RESEARCH ARTICLE
New lupeol esters as active substances in the
treatment of skin damage
Magdalena Malinowska ID1☯*, Barbara Miroslaw2☯, Elzbieta Sikora1, Jan Ogonowski1,
Agnieszka M. Wojtkiewicz3, Maciej Szaleniec3☯, Monika Pasikowska-Piwko4☯, Irena Eris4
1
2
3
4
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OPEN ACCESS
Citation: Malinowska M, Miroslaw B, Sikora E,
Ogonowski J, Wojtkiewicz AM, Szaleniec M, et al.
(2019) New lupeol esters as active substances in
the treatment of skin damage. PLoS ONE 14(3):
e0214216. https://doi.org/10.1371/journal.
pone.0214216
Editor: Mohammad Saleem, University of
Minnesota Twin Cities, UNITED STATES
Received: October 19, 2018
Accepted: March 9, 2019
Published: March 28, 2019
Copyright: © 2019 Malinowska 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 manuscript and its Supporting
Information files.
Funding: The research was carried out with the
equipment purchased thanks to the financial
support of the European Regional Development
Fund in the framework of the Operational Program
Development of Eastern Poland 2007-2013
(Contract No. POPW.01.03.00-06-009/11-00,
Equipping the laboratories of the Faculties of
Biology and Biotechnology, Mathematics, Physics
Institute of Organic Chemistry and Technology, Cracow University of Technology, Cracow, Poland,
Department of Crystallography, Faculty of Chemistry, Maria Curie-Sklodowska University, Lublin, Poland,
Jerzy Haber Institute of Catalysis and Surface Chemistry, Polish Academy of Sciences, Cracow, Poland,
Dr Irena Eris Centre for Science and Research, Piaseczno, Poland
☯ These authors contributed equally to this work.
* mmalinowska@chemia.pk.edu.pl
Abstract
The purpose of the research was to obtain new derivatives of natural triterpene lupeol and to
evaluate their potential as active substances in the treatment of skin damage. Four new
lupeol esters (propionate, succinate, isonicotinate and acetylsalicylate) and lupeol acetate
were obtained using an eco-friendly synthesis method. In the esterification process, the
commonly used hazardous reagents in this type of synthesis were replaced by safe ones.
This unconventional, eco-friendly, method is particularly important because the compounds
obtained are potentially active substances in skin care formulations. Even trace amounts of
hazardous reagents can have a toxic effect on damaged or irritated tissues. The molecular
structure of the esters were confirmed by 1H NMR, 13C NMR and IR spectroscopy methods.
Their crystal structures were determined using XRD method. To complete the analysis of
their characteristics, physicochemical properties (melting point, lipophilicity, water solubility)
and biological activity of the lupeol derivatives were studied. Results of an irritant potential
test, carried out on Reconstructed Human Epidermis (RHE), confirmed that the synthesized
lupeol derivatives are not cytotoxic and they stimulate a process of human cell proliferation.
The safety of use for tested compounds was determined in a cell viability test (cytotoxicity
detection kit based on the measurement of lactate dehydrogenase activity) for keratinocytes
and fibroblasts. The results obtained showed that the modification of lupeol structure
improve its bioavailability and activity. All of the esters penetrate the stratum corneum and
the upper layers of the dermis better than the maternal lupeol. Lupeol isonicotinate, acetate
and propionate were the most effective compounds in a stimulation of the human skin cell
proliferation process. This combination resulted in an increase in the concentration of cells
of more than 30% in comparison to control samples. The results indicate that the chemical
modification of lupeol allows to obtain promising active substances for treatment of skin
damage, including thermal, chemical and radiation burns.
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New lupeol esters as active substances in the treatment of skin damage
and Informatics, and Chemistry for studies of
biologically active substances and environmental
samples). The calculations in Discovery Studio
(BIOVIA) were supported by PL-Grid Infrastructure
(AGH CYFRONET). Calculator Plugins of
Chemicalize was used for structure property
prediction and calculation 01.2019 https://
chemicalize.com/ developed by ChemAxon (http://
www.chemaxon.com) 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.
Introduction
Human skin, as the biggest organ in the human body, acts as a protective barrier. Among
other, it prevents toxic substances from penetrating into deeper, more hydrophilic skin structures. Simultaneously, it allows drug molecules to diffuse through skin layers during the transdermal delivery [1]. The maintenance of skin integrity and function is especially important.
Unfortunately, there are many factors that negatively affect the condition of the skin, causing
its damage and dysfunctions. One of the reasons of skin barrier damage is the exposure to UV
radiation. For example, UVB light causes many harmful effects including irritation, redness
and burning of the skin. UVA is responsible for the damage of DNA and cell structure, skin
aging and discoloration caused by free radicals. Other external conditions, including air pollution, electronic device radiation (blue ray) and the presence of controversial self-care product
ingredients, may cause skin damage. Psoriasis, atopic dermatitis, skin allergies and inflammatory reactions are nowadays a problem which is affecting more and more people. Another
cause of skin damage is burn injury, which can be caused by heat, radioactivity, electricity, friction or contact with chemicals [2]. What is more, cancer treatments using radiotherapy are a
source of increasing numbers of skin damage. Some of cancer therapies create an extensive
surface area of wounds that are difficult to heal.
