Enhancement of the antioxidant and skin permeation properties of betulin and its derivatives

Article abstract of DOI:10.3390/molecules26113435

This study investigated the antioxidant activity DPPH, ABTS, and Folin–Ciocalteu methods of betulin (compound 1) and its derivatives (compounds 2–11). Skin permeability and accumulation associated with compounds 1 and 8 were also examined. Identification of the obtained products (compound 2–11) and betulin isolated from plant material was based on the analysis of¹H-NMR and¹³C-NMR spectra. The partition coefficient was calculated to determine the lipophilicity of all compounds. In the next stage, the penetration through pig skin and its accumulation in the skin were evaluated of ethanol vehicles containing compound 8 (at a concentration of 0.226 mmol/dm³), which was characterized by the highest antioxidant activity. For comparison, penetration studies of betulin itself were also carried out. Poor solubility and the bioavailability of pure compounds are major constraints in combination therapy. However, we observed that the ethanol vehicle was an enhancer of skin permeation for both the initial betulin and compound 8. The betulin 8 derivative showed increased permeability through biological membranes compared to the parent betulin. The paper presents the transformation of polycyclic compounds to produce novel derivatives with marked antioxidant activities and as valuable intermediates for the pharmaceutical industry. Moreover, the compounds contained in the vehicles, due to their mechanism of action, can have a beneficial effect on the balance between oxidants and antioxidants in the body, minimizing the effects of oxidative stress. The results of this work may contribute to knowledge regarding vehicles with antioxidant potential. The use of vehicles for this type of research is therefore justified.

Full text of DOI:10.3390/molecules26113435

molecules
Article
Enhancement of the Antioxidant and Skin Permeation
Properties of Betulin and Its Derivatives
1
,
1
2
2
2
1
Andrzej Günther * , Edyta Makuch
and Adam Klimowicz 2
, Anna Nowak , Wiktoria Duchnik , Łukasz Kucharski , Robert Pełech
1
Department of Chemical Organic Technology and Polymeric Materials, Faculty of Chemical Technology and
Engineering, West Pomeranian University of Technology, Szczecin, PL-70322 Szczecin, Poland;
emakuch@zut.edu.pl (E.M.); rpelech@zut.edu.pl (R.P.)
Department of Cosmetic and Pharmaceutical Chemistry, Pomeranian Medical University in Szczecin,
PL-70111 Szczecin, Poland; anowak@pum.edu.pl (A.N.); wduchnik@pum.edu.pl (W.D.);
lukasz.kucharski@pum.edu.pl (Ł.K.); adam.klimowicz@pum.edu.pl (A.K.)
2
*
Correspondence: andrzej.gunther@zut.edu.pl; Tel.: +48-91-449-41-48
Abstract: This study investigated the antioxidant activity DPPH, ABTS, and Folin–Ciocalteu methods
of betulin (compound ) and its derivatives (compounds 11). Skin permeability and accumulation
associated with compounds 1 and
compound
1
2–
8
were also examined. Identification of the obtained products
1
(
2–
11) and betulin isolated from plant material was based on the analysis of H- NMR
and 13C-NMR spectra. The partition coefficient was calculated to determine the lipophilicity of all
compounds. In the next stage, the penetration through pig skin and its accumulation in the skin were
ꢀ
ꢁꢂꢀꢃꢄꢅꢆꢇ
3
evaluated of ethanol vehicles containing compound
8
(at a concentration of 0.226 mmol/dm ), which
ꢀ
ꢁꢂꢃꢄꢅꢆ
was characterized by the highest antioxidant activity. For comparison, penetration studies of betulin
itself were also carried out. Poor solubility and the bioavailability of pure compounds are major
constraints in combination therapy. However, we observed that the ethanol vehicle was an enhancer
Citation: Günther, A.; Makuch, E.;
Nowak, A.; Duchnik, W.; Kucharski,
Ł.; Pełech, R.; Klimowicz, A.
of skin permeation for both the initial betulin and compound 8. The betulin 8 derivative showed
Enhancement of the Antioxidant and
Skin Permeation Properties of Betulin
and Its Derivatives. Molecules 2021,
increased permeability through biological membranes compared to the parent betulin. The paper
presents the transformation of polycyclic compounds to produce novel derivatives with marked
antioxidant activities and as valuable intermediates for the pharmaceutical industry. Moreover, the
compounds contained in the vehicles, due to their mechanism of action, can have a beneficial effect
on the balance between oxidants and antioxidants in the body, minimizing the effects of oxidative
stress. The results of this work may contribute to knowledge regarding vehicles with antioxidant
potential. The use of vehicles for this type of research is therefore justified.
2
6, 3435. https://doi.org/10.3390/
molecules26113435
Academic Editors: Irina Ivshina
and Victoria Grishko
Received: 17 May 2021
Accepted: 1 June 2021
Published: 5 June 2021
Keywords: synthesis of new betulin derivatives; antioxidant activity; chemistry of natural compounds
vehicle effect; skin barrier; stratum corneum
;
Publisher’s Note: MDPI stays neutral
with regard to jurisdictional claims in
published maps and institutional affil-
iations.
1
. Introduction
The skin has been investigated as a route to deliver active substances contained
topically in cosmetics and pharmaceuticals. Transdermal delivery of active substances
is often limited by their poor permeability [ ]. The stratum corneum (SC) acts as a
barrier for the active substance permeation into the skin. The SC is 15–20 m thick and is
1–9
µ
composed of 15–20 flattened layers, and the epidermal cell membranes are so tightly joined
that there is hardly any intercellular space through which molecules penetrate [10–12].
However, transdermal delivery of active substances offers the advantages of having a
relatively large absorption surface area and being non-invasive [10]. The permeation
of the active substance is highly dependent on the lipophilicity and molecular size of
these substances [13]. Overcoming the lipophilic barrier, which is the skin, is possible
for compounds with molecular weights of <600 Da [13]. The optimal partition coefficient
Copyright: © 2021 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4
.0/).
Molecules 2021, 26, 3435. https://doi.org/10.3390/molecules26113435
https://www.mdpi.com/journal/molecules

Molecules 2021, 26, 3435
2 of 17
value (log P), which is an indicator of the lipophilicity of the active substance, ranges from
2
to 3 [13].
A promising alternative to antioxidant agents is a natural compound, such as be-
tulin, a component of the triterpenes synthesized by plants [14 15]. Betulin (IUPAC name
lup-20(29)-en-3 ,28 -diol), belonging to the lupane-type pentacyclic triterpenes is charac-
terized by relatively high lipophilicity (partition coefficient value (log P) = 5.34 [16] and
unfortunately it is practically insoluble in aqueous media [17 18]. Therefore, betulin has
,
β
β
,
poor penetration through the skin. However, betulin and its derivatives have a broad spec-
trum of biological relevance, thus they have a high potential for application in transdermal
systems [19
,20]. To date, several formulations containing betulin and its derivatives have
been developed [21
–25]. However, there are no literature reports describing the penetration
of vehicles containing these compounds through the skin. Betulin shows a broad spectrum
of biological activity (including cytotoxic activity [19]) because its structure contains sub-
stituents and groups, which can be easily functionalized (such as the isopropenyl moiety,
alkyl group, and hydroxyl groups) [14
new betulin derivatives, especially ketones—compound
constant search for new substances with antioxidant properties.
–
16
,
26–
30]. Therefore, it seemed interesting to obtain
8
(Figure 1). This allows for the
Birch bark
isolation
purification
H
H
Jones
oxidation
H
OH
H
H
O
O
i.
H
H
O
O
H
H
H
2
3
OH
H
H
Br
HO
H
Acylation
ii.
H
H
H
1
, Betulin
O
O
iii.
O
H
H
H
O
O
O
O
O
H
H
H
HO
O
O
H
H
H
6
5
4
H
O
O
H
H
H
O
H
iv.
H
O
O
H
H
O
v.
H
H
O
HO
H
O
vi.
O
H
HO
H
H
O
7
O
H
4
H
8
9
vii.
H
OH
H
H
H
viii.
Ph Ph
Si
OH
H
O
H
HO
H
11
HO
HO
H
10
◦
Figure 1. Synthesis of betulin derivatives. Reagent and conditions: i. Jones oxidation, CrO , aq. H SO , acetone, 0 C,
3
2
4
◦ ◦
N atm); ii. DMAP, pyridine, Ac O, 0 C; iii.NBS, CCl ; iv. MeOH/THF, 10% Pd/C, H (40 psi); v. DCM, PCC, 0 C; vi.
2 2 4 2
(
◦
◦
◦
chloroform, 70%-mCPBA, 35 C; vii. THF, LiAlH , −40 C; viii. imidazole, THF, TBDPSCl, −70 C.
4
This study proposes ethanol as a vehicle for betulin and its derivative for cutaneous
applications. The active substance delivery platforms that have attracted the attention of
specialists in the medical and pharmaceutical industries are transdermal drug delivery
systems in the form of alcohols (especially ethanol) as vehicles. The main advantage of these
vehicles is their approval for use in medical, pharmaceutical, and cosmetics preparations,

