Cytotoxicity of white birch bud extracts: Perspectives for therapy of tumours

Abstract

Birch buds (Gemmae Betulae) are widely used in Russian and Chinese traditional medicine mainly as a diuretic and diaphoretic agent but also as an antiseptic, anti-inflammatory and analgesic. Despite the long history of therapeutic use of birch buds in folk medicine, the existing information on their chemical composition and pharmacological effects is insufficient. This circumstance warrants further study of the chemistry and pharmacology of birch buds. The present study was designed to investigate (a) the chemical composition of buds from two species of white birch and (b) the in vitro cytotoxic effect of extracts from these sources on selected tumour cells. Extracts from Betula pubescens Ehrh. and Betula pendula Roth. buds were obtained using three different methods: carbon dioxide supercritical fluid extraction (SFE), washing of exudate covering whole buds, and extraction of milled buds with diethyl ether. The chemical composition of extracts was investigated by GC-MS. Cytotoxicity was determined by MTT assay, and cell proliferation was determined by [³H]thymidine uptake in cancer cells and normal skin fibroblasts. The GC-MS investigation identified a total of 150 substances of different classes. The chemical composition of B. pubescens and B. pendula buds differed, with bud extracts from the former containing a relatively high quantity of sesquiterpenoids and flavonoids, while the main components of extracts from the latter were triterpenoids. The results of the biological assay indicated that birch bud extracts demonstrated time- and concentration-dependent and differential cytotoxicity. The highest cytotoxic activity demonstrated bud exudates and SFE extracts obtained from both Betula species. The rich chemical composition of birch buds suggests the possibility of a wider spectrum of biological activity than previously thought. Birch bud extracts could be a promising source of compounds with cytotoxic activity against various cancers.

Full text

RESEARCH ARTICLE
Cytotoxicity of white birch bud extracts:
Perspectives for therapy of tumours
Valery Isidorov
1☯
*, Łukasz Szoka
2☯
, Jolanta Nazaruk
3☯
1Forest Faculty, Bialystok University of Technology, Hajno
´wka, Poland, 2Department of Medicinal
Chemistry, Medical University of Bialystok, Bialystok, Poland, 3Department of Pharmacognosy, Medical
University of Bialystok, Bialystok, Poland
☯These authors contributed equally to this work.
*isidorov@uwb.edu.pl
Abstract
Birch buds (Gemmae Betulae) are widely used in Russian and Chinese traditional medicine
mainly as a diuretic and diaphoretic agent but also as an antiseptic, anti-inflammatory and
analgesic. Despite the long history of therapeutic use of birch buds in folk medicine, the
existing information on their chemical composition and pharmacological effects is insuffi-
cient. This circumstance warrants further study of the chemistry and pharmacology of birch
buds. The present study was designed to investigate (a) the chemical composition of buds
from two species of white birch and (b) the in vitro cytotoxic effect of extracts from these
sources on selected tumour cells. Extracts from Betula pubescens Ehrh. and Betula pendula
Roth. buds were obtained using three different methods: carbon dioxide supercritical fluid
extraction (SFE), washing of exudate covering whole buds, and extraction of milled buds
with diethyl ether. The chemical composition of extracts was investigated by GC-MS. Cyto-
toxicity was determined by MTT assay, and cell proliferation was determined by [
3
H]thymi-
dine uptake in cancer cells and normal skin fibroblasts. The GC-MS investigation identified
a total of 150 substances of different classes. The chemical composition of B.pubescens
and B.pendula buds differed, with bud extracts from the former containing a relatively high
quantity of sesquiterpenoids and flavonoids, while the main components of extracts from the
latter were triterpenoids. The results of the biological assay indicated that birch bud extracts
demonstrated time- and concentration-dependent and differential cytotoxicity. The highest
cytotoxic activity demonstrated bud exudates and SFE extracts obtained from both Betula
species. The rich chemical composition of birch buds suggests the possibility of a wider
spectrum of biological activity than previously thought. Birch bud extracts could be a promis-
ing source of compounds with cytotoxic activity against various cancers.
