Inclusion complexes of coenzyme Q10 with polyamine-modified β-cyclodextrins: Characterization, solubilization, and inclusion mode

Article abstract of DOI:10.1002/hlca.201100026

The inclusion-complexation behavior of coenzyme Q10 (CoQ10) with the three polyamine-modified β-cyclodextrins (CDs) 1-3 was investigated in both solution and the solid state by means of NMR, XRD, and FT-IR spectroscopy. The results showed that the apparen

Full text of DOI:10.1002/hlca.201100026

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Inclusion Complexes of Coenzyme Q10 with Polyamine-Modified
b-Cyclodextrins: Characterization, Solubilization, and Inclusion Mode
by Juan Gua), Shao-Ming Chia), Yong Zhaoa), Ping Zhenga), Qiong Ruana), Yan Zhao*a), and
Hong-You Zhu*b)
a) College of Chemistry and Chemical Engineering, Yunnan Normal University, Kunming 650092,
P. R. China (phone/fax: þ 86-871-5516061; email: zhaooyann@163.com)
b) Chemical Science and Engineer College, Yunnan University, Kunming 650091, P. R. China
(phone/fax: þ 86-871-5033922; email: hongyouzhu@Sohu.com)
The inclusion-complexation behavior of coenzyme Q10 (CoQ10) with the three polyamine-modified
b-cyclodextrins (CDs) 1 – 3 was investigated in both solution and the solid state by means of NMR, XRD,
and FT-IR spectroscopy. The results showed that the apparent solubility of CoQ10 increased linearly
upon addition of hosts 1 – 3, giving AL-type phase-solubility curves. These hosts 1 – 3 were able to
solubilize CoQ10 to high levels, up to 1.35, 1.52, and 1.44 mg/ml (calculated as CoQ10), respectively. The
host 2 with a moderate-length chain is the most suitable for inclusion complexation of CoQ10. Accroding
to the ROESY experiments, the MeO groups of CoQ10 and the tether of 2 can be co-included into the
cavity of b-CD through the induced-fit interaction between host and guest. The binding ability of
modified b-CDs 1 – 3 upon complexation with CoQ10 are discussed from the viewpoints of the size/
shape-matching relationship and the induced-fit concept between host CDs and guest CoQ10 molecule.
1. Introduction – Coenzyme Q10 (CoQ10), also known as ubiquinone or
ubidecarenone, is a fat-soluble, vitamin-like substance found in the cells of many
organisms. The systematic name of CoQ10 in the all-trans configuration (natural) is 2-
[(2E,6E,10E,14E,18E,22E,26E,30E,34E)-3,7,11,15,19,23,27,31,35,39-decamethyltetra-
conta-2,6,10,14,18,22,26,30,34,38-decaen-1-yl]-5,6-dimethoxy-3-methylcyclohexa-2,5-
diene-1,4-dione. CoQ10 acts as an antioxidant and protects mitochondrial innermem-
brane proteins and DNA against oxidative damage during oxidative stress [1]. CoQ10
also acts as a membrane stabilizer. Long-term-treatment side effects of statins include a
decrease in CoQ10 levels resulting in energy-metabolism impairment in heart, skeletal
muscle, and liver [2]. This may cause cardiomyopathy and complicate cardiovascular
disease. Supplementation of the diet with CoQ10 can reverse many of the above
symptoms [3]. Also, many studies have reported the immunostimulating action of
CoQ10 [4]. However, because of the moleculeꢀs long side chain of 10 isoprenoid units,
ꢁ 2011 Verlag Helvetica Chimica Acta AG, Zꢂrich

