RESEARCH ARTICLE

Inclusion compound formulation of hirsutenonewith beta-cyclodextrin

Byeong Kil Ahn • Sang Gon Lee • Sung Rae Kim • Dong Hoon Lee •

Myung Hwan Oh • Min Won Lee • Young Wook Choi

Received: 1 July 2013 / Accepted: 7 August 2013 / Published online: 21 August 2013

� The Korean Society of Pharmaceutical Sciences and Technology 2013

Abstract Inclusion complexation with b-cyclodextrin

(b-CD) was performed to improve the stability of hirsutenone

(HST), a naturally occurring immunomodulator that is

labile in aqueous solutions. HST-b-CD inclusion com-

plexes were prepared using a solvent evaporation method.

Briefly, solutions of HST (dissolved in isopropyl alcohol)

and b-CD (dissolved in distilled water) were mixed and

evaporated under vacuum by using a rotary evaporator.

Phase solubility studies of the product revealed 1:1 or 1:2

complex formation, with an apparent stability constant of

249.2 M-1. Differential scanning calorimetry showed a

shift of endothermic peaks and nuclear magnetic resonance

spectra displayed shift changes in H-3,5,6 protons, located

inside the b-CD cavity, in inclusion complexes. These data

provided strong evidence for inclusion complex formation.

Characterization using infrared spectroscopy was hindered

because of interfering b-CD vibrations. Inclusion complex

stability was evaluated in the solid and aqueous solution

states. Rate constants (910-2, day-1) of HST and HST-b-

CD were 13.2 and 9.79, respectively, in an aqueous solu-

tion at 25 �C; the corresponding values in the solid state

were 0.14 and 0.18. The present study therefore showed

successful formation of HST-b-CD, but the stability of

HST within this inclusion compound was not markedly

improved.

Keywords Hirsutenone � Cyclodextrin �Inclusion complex � Stability � Solvent evaporation

Introduction

Hirsutenone (HST), isolated from the bark of Alnus

japonica, is a diarylheptanoid compound known for its

natural immunomodulatory activities (Kim et al. 2005).

Previous reports suggested that HST effectively inhibited T

cell activation by blocking dephosphorylation of nuclear

factor of activated T cell transcription factors (Joo et al.

2009). By suppressing the activation of NF-jB, it reduced

tumor necrosis factor-a-stimulated responses in human

keratinocytes (Lee et al. 2009). HST also prevented the

lipopolysaccharide-induced expression of Toll-like recep-

tor 4-mediated activation of the extracellular signal-regu-

lated kinase pathway (Lee et al. 2010). Because of these

anti-inflammatory and anti-oxidant effects, HST-contain-

ing formulations have been developed to treat atopic der-

matitis. The topical application of conventional cream

containing HST or intra-peritoneal injection treatment with

HST suppressed the development of atopic dermatitis-like

skin lesions and clinical signs provoked by house dust in

NC/Nga mice. HST treatment downregulated Th2 cyto-

kines and decreased levels of IgE (Jeong et al. 2010).

Furthermore, using HST-loaded Tat peptide-admixed

elastic liposome, therapeutic improvement of atopic der-

matitis in NC/Nga mice was obtained by increasing skin

permeation of HST (Kang et al. 2011). Recently, it was

reported that HST affected human voltage-gated K?

channels including a subunit encoded by the human ether-

a-go-go related gene, providing an improved understanding

of the risk for cardiac arrhythmia and cardiovascular risk

associated with HST usage (Yun et al. 2011).

However, the instability of HST has been the main

barrier to practical formulation development. Its aqueous

stability is very poor, and its degradation mechanism is not

fully elucidated. Degradation of HST is expected to be

B. K. Ahn � S. G. Lee � S. R. Kim � D. H. Lee �M. H. Oh � M. W. Lee � Y. W. Choi (&)

College of Pharmacy, Chung-Ang University,

221 Heuksuk-dong, Dongjak-gu, Seoul 156-756, Korea

e-mail: ywchoi@cau.ac.kr

123

Journal of Pharmaceutical Investigation (2013) 43:453–459

DOI 10.1007/s40005-013-0089-9

similar to that of other diarylheptanoid compounds, whose

instability is well understood. Since the structure of HST is

similar to that of curcumin, a well-characterized diaryl-

heptanoid compound, their mechanisms of degradation

may be similar. Curcumin degrades via hydrolysis, pro-

ducing several degradation products including ferulic acid,

feruloylmethane, and vanillin (Wang et al. 1997).