Introduction
The birch (Betula L.) is one of the main arborous plants in the forests of boreal and temperate
zones as well as the mountain regions of the Northern Hemisphere [1]. It is a medicinal plant
that has been used in traditional medicine since ancient times. Its traditional use is well
PLOS ONE | https://doi.org/10.1371/journal.pone.0201949 August 14, 2018 1 / 10
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OPEN ACCESS
Citation: Isidorov V, Szoka Ł, Nazaruk J (2018)
Cytotoxicity of white birch bud extracts:
Perspectives for therapy of tumours. PLoS ONE 13
(8): e0201949. https://doi.org/10.1371/journal.
pone.0201949
Editor: David A. Lightfoot, College of Agricultural
Sciences, UNITED STATES
Received: March 1, 2018
Accepted: July 25, 2018
Published: August 14, 2018
Copyright: ©2018 Isidorov et al. This is an open
access article distributed under the terms of the
Creative Commons Attribution License, which
permits unrestricted use, distribution, and
reproduction in any medium, provided the original
author and source are credited.
Data Availability Statement: All relevant data are
within the paper and its Supporting Information
files.
Funding: This work was supported by grant of
National Science Centre (Poland) 2016/23/B/NZ7/
03360 to V.I., as well as by Bialystok University of
Technology Program S/ZWL/1/2017 to V.I. The
funders had no role in study design, data collection
and analysis, decision to publish, or preparation of
the manuscript.
Competing interests: The authors have declared
that no competing interests exist.

documented in the ethnobotanical literature [2–11]. Leaves, buds, tar and essential oils are
used to treat a wide spectrum of diseases, including inflammation, infections, urinary tract dis-
orders, skin and hair problems [12–14]. In Polish folk medicine, ethanolic maceration of fresh
buds from B.pendula was used on bleeding wounds [15]. Buds collected in the winter were
taken instead of leaves as a diuretic remedy [5]. In Russian folk medicine, ethanolic macera-
tions were used internally to treat stomach disorders and fever and externally to treat rheuma-
tism [16].
In modern Russia (as well as in the former USSR), an assemblage of birch buds, Gemmae
Betulae, is a standardised medical preparation [17]. The scientifically proven health benefits of
bud extracts are primarily due to their diuretic effect [9,11] and antimicrobial and antioxidant
properties [10,18–20]. Only anecdotal publications have been devoted to the anticancer activ-
ity of birch buds [21–23]. Despite the wide usage of birch buds in folk medicine and growing
interest of conventional medicine in this herbal material, the current information on its chemi-
cal composition is insufficient for medical purposes.
One of the main goals of this investigation was to fill this gap by determining the chemical
composition of birch buds. The Gemmae Betulae preparation is described as a mixture of
Betula pendula Roth. and Betula pubescens Ehrh. (Betulaceae) buds, but the proportions are
not regulated. However, substantial differences in the chemical compositions of the resins cov-
ering the buds of these closely related species were recently demonstrated [24]. Moreover, it is
well known that the chemical composition of any plant-derived preparation (and, as a conse-
quence, its biological activity), in many respects, depends on the procedure used for extraction.
For this reason, we used three different extraction procedures for buds from each of the white
birch species.
The second main goal was to evaluate the anticancer potential of these extracts to examine
the relationship between composition and antitumour activity, and to identify extracts worthy
of further investigation.
Materials and methods
Reagents and chemicals
Dulbecco’s modified Eagle’s medium (DMEM), Minimum Essential Medium (MEM), Roswell
Park Memorial Institute 1640 Medium (RPMI 1640), fetal bovine serum (FBS), phosphate-
buffered saline (PBS), sodium pyruvate, trypsin, penicillin and streptomycin were obtained
from Gibco (Thermo Fisher Scientific, Waltham, MA, USA). [
3
H]thymidine was purchased
from Hartmann Analytic (Braunschweig, Germany). Sodium dodecyl sulfate (SDS) was
obtained from Bio-Rad Laboratories (Hercules, CA, USA). Dimethyl sulfoxide (DMSO), 3-
(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide (MTT), glycine, sodium chlo-
ride, sodium hydroxide and cisplatin were purchased from Sigma-Aldrich (Saint Louis, MO,
USA).
Plant material
Buds of downy birch (Betula pubescens Ehrh.) and silver birch (Betula pendula Roth.) were
gathered in August–September 2015 from trees growing in the Biebrza National Park in north-
eastern Poland (53˚ 32’ N, 22˚ 43’ E). Voucher specimens (no BP-17034 and BO-17035) have
been deposited with the herbarium of the Department of Pharmacognosy, Medical University
of Bialystok (Poland). A previously described method was used to identify the birch species
[25]. Plant material was kept at -18˚C before use.
