Characterization and enhanced antioxidant activity of the cysteinyl β-cyclodextrin-baicalein inclusion complex

Article abstract of DOI:10.3390/molecules21060703

Baicalein is a type of flavonoid isolated from the roots of a medicinal plant, Scutellaria baicalensis. Although it has attracted considerable attention due to its antiviral, anti-tumor, and anti-inflammatory activities, its limited aqueous solubility inhibits the clinical application of this flavonoid. The present study aimed to prepare and characterize a host-guest complex in an effort to improve the solubility and antioxidant activity of baicalein. The host molecule is a macrocyclic β-cyclodextrin (β-CD) functionalized with cysteine for a synergetic effect. The structure of the synthesized cysteinyl β-CD was analyzed using nuclear magnetic resonance (NMR) spectroscopy and mass spectrometry. The inclusion complex with baicalein was studied by UV-vis, NMR spectroscopy, scanning electron microscopy, and X-ray powder diffractometry. The formed cysteinyl β-CD/baicalein inclusion complex efficiently improved the solubility and antioxidant ability of baicalein. Therefore, we suggest that the present cysteinyl β-CD is a potential host molecule for inclusion complexation and for bioavailability augmentation.

Full text of DOI:10.3390/molecules21060703

molecules
Article
Characterization and Enhanced Antioxidant Activity
of the Cysteinyl β-Cyclodextrin-Baicalein
Inclusion Complex
Hwanhee Kim 1, Hu Yiluo 1, Seyeon Park 2, Jae Yung Lee 3, Eunae Cho 4,* and Seunho Jung 1,4,
*
1
Department of Bioscience and Biotechnology, Microbial Carbohydrate Resource Bank (MCRB),
Konkuk University, 120 Neungdong-ro, Gwangjin-gu, Seoul 05029, Korea; hwanhee@konkuk.ac.kr (H.K.);
lannyhu0806@hotmail.com (H.Y.)
Department of Applied Chemistry, Dongduk Women’s University, Seoul 136-714, Korea;
sypark21@dongduk.ac.kr
Department of Biological Science, Mokpo National University, Jeonnam 534-729, Korea;
jaeyunglee@gmail.com
Center for Biotechnology Research in UBITA (CBRU), Institute for Ubiquitous Information Technology and
Applications (UBITA), Konkuk University, 120 Neungdong-ro, Gwangjin-gu, Seoul 05029, Korea
Correspondence: echo@konkuk.ac.kr (E.C.); shjung@konkuk.ac.kr (S.J.); Tel.: +82-2-450-3520 (S.J. & E.C.)
2
3
4
*
Academic Editor: Bernard Martel
Received: 4 May 2016; Accepted: 25 May 2016; Published: 27 May 2016
Abstract: Baicalein is a type of flavonoid isolated from the roots of a medicinal plant,
Scutellaria baicalensis. Although it has attracted considerable attention due to its antiviral, anti-tumor,
and anti-inflammatory activities, its limited aqueous solubility inhibits the clinical application
of this flavonoid. The present study aimed to prepare and characterize a host-guest complex
in an effort to improve the solubility and antioxidant activity of baicalein. The host molecule
is a macrocyclic
β-cyclodextrin (
β-CD) functionalized with cysteine for a synergetic effect. The
structure of the synthesized cysteinyl
β-CD was analyzed using nuclear magnetic resonance (NMR)
spectroscopy and mass spectrometry. The inclusion complex with baicalein was studied by UV-vis,
NMR spectroscopy, scanning electron microscopy, and X-ray powder diffractometry. The formed
cysteinyl β-CD/baicalein inclusion complex efficiently improved the solubility and antioxidant
ability of baicalein. Therefore, we suggest that the present cysteinyl
for inclusion complexation and for bioavailability augmentation.
β-CD is a potential host molecule
Keywords: cysteinyl β-cyclodextrin; baicalein; inclusion complex; antioxidant activity
1. Introduction
In an inclusion complex system, macrocyclic host molecules are of great importance, as the
cyclized and constrained conformation can provide the advantages of molecular selectivity and
recognition [
1
]. The compound
β
-cyclodextrin (
β
-CD) is
α
-1,4-linked macrocyclic oligosaccharide
].
containing seven glucose units. It can encapsulate hydrophobic compounds in its internal cavity [
2
Further, host-guest complexation can change the physico-chemical and biological characteristics
of guest compounds. Due to its complex-forming ability, various applications are found in the
pharmaceutical, food, and cosmetic industries [3].
To expand the applicability of
-CDs have been synthesized, and the inclusion complexing ability has been improved [
In addition, lipid derivatives of -CD have been investigated as promising drug-carrier systems
based on their self-assembled architecture [ ]. However, few studies of amino acid or peptide
derivatives have been reported to date [ ]. Among them, cysteine modification is expected to provide
β-CD, chemical derivatives such as hydroxypropyl and methyl
β
4,5].
β
6,7
8,9
Molecules 2016, 21, 703; doi:10.3390/molecules21060703
www.mdpi.com/journal/molecules

