Enhancement of steroid hydroxylation yield from dehydroepiandrosterone by cyclodextrin complexation technique

Article abstract of DOI:10.1016/j.steroids.2014.03.007

The cyclodextrins (CDs) complexation technique was performed for the enhancement of hydroxylation yield from dehydroepiandrosterone (DHEA) by Colletotrichum lini ST-1. Using DHEA/methyl-β-cyclodextrin (M-β-CD) or DHEA/hydroxypropyl-β-cyclodextrin (HP-β-CD) inclusion complexes as substrate (10 g/L), the hydroxylation yields were increased by 14.98% and 20.54% respectively, and the biotransformation course was shortened by 12 h. X-ray diffractometry, differential scanning calorimetry, and phase solubility analyses showed an inclusion complex was formed between CDs and DHEA at a molar ratio of 1:1, which remarkably increased the solubility of DHEA, and then improved substrate biotransformation efficiency and hydroxylation yield. Meanwhile, results of thermodynamic parameters (ΔG, ΔH, ΔS and Ks) analysis revealed the complexation process was spontaneous and DHEA/CDs inclusion complex was stable. Scanning electron microscopy and transmission electron microscopy showed that the enhancement of DHEA hydroxylation yield also depended on the improvement of cell permeability through interaction between cytomembrane and CDs. These results suggested that the CDs complexation technique was a promising method to enhance steroids hydroxylation yield by increasing steroids solubility and decreasing membrane resistance of substrate and product during biotransformation process.

Full text of DOI:10.1016/j.steroids.2014.03.007

Steroids 84 (2014) 70–77
Contents lists available at ScienceDirect
Steroids
journal homepage: www.elsevier.com/locate/steroids
Enhancement of steroid hydroxylation yield from
dehydroepiandrosterone by cyclodextrin complexation technique
Yan Wu a,b, Hui Li a, Zhen-Ming Lu a, Heng Li a, Zhi-Ming Rao b, Yan Geng a, Jin-Song Shi a,
Zheng-Hong Xu a,
⇑
a School of Pharmaceutical Science, Jiangnan University, Wuxi 214122, People’s Republic of China
b School of Biotechnology, Jiangnan University, Wuxi 214122, People’s Republic of China
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 9 April 2013
Received in revised form 8 March 2014
Accepted 13 March 2014
Available online 22 March 2014
The cyclodextrins (CDs) complexation technique was performed for the enhancement of hydroxylation
yield from dehydroepiandrosterone (DHEA) by Colletotrichum lini ST-1. Using DHEA/methyl-b-cyclodex-
trin (M-b-CD) or DHEA/hydroxypropyl-b-cyclodextrin (HP-b-CD) inclusion complexes as substrate (10 g/
L), the hydroxylation yields were increased by 14.98% and 20.54% respectively, and the biotransformation
course was shortened by 12 h. X-ray diffractometry, differential scanning calorimetry, and phase
solubility analyses showed an inclusion complex was formed between CDs and DHEA at a molar ratio
of 1:1, which remarkably increased the solubility of DHEA, and then improved substrate biotransforma-
Keywords:
DHEA
Biotransformation
Cyclodextrin
tion efficiency and hydroxylation yield. Meanwhile, results of thermodynamic parameters (DG, DH, DS
and Ks) analysis revealed the complexation process was spontaneous and DHEA/CDs inclusion complex
was stable. Scanning electron microscopy and transmission electron microscopy showed that the
enhancement of DHEA hydroxylation yield also depended on the improvement of cell permeability
through interaction between cytomembrane and CDs. These results suggested that the CDs complexation
technique was a promising method to enhance steroids hydroxylation yield by increasing steroids solu-
bility and decreasing membrane resistance of substrate and product during biotransformation process.
Ó 2014 Elsevier Inc. All rights reserved.
1. Introduction
into
7a-hydroxy-dehydroepiandrosterone (7a-OH-DHEA) or
7b-hydroxy-dehydroepiandrosterone (7b-OH-DHEA) [4,5]. Also,
Dehydroepiandrosterone (DHEA) and its derivatives are impor-
tant steroid intermediates for many hormone pharmaceuticals. The
Fusarium oxysporum was described for its ability of hydroxylation
in the 15
biotechnological aspects of double hydroxylation of DHEA in the
C7 - and C15 -positions are limited.