Considering the importance of the barrier function of the skin, there is an urgent need to
develop active ingredients with an intensive regenerative effect, which will simultaneously protect the damaged skin structure from external factors.
Triterpenes are promising agents for curing skin burns and accelerating the skin regeneration process. Their spectrum of biological activity is wide: antioxidant [3], anticancer [4,5],
antibacterial [6,7], antivirus [8,9] and regulating melanin biosynthesis [10]. They are used in
the treatment of various skin ailments [10–12]. One of the most popular triterpenes is lupeol,
generally obtained by extraction processes from natural sources such as: birch bark, white cabbage, green pepper, olive oil, strawberries, mangoes or grapes [13]. Lupeol is already known as
a compound stimulating skin cells proliferation and having influence on their migration,
improving the damaged skin reconstruction. It modifies the refraction capacities of normal
fibroblasts and increases isometric forces of fibroblasts from stretch marks. Moreover, no
stress fibers are observed and the skin is stimulated to reconstruction under lupeol treatment
[14]. The latest studies relating to the cosmetic or pharmaceutical application of lupeol-rich
extract are focused on treating and preventing a connective tissue degeneration [15,16].
It is well known that the suitability for the active substances to be used in pharmaceutical or
cosmetic applications is not only determined by its therapeutic activity, but also by their
absorption, distribution, metabolism, excretion and toxicity (ADMET). In the case of topical
application, the permeability of skin is a significant factor. The stratum corneum (the outermost layer of the skin) is highly lipophilic. It mostly consists of corneocytes and the intercellular spaces are filled with lipid compartments. Its character does not allow hydrophilic
compounds to penetrate through it. Furthermore, the penetration of highly lipophilic components is also limited [1]. The presence of lipids in the stratum corneum is very significant when
determining the affinity of highly lipophilic substances for the skin layer. The average lipids
content of human stratum corneum is about 16%. Additionally, the main class of lipids in the
stratum corneum are ceramides, cholesterol and fatty acids [17]. The structural similarity of
lupeol and cholesterol (the main, representative compound of sterols group) could facilitate
the triterpene compound to cross the skin barrier and enable the drug penetration through the
stratum corneum [18]. As previously mentioned, many triterpenes exhibit significant biological activity, but some of their physicochemical properties can restrict their pharmaceutical and
cosmetic use [12]. The need for effective treatments is increasing. Lupeol is known to have
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New lupeol esters as active substances in the treatment of skin damage
Fig 1. The structures of lupeol (1) and synthesized esters (2-lupeol acetate, 3-lupeol propionate, 4-lupeol isonicotinate, 5-lupeol succinate, 6-lupeol
acetylsalicylate).
https://doi.org/10.1371/journal.pone.0214216.g001
many benefits for damaged skin. However, it should be noted that despite the wide range of
lupeol biological activity, due to high lipophilicity and poor solubility, its bioavailability is limited [19].
In some cases, a modification of the active substances structure can increase their penetration through the skin. The purpose of our studies was to obtain new derivatives of lupeol and
to evaluate their biological activity as potential agents dedicated for treatment of skin damage.
Five lupeol esters (2–6) (Fig 1) were evaluated for their effectiveness in topical formulations.
Four of the obtained compounds (3–6) were not described in scientific studies before. In order
to compare the bioavailability of lupeol and its esters, their cytotoxicity, cell proliferation stimulation activity and the ability to penetrate the epidermis were tested.
Materials and methods
The synthesis and the physicochemical characteristic of lupeol esters
The lupeol derivatives were obtained by an esterification process. Appropriate carboxylic acid
or carboxylic acid anhydrides were used as acylating agents to obtain lupeol esters: acetate (2),
propionate (3), isonicotinate (4), succinate (5) and acetylsalicylate (6). In the first stage of the
esterification process, lupeol (1 g; 2.3 mmol, Natchem) was dissolved in tetrahydrofurane (10
cm3, Avantor), then N-methylmorpholine (7.5 cm3, Sigma Aldrich) and a stoichiometric
excess (2 eq.) of carboxylic acid or its anhydride (all from Sigma Aldrich) was added to the
reaction mixture. Next, the mixture of N,N-dicyclohexylcarbodiimide and 4-dimethylaminopyridine (DCC-DMAP, both supplied by Sigma Aldrich) was applied as a catalyst (1.15 eq).
The reactions were carried out in reflux for 4 hours.
The progress of the reactions was controlled using the Thin Layer Chromatography (TLC)
method. Polygram Sil G/UV 254, Macherey Nagel TLC plates were used. The mixture of chloroform (Chempur) and ethyl acetate (Avantor) (9:1 v/v) was applied as an eluent. The TLC
plates were sprayed with 50% water solution of phosphoric acid (Chempur) with addition of
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New lupeol esters as active substances in the treatment of skin damage
10% isopropanol (Avantor) solution of vanillin (Sigma Aldrich) (10:1 v/v). Triterpenes became
visible after heating the plate at 100˚C for 2–3 minutes.