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CoQ10 is poorly H2O-soluble [5]. It is thought that the incomplete and slow absorption
of CoQ10 from the gastrointestinal tract is attributed to its poor water solubility and
high molecular mass [6].
Because of its poor aqueous solubility, CoQ10 has presented a challenge in the
development of formulations for oral administration. There are various methods for the
improvement of aqueous solubility of lipophilic compounds in the food and
pharmaceutical fields, i.e., via an amorphous form [7], grinding [8], a solid dispersion
[9], a micelle [10], or an inclusion complex [11]. Among them, the use of cyclodextrins
(CDs) has been known in the pharmaceutical and nutrition fields because CD inclusion
complexes are stable against heat, oxidation, and UV, and better suited for these
formulations. A CD, a kind of truncated-cone polysaccharide, is mainly made up of six
to eight d-glucose monomers linked by a-1,4-glucose bonds, which exhibits a
hydrophobic central cavity and a hydrophilic outer surface. CDs are known to be
able to encapsulate model substrates to form hostꢀguest complexes or superamolecular
species resulting usually in an enhancement of drug solubility in aqueous solution and
affecting the chemical characterization of drugs [12 – 15].
However, the aqueous solubility of the native b-CD is relatively low, and the
inclusion complex of b-CD and drug dissociates before the drug reaches the organs or
tissues to which the drug is to be delivered [16]. Possessing specific functional groups as
compared with the parent b-CD, the chemically modified b-CDs can significantly alter
the original molecular-binding ability by stereochemical complements of the functional
branch located in the CD cavity [17 – 21]. Herein, we wish to report the preparation and
characterization of some H2O-soluble inclusion complexes formed by CoQ10 and
polyamine modified CDs 1 – 3 with different tethered chain lengths. It is our special
interest to explore the binding behavior of highly H2O-soluble polyamine-modified
CDs with CoQ10 and the solubilization effect of polyamine-modified CDs toward
CoQ10, which will provide a useful approach to achieve novel CoQ10-based healthcare
products with high water solubility.
2. Results and Discussion. – 2.1. Preparation of the Inclusion Complexes. The
modified b-CDs 1 – 3/CoQ10 complexes were prepared by the suspension method (see
Exper. Part).
2.2. Phase Solubility. The phase-solubility diagrams of CoQ10 in polyamine-
modified CDs 1 – 3 in H2O are shown in Fig. 1. All three diagrams are of the AL type,
indicating that the complexes are of first order with respect to the hosts 1 – 3. For 1:1
drug/CD complexes, the apparent stability constants (K1:1) of the complexes were
calculated from the phase-solubility diagrams according to Eqn. 1, where S0 is the
solubility of CoQ10 at 258 in the absence of CDs and Slope means the corresponding

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slope of the phase-solubility diagrams, i.e., the slope of the drug molar concentration vs.
CD molar concentration graph. The experiment was carried out in triplicate at 258. The
solubility of pure CoQ10 is 2.61 · 10ꢀ5 mg/ml in H2O at 258. The apparent stability
constants K1:1 are 2028, 3795, and 2857 mꢀ1 for complexes of CoQ10 with 1, 2, and 3,
respectively.
Slope
S0ð1 ꢀ SlopeÞ
K1:1
¼
ð1Þ
Fig. 1. Phase-solubility diagrams for CoQ10 in the presence of a) host 2, b) host 1, and c) host 3 in H2O at
258
2.3. Binding Ability. The hostꢀguest size matching may dominate the stability of the
complexes formed between the CDs 1 – 3 and CoQ10. The K1:1 values for the inclusion
complexation of CoQ10 by hosts increase in the following order: 1 < 3 < 2. That is, host
2 with a moderate-length chain is the most suitable for the inclusion complexation of
CoQ10. This result is reasonable because the cavity of b-CD could not encapsulate the
CoQ10 tightly, and native b-CD could slip onto the guest molecular chain like a bead
[22]. The introduction of self-included substituents at the rim of CDs may reduce the
effective cavity of the CDs and then hold CoQ10 much more tightly. The moderate-
length side chain of host 2 is partially included in its own hydrophobic cavity [23],
increasing the binding ability of host 2 towards CoQ10 to some extent. It results in a
strict size fit between host 2 and guest, which consequently exhibits the strongest
binding ability between 2 and CoQ10. The longest side chain present in host 3 is deeply
included in its own cavity, increasing the steric hindrance and reducing the hostꢀguest
interaction. It results in a lower binding ability between 3 and CoQ10. In the shortest-
chain host 1, the lowest binding ability is the result of the size unfit between the cavity
of 1 and the guest, due to the shortest chain being only shallowly self-included in the
CD cavity.