Nowadays, strains that could dihydroxylate DHEA are mainly
a-position of DHEA [6]. However, reports on the
significant bioactivity of product 7
up-regulate immunity and prevent the hypoxic cell death of neu-
rons in vitro [1,2]. While 7 ,15 -diOH-DHEA is a key intermediate
in the synthesis of aldosterone antagonists, such as the new oral
contraceptive Yasmin (6b,7b,15b,16b-dimethylene-3-oxo-17
a-OH-DHEA has been proven to
a
a
a
a
derived from genera Colletotrichum, Gibberella, Fusarium and
Nigrospora [3]. Among them, Colletotrichum lini was the strain with
the highest potential; it showed a hydroxylation yield of 5.6 g/
L–5.8 g/L under a repeated batch mode and a final substrate con-
centration of 7 g/L [7]. Similar to other steroid biotransformation,
the remarkable hydrophobic properties and low transferring rates
of steroids into and out of the cell were the main limiting factors of
DHEA biotransformation [8–10]. In order to solve these problems,
methods such as adding organic solvent or surfactant, and applying
an organic–aqueous biphasic system have been developed to im-
prove the solubility and bioavailability of substrates. However,
the toxicity of organic solvents harmed the activity and stability
of biocatalysts and consequently hindered their application on ste-
roids biotransformation [11].
a
-
pregn-4-ene-17,21-carbolactone) [3]. Steroid biotransformation is
attracting considerable attention because of its potential commer-
cial value and role as a valuable alternative to traditional chemical
processes. According to the previous report, several strains such as
Mucor racemosus and Fusarium moniliforme could transform DHEA
Abbreviations: DHEA, dehydroepiandrosterone;
dehydroepiandrosterone; 7b-OH-DHEA, 7b-hydroxy-dehydroepiandrosterone;
,15 -diOH-DHEA, ,15 -dihydroxy-dehydroepiandrosterone; CDs, cyclodex-
7a-OH-DHEA, 7a-hydroxy-
7a
a
7a
a
trins; M-b-CD, methyl-b-cyclodextrin; HP-b-CD, hydroxypropyl-b-cyclodextrin;
XRD, X-ray diffractometry; DSC, differential scanning calorimetry.
⇑
Corresponding author. Tel./fax: +86 510 85918206.
E-mail address: zhenghxu@jiangnan.edu.cn (Z.-H. Xu).
http://dx.doi.org/10.1016/j.steroids.2014.03.007
0039-128X/Ó 2014 Elsevier Inc. All rights reserved.

Y. Wu et al. / Steroids 84 (2014) 70–77
71
Cyclodextrins (CDs), a homologous group of cyclic glucans
consisting of -1,4-bound glucose units, could form inclusion
complexes with a variety of hydrophobic substrates. Gedeon
Richter Ltd. (Budapest, Hungary) firstly used macrocyclic -, b-,
and -CD for the transformation of steroids solubilized in aqueous
TLC was performed using a 0.25 mm thick layer of silica gel G
(Qingdao Marine Chemical Inc., Qingdao, China) in the system of
chloroform/methanol (15:1) and visualized by spraying the plates
with sulfuric acid/ethanol (1:1), and heated at 60–80 °C for 5 min.
The samples used for HPLC (Dionex, USA) were redissolved in
a
a
c
media in 1981 [12]. Since then, CDs have received considerable
attention for its application in steroid transformation process
[13–17]. However, no report on the use of CDs for the hydroxyl-
ation of DHEA has been documented.
acetonitrile/water (7:3) and filtered through a 0.22
tion membrane. Analysis was performed on the Agilent C18 col-
umn (5.0
lm microfiltra-
l
m, 4.6 mm ꢁ 250 mm) at the wavelength of 206 nm,
column temperature of 30 °C, with acetonitrile and water (7:3) as
In this study, a CDs complexation approach was applied for con-
the mobile phase at a flow rate of 0.5 mL/min.
verting DHEA into 7a-OH-DHEA and 7a,15a-diOH-DHEA by C. lini
ST-1. To evaluate the effects of CDs on DHEA hydroxylation, the
substrate conversion efficiency, biotransformation course and the
corresponding hydroxylation yield were examined. In addition,
the characteristics of the DHEA/CDs inclusion complexes and ef-
fects of CDs on cytomembrane permeability were also investigated
for exploring the improvement of DHEA hydroxylation yield with
the addition of CDs. These findings were of significant importance
for steroids biotransformation and could be employed for biotrans-
formation of other homologous substrates with poor solubility.