White birch buds extracts in tumors therapy
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Sample preparation and chemical analysis
Carbon dioxide supercritical fluid extraction (SFE) of buds was performed in October 2015 in
the High Pressure Technique Laboratory of the Supercritical Extraction Department in the
New Chemical Syntheses Institute (Puławy, Poland). Experimental parameters were as follows:
extraction pressure 300 bar, temperature 40˚C; yield of the product was about 7.2%. Extracts
were light-yellow and slightly viscous with the characteristic birch fragrance.
Exudates covering the buds from each of the birch species were extracted by intensive rins-
ing of the bud samples (15–20 g) for 60 s in diethyl ether (50 mL). The extracts were filtered
through the paper filter, and the solvent was evaporated to dryness.
The washed buds were milled and immediately transferred into a retort of 250 mL in vol-
ume and extracted under constant stirring, using three volumes of 50 mL of diethyl ether.
The duration of each extraction cycle at room temperature was 30 min. The combined diethyl
ether extracts were filtered through a paper filter and the solvent was removed on a rotor
evaporator.
About 5 mg of the residue of exudate and extract left on the walls (as well as 5 mg of SFE
products) was put into a vial of 2 mL in volume. After dissolving in 220 μL of pyridine, 80 μL
of BSTFA was added to the vial. The reaction mixture was sealed and heated for 0.5 h at 60
o
C
to obtain trimethylsilyl (TMS) derivatives. The whole procedure was performed in triplicate.
The resulting solutions were separated and analysed by GC–MS on a HP 7890A gas chro-
matograph with the 5975C VL MSD Triple-Axis Detector (Agilent Technologies, USA). The
apparatus was fitted with an HP-5MS capillary column (30 m ×0.25 mm i. d., 0.25 μm film
thickness), with electronic pressure control and split/splitless injector. The latter worked at
250˚C in the split (1:50) mode. The helium flow rate through the column was 1 mL/min in
constant flow mode. Injection of 1 μL of the sample was performed with the aid of a G4513a
autosampler. The injector (250
o
C) worked in split mode (1:50). The initial column tempera-
ture was 50
o
C rising to 310
o
C at 5
o
C/min. The MSD detector acquisition parameters were as
follows: the transfer line temperature was 280
o
C, the MS source temperature 230
o
C and the
MS quad temperature 150
o
C. The electron impact mass spectra were obtained at 70 eV of ion-
ization energy. Detection was performed in the full scan mode from 41 to 650 a.m.u. After
integration, the fraction of separated components in the total ion current (TIC) was calculated.
To identify the components, both mass spectral data and the calculated retention indices
were used. Mass spectrometric identification was carried out with an automatic system of GC–
MS data processing supplied by NIST and home-made mass spectra libraries. The latter con-
tains more than 1800 spectra of TMS derivatives prepared from authentic preparations of fla-
vonoids and other phenolics, as well as terpenoids, aliphatic acids, alcohols and carbohydrates.
The hexane solution of C
10
–C
40
n-alkanes was separated under the above conditions. The
linear temperature programmed retention indices (I
T
) of the registered components were cal-
culated from the results of the separation of this solution and silanized bud extracts and were
compared with the NIST collection [26] as well as with the authors’ previously published data
[24,27–29]. The identification was considered reliable if the results of the computerized search
of the mass spectra library were confirmed by the experimental I
T
values, i.e. if their deviation
from the averaged published values did not exceed ±10 u.i. (for more information see Supple-
mentary Information, S1 Text).
Cell culture
Human breast adenocarcinoma MCF-7 and MDA-MB-231 cells, human colorectal adenocar-
cinoma DLD-1 cell line, human melanoma C32 cells, human gastric adenocarcinoma AGS
cells, human glioblastoma cell lines LN-18 and LN-229 and human skin fibroblasts CCD-25Sk
White birch buds extracts in tumors therapy
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were obtained from ATCC (Manassas, VA, USA). The human endometrial adenocarcinoma
cell line (Ishikawa), human cervix adenocarcinoma HeLa and human hepatocellular carci-
noma HepG2 cells were purchased from Sigma-Aldrich. The cells were cultured in DMEM
(except for the DLD-1 cells for RPMI 1640 and for the HeLa and HepG2 cells for MEM) sup-
plemented with 10% FBS, 100 units/mL penicillin and 100 μg/mL streptomycin in a humidi-
fied 5% CO
2
atmosphere at 37˚C.
