Improving the solubility of ampelopsin by solid dispersions and inclusion complexes.

Abstract

The aim of this study was to increase the solubility of ampelopsin (AMP) in water by two systems: solid dispersions with polyethylene glycol 6000 (PEG 6000) or polyvinylpyrrolidone K-30 (PVP K30) and inclusion complexes with beta-cyclodextrin (BCD) and hydroxypropyl-beta-cyclodextrin (HPBCD). The interaction of AMP with the hydrophilic polymers was evaluated by differential scanning calorimetry (DSC), Fourier transformation-infrared spectroscopy (FTIR), scanning electron microscopy (SEM). The results from DSC, FTIR and SEC analyses of solid dispersions and inclusion complexes showed that AMP might exist as an amorphous state or as a solid solution. On the other hand, the SEM images of the physical mixtures revealed that to some extent the drug was present in a crystalline form. The influence of various factors (pH, temperature, type of polymer, ration of the drug to polymer) on the solubility and dissolution rate of the drug were also evaluated. The solubility and dissolution rates of AMP were significantly increased by solid dispersions and cyclodextrin complexes as well as their physical mixtures. The improvement of solubility using polymers was in the following order: HPBCD approximately BCD>PVP K30>PEG 6000.

Full text

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Journal of Pharmaceutical and Biomedical Analysis 38 (2005) 457–464
Improving the solubility of ampelopsin by solid dispersions
and inclusion complexes
Li-Ping Ruan, Bo-Yang Yu∗, Guang-Miao Fu, Dan-ni Zhu
School of Chinese Pharmacy, China Pharmaceutical University, Nanjing, 210009, PR China
Received 21 August 2004; received in revised form 31 January 2005; accepted 31 January 2005
Available online 2 March 2005
Abstract
The aim of this study was to increase the solubility of ampelopsin (AMP) in water by two systems: solid dispersions with polyethylene
glycol 6000 (PEG 6000) or polyvinylpyrrolidone K-30 (PVP K30) and inclusion complexes with ?-cyclodextrin (BCD) and hydroxypropyl-
?-cyclodextrin (HPBCD). The interaction of AMP with the hydrophilic polymers was evaluated by differential scanning calorimetry (DSC),
Fourier transformation-infrared spectroscopy (FTIR), scanning electron microscopy (SEM). The results from DSC, FTIR and SEC analyses
of solid dispersions and inclusion complexes showed that AMP might exist as an amorphous state or as a solid solution. On the other hand, the
SEM images of the physical mixtures revealed that to some extent the drug was present in a crystalline form. The influence of various factors
(pH, temperature, type of polymer, ration of the drug to polymer) on the solubility and dissolution rate of the drug were also evaluated. The
solubility and dissolution rates of AMP were significantly increased by solid dispersions and cyclodextrin complexes as well as their physical
mixtures. The improvement of solubility using polymers was in the following order: HPBCD≈BCD>PVP K30>PEG 6000.
© 2005 Elsevier B.V. All rights reserved.
Keywords: Ampelopsin; Polyethylene glycol 6000; Polyvinylpyrrolidone K-30; ?-Cyclodextrin; Hydroxypropyl-?-cyclodextrin; Inclusion complex; Solid
dispersion
1. Introduction
Ampelopsin (3,5,7,3?4?5?-hexahydroxyl 2,3 dihydrogen
flavonol, AMP, Fig. 1), isolated from the tender stem and
leavesoftheplantspeciesAmpelopsisGrossedentata(Hand-
Mazz) W.T. Wang, was one of the most common flavonoids.
AMPwasreportedtopossessnumerouspharmacologicalac-
tivities, such as anti-inflammatory and antimicrobial activity,
relieving cough, antioxidation, antihypertension, hepatopro-
tective effect, and anticarcinogenic effect [1–6]. There was
more than 27% of AMP in the tender stem and leaves of the
species, especially, more than 40% in the cataphyll. System-
atic research on AMP has been conducted to characterize its
pharmacological potential and possibility for drug product
∗Correspondingauthor.Presentaddress:DepartmentofComplexPresec-
tionofCMM,ChinaPharmaceuticalUniversity,Nanjing210038,PRChina.