2.5. Preparation of inclusion complex and physical mixture
The inclusion complexes between DHEA and M-b-CD or HP-b-
CD were prepared by freeze-drying (Labconco, MO, USA) according
to the methods reported by Williams [18]. Different molar ratios of
DHEA and cyclodextrin were added together as solid and then dis-
persed in aqueous solution, mixing at a room temperature for 60 h
until complexation achieved an equilibrium state. The suspension
was filtered through a 0.45 lm microfiltration membrane, and
then the filtrate was frozen at ꢂ80 °C and lyophilized in a freeze-
2. Materials and methods
dryer.
The physical mixture consisting of DHEA and M-b-CD or HP-b-
CD at the same molar ratio as the inclusion complex was admixed
together in a mortar with pestle for 5 min to obtain a homoge-
neous physical mixture.
2.1. Materials
DHEA, 7a,15
a
-diOH-DHEA were supplied by Tianjin Pharmaceu-
tical Company (99.5% of purity). 7
a-OH-DHEA (98% of purity) was
prepared in our laboratory through the previous reported method
[4]. Methyl b-cyclodextrin (M-b-CD) and hydroxypropyl-b-cyclo-
dextrin (HP-b-CD) were obtained from Zhiyuan Biotechnology Co.,
Ltd (Shangdong, China). All steroid compounds and other chemicals
were of analytical grade and obtained from commercial sources.
2.6. X-ray diffractometry
X-ray diffractometry (XRD) patterns were recorded on a BRU-
KER D8 Advance diffractometer (Bruker AXS, Germany) system
with CuKa radiaton (k = 0.15406 nm), over a range of 2h angles
from 3° to 60°. The measurement conditions were as follows: tar-
get, Cu; filter, Ni; power, 1600 W (40 kV ꢁ 40 mA); scanning rate,
4°/min; angular step, 0.010°.
2.2. Microorganism and cultivation
C. lini ST-1 was isolated from a factory district in Tianjin, China
and deposited in the China General Microbiological Culture Collec-
tion Center (CGMCC 6051; Beijing, China). C. lini ST-1 was main-
tained at 30 °C on slant consisting of the following composition
(g/L): glucose 30, FeSO4 0.01, NaNO3 3, K2HPO4 1, KCl 0.5, MgSO4-
ꢀ7H2O 0.5, and agar 20. The strain C. lini ST-1 was grown on a rotary
shaker (200 rpm) at 30 °C in flasks (500 mL) with 100 mL seed
medium of the following composition (g/L): glucose 15, yeast ex-
tract 15, soybean cake powder 10, KCl 1, (NH4)2HPO4 1, K2HPO4
1, and MgSO4ꢀ7H2O 1. The medium was adjusted to pH 7.0 with
0.5 M NaOH or HCl solution.
2.7. Differential scanning calorimetry
Differential scanning calorimetry (DSC) analysis of DHEA, CDs,
as well as their physical mixtures and inclusion complexes were
performed with a HHT-3320A thermal analyzer (Huihetang Bio-
technology Ltd., Shanghai, China) from 30 °C–370 °C at a heating
rate of 10 °C/min with argon as the carrier gas. The samples were
accurately weighed 20 mg and heated in a sealed aluminum pans.
Duplicated determinations were performed for each sample. The
temperature and heat flow were calibrated using standard tin
samples.
2.3. Transformation of DHEA
2.8. Phase solubility studies
After 24 h of growth in seed medium, the culture was inocu-
lated with 10% volume into fresh transformation medium
(30 mL) containing the following components (g/L): glucose 15,
yeast extract 15, corn steep liquor 3, KCl 1, (NH4)2HPO4 1,
K2HPO4 1, and MgSO4ꢀ7H2O 1. The medium was adjusted to pH
7.0 with 0.5 M NaOH or HCl solution. Then, the fine powder of
DHEA or DHEA/CD inclusion complex was added into the medium
after cultivating for another 24 h. Transformation reaction was car-
ried out on a rotary shaker (220 rpm) at 28 °C for 60–72 h. Samples
were then withdrawn at regular intervals and the products were
subsequently extracted by ethyl acetate and dried in vacuum.
