Genotoxic and mutagenic properties of synthetic betulinic acid and betulonic acid

Article abstract of DOI:10.1134/S1068162015040056

Abstract Betulinic acid was synthesized from birch bark extract (Betula pendula), with betulonic acid being an intermediate of the synthesis. Both compounds were isolated with a purity of 95%. Genotoxicity and mutagenicity of the prepared compounds was analyzed by the Ames test and SOS chromotest and it was found that the substances show no mutagenic and genotoxic activity.

Full text of DOI:10.1134/S1068162015040056

ISSN 1068ꢀ1620, Russian Journal of Bioorganic Chemistry, 2015, Vol. 41, No. 4, pp. 409–413. © Pleiades Publishing, Ltd., 2015.
Original Russian Text © T.S. Frolova, T.P. Kukina, O.I. Sinitsyna, 2015, published in Bioorganicheskaya Khimiya, 2015, Vol. 41, No. 4, pp. 462–467.
Genotoxic and Mutagenic Properties
of Synthetic Betulinic Acid and Betulonic Acid
,
b
T. S. Frolovaa , 1, T. P. Kukinab, and O. I. Sinitsynac
aNovosibirsk State University, Novosibirsk, 630090 Russia
bVorozhtsov Novosibirsk Institute of Organic Chemistry, Siberian Branch, Russian Academy of Sciences,
Novosibirsk, Russia
cInstitute of Cytology and Genetics, Siberian Branch, Russian Academy of Sciences, Novosibirsk, Russia
Received November 5, 2014; in final form, March 14, 2015
Abstract—Betulinic acid was synthesized from birch bark extract (Betula pendula), with betulonic acid being
an intermediate of the synthesis. Both compounds were isolated with a purity of 95%. Genotoxicity and
mutagenicity of the prepared compounds was analyzed by the Ames test and SOS chromotest and it was found
that the substances show no mutagenic and genotoxic activity.
Keywords: betulinic acid, betulonic acid, mutagenicity, genotoxicity, Ames test, SOS chromotest
DOI: 10.1134/S1068162015040056
1
INTRODUCTION
resistant to the toxic effect, that is, the effect was selecꢀ
tive [12–15].
Triterpene compounds with a lupane skeleton are
widespread secondary metabolites of various plant
products [1]. As many native lupanes and their synꢀ
thetic derivatives show different biological activities,
they are of great interest in the preparation of valuable
pharmaceuticals.
In addition to a significant antitumor effect, betuꢀ
linic acid shows antioxidant properties [16, 17] and
alcoholic extracts of plants containing betulinic acid
also reveal the antiꢀinflammatory effect [18]. This
compound was found in experiments on obesity in rats
[19] to inhibit lipase activity and induce adipocyte
lipolysis. The antimutagenic effect of extracts containꢀ
ing betulinic acid has also been observed [20].
Betulinic acid is one of the most studied lupanes
and may be isolated from plant raw materials by
extraction. Thus, the yield of isolation of the target
compound from birch bark (Betula alba L.) is 2.5%
[2–4]. Synthesis of betulinic acid from betulin, the
main component of the birch bark extracts of various
species is more promising and the yield may be as high
as 25% by weight of the birch bark [4]. Betulinic acid
shows a number of valuable properties in biomedical
tests, especially in experiments on the investigation of
cytotoxicity against cancer cells [5–7]. Thus, the
in vitro antitumor effect of betulinic acid in RKO and
SW480 colon cancer cells and a significant in vivo
inhibition of tumor growth in mice after oral adminisꢀ
tration of a drug containing betulinic acid has been
observed [8]. The inhibition of prostate tumor growth
has also been reported [9]. A combination of betulinic
acid with radioꢀ and chemotherapy has been shown in
[10] to lead to a significant cytotoxic effect in glioma
cells. The mechanism of this effect was supposed to
consist in the ability of betulinic acid to trigger apopꢀ
tosis through the release of mitochondrial apoptogenic
factors [11]. It should be noted that normal cells were
Betulonic acid was considered for a long time only as
a precursor of betulinic acid obtained in syntheses [21],
however, recent studies have shown a number of valuꢀ
able characteristics of betulonic acid such as antiviral,
anticancer, antimicrobial, hepatoprotective, and
immunostimulatory properties [22–27]. Investigation
of the influence of derivatives of betulonic acid in comꢀ
bination with rimantadine on influenza virus has shown
the possibility of reducing both side effects and dose of
the drug due to the use of this combination [28].