Molecules 2021, 26, 3435
3 of 17
high enhancing activity. and good solvating ability—which are promising candidates for
betulin and its derivatives as antioxidants supply [4].
This study presents the effect of ethanol as a vehicle on skin permeation of compounds
with antioxidant activity: betulin and its derivative 8. The use of ethanol application in
this study was to evaluate this vehicle on the permeability of the tested compounds. The
pH value of the acceptor phase in in vitro permeation tests was set at 7.4 for the simulation
of the skin surface [13
,31
–33]. The penetration through pig skin using the Franz diffusion
cell and the accumulation of betulin and compound
8
in the skin was evaluated. Due to
their mechanism of action, the compounds contained in vehicles can beneficially affect the
balance between oxidants and antioxidants in the body, minimizing the effects of oxidative
stress. The results of this work may contribute to knowledge regarding vehicles with
antioxidant potential. The use of vehicles for this type of research is therefore justified. Our
data suggest that ethanol is a promising vehicle for betulin and compound 8.
2. Results
2.1. Betulin and Its Derivatives
Figure 1 shows the scheme of the synthesis of betulin derivatives.
Table 1 presents the partition coefficient values calculated in the MestReNova program.
The lipophilicity of each compound is represented as the decimal logarithm of the partition
coefficient (log P) [34].
Table 1. Partition coefficient values calculated by theoretical method, in the MestReNova program.
Compounds
Formula
C H O
Molecular Weight [Da]
log P
5.642
5.372
4.777
6.430
7.000
6.997
6.779
6.304
6.613
5.006
9.684
1
2
3
4
5
6
7
8
9
442.73
438.7
3
0
50
2
2
3
3
4
C H O
3
0
46
C H O
454.7
3
0
46
C H O
484.77
526.8
3
2
52
C H O
3
4
54
C H BrO
4
605.7
34
53
C H O
486.78
484.77
500.76
462.76
701.16
3
2
54
3
3
4
3
C H O
3
2
52
C H O
3
2
52
1
1
0
1
C H O
30 54
C H O Si
46
72
3
The high lipophilicity (log P 4.777–9.684) of the obtained compounds
1–
11, favors their
excessive accumulation in lipid membranes. This way, the diffusion of active substances ad-
ministered epidermally, will be hindered through the living layers of the epidermis [34,35].
1
The presence of clear peaks in the H-NMR spectrum of betulin (above) and its
derivative (below) indicate the presence of certain groups of protons in the structure of the
1
13
tested compounds—Figure 2. (Supplementary data present H NMR and C NMR spectra
of compounds 1–11).
The proton signals recorded, corresponding to the hydrogen atoms from the groups
belonging to the hydrocarbon chains of the compounds 1–11, were obtained. The analysis
of the chemical shift values of carbon atoms additionally supplemented the information
and confirmed the structure of the obtained compounds.

Molecules 2021, 26, 3435
4 of 17
1
Figure 2. H-NMR spectrum of betulin (above), and its derivative (below).
2.2. Evaluation of Free Radical Scavenging Activity
Table 2 presents the results for the antioxidant activity of betulin and its derivative,
carried out using the DPPH, ABTS, and Folin–Ciocalteu methods.
The test results, presented in Table 2, show that the highest antioxidant activity (for
DPPH and ABTS methods) was observed for compound
8
: 61.83
±
0.004% RSA (for the
DPPH method) and 12.79 0.005% RSA (in the case of the ABTS method). The study
±
of DPPH radical scavenging capacity of the compounds
2,
3,
6
,
7
,
9
,
10, and 11 was
0.007% RSA, 7.04 0.007%
0.013% RSA, 4.84 0.011% RSA,
0.013% RSA, respectively. Pure betulin was characterized by antioxidant
0.007% RSA, while in the case of the studies carried out for compounds
and 5, no antioxidant activity was shown.
The antioxidant activity (determined by the ABTS method) of the compounds ob-
tained varied as follows: 12.79 0.005% RSA (for compound ) >5.65 0.015% RSA (for
10, showed no antioxidant activity by
characterized by the DPPH radical scavenging degree of: 1.50
±
±
RSA, 14.78
and 12.68
activity 2.81
±
0.014% RSA, 2.80
±
0.012% RSA, 14.11
±
±
±
±
4
±
8
±
compound 11). Pure betulin, compounds 2–7 and 9–
this method.
In contrast, the antioxidant activity obtained by the Folin–Ciocalteu method changed
3
3
as follows: 0.847
±
0.037 mmol TE/dm (for compound
3) >0.413
±
0.015 mmol TE/dm
3
(
for compound
9
) >0.367
±
0.081 mmol of TE/dm (for compound
). No antioxidant activity was obtained for compounds
6
) >0.050 0.000 mmol
±
3
of TE/dm (for compound
8
1,
2,
4
,
5, 7, 10, and 11.
2.3. Skin Penetration and Skin Extraction
Compound is characterized by the highest degree of free radical scavenging DPPH,
8
therefore the penetration through pig skin and its accumulation in the skin were evaluated.
For comparison, penetration studies of betulin were also carried out.
HPLC analysis of the compounds present in the acceptor fluid during the 24 h experi-
ment showed that the test compounds could still be detected in the acceptor chamber after
the test period.

Molecules 2021, 26, 3435
5 of 17
Table 2. Antioxidant activity of betulin and its derivatives.
*
Antioxidant Activity (DPPH Method):
Compounds
%
RSA
mmol TE/dm3
0.087 ± 0.003
0.079 ± 0.011
0.091 ± 0.001
n.a.
1
2
3
4
5
6
7
8
9
2.81 ± 0.007
1.50 ± 0.007
7.04 ± 0.007
n.a.
n.a.
n.a.
14.78 ± 0.014
2.80 ± 0.012
61.83 ± 0.004
14.11 ± 0.013
4.84 ± 0.011
12.68 ± 0.013
0.100 ± 0.002
0.087 ± 0.001
0.436 ± 0.005
0.099 ± 0.002
0.089 ± 0.001
0.098 ± 0.001
1
1
0
1
*Antioxidant Activity (ABTS method):
mmol TE/dm3
%
RSA
1
n.a.
n.a.
n.a.
2
3
4
5
6
7
8
9
n.a.
n.a.
n.a.
n.a.
n.a.
n.a.
n.a.
n.a.
n.a.
n.a.
n.a.
12.79 ± 0.005
n.a.
0.237 ± 0.002
n.a.
1
0
1
n.a.
n.a.
1
5.65 ± 0.015
0.105 ± 0.002
*Antioxidant Activity (Folin–Ciocalteu Method):
mmol TE/dm3
1
-
-
-
-
-
-
-
-
-
-
-
n.a.
n.a.
2
3
4
5
6
7
8
9
0.847 ± 0.037
n.a.
n.a.
0.367 ± 0.081
n.a.
0.050 ± 0.000
0.413 ± 0.015
n.a.
1
0
1
1
n.a.
n.a.—no activity, * Mean ± SD (n = 3).
For confirmation, the chromatogram of betulin and its derivative is shown—Figure 3.
The left side of Figure 3 shows the chromatogram for the signal with a retention time of

Molecules 2021, 26, 3435
6 of 17
9
.01 min (compound
1), while the right side of this figure shows the chromatogram for the
signal with a retention time of 8.99 min (compound 8).
Figure 3. HPLC chromatograms of reference betulin and reference compound
8 (above), HPLC chro-
matograms of betulin and betulin derivative 8 in the acceptor fluid after a 24 h experiment (below).
The remaining peaks visible in these chromatograms come from the test mixture and
are also detected in the blank. As can be seen, the obtained signals with a retention time of
9
.01 (left side of this Figure) and 8.99 (right side of this Figure) are identical, therefore the
analyzed compounds which penetrated the skin are betulin and its derivative 8.
Table 3 presents the results of the permeation and the accumulation studies of the
tested compounds.
Table 3. Permeation of active substances through skin and the amounts of extracted active ingredients accumulated in it.
The Cumulative Mass of Substance after 24 h of
Skin Accumulation of Substance:
Compounds
Betulin
2
Permeation Test: (µg)
(µg/cm Skin)
*
14.27 ± 2.20 a
21.41 ± 2.10 b
135.71 ± 9.11 a
104.06 ± 15.79 b
*
Betulin derivative 8
*
Mean ± SD (n = 3), a, b—values are significantly different between analyzed vehicles (P = 0.001).
Figure 4 shows the comparison of the in vitro permeation profiles for betulin and
derivative
8 through the skin during the 24 h experiment (Supplementary data present the
skin permeation of betulin and its derivative 8).