To improve compound stability, various pharmaceutical

techniques, including inclusion complex, micelle, liposome,

and microemulsion, can be employed. These methods can

protect the compound from a hazardous environment.

Inclusion complexation is generally achieved using cyclo-

dextrin, a group of structurally related cyclic oligosaccha-

rides that have a hydrophobic cavity and a hydrophilic

external surface. Inclusion into the hydrophobic cavity of

cyclodextrin can enhance drug molecule’s solubility and

stability (Sharma and Jain 2010). Complex formation with

various cyclodextrins increased both the water solubility of

curcumin at pH 5, and its hydrolytic stability under alkaline

conditions (Tonnesen et al. 2002). Other studies have dem-

onstrated that complex formation improved curcumin sta-

bility, including its photochemical and thermal stability in

the solid state (Zaibunnisa et al. 2009; Tomren et al. 2007;

Yallapu et al. 2010). The present study therefore adopted an

inclusion complexation approach to the stabilization of HST.

Materials and methods

Materials

HST [purity [90 % by high-performance liquid chroma-

tography (HPLC)] was supplied by the Pharmacognosy

Laboratory, College of Pharmacy, Chung-Ang University.

b-cyclodextrin (b-CD) was purchased from the Sigma-

Aldrich Chemical Company (St. Louis, MO, USA). Isopro-

pyl alcohol, methanol, and acetonitrile (all HPLC grade)

were purchased from J.T. Baker (Phillipsburg, NJ, USA).

Preparation of HST-b-CD inclusion complexes

and physical mixtures

According to conventional drug-cyclodextrin complexation

theory, drug molecules form inclusion complexes with

dissolved cyclodextrin (Kurkov et al. 2011). Briefly, HST

and b-CD were dissolved in isopropyl alcohol or distilled

water, respectively. These solutions were then mixed in a

molar ratio of 1:1 (IC1: HST 19.68 mg, b-CD 68.1 mg) or

1:2 (IC2: HST 9.84 mg, b-CD 68.1 mg) and evaporated

under vacuum using a rotary evaporator (Tokyo Rikakikai

Co., Ltd, Tokyo, Japan) at 60 �C. The physical mixtures

were prepared in a mortar at the same molar ratios of HST

and b-CD, and designated as PM1 and PM2, respectively.

Phase solubility diagram

The phase solubility study was carried out according to the

method reported by Higuchi and Connors (1965). Different

concentrations of b-CD solutions (0–15 mM) were pre-

pared in distilled water and excess HST was added to these

solutions to attain saturation. The samples were sealed and

shaken at 25 �C for 24 h prior to filtration using a 0.22 lm

syringe polyvinylidene difluoride filter, and then assayed

for the total dissolved HST content by HPLC analysis.

HPLC analysis

The quantitative determination of HST was performed by

HPLC (Moon et al. 2011). The HPLC system consisted of a

pump (L-2130), UV detector (L-2400), and a data station

(LaChrom Elite, Hitachi, Japan). A solvent gradient elution

was performed with solvent A (water) and solvent B

(acetonitrile). The starting mobile phase was 90 % A with

10 % B, and a linear gradient was run over 15 min to

achieve 60 % A, 40 % B, returning to a final proportion of

90 % A, 10 % B at 20 min. The flow rate was 1 mL/min

and the injection volume was 20 lL. HST was separated on

a HALO C18 column (2.7 lm, 4.6 9 75 mm; Advanced

Materials Technology, USA) and detected at 280 nm with

a retention time of 11.6 min.

Differential scanning calorimetry (DSC)

DSC of HST, b-CD, complexes, and the physical mixture

was performed using a TA Instruments Auto Q20 Differ-

ential Scanning Calorimeter (TA Instruments, New Castle,

DE, USA). The heating rate employed was 10 �C/min and

the samples were scanned over the temperature range of

20–300 �C. The measurements were carried out under dry

nitrogen at a flow rate of 50 mL/min.