Cytotoxicity assay
The viability of cells was determined by MTT assay [30]. Cells were detached with 0.25% tryp-
sin and seeded at 1 ×10
4
cells per well in 96-well plates. After reaching confluence, the tested
extracts and anticancer drugs used as positive controls were added. Extracts were dissolved in
DMSO, diluted with fresh medium and placed into 96-well plates at a volume of 200 μL per
well. The final DMSO concentration did not exceed 0.1%. Control cells were cultured in
medium containing 0.1% DMSO. Cisplatin was dissolved in medium. After 48 h, 100 μL of a
0.4% MTT solution in PBS was added to each well for 4 h. The medium was removed and the
formazan crystals were dissolved in 200 μL of DMSO and 25 μL of Sorensen’s glycine buffer
for 10 minutes on a plate shaker. The optical density was measured in a microplate reader
(Biochrom, Cambourne, United Kingdom) at 570 nm.
[
3
H]thymidine incorporation assay
[
3
H]thymidine incorporation was used as a measurement of cell proliferation. Cells were
seeded at 1×10
4
cells per well in 24-well tissue culture plates with 1 mL of growth medium.
After 24 h, the cells were incubated with various concentrations of extracts or cisplatin and
0.5 μCi of [
3
H]thymidine for 24 h. Then, the medium was removed and cells were washed
three times with ice-cold PBS and lysed in 1 mL of 0.1 M sodium hydroxide containing 1%
SDS. The cell lysates were transferred to scintillation vials and 3 mL of the scintillation fluid
(Perkin Elmer, Waltham, USA) was added. The amount of [
3
H]thymidine incorporated into
the DNA was determined in a scintillation counter (Perkin Elmer).
Statistical analysis
The results are presented as means ±SEM of at least two independent experiments. Differ-
ences between means for extracts-treated groups and vehicle-treated groups were analyzed
using one-way ANOVA followed by Tukey’s test. P <0.05 was considered statistically signifi-
cant. IC
50
values were calculated using nonlinear regression analysis using GraphPad Prism
version 7.04 (GraphPad Software, La Jolla, CA, USA).
Results
Composition of extracts from buds of silver birch and downy birch
In this work, we used different extraction procedures: carbon dioxide supercritical fluid extrac-
tion, washing of bud resin exudate with diethyl ether, and extraction of milled buds by diethyl
ether. It was hypothesised that the chemical composition of these extracts would differ, and
that this difference may be reflected in their biological activity.
In line with expectations, the extraction procedures had a substantial influence on the com-
position of extracts at both the quantitative and qualitative level. In total, the GC analysis
recorded 150 compounds with a relative content not less than 0.01% of the total ion current.
Extracts from downy birch and silver birch buds contained 118 and 78 substances, respec-
tively. Moreover, different extracts differed in the number of constituents: 87, 90, and 102
White birch buds extracts in tumors therapy
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compounds in SFE, exudate and milled downy birch bud extracts, respectively. Silver birch
buds contained 60, 74, and 64 compounds in the same extracts, respectively. Table 1 shows the
group composition of extracts and the relative content of the main representatives of these
groups. The relative composition of individual compounds and their analytical parameters (I
T
values, m/z of target ions, and molecular ions, M
+
) are presented as Supplementary Informa-
tion (S1 Fig,S1 Table).
Table 1. Group composition (% of TIC) of different extracts from white birches buds.
