Improved isolation of betulin and lupeol from birch bark and oxidation of their acetylated derivatives with chromyl chloride

Article abstract of DOI:10.1007/s00706-018-2165-3

Abstract: An improved method of isolation of betulin and lupeol from birch bark is developed and reported. The method afforded triterpenes with purity of 98.2% (betulin) and 96.3% (lupeol), respectively. Chromyl chloride was also investigated as an oxidating agent of O-acetylated betulin and lupeol. The transformation of isopropenyl moiety to aldehyde group was observed.

Full text of DOI:10.1007/s00706-018-2165-3

Monatshefte für Chemie - Chemical Monthly
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ORIGINAL PAPER
Improved isolation of betulin and lupeol from birch bark
and oxidation of their acetylated derivatives with chromyl chloride
Milo sˇ Luk a´ cˇ 1 • Branislav Horv a´ th2 • Martin Pis a´ r cˇ ik1 • Ferdinand Dev ´ı nsky1,3 • Ren a´ ta Hor a´ kov a´ 1
Received: 29 October 2017 / Accepted: 29 January 2018
Ó Springer-Verlag GmbH Austria, part of Springer Nature 2018
Abstract
An improved method of isolation of betulin and lupeol from birch bark is developed and reported. The method afforded
triterpenes with purity of 98.2% (betulin) and 96.3% (lupeol), respectively. Chromyl chloride was also investigated as an
oxidating agent of O-acetylated betulin and lupeol. The transformation of isopropenyl moiety to aldehyde group was
observed.
Graphical abstract
Keywords Aldehyde group Á Betulin Á Lupeol Á Oxidating agent
Introduction
activities [4]. Anticancer activities are also intensively
studied in the case of triterpene carboxylic acids: betulinic
(3b-hydroxy-lup-20(29)-en-28-oic acid) and oleanolic acid
(3b-hydroxyolean-12-en-28-oic acid) [5–7]. Betulinic acid
and its derivatives as bevirimat (3-O-(3 ,3 -dimethylsuc-
cinyl)betulinic acid) exhibit significant anti-HIV activity
[8]. Bevirimat can be easily synthesised from betulinic acid
and 2,2-dimethylsuccinic anhydride [9].
The outer bark of Betula pendula is a source rich in
triterpenes such as betulin, betulinic acid, oleanolic acid,
and lupeol [1], which possess interesting biological activ-
ities [1, 2]. Lupeol (lup-20(29)-ene-3b-ol) is the most
lipophilic triterpene that occurs in the birch bark. It exhibits
antileishmanial, immunomodulatory [3], or antineoplastic
0
0
The betulin (lup-20(29)-ene-3b,28-diol) is the most
abundant triterpene in birch bark. Therefore, it is a source
for the preparation of betulinic acid or other triterpenes. A
lot of chemical transformations of betulin were performed
by oxidating agents, from which Cr(VI) reagents such as
pyridinium dichromate, pyridinium chlorochromate, CrO3,
or K Cr O in different environments were the most
&
Milo sˇ Luk a´ cˇ
lukac@fpharm.uniba.sk
1
Department of Chemical Theory of Drugs, Faculty of
Pharmacy, Comenius University, Kalin cˇ iakova 8,
8
32 32 Bratislava, Slovakia
2
2 7
2
3
Central NMR Laboratory, Faculty of Pharmacy, Comenius
University, Odboj a´ rov 10, 832 32 Bratislava, Slovakia
studied [10–14]. Chromyl chloride is another strong oxi-
dating agent containing chromium in oxidation state VI.
CrO Cl is capable of oxidizing 2,2-disubstituted-1-alkenes
Faculty of Pharmacy, Comenius University, Odboj a´ rov 10,
8
2
2
32 32 Bratislava, Slovakia
123

