1

CB-114. Aggregation of Cyclodextrins is an Important Factor

Determining Their Complexation Behaviour

by Zsolt Bikádia), Róbert Kurdi

b), Sándor Baloghb), Julianna Szemán

c), and Eszter

Hazai*a)

a) Virtua Drug, Ltd., Csalogany st. 4c, H-1015 Budapest

(e-mail: eszter.hazai@virtuadrug.com)

b) Delta Elektronik, Ltd., Szentendrei st. 39 – 53, H-1033 Budapest

c) CycloLab Cyclodextrin Research and Development Laboratory Ltd., P.O. Box 435,

H-1525 Budapest

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Here we report a study on the complexation behaviour of carotenoids with

cyclodextrins (CDs) using solubility experiments and molecular modelling methods.

Carotenoids are an important group of naturally occurring dyes found in vegetables and

fruits. Their antioxidant property has initiated investigations on their possible use as a

drugs. However, carotenoids are lipophilic molecules with very little inherent aqueous

solubility. Cyclodextrin complexation has been widely used in order to increase the

potential applications of hydrophobic compounds. Thus, the aim of our investigation

was to design carotenoids with enhanced water solubility by cyclodextrin complexation.

Molecular modelling of carotenoid cyclodextrin complexes with 1:1 stochiometry

successfully explained the experimentally observed capability of β-cyclodextrins

(BCDs) to form complexes with carotenoids as opposed to α-cyclodextrins (ACDs) and

γ-cyclodextrins (GCDs). Furthermore, molecular dynamics calculations revealed that

the aggregation properties of CD derivates significantly influence their complexation

behaviour. Our docking calculations showed that RAMEB (random methylated β-

cyclodextrin) is the BCD derivate that possesses the lowest tendency to aggregate.

Solubility experiments yielded the same results, namely, RAMEB complexes possess

the best water solubility. Our results showed that complexation of a ligand not buried

inside of CD cavity is dependent on two factors: i) The geometry of the inclusion part of

the complex; ii) the self-aggregation property of the CD itself. The lower affinity the

3

CDs possess for self aggregation, the more likely are they involved in interactions with

carotenoids. These results suggest that self-aggregation of cyclodextrins should be

considered as an important parameter determining complexation in general.

Keywords:

Cyclodextrins

Carotenoids

Aggregation behaviour

Aqueous solubility

Complexation

4

Introduction. – Carotenoids are natural pigments, widely distributed in nature.

To date, over 750 natural compounds have been identified and characterized[1]. They

are known to be a main dietary source of vitamin A in humans. The recognition of

protective effects of carotenoids against serious disorders such as cancer, heart disease

and degenerative eye disease has stimulated intensive research on their role as

antioxidants and regulators of the immune system. The vast majority of carotenoids

have limited or undetectable solubility in aqueous solution [2][3]. Our previous study

showed that the limited solubility is the consequence of the formation of large card-pack

and head-to-tail carotenoid aggregates [4]. To increase the utility of these compounds

for evaluation in aqueous-phase model systems and potential clinical applications,

various techniques have been developed to improve the solubility and/or dispersibility

in various vehicles [3][5 – 8]. Among solubilizers, cyclodextrin complexation has been

widely used [9].

Cyclodextrins (CDs) are cyclic oligosaccharides obtained by the enzymatic

conversion of starch [10]. The parent CDs are α, β, and γ-cyclodextrins (ACD, BCD,

GCD, respectively), containing 6, 7, or 8 glucopyranose units, respectively. CDs form a

truncated cone and therefore the molecule provides a hydrophobic cavity in an aqueous

environment. When the solubility of a drug is the limiting factor for its oral delivery, its

aqueous solubility [11], stability [12] and the bioavailability [13] may be improved by

forming CD-drug inclusion complex. By complexation, the hydrophobic drug is

5

sequestered inside the cavity of the cyclodextrin. In many cases [13 – 15] complexation

of drugs by CDs improves their delivery characteristics without interfering with their

activities, because complexation is a rapidly reversible dynamic process. The parent

CDs with appropriate cavity diameter are suitable for complexing many pharmaceutical

compounds, however, they have less than optimal solubility and safety data [15]. Thus,

numerous chemically modified CDs have been developed to improve the solubility

limits of the parent CD [13 – 15].