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antioxidant effects through the inactivation of hydroxyl radicals or through the support of glutathione
synthesis [10,11].
Baicalein (5,6,7-trihydroxyflavone) is an active phytochemical isolated from Baikal skullcap root
extract. It is used against inflammatory diseases such as hepatitis, nephritis, bronchitis, asthma, and
atopic dermatitis [12]. In addition, antiviral [13], antibacterial [14], and anti-cancer activities have
been found [15]. The beneficial effects on human health are also closely related to the antioxidant
properties of baicalein because it has good hydrogen and electron donors, and the radical intermediates
are stabilized by possible resonance forms [16]. Although interest in baicalein with regard to its
chemopreventive properties has arisen, its poor aqueous solubility (90
µ
g/mL, 25 ˝C) inhibits
its bioavailability. Thus, insolubility as a practical issue remains to be solved before additional
clinical applications.
Herein, we aim to synthesize cysteine-functionalized β-CDs for inclusion complexation with
baicalein. Inclusion complexes were analyzed with nuclear magnetic resonance (NMR) spectroscopy,
scanning electron microscopy (SEM), and X-ray powder diffractometry (XRPD). The modified
properties of the complexed baicalein were also evaluated in terms of their aqueous solubility
and antioxidant activities. The desired synergetic effect by the baicalein guest compound and the
cysteine-conjugated host molecule was observed.
2. Results and Discussion
2.1. Structural Analysis of Mono-6-Cysteinyl-β-CD (Cysteinyl β-CD)
Cysteinyl
β-CD was prepared as described in the Materials and Methods (Scheme 1). After
purification, the structure of the resultant product was analyzed using matrix-assisted laser desorption/
ionization-time of flight (MALDI-TOF) mass spectrometry and NMR spectroscopy. Pseudo-molecular
ion peaks ([mono-6-cysteinyl
at m/z = 1260.42 and 1282.40 (Figure 1). The chemical structure is shown in Figure 2a. Due to cysteinyl
modification, the anomeric protons of -CD were also separated between 5.00 and 5.10. In the
range of 2.90 to 3.30, upfield-shifted methylene protons on the C6 position of glucose were detected
with the methylene signals of the side chain (Figure 2b). In its heteronuclear single quantum coherence
(HSQC) spectrum (Figure 2c), compared to C6 carbons ( 62.47) with a free OH group, the substituted
36.48, becoming correlated with the methylene germinal
H61 signals. Chiral carbons of cysteine also appear at
56.38 with the cross-peak of the attached
hydrogens ( 3.73–3.95). Methylene carbon and hydrogen in the cysteine side chain were detected
at 35.71/3.17 and 35.71/2.98, respectively. The results of these structural analyses indicate that
cysteinyl β-CD was successfully synthesized.
β
-CD + Na]+ and [mono-6-cysteinyl
β-CD
´
H + 2Na]+) were observed
β
δ
δ
δ
δ
δ
1
C6 carbons shifted significantly upfield to
δ
δ
δ
δ
δ
Figure 1. MALDI TOF mass spectrum of cysteinyl β-CD.