Molecules 2021, 26, 3435
7 of 17
Figure 4. The cumulative mass of betulin and derivative 8 penetrated into the acceptor fluid during the 24 h experiment.
The permeation of the vehicle containing betulin and its derivative
skin was assessed, in which the donor phase consisted of the vehicles tested, moreover, the
acceptor phase was the PBS solution, as it corresponds to systemic conditions [13 31 33].
As shown in Table 3, the application of compound in vehicles led to an increase in its
permeation through the skin in comparison to betulin applied in the same vehicle. After
experimenting for 24 h, the highest mean cumulative mass was observed for compound
21.41 2.10 g). The mass was slightly lower of betulin (14.27 2.20 g). The cumulative
mass of betulin which penetrated into the acceptor fluid was observed only after 5 h of
the experiment, while for derivative after 4 h. Moreover, the average cumulative masses
8 through the pig
,
,
8
8
(
±
µ
±
µ
8
for vehicles containing the tested compounds at 0.5; 1; 2; 3; 4; 5; 8, and 24 h are shown in
Figure 4 (Table 3).
After the experiment was carried out, the skin was extracted to evaluate the amount of
the accumulated tested active ingredients. The concentrations of betulin and its derivative
2
8
contained in the vehicles were respectively: 135.71
±
9.11
µ
g/cm for the skin, and
2
104.06 ± 15.79 µg/cm for the skin—Table 3.
3
. Discussion
Betulin is a pentacyclic triterpene. Pentacyclic triterpenoids (including pentacyclic
triterpenes such as betulin) are of interest to researchers due to their anti-inflammatory,
antimicrobial, antitumor, cytotoxic, and other activities [14 15 18 25 27 30 36 39], and their
,
,
,
,
–
,
–
possible use as intermediates in the synthesis of new pharmacological substances.
These compounds thus have high potential for application in transdermal systems,
however, they are highly hydrophobic (poor skin penetration), which significantly limits
their use as effective pharmacological agents [18]. The lipophilic parameter of betulin and its
derivatives (Table 1) depends on the type of substituent in C-3 and C-28 positions (Figure 1),
polar surface, polarizability, molecular volume, and molecular refraction [34,36–38]. In
the group of tested compounds, compound
3
showed the lowest lipophilicity parameter
(log P = 4.777). Modification of betulin by introducing the following moieties: an acyl group
in position C-28 in the case of compound 4, acyl groups in position C-3 and C-28 in the case

Molecules 2021, 26, 3435
8 of 17
of compound
hydrogenation of the allyl group in the case of compound
C-3 position in the case of compound
of compound , blocked –OH group by tert-butyldiphenylsilyl group (TBDPS) in the case
of compound 11, led to an increase in lipophilicity the following compounds and 11
resulting in log P in the range of 6.304–9.684. Compounds: (due to the introduction of a
ketone group at the C-3 position and an aldehyde group at the C-28 position) and (due
5, insertion of a bromine atom in the allyl position in the case of compound
6,
7
, synthesis of a ketone in the
8, formation of a lactam from the A ring in the case
9
4
–9
,
2
3
to the introduction of a ketone group at the C3 position and a carbonyl acid at the C-28
position) were characterized by a lower log P value in relation to the initial betulin.
In recent years an efficient formulation has been presented containing betulin and its
derivative, to enhance the delivery of these substances via different routes: betulinic acid
(
(
BA) loaded polylac-tide-co-glycolide-monomethoxy polyethylene glycol nanoparticles
PLGA-mPEG NPs), inhalable lactose based nanosystems of betulin delivery, a liposome
formulation of BA (oral administration of this formulation slowed down tumor growth in
mice) mulauer, copolymers (which can be used as carriers in drug delivery systems in the
form of microspheres etc.), but there is still room for improvement [17,21–24,39].
Currently, one of the most common ways to increase the effectiveness and bioavail-
ability of highly hydrophobic compounds that penetrate poorly through the skin is the
application of these compounds in vehicles (in the form of alcohols—ethanol, n-propanol,
isopropyl alcohol, n-butanol, n-pentanol, and propylene glycol [33,39,40].
The main benefit of ethanol vehicles is the approval for use in medical, pharmaceutical,
and cosmetics preparations, high enhancing activity, and good solvation ability [
addition, the good permeability of vehicle containing compound through the skin and its
proper accumulation in the skin (Table 3, Figure 4) as well as the high antioxidant capacity
of the initial compound (Table 2) may also limit the effects of free radicals, which are
highly toxic molecules due to the presence of one or more unpaired electrons. Compound
4,13]. In
8
8
8
has a ketone group in the C-3 position, which may increase the reactivity of this compound
with the DPPH radical [4,13,41–46].
Our previous studies on the effect of vehicles on skin permeability of a new eugenol
derivative (eugenyl dichloroacetate—EDChA) with antioxidant activity have shown that
the application of a eugenol (E) derivative of the cream and gel as vehicles did not lead to
an increase in the skin permeation of EDChA, in comparison to the initial E applied in the
same vehicles [47].
However, the highest permeation rate of cream vehicles containing E and EDChA
into the acceptor fluid (µg/h) was observed between 4 and 5 h, while for gel vehicles
between 3 and 4 h [47]. In addition, after skin extraction, the obtained results showed that
the concentration of substances contained in the cream vehicles decreased in the following
order: eugenyl dichloroacetate > eugenol. Moreover, the concentration of substances
contained in the gel vehicles decreased in the following order: E > EDChA [47].
4. Materials and Methods
4.1. Chemicals
The following compounds were used for the preparation of betulin, and the synthesis
of its derivatives: 4-dimethylaminopyridine (DMAP), acetic anhydride (Ac O), carbon
2
tetrachloride (CCl ), tetrahydrofuran (THF), dichloromethane (DCM), palladium on carbon
4
(10% Pd/C), pyridinium chlorochromate (PCC), 3-chloroperbenzoic acid (mCPBA), lithium
aluminum hydride (LiAlH ), tert-butyl(chloro)diphenylsilane (TBDPSCl), purchased from
4
Sigma Aldrich (Steinheim am Albuch, Germany), KOH, hexane, methanol, ethyl acetate,
´
acetone, MgSO , ethanol, purchased from Chempur (Piekary Sla˛skie, Poland). All reagents
4
were of analytical grade.
For the determination of the antioxidant activity and to assess the skin permeation of
the compounds obtained: acetonitrile for HPLC was from J.T. Baker (Berlin, Germany). 2,2-
0
diphenyl-1-picrylhydrazyl (DPPH), 2,2 -azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)
(
ABTS) and 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (TE), were purchased