The possible factors and the various molecular forces which may play a role in

cyclodextrin complexation has been widely discussed [1], and several hypotheses have

been proposed to account for cyclodextrin complex formations [14][16][17]. Computer

simulations are widely used to rationally explain the experimental findings concerning

inclusion and recognition [18]. Faucci and co-workers have recently developed a

mathematical model for predicting the stability of drug-cyclodextrin complexes with the

aid of molecular modelling techniques [19]. Their model was appropriate for predicting

the stability of CD- small drug complexes, which are able to totally intrude into the

hydrophobic cavity of cyclodextrins. However, in spite of the efforts, the relative

contributions of the different forces involved in the process of complexation are still not

known. Evidence has been shown that interactions among cyclodextrins are also

important factors in determining cyclodextrin complexation behaviour. Recently,

experimental evidence proved the self-aggregation of CDs in water well below the

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maximum solubility [20]. Moreover, the self-assembly of CDs has been shown to affect

their aggregation properties.

Previous studies on short-chain analogues of carotenoids, beta ionone retinoids

[21][22] demonstrated the formation of stable inclusion complexes with CDs. It was

shown that the terminal cyclohexene fragment of beta-ionone, which is present in most

carotenoids, has the requisite size for incorporation into the CD cavity. Thus, it seems

likely that carotenoids also form complexes. Recently, experimental evidence of a real

carotenoid-cyclodextrin inclusion complex formation was published [21]. However,

because of the size and shape of carotenoids and CDs, carotenoids will not be totally

buried in the cyclodextrin cavity. Therefore, it is reasonable to assume that even when

forming inclusion complex, a part of the carotenoid molecule is still able to form

interactions with either another carotenoid or cyclodextrin molecule.

In the present work, molecular docking and molecular dynamics calculations

were utilized as a tool for designing carotenoid-cyclodextrin complexes with enhanced

carotenoid water solubility. Typical carotenes and carotenoids were selected for

investigation: open-chain carotene lycopene, β-carotene with two β-rings and

carotenoids lutein, zeaxanthin and a slightly water soluble carotenoid derivative,

crocetin (Fig. 1). Our experimental results showed that among the parent CDs, only

BCD raises carotenoid solubility at an excess cyclodextrin concentration. Molecular

dynamics calculation was carried out to get deeper insight into the complexation

7

mechanism. Our calculations revealed that carotenoids are able to form complexes with

CDs with greater than 1:1 stochiometry. Moreover, we showed that the tendency of CDs

to aggregate inversely correlates with their ability to form water soluble complexes with

carotenoids. Finally, our results showed that the CD of choice for complexation with

carotenoids possesses a low affinity for self-aggregation and forms large contact surface

area complex.

Results and Discussion. – The complexation of various molecules with CDs is

of high industrial importance as many applications exist in pharmaceutical and

environmental research. Numerous investigations have been carried out on the

mechanism of host-guest inclusion processes in order to get some information about the

forces which lead to the association of a molecule into the cavity of CDs. In our study,

the interactions between the guest molecules and the interior of various CDs were

investigated by both experimental methods and molecular modelling calculations. The

goal of our project was to find the most appropriate CD derivate which enhances the

solubility of the investigated carotenoids. Representative carotenes and carotenoids

selected for investigation were lycopene, β-carotene, lutein, zeaxanthin and crocetin

(Fig. 1).