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Figure 2.
β-CD (bottom); (c) HSQC spectrum of cysteinyl β-CD. Solvent: D2O.
(a) Chemical structure of cysteinyl β-CD; (b β-CD (top) and cysteinyl
) 1H-NMR spectra of
Scheme 1. Schematic illustration of synthesis of cysteinyl β-CD.
2.2. Phase Solubility Diagram
The substituted cysteine may provide additional effects related to electrostatic interaction and
hydrogen bonding sites as well as easily oxidized thiol groups. Together with the attached -CD cavity,
cooperative recognition will lead to more sophisticated complexation for guest molecules. In relation
to this, the inclusion capacity of cysteinyl -CD for baicalein was investigated. The phase solubility
diagram is shown in comparison with that of unmodified -CD (Figure 3). According to Higuchi
and Connors [17], an AN-type phase diagram for the cysteinyl -CD/baicalein complex is obtained.
Although cysteinyl -CD-induced changes in the dielectric constant of the aqueous complexation
solvent or self-association of the complexes will give rise to negative deviation from linearity, the curve
indicates the formation of a soluble complex between cysteinyl -CD and baicalein. The solubilizing
β
β
β
β
β
β
efficiency (SE) is determined as the ratio between the solubility of the drug in the presence of a certain
host concentration and the drug alone in water. Here, the solubilizing efficiency (SE 9.21) of cysteinyl
β
-CD was much better than that of the original
After attaching cysteine on -CD, the intrinsic aqueous solubility of
100 fold (cysteinyl
-CD: >180 g/100 mL, 25 ˝C vs. -CD: 1.82 g/100 mL, 25 ˝C). Furthermore, the
complexation with baicalein may be more effective, although β-CD also has a capturable cavity.
β
-CD (SE 2.35) in the presence of 10 mmol
β-CDs.
β
β-CD could be improved by about
β
β

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Figure 3. Phase solubility diagrams of baicalein with cysteinyl β-CD and β-CD in aqueous solution
at 25 ˝C.
2.3. NMR Studies
To examine non-covalent interactions at the molecular level, NMR spectroscopic analysis is
performed. Figure 4 shows the 1H-NMR spectra, where the proton signals of baicalein are shifted
in the upfield or downfield direction by the inclusion complex with cysteinyl
displacement indicates an environment of electronegative atoms [18], and an upfield shift displacement
is attributed to the variation in the local polarity after insertion into a -CD cavity [19]. The protons
β-CD. A downfield
β
(2–6) in the benzyl ring are shifted toward the upfield direction, indicating the incorporation in the
cavity. For the 31 and 81 protons in the chromene moiety, a pronounced downfield-shifted and split
pattern is observed in the presence of cysteinyl
β-CD. This result places stress on the chromene
positioning toward the cysteine on the primary part of β-CD together with the benzyl insertion.
Figure 4.
of cysteinyl
(
a
) 1H-NMR spectrum of baicalein (Solvent: D2O:MeOD, 70:30, v/v); (
-CD/baicalein complex (Solvent: D2O:MeOD, 70:30, v/v); ( ) Enlarged spectrum of the
-CD/baicalein complex). The inset shows the
b
) 1H-NMR spectrum
β
c
aromatic region (top: baicalein, bottom: cysteinyl
chemical structure of baicalein.
β

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In addition, two-dimensional nuclear Overhauser effect (NOE) signals were detected in a NOESY
experiment. Given that two protons located in close proximity can induce an NOE cross-peak, this
approach is useful for analyzing the intermolecular interaction of inclusion complexes [20]. Both
the β
-CD cavity (H3,5) and cysteine protons (H71) share NOEs with benzyl-ring protons (2–6) and
chromene protons (31,81) (Figure 5). Taken together, the results of these NMR spectroscopic analyses
suggest that the plausible complex is the A mode rather than the B mode (Figure 5c). The benzyl moiety
penetrates into the β-CD cavity from the narrow rim side, and the 5,6,7-trihydroxy-4H-chromene-4-one
is located near the cysteine residue. The proposed structure would be effective for the cooperative
complexation of baicalein and cysteinyl β-CD.
Figure 5. (a) NOESY spectrum of cysteinyl β-CD/baicalein complex (Solvent: D2O); (b) Enlarged spectrum
of the area in the red rectangle; (c) The proposed model of the cysteinyl β-CD/baicalein complex.
2.4. SEM Analysis
SEM analysis is frequently used as a tool to visualize the surface morphological changes in
inclusion complexation studies [21
the physical mixture, and the inclusion complex. Baicalein shows a rod shape of approximately 10
m in length, whereas amorphous particles 2 m in size are observed in cysteinyl -CD. The physical
mixtures showed a combined morphology of baicalein and cysteinyl -CD. After complexation, the
,22]. Figure 6 shows the SEM images of baicalein, cysteinyl β-CD,
µ
µ
β
β
original morphologies of both compounds disappeared and fibrous structures were newly observed.
Thus, the data obtained from the SEM analysis suggest that the inclusion complex formation process
between baicalein and cysteinyl β-CD occurs in a solid state.