Tel.: +86 25 8539 1042; fax: +86 25 8331 3080.
E-mail address: boyangyu@cpu.edu.cn (B.-Y. Yu).
development. However, AMP was poorly soluble in water
(0.2mg/ml at 25◦C). In aqueous or organic solution, AMP
decomposed under exposure to light, and colored photolysis
product was formed. The permeability through rat intestine
mucosa determined in our laboratory was 9.3×10−6cm/s,
and oral drug absorption in humans based on the intestinal
permeability in rats could be less than 10% [7]. Use of AMP
in most pharmaceutical preparations and some research ex-
periments was thereby limited due to its low water solubility,
lowintestinepermeabilityanddegradationinsolution.Toour
knowledge, no information is available on the improvement
ofthesedrug-likepropertiesofAMP.Inthispaper,increasing
the solubility of AMP has been addressed.
Various techniques have been used to improve the solu-
bility/dissolution rate of poorly water soluble drugs. Among
them,thesoliddispersiontechnique[8–10]andthecomplex-
ationwithcyclodextrins[11–13]aremostfrequentlyused.In
solid dispersion, hydrophilic polymers have been commonly
usedascarriers.Polyvinylpyrrolidon(PVP)andpolyethylene
0731-7085/$ – see front matter © 2005 Elsevier B.V. All rights reserved.
doi:10.1016/j.jpba.2005.01.030

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Fig. 1. Chemical structure of AMP.
glycol (PEG) have been widely employed for their low cost
andhighaqueoussolubility.Bothpolymersarefreelysoluble
inwaterandareavailableinvariousmolecularweights,rang-
ingfrom10,000to700,000forPVPandfrom200,toinexcess
of 300,000 for PEG. The molecular size of both polymers fa-
vors the formation of interstitial solid solutions [14–16]. Cy-
clodextrins as pharmaceutical excipients are mainly used as
solubilizingandstabilizingagentsforlipophilicsubstancesin
aqueouspreparations.Anumberofmoleculesaresolubilized
in cyclodextrin solutions through formation of an inclusion
complex [17,18]. HPBCD is one of the most accepted rep-
resentatives of hydroxylalkylated derivatives as hydrophilic
drug carrier, because of its amorphousness, high water solu-
bility and solubilizing ability, low cost and low toxicity.
The main objective of this work was to investigate the
possibility of improving the solubility and dissolution rate
of AMP including by (a) solid dispersion with PEG 6000
or PVP K30; and (b) complexation with BCD or HPBCD.
In order to characterize the prepared dispersions and inclu-
sion complexes, analyses using DSC, FTIR, SEM as well as
dissolution and solubility studies were carried out.
2. Materials and methods
2.1. Materials
AMP was extracted and purified in our laboratory. PVP
K30 was purchased from BASF (Germany) and PEG 6000
was supplied from Shanghai Guangming Chemical Plant
(China). BCD was kindly donated by Guangdong Yunan Cy-
clodextrenPlant(China)andHPBCDwithadegreeofsubsti-
tution 0.66 was purchased from Yiming Fine Chemical Co.,
Ltd. (China). All other materials were of analytical or HPLC
grade commercially available.
2.2. AMP assay
The concentration of AMP was measured by a reverse-
phase HPLC method described below. A Shimadzu HPLC
system was equipped with a SPD-10A SHIMADZU UV–vis
detector, a 20?l loop injection valve, a reversed phase Ver-
sapackC18(25cm×4.6mm;10?mparticle)columnincon-
junctionwithaprecolumninsert.Themobilephasewascom-
posed of CH3OH and H2O (60:40), and pH was adjusted to 3
with phosphoric acid. The mobile phase was filtered through
anylonmembranefilter(0.45?m)anddegassedbyultrason-
ication before use. The flow rate was 1.0ml/min, and the de-
tection wavelength was 292nm. The retention time of AMP
was approximately 4min. Quantification of the compound
was carried out by measuring the peak area in relation to the
concentration of standards chromatographed under the same
conditions. The standard curve was linear (r2>0.9998) over
the range of concentrations of interest (50–400?g/ml). The
relative standard deviation of the inter- and intra-day assay
was less then 5% (n=3).