Solubility of carotenoids in parent ACD, BCD and GCD solutions. First, the

complexation behaviour of different carotenoids with parent CDs were examined (Table

8

1) in order to determine the appropriate cavity size. With the exception of lutein, none

of the investigated carotenoids (lycopene, beta-carotenoid, zeaxanthin, crocetin, Fig. 1)

were able to form soluble complexes with any of the parent CDs. Lutein was the only

carotenoid forming BCD complex that showed a slight solubility, while it was not

soluble in ACD and GCD. These data demonstrate that underivatized CDs are unable to

significantly enhance carotenoid solubility. It was anticipated that molecular modelling

helps predicting which CD is the most likely to form complexes of greater solubility.

Therefore, molecular modelling calculations have been carried out in order to identify

the molecular mechanism underlying the complexation mechanism.

Molecular Docking Studies of Carotenoids to Parent CDs. Molecular modelling

studies were performed in order to determine the relationship between the docking

energies of drug-cyclodextrin complexes and their corresponding solubility. The

docking energies were calculated as the sum of the van der Waals forces and

electrostatic interaction energies. The only carotenoid that showed solubility in any of

the parent CDs was lutein, therefore, this molecule was applied in our docking

calculations. The best energy docking geometries of lutein with ACD, BCD and GCD

can be seen on Fig. 2. In the case of ACD, the size of the lutein ring does not entirely

allow the carotenoid ring to intrude into the cavity, therefore, the ring-OH is not able to

form a hydrogen bridge with a hydroxy group of CDs. Non-specific hydrophobic

interactions are formed between lutein and ACD. In contrast, in case of BCD and GCD,

9

the lutein ring is buried inside the cavity, and is able to form a hydrogen bond with a

hydroxy group of CD. However, the docking energy of lutein complexes with ACD,

BCD and GCD do not differ substantially (Table 2), indicating that analyzing the

docking energy itself is not sufficient in explaining which CD forms complex with

lutein. Mura and co-workers explained the solubility of inclusion complexes with three

theoretical parameters: intermolecular interaction fields, docking energy and contact

surface [19]. They acquired the most accurate theoretical model for the computational

prediction of the aqueous solubility of the inclusion complex. Therefore, the contact

surface of the complexes as a measure of the degree of fit of the guest molecule into the

host molecule (Table 3), was examined. This was possible first by evaluating the

solvent accessible surface of the single molecules and than by the complexes formed.

After that the decrease in the solvent accessible surface of the complex as compared to

the single molecules was analyzed. Our results showed that the greatest contact surface

is observed in the case of lutein BCD complex. The ring of the lutein cannot totally

intrude into the cavity of ACD. In contrast, there is an unfilled volume in the GCD

cavity, and lutein ring forms interactions with only a part of the CD-ring. Indeed,

solubility experiments showed that lutein dissolves in aqueous BCD solutions, but not

in ACD or GCD (Table 1).

Contact surface calculations of all investigated carotenoid-BCD complex were

carried out. This calculation was based on the assumption that a molecule possessing

10

similar or higher contact surface than lutein, is able to form water soluble complex with

a BCD derivate. The resulted contact surfaces of the calculated minimum-energy

complexes are shown in Table 4. These data show that zeaxanthin and lutein are the

most promising carotenoids to form complexes with BCDs. Other investigated

carotenoids without a substituent on their six-membered ring and open-chain

carotenoids do not fill up the available space inside the CD cavity.

Stepwise Docking Calculations of Carotenoids to Parent CDs and CD

Derivatives. Many examples are known that the use of derivatized CDs greatly enhance

solubility. Therefore, interaction energies of carotenoids with different BCD derivates

were examined. The docking energies of carotenoids to the different BCD derivates

assuming 1:1 complexes yielded about identical energy values and about identical

contact surface as well (data not shown). This finding is reasonable if we consider that

the substituted groups lie at the surface of the CDs, therefore, are not expected to

influence the interaction with the ligand. Thus, the following factors should be taken

into consideration when explaining the different solubility behaviour of CD derivates as

compared to the parent CD: a) One possibility is that carotenoids form complex with

CDs with greater than 1:1 stochiometry. In this case it is possible that not only the CD

cavity, but also the polar surface of the CD molecule interacts with the carotenoids. b)

The other possibility is that CD self-aggregation influences complexation behaviour.