M. Luká cˇ et al.
to aldehydes [15]. This moiety is present in the molecules
of betulin and lupeol and its oxidation with chromyl
chloride was investigated in this work. Following the
results of the present study, our aim is to expand research
in this area to develop an improved method for the isolation
of betulin and lupeol from birch bark.
triterpenes present in birch bark (lupeol, betulinic acid, and
oleanolic acid) are more soluble in benzene than betulin
and can be easily removed by crystallization from this
solvent. A similar method of the removal of lupeol from
ˇ
birch bark extract was used by Siman et al. [16]. However,
they used three crystallisation cycles and crystallised
betulin was isolated by centrifugation. The final crystalli-
sation from ethanol was used for removing impurities
which are more polar than betulin. These procedures led to
the isolation of betulin in a relatively good yield (14.5%)
and high purity, 98.2%.
Results and discussion
Betulin and lupeol were isolated from the outer part of
birch bark (Scheme 1). The isolation was performed by
Soxhlet extraction with azeotropic mixture cyclohexane/
ethyl acetate instead of using alcohols which are commonly
used extraction agents [11, 16]. Acetone or ethyl acetate
was also used as an extraction solvent as described previ-
ously [17, 18]. The choice of the aforementioned mixture is
due to the minimisation of the extraction of more polar
components from the bark. The raw extract was purified by
crystallization from benzene and ethanol. More nonpolar
According to our best knowledge, the purification of
betulin from crude extract was mainly performed by col-
umn chromatography [19–24]. Column chromatography-
free processes are less commonly reported [2, 16, 25]. We
compared our purification process with the latest one
ˇ
published by Siman [16], which provides thorough infor-
mation of the efficiency of extraction, purification process,
and the purity of the product. The comparison shows that
the main advantage of our purification process is the
Scheme 1
1
23

An improved method of isolation of betulin and lupeol from birch bark was developed and discussed…
simplification of the method. We reduced the number of
purification steps. We used only three crystallizations
instead of four crystallization steps (preparation of frac-
with 1 equivalent or 1.5 equivalents of chromyl chloride.
Reaction was stirred for 15 or 60 min, as indicated in the
experimental procedure below. Longer time and higher
amount of oxidizing agents produced lower yield. The
reaction provided 130 mg (23.9%) of 3b,28-diacetoxylu-
pan-29-al-11.0% of 20R-epimer and 12.9% of 20S-epimer.
Shorter time and equimolar amount of chromyl chloride
afforded better yield. The isolated amount of aldehyde was
292 mg (53.8%). 3b,28-Diacetoxylupan-29-al was isolated
as 20R-epimer (143 mg, 26.3%), 20S-epimer (83 mg,
15.3%). The mixture of both epimers (66 mg, 12.2%) is
composed of 21.5% of 20R-epimer and 79.5% of 20S-
epimer. A calculation shows that the ratio of the isolated
epimers was 54%:46% (20R-epimer to 20S-epimer). The
results from the both experiments show that the oxidation
of 3b,28-di-O-acetylbetulin with chromyl chloride is not a
stereospecific reaction.
tions B0 and B2) and one extraction with Ca(OH)2
ˇ
(
preparation of fraction B1) as described Siman et al. [16]
for the preparation of betulin fraction B2. The other
advantage is that the crystals were separated by filtrations
only. The centrifugation was not necessary, as requested by
ˇ
the procedure published by Siman et al. [16]. The purity of
betulin obtained with both methods was identical,
amounting to 98.2%. We obtained only slightly lower
overall yield of betulin (14.5%) as compared with the yield
ˇ
(
overall yield of B2 was 17.1%) published by Siman [16].
However, our less time-consuming and simpler purification
process provides significant benefits.
Lupeol was isolated from solutions which remained
after the separation of betulin. The solutions were ethyl
