J Incl Phenom Macrocycl Chem
DOI 10.1007/s10847-014-0385-7
ORIGINAL ARTICLE
Investigation of monoterpenes complexation with hydroxypropyl-
b-cyclodextrin
Miriana Kfoury • Lizette Auezova •
Sophie Fourmentin • Hélène Greige-Gerges
Received: 12 November 2013 / Accepted: 22 January 2014
Ó 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.
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: laouezova@ul.edu.lb; auezova_l@hotmail.com
M. Kfoury  S. Fourmentin (&)
Univ Lille Nord de France, 59000 Lille, France
e-mail: lamotte@univ-littoral.fr
M. Kfoury  S. Fourmentin
ULCO, UCEIV EA 4492, 59140 Dunkerque, France
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
123

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
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J Incl Phenom Macrocycl Chem
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
J Incl Phenom Macrocycl Chem

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
a initially used to prepare the solid inclusion complex.
(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:
EMC ðmgÞ
EE% ¼  100; ð1Þ
TMC ðmgÞ
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

2500
F-F

Results and discussion

2000

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

1500
eight monoterpenes were prepared and characterized both
1000
in aqueous solution and in solid state.

500 Investigation of inclusion complexes in aqueous

solution
0
290 310 330 350
Effect of HP-b-CD on the fluorescence behavior
(nm)
of monoterpenes

b Figure 1 shows the fluorescence emission spectra of pule-

3000
gone and b-pinene (as example) in aqueous solution for
F-F

2500 different HP-b-CD concentrations. All studied monoter-

penes, with the exception of thymol, were found to exhibit
2000
[HP- -CD] M no or low fluorescence yield in the absence of HP-b-CD.
1500 Thymol showed noticeable emission intensity, due to its
aromatic ring moiety. Upon addition of HP-b-CD, the
1000
fluorescence intensity of monoterpenes was enhanced and
500 increased gradually with its concentration. This intensity
increment can be obviously seen from the plots (F - F0)
0 against HP-b-CD concentration (Fig. 1), where F and F0
280 300 320 340
(nm) are the fluorescence intensities of inclusion complex and of
free G, respectively. The best fluorescence enhancement,
Fig. 1 Fluorescence spectra of a pulegone (1.5 9 10-5 M) and b b- F0 /F0 (F0 , fluorescence intensity in the presence of the
pinene (1.5 9 10-5 M) in absence and presence of HP-b-CD. highest HP-b-CD concentration), was obtained for the HP-
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 b-CD/linalool inclusion complex (Table 1). It might be due
(F - F0), as a function of HP-b-CD concentration to the better fit of linalool inside the HP-b-CD cavity in

123
 J Incl Phenom Macrocycl Chem

Table 1 Emission wavelengths of monoterpenes in absence (k1) and approaches and SH-GC (Kf3), fluorescence enhancement ratios (F0 /
presence (k2) of HP-b-CD, Kf values of HP-b-CD/monoterpene F0), logP values, and encapsulation efficiency (EE%)
complexes calculated from Benesi–Hildebrand (Kf1), Scatchard (Kf2)
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
b
Geraniol 311 309 1,320 1,064 712 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
a
Pulegone 311 309 867 798 676 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-CD

a
See [18]
b
See [17]
c
See [37]
d
See [38]

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
HP-b-CD=G; ð2Þ
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-CD þ G
with overall Kf:
½HP-b-CD/G
Kf ¼ : ð3Þ
½HP-b-CD½G
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 1 1 1
¼  þ : ð4Þ
DF Kf  k  Q  ½G ½HP -b- CD k  Q  ½G]
[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

123
J Incl Phenom Macrocycl Chem

a 0.012 R = 0.9845

0.01

0.008
1/F-F

0.006

0.004

0.002

0
0 200 400 600 800 1000 1200
1/[HP- -CD] (M-1)

b 100000 2000000
R = 0.9573

1600000
75000
(F-F0 )/HP- -CD]

Area (µV.s)
1200000
50000
800000

25000
400000

0 0
0 50 100 150 200 0.000 0.002 0.004 0.006 0.008 0.010 0.012
(F-F0 ) [HP- -CD] M

Fig. 2 a Benesi–Hildebrand and b Scatchard plots for HP-b-CD/ Fig. 3 Representation of the experimental points (filled diamonds)
pulegone inclusion complex obtained for HP-b-CD/thymol compared with theoretical titration
curve (dashed lines) for a 1:1 complex

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  F0 Þ
¼ ðF1  F0 Þ  Kf  ðF  F0 Þ  Kf ;
½HP -b- CD
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
ð5Þ 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

123

a R = 0.8872
2000
1500
K (M-1 )
1000
500
0 Solid inclusion complexes investigation
2 2.5 3 3.5 4 4.5 5
Log P
b 2000 R = 0.9232
1500
K (M-1)
1000
500
0
2 2.5 3 3.5 4 4.5 5 respectively.
Log P
Fig. 4 Relationship between a Benesi–Hildebrand and b Scatchard
formation constants (Kf) and hydrophobicity parameter (logP) of
monoterpenes
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,
123
J Incl Phenom Macrocycl Chem
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].
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,
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
J Incl Phenom Macrocycl Chem

Fig. 5 FTIR spectra of a HP-b- a 100

CD, b limonene, c linalool and
their relative inclusion

%T
complexes, d HP-b-CD/ 50
limonene and e HP-b-CD/
linalool 3437
0
3400 2400 1400 400
Wavenumber (cm-1)

b 90 c 90

60
%T

%T
60 1644
30 1644
834
889 919
0 30
3400 2400 1400 400 3400 2400 1400 400
Wavenumber (cm-1) Wavenumber (cm-1)

d 90
e
80

%T
%T

1652 852
60 1647
50
852
3362
945
3386
30 20
3400 2400 1400 400 3400 2400 1400 400
Wavenumber (cm-1) Wavenumber (cm-1)

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

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

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 J Incl Phenom Macrocycl Chem

a components. The endothermic peak corresponding to the

free G disappeared, suggesting a complex formation.
Similar observations were done for b-CD/thymol inclusion
Exo complex [47]. Moreover, the broad endothermic peak,
Heat flow (w/g)

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

indicating that G molecules had replaced water molecules
b
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
c explained by the diffusion of these highly volatile G into
d the CD cavity during the heating process provided by the
DSC analysis [48]. The DSC results confirmed that mon-
50 100 150 200 250 300 oterpenes were successfully included into the cavity of HP-
b-CD.

e Encapsulation efficiency

For each monoterpene, an HPLC method was developed to

Exo determine the EE% of HP-b-CD. EE% is a quantitative
Heat flow (w/g)

parameter indicating the amount of included monoterpene

f
g were entrapped in HP-b-CD cavity much more effectively
h a- and b-pinene; EE% from 15.4 to 24.9 %). The lower
50 100 150 200 250 300
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
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
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)
(EE% [82.1) than monoterpene hydrocarbons (limonene,
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.

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
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

123
J Incl Phenom Macrocycl Chem
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).
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