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COMPLEXATION WITH HYDROXYPROPYLγ-CYCLODEXTRIN OF SOME PENTACYCLIC
TRITERPENES. CHARACTERISATION OF
THEIR BINARY PRODUCTS
CODRUŢA M. ŞOICA1*, CRISTINA A. DEHELEAN2, CAMELIA I.
PEEV3, GEORGETA CONEAC4, ALEXANDRA T. GRUIA5
University of Medicine and Pharmacy “Victor Babeş” Timişoara, P-ta
Eftimie Murgu nr.2, 300041
1
Department of Pharmaceutical Chemistry
2
Department of Toxicology
3
Department of Pharmacognosy
4
Department of Pharmaceutical Technology
5
County Hospital Timişoara, Immunology Department
*corresponding author: codrutasoica@yahoo.com
Abstract
Pentacyclic triterpenes possess anticancer, anti-inflammatory and antiviral
activity. The compounds can be obtained by simple extraction with organic solvents. The
major problem of this type of triterpenes is their low water solubility which can be
increased by physical procedures like cyclodextrin complexation. The aim of present study
was to analyse the products obtained between pentacyclic triterpenes (betulinic acid,
betulin) and hydroxipropil-γ-cyclodextrin (HPGCD).
HPGCD is a hydrophilic semi synthetic cyclodextrin cited in literature as a
complexation agent for compounds like betulinic acid, so it was used as a host-molecule to
improve its solubility in water. In order to obtain the inclusion complexes, 1:2 molar ratio
and two preparation methods (physical mixing, kneading) were used. The inclusion
complexes were analysed by in vitro dissolution tests and X-ray diffraction and then
submitted to MTT test on mesenchimal stem cells.
Rezumat
Triterpenele pentaciclice prezintă activitate antitumorală, antiinflamatorie şi
antivirală. Compuşii pot fi obţinuţi prin simpla extracţie cu solvenţi organici. Principalul
dezavantaj al acestui tip de triterpene este solubilitatea scăzută în apă, care poate fi mărită
prin metode fizice cum este complexarea cu ciclodextrine. Scopul prezentului studiu a fost
analiza produşilor triterpenelor pentaciclice (acid betulinic, betulină) cu hidroxipropil-γciclodextrina (HPGCD).
HPGCD este o ciclodextrină semisintetică hidrofilă citată în literatură ca agent
de complexare pentru compuşi de tipul acidului betulinic, motiv pentru care a fost folosită
ca moleculă-gazdă pentru optimizarea solubilităţii în apă a acestuia. Pentru obţinerea
complecşilor de incluziune au fost folosite două metode de preparare (amestecarea
mecanică şi malaxarea), în raport molar de 1:2. Complecşii de incluziune au fost analizaţi
prin teste de dizolvare in vitro şi difracţie cu raze X şi apoi supuşi testului MTT pe celule
stem mezenchimale.
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

triterpenes
cyclodextrin


complexation
antitumor
INTRODUCTION
Betulinic acid (BA), 3-beta-hidroxi-20(29)-lupane-28-oic acid is
an important therapeutic compound, tested on cell lines type: MHH1,
MHH3, A172, SK17, SK19, MCF-7 etc.; it is a selective inductor of
apoptosis in tumor cells, inhibits NF-kB activation, reduces inflammation
and modulates immune answer. [1,2] BA has a selective activity on
melanoma cells and does not affect normal cells [3].
Betuline (Bet), lup-20(29)-ene-3β, 28-diol is a natural compound
obtained from vegetal sources (birch tree outer bark, etc.) by sublimation or
extraction with chemical solvents (methanol, dichloromethane, chloroform) [4,5].
Betuline is a glucocorticoid-type anti-inflammatory agent which
can be used as precursor in betulinic acid synthesis [5].
Betuline was used in popular medicine for the treatment of skin
diseases [5,6] .