Infrared spectroscopy (IR spectroscopy)

IR spectroscopy was performed with a Nicolet 6700

spectrometer (Thermo Scientific, USA) using the potas-

sium bromide disk method. Sample was mixed with dry

powdered potassium bromide and compressed into a

transparent disk under high pressure. The disk was placed

in the IR spectrometer and the spectrum was scanned over

the wave number range of 4,000–400 cm-1.

Nuclear magnetic resonance spectroscopy (NMR

spectroscopy)

1H NMR experiments were performed at 300 MHz using a

Gemini 2000 spectrometer (Varian, USA). The probe

temperature was regulated at 25 �C. DMSOd-6 was used for

454 B. K. Ahn et al.

123

pure HST analysis and 99.96 % D2O was used for the

analysis of b-CD and inclusion complexes.

Stability test

Stability tests were performed in the solid and solution

states. For solid-state analyses, HST and inclusion com-

plexes were transferred to Teflon-capped vials, sealed with

parafilm, and stored at a constant temperature of either 25

or 40 �C. To test stability in solution, HST was analyzed in

a cosolvent system composed of distilled water and

methanol (9:1 v/v). Inclusion complexes were dissolved in

distilled water. Aqueous solution samples were then

transferred to vials and stored as described above. At

appropriate time points, samples were dissolved in meth-

anol for HPLC analysis.

Results

Phase solubility diagram

The phase solubility diagrams for the complex formation

between HST and b-CD are represented in Fig. 1. These

plots showed that the aqueous solubility of HST increased

linearly as a function of b-CD concentration, up to 15 mM.

The apparent stability constant for complex formation (KC)

was calculated from the straight line of the phase solubility

diagram using the following equation:

KC = Slope/S0 (1-slope), where S0 is intrinsic solubil-

ity. The estimated values for the slope and KC were 0.62

and 249.2 M-1, respectively.

DSC

DSC thermograms of pure HST,b-CD, physical mixtures, and

inclusion complexes are shown in Fig. 2. DSC thermograms

of HST revealed a sharp endothermic peak at 103.65 �C andFig. 1 Phase solubility diagram of HST-b-CD complex in water at

25 �C (n = 3)

Fig. 2 DSC thermograms of HST, b-CD, PM1, PM2, IC1, and IC2

Fig. 3 IR spectra of a HST, b b-CD, c PM1, d PM2, e IC1, and f IC2

Inclusion compound formulation of hirsutenone 455

123

small endothermic peaks at 128.38–178.29 �C. b-CD yielded

a broad endothermic peak at 118.58 �C. The HST endother-

mic peak persisted in the physical mixture. In 1:1 and 1:2

inclusion complexes, large broad peaks appeared in 78.57 and

101.94 �C, respectively.

IR spectroscopy

The IR spectra of HST, b-CD, physical mixtures, and

inclusion complexes are represented in Fig. 3. The charac-

teristic peaks of HST were as follows: 3,368 cm-1 (phenolic

OH stretching vibration), 1,607 cm-1 (stretching vibration

of benzene ring skeleton), 1,520 cm-1 (mixed C=O and C=C

vibration), 1,445 cm-1 (olefinic C–H bending vibration),

and 1,283 cm-1 (aromatic C–O stretching vibration).

Spectra of b-CD showed prominent peaks at 3,383 cm-1

(OH group stretching vibration), 2,923 cm-1 (C–H sym-

metric, asymmetric stretching vibration), 1,644 cm-1 (HOH

deformation bands of water), and 1,156 cm-1 (C–O–C

vibration).

NMR spectroscopy

Complex formation was evaluated by 1H NMR spectros-

copy. The spectra of pure HST are shown in Fig. 4. It

exhibited proton signals at d6.83–6.86 (1H, multiplet, H-5),

d6.64–6.80 (4H, multiplet, H-20,200,50,500), d6.55–6.57 (2H,

multiplet, H-60,600), d6.02 (1H, doublet, H-4), and

d2.49–2.86 (8H, multiplet, H-1,2,6,7). The proton signals

of pure b-CD were exhibited at d3.57–4.02 (H2–H6). As

shown in Fig. 5, complex formation (IC1, IC2) was

Fig. 4 Structure and 1H NMR spectrum of pure HST

Fig. 5 1H NMR spectra of IC1, IC2, and b-CD

Table 1 Chemical shift d and Dd of protons in free b-CD and

inclusion complex

Proton db-CD dIC1 DdIC1 dIC2 DdIC2

H1(1H,d) 5.10 5.08 -0.02 5.08 -0.02

H2(1H,dd) 3.65 3.64 -0.01 3.64 -0.01

H3(1H,t) 3.99 3.90 -0.09 3.94 -0.05

H4(1H,t) 3.61 3.61 0 3.61 0

H5(1H,dt) 3.88 3.72 -0.16 3.77 -0.11

H6(1H,t) 3.90 3.83 -0.07 3.86 -0.04

1 H NMR chemical shifts variations Dd were calculated according to

the formula

d doublet, t triplet

Dd = d(complex)-d (b-CD)