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1
2.4. NMR Analysis. H-NMR Spectrometry has been widely used to evidence
inclusion complexes of CDs in solution [24]. For the sake of comparison, the spectra of
modified CDs 1 – 3 and CoQ10 as well as their inclusion complexes at the same
concentration are shown in Fig. 2 and some of the corresponding data in the Table. As
illustrated in Fig. 2,g, the majority of the CoQ10 protons appear at d 1.52 – 3.18, well
separated from those of the CD H-atoms. Owing to its fairly poor water solubility,
CoQ10 is transparent to 1H-NMR under most conditions when D2O is used as solvent.
Assessment of the CoQ10 complexes by 1H-NMR now clearly demonstrates the
presence of the framework H-atoms of the CoQ10 molecule consistent with the
significant solubilization.
Table. Chemical Shifts (d) of the Modified b-CDs 1 – 3, and of the 1 – 3/CoQ10 Complexes in D2O, and of CoQ10
in CDCl3 at the Same Concentration and the Corresponding Changes (Dd ¼ d(host/CoQ10) ꢀ d(host))
CoQ10
1
1/CoQ10
Dd
2
2/CoQ10
Dd
3
3/CoQ10
Dd
HꢀC(1) (CD)
HꢀC(2) (CD)
HꢀC(3) (CD)
HꢀC(4) (CD)
HꢀC(5) (CD)
CH2(6) (CD)
5.02 5.01
3.60 3.58
3.90 3.82
3.51 3.50
3.80 3.70
3.82 3.75
4.28
ꢀ 0.01 5.04 5.03
ꢀ 0.02 3.61 3.59
ꢀ 0.08 3.92 3.83
ꢀ 0.01 3.54 3.53
ꢀ 0.10 3.81 3.73
ꢀ 0.07 3.84 3.78
ꢀ 0.01 5.03 5.02
ꢀ 0.02 3.61 3.59
ꢀ 0.09 3.94 3.84
ꢀ 0.01 3.53 3.52
ꢀ 0.08 3.81 3.73
ꢀ 0.06 3.82 3.76
ꢀ 0.01
ꢀ 0.02
ꢀ 0.10
ꢀ 0.01
ꢀ 0.08
ꢀ 0.06
þ 0.19
þ 0.19
Me(7)O (CoQ10) 3.99
Me(8)O (CoQ10) 4.01
þ 0.29
þ 0.28
4.20
4.22
þ 0.21
þ 0.21
4.18
4.20
4.29
As can be seen from the Table, in the presence of CoQ10, a negligible effect is
observed on the d(H) of the CD H-atoms HꢀC(1), HꢀC(2), and HꢀC(4). In contrast,
the d(H) of HꢀC(3), HꢀC(5), and CH2(6) exhibit substantial changes, undergoing a
relatively strong shielding exceeding 0.06 ppm. On the other hand, substantial
differences of the guest data are also found in the presence of CD. The d(H) of the
two MeO groups of CoQ10 are remarkably downfield shifted (Dd ¼ 0.19 – 0.29) in the
complex, which suggested that the two MeO groups are shielded largely in the complex,
and they must have penetrated deeply into the cavity of the CD.
2D-NMR Spectroscopy has recently become an important method for the
investigation of the interaction between host CDs and guest molecules, since the
NOE cross-peaks between the protons that are closer than 0.4 nm in space will be
observed in the NOESY or ROESY plots, and the relative intensities of these cross-
peaks depend on the spaces between the corresponding protons. To gain more
conformation information, we recorded the 2D ROESY plot of the inclusion complex
of the host 2 with CoQ10 (Fig. 3). The spectrum displays clear NOE cross-peaks (peaks
C) between HꢀC(3) of host 2 and the MeO groups of CoQ10, which indicated distinctly
that the CoQ10 molecule is included in the hydrophobic cavity from the secondary side
of the modified b-CD. Moreover, the obvious cross-peaks (peaks A) assigned to NOE
correlations between HꢀC(5) and CH2(6) of host 2 and the H-atoms of the
diethylenetriamine (¼ N-(2-aminoethyl)ethane-1,2-diamine) moiety of host 2 indicat-
ed that the tether of 2 is inserted in the CD cavity from the first side of the modified b-
CD. It is interesting to note that the correlation peaks B between the H-atoms of the

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Fig. 2. 1H-NMR Spectra of modified b-CDs in the absence and presence of CoQ10 in D2O and of CoQ10
in CDCl3 at 258. a) Host 1, b) host 1/CoQ10 complex, c) host 2, d) host 2/CoQ10 complex, e) host 3, f) host
3/CoQ10 complex, and g) CoQ10. Protons of the glucose moieties: H1, H2, H3, H4, H5, and H6 ¼
HꢀC(1), HꢀC(2), HꢀC(3), HꢀC(4), HꢀC(5), and CH2(6). Protons of CoQ10: see atom numbering in
the Formula.