acetate/cyclohexane (B1) and benzene (B2). Triterpenes
contained in these solutions were betulin, lupeol, betulinic
acid, and oleanolic acid. Triterpene acids were separated
from the raw mixture of triterpenes by washing of benzene
solution with aqueous solution of sodium hydroxide.
Betulinic and oleanolic acids formed sodium salts which
were soluble in water. Some other impurities were pre-
cipitated from benzene solution in the form of dark brown
solid compounds. Lupeol is the most lipophilic triterpene
from birch bark. Therefore, it was subsequently purified by
filtration of its solution (10% ethyl acetate in cyclohexane)
through the bed of silica gel. The last step of purification
was performed by crystallization of lupeol from acetone.
Triterpene was obtained in the yield 0.98%, as calculated
with respect to birch bark, and in good purity of 96.3%. Its
purity was higher than that of some commercially available
agents. Less time-consuming and column chromatography-
free process with good purity of obtained lupeol represents
the main benefit resulting from this purification method in
comparison with the previously described isolation and
preparation processes [21, 24, 26–28]. Our procedure
provides an easy and cheap isolation method for obtaining
lupeol. It is a superior alternative to expensive sources
provided by commercial suppliers.
Direct of oxidation of 3b,28-di-O-acetylbetulin was
previously performed by iron(III) picolinate [29]. This
oxygenation results in different amounts of epimers. Their
ratio was approximately 1:2 (20R-epimer to 20S-epimer).
The overall yield of the reaction was lower (44.8%). 3b,28-
Diacetoxylupan-29-al was prepared for the first time by an
oxidation of a double bond with potassium permanganate
[30]. However, the resolution of epimers was not carried
out. The transformation of the double bond to an aldehyde
in lup-20(29)-ene moiety was also performed by several
other methods [31–34]. However, it had to be made in
several reaction steps.
The oxidation of isopropenyl group of acetylated lupeol
(3b-O-acetyllupeol) by chromyl chloride was also investi-
gated (Scheme 2). The reaction was performed with good
yield (64.5%). It was slightly higher than that observed in
the case of 3b,28-di-O-acetylbetulin. The procedure was
also nonstereo-specific. Both epimers of 3b-acetoxylupan-
29-al were obtained. Their ratio was approximately 3:2
(20R-epimer to 20S-epimer). However, only small amount
of epimeric pure 3b-acetoxylupan-29-al (20R-epimer;
m = 94 mg) was obtained by the purification on silica gel.
The main fraction consists from the mixture of both epi-
mers. 20S-Epimer could not be separated because its Rf
value was close to that of 20R-epimer.
Oxidation of betulin and its diacetate with chromyl
chloride was performed three times (Scheme 2). The first
oxidation of unprotected betulin was unsuccessful. Betulin
is very slightly soluble in dichloromethane and therefore, it
is poorly oxidisable. The remaining two oxidations were
made on 3b,28-di-O-acetylbetulin which is well soluble in
dichloromethane. Acetyl groups protected hydroxyl groups
before oxidation with the reagent. Acetylated betulin was
prepared by conventional reaction of alcohol with acetic
anhydride in basic environment of pyridine and in the
presence of catalytic amount of 4-(dimethylamino)pyridine
3b-Acetoxylupan-29-als has been prepared previously
[35]. Firstly, 3b-O-acetyllupeol was oxidised by m-
chloroperbenzoic acid [36]. Then, the epoxide was treated
with boron trifluoride [35]. This method also produced
epimers in a ratio 3:2 (20R-epimer to 20S-epimer; deter-
1
mined by H NMR) which is similar to our observation.
The authors prepared enantiomeric pure 3b-acetoxylupan-
29-als by the reduction of aldehyde to an alcohol. Epimeric
3b-acetoxylupan-29-ols were separated by multiple chro-
matography on silica gel. Then, enantiomerically pure
alcohols were repeatedly oxidised to aldehydes.
(
DAMP) [2]. Oxidation of protected betulin was performed
123