Molecules 2021, 26, 3435
9 of 17
from Sigma Aldrich (St. Louis, MO, USA); Folin–Ciocalteu reagent and gallic acid (GA),
were from Merck (Darmstadt, Germany), potassium persulfate and chloride, sodium
chloride, methanol, ethanol, acetone and phosphate buffered saline (PBS, pH = 7.4) were
´
purchased from Chempur (Piekary Sla˛skie, Poland). All reagents were of analytical grade.
4.2. Plant Material
The plant material (the papilionaceous birch bark) was collected in June in Poland in
◦
0
00
◦
0
00
the year 2019 (N53 20 12 , E15 02 59 ) from the natural state, near-located places. Ten
samples were harvested and combined into one collective sample.
Samples were deposited in the plant material storage room (No. BC-2019/015) at
the Faculty of Chemical Technology and Engineering, Department of Chemical Organic
Technology and Polymeric Materials, West Pomeranian University of Technology, Szczecin.
The deposited material consisted of samples of dried papilionaceous birch bark (Betula
pendula Roth).
4.3. Preparation of Betulin and Characterization of Betulin and Its Derivatives
The betulin and novel compounds were obtained in high yields and were identified
by H and C-NMR, moreover, the partition coefficient for all compounds was calculated
in the MestReNova program (Figure 1, Table 1).
1
13
The structures of betulin and the obtained compounds were confirmed based on the
analysis of nuclear magnetic resonance (NMR) spectroscopy spectra. Measurements were
1
performed using a Bruker DPX-400 spectrometer. H-NMR spectra were recorded under
the following conditions: 400.13 MHz, a 12 kHz spectrum width, 65.5 K data points, a
0
.488 Hz/point resolution, a data acquisition time of 4.09 s, a repetition time of 1 s, a 7.8 µs
13
pulse width, and 16–32 scans. C-NMR spectra were as follows: 100.62 MHz, a spectrum
width of 24 kHz, 65.5 K data points, a resolution of 1.46 Hz/point, a data acquisition time
of 1.37 s, a repetition time of 1 s, a pulse width of 9.2 µs, and 1–8 scans.
4.3.1. Preparation of the Birch Bark and Isolation of Betulin from the Birch Bark
The bark was boiled in a 1% aqueous KOH solution for 2 h. The bark was then filtered
on a Büchner funnel and washed with hot water. The bark was dried in a vacuum dryer at
80 °C for 2 days.
The isolation of betulin was carried out from the shredder birch bark of the Betula
verrusoca tree. An amount of 100 g of dried bark was first extracted with hexane (500 mL)
in a Soxhlet apparatus, for 24 h (2 12 h), then with methylene chloride (500 mL) for 24 h
12 h). After evaporation to dryness, the hexane extract yielded 3.25 g of dry extract,
×
(
2
×
while the methylene chloride extract yielded 11.17 g of dry extract.
The 11.17 g of dry extract obtained was heated under a reflux condenser in methanolic
KOH 2% solution (100 mL) for 10 h. The flask was allowed to stand overnight at room
temperature. The precipitate was drained on a Büchner funnel and washed copiously
◦
with hot water. The obtained impure betulin (8.4 g) was dried in a vacuum oven at 80 C
overnight. The betulin was then purified on a chromatography column in the system
hexane:ethyl acetate (H:OAc) 40:1
→
5:1 v/v, monitoring the extraction with TLC (H:OAc
5:1). Finally 7.23 g of pure betulin was obtained (1).
4.3.2. Synthesis of Betulonic Aldehyde (2) and Betulonic Acid (3)
An amount of 1 g (2.26 mmol) of betulin (
in 150 mL of acetone, at 0 C (ice bath) under N . Freshly prepared Jones reagent (3.9 mL,
1
) was placed in 3-neck round-bottomed flask,
2
◦
0.39 M, 7.68 mmol) was added dropwise from an additional funnel to the stirred suspension
of betulin ( ) over 15 min. After 1 h, stirring was continued at room temperature for 2 h,
1
then 25 mL of methanol was added until a dark green color appeared. The reaction mixture
was then poured onto 100 mL of water with ice. Methanol and acetone were removed under
reduced pressure. The aqueous residue was washed 3 times with 40 mL of ethyl acetate.
The combined organic layers were first washed with brine (20 mL) then with water (20 mL)

Molecules 2021, 26, 3435
10 of 17
and finally dried with anhydrous MgSO4. The solvent was evaporated under reduced
pressure to give a crude white solid, which was purified by column chromatography to
deliver first 59.7 mg (6%) of betulonal as a white solid, then 860 mg (86%) of betulonic acid
as a white solid.
4.3.3. Synthesis of Compounds 4 and 5 by Acetylation of Betulin
An amount of 5 g (11.3 mmol) of betulin (
in a round-bottomed flask, in 200 mL of dry pyridine, at 0 C (cooling bath). Then, 24.5 mL
1
) and DMAP (3.05 g, 25 mmol) were placed
◦
(
0.25 mmol) of Ac O was slowly added dropwise (over 1 h) to the stirred solution from the
2
additional funnel. The solvent was evaporated under reduced pressure to give a crude dark
solid, which was purified by column chromatography in the system hexane:ethyl acetate
(
H:OAc) 40:1
→
5:1 v/v, to deliver first 0.55 g (11%) of betulin 3,28-diacetate 3 as a w white
crystalline powder, then 1.86 g (37%) of betulin 28-acetate as a white crystalline powder.
4.3.4. Synthesis of Compound 6 (30-bromolup-20(29)-ene-3β,28-diyl diacetate)
A solution of (0.5 mmol, 265 mg) in dry CCl (5 mL) was treated with freshly
5
4
recrystallized NBS (0.15 g, 0.85 mmol,) and refluxed for 3 h. The solid was filtered off and
the mother liquor was evaporated under vacuum. The dark solid was recrystallized from
EtOH to obtain 0.183 g (69%) as brown needles.
4.3.5. Synthesis of Compound 7 (3β,28-lupanediol 28-acetate)
To a solution of (3.09 mmol, 1.5 g) in 60 mL mixture of MeOH/THF (2:1 v/v), 10%
4
Pd/C (167 mg) was added and hydrogenated under H (40 psi) for 24 h. Next, it was
2
filtered, and the solvent was evaporated under reduced pressure to give 1.49 g, (99+%) as a
white crystalline solid.
4.3.6. Synthesis of Compounds 8 (28-acetoxy-3-lupanone)
To a cooled (ice bath) solution of monoacetate (1.40 g, 2.88 mmol) in 50 mL of DCM,
7
PCC (3.68 g, 17.07 mmol) was slowly added in small portions over 5 min. The reaction
was monitored by TLC. After 4 h, the flask with the reaction mixture was placed in the
freezer overnight. The solvent was evaporated under reduced pressure and the obtained
residue was purified by column chromatography (H:OAc 1:1 v/v) to give 1.30 g (92%) of
the ketone 8 as a white solid.
4.3.7. Synthesis of Compound 9
To a solution of ketone
0%-mCPBA (3.23 g, 15 mmol). The reaction mixture was heated to 30–35 C and after
h another portion of 70%-mCPBA (0.91 g, 4.22 mmol) was added. The reaction was
8
(1.20 g, 2.48 mmol) in 50 mL of chloroform was added
◦
7
4
monitored by TLC, there was still presence of the substrate. After 24 h (the substrate was
still present on TLC), another portion of 70%-mCPBA (0.58 g, 2.69 mmol) was added. After
1
2 h (there was only a trace of the substrate, TLC), 30 mL solution of 10%-Na S O was
2 2 3
slowly added (the temperature of the mixture increases). The mixture was transferred to
a separatory funnel and the organic layer was washed with 30 mL of saturated NaHCO3
solution. The solvent was evaporated under reduced pressure and the obtained residue
was purified by column chromatography (H:OAc 20:1
as a white solid.
→
3:1 v/v) to give 1.06 g (85%) of 9
4
.3.8. Synthesis of Compound 10
◦
40 C acetonitrile with dry ice), LiAlH
4
To a cooled 20 mL amount of THF (about
−
(
606 mg, 16 mmol) was added followed by
for 1.5 h until the substrate disappeared (TLC). Once the reaction was complete, 10 mL
of ethyl acetate was added followed by slowly added 30 mL of saturated NH Cl solution
9 (800 mg, 1.6 mmol). The reaction was stirred
4
(until bubbling disappeared). The organic layer was separated and filtered through a
thin layer of silica gel. The aqueous layer was extracted with 20 mL of ethyl acetate. The