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Figure 6. SEM photographs. (a) Baicalein; (b) Cysteinyl β-CD; (c) Physical mixture; (d) Cysteinyl
β-CD/baicalein inclusion complex.
2.5. XRPD
Further evidence for the cysteinyl
β-CD/baicalein inclusion complex is provided by XRPD
experiments. XRPD is a suitable method for the evaluation of inclusion complexation in powder or
microcrystalline states [23]. Figure 7 shows the disappearance of the sharp peak, indicating a lack of
crystallinity with the formation of the inclusion complex. In the diffractogram of the physical mixture,
a superimposed pattern of each compound is clearly displayed. Accordingly, the formation of the
cysteinyl β-CD/baicalein inclusion complex is confirmed.
Figure 7. X-ray diffractograms. (a) Baicalein; (b) Cysteinyl β-CD; (c) Physical mixture; (d) Cysteinyl
β-CD/baicalein inclusion complex.

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2.6. Antioxidant Effect
Baicalein has been reported to scavenge reactive oxygen species which cause damage to lipids,
proteins, and DNA in cellular systems [24]. Based on the structure, one-step hydrogen atom transfer
or an electron transfer followed by a proton transfer can be the antioxidant mechanism [16]. The
antioxidant effects of samples were investigated based on a DPPH assay. Because DPPH is a stable free
radical generating a violet color, a progressive discoloration represents antioxidant activity. The amount
of DPPH free radical scavenging is estimated by observing the decrease in the absorbance at 518 nm [25].
The antioxidant activity of the cysteinyl β-CD/baicalein inclusion complex is clearly differentiated
from that of free baicalein (Figure 8a). After efficient complexation, the limited antioxidant activity
of baicalein was highly enhanced together with the aqueous solubility. The few negative values in
the case of
β-CD or 2,6-di-O-methyl-β-CD (DM
β-CD) can be due to the enhanced solubility from the
complex formation with the violet-colored DPPH in water [26]. Furthermore, cysteinyl
β-CD shows
little antioxidant activity. The synergetic antioxidant activity may be affected by the complexation
mode (Figure 5c). The trihydroxy groups, the specific sites for the antioxidant effect, remain available to
scavenge radicals, and the sulfhydryl group of cysteine can support this activity. Given that the β-CD
cavity masks the irrelevant benzyl ring, the antioxidant activity of baicalein cannot be reduced, and,
rather, a positive effect is considered by baicalein solubilization. Recently, the solubility of baicalein
was investigated with the addition of
where DM -CD showed the highest solubilizing effect [27]. Thus, the antioxidant activity of DM
-CD was also compared, and that of cysteinyl -CD showed three-fold higher values compared to
the antioxidant effect of DM -CD under an identical treatment. The corresponding absorption spectra
α-, β-, γ-CD, hydroxypropyl-β-CD (HP β-CD), and DM β-CD,
β
β
β
β
also support the antioxidant results (Figure 8b). Hence, we find that the synthesized cysteinyl
effectively utilized to improve the solubility and antioxidant activity of baicalein.
β-CD is
Figure 8.
(a) Consumption of DPPH in the presence of baicalein, β-CDs and their complexes; (b)
Absorption spectra of baicalein in aqueous solution with different β-CDs.