Solubility studies were performed according to the method of
Higuchi and Connors [19]. Different concentrations of M-b-CD or
HP-b-CD and an excess of DHEA were added to 30 mL aqueous
solution in 250 mL shake flasks, then the shake flasks were oper-
ated at 28 °C, 220 rpm. After 24 h of equilibration, the suspensions
were filtrated through a 0.45 lm microfiltration membrane; and
the DHEA concentrations in the filtrate were assayed by HPLC.
The apparent stability constant (Ks) of the DHEA/M-b-CD or
DHEA/HP-b-CD complex was calculated from the phase solubility
diagram according to the equation below:
2.4. Analysis of transformation products
slope
S0ð1 ꢂ slopeÞ
Ks ¼
ð1Þ
Analysis of DHEA transformation products were carried out by
thin layer chromatography (TLC) and high performance liquid
chromatography (HPLC).
where Ks is the stability constant of the 1:1 complex; S0 is the
intrinsic solubility of DHEA in the absence of CDs; slope is the

72
Y. Wu et al. / Steroids 84 (2014) 70–77
Fig. 1. Effect of the molar ratio of (A) M-b-CD and (B) HP-b-CD to substrate on
DHEA conversion efficiency.
saturation concentration of DHEA measured without CDs
(intercept).
The stability constants (Ks) at different temperatures were used
to determine different thermodynamic parameters. The entropy
(D
S) of complexation was calculated using the Van’t Hoff equation
as follows:
lnðKÞ ¼ S=R ꢂ
D
D
H=RT
ð2Þ
A plot of ln(K) versus 1/T indicate that the intercept is the value
of
D
S/R. The free energy changes ( G) and enthalpies ( H) of the
D
D
complexation between DHEA and CDs were determined using the
following equations:
D
D
G ¼ ꢂRT ln K
G ¼ H ꢂ T
ð3Þ
ð4Þ
D
DS
Fig. 2. Conversion process of DHEA with M-b-CD (A) and HP-b-CD (B).
2.9. Effects of CDs on cells permeability
outer surfaces and
[13,15,19,20]. Among the six CDs investigated (
a
hydrophobic cavity at the center
-CD, b-CD, -CD,
The surface morphology of the C. lini ST-1 incubated with CDs
was determined by scanning electron microscope (SEM) and trans-
mission electron microscopy (TEM).
a
c
CM-b-CD, M-b-CD, and HP-b-CD), M-b-CD and HP-b-CD were
found to be the most effective at improving DHEA hydroxylation
production in C. lini ST-1 biotransformation (data not shown).
Therefore, M-b-CD and HP-b-CD with different concentrations
were chosen and applied for DHEA hydroxylation.
3. Results and discussion
3.1. DHEA biotransformation by CDs complexation technique
As seen in Fig. 1A and B, it was quite evident that the conversion
efficiency and hydroxylation yield (7a-OH-DHEA and 7a,15a-
3.1.1. Effects of molar ratio (CDs:DHEA) on DHEA conversion efficiency
CDs has been widely used in steroids biotransformation to im-
prove the substrate aqueous solubility, due to their hydrophilic
diOH-DHEA) increased with the increasing CD concentrations in
some extent. However opposite results were found when the CD
to DHEA ratio in the media was more than 2:1. The data suggested

Y. Wu et al. / Steroids 84 (2014) 70–77
73
that a high CD concentration was toxic to C. lini ST-1 hydroxylase
and might prevent or delay the proceeding of hydroxylation. In
addition, at higher concentration of CD, DHEA molecular was en-
closed by CD and ‘‘less accessible’’ for biotransformation, which
limited the conversion efficiency in return. At the optimum molar
ratio of 1:1, the substrate conversion efficiency and product accu-
mulation were 99.35% and 7.79 g/L with M-b-CD as well as 99.45%
and 7.90 g/L with HP-b-CD respectively. By contrast, the control
samples without CDs achieved only 86.64% and 6.92 g/L.
without CDs were withdrawn at regular intervals and assayed by
HPLC.