Molecules 2021, 26, 3435
11 of 17
solvents were evaporated under reduced pressure and the obtained residue was purified
by column chromatography (H:OAc 1:1 v/v) to give 736 mg (96%) of 10 as a white solid.
4
.3.9. Synthesis of Compound 11
◦
70 C isopropyl alcohol and dry ice) of 10 (500 mg,
To a cooled solution (about
−
1
.08 mmol) in 20 mL THF was added imidazole (235.39 mg, 3.46 mmol) and TBDPSCl
(
445.48 mg, 1.62 mmol, d = 1.057 g/mL). When the temperature reached ambient temper-
ature, the mixture was further stirred for 24 h. Next, the solvent was evaporated under
reduced pressure and the obtained oily residue was purified by column chromatography
(
H:OAc 20:1→5:1 v/v) to give 370 mg (49%) of ketone 11 as a light straw-colored oil.
4.4. Evaluation of Free Radical Scavenging Activity
The studies on the antioxidant activity of betulin and its derivatives were carried
out using the DPPH [13
The absorbance at 517 nm (in the case of the DPPH method), 734 nm (in the case of
the ABTS method), and 765 nm (in the case of the Folin–Ciocalteu method) working
solutions was adjusted to 1.00 0.02. Measurements were taken using a Spectroquant,
,47,48], ABTS [13,47] and the Folin–Ciocalteu [13,47,49] methods.
λ
±
model Pharo 300 (Merck, Germany), in triplicate for each tested sample. The efficiency of
neutralization of DPPH radicals by betulin and its derivatives was expressed in the form of
TEAC (Trolox Equivalent Antioxidant Capacity) factor, determining the concentration of
Trolox with identical antioxidant capacity. TEAC values were calculated from absorbance
measurements using the calibration curve method. The results were expressed as TEAC
3
in millimoles of trolox per volume of sample (mmol TE/dm ). In the case of the Folin–
3
Ciocalteu method, the antioxidant activity was expressed in mmol GA/dm .
In the next stage of the compound characterized by the highest degree of free radical
scavenging DPPH (compound 8), penetration through the pig skin and its accumulation
in the skin were evaluated. For comparison, penetration studies of betulin were also
carried out.
4.4.1. Evaluation of Free Radical Scavenging Activity Using DPPH Method
The antioxidant activity of the tested compounds was evaluated using a modified
DPPH method. The procedure was as follows: to 2.85 mL of the DPPH radical solution (at
3
concentrations of 0.3 mmol/dm ) dissolved in 96% (v/v) ethanol to 0.15 mL of the tested
3
compounds (at the concentrations of 0.226 mmol/dm ) was added, and then the tube was
incubated for 10 min at room temperature, followed by spectrophotometric measurements
at λ = 517 nm.
The antioxidant activity of the tested samples was calculated according to the follow-
ing formula:
%
RSA = [(A − Ap)/A ] × 100% = (1 − Ap/A ) × 100%,
(1)
0
0
0
where: %RSA—antioxidant activity, A —mean value of the absorbance of the ethanol
0
solution of DPPH containing 0.15 mL of the ethanol, A
p
—mean value of absorbance of the
ethanol solution of the DPPH radical containing 0.15 mL of the tested compound.
4.4.2. Evaluation of Free Radical Scavenging Activity Using ABTS Method
The antioxidant activity of the tested compounds was evaluated by the ABTS method,
3
using a solution of ABTS (at concentrations of 7 mmol/dm ) in an aqueous solution of
3
potassium persulfate (at concentrations of 2.45 mmol/dm ) as a stock solution. The solution
◦
was incubated for 24 h, at the temperature of 4 C and then diluted with 50% (v/v) methanol.
The antioxidant activity was measured as follows: to 2.5 mL of a stock solution of ABTS to
3
0
.25 mL of the tested compound (at the concentrations of 0.226 mmol/dm ) was added,
and then the tube was incubated for 6 min at room temperature. Spectrophotometric
measurements were carried out at λ = 734 nm.

Molecules 2021, 26, 3435
12 of 17
The antioxidant activity was calculated from the equation:
RSA = [(A − Ap)/A ] × 100% = (1 − Ap/A ) × 100%,
%
(2)
0
0
0
where: %RSA—antioxidant activity, A —absorbance of the stock solution ABTS containing
0
0.25 mL of the ethanol, Ap—absorbance of the stock solution ABTS containing 0.25 mL of
the tested compound.
4.4.3. Evaluation of Total Polyphenol Content Using Folin–Ciocalteu Method
The total content of phenolic compounds found in the tested samples was evaluated
using the Folin–Ciocalteu method. Two milliliters of Folin–Ciocalteu reagent in 1.8 mL
of water was dissolved in a dark bottle, then the solution was incubated for 60 min (at
room temperature). The antioxidant activity was measured as follows: 1.35 mL of distilled
3
water and 1.35 mL of sodium carbonate solution (at concentrations of 0.01 mol/dm )
were introduced into the tube, with 0.15 mL of the prepared Folin–Ciocalteu solution
and 0.15 mL of an ethanol solution containing the tested compound (at a concentration
3
of 0.226 mmol/dm ), and then the tube was incubated for 15 min at room temperature,
followed by spectrophotometric measurements at λ = 765 nm.
4.5. Skin Electrical Impedance
A fresh portion of skin from the abdomen impedance was measured using an LCR
080 m (Conrad Electronic, Hirschau, Germany) operating in parallel mode at 120 Hz
4
(
kΩ error < 0.5%). The skin came from a local slaughterhouse. A pig skin sample was
2
placed in a Franz diffusion cell, the permeation area of the tested compounds was 1 cm .
First of all, both chambers were filled with PBS buffer, then the tips of the measuring
probes were immersed in the donor chamber and the acceptor chamber. Pig skin samples
with a specific impedance above 3 kU were used in the permeation of betulin and its
derivative experiments. The values of pig skin electrical impedance were similar to the
electrical resistance of human skin reported by others [31,33,50].
4.6. Skin Penetration
The effect of various alcohols (methanol, ethanol, and isopropanol) as vehicles on
skin permeability for compound
8 was compared. The lag time on pig skin permeability
of betulin derivative was tested and compared. Preliminary studies showed that the
8
lag time increases with increasing chain-length of the alcohol, and it is respectively: 3, 4,
and 6 h. The reason for methanol application in the preliminary studies was to evaluate
the effect of the chain length on the lag time of the betulin derivative. Methanol will
therefore not be used in commercial vehicles for skin application. The cumulative mass of
compound
8 in ethanol as a vehicle, which penetrated into the acceptor fluid was observed
after 4 h of experimentation. Therefore, in this study, the penetration through the pig skin
and its accumulation in the skin of ethanol vehicles containing compounds
concentrations of 0.226 mmol/dm ) were evaluated.
1 and 8 (at the
3
Betulin derivatives generally have greater antioxidant activities than pure betulin [51–54].
These compounds may play an important role in the regeneration of the epidermis and
the underlying layers [55–57]. Therefore, the assessment of skin penetration of betulin
derivatives is important. Penetration of these derivatives may vary depending on their
structure and lipophilicity [36,37,58–61].
The skin penetration studies were conducted in a Franz diffusion cell (SES GmbH An-
alyze Systeme, Germany) consisting of a 2 mL donor chamber and 8 mL acceptor chamber,
◦
which was kept at a constant temperature of 37
±
0.5 C with the VEB MLW Prüfgeräte-
Werk type 3280 thermostat. Porcine skin was used for the study due to its permeability
properties being similar to human skin. The permeation area was 1 cm [62,63].
2
Before starting the test, a fresh portion of the abdominal skin (with a thickness of
0
.5 mm) was washed several times with PBS solution, moreover the Franz diffusion cells
◦
were allowed to equilibrate at 37 C for 15 min. In the next stage, the skin was placed