The CD molecules already in interaction with other CDs are not freely available for

11

interaction with carotenoids. c) Our previous study showed that carotenoids form large

card-pack and head-to-tail carotenoid aggregates [4]. Although this fact is likely to

influence carotenoid-CD complexation in general, it does not explain diffences in

solubility in parent CD and CD derivates for a given carotenoid.

a) The first possibility was examined as follows: another CD molecule was

docked into the lutein- CD 1:1 complex (data not sown). The results showed that the

second CD forms interactions with the first CD molecule rather than with the

carotenoid. Subsequent dockings yielded differently arranged CDs around the

previously docked CDs rather than around the carotenoid molecule. These results

indicate that interaction between the polyene chain of the carotenoid and the polar

surface of the CD are of non-specific nature. Derivatization of BCD does not influence

substantially this non-specific interaction. These results point to the possibility of CD-

CD complexation influencing solubility rather than more CD molecules interacting with

one carotenoid molecule. b) According to the docking calculations, the effect of CD

self-aggregation should be taken into account in prediction of complexation behaviour

of CD derivates. Indeed, the self-aggregation of CDs in water has recently been

postulated [20].

Molecular dynamics of carotenoid-CD system. Molecular dynamics simulations

were carried out in order to simulate the dynamical behaviour of CDs in the presence of

carotenoids. A simulation box including water, different CDs at a concentration of

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0.05M, was constructed (‘in silico CD solution’). The simulation was carried out in the

in silico CD solution including a carotene ligand. This way both the self-aggregation

properties of CDs and the possibility of formation of carotene-CD complex with more

than 1:1 stoichiometry could be simultaneously analyzed. Three parallel runs were

carried out for a simulation time of 3 ns. The organization and geometry of BCDs and

carotenoid was analyzed at the end of the run for i) stoichiometry of lutein-BCD

complex ii) BCD aggregation. Every run differed in the stoichiometry of lutein–BCD

complex, with 3 – 12 CD being in interaction distance of lutein. One example of a

complex geometry after 3 ns molecular dynamic run is shown in Fig. 3.

The molecular dynamic simulation revealed that in BCD solution specific

interactions might form between the lutein ring and BCD cavity. Furthermore,

interactions between the polyene chain of lutein and BCDs can be formed, however,

these interactions are non-specific in nature. The exact stoichiometry of the complex

cannot be determined. Besides interacting with the carotenoid, CDs aggregated during

the simulation. The beginning and the end of a molecular dynamics simulation is shown

in Fig. 4. It can clearly be seen that the self-aggregation of CDs influenced the number

of BCDs available for interaction with lutein. The aggregation of CDs in water is also

confirmed by experimental data. Namely, experimental evidence has recently been

reported of BCDs forming aggregates in aqueous solution at low concentrations well

below the maximum solubility [20]. Additionally, it has been shown that aggregation of

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CDs significantly influences their complexation behaviour. Analyzing BCD aggregates

reveals that BCDs are held together through a large number of hydrogen bonds. It is

reasonable to assume that substitution of the hydroxy groups (as is in the case of

dimethylated β-cyclodextrin, DIMEB and random methylated β-cyclodextrin, RAMEB)

hinders the ability of CDs to form aggregates. Thus, at same CD concentration, a higher

number of RAMEB and DIMEB molecules are available for interaction with lutein than

in case of BCDs. This possibility can be further examined by calculating the interaction

energy of CDs with CDs by molecular docking.

Molecular Modelling of CD Dimerization. The interaction energy of CD

derivates in dimers was calculated using Autodock program. The results of docking

studies are shown in Fig. 5 and the estimated free energy of dimerization in Table 5.