In each of the synthesis, the reaction mixtures were poured into 300 cm3 of 10% aqueous
solution of hydrochloric acid (Avantor). The organic phase was neutralized by washing it with
20% aqueous solution of sodium bicarbonate (Avantor) for three times (50 cm3 each time).
After that, the organic phase was dried with magnesium sulfate (Avantor) and concentrated to
1/3 of the original volume. The pure reaction products were precipitated from the solution as a
white solids. After the solvent evaporation, the solids were dried to a constant mass, at room
temperature and then crystallized from methanol and chloroform.
The melting points of the crystalline triterpenes were determined by Stuart SMP10 melting point apparatus. The UV-VIS spectra of the esters were measured using Macherey
Nagel Nanocolor UV-VIS Spectrophotometer (in the wavelength range from 200 to 400
nm). The applied concentration of each triterpene amounted to 2 mg/cm3. Ethanol was
used as a solvent and as a blank sample. The 1H NMR and 13C NMR analysis were recorded
in CDCl3 using Mercury-VX Varian, 300 and 75 MHz, respectively. IR spectra were measured for chloroform solutions by Nicolet S10 Spectrometer. The 2 μL injections of the 0.1
mg/cm3 samples dissolved in acetonitrile were analysed by RP-HPLC-MS method, using
Ascentis Express RP-Amide column (2.7 μm, 7.5 cm × 2.1 mm), in a gradient mode (80–
98% acetonitrile/water/0.1% formic acid for 7 min followed by isocratic 98% of acetonitrile
for next 20 min, flow rate 0.4 cm3/min, temperature 40˚C). The MS detection (Agilent VL)
was conducted in positive APCI ion mode with a mass range of 300–450 m/z, drying gas
flow rate of 6 dm3/min, temperature 350˚C, nebulizer pressure 60 psig, vaporizer temperature 500˚C, capillary voltage 5000V (positive and negative) and corona current in the range
of 5.0 μA.
Crystal data for 2, 3 and 5 were collected at 120 K on a SuperNova diffractometer equipped
with the microfocus X-ray source and AtlasS2 detector, using the Cu Kα radiation (λ =
1.54184 Å). The CRYSALIS program system [20] was used for data collection, cell refinement
and data reduction. The absorption corrections were applied by multi-scan method of Blessing
[21]. Using Olex2 and ShelXS [22,23] the structures were solved using direct methods and
refined with the ShelXL refinement package [23]. Due to very poor crystals of lupeol succinate
(5), the disordered oxygen atoms were refined isotropically. The hydrogen atoms were introduced at calculated positions and refined riding on their carrier atoms. The crystals were enantiomerically pure. The absolute configuration of the chiral molecules was determined by using
the Flack x [24] and Hooft y [25] parameters; however, they were of minor importance because
the compounds were weak anomalous scatters. Crystal data and structure refinement for three
lupeol esters: acetate, propionate and succinate are presented in Table 1. Crystal data for lupeol
isonicotinate (4) and lupeol acetylsalicylate (6) were not obtained because of the non-crystalline form of the compounds.
Table 1. Lupeol esters physicochemical properties (MP–melting point, RT- retention time, RF—retardation factor).
No
Name
MP [ºC]
Purity
Reaction yield [%]
1
Lupeol
213–215
96.8
-
208.8
UV/VIS max [nm]
8.9
RT [min]
0.65
RF [–]
2
Lupeol acetate
216–219
95.1
85.4
207.8
9.0
0.72
3
Lupeol propionate
220–222
93.3
70.9
208.5
10.2
0.78
4
Lupeol isonicotinate
179–183
95.7
66.4
209.8
10.4
0.81
5
Lupeol succinate
221–223
96.2
69.2
208.3
8.9
0.68
6
Lupeol acetylsalicylate
226–229
92.2
53.0
209.3
9.4
0.77
https://doi.org/10.1371/journal.pone.0214216.t001
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New lupeol esters as active substances in the treatment of skin damage
The evaluation of lipophilicity and potential therapeutic efficiency
Lipophilicity as one of the most important physicochemical property that influences skin barrier permeation should be always considered when new dermatological active ingredients are
synthesized [26]. The lipophilic character of the tested triterpenes was characterized experimentally and theoretically. The experimental lipophilicity of the tested esters was determined
through the calculation of chromatographic partition coefficients (RM), using reversed phase
thin layer chromatography (RP-TLC) [27]. Assays were performed in duplicate on reversephase thin-layer aluminum sheets: RP-18F254S (Merck), 20 × 20 cm. The developing system
was a mixture of 1,4-dioxane-acetate buffer (pH = 4.8), with concentrations of 1,4-dioxane in
the range of 60–90% (v / v) with a graduation rate of 5%. Buffer pH was measured with the
Seven Multi pH meter (Mettler Toledo). The chloroform solutions of the lupeol esters were
applied to a TLC plate and developed at 23 ± 1˚C. After drying, the chromatograms were generated by spraying with 20% sulfuric acid in methanol and heating at 100˚C for 2–3 min.
Retention factors (Rf) of the visible spots obtained in this process were calculated according to
Pyka and Miszczyk [28].
