Host-guest system of nimbin and β-cyclodextrin or its derivatives: Preparation, characterization, inclusion mode, and solubilization

Article abstract of DOI:10.1021/jf101079e

The inclusion complexation behavior, characterization, and binding ability of nimbin with β-cyclodextrin (β-CD) and its derivatives were investigated in both solution and the solid state by means of XRD, DSC, ¹H and 2D NMR, and UV-vis spectroscopy. The results showed that the water solubility and thermal stability of nimbin were obviously increased in the inclusion complex with cyclodextrins. This satisfactory water solubility and high thermal stability of the nimbin/CD complexes will be potentially useful for their application as herbal medicines or healthcare products.

Full text of DOI:10.1021/jf101079e

J. Agric. Food Chem. 2010, 58, 8545–8552 8545
DOI:10.1021/jf101079e
Host-Guest System of Nimbin and β-Cyclodextrin or Its
Derivatives: Preparation, Characterization, Inclusion Mode,
and Solubilization
†
L
I
-JUAN
Y
ANG,† B
O
Y
ANG,† WEN
C
HEN,† RONG
,†,‡
H
UANG,† SHENG-JIAO
Y
AN
,
AND
J
UN
LIN*
†Key Laboratory of Medicinal Chemistry for Natural Resources, Ministry of Education, School of
Chemical Science and Technology, Yunnan University, Kunming, 650091, People’s Republic of China,
and ‡State Key Laboratory of Elemento-Organic Chemistry, Nankai University, Tianjin 300071,
People’s Republic of China
The inclusion complexation behavior, characterization, and binding ability of nimbin with β-cyclodextrin
(β-CD) and its derivatives were investigated in both solution and the solid state by means of XRD, DSC,
1H and 2D NMR, and UV-vis spectroscopy. The results showed that the water solubility and thermal
stability of nimbin were obviously increased in the inclusion complex with cyclodextrins. This satisfactory
water solubility and high thermal stability of the nimbin/CD complexes will be potentially useful for their
application as herbal medicines or healthcare products.
KEYWORDS: Nimbin; β-cyclodextrin; inclusion complex; binding ability; characterization; inclusion
mode
INTRODUCTION
Scheme 1. Structure of Nimbin
The neem tree, Azadirachta indica A. Juss, is a tropical plant
that has long been used in agriculture and medicine. According to
folklore, the bark, leaves, and fruit have been used as medicines
for the treatment of a variety of human ailments, particularly
against diseases of bacterial and fungal origin (1-3). Many
studies have demonstrated that its seed contains abundant
limonoids and simple terpenoids that are responsible for its
biological activity (4-8). Among these limonoids, nimbin
(Scheme 1), a representative of ring C seco-tetranortriterpenoids,
is considered to be one of the most significant active ingredients
due to its various effects on insects and is a potential molecule for
improving the pesticidal properties of the products and a good
chemomarker for evaluation of germplasm (9). Furthermore,
nimbin is widely used in herbal medicine/healthcare products
such as anti-inflammatories, antipyretics, antifungals, antihist-
amines, antiseptics, and antioxidants (4,10). For example, nimbin
has been shown to be 4 times as effective as the steroid hydro-
cortisone in the treatment of eczema and psoriasis, but without
side effects such as thinning of the skin (11). Additionally,
docking studies carried out with different herbal ligands suggest
that nimbin is one of the best herbal candidates to replace the
synthetic drugs thiolactomycin and cerulenin (12). However, the
use of nimbin and other limonoids as biopesticides or herbal
medicines is greatly limited by its low water solubility and
bioavailability (13, 14). Although much effort has been made to
improve its water solubility and stability by introducing some
nontoxic solubilizers (15, 16), nimbin and other limonoids still
cannot be sufficiently dissolved in water. Therefore, the search for
an efficient and nontoxic carrier for nimbin has become impor-
tant to further its clinical application.