The dynamics of the accumulation of hydroxy product showed
in Fig. 2A led to an assumption that the formation of 7
DHEA from DHEA occurs at the subsequent introduction of hydro-
xyl groups in the 7 -position and then in the 15 -position of the
steroid molecule [3]. Moreover, the yields of 7 -OH-DHEA and
,15 -diOH-DHEA accumulated constantly with the substrate
DHEA consuming. The highest yields of hydroxylation products
(7 -OH-DHEA and 7 ,15 -diOH-DHEA) were 8.06 g/L with M-b-
a,15a-diOH-
a
a
a
7a
a
a
a
a
CD and 8.45 g/L with HP-b-CD (Fig. 2B and C), which were en-
hanced by 14.98% and 20.54% compared with the control (7.01 g/
L, Fig. 2A). Meanwhile, conversion course of the maximum product
3.1.2. Effects of CDs on hydroxylation process
To monitor the changes of substrate and product concentrations
during bioconversion process, the conversion samples with or
Fig. 3. (A) X-ray diffractograms of (a) DHEA, (b) M-b-CD, (c) inclusion complex, and (d) physical mixture. (B) X-ray diffractograms of (a) DHEA, (b) HP-b-CD, (c) inclusion
complex, and (d) physical mixture.

74
Y. Wu et al. / Steroids 84 (2014) 70–77
accumulation was shortened from 60 h to 48 h comparing to the
process absence of CDs.
Interestingly, the proportion of the two biotransformation prod-
ucts was changed in the presence of CDs. As shown in Fig. 2B and C,
the rate of converting DHEA to 7
stantially in preliminary 30 h with the addition of CDs. And the
-OH-DHEA yield was enhanced by 37.12% with M-b-CD and
43.21% with HP-b-CD comparing to the absence of CDs. However,
the rate of converting 7 -OH-DHEA to 7 ,15 -diOH-DHEA in
a-OH-DHEA was increased sub-
7a
a
a
a
presence of CDs was increased slightly and similar to the rate in
the absence of CDs after 18 h. The first hydroxylation product of
7a
-OH-DHEA in the presence of CDs was not converted into
,15 -diOH-DHEA in the latter transformation period, and the
,15 -diOH-DHEA was decreased by 23.41% with M-b-
CD and 20.24% with HP-b-CD. These results suggested that the
presence of CDs suppressed the transformation of 7 -OH-DHEA
to 7 ,15 -diOH-DHEA in some extent, and a single target product
7a
a
yield of 7
a
a
a
a
a
would be achieved by regulating the molar ratio of DHEA to CDs.
In order to illustrate the effects of CDs on DHEA biotransforma-
tion elaborately, two aspects including CDs solubilized steroids and
CDs affected cell structure of C. lini ST-1 were investigated in the
following experiments.
3.2. Characterization of the CDs/DHEA inclusion complex
3.2.1. X-ray powder diffractometry
X-ray technique is a useful method to characterize the forma-
tion of an inclusion complex between steroid and CDs. In this
study, the XRD patterns of M-b-CD, DHEA, DHEA/M-b-CD inclusion
complex and their physical mixtures were investigated (Fig. 3A). In
addition, the diffraction patterns of HP-b-CD, DHEA, DHEA/HP-b-
CD inclusion complex and their physical mixtures were also deter-
mined (Fig. 3B).
The X-ray pattern of DHEA (Fig. 3A-a and B-a) exhibited numer-
ous sharp peaks that indicated its crystalline nature. On the other
hand, the X-ray pattern of M-b-CD (Fig. 3A-b) and HP-b-CD
(Fig. 3B-b) showed no crystalline peak, which suggested their
amorphous nature. The diffractograms of physical mixture
(Figs. 3A-d and Fig. 3B-d) were superimposed figures of DHEA
and M-b-CD or HP-b-CD but with lower intensity compare with
each pure components. This was due to the dilution of DHEA in
physical mixtures [18]. Meanwhile, X-ray spectrum of the two
inclusion complexes (Fig. 3A-c and B-c) showed no characteristic
diffraction peaks of DHEA, which was distinct from the corre-
sponding physical mixtures. This difference indicated the lack of
crystallinity for the physical mixture and it might provide evidence
for the formation of an inclusion complex between DHEA and CDs.