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diethylenetriamine moiety of host 2 and the MeO groups of CoQ10 appear, indicating
that the CoQ10 molecule and the tether of 2 can be co-included in the cavity through
the induced-fit interaction between host and guest. In combination with the 1:1
inclusion stoichiometry observed in the phase-solubility diagram, a possible inclusion
mode for the 2/CoQ10 complex is proposed (Fig. 4).
Fig. 3. ROESY Plot of the host 2/CoQ10 complex in D2O at 258
Fig. 4. Possible binding mode of the host 2/CoQ10 complex
2.5. X-Ray Diffraction Analysis of the Inclusion Complex. The powder X-ray
diffraction (XRD) patterns of CoQ10, modified b-CD 2, as well as of its inclusion
complex are illustrated in Fig. 5. As can be recognized from Fig. 5, CoQ10 is in

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crystalline form (Fig. 5,a), while host 2 is amorphous (Fig. 5,b). In contrast, the guest
in the 2/CoQ10 complex (Fig. 5,c) is no longer present as a crystalline material but
exists in the amorphous state. In addition, the pattern of the 2/CoQ10 complex also
exhibits an appreciable difference from that of 2 in terms of not only the peak shape
but also the relative intensities in the 128 – 208 (2q) region, indicating that the
complexation of CoQ10 makes the CD reorient to some extent. These results indicate
that the XRD of the 2/CoQ10 complex is quite different from a superimposition of
amorphous 2 and crystalline CoQ10. Therefore, a new species, in this case the 2/CoQ10
complex, is formed between 2 and CoQ10.
Fig. 5. XRD Patterns of a) CoQ10, b) host 2, and c) host 2/CoQ10 complex
2.6. Infrared Spectroscopy. The IR Spectra of CoQ10, host 2, and its inclusion
complex are shown in Fig. 6. The spectrum of CoQ10 (Fig. 6,a) shows strong
absorption bands at 2964 – 2852, 1647 – 1025, and 875 – 600 cmꢀ1. The absorption bands
at 2852, 1647, 1610, 1450, 1383, 1263, and 1205 cmꢀ1 disappear in the inclusion complex.
In the IR spectrum of the complex (Fig. 6,c), two peaks in the 2964 – 2912 cmꢀ1 region
are merged into one peak. Furthermore, the absorption intensities of the inclusion
complex are also significantly weaker than those of the free CoQ10 molecule. The IR
spectrum of host 2 (Fig. 6,b) is characterized by intense bands at 3300 – 3500 cmꢀ1,
assigned to absorption by H-bonded OH groups, as well as the bands at 2800 –
3000 cmꢀ1 corresponding to the vibrations of the CH and CH2 groups, which are
equivalent to the bands of native b-CD [25]. After association with CoQ10, the
spectrum of the inclusion complex (Fig. 6,c) shows increased intensity and a shift of the
peak from 3387 cmꢀ1 to 3385 cmꢀ1 corresponding to the H-bonded OH groups. This
suggests that after complex formation, some of the existing H-bonds formed between
OH groups of the CD are broken. The above results suggest that the CoQ10 molecule is
included into the b-CD cavity of host 2.