It is well-known that cyclodextrins (CDs) are truncated-cone
polysaccharides mainly composed of six to eight
D-glucose
monomers linked by R-1,4-glucose bonds. They have a hydro-
phobic central cavity and a hydrophilic outer surface and can
encapsulate various inorganic/organic molecules to form host-
guest complexes or supramolecular species. This usually enhances
drug solubility in aqueous solution and affects the chemical
characteristics of the encapsulated drug in the pharmaceutical
industry (17-19). This fascinating property enables CDs to be
successfully utilized as drug carriers (20-22), separation reagents
(23), enzyme mimics (24), and photochemical sensors (25), etc. The
same effect has been demonstrated in the agriculture industry
(26-33). Recently, some studies and patents of inclusion complex
formation between CDs and limonoids have been reported (34).
In this paper, we aim to report the preparation and char-
acterization of some water-soluble inclusion complexes
formed by nimbin and β-cyclodextrin (β-CD) and its deriva-
tives: 2-hydroxypropyl-β-cyclodextrin (HPβCD), heptakis-
(2,6-di-O-methyl)-β-CD (DMβCD), and heptakis(2,3,6-tri-
O-methyl)-β-CD (TMβCD) (Scheme 2). We were particularly
*Corresponding author (phone/fax þ86-871-5033215; e-mail: linjun@
ynu.edu.cn).
© 2010 American Chemical Society
Published on Web 07/20/2010
pubs.acs.org/JAFC

8546 J. Agric. Food Chem., Vol. 58, No. 15, 2010
Scheme 2. Structure of β-CD and Its Derivatives
Yang et al.
with presaturation of the residual water resonance and a mixing (spin-lock)
time of 350 ms at a field of ∼2 kHz, using the TPPI method, using a 1024 K
time domain in F2 (FID resolution 5.87 Hz) and 460 experiments in F1.
Processing was carried out with zero-filling to 2K in both dimensions using
sine (F2) and qsine (F1) window functions, respectively.
Powder X-ray Diffraction. XRD patterns were obtained using a D/
Max-3B diffractometer with Cu KR radiation (40 kV, 100 mA), at a scanning
rate of 5°/min. Powder samples were mounted on a vitreous sample holder
and scanned with a step size of 2θ = 0.02° between 2θ = 3° and 50°.
Thermal Analyses. Differential scanning calorimetry (DSC) and
thermogravimetric (TG) measurements were performed with a 2960 SDT
V3.0F instrument and a Netzsch STA 449F3, respectively, at a heating rate
of 10 °C/min from room temperature to 400 °C in a dynamic nitrogen
atmosphere (flow rate = 70 mL/min).
Solubilization Studies. An excess amount of complex was placed in
2 mL of water (ca. pH 6.0), under nitrogen, sheltered from light, and the mix-
ture was stirred for 1 h at 20 ( 2 °C. The solution was then filtered on a
0.45 μm cellulose acetate membrane. The filtrate was evaporated under re-
duced pressure to dryness, and the residue was dosed by the weighing method.
interested in exploring the solubilization effect of CDs on nimbin
and the binding ability of the resulting inclusion complexes, which
would provide a useful approach for obtaining novel nimbin-based
healthcare products with high water solubility, high bioavailability,
and low toxicity.
MATERIALS AND METHODS
RESULTS AND DISCUSSION
Materials. Nimbin (FW = 540, PC>95%) was obtained by micro-
wave-assisted extraction from dry neem leaf in Yunnan Province, China.
β-CD (average substitution degree = 1135), 2-hydroxypropyl-β-cyclodextrin
(HPβCD, average substitution degree = 1380), heptakis(2,6-di-O-methyl)-
β-CD (DMβCD, average substitution degree=1331), and heptakis(2,3,6-
tri-O-methyl)-β-CD (TMβCD, average substitution degree = 1429) were
purchased from ABCR GmbH & Co. KG and used without further
purification. Other reagents and chemicals were of analytical reagent grade.
All experiments were carried out using ultrapure water.
Spectral Titration. Quantitative investigation of the inclusion
complexation behavior of β-CD and its derivatives with nimbin
was carried out in a water/ethanol (v/v = 4:1) mixed solution
using a spectrophotometric titration method owing to the rather
low water solubility of nimbin. As illustrated in Figure 1, the
absorbance intensity of nimbin gradually increased with the
stepwise addition of β-CD, HPβCD, DMβCD, and TMβCD.