Significant differences in interaction energy of CDs were observed. Namely,

aggregation of BCDs yielded the lowest energy, followed by hydroxypropylated β-

cyclodextrin (HPBCD), DIMEB and RAMEB.

The degree of average substitution of BCDS is as follows: HPBCD 3, DIMEB

14, RAMEB 14 (random distribution). In the cases of DIMEB and RAMEB,

substitution of CD decreases its ability to donate hydrogen bonds, whereas

hydroxypropyl substitution (HPBCD) does not strongly influence the ability forming

intermolecular hydrogen bonds. As CDs form hydrogen bonds during self-aggregation,

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it is reasonable to assume that the number of hydrogen bond donors available in CDs

determine their self-aggregation properties. Indeed, the best energy of interaction was

observed for BCD, followed by HPBCD, DIMEB and RAMEB. The interaction energy

of CDs inversely correlates with their tendency to self-aggregate which in turn inversely

correlates with complexation behaviour. Based on this, the investigated CDs are

predicted to form water soluble complexes with carotenoids in the following order:

RAMEB followed by DIMEB, HPBCD and BCD.

Aqueous Solubility of Carotenoids in Complex with CD Derivatives. Lycopene

and β-carotene was not soluble in the aqueous solutions of any of the investigated CDs,

while crocetin and zeaxanthin showed an improved aqueous solubility in RAMEB

(Table 6). Lutein was slightly water-soluble in all investigated BCD derivates. The

solubility rank order of lutein in the investigated BCD derivates was as follows:

RAMEB was the most effective solubilizing agent for lutein, followed by DIMEB,

HPBCD and BCD in accordance with the calculations.

Overall, RAMEB was by far the most efficient BCD derivate that is capable of

forming water-soluble complex with the investigated carotenoids. The calculated

interaction energy of CD dimerization inversely correlated with the results of solubility

experiments (Fig. 6). Thus, our study shows that formation of CD complexes of

carotenoids depends on the aggregation properties of CDs themselves. The less the CDs

aggregate, the more likely are they involved in interactions with carotenoids. The degree

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of substitution of a BCD derivate correlates with its ability to form complex with

carotenoids.

Conclusion. – In this article, we report a study on the complexation behaviour of

carotenoids with CDs using molecular modelling and experimental methods. The aim of

the investigation was to assist designing carotenoids with enhanced water solubility by

different CD derivates. Our results indicate that the investigated carotenoids interact

with BCD derivates but not with ACD or GCDs. Molecular docking studies with

carotenoid cyclodextrin complex with 1:1 stochiometry could explain the capability of

ΒCDs to form complexes with carotenoids as opposed to ACDs and GCDs. However,

docking studies assuming 1:1 carotenoid-CD complex cannot differentiate between the

different complexation behaviour of BCD derivates. Molecular dynamics calculations

revealed that the aggregation properties of CD derivates significantly influence their

complexation behaviour. The less the CDs aggregate, the more likely are they involved

in interactions with carotenoids. Docking calculations identified RAMEB as the BCD

derivate with lowest tendency to aggregate. Indeed, solubility experiments confirmed

that RAMEB complexes show by far the best water solubility. To summarize, our

results showed that complexation of a ligand not buried inside of CD cavity is

dependent on two factors: i) The geometry of the inclusion part of the complex. ii) The

self-aggregation property of the CD itself. Moreover, our results suggest that self-

16

aggregation of cyclodextrins should be considered as an important parameter

determining complexation in general.

Experimental Part

General. Abbreviations. Cyclodextrin: CD; α-cyclodextrin: ACD; β-

cyclodextrin: BCD; γ-cyclodextrin: GCD; Random methylated β-cyclodextrin:

RAMEB; dimethylated β-cyclodextrin: DIMEB; hydroxypropylated β-cyclodextrin:

HPBCD

Docking Calculations. MMFF94 force field [23] with conjugate gradient method

was used for energy minimization of carotenoids using Spartan’04 program. Initial

coordinates of α-, β- and γ-CDs were extracted from published x-ray structures of CD-

protein complexes (PDB entries: 1CXF, 3CGT, 1P2G, respectively). Gasteiger partial

charges were added to ligand and CD atoms with the aid of Autodock tools [24]. Non-

polar hydrogen atoms were merged, rotatable bonds and solvation parameters were

defined with Autodock tools. Each carotenoid was docked into each CD using the same

parameter set.

