Journal.pone.0154933 [631793]

RESEARCH ARTICLE
Effective Method of Purification of Betulin
from Birch Bark: The Importance of Its Purityfor Scientific and Medicinal Use
Pavel Šiman1,A lžběta Filipová1, Alena Tichá2, Mohamed Niang1, AlešBezrouk3,
Radim Havelek1*
1Charles University in Prague, Faculty of Medicine in Hradec Králové, Department of Medical Biochemistry,
CZ-50003, Hradec Králové, Czech Republic, 2University hospital Hradec Králové, Department of Research
and Development, CZ-50005, Hradec Králové, Czech Republic, 3Charles University in Prague, Faculty of
Medicine in Hradec Králové, Department of Medical Biophysics, CZ-50038, Hradec Králové, Czech Republic
*[anonimizat]
Abstract
A new and relatively simple method for purification of betulin from birch bark extract was
developed in this study. Its five purification st eps are based on the differential solubility of
extract components in various solvents and their crystallization and/or precipitation, ontheir affinity for Ca(OH)
2in ethanol, and on the affinity of some impurities for silica gel in
chloroform. In addition, all used solvents can be simply recycled. Betulin of more than99% purity can be prepared by this method with minimal costs. Various observationsincluding crystallization of betulin, changes in crystals during heating, and attempt of local-ization of betulin in outer birch bark are also described in this work. The original extract,
fraction without betulinic acid and lupeol, amor phous fraction of pure betulin, final crystal-
line fraction of pure betulin and commercial betulin as a standard were employed to deter-mine the antiproliferative/cytotoxic effect. W e used WST-1 tetrazolium-based assays with
triple negative breast cancer cell line BT-549. T he decrease in cell survival showed clear
relationship with the purity of the samples, being most pronounced using our final product
of pure crystalline betulin. WST-1 proliferation/cytotoxicity test using triple negative breastcancer cell line BT-549 clearly showed the importance of purity of betulin for biologicalexperiments and, apparently, for its medicinal use.
Introduction
B e t u l i ni sap e n t a c y c l i ct r i t e r p e n eo fl u p a n et y p e :l u p – 2 0 ( 2 9 ) – e n – 3 β,28-diol (CAS no. 473-
98-3) –seeFig 1 . It occurs in a number of plants, especially in many species of birch, where it
c a nb ef o u n di nl a r g ea m o u n ti nt h eo u t e rb a r k .T h eq u a n t i t yo fb e t u l i nc a nb eu pt o2 0 –30%
(or even nearly 45% [ 1]) of the dry outer bark weight depending on the tree species and its
regional location [ 2–4]. A lesser amount of betulin can also be found in the root skin and
leaves of birches [ 5].
PLOS ONE | DOI:10.1371/journal.pone.0154933 May 6, 2016 1/1 4a11111
OPEN ACCESS
Citation: Šiman P, Filipová A, Tichá A, Niang M,
Bezrouk A, Havelek R (2016) Effective Method ofPurification of Betulin from Birch Bark: TheImportance of Its Purity for Scientific and MedicinalUse. PLoS ONE 11(5): e0154933. doi:10.1371/journal.pone.0154933
Editor: Horacio Bach, University of British Columbia,
CANADA
Received: November 26, 2015
Accepted: April 21, 2016
Published: May 6, 2016
Copyright: © 2016 Šiman et al. This is an open
access article distributed under the terms of theCreative Commons Attribution License , which permits
unrestricted use, distribution, and reproduction in anymedium, provided the original author and source arecredited.
Data Availability Statement: All relevant data are
within the paper.
Funding: The authors are grateful for the financial
support offered through the PRVOUK P37/01programme initiated by Charles University in Pragueand MH CZ –DRO (UHHK 00179906).
Competing Interests: The authors have declared
that no competing interests exist.

As a member of the plant pentacyclic triterpene family, betulin has antifungal and antimi-
crobial effects [ 6–8]. Thus as with lupeol and betulinic acid, it may protect the tree against fun-
gal and bacterial attack through the bark.