In all experiments, the pH of the solution did not change
appreciably during the experimental procedure. As the size-fit,
shape-fit, and charge-fit effects are the dominant controlling
factors on the formation of inclusion complexes of β-CDs (17),
these results indicate that the binding behavior is mainly depen-
dent on the individual structural features of the host and guest.
Assuming a 1:1 stoichiometry for the nimbin/β-CD inclusion
complex, the inclusion complexation of nimbin with β-CD could
be expressed by eq 1, and the stability constant (KS) can be
calculated from eq 2, where [nimbin/β-CD], [nimbin], [β-CD],
[nimbin]0, and [β-CD]0 refer to the equilibrium concentration of
the nimbin/β-CD inclusion complex, the equilibrium concentra-
tion of nimbin, the equilibrium concentration of β-CD, the
original concentration of nimbin, and the original concentration
of β-CD, respectively, and Δε is the differential molar extinction
coefficient of nimbin in the absence and presence of β-CD.
According to Lambert-Beer law, we can obtain that the con-
centration of the nimbin/CD complex is equal to ΔA/Δε (eq 2).
We can then derive eq 3 from eq 2. Finally, KS can be obtained
from the analysis of the sequential changes of absorption (ΔA) at
various β-CD concentrations, with a nonlinear least-squares
method according to the curve-fitting eq 3.
Preparation of Nimbin/β-CD, Nimbin/HPβCD, Nimbin/DMβCD,
and Nimbin/TMβCD Complexes. Nimbin (0.03 mM, 16.2 mg) and CD
(0.01 mM) were completely dissolved in a mixed solution of ethanol and
water (ca. 7 mL, v/v = 1:5, given the poor water solubility of nimbin,
ethanol was used), and the mixture was stirred for 5 days at room
temperature. After evaporation of the ethanol from the reaction mixture,
the uncomplexed nimbin was removed by filtration. The filtrate was
evaporated under reduced pressure to remove the solvent and dried in
vacuum to give the nimbin/CD complexes. Nimbin/β-CD complex (yield
91%): 1H NMR (500 MHz, D2O, TMS) δ 0.6-2.42 (m, 18H, nimbin
protons), 3.40-3.90 (m, >50H, H-2-6 of β-CD and some protons of
nimbin), 4.97-4.99 (s, 7H, H-1 of β-CD). Nimbin/HPβCD complex (yield
90%): 1H NMR (500 MHz, D2O, TMS) δ 0.6-2.4 (m, 39H, 18H nimbin
protons and 7 ꢀ CH3 of HPβCD), 3.15-3.95 (m, >110H, H-2-6 and
CH2- and CH3-2,3,6 of HPβCD and some protons of nimbin), 5.10-5.12 (s,
7H, H-1 of HPβCD). Nimbin/DMβCD complex (yield 82%): 1H NMR
(500 MHz, D2O, TMS) δ 0.6-2.4 (m, ca. 18H nimbin protons), 3.00-3.75
(m, >90H, H-2-6 and OCH3-2,6 of DMβCD and some protons of
nimbin), 5.03-5.04 (s, 7H, H-1 of DMβCD). Nimbin/TMβCD complex
(yield 80%): 1H NMR (500 MHz, D2O, TMS) δ 0.6-2.4 (m, ca. 18H
nimbin protons), 3.15-3.95 (m, >110H, H-2-6 and OCH3-2,3,6 of
TMβCD and some protons of nimbin), 5.21-5.23 (s, 7H, H-1 of TMβCD).
Determination by UV Spectra. Absorption spectra measurements
were carried out with a Shimadzu UV 2401 using a conventional 1 cm path
(1 cm ꢀ 1 cm ꢀ 4 cm) quartz cell in a thermostated compartment, which
was kept at 25 °C through a Shimadzu TB-85 Thermo Bath unit. Given the
poor water solubility of nimbin, a water/ethanol (v/v = 4:1) solution was
used in the spectral measurements. The concentration of nimbin was held
constant at 0.05 mM. Then, an appropriate amount of CDs was added
with the final concentrations varied from 0 to 2.80-6.00 mM (β-CD, 0,
0.18, 0.36, 0.71, 1.01, 1.44, 2.94, 6.00 mM; HPβCD, 0, 0.24, 0.47, 0.67, 1.37,
2.80 mM; DMβCD, 0, 0.47, 0.67, 0.96, 1.37, 1.96, 2.80 mM; TMβCD, 0,
0.24, 0.48, 0.96, 1.37, 1.96, 4.00 mM). The absorption spectra measure-
ment was taken after 1 h. The measurements were done in the 220-400 nm
spectral range. All experiments were carried out in triplicate.