Similar results were found by Wang et al. [20] and Forgo et al. [14].
Fig. 4. (A) DSC thermograms of (a) DHEA, (b) MCD, (c) complex mixture, and (d)
inclusion complex. (B) DSC thermograms of (a) DHEA, (b) HPCD, (c) complex
mixture, and (d) inclusion complex.
was identical to the thermogram of CDs. The difference between
inclusion complex and physical mixture indirectly indicated that
the product obtained by the freeze drying method maybe not sim-
ply a physical mixture but the CDs/DHEA inclusion complex. This
finding agreed with the XRD analysis results.
3.2.3. Phase solubility study
Phase solubility study was carried out by gently shaking the
aqueous containing an excess amount of DHEA and M-b-CD or
HP-b-CD at 0 mM to 60 mM concentration (Fig. 5). The solubility
3.2.2. Differential scanning calorimetry
The DSC analysis of DHEA, CDs, their physical mixtures and
inclusion complexes were performed (Fig. 4A and B). The DSC ther-
mogram of DHEA (Fig. 4A-a and B-a) demonstrated a sharp endo-
thermic peak at about 150 °C, which was consistent with its
melting point. According to previous studies, the melting point of
pure DHEA was proved to be 150.9 °C in literature [21]. The DSC
curve of M-b-CD (Fig. 4A-b) and HP-b-CD (Fig. 4B-b) displayed a
broad endothermic peak between 50 °C to 125 °C and 75 °C to
150 °C, respectively, with no characteristic thermal signal. A simi-
lar result was also reported for characterization of the inclusion
complex of 16,17a-epoxyprogesterone with M-b-CD [22], which
was attributed to the release of water molecular from the cavity.
Both the endothermic peak of DHEA and CDs were observed in
the physical mixture curve (Fig. 4A-c and B-c). However, the sharp
endothermic peak of DHEA disappeared in the inclusion complex
system in the present study (Fig. 4A-d and B-d) and the curve
Fig. 5. Phase solubility diagram of DHEA with CDs in aqueous solution at 30 °C.

Y. Wu et al. / Steroids 84 (2014) 70–77
75
of DHEA linearly increased with increasing CDs concentration. The
plot showed a typical AL-type solubility curve classified by Higuchi
and Connors [19], indicating that the complex of DHEA and CDs
was formed at a molar ratio of 1:1. The inherent maximum solubil-
ity of DHEA in aqueous was 0.015 mM, and the maximum solubil-
ity increased to 18.54 mM and 15.05 mM with the addition of
60 mM M-b-CD and HP-b-CD, respectively. The apparent Ks values
of the DHEA/M-b-CD and DHEA/HP-b-CD complex were calculated
to be 28989 Mꢂ1 and 20284 Mꢂ1, which suggested the feasibility
of generating DHEA/CDs complexes. Moreover, DHEA/M-b-CD
complex was relatively more stable than DHEA/HP-b-CD complex
when comparing their Ks values. This result could attribute to
the steric hindrance caused by the presence of hydroxypropyl sub-
stituent. Similar results were also observed in the characterization
inclusion was spontaneous, whereas the entropy changes sug-
gested that the hydrophobic effects was a strong driving force for
DHEA/M-b-CD or DHEA/HP-b-CD inclusion action [22]. The com-
plexation process of inclusion complex formed between DHEA
and CDs was spontaneous and stable through the thermal analysis.
3.3. Effect of CDs on cell structure
Cell permeability is another important factor limiting the ste-
roid bioconversion process, besides the solubility of steroid sub-
strates. As previous literature reported, the permeability of
microbial cells could be affected by CDs [26]. Therefore, the
changes in cell surface structure (cell walls and cytoplasmic mem-
brane) of C. lini ST-1 were examined by SEM and TEM, after culti-
vating of the microorganism with and without CDs for 12 h.
The scanning electron micrographs showed that the control
cells had distinct outlines and smooth surface (Fig. 7A). Similarity,
the TEM observations showed that the control cells were sur-
rounded by the cell membranes with compact surface, without re-
lease of intracellular components and notable ruptures on cell
surface (Fig. 8A and B). Compared with the untreated control,
remarkable modifications of cell wall and cell membranes were
found after exposure to CDs for 12 h. As shown in Fig 7B and C,
the cells treated with M-b-CD or HP-b-CD appeared rather rough
with numerous unknown components making the cell outline un-
even by the observation of SEM. The mycelial morphology and out-
ermost cell surface changed, but no destructive alteration in cell
integrity was observed.
of the inclusion complex of 16,17a-epoxyprogesterone with M-b-
CD and HP-b-CD [22].