Compounds B.pubescens B.pendula
SFE exudate extract SFE exudate extract
Sesquiterpenoids 56.14 38.79 29.81 4.63 5.06 4.34
including:
β-caryophyllene 0.36 0.11 0.13 0.09 0.17 0.40
birkenal 0.43 1.01 0.42 -
a
- -
6-hydroxy-β-caryophyllene 12.54 5.93 5.80 1.48 2.85 238
6-hydroxy-β-caryophyllene acetate 2.28 1.07 0.79 0.31 trace -
14-hydroxy-β-caryophyllene 3.49 1.45 1.57 045 0.77 062
14-hydroxy-β-isocaryophyllene 2.12 078 0.75 - 0.39 0.71
14-hydroxy-β-caryophyllene acetate 10.72 5.47 3.97 1.60 trace -
Triterpenoids 2.43 1.33 2.02 67.53 78.97 79.55
including:
dammaradien-3-one - - - 5.01 5.00 4.08
dipterocarpol 0.12 - 0.07 7.06 8.90 8.33
lupen-20(29)-en-28-al
b
- - - 16.01 28.35 35.29
betulinic acid - - 0.06 - 0.02 1.74
Flavonoids 23.58 48.05 56.93 1.31 4.99 4.13
including:
apigenin - 0.68 1.08 - - -
sakuranetin 6.14 14.60 12.50 0.13 0.33 0.63
kumatakenin 1.96 4.5 3.25 - - -
3’-methoxyapigenin 0.54 4.69 6.45 - 0.34 0.46
rhamnocitrin 0.36 3.19 4.78 - 0.10 0.12
pectolinaringenin 0.57 4.60 7.94 - 0.45 0.19
cirsimaritin - 1.44 1.75 0.46 0.99 0.86
catechin - - trace 0.29 0.35 0.28
Phenylpropenoids 4.77 4.85 7.48 0.87 0.16 0.83
including:
6-hydroxycaryophyllene p-coumarate 1.70 2.29 3.54 - - -
14-hydroxycaryophyllene p-coumarate 1.01 0.94 2.25 - - -
6-hydroxycaryophyllene ferulate 0.16 0.18 0.16 - - -
14-hydroxycaryophyllene caffeate 0.13 0.20 0.22 - - -
n-docosyl p-coumarate 0.70 0.50 0.47 0.87 0.16 0.83
Aromatics - - 0.57 0.37 0.38 0.24
Aliphatic C
16
-C
28
acids and esters 6.27 0.73 1.13 11.28 8.21 8.60
Aliphatic C
20
-C
28
alcohols 1.15 0.38 0.53 0.88 1.60 0.76
Alkanes 3.95 3.08 1.46 5.80 0.76 2.58
a
—not detected
b
identified tentatively based on the MS fragmentation patterns.
https://doi.org/10.1371/journal.pone.0201949.t001
White birch buds extracts in tumors therapy
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As can be seen from the data in Table 1, sesquiterpenoids were the main group of com-
pounds in downy birch, while in silver birch buds, triterpenoids prevailed. The second main
group found in downy birch extracts was flavonoids (24–57% of TIC), but these were found at
substantially lower levels in silver birch buds (1.3–4.9% of TIC). Flavonoids in birch buds were
presumably methoxylated flavones and 3-hydroxyflavones. Catechin, a flavan-3-ol, was
detected in noticeable quantities in silver birch buds only. Some of these compounds have pre-
viously been identified in silver birch buds [31], but to the best of our knowledge, kumatake-
nin, 3’-methoxyapigenin, and cirsimaritin have not previously been found in birch buds.
Species-specific differences in the composition of phenylpropanoids were also observed.
Esters of sesquiterpene alcohols and hydroxycinnamic acids were detected only in downy
birch buds, although n-docosyl p-coumarate was characteristic of both species.
Cytotoxic and antiproliferative activity of bud extracts
The cytotoxicity of B.pendula and B.pubescens bud extracts on cancer cells and normal fibro-
blasts was evaluated by MTT assay. The commonly used anticancer drug cisplatin was used as
a reference agent. Cells were treated with extracts for 24, 48 and 72 hours. The concentrations
of extracts inducing 50% reduction in cell viability (IC
50
) are shown in Table 2. The IC
50
values
were determined from concentration-response curves presented in Supplementary Informa-
tion (S2 Fig). All examined extracts induced high concentration- and time-dependent
decreases in cell viability. Generally, ether extracts from both birch species exerted lower inhi-
bition of cell viability than SFE or exudates. The most sensitive cell lines compared to fibro-
blasts were LN-18, MDA-MB-231 and HeLa. The cytotoxic effect of cisplatin was also
concentration and time dependent but was stronger than all Betula bud extracts. However, the
IC
50
for the reduction of cell viability was generally lower in fibroblasts than in cancer cells.