The most frequent used cyclodextrins in the pharmaceutical fields
are β- and γ-cyclodextrin and their hydrophilic derivatives: HPBCD,
HPGCD, and kneading is the most accessible method for the preparation of
cyclodextrin inclusion complexes. [7,8,9] Betulinic, ursolic or oleanolic
acid, with antitumoral activity, can be associated with different
cyclodextrins in order to improve their physicochemical properties. The best
results concerning water solubility were obtained with HPGCD, which
represents an important progress toward a better bioavailability of those
triterpenic compounds. [10]
Hydroxypropyl-γ-cyclodextrin (HPGCD) was used as a hostmolecule to improve its solubility in water, via inclusion complex
formation. In order to obtain the inclusion complexes, 1:2 molar ratio and
kneading preparation method was used. The inclusion complexes were
analyzed by in vitro dissolution tests, thermal analysis and X-ray diffraction.
Biological activity was confirmed using an in vitro preliminary test.
MATERIALS AND METHODS
Betulinic acid and betulin (figure 1) [5,11] were purchased from
Sigma-Aldrich Ltd.
HPGCD (figure 2) was purchased from Cyclolab R&D
Ltd.(Budapest, Hungary). Solvents (ethanol, methanol, n-octanol) are of
analytical grade requested by the Romanian Pharmacopoeia 10th Ed. [12].
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CH3
CH3
H2C
CH3
H2C
CH3
COOH
CH3
CH3
CH3
HO
H3C
CH3
Betulinic acid
CH2OH
CH3
HO
H3C
CH3
Betulin
Figure 1
Betulinic acid and betulin structural formula
Cyclodextrin
HPBCD
R
-CH2CHOHCH3
Figure 2
HPGCD structural formula
Preparation of solid products
The methods applied for the preparation of the inclusion complexes
were [9]:
 simple powder mixing, using a mortar and a pestle;
 kneading with a 50% ethanolic solution until the bulk of solvent
evaporated; the mixture was then dried at room temperature for 24
hours and then was put in the oven, at 105ºC for several hours. The
final product was pulverized and sieved.
The binary products were prepared using 1:2 molar ratio.
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185
In vitro dissolution tests
The dissolution studies were carried out using a modified paddle
Erweka DT apparatus, in 100 mL buffer solution of pH = 5.5, at 37±1ºC, for
120 min. Samples were taken after 5, 10, 15, 30, 60 and 120 min and the
dissolved quantity of BA and Bet was determined spectrophotometrically at
210 nm. All studies were performed at least in triplicate.
X-ray Diffraction
XRD spectra were recorded with a DRONUM-1 X-ray
diffractometer (Russia) system with CuKα1 radiation (λ = 1.54178 Ǻ) over
the interval 2-44º/2θ. The measurement conditions were as follows: target,
Cu; filter, Ni; voltage, 35kV; current, 20 mA; time constant, 1S; angular
range 2º < 2θ < 44º.
The biological activity confirmation.
MTT test on human
mesenchymal stem cells
The test and method followed literature data [13,14]. Bone marrow
was obtained from 8 patients with different hematological disorders. The
agreement was certified by signature from each patient according to the
rules established by the University Ethics Committee. Human mesenchimal
stem cells (huMSCs) were isolated by adherence on plastic in tissue culture
flasks. After isolation in uncoated flasks on DMEM low-glucose medium
(1g/l, Stemcell Technologies, Inc) supplemented with 10% FCS (fetal calf
serum) (Promocell, Germany). Cell culture medium was changed every 3
days. After 14-21 days of plating and upon reaching 80% confluence cells
were harvested using 0,25% trypsin and 1 mM EDTA and replated in
uncoated flaks, at a density of 3x103 cells/cm2, using the same culture
medium.
In vitro toxicological analyses were performed by a standardized
colorimetric MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide) assay on human mesenchymal stem cells.
For the MTT assay, human MSCs at different passages (1,5,7) and
plated into flat bottom 96 well plates at a concentration of 50000 cells/well
in 150 μl of medium. The plates were incubated for 24 hours at 37 °C and
5% CO2 environment. MTT assay was performed in order to evaluate the
inhibitory effects of the solutions. Tested compounds were dissolved in
phosphate buffer solution (PBS) and 10 μL/well were added to the cells [12,
13]. Final conditions for the cell tests were: 5000 cells/well, low glucose
medium, FCS 10%, penicillin and streptomicin 1%, FGF (fibroblast growth
factor) 10 ng/mL medium. All the liquid solutions were sterile filtered
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(through 0.22 μm pore filters) before being added to the cells. Absorbances
were read at 570 nm (690 nm reference filter) using a standard ELISA
reader. Results were normalized to medium-only containing blanks and
processed in the same way as the samples. Concentration in dry extract of
each sample was 1 mg/1 ml DMSO (dimethyl sulfoxide) and the tested
quantity was 1μl diluted with medium.