3.2.4. Thermal analysis
The effects of temperature on the solubilizing ability of CDs in
aqueous were studied (Fig. 6A and B) and relevant thermodynamic
parameters (DH, DS, and DG) of complexation were reported in
Table 1. Although the solubility of DHEA showed marked improve-
ment with the increasing temperature, the apparent Ks value de-
creased surprisingly. This was similar to the results observed by
Cerchiara [23] and Badawy [24]. It was deduced that the reduction
of the interaction forces, such as Van der Waals, hydrogen bonding
force and hydrophobic interaction between the guest molecule and
CDs were probably the main causes [22,25]. The negative enthalpy
demonstrated that the complexation of DHEA with M-b-CD or HP-
b-CD was exothermic due to the water molecule in the inner CDs
cavity was substituted by DHEA, and the energy release supported
the formation of an inclusion complex. Furthermore, the negative
value of the Gibbs energy indicated that the formation process of
The TEM data displayed that the cell walls of C. lini ST-1 cul-
tivated with M-b-CD loosened and apparently thickened, whereas
the membranes became folded and inconsecutive (Fig. 8C and D).
The cell also showed reduced electron density, and some ruptures
that could cause slight leakages of cellular cytoplasmic contents
Fig. 6. Phase solubility diagrams for DHEA with (A) M-b-CD and (B) HP-b-CD at different temperatures.
Table 1
Thermodynamic parameters of the inclusion process of DHEA in M-b-CD or HP-b-CD.
T (K)
DHEA/M-b-CD complex
DHEA/HP-b-CD complex
K1:1 (Mꢂ1
)
D
G (kJ Mꢂ1
)
D
H (kJ Mꢂ1
)
D
S (JMKꢂ1
)
K1:1 (Mꢂ1
)
D
G (kJ Mꢂ1
)
D
H (kJ Mꢂ1
)
D )
S (JMꢂ1 Kꢂ1
293
298
303
310
313
40442.2
34144.5
28988.9
23257.3
21226.0
ꢂ25.841
ꢂ25.86
ꢂ25.88
ꢂ25.92
ꢂ25.93
ꢂ21.78
ꢂ21.73
ꢂ21.68
ꢂ21.61
ꢂ21.59
27.95
23.745
20.28
16.40
15.02
ꢂ24.94
ꢂ24.96
ꢂ24.99
ꢂ25.02
ꢂ25.03
ꢂ20.98
ꢂ20.93
ꢂ20.89
ꢂ20.82
ꢂ20.79
13.87
13.52

76
Y. Wu et al. / Steroids 84 (2014) 70–77
Fig. 7. Effect of CDs on cells structure of C. lini by SEM: (A) cells grown without CD (control); (B) cells grown with M-b-CD; (C) cells grown with HP-b-CD.
Fig. 8. Effect of CDs on cells structure of C. lini by TEM: (A and B) cells grown without CD (control), (C and D) cells grown with M-b-CD and (E and F) cells grown with HP-b-CD.
were also observed on the cell membranes of C. lini ST-1. By
contrast, the effects of HP-b-CD on the cell surface structure were
weaker than those of M-b-CD at the same concentration. As
shown in Fig. 8E and F, although the cell walls damaged by the
addition of HP-b-CD became thinner, the cell membrane appeared
integrated and showed no visible leakages of cytoplasmic
contents.
Acknowledgements
This work is financially supported by National High Technology
Research and Development Program of China (863 Program) (No.
2011AA02A211), the National Natural Science Foundation of China
(No. 21206055) and the Natural Science Foundation of Jiangsu
Province (Nos. BK2012127, BK2012117).
According to these findings, we assumed that the cultivation of
C. lini ST-1 in the medium with M-b-CD or HP-b-CD affected the
cell walls and membranes in some extent. This might be beneficial
to increase cell permeability as well as facilitate the absorption and
subsequent transport of the steroid substrate from the cell wall to
underlying membrane structure.
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