The results were compared to the theoretical prediction methods such as ACD Labs
Chemsketch 2012, v.14.01 [29], PubMed [30], Discovery Studio ver. 4.1 (BIOVIA) [31] databases and ChemAxon programme [32]. The PubMed database was not suitable to find values
of two of the lupeol esters, propionate and acetylsalicylate, as these compounds were not
described in scientific papers before. The theoretical water solubility (logSw) of the compounds obtained was used as a complementary parameter. LogSw was calculated according to
Cheng and Merz predictive model which gives information about compound ADMET [33].
ADMET calculations were prepared using PreADMET 2004, v. 1.0 [34]. The calculations of
toxicity of the described triterpenes were evaluated by Acute toxicity to Daphnia. Their solubility in buffer was calculated for pH 7.4 buffer system by SK atomic types (SKlogD distribution
coefficient).
The evaluation of cytotoxicity and proliferation activity
A Skin Irritation Test was prepared according to SOP (Standard Operation Protocol) of In
vitro Skin Irritation Test (ECVAM): Human Skin Model, EpiDerm-200, Version: 7.0, 30th Oct
2007. Reconstructed Human Epidermis (RHE) model represents place where a potential irritating substance acts and it shows the inflammation process which can appear after the exposure of the tissue to the irritating chemicals in vivo conditions. The method is based on cells
viability assay and give information if tested compound shows irritating effect and if it can be
classified into 2nd category according to UN GHS (United Nations Globally Harmonized System) and EU CLP (Globally Harmonized System of Classification and Labelling of Chemicals)
requirements [35].
Standard EpiDermFT kit (MatTek Corporation) consisted of 24 tissues. Costar Snapwell
single well tissue culture plate inserts had the surface area of about 1.0 cm2 and the diameter of
about 1.2 cm. Moreover inserts were equipped with pores (size 0.4 μm diameter). Before test,
all tissues were visually inspected if there are any physical imperfections. The media used
throughout the production process were checked for sterility. Furthermore, all cells were
screened and were negative for HIV, hepatitis B and hepatitis C using PCR (all the tests made
by MatTek). The culture medium was fed through a microporous membrane. The tissue contained 8–12 cell layers plus stratum corneum (basal, spinous, and granular layers) [35].
In vitro skin irritation test was prepared according to the procedure described by Kandarova et al. [35]. On the first day, tissues were topically exposed to the oil solutions of tested triterpenes. White crystals of each compound were dissolved in caprylic and capric triglyceride
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New lupeol esters as active substances in the treatment of skin damage
Fig 2. General scheme of the experiment with RHE (Reconstructed Human Epidermis) model and lupeol derivates
(PBS–phosphate buffer, i-PrOH–isopropanol, RT–room temperature, MTT—3-(4,5-methylthiazol-2-yl)2,5-diphenyltetrazolium bromide, OD–optical density).
https://doi.org/10.1371/journal.pone.0214216.g002
(Crodamol GTCC, Croda). The esters concentration in the solutions was 0.5% (m/m). Three
tissues were used for each tested substance, as well as for the positive and negative control. Positive control solution consisted of 5% aq. solution of SDS (Sodium Dodecyl Sulfate, Mattek),
and a negative control solution consisted of Caprylic and Capric Triglycerides (Crodamol
GTCC, Croda). Chemical exposure time was 60 min, during which, for 35 min, the tissues
were kept in an incubator at 37˚C (chamber Heraeus, Kendro). The test substances were then
removed from the tissue surface by an extensive washing procedure using DPBS (phosphatebuffered saline, pH = 7.4, Mattek). The tissue inserts were blotted and transferred to fresh
medium. A scheme of the procedure is shown in Fig 2.
After 24 hours of incubation, the medium was exchanged and the tissues were incubated
for an additional 18 hours. After 42 hours of incubation time, the tissues were transferred into
a yellow solution of MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide,
Mattek) and incubated for a further 3 hours. The resultant purple-blue formazan salt, formed
mainly by mitochondrial metabolism, was extracted after 2 hours with isopropanol, pure (Mattek). The optical density (OD570) of the extracted formazan solution was determined using a
spectrophotometer (Biotek, PowerWave XS). Cells viability assay was calculated referring to
the negative control tissues. A substance is classified as an irritant if the tissue viability relative
to the negative control treated tissues is reduced below 50%.
The cytotoxicity assay was used to determine the toxicity of lupeol esters. Cell viability was
assessed by the Cytotoxicity Detection Kit (Roche), a colorimetric assay based on the measurement of lactate dehydrogenase (LDH) activity released from the cytosol of damaged cells into
the supernatant. The method is based on the reduction of MTS tetrazolium compound by viable cells to generate a colored product soluble in cell culture media. The assay was performed
according to the manufacturer’s protocol. Cells were seeded in 96-well plates (fibroblasts) or in
12-well plates (keratinocytes). Keratinocytes isolated from the epidermis from 3 different
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New lupeol esters as active substances in the treatment of skin damage
donors were plated in KGM-Gold medium (Lonza) at a density of 5–20 x 103 per cm2. Fibroblasts isolated from the dermis from 3 different donors were plated in DMEM medium supplemented with 10% fetal bovine serum at a density of 5–20 x 103 per cm2. After 24 hours, the
medium was changed to fresh with the addition of lupeol esters at concentrations of 100μM in
dimethyl sulfoxide (DMSO, Sigma Aldrich). Cells were than incubated with test substances or
vehicle control for 48 hours (37˚C, 5% CO2, 95% humidity). Maximum LDH release was
induced with 1% (v/v) Triton X100 in assay medium. The absorbance of the samples was measured at 490 nm with the reference wavelength at 620 nm using the VersaMax ELISA Microplate Reader (Molecular Devices). The experiments were run in triplicate. The cytotoxicity of
the tested compounds was determined by the estimation of cells viability of incubated samples
compared to the control.