Despite such a broad spectrum of valuable properꢀ
ties of betulinic acid and betulonic acid, the safety of
their use in therapy has not been fully examined.
According to the Bridges principles [29], the first
stages of testing the safety of chemical compounds are
rapid bacterial test systems, namely, the Ames test and
SOS chromotest. Nevertheless, there are no data on
testing betulinic acid and betulonic acid with the use of
mentioned tests. Therefore, in the present work, betuꢀ
linic acid was synthesized from betulin, one of the
main components of the birch bark extract, to prepare
the desired compound in an amount sufficient for
microbiological tests. The samples of betulonic acid,
Abbreviations: NQO, 4ꢀnitroquinolinꢀ1ꢀoxide; МТBE, methyl
tertꢀbutyl ether; tꢀBuO H, tertꢀbutyl hydroperoxide.
2
1
Corresponding author: eꢀmail: frolova@bionet.nsc.ru.
409

410
FROLOVA et al.
Table 1. Genotoxicity of betulinic acid and betulonic acid
titative colorimetric method for analyzing the activity
of genotoxins. The test is based on the induction of
SOS response of E. coli cells, with this response, in
turn, inducing a number of functions as a reaction to
DNA damage or termination of its synthesis. The
Compound (mM)
DMSO
I
(c)m
1
σ
structural gene of ꢀgalactosidase (lacZ) being conꢀ
trolled by the promoter of DNA damageꢀinducible
gene (dinꢀgene), the SOS chromotest consists in the
β
NQO (1.6)
2.96 0.43a
0.71 0.15
Betulonic acid (10)
Betulinic acid (10)
analysis of ꢀgalactosidase activity and makes it possiꢀ
β
0.83 0.15
ble to measure the ability of various physical and
chemical mutagens to induce expression of dinꢀgenes
a
At the probability
P
≥
0.95, the values are significantly different
in E. coli [34, 35]. In E. coli PQ37 strain (F–, thr
hisꢀ4 pyrD, thi galE, galK EIE galT, srl300::Tn10,
rpoB rpsL urvA rfa trp::Muc– sfiA::Mud Ap lac),
cts lac U169, PhoC) used in the SOS chromotest, the
structural gene of ꢀgalactosidase (lacZ) was introꢀ
duced under the control of the promoter of sfiA din
gene, so that ꢀgalactosidase activity was dependent on
, leu,
from those of the control.
,
,
,
,
,
,
,
(
,
Table 2. Genotoxicity of metabolites of betulinic acid and
betulonic acid
,
Δ
β
Compound (mM)
I(c)m
σ
β
the expression of this gene and induced under the influꢀ
ence of many genotoxic agents on the bacterial cells.
DMSO
Benzo[
1
α
]pyrene (2)
2.77 0.14a
1.15 0.12
The analysis consisted in determination of the relꢀ
ative ꢀgalactosidase activity in E. coli PQ37 cells
β
Betulonic acidact (10)
Betulinic acidact (10)
induced by the influence of a supposed genotoxic facꢀ
tor. The analyzed compounds were tested by the conꢀ
ventional technique [36] with some modifications
reported in [37].
0.83 0.08
a
At the probability
P
≥
0.95, the values are significantly different
from those of the control.
Certain doses of potential genotoxic factors may
inhibit the protein synthesis, thus, leading to incomꢀ
plete determination of ꢀgalactosidase induction. The
β
which was an intermediate of the synthesis, were also
examined.
ability of compounds to induce DNA damages resultꢀ
ing in prevention of replication is referred to as genoꢀ
toxicity. In order to correct this influence, the syntheꢀ
RESULTS AND DISCUSSION
sis of the total protein, ꢀgalactosidase activity, and
β
alkaline phosphatase activity (the latter is a constituꢀ
tively expressed enzyme) was determined in the incuꢀ
Synthesis of Betulinic Acid
Synthesis was performed via the described techꢀ
nique [4] with some modifications concerning purifiꢀ
cation of the target compound. The birch bark extract
mainly composed of betulin and lupeol was used as an
initial sample. In the first stage of synthesis, betulonic
acid was prepared using Jones reagent (chromium(IV)
trioxide in a mixture of dilute sulfuric acid and aceꢀ
tone), followed by reduction with sodium borohydride
to yield betulinic acid. The target compounds were
purified by recrystallization.
bation period. The ratio of ꢀgalactosidase activity to
β
alkaline phosphatase activity obtained at the same
exposure doses indicates the induction of sfiA gene
even in the case of inhibition of the protein synthesis.