Other triterpenes can be found in birch bark. Lupeol and betulinic acid appear as the most
abundant of the other triterpenes. In much lesser amount there may be betulon, erythrodioland oleanolic acid [ 9–11]. All the terpenes mentioned above can be easily extracted in the form
of a triterpene-rich extract that can be used for biological studies in the same way as the pure
substances [ 10]. The content of betulin and the ratio of triterpenes depend on the botanical
species and the locality of the tree [ 3,4]. The common European species Betula pendula Roth.,
Betulaceae (syn. B.alba) can contain 10 –20% of lupeol and betulinic acid in relation to content
of betulin in the outer bark, and both these triterpenes are the main impurities of crude betulin[12]. Lupeol and betulinic acid show similar efficiency in most of their biological activities [ 13–
16]. Moreover, betulin and betulinic acid are valuable templates for many semisynthetic deriva-
tives that can be even more effective drugs [ 17–22].
It is well known that betulin and other triterpenes exhibit a wide range of important biologi-
cal effects on animal and human health [ 5]. Along with the antimycotic and antimicrobial
activity mentioned above, anti-inflammatory [ 23,24], antiviral (including anti-HIV) [ 16,20,
25], hepatoprotective [ 26,27], gastroprotective [ 28,29], anti-proliferative and anti-cancer
[17,23,29–31] properties have previously been demonstrated. Betulin also moderates the bio-
synthesis of cholesterol and fatty acids, and so ameliorates diet-induced obesity and reduces
the size and improves the stability of atherosclerotic plaques (evidenced by reduced accumula-tion of macrophages) [ 32]. It can be also used in the treatment of type II diabetes via promotion
of insulin sensitivity of cells [ 32].
Triterpenes may be applied and developed as novel drugs with broad clinical applications
[17,20]. Besides this, cosmetic applications have also been reported, and betulin and birch bark
extracts are used as additives in cosmetology and food products [ 21]. Thus, betulin of high
purity can be found widely-used in the pharmaceutical and cosmetic industries. Betulin and itssemisynthetic derivatives have very high potential for application, mainly in medicine [ 16,30].
Antiproliferative and/or cytotoxic effect of betulin has been described for many cell lines of
human cancers and also on some animal (mouse) cell lines. For most cancer cell lines betulin
exhibited cytotoxic potential with IC
50(inhibitory effect 50%) values in range 5 –10μg/ml.
Only some types of cancer cells resist this treatment and had IC 50values about 100 μg/ml and
more. It can be summarized that many of malignant cell cultures and cancers are very sensitive
to betulin [ 23,29,31,33].
Moreover betulin is nontoxic compound for whole living organism. The minimal lethal
(LD16) and median lethal (LD50) doses in mice are 6500 mg/kg and 9000 mg/kg, respectively
[28]. Therefore, according to the international classification, it can be assigned to the 4th class
of low-toxic substances [ 28]. Its low toxicity was documented for experimental animals as
mice, rats or dogs. Changes in the ratio of leucocytes and platelets during subchronic toxicity
Fig 1. Structure of betulin, lupeol and betulinic acid.
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studies was observed after i.p. and s.c. application of more than 100 mg/kg of triterpenoid
extract of birch bark (with more than 80% content of betulin) [ 10]. In pharmacological safety
studies this extract showed no histopathological or other deleterious effect at doses up to 540mg/kg (i.p.) and 300 mg/kg (s.c.), respectively [ 10].
Birch bark is the best raw material for betulin isolation or production, not only for its con-
tent of triterpenes but also for the large amount of bark being produced as waste by the timber
industry, especially the paper industry [ 34]. At present, birch bark is mainly burnt for com-
bined heat and power production instead of its more valuable use as a source of triterpenes,
antioxidants and suberin [ 16,20,35].
Various methods of isolation and purification of betulin have been described, mostly from
birch bark. Simple extraction by organic solvents is the easiest way to obtain biologically active
material. As solvent we can use methanol, ethanol, propan-2-ol, n-heptane or n-hexane, ethyl
acetate and its mixtures with ethanol and water, dichloromethane, a mixture of chloroform/dichloromethane/methanol, a mixture of ethanol and aqueous alkali, butan-1-ol, toluene, petro-
leum ether, limonene and others [ 10,12,16,22,23,28,33,36–38]. Ionic liquids based on imidazole
are also effective [ 39]. The extraction is often forced and accelerated by milling or crushing of
the bark. Other techniques include ultrasonic disruption and activation of the bark by steam,superheated steam or microwaves [ 1,11,40,41]. Supercritical extraction with carbon dioxide,
with a mixture of methanol, ethanol or acetone, or extraction after esterification of betulin to its
diacetate or dipropionate are more difficult methods than simple extraction [ 1,34,41,42]. A sub-
limation method has also been described [ 43], in which atmospheric pressure or high vacuum
and high temperatures were used. Recrystallization and/or various column chromatography
methods are the major methods for purification of betulin from extracts [ 22,29,36,37,44,45].