M. Luká cˇ et al.
Scheme 2
The method using oxidation of isoprenyl group with
chromyl chloride to aldehyde has several advantages in
comparison with previously discussed methods [29–36].
The yields of aldehydes are in the most cases higher than
those cited in the literature and the aldehydes are prepared
in one reaction step instead of multistep reactions.
Isolation of betulin
Betulin was extracted from outer white bark of Betula
pendula. The bark was collected from fallen trees in the
locality: Detva, Slovakia, 25.12.2016 (coordinates WGS-
0
00
0
00
84: 48°34 25 N, 19°23 55 E). The bark was air dried at
5 °C, then cut in small pieces in a kitchen blender. 50 g of
2
3
bark were extracted in 500 cm Soxhlet extractor. The
extraction was performed with an azeotropic mixture of
ethyl acetate and cyclohexane (cyclohexane to ethyl acet-
ate - 46 wt% : 54 wt%). The bark was extracted for 3 h.
The resulting extract was concentrated to a volume of
Conclusion
We evaluated an improved separation method of betulin
and lupeol from birch bark. The main benefit resulting
from the application of this procedure is the isolation of
lupeol in good purity and its recovery. This triterpene is
also available by commercial suppliers. However, it is
expensive and is often provided with lower purity than that
obtained by us. Our procedure provides an easy, efficient,
and cheap isolation method.
3
200 cm by distillation in the rotary evaporator. The con-
centrate was cooled to - 18 °C, and the precipitate (frac-
tion B1) was filtered through a filter glass with pore size
16–40 lm. The precipitate was dissolved in the boiling
3
3
mixture of 110 cm of benzene and 11 cm of ethanol and
the solution was filtered through the filter glass with pore
size 16–40 lm. The filtrate was concentrated by distillation
until the first precipitate was observed. This concentrate
was cooled to 4 °C. The precipitate (fraction B2) was fil-
tered through the filter glass with pore size 16–40 lm and
The acetylated triterpenes were also investigated in an
oxidation reaction with oxidizing agent which enables a
direct transformation of alkene to aldehyde. Lup-20(29)-
ens were oxidised with chromyl chloride to lupan-29-als
with a good yield. Reactions were not stereoselective and
both enantiomers were obtained. Epimers of 3b,28-diace-
toxylupan-29-als were separated by column chromatogra-
phy on silica gel. However, the separation of epimeric pure
3
washed by 25 cm of cold benzene. The solid compound
was dried at 110 °C for 2 h. B2 was obtained in the amount
8.14 g. The fraction B2 (1 g) was crystallised from 30 cm
3
of technical grade ethanol (w = 96%). The solution was
3
b-acetoxylupan-29-als is more complicated.
cooled in the fridge at - 18 °C over night. Crystals
(
fraction B3) were filtered through the filter glass with pore
3
size 16–40 lm and washed with small amount (3 cm ) of
cold technical grade ethanol. The betulin was dried at
Experimental
1
50 °C for 75 min and the final amount of 890 mg was
All chemicals used in the synthesis were obtained from
13
obtained. The overall yield of betulin was 14.5%.
1
commercial suppliers and were of p.a. purity. H and
C
NMR spectra were measured on a Varian MERCURY plus
spectrometer operating at frequencies of 300 and 75 MHz,
Isolation of lupeol
1
respectively. C NMR spectra were decoupled against
3
The solution after filtration of fraction B1 was evaporated
and the solid rest was dissolved in benzene solution which
protons. The spectra were measured in CDCl . The
3
chemical shifts were referenced with respect to an internal
1
remained after the filtration of B2. Benzene solution was
3
washed with 10% NaOH solution (2 9 50 cm ), H O
13
TMS (d( H) = 0 ppm, d( C) = 0 ppm).
2
3
1 9 50 cm ) and brine (1 9 25 cm ), then dried with
3
(
1
23