The
(induction of
tion of the ratio of
I
(c) induction factor of lacZ gene expression
ꢀgalactosidase activity) is the proporꢀ
ꢀgalactosidase activity to alkaline
β
β
phosphatase activity at a specified dose of a damage
factor to the same ratio obtained in the absence of the
factor. This value shows the magnitude of the damagꢀ
ing effect of a given agent.
1
13
Measured melting points and H and C NMR
spectra of betulonic acid and betulinic acid coincided
with those reported in [30–32]. The prepared samples
were shown by HPLC and LCꢀMS analysis to be charꢀ
acterized by a purity of 95%.
The SOS chromotest was performed both with and
without metabolic activation providing actually full
spectrum of possible metabolites.
Investigation of genotoxic properties of betulinic
The values of
βꢀgalactosidase induction factor
acid and betulonic acid by the SOS chromotest. Cytoꢀ obtained after incubation of the test strain with betuꢀ
toxic properties of many compounds are based on linic acid, betulonic acid, and their metabolites are
their ability to induce DNA damages leading to preꢀ summarized in Tables 1 and 2. The studied comꢀ
vention of replication [33]. The SOS chromotest was pounds were used in the highest possible concentraꢀ
developed to detect these damages and determine tions taking into account their solubility limits in
their causative agents. The SOS chromotest is a quanꢀ DMSO.
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 41
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GENOTOXIC AND MUTAGENIC PROPERTIES
411
Table 3. Mutagenicity of betulinic acid and betulonic acid
Nm
S. typhimurium S. typhimurium
Standard mutagens such as 4ꢀnitroquinoline
1ꢀoxide (NQO) and benzo[ ]pyrene were used as posꢀ
α
σ
itive controls in concentrations enough to induce an
appreciable SOS response of E. coli PQ37 cells. It
should be noted that there was no significant differꢀ
ence between the value of the induction factor
obtained with the negative control and those obtained
with betulinic acid and betulonic acid. Meanwhile, the
Compound (mM)
TA98
TA102
DMSO
NQO (1.6)
ꢀBuO2H (0.011)
21
4
93 11*
–
322 27
–
760 36a
327 28
3220 16
reference NQO and benzo[ ]pyrene showed high valꢀ
α
ues of the induction factor (Tables 1 and 2).
t
Investigation of mutagenic properties of betulinic
acid and betulonic acid by the Ames test. The standard
test histidineꢀdependent Salmonella typhimurium TA
98 (hisD3052) and TA102 (hysG428) strains develꢀ
oped by Ames [38] and containing various mutations
in the histidine operon [39–41] were used in the Ames
test to detect the potential mutagenic activity of synꢀ
thesized compounds [41]. The induction of mutations
was tested by taking into account the reverse mutations
from histidine auxotrophy to prototrophy resulting
from a nitrogenous base substitution or reading frame
shift caused by the influence of mutagenic factors. The
base substitutions were detected by TA102 strain,
while the reading frame shift was revealed by TA98
strain [39]. Thereby, the Ames test makes it possible to
detect the mutagenic activity of various chemical
compounds such as hydroperoxides, phenylhydrazine
quinones, aldehydes, and other agents inducing base
damages as well as the mutagenic activity of various
radiations [40]. TA102 strain is a standard strain used
to evaluate the mutagenic effect of oxidants.
Betulonic acid (10)
Betulinic acid (10)
18
19
5
4
a
At the probability
P
≥
0.95, the values are significantly different
from those of the control.
Table 4. Mutagenicity of metabolites of betulinic acid and
betulonic acid (S. typhimurium TA98 starin)
Compound (mM)
Nm
σ
DMSO
Benzo[
60 11
α
]pyrene (2)
154 17a
61 10
Betulonic acidact (10)
Betulinic acidact (10)
56 10
a
At the probability
P
≥
0.95, the values are significantly different
from those of the control.