Reverse-phase high performance liquid chromatography (RP-HPLC;
[2,3,23,29,38,42,46,47]) and gas chromatography with mass spectrometry detection (GC-MS;
[
12,48]) are widely used methods for analysis of betulin and other triterpenes in samples. How-
ever, thin layer chromatography (TLC, especially in high performance modification —HPTLC)
is also well applicable for assessment of purity and visualisation of impurities [ 9,44,45].
The importance of betulin and other triterpenes in present medicine is obvious and may get
an increase. But most purification methods developed so far cannot reach criteria for purity ofthe drug along with ecological and economical demands. Also the importance of purity of used
triterpenes for research tests and for medical applications has not yet been sufficiently sup-
ported. This work may give some contribution for solving these problems.
Materials and Methods
Ethics Statement
Outer white bark for extraction of betulin was obtained by careful peeling from a relatively
freshly felled tree Betula pendula in the locality of Hradec Kralove, Czech Republic. The study
was carried out on private land and owner of the land gave permission to conduct the study(collection of outer white bark of freshly felled tree Betula pendula ) on this site. The Betula pen-
dula tree is not listed as endangered, protected or rare species.
Extraction and purification
Notes:
1. A small part of each particular fraction was used for sample analysis and other experimental
purposes. Therefore, the amount of fraction used in the next purification step is smaller
than the amount of respective fraction obtained in the previous purification step.
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2. All fractions mentioned below are marked by letter (B —betulin fraction, L —lupeol fraction,
A—betulinic acid fraction) and number (number of purification step; 0 —for basic extrac-
tion from bark).
Extraction. Outer white bark for extraction of betulin was obtained by careful peeling
from a relatively freshly felled tree Betula pendula in the locality of Hradec Kralove, Czech
Republic (coordinates: 50.2429658N, 15.8921664E; spring 2014; botanist: Prof. RNDr. LubomirOpletal, CSc., Department of Pharmaceutical Botany and Ecology, Faculty of Pharmacy in Hra-
dec Kralove, Charles University in Prague, Czech Republic) on this site. The Betula pendula
tree is not listed as endangered, protected or rare species. The bark was air-dried indoors in lab-oratory, shielded from the sun, at room temperature for approximately one month up to con-stant weight. About 50 g of this bark was cut into small pieces and crushed in a kitchen
blender. 300 mL of technical grade ethanol was used for Soxhlet extraction. The bark was
extracted only by seven cycles of extraction. The resulting extract was then concentrated by dis-tillation until the first precipitate appeared. This concentrate was cooled to laboratory tempera-
ture, and the thick precipitate was filtered on filter glass and quickly washed with cold pure
ethanol. Brownish fraction B0was obtained after drying the precipitate in air.
The first step of purification —removal of betulinic acid. About 0.5 g of freshly precipi-
tated Ca(OH)
2obtained from CaCl 2and NaOH was first washed with absolute ethanol. This
wet hydroxide was then immediately added to a solution of 5 g of fraction B0dissolved in 100
mL of hot absolute ethanol. The original brownish colour darkened to brownish-red. This mix-ture was boiled on a water bath with shaking for about 15 minutes, and the hot precipitate was
quickly filtered on filter glass. All the ethanol from the clear brownish-red filtrate was carefully
evaporated and the resulting brownish powder of fraction B1was dried in air.
The dark brown filter residue of Ca(OH)
2with many impurities was washed with cold etha-
nol and dried. This powder was then treated with 20% HCl to remove hydroxide and the resi-
due—fraction A1—was washed with distilled water and dried in air.