KS
nimbin þ CD
s
nimbin CD
ð1Þ
F
s
R
3
½nimbin CDꢁ
3
KS
¼
¼
½nimbinꢁ½CDꢁ
ΔA=Δε
ð2Þ
ð½nimbinꢁ0 - ΔA=ΔεÞð½CDꢁ0 - ΔA=ΔεÞ
ꢀ
1H and 2D NMR. All NMR experiments were carried out in D2O.
Tetramethylsilane was used as a reference. Samples were dissolved in
99.98% D2O and filtered before use. 1H NMR spectra were acquired on a
Bruker Avance DRX spectrometer at 500 MHz and 298 K. The one-
dimensional spectra of both solutions were run with FID resolution of
0.18 Hz/point. The residual HDO line had a line width at half-height of
2.59 Hz. Two-dimensional (2D) ROESY spectra were acquired at 298 K
ΔA ¼ Δεð½nimbinꢁ0 þ ½CDꢁ0 þ 1=KSÞ
ꢁ
qffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
.
2
(
Δε2ð½nimbinꢁ0 þ ½CDꢁ0 þ 1=KSÞ - 4Δε2½nimbinꢁ0½CDꢁ0
2
ð3Þ

Article
J. Agric. Food Chem., Vol. 58, No. 15, 2010 8547
Figure 1. UV-vis spectral changes in nimbin (0.05 mM) and the nonlinear least-squares analysis (inset) of the differential intensity (ΔA at 216 nm)
used to calculate the complex stability constant (KS) upon addition of β-CD (1:, 0-6.00 mM, from a to h), HPβCD (2, 0-2.80 mM, from a to f), DMβCD
(3, 0-4.00 mM, from a to h), and TMβCD (4, 0-4.00 mM, from a to g) in a water/ethanol (v/v = 4:1, ca. pH 10.5) mixed solution.
Table 1. Stability Constant (KS) and Gibbs Free Energy Change (-ΔG°)
for the Inclusion Complexation of CDs with Nimbin Guest in a Water/Ethanol
(v/v = 4:1, ca. pH 10.5) Mixed Solution
and nimbin. From Table 1, we can see that the binding constants
for the complexation of nimbin with β-CD, HPβCD, DMβCD,
and TMβCD were in the following order: HPβCD > β-CD >
DMβCD > TMβCD. By comparing the enhancement effect of all
kinds of β-CDs for nimbin, HPβCD and β-CD gave a stronger KS
value than DMβCD and TMβCD, which demonstrated that
HPβCD and β-CD can complex better with the guest nimbin than
the methylated CDs. Considering the structural features of the host
and guest, we deduced that the hydrogen bond between the hydroxyl
arms of HPβCD or the hydrogen atoms of β-CD and the oxygen
atoms of nimbin may strengthen the host-guest association. In
contrast, the methylated CDs (DMβCD and TMβCD) showed
a weaker KS value due to the larger and deeper CD cavity, which
caused the intermolecular hydrogen bond to become weaker (39,40).
1H and 2D NMR Analysis. To explore the possible inclusion
host
KS (M-1
)
log KS
-ΔG° (kJ mol-1
)
β-CD
1279
1360
390
3.10
3.13
2.59
2.54
17.73
17.88
14.79
14.53
HPβCD
DMβCD
TMβCD
351
Using a nonlinear least-squares curve-fitting method (35), we
obtained the complex stability constant for each host-guest
combination. Figure 1 (inset) illustrates a typical curve-fitting
plot for the titration of nimbin with β-CD, HPβCD, DMβCD,
and TMβCD, which shows the excellent fit between the experi-
mental and calculated data and the 1:1 stoichiometry of the
nimbin/CDs inclusion complexes. In repeated measurements, the
KS values were reproducible within an error of(5%. The stability
constant (KS) and Gibbs free energy change (-ΔG°) for the
inclusion complexation of CDs with nimbin are listed in Table 1.