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Fig. 6. FT-IR Spectra (KBr) of a) CoQ10, b) host 2, and c) host 2/CoQ10 complex
2.7. Solubility of Complexes.The water solubilities of the modified b-CDs/CoQ10
complexes were assessed by the preparation of their saturated solutions [26]. An excess
amount of complex was put into 2 ml of H2O, and the mixture was stirred for 1 h.
Having removed the insoluble substance by filtration, the filtrate was concentrated and
the residue dosed by the weighing method. The results showed that the water solubility
of the 1/CoQ10, 2/CoQ10, and 3/CoQ10 complexes, comparing with that of CoQ10
(2.61 · 10ꢀ5 mg/ml) was dramatically increased to ca. 1.35, 1.52, and 1.44 mg/ml
(calculated as CoQ10 residue), respectively. In the control experiment, a clear solution
was obtained after dissolving 1/CoQ10 (5.6 mg), 2/CoQ10 (15.8 mg), or 3/CoQ10
(22.3 mg) complex, which is equivalent to 1.35, 1.52, or 1.44 mg of CoQ10, in 1 ml of
H2O at room temperature. This subsequently confirmed the reliability of the obtained
satisfactory water solubility of CD/CoQ10 complexes, which will be beneficial for the
medical utilization of this compound.
3. Conclusion. – The complexes between CoQ10 and polyamine-modified b-CDs
1 – 3 were obtained in H2O solutions by the ꢃco-precipitationꢀ method. The results
showed that the modified b-CDs dramatically enhanced the water solubility of CoQ10.
The host 2 with a moderate-length chain among these modified CDs studied was the
most suitable for inclusion complexation of CoQ10. Considering that all of the CDs
employed in this work were easily prepared, they should be regarded as an important
choice in the design of carriers for biological and medicinal substrates.

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This work was supported by the National Natural Science Foundation of China (No. 21062030) and
the Yunnan Province Natural Science Foundation (Grant No. 2008ZC040M), which are gratefully
acknowledged.
4. Experimental Part
4.1. General. The CoQ10 used in this work was obtained from Kunming Yunnan University Medical
Development Co. Ltd. (Yunnan Province, P. R. China). 6A-[(2-Aminoethyl)amino]-6A-deoxy-b-cyclo-
dextrin (1), 6A-{{2-[(2-aminoethyl)amino]ethyl}amino}-6A-deoxy-b-cyclodextrin (2), and 6A-{{2-{{2-[(2-
aminoethyl)amino]ethyl}amino}ethyl}amino}-6A-deoxy-b-cyclodextrin (3) were synthesized according
to the reported procedures [27][28]. UV/VIS Spectra: Shimadzu-UV-3600 spectrophotometer. FT-IR
Spectra: Bruker FL-IR spectroscopy device; samples mixed with KBr and compressed as disks; 16 scans
1
were signal-averaged at a resolution of 8 cmꢀ1. H-NMR Spectra: Bruker-Avance-DRX500 spectrom-
eter; at 298 K in D2O or CDCl3; ROESY Experiments: Bruker-Avance-DRX500 instrument; sample
equilibration for at least 24 h before measurement; all 2D-NMR experiments in D2O.
4.2. X-Ray Diffraction. Powder X-ray diffraction (XRD): D8-Advance diffractometer; CuKa (k ¼
1.5460 ꢄ) with 30 mA, 40 kV, and a scanning rate of 58/min. Powder samples were mounted on a
sample holder and scanned with a step size of 2q ¼ 0.028 between 2q ¼ 38 and 608.
4.3. Modified b-CDs 1 – 3/CoQ10 Complexes. To generate the CD/drug complex, CoQ10
(0.045 mmol, 38.83 mg) and 1 (0.03 mmol, 35.28 mg) were completely dissolved in THF/H2O 1:3 and
stirred for 3 d at r.t. After evaporation of the THF from the mixed soln., the uncomplexed CoQ10 was
removed by filtration. The filtrate was again concentrated to remove H2O and the residue dried under
vacuum: 1/CoQ10 (29.5 mg, 48%).
Complex 2/CoQ10 and complex 3/CoQ10 were prepared similarly as 1/CoQ10 in ca. 49% yield from
2 and CoQ10, and in ca. 47% yield from 3 and CoQ10, resp.
4.4. Phase-Solution Diagram. Phase-solubility measurements were carried out by the method of
Higuchi and Connors [29]. An excess amount of CoQ10 (150 mg) was added to 2 ml of deionized H2O
containing increasing amounts of polyamine-modified CDs (0 – 0.0274 m). The suspensions were stirred
at 258 for 4 d, until equilibrium was reached. Suspensions were filtered through 0.4 mm membrane filters
to remove undissolved solid. An aliquot from each vial was diluted and analyzed with a UV/VIS
spectrophotometer (UV-2550, Shimadzu) at 254 nm. Polyamine-modified CDs did not interfere in the
spectrophotometric assay of CoQ10.
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Received January 21, 2011