An improved method of isolation of betulin and lupeol from birch bark was developed and discussed…
3
anhydrous Na SO . Benzene was evaporated and the rest in
2
was added. The mixture was stirred for 5 min, 1 cm of
distilled water was added, and the mixture was stirred for
additional 15 min. The reaction mixture was diluted with
4
the flask was suspended in a hot solution of cyclohexane
3
and ethyl acetate (50 ? 5 cm ). The suspension was fil-
3
tered through the bed of silica gel (3 cm layer of Kieselgel
3
0G, diameter 4 cm) and washed with 100 cm of 10%
50 cm of dichloromethane and dried with 3 spoons of
6
anhydrous Na SO . The suspension was filtered through a
2
4
ethyl acetate in cyclohexane. Then, the solution was
bed of neutral aluminium oxide (1.5 cm high). Colourless
filtrate was mixed with silica gel and evaporated to
dryness.
evaporated. The fraction L1 (m = 1.7 g) was obtained.
3
This fraction was crystallised from 35 cm of acetone at –
1
8 °C. Crystals were filtered through the filter glass with
3
pore size 16–40 lm, washed with 5 cm of cold acetone
and subsequently dried. Acetone solution was concentrated
Purification of 3b,28-diacetoxylupan-29-al
3
to a volume of 20 cm and cooled to - 18 °C. The crystals
were filtered through the filter glass with pore size
The adsorbed raw reaction mixture was chromatographed
3
on silica gel (20 g). The column was eluted with 100 cm
3
1
6–40 lm, washed with 5 cm of cold acetone and dried.
of petrol ether and mixtures of ethyl acetate and petrol
3
ether (100 cm of 2%, 100 cm of 4%, 100 cm of 6%,
3
3
The obtained crystals were added to the first portion of
crystals. The fraction L2 was obtained. Pure lupeol was
obtained by the recrystallization of fraction L2 from ace-
tone with the same procedure as was used for the fraction
L2. The lupeol was dried at room temperature for 1.5 h and
at 145 °C for 15 min. The final amount of lupeol was
3
3
3
100 cm of 7%, 100 cm of 8%, 200 cm of 10%, and
3
100 cm of 12.5% of solution of ethyl acetate in petrol
ether). The first collected fraction provided 80 mg of
unreacted 3b,28-di-O-acetylbetulin. The second collected
fraction afforded 143 mg (26.3%) of 3b,28-diacetoxy-
4
80 mg (0.96%).
(20R)-lupan-29-al (R = 0.14 in 5% solution of ethylac-
f
etate in petrol ether). The third collected fraction consisted
from the mixture of 3b,28-diacetoxy-(20R)-lupan-29-al
and 3b,28-diacetoxy-(20S)-lupan-29-al in the ratio
21.5%:79.5%. The ratio of epimers was determined
according to the integrals of hydrogens of aldehydic groups
(R-epimer: d = 9.85 (d, J = 1.9 Hz, 1H) ppm; S-epimer:
Determination of purity of betulin and lupeol
1
The purity of betulin and lupeol was determined using H
NMR spectroscopy. Dimethyl sulfone (Standard for quan-
Ò
titative NMR, TraceCERT ) was used as the internal
1
standard. The purity was estimated to 90.8 ± 0.8% for
betulin fraction B2, 98.2 ± 0.4% for betulin fraction B3,
and 96.3 ± 0.8% for lupeol, respectively. The large dif-
ferences in molecular weight of the standard and triterpe-
nes and low solubility of betulin caused that a relatively
high level of uncertainty had to be considered according
the recommendation of producer.
d = 9.61 (s, 1H) ppm) in H NMR spectra. The amount of
third fraction was 66 mg (12.2%). The fourth collected
fraction provided 83 mg (15.3%) of 3b,28-diacetoxy-
(20S)-lupan-29-al (R = 0.09 in 5% solution of ethylac-
f
etate in petrol ether). Analytical data of 3b,28-diacetoxy-
lupan-29-als were in accordance with the literature [29].
Purification of 3b-acetoxylupan-29-al
3
b,28-Di-O-acetylbetulin and 3b-O-acetyllupeol
Acety-
lation of betulin and lupeol was performed according to the
literature [2]. Analytical data of acetylated betulin were in
accordance with the literature [2]. Analytical data of
acetylated lupeol were in accordance with the literature
The adsorbed raw reaction mixture was chromatographed
3
on silica gel (30 g). The column was eluted with 100 cm
of petrol ether and mixtures of ethyl acetate and petrol
3
ether (100 cm of 1%, 100 cm of 2%, 100 cm of 3%,
3
3
[
37].
3
3
1
00 cm of 4%, 400 cm of 5% of solution of ethyl acetate
in petrol ether). The first collected fraction provided 94 mg
19.6%) of 3b-acetoxy-(20R)-lupan-29-al (R = 0.33 in 5%
Oxidation of acetylated betulin and lupeol
with chromyl chloride
(
f
solution of ethylacetate in petrol ether). The second col-
lected fraction consisted from the mixture of 3b-acetoxy-
3
3
b,28-Di-O-acetylbetulin (1 mmol, 527 mg) or 469 mg
b-O-acetyl-lupeol (1 mmol) was dissolved in 20 cm of
(
20R)-lupan-29-al
and
3b-acetoxy-(20S)-lupan-29-al
Rf = 0.30 in 5% solution of ethylacetate in petrol ether) in
3
(
anhydrous dichloromethane. After cooling to 2 °C the
the ratio 43.8% : 56.2%. The ratio of epimers was deter-
mined according to the integrals of hydrogens of aldehydic
groups (R-epimer: d = 9.87 (d, J = 1.9 Hz, 1H) ppm; S-
3
solution of 80 mm freshly distilled CrO Cl (1 mmol) in
2
2
3
0 cm of anhydrous dichloromethane was added dropwise
1
over a period of 10 min. The reaction mixture was stirred
at 2 °C for 15 min after adding of the whole amount of
chromyl chloride solution. Subsequently, 0.7 g zinc dust
1
epimer: d = 9.63 (s, 1H) ppm) in H NMR spectra. The
amount of second fraction was 218 mg (44.9%). Analytical
123

M. Luká cˇ et al.
data of 3b,-acetoxylupan-29-als were in accordance with
the literature [35].
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1
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ˇ
6. Siman P, Filipov a´ A, Tich a´ A, Niang M, Bezrouk A, Havelek R
Acknowledgements We are grateful to L’ ubica Luk a´ cˇ ov a´ , M.Sc., and
Anna Chabadov a´ for the help in collection of birch bark. This work
was supported by the Slovak Research and Development Agency
Grant No. APVV-0516-12 and by the Grant Agency of Ministry of
Education and Academy of Science of Slovak republic (VEGA),
Project No. 1/0298/2016.
(
2016) PLoS One 11:e0154933
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