2.3. Determination of solubility
Drugsolubilitywasdeterminedbyaddingexcessamounts
of AMP to water or buffer solutions with different pHs (pH
1.2,2.8,4.6,6.0,7.4)at25±0.5◦C.Thesuspensionsformed
were equilibrated under continuous agitation for 1 week and
then filtered through a 0.45?m membrane filter to obtain a
clear solution for HPLC assay. Phase-solubility studies were
carried out in the same way as solubility studies. Excess
amount of AMP was added to aqueous solutions contain-
ing various concentrations of PVP K30, PEG 6000, HPBCD
(5, 10, 15 and 20% w/v, respectively) and BCD saturation
solution. The suspensions were shaken at 25±0.5◦C for 7
days, and then the samples were filtered through a 0.45?m
membrane filter. The AMP concentrations were determined
using the HPLC method. Each sample was determined in
duplicate.
2.4. Preparations of physical mixtures, solid dispersions
and inclusion complexes
2.4.1. Physical mixtures
AMP and excipients (PVP K30, PEG 6000, BCD or HP-
BCD) were accurately weighed at a ratio of 1:10, pulverized
and then mixed thoroughly in a mortar with a pestle until a
homogeneousmixturewasobtained.Themixturewaspassed
through a 200?m sieve for further experiments.
2.4.2. Solid dispersions
Solid dispersions of AMP in PVP K30 or PEG 6000 con-
tainingthreedifferentratios(1:5,1:10,1:15w/wforPVPand
1:10, 1:15, 1:20 w/w for PEG) were prepared by the solvent
method. Briefly, AMP and the polymer were dissolved in a
minimum amount of purified ethanol. The solvent was then
removed by evaporation under reduced pressure at 40◦C.
The resulting residue was dried under vacuum for 3h and
stored overnight in a desiccator. After drying, the residue
was ground in a mortar, and then passed through a 200?m
sieve. The resultant powders were stored in a desiccator until
further investigation.
2.4.3. Inclusion complexes
AMP and ?-CD or HP-?-CD at 1:1 molar ratio were dis-
solved in distilled water. The resulting mixtures were stirred
at 25◦C for 7 days. After dissolution was completed, the so-
lutions were filtered through a 0.45?m membrane filter. The

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459
clear solutions were subjected to evaporation at 40◦C and
under vacuum. Then, the drying powders were treated as the
preparation of solid dispersions.
2.5. Differential scanning calorimetry analysis
Thermalcharacteristicsofthepurematerials,thephysical
mixtures, the solid dispersions and inclusion complexes of
AMP with excipients were determined by a differential scan-
ning calorimeter (DSC 204, Netzsch, Germany). The scan-
ning rate was 10◦C/min, and the scanning temperature range
was between 30 and 350◦C.
2.6. FTIR spectroscopy
FTIR spectra of the samples were obtained on a SHI-
MADZUFTIR−8400sspectrometer(Japan).Sampleswere
prepared into KBr disks (2mg sample in 200mg KBr). The
scans were obtained from 4000 to 400cm−1at resolution of
1cm.
2.7. Scanning electron microscopy (SEM)
The images of the samples were analyzed by SEM
(AKASHI SX-40, Japan). Samples were mounted on a
double-faced adhesive tape, sputtered with platinum prior
to analysis. The pictures were taken at a magnification of
600-fold or 2000-fold.
2.8. In vitro dissolution
The dissolution studies of AMP and different drug–
polymercombinations were
37±0.5◦C according to the dispersed amount method [19].