Affinity (grid) maps of 100×40×40 grid points and 0.375 Å spacing were

generated using the Autogrid program [24]. Preliminary studies showed that without

restrictions Autodock results in an artificial complex, where a cyclodextrin is trapped

17

between the end groups of carotenoids. Box centres were defined to rule out this kind of

complex conformation. AutoDock parameter set- and distance-dependent dielectric

functions were used in the calculation of the van der Waals and the electrostatic terms,

respectively. Docking simulations were performed using the Lamarckian genetic

algorithm (LGA) and the Solis & Wets local search method [25] of Autodock. Initial

position, orientation, and torsions of the ligand molecule were set randomly. All

rotatable torsions were released during docking. Each docking experiment was derived

from 100 different runs that were set to terminate after a maximum of 1,500,000 energy

evaluations. The population size was set to 250. During the search, a translational step

of 0.2 Å, and quaternion and torsion steps of 5 were applied. CD-CD docking

calculations were performed for BCD, RAMEB, DIMEB and HPBCD. The free energy

of binding was calculated with Autodock [24].

Contact Surface Calculations. The program VEGA ZZ [26] was used to

calculate the contact surface area of carotenoids-cyclodextrin complexes. A probe

radius 1.0 Å and a density 20 were applied. Surface areas were calculated for CDs and

carotenoids independently and in the docked complex. The contact surface area is

derived from the difference between the sum of independent surfaces and the area of the

complex surface.

Molecular Dynamics Calculations. Molecular dynamics calculations were

carried out using the GROMACS program package [27] to simulate the behaviour of

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lutein molecule in aqueous solutions of different CDs. The topology files of the solvent

molecules (cyclodextrin) and the solute (lutein) were generated using the Dundee

Prodrg server [28]. Water-CD solvent boxes at 0.05M CD concentration were generated

for ΑCD, BCD and GCD using the editconf and genbox commands of GROMACS. The

solvent boxes were energy minimized by unrestrained MD calculations. The simulation

time was 1 ns. These simulation boxes were applied to solvate lutein and to calculate

the dynamic behaviour of lutein-cyclodextrin systems. Complexes were energy

minimized using the GROMOS force field [29] implemented in the program package.

Unrestrained molecular dynamics simulations (MD) were performed for 3 ns for each

lutein-CD system.

Solubility Experiments. The used cyclodextrins and cyclodextrin derivatives

were the products of CycloLab Ltd. (Budapest, Hungary). The fine chemical grade CD

derivatives are tested, identified and characterized with analytical methods. The average

degree of substitution of statistically substituted derivatives is calculated from NMR

data. For characterization of isomer distribution various methods (TLC, MS, NMR, for

some derivatives, GC, HPLC or capillary electrophoresis) are used. The average

molecular weights of parent and derivatized CDs were determined (ACD: 972.9, BCD:

1135.0, GCD: 1297.2, RAMEB: 1303, DIMEB: 1331.4, HPBCD: 1309.0) The phase

solubility studies were performed in deionised water at 25°. Excess amounts (more than

19

those assumed to be dissolved by the cyclodextrins) of the studied carotenoids were

applied to the cyclodextrin-containing aqueous solutions. The cyclodextrin

concentration generally varied between 1.0 and 20.0g/100 ml. The aqueous suspensions

were stirred with magnetic stirrer (200 rpm) for 24 hours.After the sufficient

equilibration the suspensions were filtered across a 0.45µm membrane and assayed with

or without appropriate dilution for dissolved carotene concentration by UV-VIS

spectrophotometry.