RESULTS AND DISCUSSION
In vitro dissolution tests
Aqueous solubility of BA and Bet was increased by HPGCD,
which also influenced the dissolution rate of the active substances. The
physical mixtures prepared with HPGCD yielded a higher dissolution
profile as compared to triterpenic compounds and the kneaded products
were better than the physical mixtures. Figures 3 and 4 show the dissolution
profiles for BA and Bet and their kneading products (KP) with HPGCD, in
buffer solution.
3,5
Absorbance (210 nm)
3
2,5
2
1,5
1
0,5
0
0
20
40
60
80
100
120
Time (min)
AB
AB+HPGCD
Figure 3
In vitro dissolution of BA from HPGCD kneaded products
140
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0,25
Absorbance (210 nm)
0,2
0,15
0,1
0,05
0
0
20
40
60
80
100
120
140
Time (min)
Bet
Bet+HPGCD
Figure 4
In vitro dissolution of Bet from HPGCD kneaded products
X-ray diffraction analysis
X-ray diffraction analysis confirms the previous results. As we can
see in figure 5, BA and Bet present some peaks, characteristic for crystalline
compounds, whereas they are practically absent in the respective KP
products. The disappearance of all crystalline peaks leads to the conclusion
that an amorphisation phenomenon takes place, which can be presumed as
an inclusion complex formation between triterpenic substances and
HPGCD.
MTT test on mesenchimal stem cells
Preliminary in vitro tests indicate that the morphological and
numerical changes of the cells using MTT method showed the increase of
the antiproliferative and inhibitory activity after the administration of the
betulinic acid, compared to betulin. That observation indicated that betulinic
acid is more active than betulin but it could also be more noxious for
sensitive cells. The addition of HPGCD increased the antiproliferative and
inhibitory activity in both cases for betulin and betulinic acid also but not
very representative. This aspect can be sustained by the hypothesis that
increasing of water solubility for drugs at proper concentrations leads to the
improvement of their dissolution rate in cells medium. The MTT results for
huMSCs are presented in table I.
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BA
Bet
HPGCD
BA + HPGCD KP 1:2
Bet + HPGCD KP 1:2
Figure 5
Diffractograms of BA, Bet, HPGCD and their kneaded products
Type of sample
Betulinic acid
Betulin
Betulinic acid + HPGCD
Betulin + HPGCD
Cells only
**NA=nothing added
Table I
The MTT assay for betulinic acid and betulin
alone or mixed with HPGCD on huMSCs cells
Amount (μl)
Absorbance 570/690 nm
1
0.110; Replicate 0.111
1
0.129; Replicate 0.130
1
0.078; Replicate 0.074
1
0.114; Replicate 0.116
**
NA
0.187; Replicate 0.189
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189
CONCLUSIONS





HPGCD (hydroxipropil-γ-cyclodextrin) is able to form inclusion
complexes with compounds that fit its cavity dimensions. The solubility
of triterpenic compounds is increased in the presence of HPGCD.
Products prepared in molecular ratio 1:2 are suitable for increasing
hydrosolubility process. The formation of inclusion complexes has
been proved by X-ray diffraction analysis, which confirmed the
efficacy of the kneading method in this purpose.
In vitro dissolution tests revealed that the kneading method
significantly improved the rate of dissolution especially at a molar
ratio of 1:2.
The presence of HPGCD significantly influences different
parameters of the drug such as solubility and dissolution rate; this
could prove advantageous in future, offering the possibility of new
pharmaceutical preparations with higher bioavailability and smaller
therapeutic dosage.
The addition of HPGCD increased the antiproliferative activity of
pentacyclic triterpenes like betulinic acid and betulin.
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Acknowledgements to grant CNCSIS PN II 1257/2007 contract
212/2007, for financial support.