Samples equivalent to 100mg of AMP were added to 100ml
of water in a 200ml beaker, which was stirred with a rotating
performedin water at
paddle at 100rpm. At predetermined intervals, 1ml samples
were withdrawn, filtered (pore size 0.45?m), and analyzed
with HPLC for AMP. The same volume of fresh medium
was replaced and the correction for the cumulative dilution
was calculated. Dissolution experiments were carried out in
triplicate.
3. Results and discussion
3.1. Solubility studies
The solubility of AMP in water and the solutions with
different pHs was shown in Table 1. The solubility of AMP
in water at 25◦C was found to be 200.3?g/ml. The pH of
solutions had little effect on the solubility of AMP, but had
significant effect on the stability of AMP. When pH was be-
tween 1.2 and 4.6, the solution was stable. When pH was
6.0, there was some degradation observed. The solubility at
pH 7.4 could not be determined because it had decomposed
before it reached saturation.
The effect of different carriers and temperatures on the
aqueoussolubilityofAMPwasdisplayedinFig.2.Solubility
experimentsshowedthatthesaturationconcentrationofAMP
inwaterisnotablyaffectedbytemperature,from0.2mg/mlat
25◦Cto0.9mg/mlat37◦C.Inthephase-solubilitydiagrams
(Fig. 2), the results that the concentration of AMP in water
increased as a function of PEG 6000, PVP K30 and HPBCD
concentration were observed at both temperatures. The rela-
tionship appeared to be linear (r2>0.9). The increase of the
solubilitywithincreasingtemperaturerevealedtheendother-
micnatureofthisprocess.Theimprovementofthesolubility
due to presence of hydrophilic excipients might be attributed
to the improved wetting of AMP by forming intermolecu-
lar hydrogen bonding between AMP and PVP K30 or PEG
6000, but forming 1:1 cyclodextrin complex between AMP
Table 1
Solubility of AMP at 25◦C in water and aqueous solutions with different pHs
MediaWater (pH 6.1)
Solubility (?g/ml)200.3
Data are the mean of two determinations.
aNot determined.
pH 1.2
202.0
pH 2.8
223.3
pH 4.6
217.9
pH 6.0
214.5
pH 7.4
NDa
Fig. 2. Phase-solubility diagrams for AMP in the presence of PVP K 30, PEG 6000, HPBCD in water at 25±0.5◦C (n=2) (a) and 37±0.5◦C (n=2) (b).

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Fig. 3. DSC curves of single components and binary systems of AMP and
PVP,PEG,BCDandHPBCD.(A)AMP,(B)PVPK30,(C)physicalmixture
of AMP/PVP, (D) solid dispersion of AMP/PVP K30, (E) PEG 6000, (F)
physical mixture of AMP/PEG, (G) solid dispersion of AMP/PEG 6000,
(H) BCD, (I) physical mixture of AMP/BCD, (J) inclusion complex of
AMP/BCD, (K) HPBCD, (L) physical mixture of AMP/HPBCD, (M) in-
clusion complex of AMP/HPBCD.
and HPBCD. Similar observations have previously been re-
ported[20,21].InaBCDsaturationsolution,AMPsolubility
was 2.8mg/ml at 25◦C and 9.6mg/ml at 37◦C, respectively.
Thus, enhancement of solubility for AMP was 14.1-fold at
25◦C and 10.7-fold at 37◦C by BCD.
3.2. Differential scanning calorimetry (DSC)
DSC curves obtained for pure material, solid dispersions,
inclusion complexes and their corresponding physical mix-
tures were displayed in Fig. 3. Pure AMP powder had three
endothermic peaks and two exothermic peaks correspond-
ing to the loss of water (104.5 and 150.3◦C), recrystallizing
(198.7◦C), the melting point of AMP (240.1◦C) and oxidiz-
ing (243.8◦C) respectively (Fig. 3A). In the thermogram of
PEG 6000 (Fig. 3E), a sharp peak (65.4◦C) was observed,
whichwasassociatedwithmeltingendothermofPEG.Inthe
DSC curves of pure PVP (Fig. 3B), BCD (Fig. 3H) and HP-
BCD (Fig. 3K), the peaks corresponding to the evaporation
of water appeared in the temperature range of 50–150◦C.