Results and discussion
The synthesis and the physicochemical characteristic of lupeol esters
Four new lupeol derivatives (propionate, succinate, isonicotinate and acetylsalicylate) and
lupeol acetate were obtained by lupeol esterification using various acids or acid anhydrides.
As mentioned previously, compounds 3–6 (Fig 1) are novel structures. Compound 2
(lupeol acetate) has been described in the literature as biologically active lupeol derivative. It
shows similar activity as lupeol but it exhibits better bioavailability. Lupeol acetate significantly
decreases rheumatoidal arthritis symptoms by inhibition of inflammatory cytokines expression [19]. This anti-inflammatory activity is very significant during skin regeneration process.
Chemical modification of lupeol can alter not only biological activity of the obtained derivative
but can also improve its bioavailability and effectiveness. The general procedure of lupeol
esterification described by Vasnev et al. [36] requires the application of catalysts and solvent
with relevant high cytotoxicity. The conventional reaction is carried out in dichloromethane
and pyridine. DMAP (4-dimethylaminopyridine) plays, in the reaction, the role of a catalyst.
Considering the application of the synthetized compounds as the active ingredients in skin
care products, these hazardous substances were replaced by another less harmful and safer
ones.
Table 1 presents the values of the melting points, purities, reaction yields and maximum
absorbance wave lengths of the tested compounds. The esterification yields ranged from 64.8
to 87.9% which is satisfactory in comparison to similar synthesis of lupeol derivatives [37,38].
All of the obtained esters had form of white solids. The purity of the obtained compounds was
relatively high (92.9–96.2%), especially as the substrate, natural lupeol (96.8%) was most probably contaminated with other triterpenes and secondary birch metabolites. An extraction of
the natural lupeol from plant material may result in the presence of other lipophilic triterpene
compound like 3-epi-lupeol, α-amyrin or β-amyrin [39]. Melting points of the obtained esters
were higher than the temperature of the alcohol substrate. The narrow range of the melting
points values confirmed a high purity of the synthesised substances.
The molecular structure of the compounds obtained were confirmed using spectroscopic
methods (1HNMR, 13CNMR, IR) and by MS-APCI and CHN methods (see: S1 File).
MS-APCI analysis shows that there are insignificant impurities present in lupeol esters which
are not visible at lupeol chromatogram. Intense ion mass signals of 409.3 ([M+H-H2O]+ of
lupeol) and 423.3 m/z, which are also present in lupeol standard (analytical standard, Sigma
Aldrich) are described by Khan et al. as characteristic for lupeol triterpene [40].
The X-ray crystal structure analysis of esters 2, 3 and 5 gave an unambiguous confirmation
of the successful syntheses of new enantiopure esters of lupeol. The R–C (= O)–O–ester parts
of the molecules are perpendicular to the mean plane of the triterpenoid fragment in (2) and
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New lupeol esters as active substances in the treatment of skin damage
(3), whereas in 5 the alkyl chain has a bent conformation with the carboxylic group being disordered in the crystal over two positions (Fig 3).
The positional disorder observed in compound 5 (lupeol succinate) is probably associated
with the necessity to adjust the molecular conformation to the packing during crystallization
of only one enantiomer. In all of the analysed crystals a “head to head” packing is observed as
in the crystal of lupeol [1]. However, the introducing of the less hydrophilic ester groups
instead of hydroxyl one, causes that the molecules interact mainly through weak C–H. . .O contacts. This change in intermolecular interactions explains a poor quality of ester crystals and
simultaneously decreases their solubility in polar solvents. Only in lupeol succinate (5) the
stronger intermolecular interactions are observed. The molecules form dimers through O–
H. . .O hydrogen bonds between the disordered carboxylic groups. These results correlate well
with the lipophilicity determination data, which show that lupeol succinate (5) is the less
Fig 3. Molecular structures of 2 (lupeol acetate), 3 (lupeol propionate) and 5 (lupeol succinate) with shown two
positions of the disordered ester group.
https://doi.org/10.1371/journal.pone.0214216.g003
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New lupeol esters as active substances in the treatment of skin damage
Table 2. Crystal data and structure refinement for 2- lupeol acetate, 3- lupeol propionate and 5-lupeol succinate.