Binding Ability. Extensive studies have revealed that the size/
shape-fit concept plays a crucial role in the formation of inclusion
complexes of host CDs with guest molecules of various structures.
On the basis of this concept, several weak intermolecular forces
such as ion-dipole, dipole-dipole, van der Waals, electrostatic,
hydrogen bond, and hydrophobic interactions are known to
cooperatively contribute to the inclusion complexation. CDs
possess a cyclic truncated cone cavity with a height of 0.79 nm,
an inner diameter of 0.62-0.78 nm, and a cavity volume of
0.262 nm3 for β-CD (34a, 36-38). The host-guest size match
may dominate the stability of the complexes formed between CDs
1
mode of the nimbin/CD complex, we compared the H NMR
spectra of nimbin in the presence of host CDs (Figure 2). Owing to
its poor water solubility, nimbin is transparent to 1H NMR under
most conditions when D2O is used as a solvent. Assessment of the
nimbin complex by 1H NMR clearly demonstrated the presence
of the framework protons of the nimbin molecule, consistent with
the significant solubilization. As illustrated in Figure 2, the
majority of nimbin protons displayed chemical shifts at δ
1.0-2.5, which were distinct from the CD protons (usually at δ
3.0-5.0). By comparing the integration area of these protons with
that of the CD’s H-1 protons, we calculated the inclusion
stoichiometry of the nimbin/CD complexes, that is, 1:1 for the
nimbin/β-CD and nimbin/TMβCD complexes. The 1:1 inclusion
stoichiometry was also observed in the nimbin/HPβCD and
nimbin/DMβCD complexes.

8548 J. Agric. Food Chem., Vol. 58, No. 15, 2010
Yang et al.
Figure 2. 1H NMR spectra of nimbin in the absence and presence of β-CD and TMβCD in D2O at 25 °C, respectively: (a) β-CD; (b) nimbin/β-CD complex;
(c) TMβCD; (d) nimbin/TMβCD complex (asterisk highlights the water peak).
Table 2. Chemical Shifts (δ) of the β-CD, TMβCD, Nimbin/β-CD, and
Nimbin/TMβCD Complexes
these chemical shifts, we found that the shifts of the H-5
(0.06 ppm) and H-6 (0.05 ppm) protons were larger than those
of the H-3 protons (0.03 ppm), indicating that nimbin may
enter the cavity of TMβCD from the narrow side. It was also
revealed that nimbin should penetrate into the HPβCD cavity
from the wide side but enter the cavity of DMβCD from the
narrow side (see the Supporting Information).
δ
nimbin/β-CD
complex
nimbin/TMβCD
complex
β-CD
TMβCD
H-1
H-2
d
4.99
3.57
3.88
3.50
3.80
3.78
4.98
3.57
3.83
3.50
3.80
3.75
5.19
3.25
3.61
3.66
3.76
3.55
3.42
3.51
3.28
5.22
3.29
3.64
3.71
3.81
3.60
3.47
3.57
3.35
Two-dimensional (2D) NMR spectroscopy provides impor-
tant information about the spatial proximity between host and
guest atoms by observation of intermolecular dipolar cross-
correlations (41). Two protons that are closely located in space
can produce a nuclear Overhauser effect (NOE) cross-correlation
in NOE spectroscopy (NOESY) or ROESY. The presence of
NOE cross-peaks between protons from two species indicates
spatial contacts within 0.4 nm (42). To gain more conformational
information, we obtained 2D ROESY of the inclusion complexes
of nimbin with CDs. The ROESY spectrum of the nimbin/CD
complex (Figure 3) showed appreciable correlation of the H-22
proton of nimbin with the H-3 protons of β-CD (peak a), as well
as key correlations between the H-19 protons of nimbin and the
H-3 and H-4 protons of β-CD (peak b). These results indicate that
the E ring (furan ring) and C/D rings of nimbin are included in the
β-CD cavity. The ROESY spectrum of the nimbin/TMβCD
complex (Figure 4) also showed significant correlations between
the H-23 proton of nimbin and the H-3 protons of TMβCD and
between the H-22 proton of nimbin and the H-3, H-4, and H-5
protons of TMβCD (peak c), as well as key correlations between
the H-19 protons of nimbin and the H-3, H-4, and H-5 protons of
TMβCD (peak d). These results indicate that the E ring (furan
ring) and C/D rings of nimbin are also included in the TM-β-CD
cavity. It can also be shown that nimbin should be included in the
HPβCD and DMβCD cavity in similar ways (see the Supporting
Information).