To determine whether Betula bud extracts impair cell proliferation after 24 hour exposure,
[
3
H]thymidine incorporation assay was performed. IC
50
values were determined from the con-
centration-response curves presented in Supplementary Information (S3 Fig). As can be seen
from the data in Table 3, antiproliferative activity of ether extracts was less pronounced than in
SFE or exudates. Nevertheless, IC
50
values of both ether extracts and the B.pubescens bud exu-
date for fibroblasts were higher than the values obtained in cancer cells. Of note, these values
were lower than IC
50
for reduction in cell viability, suggesting antiproliferative activity. Further
comparison of IC
50
for [
3
H]thymidine incorporation and IC
50
for reduction of cell viability
revealed that all examined extracts exhibited stronger antiproliferative activity than cytotoxic
activity in HeLa and AGS cells. Moreover, almost all tested extracts more efficiently inhibited
cell proliferation than induced cell death in MCF-7, Ishikawa, DLD-1 cells (except B.pendula
exudate) and fibroblasts (except B.pendula ether extract). Cisplatin inhibits cell proliferation
stronger than the tested extracts. IC
50
values for the reduction of [
3
H]thymidine incorporation
were a few times lower than the IC
50
for the reduction of cell viability, but fibroblasts were also
one of most susceptible cell types for antiproliferative action of cisplatin.
Discussion
In this study, we have demonstrated the potent cytotoxic and differential activities of extracts
from buds of B.pendula and B.pubescens. The group of compounds that could significantly
influence anticancer activity were the triterpenes, which were dominant in all extracts of B.
pendula. Many studies have confirmed that this group of natural components has various
potential modes of anticancer action [32]. It has previously been demonstrated that dammar-
ane-type triterpenes from Betula spp. possess cytotoxic activity towards Ehrlich carcinoma
ascite cells. The action of these compounds was associated with an effect on the microviscosity
White birch buds extracts in tumors therapy
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of tumour-cell membranes [33]. Another important group of compounds with anticancer
activity are the flavonoids, especially the methoxylated derivatives of flavones. It was previously
shown that O-methylation enhanced cytotoxicity on human leukaemia cells [34]. Such com-
pounds were found in most extracts of both birch species but were at higher levels in B.pubes-
cens. Sesquiterpenoids may also contribute to the cytotoxic activity of birch bud extracts, if not
directly, at least indirectly. For example, Legault and Pichette [35] demonstrated that β-caryo-
phyllene increased the anticancer activity of other substances.
Table 2. Concentrations of extracts (μg/ml ±SEM) required for 50% reduction in cells viability (IC
50
) after 24, 48 and 72 hours of treatment.
24 hours of treatment
Cell lines B.pubescens B.pendula cisplatin
SFE exudate ether extract SFE exudate ether extract
LN-18 50.23±2.32 58.44±2.30 81.75±3.55 48.32±2.12 69.21±3.05 83.92±3.62 4.51±0.14
LN-229 62.18±2.89 61.58±2.69 109.22±4.18 55.99±3.43 71.15±3.14 142.50±6.49 33.52±2.80
MCF-7 109.9±4.12 88.69±5.22 148.45±6.03 93.89±5.37 102.25±4.67 157.70±6.50 15.34±0.58
MDA-MB-231 54.33±3.15 59.53±2.98 170.00±7.61 49.07±2.16 60.70±2.91 125.64±7.00 34.51±1.32
HeLa 58.62±2.45 63.69±2.26 118.00±6.10 44.79±2.36 75.42±3.12 143.30±5.21 19.60±0.86