ID
2
3
Empirical formula
C32H52O2
C33H54O2
5
C34H54O4
Crystal system
orthorhombic
monoclinic
monoclinic
Space group
P212121
C2
C2
14.269(2)
a/Å
8.0837(2)
14.341(2)
b/Å
21.6854(5)
6.5377(7)
6.6401(8)
c/Å
47.385(1)
30.880(3)
31.787(5)
β/˚
90
96.70(1)
96.31(1)
Volume/Å3
8306.5(3)
2875.4(5)
2993.6(7)
Z, Z’
12, 3
4, 1
4, 1
ρcalcg/cm3
1.124
1.115
1.169
μ/mm-1
0.508
0.502
0.576
F(000)
3120.0
1072.0
1160.0
Crystal size/mm3
0.4 × 0.08 × 0.05
0.3 × 0.15 × 0.05
0.3 × 0.3 × 0.05
Reflections collected
58157
20119
9772
Independent reflections
15034 [Rint = 0.0982,
Rsigma = 0.0804]
5193 [Rint = 0.1028,
Rsigma = 0.0751]
4597 [Rint = 0.0749,
Rsigma = 0.0982]
Data/restraints/parameters
15034/0/967
5193/1/324
4597/1/366
2
1.016
1.080
1.047
Final R indexes [I> = 2σ (I)]
R1 = 0.0564,
wR2 = 0.1382
R1 = 0.0956,
wR2 = 0.2182
R1 = 0.0938,
wR2 = 0.2500
Final R indexes [all data]
R1 = 0.0685,
wR2 = 0.1500
R1 = 0.0876,
wR2 = 0.2385
R1 = 0.1129,
wR2 = 0.2720
Goodness-of-fit on F
Largest diff. peak/hole / e Å-3
0.36/-0.26
0.52/-0.34
0.61/-0.44
Flack x parameter
0.1(2)
0.1(5)
0.2(5)
Hooft y parameter
0.1(2)
0.6(3)
0.1(4)
CCDC No.
1487997
1487998
1487999
https://doi.org/10.1371/journal.pone.0214216.t002
hydrophobic in the studied group of esters, whereas acetate (2) and propionate (3) are more
lipophilic than lupeol (1).
The obtained crystal data and structure refinement for lupeol acetate (2), lupeol propionate
(3) and lupeol succinate (5) are presented in Table 2.
The evaluation of lipophilicity and potential therapeutic efficiency
RP-HPLC-MS method not only determined the purity of the triterpenes and confirmed the
preservation of the lupeol core after chemical processing, but also revealed differences in esters
hydrophobicity (Rf and RT values). Propionate and acetylsalicylate lupeol derivatives and to a
lesser extent the lupeol acetate exhibit a higher hydrophobicity than the parental lupeol. Such a
tendency was confirmed by the logP values (Table 3).
Data in Table 3 show the lupeol esters lipophilicity, expressed as a logarithm of octanol–
water partition coefficient (logPOW) determined by different methods: experimental (RP-TLC
method) [27] and theoretical (ACD/logP [29], XlogP3 PubMed [30], AlogP and Chemaxon
logP [32]). The values of logD were theoretically determined with Chemaxon and ACD
Chemsketch [29].The values of logSw have been also theoretically evaluated [33]. The example
relation between pH and logP as well as logD values are presented in S1 File. The calculations
were prepared using Calculator Plugins of Chemicalize.
The data shown in the Table 3 indicate that in all cases the lipophilicity of the obtained
lupeol esters is higher than the starting triterpene alcohol. The lowest values of octanol / water
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New lupeol esters as active substances in the treatment of skin damage
Table 3. Lupeol esters lipophilic properties (RM0, ACD/logP, XlogP3, AlogP and logSw), RT- retention time, Rf—Retardation factor.
logP (RM0)
RP-TLC method
ACD/logP
XlogP3 PubMed
AlogP
ACD/logD
pH 5.5 / pH 7.4
Chemaxon logD
pH 7.4
logSw
No
Name
1
Lupeol
7.67+0.07
10.98+/- 0.38
9.9
7.403
9.41/
9.41
7.45
-8.757
2
Lupeol acetate
8.12+0.03
11.87+/- 0.40
10.4
7.782
10.92/
10.92
7.889
-9.565
3
Lupeol propionate
8.23+0.09
12.41+/- 0.40
-
8.449
-
8.59
-9.994
4
Lupeol isonicotinate
7.98+0.04
12,58+/- 0,41
-
7,627
-
8.73
-9.998
5
Lupeol succinate
8.34+0.08
11.49+/- 0.53
10.3
7.627
8.99/
7.19
4.72
-8.479
6
Lupeol acetylsalicylate
9.76+0.08
13.17+/- 0.57
-
9.214
-
9.55
-10.005
https://doi.org/10.1371/journal.pone.0214216.t003
partition coefficient logarithm were obtained for lupeol succinate (from 7.623 to 11.49, according to applied method) and the most lipophilic compound was lupeol acetylsalicylate (logP values ranged from 9.214 to 13.17). Experimental values comply with the theoretical ones.
Simultaneously, lupeol exhibited the lowest logP values (from 7.403 to 10.980) which shows
that its structure modification influences its compound character.