The second step of purification —removal of lupeol. 50 mL of pure benzene was added
to 4.7 g of fraction B1, stirred and then refluxed for 20 minutes. After cooling to 4°C the result-
ing gel-like precipitate was centrifuged (30 min., 2500 r.p.m., 4°C) and the supernatant sepa-rated and retained. This procedure was repeated twice, and following the third purification by
benzene the precipitate was able to be filtrated on a glass filter, and centrifugation was not nec-
essary. After washing of the final precipitate with cold benzene and drying, pale brownish frac-tion B2was obtained.
The three combined benzene fractions (including the benzene washings) were carefully dis-
tilled off and nearly all benzene was recycled by distillation and used again for the next purifi-
cation process of betulin. After evaporation, 1.12 g of a brownish fraction L2was obtained.
The third step of purification —removal of more polar impurities. In the third step, 3 g
of fraction B2was dissolved in 120 mL of boiling 96% ethanol and quickly filtered on paper filter
(filter for quantitative analysis KA-1-M, Paper mills Pern štejn, Czech Republic). 20 mL of distilled
water was then added to the hot filtrate, and the resulting fine precipitate dissolved by boiling to
give a clear pale beige solution. This was left to stand overnight at 7°C. The thick precipitate
which had formed was centrifuged (30 min., 2500 r.p.m., 4°C) and then dissolved in 70 mL ofboiling 96% ethanol. After 5 hours at 7°C the resulting fine crystalline precipitate was filtered on a
glass filter, washed with cold ethanol and dried in air. Pale beige fraction B3was obtained.
The liquids from the centrifugation and crystallisation procedures were collected and evapo-
rated, and the solid residue was kept for the next isolation as a valuable additive to “future ”
fraction B1.
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The fourth step of purification —removal of all residual impurities. 1 g of fraction B3
was dissolved in 50 mL of chloroform at room temperature and this clear, slightly yellowish
solution was filtered on a column of silica gel (silica gel for column chromatography Silpearl,
Glassworks Kavalier, Czech Republic) in chloroform. The height of the column filling was onlyabout 1.5 cm and the diameter of the column was 1 cm. All colored impurities and maybe allresidual impurities were visually captured in the top layer of approximately 2 –3 mm of the sil-
ica gel. The column was then washed with 20 ml of pure chloroform. All eluent was carefully
distilled off and bright white amorphous fraction B4was obtained.
All the used chloroform —up to 70 mL (except for a very small amount in the silica gel) —
was recycled by distillation for use in the next purification process of betulin.
The fifth step of purification —crystallization. In the last step, 0.85 g of fraction B4was
dissolved in 20 mL of boiling absolute ethanol and the clear colourless solution was allowed to
stand overnight at 7°C. Betulin crystallized out as flat transparent colourless small crystals,
about 0.2 mm length on average. The crystalline precipitate was washed with a small amountof ice-cold absolute ethanol and dried in air. Fraction B5, betulin of high purity, was obtained.
Extraction and purification of betulin described above is shown in the Fig 2 .
Analysis of samples
Gas chromatography with mass spectroscopy detection was chosen for quantification of the
isolated triterpenes. Samples were derivatized with a mixture of pyridine (Sigma Aldrich, USA)
with N,O-bis(trimethylsilyl)trifluoroacetamide (Supelco, USA) (1:1 v/v) at 45 min., 75°C. Thetrimethylsilyl derivatives of the triterpenes were determined by gas chromatography-massspectrometry (Agilent Technologies —GC 7890A, MS 7890A, USA) and capillary column J and
W DB-5 MS 60m x 250 um x 0.25 um (Agilent Technologies, USA). Injector temperature was
280°C and the oven was programmed as follows: initial temperature 70°C, hold time 1 min.,rate 15°C/min., to 300°C. The pressure at the column head was 50 kPa. The mass spectrometer
was used in electron impact mode (electron energy 70 eV, temperature of source 230°C and
quadrupole 150°C). Sample concentrations were quantified using external standard method
Fig 2. Extraction and purification process.
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and this method is resistant to derivatization differences. The ratio of betulin, betulinic acid
and lupeol were quantified by calculation of calibration curve.