dd
dd
dd
m
dd
s
H-3
H-4
H-5
H-6
H-Me2
H-Me3
H-Me6
s
s
To further explore the inclusion mode, the chemical shifts of
β-CD protons in the absence and presence of nimbin were
examined (Table 2). Inclusion complexation with nimbin had a
negligible effect on the δ values of the H-1, H-2, H-4, and H-5
protons of β-CD (<0.01 ppm). In contrast, those values of the
H-3 and H-6 protons exhibited relatively weak but significant
changes (0.03-0.05 ppm), which could be caused by the
hydrogen bond between the hydroxyl arms of β-CD and the
oxygen atoms of nimbin. It is noteworthy that the H-3 protons
shifted ca. 0.05 ppm but that the H-5 protons showed no
appreciable shifts after inclusion complexation. Because both
the H-3 and H-5 protons are located in the interior of the β-CD
cavity and the H-3 protons are near the wide side of the cavity
whereas the H-5 protons are near the narrow side, this
phenomenon may indicate that nimbin should penetrate into
the β-CD cavity from the wide side. In contrast, all of the
TMβCD protons showed appreciable shifts after inclusion
complexation with nimbin (0.03-0.07 ppm). By comparing

Article
J. Agric. Food Chem., Vol. 58, No. 15, 2010 8549
Figure 3. ROESY spectrum of the nimbin/β-CD complex in D2O.
Figure 4. ROESY spectrum of the nimbin/TMβCD complex in D2O.

8550 J. Agric. Food Chem., Vol. 58, No. 15, 2010
Yang et al.
Figure 5. Possible inclusion mode and significant NOESY (T) correla-
tions of the nimbin/β-CD, nimbin/HPβCD, nimbin/DMβCD, and nimbin/
TMβCD inclusion complexes.
Figure 7. DSC thermograms of (a) nimbin, (b) β-CD, (c) TM-β-CD,
(d) nimbin/β-CD inclusion complex, and (e) nimbin/TM-β-CD inclusion
complex.
Thermal Analysis. The thermal properties of the nimbin/β-CD
and nimbin/TMβCD complexes were investigated by TG methods
(see the Supporting Information). A systemic analysis of the TG
curves showed that nimbin decomposes at ca. 210 °C, β-CD at ca.
200 °C, and TMβCD at ca. 170 °C. However, the thermal stability
of their inclusion complexes differed; that is, the decomposition
temperatures were ca. 230 and 250 °C for the nimbin/β-CD and
nimbin/TMβCD complexes, respectively. These results indicate
that nimbin’s usual thermal properties were altered after inclusion
complexation. In similar tests, the nimbin/HPβCD or nimbin/
DMβCD complex also shows a high decomposition temperature
up to 220 or 250 °C, respectively.
Figure 6. XRD patterns of (a) nimbin, (b) β-CD, (c) TMβCD, (d) nimbin/
β-CD inclusion complex, and (e) nimbin/TMβCD inclusion complex.