Potential therapeutic agents are evaluated at early stages of drug development based on
computational modeling, high throughput screening and cell-based assays that predict their
pharmacologic activity. Predicting the compound absorption, distribution, metabolism, excretion and toxicity (ADMET) is much more complicated. The rule of five which is widely used
to predict these processes for transdermal way of drugs application [41]. The studied triterpenes comply the three requirements of the Rule of Five: molecular weight MW (< 500), number of H-bond donors NHD (< 5) and number of H-bond acceptors NHA (< 10). The
predicted values of octanol partition coefficient logarithm logP exceed the expected value
logP < 5 (Table 2). All of the tested compounds are insoluble in water (logSw between -8.5
and -10.0) but highly soluble in organic solvents. The esterification of lupeol hydroxyl group
increased the logP parameter. The permeation tests of lupeol, however, confirm that lupeol
can absorb into skin structures [14,42]. The stratum corneum permeation tests show that the
permeability for lipophilic compounds is not homogenous and generally decreases rapidly
with depth. It has been diagnosed by Raman spectroscopy that the lipophilic compounds, like
terpenes, act within stratum corneum. The absorption profile of terpene beta-carotene in the
near IR region extends to about 800 nm [43]. The logP restriction which is described by the
Rule of Five concerns molecules dedicated to the deeper skin layers including the dermis layer
or drugs which are supposed to permeate into the bloodstream [40]. Compounds with high
lipophilicity will not absorb into dermis but will act within epidermis layer. Therefore, high
lipophilic character is crucial for biologically active substances that should act within the stratum corneum structures [1] and the studied triterpene compounds may penetrate the upper
skin layers. Notably, the esterification of lupeol hydroxyl group causes the increase of logP
wherein it is dependent on acyl group structure. Taking into consideration the fact that the
stratum corneum is a highly lipophilic medium, triterpene compounds are suitable active substances that acts in upper skin layers. The values of logD for lupeol isonicotinate (4) and lupeol
succinate (5) are dependent on pH values as shown for theoretical estimations (see S1 File).
The calculations of these values can be based on the consideration of microscopic dissociation
constants (microconstants), the partition coefficients of the microspecies for the compound
and the counterion concentration [44]. However, our calculations suggests, that only in case of
lupeol succinate (5) the slightly acidic pH of skin (5.0–5.5) will have significant influence on its
PLOS ONE | https://doi.org/10.1371/journal.pone.0214216 March 28, 2019
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New lupeol esters as active substances in the treatment of skin damage
Table 4. The selected ADMET calculations for lupeol (1) and its derivatives (2-lupeol acetate, 3-lupeol propionate, 4-lupeol isonicotinate, 5-lupeol succinate,
6-lupeol acetylsalicylate).
ID
1
3
2
3
4
5
6
PBS (pH = 7.4) solubility [mg/dm ]
1.95
2.00
0.82
0.17
24.38
0.03
Water solubility [mg/dm3] x10-3
1.42
0.67
0.18
0.32
1.63
0.01
SKlogD value
7.48
7.78
8.42
6.43
6.43
9.16
Toxicity (48hrs, EC50 in mg/dm3) x 10−3
3.05
1.70
1.33
1.06
1.55
0.29
https://doi.org/10.1371/journal.pone.0214216.t004
hydrophobicity (i.e. increased value of logD due to partial protonation of free carboxyl group
of succinate).
ADMET calculations were prepared using PreADMET 2004, v. 1.0. Numerous in vitro
methods have been used in the drug selection process for assessing the transdermal absorption
of lupeol triterpene and the obtained esters. Among others, the in silico skin permeability
model could predict and identify potential drugs for transdermal delivery. The calculations of
toxicity of the described triterpenes were evaluated by Acute toxicity to Daphnia. Tested compounds, as potential active ingredients in cosmetic and pharmaceutical industry, offer a wide
range of benefits, but they also have a lot of limitations, such as potential environmental toxicity. When we consider new structures, which have not been present in natural environment
before, it is important to ensure that level of their toxicity should be below the levels presenting
an unacceptable risk to humans. Computational modeling approach such as Quantitative
Structure—Activity Relationships (QSARs) are widely used to evaluate this property and
Daphnia is a basic model considered for these calculations. The prime aim of the calculations
is to generate models for accurate and reliable predictions of unknown compounds properties.
Models are based on the experimental acute toxicity data of various compounds against Daphnia. After collection of the experimental data, they were carefully screened for particular endpoints and same exposure time in order to get reliable predictions from the standardized data
[45].
Moreover, the solubility in phosphoric buffer (PBS) pH 7.4 was calculated for all of the
compounds. The SKlogD values were calculated from the computed logP and pKa, according
to the equation logD = logP − log (1+10pH−pKa) and pH = 7.4 [32] (Table 4).
Extremely low values of water solubility confirm lipophilic character of the tested triterpenes. On the other hand, higher buffer solubility (especially 24.38 mg/dm3 for lupeol succinate) provides the possibility of the application of this ester as aqueous phase ingredient in
emulsion formulation. Low toxicity of the obtained lupeol esters leads to the conclusion that
they are potentially safe components for cosmetic or pharmaceutical formulations for topical
applications. All of the tested compounds exhibit excellent ability of binding to plasma protein.
The plasma protein binding in all the cases was 100% and the values were calculated according
to human albumin. The ability values confirm the therapeutic efficiency of the tested triterpene
compounds.