As the SOS chromotest, the Ames test allows evalꢀ
uating the direct mutagenic effect of a substance and
its possible metabolites. The highest possible concenꢀ
trations of studied compounds (in terms of their soluꢀ
bility limits in DMSO) were used.
spectra was determined via the standard methods by
experiments in ꢀmodulation mode (JMOD) with offꢀ
J
resonance suppression. Melting points were measured
with the use of a Kofler instrument. A Milichrom liqꢀ
uid chromatograph was used to perform analytical
HPLC.
The Ames test was performed via the original
method [39]. The NQO and
benzo[ ]pyrene promutagen were used as standard
tꢀBuO2H mutagens and
α
mutagens. Data in Tables 3 and 4 show that the test
S. typhimurium TA98 and TA102 strains being used,
betulinic acid, betulonic acid, and their metabolites do
not induce any base substitution mutations and frameꢀ
shift mutations, whereas the standards show a proꢀ
nounced mutagenic effects.
Preparation of Betulin Concentrate
The bark of Betula pendula has been harvested in
April 2012 near the city of Novosibirsk (Akademgoroꢀ
dok). The bark was air dried at room temperature and
separated manually to obtain the outer bark and bast.
The outer bark (100 g) was minced with scissors to
Thus, according to the Ames test and SOS chroꢀ
motest, neither betulinic acid and betulonic acid nor
their metabolites show noticeable genotoxic and
mutagenic effects.
obtain pieces of
methyl tertꢀbutyl ether (MTBE) in a fluid percolator,
with the extractant being preheated to 47 . The solꢀ
vent was added to a raw material so that the solvent
1
×
5 mm size and extracted with
°
С
EXPERIMENTAL
Chemicals of reagent grade (Russia) were used in layer was from 0.5 to 1.0 cm above the raw material and
the synthesis of betulinic acid and betulonic acid. the ratio of the former to the latter was from 1.5 to 2.
NMR spectra of compounds dissolved in pyridineꢀd5 After one hour of infusion, the extract was drained
were recorded on a AVꢀ400 spectrometer at frequenꢀ through the bottom valve and a new portion of the solꢀ
1
cies of 400.13 (for H NMR) and 100.78 MHz (for vent was added. The process was repeated four times to
13
13C NMR). The multiplicity of signals in C NMR achieve the complete extraction of lipophilic compoꢀ
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 41
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412
FROLOVA et al.
nents and the combined ether extracts were evapoꢀ B. Ames (University of California, USA) were
rated to dryness on a rotary evaporator under reduced employed in the Ames test.
pressure (from 20 to 30 mmHg) provided by a water jet
pump. The yield of extract was 38.4 g (38.4% by weight
of the airꢀdry raw material). The content of betulin in
SOS Chromotest
the resulting extract determined by HPLC was 80.2%,
that is, 30.8% by weight of the airꢀdry raw material.
The SOS chromotest was performed via the stanꢀ
dard technique [34] with some modifications [37]. An
overnight E. coli culture (PQ37 strain) was grown to an
OD550 of 0.45 (2
and diluted tenfold with LB medium. Aliquots
(600 L) of the suspension of bacterial cells were
placed into a testꢀtube containing a solution (10 L) of
the test compound, with the exception being
benzo[ ]pyrene in the experiment with metabolic
activation. The samples were incubated with shaking
at 37 for 2 h, followed by analysis of the enzyme
activity.
×
108 cells/mL) with shaking at 37
°
С
Synthesis and Isolation of Betulinic Acid
and Betulonic Acid
μ
μ
The synthesis and isolation was performed by the
previously proposed method [4]. The standard Jones
reagent in acetone (5 mL) containing chromium triꢀ
oxide was added dropwise to a stirred solution of 0.5 g
of the birch bark extract (mainly composed of betulin
α
°
С
and lupeol [2]) in acetone (20 mL) at
was stirred with a magnetic stirrer for 1 h, diluted with
water to 80 mL, extracted with MTBE ( 20 mL) and
the combined organic layers were extracted with 2%
aqueous sodium hydroxide solution ( 20 mL) and
washed with water ( 20 mL) to neutral reaction.
0°С. The mixture
In the test with metabolic activation, the bacterial
culture was diluted tenfold with the activation mixꢀ
4
×
ture, ꢀgalactosidase activity and alkaline phosphatase
β
activity was then measured and the induction factor
was calculated as the proportion of the ratio of
3
×
3
×
βꢀgalactosidase activity to alkaline phosphatase activꢀ
The evaporated dry organic layer contained a lupeꢀ
none concentrate. The combined aqueous extracts
were acidified to pH 2 with 10% hydrochloric acid
ity observed at the studied dose of a damage factor to
the same ratio obtained in the absence of the factor.