Betulin of purity >98%, lupeol of purity >94% and betulinic acid of purity >98% from
Sigma-Aldrich were used as standards for qualitative determination of the peaks. The standardsubstances were used for evaluation of retention time of extracts and calculation of amounts of
each presented substances. Mass spectra were the same in standards and samples.
Cell cultures
The WST-1 experiments were carried out with the triple negative breast cancer cell line BT-549 (ATCC, USA). Triple negative breast cancer is a carcinoma negative for estrogen and pro-
gesterone receptors and without overexpression of HER/2 protein. We chose this cell linebecause it would be original research, and also for its high invasiveness as a primary tumor —
invasive ductal carcinoma. In the studied publications, betulin has not yet been used for in
vitro treatment of this cell culture. BT-549 cells were propagated in Dulbecco's Modified Eagle's
medium DMEM (Sigma-Aldrich, USA) supplemented with 10% FBS (Life Technologies,
USA), 2% glutamine (Life Technologies, UK), 1% penicillin/streptomycin (Life Technologies,
UK) and 1% insulin (Sigma-Aldrich, USA). The cell cultures were maintained at 37°C in ahumidified incubator in an atmosphere of 5% CO
2−95% air. BT-549 cells in the maximum
range of 20 passages and in an exponential growth phase were used for this study.
WST-1 proliferation test
Cell proliferation was determined by the WST-1 quantitative colorimetric assay. The solutionof betulin was prepared by dissolving 1 mg in 0.5 mL of hot ethanol and 0.5 mL of DMSO. This
basic stable solution was then diluted by the complete cultivation medium for BT-549 cells to aconcentration of 20 μg/mL (50x), and this final solution was added by programmable micro-
plate dispenser MultiFlo (BioTek Instruments, USA) to minimally the same volume of 24
hours pre-seeded cells in the complete cultivation medium. The 10.000 cells per mL, 400 cells
per well in a 384-well plates (Greiner Bio-One, Austria) were treated with betulin at variousconcentrations from 1 to 10 μg/mL (2.26 –22.6μmol/L) for 24 hours in a humidified atmo-
sphere with 5% CO
2and 37°C. After the incubation period, WST-1 was added and the cells
were incubated for 3 h. Absorbance of the samples at 440 nm against a background control(medium alone) as a blank was measured using a microplate reader Tecan Infinite M200
(Tecan, Switzerland). The confluence of the cells examined in previous pilot experiments
reached under 80% in negative controls over the whole assay time course. Results from cellscultured in medium without betulin were used as a negative control. The results from experi-
ments using doxorubicin treatment for 24 hours at 1 and 2 μM were considered as a positive
control. The maximal concentration of ethanol and DMSO in the cell cultures was 0.5%, whichis the “safe”non-toxic concentration of both solvents for these cells. This assertion was con-
firmed experimentally before performing WST-1 tests.
Statistical analysis
Measurement data were processed and statistically evaluated with the help of MS Excel 2007(Microsoft Corp, Redmond WA, USA), NCSS 2007 (Hintze, J. (2007). NCSS 2007. NCSS LLC,
Kaysville, Utah, USA. www.ncss.com ), and GraphPad Prism 5 biostatistics (GraphPad Soft-
ware, USA). We used Kolmogorov-Smirnov test to test normality of the data distribution. Wecompared the tested samples with control samples with the help of the two-sample t-test (and
Levene ’s test to check the homoscedasticity assumption). To adjust for multiple comparisons
and keep the family-wise αat 0.05 we used the Bonferroni correction. The resulting αfor a
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single comparison was 0.001. The IC 50values of the viability datasets (obtained by use of WST-
1 assay) were determined using a non-linear regression.
Results and Discussion
Extraction and purification
A relatively simple environmentally friendly chemical method was developed for isolation of
betulin from birch bark and its subsequent purification to achieve high-purity (higher than99%) betulin. Ethanol, water and CaCl
2are harmless compounds. Two solvents —benzene and
chloroform —are ecologically undesirable, but both are nearly 100% recycled via distillation
and can be repeatedly used for further purifications of betulin. NaOH and HCl are used in rela-
tively small amounts and practically in stoichiometric ratio, and therefore are not dangerousfor the environment. Solid waste from the bark after extraction is quite environmentally harm-
less and can be used also as a relatively pure source of suberin. Two of the by-products are a
potentially valuable source of betulinic acid (fraction A1) and lupeol (fraction L2). Taken
together, this is why this method can be declared as “green ”.