Molecules 2021, 26, 3435
13 of 17
in Franz diffusion cells, and the integrity of the skin was checked one hour after its
installation [5,31,47].
Ethanol solutions of betulin and its derivative 8
(at a concentration of 0.226 mmol/dm3)
were placed in the donor chamber, then the chamber was closed with a plastic stopper to
prevent excessive evaporation of the tested compounds. The process was carried out for
2
4 h, and aliquots of 0.3 mL of the solution in the acceptor chamber were taken at specified
intervals (30 min, 1 h, 2 h, 3 h, 4 h, 5 h, 8 h, and 24 h), and then supplemented with a fresh
portion of the buffer of the same pH. The samples obtained in this way were analyzed by
HPLC, and then after the completion of the permeation experiment, the skin was extracted
to estimate the residual amount of tested active ingredients accumulated in it.
4.7. Skin Extraction
To estimate the residual amount of betulin and its derivative accumulated in the
skin, its extraction was carried out. The Franz diffusion chambers (ppkt 4.5. Skin pene-
tration) were dismantled and the skin surface was washed three times with an aqueous
solution of sodium lauryl sulfate (at a concentration of 0.5% w/w) to rinse off the excess of
these compounds.
2
A patch (1 cm diffusion surface) was cut from the skin prepared in this way, dried
at room temperature, and then weighed and cut into smaller pieces. Then, 2 mL of
◦
concentrated methanol was added, and extraction was carried out for 24 h at 4 C. After
2
®
4 h of incubation, the skin was homogenized (for 3 min) using a homogenizer (IKA T18
digital ULTRA TURRAX, Germany). The obtained extracts were then centrifuged at
500 rpm for 5 min. The supernatant was analyzed by HPLC to determine the content of
active ingredients.
The cumulative mass of active substance (
3
µg) permeating into the receptor chamber
was calculated based on the concentrations of compounds in the receptor fluid determined
by HPLC. The accumulation of compounds in the skin was calculated by applying the
2
amount of compound obtained after skin extraction; the results are given in
skin [13,31].
µ
g/cm of
4.8. HPLC Analysis
The content of the test compounds which penetrated into the acceptor fluid during
the 24 h experiment and the amounts of extracted active ingredients which accumulated
in the skin were determined using the high-performance liquid chromatography (HPLC)
method, with the HPLC system from Knauer with a UV detector (Berlin, Germany).
The samples were separated on a 125
×
4 mm column containing Hyperisil ODS;
particle size 5 m. The flow rate of the mobile phase consisting of acetonitrile, water, and
µ
MeOH (28:64:8, by vol) was 1 mL/min. Twenty microliters of each analyzed sample were
injected onto the column. The detection wavelength was 210 nm. Injections were repeated
at least three times for each sample and the results were averaged. The concentration of
betulin and its derivative was calculated based on the peak area measurements using the
calibration curve method.
4.9. Statistical Analysis
The skin permeation results of betulin and its derivative and the accumulation of these
compounds in the skin are presented as the mean
±
standard deviation (SD). Statistical
calculations were made using Statistica 13 PL software (StatSoft, Polska) and the Tukey
post-hoc test. The results were evaluated using a one-way analysis of variance (ANOVA).
Probabilities p < 0.05 were considered statistically significant.
5
. Conclusions
During the research, betulin (compound
were obtained and characterized. The penetration of compounds
through the skin was observed (under physiological conditions 37 °C, pH = 7.4), and it was
1) and its derivatives (compounds
2–11)
1
and from the vehicle
8

Molecules 2021, 26, 3435
14 of 17
demonstrated that the betulin derivative penetrates through biological membranes more
readily than the initial betulin. Moreover, betulin and its derivative
the skin.
8 also accumulated in
The obtained results indicate the possibility of using these compounds as an ingredient
in cosmetics and pharmaceutics applied to the skin. Betulin and some betulin derivatives
inhibit the formation of reactive oxygen species (ROS) [64], therefore vehicles containing
these compounds look promising as antioxidant delivery systems.
The biological activity of betulin is well documented. Furthermore, the vehicles are
approved for use in active substances delivery systems. The use of such a solvent as a
vehicle in biological systems enables a controlled release of betulin and its derivative
which is dictated by the penetration rate of the vehicles.
8,
Supplementary Materials: The following are available online, H NMR and 13C NMR spectra of
1
compound 1–11.
Author Contributions: A.G., E.M., and A.N.; Writing—review & editing, A.G., Ł.K., and A.N.;
Methodology, A.G., A.N., E.M., W.D., and Ł.K.; Reviewing R.P. and A.K.; Formal analysis, A.G., A.N.,
E.M., and Ł.K.; Investigation A.G., A.N., Ł.K., and E.M.; Writing-original draft, A.G., A.N., and E.M.;
Supervision, A.K. and R.P. All authors read and approved the final manuscript.
Funding: This research received no external funding.
Institutional Review Board Statement: We would like to inform that after consulting with Local
Bioethics Committee for Animal Testing we received the information that using in such experiment
commercially available pig skin did not require consent of the committee.
Data Availability Statement: The data presented in this study are available in this article.
Conflicts of Interest: The authors declare no conflict of interest.
Sample Availability: Samples of the compounds are available from the authors.
References
1
.
Fang, J.Y.; Leu, Y.L.; Hwang, T.L.; Cheng, H.C.; Hung, C.F. Development of sesquiterpenes from Alpinia oxyphylla as novel skin
permeation enhancers. Eur. J. Pharm. Sci. 2003, 19, 253–262. [CrossRef]
2
.
Torin Huzil, J.; Sivaloganathan, S.; Kohandel, M.; Foldvari, M. Drug delivery through the skin: Molecular simulations of
barrier lipids to design more effective noninvasive dermal and transdermal delivery systems for small molecules, biologics, and
cosmetics. Wiley Interdiscip. Rev. Nanomed. Nanobiotechnol. 2011, 3, 449–462. [CrossRef] [PubMed]
Puglia, C.; Bonina, F. Lipid nanoparticles as novel delivery systems for cosmetics and dermal pharmaceuticals. Expert Opin. Drug
Deliv. 2012, 9, 429–441. [CrossRef]
3
4
.
.
Ossowicz, P.; Klebeko, J.; Janus, E.; Nowak, A.; Duchnik, W.; Kucharski, Ł.; Klimowicz, A. The effect of alcohols as cosmetic
formulations on the percutaneous absorption and skin retention of ibuprofen modified with l-valine alkyl esters. RSC Adv. 2020
0, 41727–41740. [CrossRef]
,
1
5.
6.
7.
Janus, E.; Ossowicz, P.; Klebeko, J.; Nowak, A.; Duchnik, W.; Kucharski, Ł.; Klimowicz, A. Enhancement of ibuprofen solubility
and skin permeation by conjugation with L-valine alkyl esters. RSC Adv. 2020, 10, 7570–7584. [CrossRef]
Khezri, K.; Saeedi, M.; Maleki Dizaj, S. Application of nanoparticles in percutaneous delivery of active ingredients in cosmetic
preparations. Biomed. Pharmacother. 2018, 106, 1499–1505. [CrossRef]
Pinzaru, I.; Tanase, A.; Enatescu, V.; Coricovac, D.; Bociort, F.; Marcovici, I.; Watz, C.; Vlaia, L.; Soica, C.; Dehelean, C. Proniosomal
gel for topical delivery of rutin: Preparation, physicochemical characterization and in vitro toxicological profile using 3d
reconstructed human epidermis tissue and 2d cells. Antioxidants 2021, 10, 85. [CrossRef]
8
9
1
.
.
Ramadon, D.; McCrudden, M.T.C.; Courtenay, A.J.; Donnelly, R.F. Enhancement strategies for transdermal drug delivery systems:
Current trends and applications. Drug Deliv. Transl. Res. 2021, 1–34. [CrossRef]
Tambunlertchai, S.; Geary, S.M.; Salem, A.K. Skin Penetration Enhancement Strategies Used in the Development of Melanoma
Topical Treatments. AAPS J. 2021, 23, 19. [CrossRef]
0. Fox, L.T.; Gerber, M.; Du Plessis, J.; Hamman, J.H. Transdermal drug delivery enhancement by compounds of natural origin.
Molecules 2011, 16, 10507–10540. [CrossRef]
1
1
1
1. Hadgraft, J. Skin the final frontier. Int. J. Pharm. 2001, 224, 1–18. [CrossRef]
2. Hsieh, D.S. Drug Permeation Enhancement—Theory and Applications; Marcel Dekker: New York, NY, USA, 1993.
3. Makuch, E.; Nowak, A.; Günther, A.; Pełech, R.; Kucharski, Ł.; Duchnik, W.; Klimowicz, A. Enhancement of the antioxidant and
skin permeation properties of eugenol by the esterification of eugenol to new derivatives. AMB Express 2020, 10, 187. [CrossRef]
[PubMed]