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3. Materials and Methods
3.1. Chemicals
The β-CD (97%), 1-(p-toluenesulfonyl)imidazole (98%), and baicalein (>99%) were obtained from
Tokyo Chemical Industry Co., Ltd. Triethylamine (TEA), DM β-CD, L-cysteine (97%), 2,2-diphenyl-1-
picrylhydrazyl (DPPH), ethanol (>99.8%) were purchased from Sigma–Aldrich Chemicals Co. The
used water was triply distilled. D2O (99.96% at D) and CD3OD (99.8% at D) were from Cambridge
Isotope Laboratories, Inc. (Andover, MA, USA).
3.2. Synthesis of Cysteinyl β-CD
The cysteinyl
most important intermediate for mono-functionalization on the primary side of
-CD was synthesized as described in our previous report [29]. The -CD (5.0 g, 4.4 mmol) was
β
-CD was synthesized from mono-6-O-p-toluenesulfonyl-
β-CD (tosyl
β-CD), the
β-CD [28]. The tosyl
β
β
˝
dissolved in water by heating it to 60 C, and the cooled solution was mixed with 1-(p-toluenesulfonyl)
imidazole (3.9 g, 17.7 mmol). After 6 h, a NaOH aqueous solution was added over a time of 20 min,
and the unreacted precipitates were removed. Ammonium chloride (6.1 g, 115 mmol) was added to
stop the reaction, and the mixture was concentrated to half of the original volume by blowing air. Tosyl
β-CD began to precipitate, and the precipitates were washed and dried. Tosyl β-CD (921 mg, 0.7 mmol)
and L-cysteine (251 mg, 2.1 mmol) were dissolved in a 40% (v/v) TEA aqueous solution (10 mL) at
85 ˝C while stirring for two days under N2. After 48 h, excess solvent was removed by evaporation
under reduced pressure, and the concentrated sample was precipitated using ethanol (100 mL). The
precipitate was collected, and the cysteinyl
β-CD was purified using DEAE-Sephadex A-25 and a
Bio-gel P-2 column. The lyophilized product was obtained at a 30% yield starting with β-CD.
Mono-6-cysteinyl-
-CD. 1H-NMR (500MHz, D2O): 5.10–5.00 (m, 7H, H1), 3.95–3.73 (m, H3,6,71,5),
β
δ
1
1
1
3.65–3.50 (m, H2,4), 3.29–3.23 (m, 1H, H6 a), 3.19–3.12 (m, 1H, H8 a), 3.08–3.01 (m, 1H, H6 b), 3.00–2.93
(m, 1H, H81b);113C-NMR (500 MHz, D2O):
δ
175.26 (C91), 104.64 (C1,11), 86.49 (C411), 83.54 (C4), 75.60
1
1
1
(C3), 75.29 (C3 ), 74.63 (C5 ), 74.58 (C5), 74.50 (C2), 74.44 (C2 ), 62.47 (C6), 56.38 (C7 ), 36.48 (C6 ), 35.71
(C8 ); MALDI-TOF MS: 1260.42 [mono-6-cysteinyl β-CD + Na]+.
1
3.3. MALDI-TOF Mass Spectrometry
MALDI-TOF MS (Voyager- DETM STR Bio-Spectrometry, Applied Biosystems, Framingham, MA,
USA) was carried out to obtain the mass spectrum of cysteinyl
2,5-Dihydroxybenzoic acid (DHB) was used as the matrix.
β-CD in the positive ion mode. The
3.4. NMR Spectroscopy
For the NMR spectroscopic analysis, a Bruker Avance 500 spectrometer (Bruker, Karlsruhe,
1
Germany) was used to record the H-NMR, 13C-NMR, HSQC, and NOESY spectra. The NOESY
spectra were recorded with 256/2048 complex data points using a pulse train to achieve a spin-lock
field with a mixing time of 600 ms for the complex.
3.5. Phase Solubility Studies
Baicalein was dissolved in methanol to obtain a 20 mM stock solution. In this case, 500
µL of the
stock solution was added to an aqueous solution (500 L) of -CD and cysteinyl -CD with various
µ
β
β
concentrations (0.0–10.0 mM) in capped vials. In each case, the mixture was magnetically stirred
at 25 ˝C and shielded from light to prevent the degradation of the baicalein. After 72 h, methanol
was evaporated, lyophilized, and dissolved in 500 µL water. After filtering (PTFE 0.2 µm filter,
ADVANTEC), the solubilized baicalein was evaluated at 270 nm using a spectrophotometer (UV 2450,
Shimadzu Corporation, Shimadzu, Japan).