The results of extraction and of purification steps are summarized in Table 1 . Chromato-
grams of four most interesting fractions are shown in Fig 3 .
The total efficiency of the purification process in our case (result: small crystals of fraction
B5) was about 20% of the initial weight of dry extract B0. This “total”efficiency was calculated
as a multiple of the efficiencies of the particular purification steps. The real efficiency withrespect to the content of betulin in fraction B0is somewhat higher. However the betulin from
all“wastes ”and by-products can be reused in the next purification process as an additive to the
initial extract or to solutions of later phases of purification. Theoretically, in a continuous and
“endless ”(semi)industrial process, the efficiency could reach substantially over 90% of betulin
content in the dry extract if no losses on filtration material or on the surfaces of the vessels are
considered. Unfortunately, a careful comparison of our method with the methods published in
papers referenced earlier [ 1,10,11,12,16,22,23,28,33,36–45], even considering patents, is not
possible as there are insufficient data on efficiency of the extraction and purification process
and/or the purity of the product.
The method described in this paper was performed on a laboratory scale only, with gram
quantities. However there is no reason why this method is not applicable on a pilot-plant or
commercial scale. It is only a matter of the technological equipment and amount of material
Table 1. Amount of betulin, betulinic acid, lupeol and other triterpenes in fractions.
Composition of fractionsaPurificationb
Fraction betulin % BA % lupeol % others % Yield %
B0 83.0 1.4 15.2 0.4 (26% of bark)
B1 83.5 0.1 15.9 0.5 94
A1 45.7 53.3 0.2 0.8 (about 0.7 g)
B2 98.2 0.1 1.1 0.6 70
L2 55.4 0.2 44.1 0.3 (1.12 g)
B3 98.7 0.1 1.0 0.2 43
B4 99.2 0.0 0.7 0.1 91
B5 99.8 0.0 0.2 0.0 71
aDetermined by areas of GC-MS chromatogram peaks. Results are shown as % of abundance ratio obtained from calibration curves (BA —betulinic acid).
bYield of extraction and puri fication steps in % of the initial amount of the previous fraction.
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used. Collectively, all materials are cheap and easily available, including the initial raw material:
birch bark, a waste product of the paper and wood industries.
Crystals
Fraction B5crystallized out the ethanol solution as flat transparent colourless small crystals,
about 0.2 mm length on average (see Fig 4A ). If the hot saturated ethanolic solution was
allowed to cool slowly to room temperature, much bigger crystals formed, up to 12 mm long
Fig 3. GC-MS chromatograms of four important fractions. B0 —original extract, A1—by-product enriched
with betulinic acid, L2—by-product enriched with lupeol, B5—pure betulin; BA —betulinic acid; relative
detector response (Y-axis) against retention time in minutes (X-axis).
doi:10.1371/journal.pone.0154933.g003
Fig 4. Images of crystals of betulin, fraction B5. A—microphotograph of crystalline fraction; B —large
crystals obtained by slow cooling; C and D —macro- and microphotographs of crystals after free evaporation
of ethanol.
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(Fig 4B ). The filtrate was also allowed to stand for spontaneous evaporation in air. Acicular
crystals (needle-like, slender crystals) were formed (see Fig 4C —macroscopic photo and 3D —
microscopic photo).
Betulin crystallizes from ethanol solution in orthorhombic symmetry with one molecule of
ethanol per one molecule of betulin bonded by hydrogen bond [ 28,49]. Such crystals are quite
transparent. About 100 mg of crystals of fraction B5were heated to 135°C in a drier for 30
minutes. The resulting brightly white and opaque material was then weighed and from the dif-
ference it was estimated that the molecular weight of evaporated material was about 55 Da. Ifwe assume some loss of weight from sublimation of betulin, the result corresponds well with
the literature data mentioned above (ethanol: 46 Da).
One of the classic physicochemical characteristics of pure substances is their melting point.
Unfortunately, we can find a wide range of melting points assigned to betulin in the literature:
251–261°C [ 28]. Most authors report a value around 255°C. Pure betulin from Sigma-Aldrich
(declared >98% by HPLC) was taken as a standard for comparison. The melting point of the
standard was determined at 254 –255°C, and for fraction B5we measured 255 –256°C.