Molecules 2021, 26, 3435
15 of 17
1
1
1
4. Rastogi, S.; Pandey, M.M.; Kumar, A.; Rawat, S. Medicinal plants of the genus Betula—Traditional uses and a phytochemical–
pharmacological review. J. Ethnopharmacol. 2020, 159, 62–83. [CrossRef]
5. Venza, M.; Visalli, M.; Beninati, C.; De Gaetano, G.V.; Teti, D.; Venza, I. Cellular Mechanisms of Oxidative Stress and Action in
Melanoma. Oxid. Med. Cell. Longev. 2015, 2015, 481782. [CrossRef]
6. Chrobak, E.; Kadela-Tomanek, M.; B e˛ benek, E.; Marciniec, K.; Wietrzyk, J.; Trynda, J.; Pawełczak, B.; Kusz, J.; Kasperczyk, J.;
Chodurek, E. New phosphate derivatives of betulin as anticancer agents: Synthesis, crystal structure, and molecular docking
study. Bioorg. Chem. 2019, 87, 613–628. [CrossRef] [PubMed]
1
7. Drag-Zalesinska, M.; Kulbacka, J.; Saczko, J.; Wysocka, T.; Zabel, M.; Surowiak, P.; Drag, M. Esters of betulin and betulinic acid
with amino acids have improved water solubility and are selectively cytotoxic toward cancer cells. Bioorg. Med. Chem. Lett. 2009
,
1
9, 4814–4817. [CrossRef]
1
1
2
2
8. Koch, B.P.; Rullkötter, J.; Lara, R.J. Evaluation of triterpenols and sterols as organic matter biomarkers in a mangrove ecosystem
in northern Brazil. Wetl. Ecol. Manag. 2003, 11, 257–263. [CrossRef]
9. Niewolik, D.; Krukiewicz, K.; Bednarczyk-Cwynar, B.; Ruszkowski, P.; Jaszcz, K. Novel polymeric derivatives of betulin with
anticancer activity. RSC Adv. 2019, 9, 20892–20900. [CrossRef]
0. Sousa, J.L.C.; Freire, C.S.R.; Silvestre, A.J.D.; Silva, A.M.S. Recent developments in the functionalization of betulinic acid and its
natural analogues: A route to new bioactive compounds. Molecules 2019, 24, 355. [CrossRef]
1. Saneja, A.; Kumar, R.; Singh, A.; Dhar Dubey, R.; Mintoo, M.J.; Singh, G.; Mondhe, D.M.; Panda, A.K.; Gupta, P.N. Development
and evaluation of long-circulating nanoparticles loaded with betulinic acid for improved anti-tumor efficacy. Int. J. Pharm. 2017
31, 153–166. [CrossRef]
,
5
2
2
2
2. Karlina, M.V.; Pozharitskaya, O.N.; Shikov, A.N.; Makarov, V.G.; Mirza, S.; Miroshnyk, I.; Hiltunen, R. Drug synthesis methods
and manufacturing technology biopharmaceutical study of nanosystems containing betulin for inhalation administration. Pharm.
Chem. J. 2010, 44, 501–503. [CrossRef]
3. Pozharitskaya, O.N.; Karlina, M.V.; Shikov, A.N.; Kosman, V.M.; Makarov, V.G.; Casals, E.; Rosenholm, J.M. Pharmacokinetics
and Tissue Disposition of Nanosystem- Entrapped Betulin After Endotracheal Administration to Rats. Eur J Drug Metab Pharm.
2
017, 42, 327–332. [CrossRef] [PubMed]
4. Mullauer, F.B.; van Bloois, L.; Daalhuisen, J.B.; Ten, M.S.; Storm, G.; Paul, J.; Schiffelers, R.M.; Kessler, J.H. Betulinic acid delivered
in liposomes reduces growth of human lung and colon cancers in mice without causing systemic toxicity. Anti-Cancer Drugs 2011
2, 223–233. [CrossRef]
,
2
2
2
2
2
2
3
5. Niewolik, D.; Bednarczyk-cwynar, B.; Ruszkowski, P.; Sosnowski, T.R.; Jaszcz, K. Bioactive Betulin and PEG Based Polyanhydrides
for Use in Drug Delivery Systems. Int. J. Mol. Sci. 2021, 22, 1090. [CrossRef] [PubMed]
6. Csuk, R.; Barthel, A.; Sczepek, R.; Siewert, B.; Schwarz, S. Synthesis, encapsulation and antitumor activity of new betulin
derivatives. Arch. Pharm. Chem. Life Sci. 2011, 1, 37–49. [CrossRef] [PubMed]
7. Sharaf, A.; De Michele, R.; Sharma, A.; Fakhari, S. Transcriptomic analysis reveals the roles of detoxification systems in response
to mercury in Chromera velia. Biomolecules 2019, 9, 647. [CrossRef]
8. Bi, Y.; Xu, J.; Wu, X.; Ye, W.; Yuan, S.; Zhang, L. Synthesis and cytotoxic activity of 17-carboxylic acid modified 23-hydroxy
betulinic acid ester derivatives. Bioorg. Med. Chem. Lett. 2007, 17, 1475–1478. [CrossRef]
9. Yamansarov, E.Y.; Saltykova, I.V.; Kovalev, S.V.; Petrov, R.A.; Shkil’, D.O.; Seleznev, E.I.; Beloglazkina, E.K.; Majouga, A.G.
Synthesis and cytotoxicity of new alkyne derivatives of pentacyclic triterpenoids. Russ. Chem. Bull. 2019, 68, 855–861. [CrossRef]
0. Marciniec, K.; Pawełczak, B.; Latocha, M.; Skrzypek, L.; Macia˛zek-Jurczyk, M.; Boryczka, S.; Supuran, C.T. Synthesis, anti-breast
cancer activity, and molecular docking study of a new group of acetylenic quinolinesulfonamide derivatives. Molecules 2017
2, 300. [CrossRef]
,
2
3
1. Nowak, A.; Cybulska, K.; Makuch, E.; Kucharski, Ł.; Ró z˙ ewicka-Czaba n´ ska, M.; Prowans, P.; Czapla, N.; Bargiel, P.; Petriczko, J.;
Klimowicz, A. In Vitro Human Skin Penetration, Antioxidant and Antimicrobial Activity of Ethanol-Water Extract of Fireweed
(Epilobium angustifolium L.). Molecules 2021, 26, 329. [CrossRef]
3
3
3
2. Redasani, V.K.; Bari, S.B. Prodrug Design: Perspectives, Approaches and Applications in Medicinal Chemistry, 1st ed.; Academic Press:
London, UK, 2015.
3. Wang, X.; Wang, W.; Jin, S.; Muhammad, N.; Guo, Z. Stimuli-responsive therapeutic metallodrugs. Chem. Rev. 2019
19, 1138–1192. [CrossRef]
,
1
4. Achrem-Achremowicz, J.; K e¸ pczy n´ ska, E.; Zylewski, M.; Janeczko, Z. Synthesis of betulin derivatives and the determination of
their relative lipophilicities using reversed-phase thin-layer chromatography. Biomed. Chromatogr. 2010, 24, 261–267. [CrossRef]
[
PubMed]
5. Sangster, J. Octanol-Water Partition Coefficients: Fundamentals and Physical Chemistry; John Wiley & Sons: Chichester, UK,
997; p. 170.
3
3
3
1
6. Kadela-Tomanek, M.; Jastrz e˛ bska, M.; Chrobak, E.; B e˛ benek, E.; Boryczka, S. Chromatographic and computational screening of
lipophilicity and pharmacokinetics of newly synthesized betulin-1,4-quinone hybrids. Processes 2021, 9, 376. [CrossRef]
7.
B e¸ benek, E.; Bober-Majnusz, K.; Siudak, S.; Chrobak, E.; Kadela-Tomanek, M.; Wietrzyk, J.; Boryczka, S. Application of TLC to
Evaluate the Lipophilicity of Newly Synthesized Betulin Derivatives. J. Chromatogr. Sci. 2020, 58, 323–333. [CrossRef] [PubMed]