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21

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Received August 10, 2006

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Table 1. Solubility Data of Carotenoids with a 5% Solution of Parent CDs. The only

complex showed higher aqueous solubility than limit of detection (LOD) is lutein-BCD.

Aqueous solubility (µg/ml)

Carotene/CD ACD GCD BCD

Lutein < LOD < LOD 0.55 ± 0.05

Lycopene < LOD < LOD < LOD

Beta-carotenoid < LOD < LOD < LOD

Zeaxanthin < LOD < LOD < LOD

Krocetin < LOD < LOD < LOD

24

Table 2. Docked energy of lutein to parent CDs. The calculated difference is negligible.

Cyclodextrin-lutein complex Docking energy / kcal/mol

ACD-lutein –5.43

BCD-lutein –5.41

GCD-lutein –5.44

25

Table 3. Calculated Contact Surface Areas of Lutein-CD Complexes. Lutein-BCD

complex posses the largest contact surface area.

Surface area

[Å2]

Sum of CD and

lutein [Å2]

CD-Lutein

complex [Å2]

Contact

surface [Å2]

ACD 895.0 1 787.1 1 518.3 268.8

BCD 1 058.5 1 950.6 1 626.9 323.7

GCD 1 177.1 2 069.2 1 793.4 275.8

Lutein 892.1

26

Table 4. Docked Energies and Contact Surface Areas of Carotenoid-BCD Complexes.

Zeaxanthin and lutein were calculated to form highest surface area complexes with

BCD.

Crocetin Lutein Lycopene β-Carotene Zeaxanthin

Docked energy [kcal/mol] –5.32 –5.71 –5.74 –5.58 –5.35

Complex surface [Å2] 1345.0 1626.9 1666.5 1624.5 1597.0

Ligand surface [Å2] 563.3 892.1 915.0 858.7 883.1

BCD surface [Å2] 1058.5 1058.5 1058.5 1058.5 1058.5

Contact surface [Å2] 276.8 323.7 307.0 292.7 344.6

27

Table 5. Estimated Free Energy of Binding of Cyclodextrin Dimerization. The

calculated energy correlates with hydrogen bond forming ability of CDs.

Estimated free energy of binding [kcal/mol]

BCD –2.60

HPBCD +1.81

DIMEB +3.44

RAMEB +5.49

28

Table 6. Solubility Data of Carotenoids with 5% Solution of BCD Derivatives. RAMEB

complexes show by far the best aquaeous solubility.

Aqueous solubility [µg/ml]

Carotene/CD HPBCD DIMEB RAMEB

Lutein 0.87 ± 0.09 10.3 ± 0.6 29.0 ± 1.6

Lycopene < LOD < LOD < LOD

Beta-carotenoid < LOD < LOD < LOD

Zeaxanthin < LOD < LOD 20.0 ± 1.2

Krocetin < LOD < LOD 3.6 ± 0.3

29

Captions

Fig. 1. Chemical structures of the investigated carotenoids

Fig. 2. Best energy docking results of lutein to a) ACD b) BCD, and c) GCD from two

viewpoints (left and right panels)

Fig. 3. The final geometry of a lutein-BCD system after 3 ns molecular dynamics

simulation. Lutein molecule is buried inside an aggregate formed by BCDs.

Fig. 4. Initial structure and the resulting geometry of cyclodextrin solvent after a 3 ns

MD run. Formation of cyclodextrin aggregates can be observed, which influences their

ability to form complexes with carotenoids.

Fig. 5. Best energy results of cyclodextrin-cyclodextrin dockings, a) RAMEB, b)

DIMEB, c) HPBCD, d) BCD. The number of hydrogen bonds formed between CDs

increases in the order of RAMEB < DIMEB < HPBCD < BCD.

Fig. 6. Lutein solubility in BCD derivatives in function of calculated free energy of CD

dimerization