During heating of a pure B5fraction sample under a cover glass changes in the crystals were
observed:
130–140°C —darkening of the crystals (in transmitted light in microscope, macroscopically
whitening) as crystal-bound ethanol is evaporated, and the crystals visibly fragment (see
chapter about crystals above);
160–170°C —betulin begins visibly to sublime;
180–200°C —new transparent microcrystals arise on the surface of the original crystals (see Fig 5 );
255–256°C —general melting.
After cooling down no crystals were formed and the melt solidified as an amorphous glass.
Amorphous microscopic beads were also formed as a bright white powder during sublima-
tion under atmospheric pressure in a sublimation apparatus.
Birch bark
Birch bark consists of brown inner bark ~75% and white outer bark ~25%. The outer bark con-tains fats, fatty acids, resins, suberin and in particular betulin —up to 30% [ 20].
The outer bark consists of numerous tightly packed layers of periderm cells on the surface
of the stem [ 50] (see also Fig 6A ).
Fig 5. Crystals of fraction B5 during heating.
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A simple approach for microscopic localization of triterpenes in the outer bark was per-
formed. A lateral cut of the bark was made. Nile red dissolved in dimethyl phthalate was added
to this cut and almost immediately was removed again by suction. Fluorescence microscopy
was used to obtain a microscopy image ( Fig 6B ). The fluorescent dye nile red is very lipophilic,
and is consequently used for labelling of lipophilic structures, e.g. adipose tissue [ 51,52]. Thus
the brighter regions on the photograph may indicate the locality of hydrophobic triterpenes,
especially betulin.
The structure of the outer bark revealed after labelling by nile red may be an interesting
result. If mainly triterpenes were labelled by this lipophilic fluorescence dye, we can conclude
that betulin is localized in small longitudinal clads through the outer bark. Bearing in mind the
existence of triterpene clads and the non-homogeneity of localization of the betulin particles,proper consideration must be given to the mechanical processing of the birch bark before the
extraction process. This observation is supported by the fact that when cut into small pieces
about 5×5 mm, the bark yielded on average only about 19% extractives, compared with about26% under the same conditions of extraction after the bark had been crushed in a kitchenblender as described above. Even more so, extraction from paper-like leavings on surface of the
bark of thickness about 4 μm gave more than 45% of extractives.
WST-1 test
WST-1 antiproliferation/cytotoxic test in the broad concentration range 1 –10μg/mL was cho-
sen as a control test of biological efficiency of the product. The results of some representativemeasurements are shown in Fig 7 . The points representing concentrations 1 and 2 μg/mL were
excluded from data evaluation process using GraphPad statistic software. Betulin in a case of
low (subcytotoxic) concentrations, similarly as many other compounds, causes increase inmitochondrial dehydrogenase activities above that of nontreated controls rather than decrease.
In our experiment these low concentrations roughly deteriorated accuracy of calculated curves.
The WST-1 test using the human breast cancer BT-549 cell line clearly demonstrated the
antiproliferative and/or cytotoxic effect of betulin against this cancer cell line in vitro . The
results were well reproducible. Four independent experiments showed an IC
50value between
4.3–4.6μg/mL for pure betulin B5and 4.6 –4.9μg/mL for standard betulin B-Sfrom Sigma-
Aldrich. The amorphous fraction B4had practically the same IC 50value as crystalline fraction
B5(4.0 and 4.3 μg/mL; two simultaneous measurements). The decrease of BT-549 cell viability
compared to the negative controls was statistically significant (P <0.001) at 3 μg/mL for B4
(P<1/C110−6),B5(P<1/C110−6) and for B-S(P<0.000564), at 5 μg/mL for B3(P<5/C110−6), and
at 6μg/mL for B0(P<1/C110−6). Comparison of effects between the various intermediate prod-
ucts of the purification process showed a clear relationship with the purity of the samples. In
accordance with expectation, the original extract B0demonstrated the smallest
Fig 6. Structure of outer bark. A—bright-field microscopy, B —fluorescence microscopy of outer bark
stained by nile red.
doi:10.1371/journal.pone.0154933.g006
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PLOS ONE | DOI:10.1371/journal.pone.0154933 May 6, 2016 10 / 14

antiproliferative/cytotoxic effect with IC 50value nearly doubles that of B5(about 9 μg/mL,
three measurements). Fraction B3—almost pure betulin without betulinic acid and lupeol —
had an IC 50value also substantially higher (nearly 5.7 and 6.1 μg/mL, two measurements).