Molecules 2021, 26, 3435
16 of 17
3
3
8. Bildziukevich, U.; Vida, N.; Rárová, L.; Kolá rˇ , M.; Šaman, D.; Havlí cˇ ek, L.; Drašar, P.; Wimmer, Z. Polyamine derivatives of
betulinic acid and β-sitosterol: A comparative investigation. Steroids 2015, 100, 27–35. [CrossRef]
9. Kuo, S.C.; Chuang, S.K.; Lin, H.Y.; Wang, L.H. Study of the aerosol fragrances of eugenol derivatives in Cananga odorata
using diffuse reflectance infrared Fourier transform spectroscopy and gas chromatography. Anal. Chim. Acta. 2009, 653, 91–96.
[
CrossRef] [PubMed]
0. Chandra, A.; Sharma, P.K.; Irchhiaya, R. Effect of alcohols and enhancers on permeation enhancement of ketorolac. Asian J. Pharm.
009, 3, 37. [CrossRef]
4
2
4
4
1. Sies, H. Oxidative stress: Concept and some practical aspects. Antioxidants 2020, 9, 852. [CrossRef]
2. García-Sánchez, A.; Miranda-Díaz, A.G.; Cardona-Muñoz, E.G. The Role of Oxidative Stress in Physiopathology and Pharma-
cological Treatment with Pro- And Antioxidant Properties in Chronic Diseases. Oxid. Med. Cell. Longev. 2020, 2020, 2082145.
[CrossRef] [PubMed]
4
4
4
4
4
4
4
3. Kim, T.Y.; Leem, E.; Lee, J.M.; Kim, S.R. Control of reactive oxygen species for the prevention of parkinson’s disease: The possible
application of flavonoids. Antioxidants 2020, 9, 583. [CrossRef]
4. Chakraborti, S.; Chakraborti, T.; Chattopadhyay, D.; Shaha, C. Oxidative stress in Microbial Diseases; Springer: Singapore, 2019.
[
CrossRef]
5. Pisoschi, A.M.; Pop, A. The role of antioxidants in the chemistry of oxidative stress: A review. Eur. J. Med. Chem. 2015, 97, 55–74.
CrossRef]
[
6. Liguori, I.; Russo, G.; Curcio, F.; Bulli, G.; Aran, L.; Della-Morte, D.; Gargiulo, G.; Testa, G.; Cacciatore, F.; Bonaduce, D. Oxidative
stress, aging, and diseases. Clin. Interv. Aging 2018, 13, 757–772. [CrossRef]
7. Makuch, E.; Nowak, A.; Günther, A.; Pełech, R.; Kucharski, Ł. The effect of cream and gel vehicles on the percutaneous absorption
and skin retention of a new eugenol derivative with antioxidant activity. Front. Pharmacol. 2021, 12, 1249. [CrossRef]
8. Brand-Williams, W.; Cuvelier, M.E.; Berset, C.L.W.T. Use of a free radical method to evaluate antioxidant activity. Lebensm. Wiss.
Technol. 1995, 28, 25–30. [CrossRef]
9. Nowak, A.; Klimowicz, A.; Duchnik, W.; Kucharski, Ł.L.; Florkowska, K.; Muzykiewicz, A.; Wira, D.; Zielonkabrzezicka, J.;
Siedłowska, A.; Nadarzewska, K. Application of green-extraction technique to evaluate of antioxidative capacity of wild
population of fireweed (Epilobium angustifolium). Herba Pol. 2020, 65, 18–30. [CrossRef]
5
5
0. Davies, D.J.; Ward, R.J.; Heylings, J.R. Multi-species assessment of electrical resistance as a skin integrity marker for in vitro
percutaneous absorption studies. Toxicol. Vitr. 2004, 18, 351–358. [CrossRef] [PubMed]
1. Sousa, J.L.C.; Gonçalves, C.; Ferreira, R.M.; Cardoso, S.M.; Freire, C.S.R.; Silvestre, A.J.D.; Silva, A.M.S. Functionalization of
betulinic acid with polyphenolic fragments for the development of new amphiphilic antioxidants. Antioxidants 2021, 10, 148.
[CrossRef] [PubMed]
5
5
2. Amiri, S.; Dastghaib, S.; Ahmadi, M.; Mehrbod, P.; Khadem, F.; Behrouj, H.; Aghanoori, M.R.; Machaj, F.; Ghamsari, M.; Rosik, J.
Betulin and its derivatives as novel compounds with different pharmacological effects. Biotechnol. Adv. 2020, 38, 107409.
[CrossRef]
3. Gomes Castro, A.J.; Cazarolli, L.H.; Bretanha, L.C.; Sulis, P.M.; Rey Padilla, D.P.; Aragón Novoa, D.M.; Dambrós, B.F.;
Pizzolatti, M.G.; Mena Barreto Silva, F.R. The potent insulin secretagogue effect of betulinic acid is mediated by potassium
and chloride channels. Arch. Biochem. Biophys. 2018, 648, 20–26. [CrossRef] [PubMed]
5
4. Silva, F.S.G.; Oliveira, P.J.; Duarte, M.F. Oleanolic, Ursolic, and Betulinic Acids as Food Supplements or Pharmaceutical Agents
for Type 2 Diabetes: Promise or Illusion? J. Agric. Food Chem. 2016, 64, 2991–3008. [CrossRef] [PubMed]
5. Melnikova, N.; Knyazev, A.; Nikolskiy, V.; Peretyagin, P.; Belyaeva, K.; Nazarova, N.; Liyaskina, E.; Malygina, D.; Revin, V.
Wound healing composite materials of bacterial cellulose and zinc oxide nanoparticles with immobilized betulin diphosphate.
Nanomaterials 2021, 11, 713. [CrossRef]
5
5
5
5
5
6
6. Sułkowska-Ziaja, K.; Grabowska, K.; Apola, A.; Kryczyk-Poprawa, A.; Muszy n´ ska, B. Mycelial culture extracts of selected
wood-decay mushrooms as a source of skin-protecting factors. Biotechnol. Lett. 2021, 43, 1051–1061. [CrossRef]
7. Zagórska-Dziok, M.; Ziemlewska, A.; Bujak, T.; Nizioł-Łukaszewska, Z.; Hordyjewicz-Baran, Z. Cosmetic and Dermatological
Properties of Selected Ayurvedic Plant Extracts. Molecules 2021, 26, 614. [CrossRef]
8. Bober, K.; B e¸ benek, E.; Boryczka, S. Application of TLC for evaluation of the lipophilicity of newly synthetized esters: Betulin
derivatives. J. Anal. Methods Chem. 2019, 3, 1297659–1297667. [CrossRef] [PubMed]
9. B e˛ benek, E.; Kadela-Tomanek, M.; Chrobak, E.; Wietrzyk, J.; Sadowska, J.; Boryczka, S. New acetylenic derivatives of betulin and
betulone, synthesis and cytotoxic activity. Med. Chem. Res. 2017, 26, 1–8. [CrossRef]
0. Dubinin, M.V.; Semenova, A.A.; Ilzorkina, A.I.; Penkov, N.V.; Nedopekina, D.A.; Sharapov, V.A.; Khoroshavina, E.I.;
Davletshin, E.V.; Belosludtseva, N.V.; Spivak, A.Y. Mitochondria-targeted prooxidant effects of betulinic acid conjugated with
delocalized lipophilic cation F16. Free Radic. Biol. Med. 2021, 168, 55–69. [CrossRef]
6
6
1. Cichewicz, R.H.; Kouzi, S.A. Chemistry, Biological Activity, and Chemotherapeutic Potential of Betulinic Acid for the Prevention
and Treatment of Cancer and HIV Infection. Med. Res. Rev. 2004, 24, 90–114. [CrossRef]
2. Haq, A.; Michniak-Kohn, B. Effects of solvents and penetration enhancers on transdermal delivery of thymoquinone: Permeability
and skin deposition study. Drug Deliv. 2018, 25, 1943. [CrossRef] [PubMed]

Molecules 2021, 26, 3435
17 of 17
6
3. Kope cˇ ná, M.; Machá cˇ ek, M.; Prchalová, E.; Št eˇ pánek, P.; Drašar, P.; Kotora, M.; Vávrová, K. Galactosyl Pentadecene Reversibly
Enhances Transdermal and Topical Drug Delivery. Pharm. Res. 2017, 34, 2097–2108. [CrossRef] [PubMed]
4. Szuster-Ciesielska, A.; Plewka, K.; Daniluk, J.; Kandefer-Szerszen, M. Betulin and betulinic acid attenuate ethanol-induced liver
stellate cell activation by inhibiting reactive oxygen species (ROS), cytokine (TNF-alpha, TGF-beta) production and by influencing
intracellular signaling. Toxicology 2011, 280, 152–163. [CrossRef]
6