Ishikawa 93.56±5.24 88.62±3.12 123.78±5.28 85.19±4.21 96.17±4.48 125.82±5.76 36.82±1.26
AGS 84.14±3.65 65.42±3.56 141.12±6.32 69.51±3.32 88.82±3.44 145.83±8.14 26.41±1.05
HepG2 81.31±4.01 76.15±4.05 152.80±6.98 80.12±4.58 93.42±5.12 149.32±7.55 9.55±0.62
DLD-1 97.57±3.95 75.83±2.66 137.30±6.59 97.36±4.24 90.89±4.56 148.30±6.32 32.25±1.11
C32 63.24±3.88 79.74±4.07 122.42±7.01 60.13±2.11 79.62±3.43 122.41±4.67 15.90±0.44
Fibroblasts 87.99±4.22 70.47±3.02 151.50±8.96 87.64±.45 88.14±4.01 137.33±6.53 14.52±0.87
48 hours of treatment
LN-18 24.01±1.33 28.40±1.62 55.74±2.30 29.84±1.18 44.63±1.97 60.08±2.89 3.20±0.11
LN-229 36.72±1.89 53.08±0.67 66.58±3.01 43.55±2.55 51.92±2.61 93.28±4.22 12.53±0.44
MCF-7 92.23±4.16 78.92±3.68 106.80±5.19 84.21±4.45 84.69±3.88 109.30±6.04 7.60±0.32
MDA-MB-231 32.39±1.92 52.73±4.12 60.46±3.66 28.60±1.24 49.80±2.38 70.63±2.97 28.10±1.36
HeLa 42.89±2.45 43.93±2.17 109.60±7.23 41.97±1.95 57.89±3.25 116.10±4.87 11.39±.63
Ishikawa 74.61±3.80 78.87±3.27 115.90±5.10 60.00±2.62 86.37±3.76 115.23±6.12 27.10±1.40
AGS 34.68±1.67 50.58±2.89 135.10±5.84 41.75±2.30 59.23±2.45 140.20±6.12 21.25±1.12
HepG2 49.75±2.25 51.89±2.08 123.40±7.14 55.14±.70 63.84±.31 123.00±7.55 4.25±.12
DLD-1 73.82±3.30 64.44±3.11 109.60±4.23 71.13±4.02 75.25±3.29 120.70±7.90 25.32±0.97
C32 51.00±3.14 55.34±2.21 102.45±4.67 43.68±2.90 62.22±3.05 97.68±4.72 6.32±0.24
Fibroblasts 52.31±2.91 55.26±2.90 84.52±3.61 57.42±3.50 73.02±4.21 97.18±6.20 5.65±0.30
72 hours of treatment
LN-18 15.94±0.89 23.98±1.02 21.21±0.90 15.84±0.67 24.63±1.08 24.61±1.32 2.86±0.12
LN-229 33.73±1.34 42.66±1.87 42.46±1.64 39.26±1.35 40.68±.52 62.26±3.40 5.61±0.18
MCF-7 45.76±1.89 64.03±3.06 73.77±3.40 45.00±2.50 62.42±2.69 75.12±4.04 5.21±0.15
MDA-MB-231 23.94±0.98 49.33±1.67 43.21±1.82 21.84±0.75 48.29±1.64 59.73±2.68 10.25±0.34
HeLa 29.56±1.32 36.85±1.44 46.57±2.16 32.55±2.05 35.32±1.59 58.81±2.68 11.38±0.46
Ishikawa 57.64±2.98 69.79±3.68 79.83±3.22 44.29±3.11 57.19±3.04 86.58±4.53 6.48±0.14
AGS 31.02±1.04 36.23±1.25 87.07±3.16 26.92±0.88 37.14±1.08 80.49±3.76 11.59±0.45
HepG2 41.02±2.34 38.84±1.78 84.53±3.45 42.71±1.85 48.86±2.32 92.33±3.61 3.11±0.11
DLD-1 55.99±2.44 58.58±2.18 74.26±3.21 56.63±2.80 57.29±2.46 77.55±3.12 9.73±0.36
C32 44.72±2.65 54.26±2.14 88.01±4.57 38.43±2.67 56.58±4.12 65.17±2.77 3.12±0.24
Fibroblasts 42.88±2.55 47.83±2.24 59.85±2.69 42.91±2.18 58.62±2.45 61.89±3.26 2.84±0.13
Lower IC
50
values compared to fibroblasts are in bold.
https://doi.org/10.1371/journal.pone.0201949.t002
White birch buds extracts in tumors therapy
PLOS ONE | https://doi.org/10.1371/journal.pone.0201949 August 14, 2018 7 / 10

According to the literature, esters of cinnamic acids with aliphatic alcohols show different
biological activities [36,37]. For example, phenylpropenoids of two terpene alcohols (geraniol
and farnesol) had an inhibitory effect on nitric oxide production and exhibited antitumour
activity [38]. In vitro experiments showed that synthetic esters of ferulic and caffeic acids have
the ability to inhibit the development of colon, gastric and breast cancer cells [37].
Among the examined cancer cell lines, LN-18, MDA-MB-231 and HeLa were more sensi-
tive to the cytotoxic action of tested extracts than fibroblasts. This indicates that, similar to the
anticancer drug used as reference, extracts from Betula buds do not show general selectivity
towards cancer cells. Nevertheless, further studies are needed for the isolation and identifica-
tion of pure compounds with anticancer activity.
Supporting information
S1 Text. Analytical procedure.