These results do not exactly correspond to the content of betulin in the fractions and the influ-ence of some inhibitor(s) to betulin efficiency may have to be considered. Such inhibitor(s)possibly remains in the fractions until purification is performed by filtration through silica gel
(step 4), and the small rests of colored impurities in the fraction are removed. For example,
some antioxidants (phenols, sesquiterpenes and the like) occurred in birch bark [ 42,48] and in
fractions up to fraction B3may prevent the cytotoxic effect of betulin. This is one of questions
for separate study to explore mode of betulin activity against the cancer cells.
On the basis of these observations we can conclude that the purity of betulin is extremely
important for any scientific experiments and measurements in the field of the biological effects
of this triterpene. Similarly, the purity may be very important for the medical use of betulin
and other triterpenes.
Conclusions
A relatively simple and cheap method for isolation of betulin from birch bark and its purifica-tion to very high purity >99% was developed. For isolation Soxhlet extraction to ethanol was
chosen due to its high efficiency and technical simplicity. The purification procedure consisted
of five steps:
1st step —removal of acids and other impurities with Ca(OH)
2;
2nd step —removal of lupeol by benzene extraction;
3rd step —recrystallization from ethanol solution;
4th step —removal of residual (partly colored) impurities on silica gel in chloroform;
5th step —recrystallization from ethanol solution.
The purification method is environmentally friendly despite the use of benzene and chloro-
form. Moreover both biologically and environmentally hazardous solvents are almost 100%
Fig 7. Antiproliferative/cytotoxic effect. B0 —original extract, B3—fraction 3 without betulinic acid and
lupeol, B4—amorphous fraction of pure betulin, B5—crystalline fraction of pure betulin, B-S—pure betulin
obtained from Sigma-Aldrich. The results are given as relative values to the untreated control in percent.
Cells treated with topoisomerase II inhibitor doxorubicin at 1 and 2 μM were used as positive control for
decreased cell survival. Bars indicate SD in three independent experiments. *Significantly different from
control (P /C200.001).
doi:10.1371/journal.pone.0154933.g007
Importance of Betulin Purity for Scientific and Medicinal Use
PLOS ONE | DOI:10.1371/journal.pone.0154933 May 6, 2016 11 / 14

recycled by distillation and can be reused in further purification processes or used for other
purposes. From small (gram-scale for laboratory use) to large industrial-scale amounts of betu-
lin with more than 99% purity can be prepared by this method. On top of this, three valuableby-products are generated —solid bark waste as a potential source of suberin, the fraction after
removal of acids as a rich source of betulinic acid, and the fraction after processing by benzene
as a rich source of lupeol.
WST-1 proliferation/cytotoxicity test using triple negative breast cancer cell line BT-549
clearly showed the importance of purity of betulin for scientific experiments and for medicinal
use. This is an original and high important result.
Various interesting observations regarding pure betulin and birch bark were also described
in this work.
Acknowledgments
The authors are grateful for the financial support offered through the PRVOUK P37/01 pro-
gramme initiated by Charles University in Prague and MH CZ —DRO (UHHK 00179906). The
authors thank Mrs. Simona Vrchotová (Dept. of Histology and Embryology, Faculty of Medi-cine in Hradec Králové) for the preparation of bark cuts for microscopic localization of betulin.
The authors are also grateful to Ian McColl MD, PhD for language assistance with the manu-
script. Authors wish to acknowledge Prof. Lubomir Opletal for botanical identification ofBetula pendula Roth. (Betulaceae).
Author Contributions
Conceived and designed the experiments: P ŠAF RH. Performed the experiments: P ŠAF. Ana-
lyzed the data: P ŠAT AB. Contributed reagents/materials/analysis tools: P ŠAF AT. Wrote the
paper: P ŠMN RH.
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