 41, 951–958 (2008)

38. Griffin, S., Wyllie, S.G., Markham, J.: Determination of octanol–

water partition coefficient for terpenoids using reversed-phase

high-performance liquid chromatography. J. Chomatogr. A 864,

221–228 (1999)

39. Singh, R., Bharti, N., Madan, J., Hiremath, S.N.: Characterization

of cyclodextrin inclusion complexes: a review. J. Pharm. Sci.

Technol. 2(3), 171–183 (2010)

40. Menezes, P.P., Serafini, M.R., Santana, B.V., Nunes, R.S.,

Quintans Jr., L.J., Silva, G.F., Medeirosc, I.A., Marchioro, M.,

Fraga, B.P., Santos, M.R.V., Araujo, A.A.S.: Solid-state-b-

cyclodextrin complexes containing geraniol. Thermochim. Acta

548, 45–50 (2012)

41. Moon, T.W., Lee, J.W., Jhee, K.H., Khang, K.W., Jeong, H.S.,

Yang, S.A., Kim, H.J.: Supramolecular encapsulation of pulegone

from oriental herb, Schiznepeta tenuifolia, Briquet by b- and c-

cyclodextrins. Bull. Korean Chem. Soc. 29(8), 1579–1582 (2008)

42. Gajare, P., Patil, C., Kalayne, N., Pore, Y.: Effect of hydrophilic

polymers in pioglitazone complexation with hydroxypropyl-b-

cyclodextrin. Dig. J. Nanomater. Biostruct. 4(4), 891–897 (2009)

43. Jun, S.W., Kim, M.S., Kim, J.S., Park, H.J., Lee, S., Woo, J.S.,

Hwang, S.J.: Preparation and characterization of simvastatin/

hydroxypropyl-b-cyclodextrin inclusion complex using super-

critical antisolvent (SAS) process. Eur. J. Pharm. Biopharm. 66,

413–421 (2007)

44. Zhang, A., Liu, W., Wang, L., Wen, Y.: Characterization of

inclusion complexation between fenoxaprop-p-ethyl and cyclo-

dextrin. J. Agric. Food Chem. 53(18), 7193–7197 (2005)

45. Wang, T., Li, B., Si, H., Li, L., Chen, L.: Release characteristics

and antibacterial activity of solid state eugenol/b-cyclodextrin

inclusion complex. J. Incl. Phenom. Macrocycl. Chem. 71,

207–213 (2011)

46. Del Toro-Sanchez, C.L., Ayala-Zavala, J.J., Machi, L., Santacruz,

H., Villegas-Ochoa, M.A., Alvarez-Parrilla, E., Gonzalez-Agui-

lar, G.A.: Controlled release of antifungal volatiles of thyme

essential oil from b-cyclodextrin capsules. J. Incl. Phenom.

Macrocycl. Chem. 67, 431–441 (2010)

47. Moutzinos, I., Kalogeropoulos, N., Papadakis, S.E., Konstanti-

nou, K., Karathanos, V.T.: Encapsulation of nutraceutical mon-

oterpenes in b-cyclodextrin and modified starch. J. Food Sci.

73(1), 89–94 (2008)

48. Soares-Sobrinho, J.L., de La Roca Soares, M.F., Rolim-Neto,

P.J., Torres-Labandeira, J.J., : Physicochemical study of solid-

state benznidazole–cyclodextrin complexes. J. Therm. Anal.

Calorim. 106, 319–325 (2011)

49. Dos Santos, C., Buera, M.P., Mazzbore, M.F.: Influence of ligand

structure and water interactions on the physical properties of b-

cyclodextrins complexes. Food Chem. 132, 2030–2036 (2012)

50. Shukla, D., Chakraborty, S., Singh, S., Mishra, B.: Preparation

and in vitro characterization of risperidone–cyclodextrin inclu-

sion complexes as a potential injectable product. DARU 17(4),

226–235 (2009)


123

Investigation of monoterpenes complexation with hydroxypropyl-β-cyclodextrin

Documents

Transcript of Investigation of monoterpenes complexation with hydroxypropyl-β-cyclodextrin