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3.6. Aqueous Solubility of β-CD and Cysteinyl β-CD
The intrinsic solubility measurement was performed with some modification in the previous
protocol [30]. The β-CD and cysteinyl β-CD were added to 250 µL of water, and the mixture was
sonicated for 1 min and then stirred for 1 h at 25 ˝C. After lyophilizing the soluble fraction, the mass
was measured as a function of the volume. Because of increasing viscosity, a more accurate estimation
of solubility was difficult.
3.7. Preparation of the Cysteinyl β-CD/Baicalein Complex
Inclusion complexes of baicalein and cysteinyl
method [31 Baicalein was dissolved in methanol and then added to an aqueous solution of an
appropriate concentration of cysteinyl
β-CD were prepared using the suspension
]
.
˝
-CD. The resulting mixture was stirred at 25 C for 72 h. After
β
equilibration, methanol was evaporated and lyophilized. The dissolved sample was lyophilized and
the sample was then dissolved in 500 µL water, after which it was filtered and freeze-dried.
3.8. SEM
The surfaces morphology of the materials was examined using a field emission scanning electron
microscope (JSM-6700F, JEOL, Ltd., Tokyo, Japan). The samples were precisely fixed on a brass stub
using double-sided adhesive carbon tape, and were then made to be electrically conductive by sputter
coating with a thin layer of gold in a vacuum. Images were taken at an accelerating voltage of 10 kV,
and a magnitude level of 2300ˆ was used.
3.9. XRPD
XPRD patterns for baicalein, cysteinyl β-CD, the physical mixtures and the inclusion complexes
were traced employing high resolution X-ray diffractometer (Bruker D8 DISCOVER, Karlsruhe,
˝
Germany) at a scanning rate of 2 /min. The voltage/current used was 40 kV/40 mA and the angular
˝
˝
range (2θ) covered was between 6 and 50 .
3.10. Antioxidant Activity
The antioxidant activities of the samples are measured in terms of their radical scavenging ability
(RSA), using the DPPH method [32]. A volume of 1 mL (ethanol:water, 50:50 v/v) of 50
used. The reaction was started with an addition of 10 L of each sample without and with a 2 mM
-CD concentration. The resulting mixtures were incubated in the dark at 25 ˝C. After the system
µM DPPH was
µ
β
reached equilibrium at the end of 30 min, the absorbance levels of the solutions were measured by
UV-Vis spectroscopy at 518 nm. The results were expressed as the percentage of DPPH eradication
calculated according to the following equation:
RSA “ p1 ´ AS{ABq ˆ 100
(1)
where the absorbance of the sample is AS and that of the blank sample is AB.
4. Conclusions
In the present study, mono-6-cysteinyl-
mono-6-O-p-toluenesulfonyl- -CD with L-cysteine in water. The complexation characteristics of
cysteinyl -CD and baicalein were analyzed using UV-vis, NMR spectroscopy, SEM, and XRPD.
The driving forces for the complexation are considered as non-covalent interactions such as hydrogen
bonding, hydrophobic interaction, and van der Waals interactions between mono-6-cysteinyl- -CD
and baicalein. The results of NMR spectroscopic analyses show a complexation mode where the
benzyl moiety penetrates into the -CD cavity and the chromene part is located near the cysteine. This
cooperative complexation can enhance the solubility and antioxidant activity of baicalein. Cysteinyl
β-CD was successfully synthesized by reacting
β
β
β
β

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β-CD and this synergetic approach for inclusion complexation will be useful for the design and
development of novel host molecules.
Acknowledgments: This paper was supported by the KU Research Professor Program of Konkuk University. This
research was also supported by the National Research Foundation of Korea, funded by the Korean Government
(NRF-2015R1D1A1A01058686) and the Bio & Medical Technology Development Program of the NRF funded by
the Korean government, MSIP (2015M3A9B8031831). SDG.
Author Contributions: Seunho Jung conceived and designed the experiments; Hwanhee Kim and Hu Yiluo
performed the experiments. Hwanhee Kim and Eunae Cho analyzed the data and wrote the paper. Jae Yung Lee
contributed the reagents and the analysis tools. Seyeon Park supervised and provided consulting during the
project. All authors reviewed the manuscript.
Conflicts of Interest: The authors declare no conflict of interest.
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Sample Availability: Samples of the compounds are Not available from the authors.
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