(PDF)
S1 Table. Chemical composition of birch bud extracts. S1Table presents the chemical com-
position of extracts (SFE, exudate, and ether extract of milled buds) and some of the analytical
parameters: I
T
values, m/z of target peaks, and molecular ion, M
+
(if was registered).
(PDF)
S1 Fig. Chromatograms of SFE extracts of silver birch (upper) and downy birch.
(PDF)
S2 Fig. Concentration-response curves for the cytotoxic effect of birch bud extracts. The
effect of B.pendula exudate (red closed squares), B.pubescens exudate (red open squares), B.
pendula SFE (blue closed triangles), B.pubescens SFE (blue open triangles), B.pendula ether
extract (green closed circles) and B.pubescens ether extract (green open circles) on cell viability
after 24, 48 or 72 hours of treatment. The results are presented as a mean ±SEM of three inde-
pendent experiments done in triplicates. P<0.05.
(PDF)
S3 Fig. Concentration-response curves for the antiproliferative effect of birch bud extracts.
The effect of B.pendula exudate (red closed squares), B.pubescens exudate (red open squares),
Table 3. Concentrations of extracts (μg/ml ±SEM) required for 50% reduction in [
3
H]thymidine incorporation (IC
50
) after 24 hours of treatment.
Cell lines B.pubescens B.pendula cisplatin
SFE exudate ether extract SFE exudate ether extract
LN-18 59.96±3.22 33.34±1.18 50.54±2.44 66.77±5.03 52.33±2.96 73.32±4.61 2.16±.18
LN-229 70.89±3.18 56.93±2.34 98.56±5.32 77.39±3.40 83.32±5.13 108.20±6.03 2.88±0.17
MCF-7 78.17±3.37 59.25±2.43 110.00±6.90 68.09±2.45 98.24±4.22 115.80±4.99 3.42±0.12
MDA-MB-231 65.36±2.32 42.84±1.55 124.20±7.72 59.13±2.11 75.68±4.58 106.70±6.02 9.12±0.32
HeLa 40.69±1.50 20.80±1.20 47.82±2.11 38.30±1.29 49.58±2.80 62.50±3.20 2.64±0.11
Ishikawa 77.61±3.21 35.01±2.90 83.99±3.58 42.11±2.33 97.21±5.91 96.46±4.18 5.56±0.18
AGS 43.23±1.59 36.23±1.68 91.72±3.90 49.97±2.58 55.28±3.14 127.20±5.22 4.84±0.14
HepG2 77.54±3.02 33.96±1.69 88.51±4.11 78.07±4.90 87.82±3.86 107.30±5.85 3.82±0.21
DLD-1 70.85±3.10 30.01±1.42 78.84±3.50 45.95±2.77 86.33±4.52 103.70±4.62 4.38±0.19
C32 60.14±2.47 40.40±3.21 72.55±3.66 61.17±3.14 82.10±3.51 98.92±4.06 2.21±0.11
Fibroblasts 79.89±3.12 46.52±2.50 116.60±6.11 63.34±2.90 69.25±3.23 142.8±8.45 2.47±0.13
Lower IC
50
values compared to fibroblasts are in bold.
https://doi.org/10.1371/journal.pone.0201949.t003
White birch buds extracts in tumors therapy
PLOS ONE | https://doi.org/10.1371/journal.pone.0201949 August 14, 2018 8 / 10

B.pendula SFE (blue closed triangles), B.pubescens SFE (blue open triangles), and B.pendula
ether extract (green closed circles) and B.pubescens ether extract (green open circles) on [
3
H]
thymidine incorporation after 24 hours of treatment. The results are presented as a mean ±
SEM of two independent experiments done in triplicates. P<0.05.
(PDF)
Acknowledgments
The authors would like to thank Edward Roy and his staff (New Chemical Synthesis Institute,
Puławy) for SFE extraction of birch buds. This work was supported by grant of National Sci-
ence Centre (Poland) 2016/23/B/NZ7/03360 and by Bialystok University of Technology Pro-
gram S/ZWL/1/2017.
Author Contributions
Conceptualization: Valery Isidorov.
Investigation: Valery Isidorov, Łukasz Szoka.
Methodology: Łukasz Szoka, Jolanta Nazaruk.
Supervision: Valery Isidorov.
Writing – original draft: Valery Isidorov, Jolanta Nazaruk.
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