456 B. K. Ahn et al.

123

associated with significant chemical shifts in proton signals

at the H3, H5, and H6 protons. The changes in chemical

shift of b-CD’s proton region are listed in Table 1.

Stability of HST

First-order plots of the remaining HST versus time were used

to calculate the rate constants and half-lives of pure HST and

inclusion complexes. The solid-state rate constants and half-

lives of pure HST and the inclusion complexes are listed in

Table 2. All of the samples were relatively stable in the solid

state, regardless of storage temperature. However, in solu-

tion state as shown in Table 3, both HST and HST-b-CD

were very unstable. This study revealed short half-lives of

\10 days in all samples, indicating no practical improve-

ment of the aqueous stability of HST within inclusion

complexes.

Discussion

Various methods have been employed to achieve inclusion

complexation, including kneading, solvent evaporation,

freeze–drying, and spray drying (Del Valle 2004). In the

present study, solvent evaporation was employed to pre-

pare HST-b-CD inclusion compounds, because of the

convenience of this method at the laboratory scale. Con-

struction of a phase solubility diagram is useful for investi-

gating inclusion complexation of drugs with cyclodextrins in

water, enabling analysis of both the solubilizing capability of

the host molecule, and calculation of the stability constant of

complexes. The aqueous solubility of HST increased line-

arly as a function of b-CD concentration. Previous literature

indicates that HST solubility in the presence of b-CD can be

classified as the AL type (Higuchi and Connors 1965). The

linear host-guest (b-CD-HST) correlation with a slope of\1

suggested the formation of 1:1 complexes. The KC value

provides a useful index to estimate the degree of binding

strength and the changes of physicochemical properties of

the guest molecule. The KC value of the HST-b-CD complex

was estimated to be 249.2 M-1. It is generally accepted that

KC values between 200 and 5,000 M-1 indicate strong

interactions between the host and guest molecules, forming

very stable complexes (Higuchi and Connors 1965; Yadav

and Prakash 2009).

Many methods can be used to analyze the inclusion

complex, including X-ray diffraction (XRD), DSC, IR

spectrometry, scanning electron microscopy (SEM), and

NMR spectrometry (Singh et al. 2010). Since HST exists as

a viscous solid state in nature, XRD was inappropriate for

analysis of this compound. DSC is as a useful tool for

investigation of the thermal properties of b-CD complexes.

When guest molecules are included in the b-CD cavity,

their melting, boiling, and sublimation points generally

shift to a different temperature or often disappear within

the temperature range at which b-CD decomposes. The

sharp endothermic peak in HST spectra at 103.65 �C cor-

responded to its melting point. Small endothermic peaks

from 128 to 178 �C may reveal the presence of impurities.

The spectra of b-CD showed a very broad endothermic

peak at 118.58 �C because of elimination of the water of

crystallization (Sinha et al. 2005). In the physical mixture,

we observed mixed HST and b-CD endothermic peaks,

suggesting little or no interaction between the two com-

pounds. In contrast, the IC1 and IC2 inclusion complexes

showed broad endothermic peaks at 78.57 and 101.94 �C,

respectively. We observed peak shifts and the absence of

HST peaks, which provided crucial evidence that com-

plexation had occurred.