The SOS chromotest was carried out twice in tripliꢀ
cate and the result was shown as the I(c)m mean value
of the induction factor calculated via the standard
method [34].
and extracted with hexane (
2
×
20 mL) to remove aliꢀ
phatic impurities, followed by extraction with MTBE
(
(
3
×
20 mL), washing the ether extract with water
3 20 mL) to neutral reaction, and evaporation to
×
yield a concentrate of betulonic acid. Recrystallization
from ethanolꢀhexane mixture resulted in 0.4 g of betꢀ
ulonic acid.
Ames Test
The Ames test was performed according to the
method proposed in [39]. An overnight S. typhimurium
An alcoholic solution of potassium borohydride
was added to a solution of 0.2 g of betulonic acid in
10% aqueousꢀalcoholic solution of potassium hydroxꢀ
culture (TA98 (rfa,
strains) was grown to an OD550 of 0.45 (2
108 cells/mL) at 37
for 16 h and the culture
(100 L) was placed into a testꢀtube containing a soluꢀ
tion (10 L) of the test compound. Two milliliters of
Δ
uvrB, +R) and TA102 (rfa, +, +R)
×
ide (2 mL) at
1 h, acidified with 5% hydrochloric acid and extracted
with MTBE ( 20 mL). The resulting extract was
washed with water ( 20 mL) to neutral reaction and
8°С. The reaction mixture was stirred for
°С
μ
3
×
μ
3
×
0.6% top agar containing histidine and biotin (for
TA98 strain) or only histidine (for TA102 strain) were
added, the mixture was thoroughly stirred by rotaꢀ
tional movements of the testꢀtube between the hands
and poured into Petri dishes containing 3% minimal
glucose agar. In experiments with metabolic activaꢀ
tion, a mixture (0.5 mL) of S9 was added to each 2 mL
of top agar. The dishes were kept to dry the agar, therꢀ
evaporated to dryness to yield a concentrate of betuꢀ
linic acid. Recrystallization from ethanolꢀhexane
mixture resulted in 0.18 g of betulinic acid (28% of the
initial raw material).
The standard mutagens such as NQO, tꢀBuO2H
and benzo[ ]pyrene, substrates such as ꢀnitropheꢀ
nylphosphate and ꢀnitrophenylꢀ ꢀgalactoside
,
α
p
o
β
(Sigma), ampicillin (AppliChem, Germany), Bacto
agar of special purity grade (Difco, USA), and other
chemicals of reagent grade (Russia) were used in
experiments. The induced rat liver S9 microsomal
fraction was kindly provided by V.I. Kaledin (Institute
of Cytology and Genetics).
mostated at 37°С for 48 h and the colonies of reverꢀ
tants germinated into the underlying layer of the
minimal glucose agar were counted. The Ames test
was performed twice in triplicate and the result was
shown as the Nm mean value of the number of reverꢀ
tants per dish.
Three bacterial strains were used in the work. E. coli
Statistical analysis was performed via the procedure
PQ37 strain (sfiA::Mud(Ap lac
uvrA, galE, galY, PhoC, rfa) [35] provided by P. Quilꢀ mine the significant differences between the mean valꢀ
lardet (Institut Pasteur, France) was used in the SOS ues of samples. The measurement error ( ) was deterꢀ
chromotest. S. typhimurium TA98 (rfa uvrB, +R) and mined as a standard deviation from the mean of indeꢀ
TA102 (rfa, +, +R) strains [37] from the collection of pendent measurements of the same sample [43].
)
cts,
lac
Δ
U169, mal+, described in [42]. The Student’s
tꢀtest was used to deterꢀ
σ
,
Δ
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 41
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GENOTOXIC AND MUTAGENIC PROPERTIES
413
22. Tolstikova, T.G., Sorokina, I.V., Tolstikov, G.A., Tolꢀ
ACKNOWLEDGMENTS
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,
The authors would like to thank V.I. Kaledin who
kindly provided the induced rat liver S9 microsomal
fraction.
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Translated by D. Lonshakov
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 41
No. 4
2015