The evaluation of cytotoxicity and proliferation activity
The test of the compound irritancy was performed for three fragments of RHE model for each
compound to obtain an estimation of its cytotoxic activity. Cells viability assay was calculated
from mean optical density (OD570) value in colorimetric MTT test. Table 5 presents the cells
viability assay for the tested triterpenes as well as for the positive and negative control. Repeatability of the obtained results for the positive control referring to the negative control was no
higher than 20%. For the analysis in triplicates of the tested solutions, the repeatability was
determined with standard deviation values.
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New lupeol esters as active substances in the treatment of skin damage
Table 5. Viability assay of the cells in vivo conditions after exposure to tested triterpenes 1–6.
Method
Tested cells
Viability [%] / SD
1
2
3
4
5
6
NC
PC
Viability
Epiderm
89.2
/6.0
131.2/
19.6
133/
17.6
134.1/
10.2
120.0/
12.1
120.9/
7.5
100.0/
10.2
28.1/
10.2
LDH
keratinocytes
91.6/
0.4
98.1/
2.5
94.7/
3.1
101/
0.5
93.2/
4.0
91.5/
2.1
101.0/
2.3
-
fibroblasts
100.0/
0.5
100.9/
0.4
101.1/
0.2
101/
0.69
102.0/
0.6
101/
0.7
100.6/
0.7
-
(NC-negative control, PC–positive control), LDH cytotoxicity assay (SD-standard deviation, n = 3)
https://doi.org/10.1371/journal.pone.0214216.t005
The data shown in Table confirm that all of the synthesized compounds have no irritating
properties. Positive control (PC, 28.1% cells viability) used in cell viability assay caused cells
destruction process. Negative control (NC—100% cells viability) was a reference sample which
had no effect on cell destruction as well as their proliferation. The cells viability observed for
lupeol was slightly lower than for NC. Whereas the proliferation activity for the esters reached
over 30% of the initial cell concentration. The most effective in causing cell proliferation were
compounds 2–4. (Table 5). Cytotoxicity LDH assay proves safety of use for all tested compounds. There were no toxic properties for human keratinocytes and fibroblasts observed for
lupeol esters solutions.
Conclusion
The chemical modification of lupeol is an efficient way to obtain new compounds with desirable properties. The esterification of lupeol’s hydroxyl group resulted in the increased lipophilicity of the obtained esters with respect to the initial compound, enabling the compounds to
precisely target the hydrophobic skin sub-structures. None of the studied triterpenes showed
cytotoxic activity. The chemical modification also changed the biological activity of the alcohol, giving the new compounds better skin recovery properties, stimulating human skin cells
proliferation. Lupeol isonicotinate, acetate and propionate (2–4) were the most effective compounds in stimulating human skin cell proliferation resulting in more than 30% increase of
cell concentration in comparison to control samples. The lupeol esters presented here show
promising activity in stimulating skin repairing processes and can be applied as active substances in formulations for topical application especially in treatment of skin burns.
Supporting information
S1 File. Physicochemical properties of the obtained compounds: physical form, melting
point, reaction yield, compound purity, absorption maximum, data for IR and NMR spectra interpretation, data for elementary analysis and MS-APCI analysis, logP and logD values at various pH.
(DOCX)
Acknowledgments
The research was carried out with equipment purchased thanks to the financial support of the
European Regional Development Fund in the framework of the Operational Program Development of Eastern Poland 2007–2013 (Contract No. POPW.01.03.00-06-009/11-00 Equipping
the laboratories of the Faculties of Biology and Biotechnology, Mathematics, Physics and
PLOS ONE | https://doi.org/10.1371/journal.pone.0214216 March 28, 2019
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New lupeol esters as active substances in the treatment of skin damage
Informatics, and Chemistry for studies of biologically active substances and environmental
samples).
The calculations in Discovery Studio (BIOVIA) were supported by PL-Grid Infrastructure
(AGH CYFRONET).
Calculator Plugins of Chemicalize was used for structure property prediction and calculation, (accessed 01.2019, https://chemicalize.com/) developed by ChemAxon (http://www.
chemaxon.com).
The Authors would like to thank Prof. Justyna Drukala, Dr. Julia Borowczyk-Michalowska
and Ms. Joanna Stalinska (Department of Cell Biology, Faculty of Biochemistry, Biophysics
and Biotechnology, Jagiellonian University) for determining keratinocytes and fibroblasts
viability.
Author Contributions
Conceptualization: Magdalena Malinowska, Elzbieta Sikora.
Formal analysis: Magdalena Malinowska, Barbara Miroslaw, Agnieszka M. Wojtkiewicz,
Maciej Szaleniec.
Funding acquisition: Magdalena Malinowska.
Investigation: Magdalena Malinowska.
Methodology: Magdalena Malinowska, Elzbieta Sikora, Jan Ogonowski, Monika PasikowskaPiwko.
Resources: Monika Pasikowska-Piwko.
Software: Barbara Miroslaw, Maciej Szaleniec.
Supervision: Elzbieta Sikora, Jan Ogonowski, Irena Eris.
Writing – original draft: Magdalena Malinowska.
Writing – review & editing: Magdalena Malinowska, Barbara Miroslaw, Elzbieta Sikora,
Agnieszka M. Wojtkiewicz, Maciej Szaleniec, Monika Pasikowska-Piwko.
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