The DSC thermogram gave further information about the
thermal properties of the nimbin/β-CD and nimbin/TMβCD
complexes. As shown in Figure 7, the DSC curve of nimbin
contained an endothermic peak at 193 °C. In contrast, the DSC
curve of pristine β-CD or TMβCD had an endothermic peak at 99
or 162 °C, respectively. However, in the DSC curves of the CD/
nimbin complexes, the endothermic peaks at about 193 °C
corresponding to the free nimbin disappeared, coinciding with
the appearance of a new exothermic peak at 82 °C (or 107 °C) in
the case of the nimbin/β-CD (or nimbin/TMβCD) system. In
similar tests, the nimbin/HPβCD or nimbin/DMβCD complex
also shows the appearance of a new exothermic peak at 51 or
73 °C, and the endothermic peak at about 193 °C disappears (see
the Supporting Information). This suggested that the nimbin/
TMβCD complex is more stable than the nimbin/β-CD, nimbin/
HPCD, or nimbin/DMCD complex.
These results not only further confirm the formation of
nimbin/CDs complexes but also indicate that the four resultant
nimbin/CDs complexes start to decompose only at a temperature
above 220 °C, which means that these complexes are fairly stable
from a thermal viewpoint.
Solubilization. The water solubility of the nimbin/CD complex
was assessed by the preparation of its saturated solution (43).
An excess amount of complex was placed in 2 mL of water (ca.
pH 6.0), and the mixture was stirred for 1 h. After removal of the
insoluble substance by filtration, the filtrate was evaporated
On the basis of these observations, together with the 1:1 sto-
ichiometry, we deduced the possible inclusion modes of nimbin
with CDs as illustrated in Figure 5.
XRD Analysis. The XRD patterns of nimbin, β-CD, and
TMβCD as well as their inclusion complexes are illustrated in
Figure 6 (the XRD of HPβCD and DMβCD and their inclusion
complexes with nimbin can be seen in the Supporting Informa-
tion). As indicated in Figure 6, nimbin is amorphous (Figure 6a),
but β-CD (Figure 6b) and TMβCD (Figure 6c) are in crystalline
form. In contrast, the XRD of the nimbin/β-CD and nimbin/
TMβCD complexes (Figure 6d,e) showed halo patterns, which
were quite different from the superimposition of crystalline β-CD
(or TMβCD) and the amorphous nimbin, indicating the forma-
tion of the inclusion complex between β-CD (or TMβCD) and
nimbin. The similar phenomenon for HPβCD and DMβCD and
their inclusion complexes can be found (see the Supporting
Information). In addition, most of the crystalline diffraction
peaks of β-CD, TMβCD, HPβCD, or DMβCD disappeared
after complexation with nimbin, indicating that the complexation
of nimbin reoriented the CD molecules to some extent. Further-
more, the less sharp peaks in the XRD of the nimbin/β-CD
complex may imply that nimbin/β-CD, nimbin/HPβ-CD, and
nimbin/DMβ-CD possess a more amorphous structure than the
nimbin/TMβCD complex.

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J. Agric. Food Chem., Vol. 58, No. 15, 2010 8551
under reduced pressure to dryness and the residue was dosed by
the weighing method. The results show that the water solubility of
the nimbin, compared with that of native nimbin (ca. 50 μg/mL),
was remarkably increased to approximately 4.7, 3.8, 1.3, and
2.4 mg/mL by the solubilizing effects of β-CD, HPβCD, DMβCD,
and TMβCD, respectively. In the control experiment, a clear
solution was obtained after dissolution of the nimbin/β-CD
(19.7 mg), nimbin/HPβCD (15 mg), nimbin/DMβCD (10.6 mg),
or nimbin/TMβCD (14.8 mg) complex, respectively, which was
equivalent to 4.7, 3.8, 1.3, or 2.4 mg of nimbin, in 1 mL of water at
room temperature. This confirmed the reliability of the obtained
satisfactory water solubility of the nimbin/CD complex, which
will be beneficial for the medical utilization of this compound.
Conclusions. The inclusion complexation behavior, character-
ization, and binding ability of nimbin with β-CD and its deriva-
tives (HPβCD, DMβCD, TMβCD) were investigated. The results
showed that β-CD and its derivatives can enhance the water
solubility of nimbin. Given the shortage of applications of nimbin
and the easy and environmentally friendly preparation of the
nimbin/β-CD complex, this inclusion complexation should be
regarded as an important step in the design of a novel formulation
of nimbin for biopesticide and herbal medicine or healthcare
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