IR spectroscopy was used to confirm the formation of

inclusion complexes by the loss or shifting of functional

group peaks included in the b-CD cavity. The aspects that

are usually taken as an evidence of complex formation

include the peak at 1,607 cm-1 for stretching vibration of

benzene ring skeleton in HST. However, the peak of

b-CD’s molecular vibrations in the 1,700–1,550 cm-1

range unfortunately masks this peak. HOH deformation

bands of water in b-CD at 1,644 cm-1 overlapped with

other peaks associated with complex formation. The peak

Table 2 Rate constants and half-lives of solid HST and inclusion

complexes

Sample Temperature (�C) Rate constant

(910-2, day-1)

Half life

(day)

HST 25 0.138 501.5

IC1 0.184 376.1

IC2 0.345 200.6

HST 40 0.368 188.1

IC1 0.345 200.6

IC2 0.438 158.4

Table 3 Rate constants and half-lives of HST and inclusion com-

plexes in solution

Sample Temperature (�C) Rate constant

(910-2, day-1)

Half life

(day)

HST 25 13.2 5.24

IC1 9.79 7.08

IC2 11.0 6.32

HST 40 15.0 4.61

IC1 13.2 5.26

IC2 9.72 7.13

HST hirsutenone in water and methanol (9:1 v/v) cosolvent system,

IC1, IC2 inclusion complexes dissolved in pure water

Inclusion compound formulation of hirsutenone 457

123

at 1,520 cm-1 for mixed C=O and C=C vibration was

decreased in ICs, because of the low HST content in the

complexes. Thus, IR spectroscopy failed to explain HST-b-

CD complex formation in detail. This phenomenon is

consistent with a previous report (Mohan et al. 2012).

NMR spectroscopy is one of the most effective approaches

to confirm inclusion complexation. This provides clear data

relating to complexation in the solution phase (Ali and

Upadhyay 2008; Bernini et al. 2004). Significant shift

changes were observed in H-3,5,6, located inside the b-CD

cavity, in the presence of HST (Table 1; Fig. 5). Mean-

while, H-2,4 (located outside the b-CD cavity) showed no

significant shift changes in the presence of HST. This

indicated that HST was included in the b-CD cavity when

the inclusion complex was formed. Taken together with the

DSC results, the NMR spectroscopy data confirmed suc-

cessful HST-b-CD complexation.

The stability of HST and the inclusion complexes was

analyzed by measuring the amount of HST remaining over

time. All of the compounds showed great stability as solids,

with half-lives of [150 days. However, the much shorter

half-life of HST in solution (*5 days) was not improved

dramatically by complex formation at either temperature

examined. As the chemical structure of HST is very similar

to that of curcumin, its degradation mechanism may

involve hydrolysis. Curcumin is hydrolyzed and broken

into two molecules of feruloylmethane and ferulic acid

(Shen and Ji 2012). Cyclodextrins are known to accelerate

or decelerate various kinds of reactions. When an ester

group of the guest molecule is located close to secondary

hydroxyl groups, its hydrolysis is accelerated. On the

contrary, hydrolysis is decelerated when the ester group is

included deep inside the cavity. Originally, this work was

proposed by the supposition that the host molecule could

include the guest molecule efficiently into its inner cavity.

However, in case of HST, b-CD seems to include the

aromatic ring of HST, but leaves the aliphatic double bonds

located at C3 and C5. This region is expected to be sus-

ceptible to hydrolytic attack (Fig. 6). Due to this discrep-

ancy between original and illustrated proposal, aqueous

instability problem of HST was unavoidable. Moreover, as

shown in Table 2, poorer stability of HST in solid state at

25 �C was observed in inclusion complex. This result

might be attributed to the following reasons: the presence

of water in the reaction vehicle during preparation proce-

dure and the increased surface area of inclusion complex

compared to that of the concentrated raw material deteri-

orated the susceptibility of the molecule. As a result,

inclusion complex formation failed to protect HST from

aqueous hydrolysis, even though HST itself was relatively

stable in the solid state. Further approaches to stabilization

of HST in aqueous solutions are therefore necessary.

Conclusion

The present study successfully prepared HST inclusion

compounds by the solvent evaporation method at a lab

scale using b-CD as a host molecule with the aim of

enhancing HST stability. The findings indicated that even

though both HST and inclusion compounds were relatively

stable in the solid state, the aqueous stability of HST was

not critically improved using this approach.

Acknowledgments This article dose not contain any studies with

human and animal subjects performed by any of the authors. And all

authors ( BK ahn, SG Lee, SR Kim, DH Lee, MH Oh, MW Lee and

YW Choi) declare that they have no conflict of interest. This study

was supported by a grant of the Korea Healthcare Technology R&D

Project, Ministry for Health, Welfare & Family Affairs, Republic of

Korea (A091121).

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