Besides the endothermic peaks corresponding to the loss of
water, the thermogram of BCD displayed melting endotherm
with a shoulder, which indicated the presence of more than
one crystal form.
The physical mixture of AMP with PVP K30 showed
spectra corresponding to superposition of their parent prod-
ucts (Fig. 3C). In the curve of solid dispersion with PVP
(Fig. 3D), it exhibited a very broad endotherm ranging be-
tween 51.5 and 145◦C with a peak at 88.9◦C. The charac-
teristic features of AMP peak were lost. This indicated that
AMP was no longer present as a crystalline material, but was
converted into the amorphous state [22]. Solid dispersions of
AMP with PEG showed almost the same thermal behavior
as their physical mixtures of the same composition (Fig. 3F
and G), the absence of AMP peaks suggested that AMP was
completely soluble in the liquid phase of PEG 6000. The
samephenomenonhadpreviouslybeenreported[22,23].The
physical mixture with BCD (Fig. 3I) showed some charac-
teristic feature peaks of AMP, such as peak 145.5, 199.1 and
244.3◦C,however,themeltingpeakofAMPwasdisappeared
and another endothermic peak (213.9◦C) was observed. The
physical mixture with HPBCD (Fig. 3L) only appeared one
characteristic peak of AMP, i.e., 197.7◦C. However, all the
characteristic peaks of AMP were lost in the inclusion com-
plexeswithBCDandHPBCD.Thedisappearanceofthether-
mal features of the drug indicated that the drug penetrated
into the cyclodextrin cavity replacing the water molecules
[18,24].
3.3. Fourier transform-infrared spectroscopy
Fig. 4 shows the spectra of pure materials and respec-
tive AMP-carrier systems. The spectrum of AMP (Fig. 4A)
showed three characteristic bands of the OH group which
were found at 3477, 3352, and 3236cm−1. The carbonyl
stretching mode appeared at 1639cm−1. Other characteris-
tic bands were found at 1599cm−1, 1458cm−1(stretching
vibration of C C in the aromatic ring), 1155cm−1(C O C
stretch), 1128cm−1(C OH stretch) and 833cm−1(benzene
ring with tetra-substitutions).
Physical mixtures of AMP/PVP (Fig. 4C) and AMP/PEG
(Fig. 4F) exhibited spectra corresponding to a superposition
of their parent components. In the spectra of solid disper-
sion (Fig. 4D and G), the absorption bands which could be
assigned to the free OH and the OH involved in intramolec-
ular hydrogen bonding changed or disappeared. The reason
for this observation might be interpreted as a consequence
of hydrogen bonding between OH of AMP and >N
and

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C O of PVP K30, or the lone pairs of the oxygen atom in
PEG6000.ThecarbonylstretchingpeakofAMPwasshifted
from1639cm−1towardslowerfrequencies,thepeakoftetra-
substituted benzene ring was shifted from 833cm−1towards
higherfrequenciesinthesoliddispersions.Theseresultsalso
suggested that hydrogen bonding interactions between AMP
and PVP or PEG. IR spectra did not reveal dramatic changes
in characteristic peaks of AMP frequency, suggesting the ab-
sence of chemical interactions between AMP and the excip-
ients, as also reported by Ford [25].
The spectra of pure BCD and HPBCD (Fig. 4H and
K) illustrated the vibration of free OH between 3100 and
Fig. 4. FTIR spectra of pure materials, solid dispersions, inclusion complexes and corresponding physical mixtures. (A) AMP, (B) PVP K30, (C) physical
mixture of AMP/PVP, (D) solid dispersion of AMP/PVP K30, (E) PEG 6000, (F) physical mixture of AMP/PEG, (G) solid dispersion of AMP/PEG 6000, (H)
BCD, (I) physical mixture of AMP/BCD, (J) inclusion complex of AMP/BCD, (K) HPBCD, (L) physical mixture of AMP/HPBCD, (M) inclusion complex of
AMP/HPBCD.