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A multiscale study of the penetration-
enhancing mechanism of menthol
Liping Chen
a,b
, Lina Ma
a,b
, Shufang Yang
a,b
, Xiaowen Wu
a,b
,
Xingxing Dai
a,b
, Shifeng Wang
c
, Xinyuan Shi
a,b,
*
a
School of Chinese Materia Medica, Beijing University of Chinese Medicine, Beijing 102488, China
b
Key Laboratory for Production Process Control and Quality Evaluation of Traditional Chinese
Medicine, Beijing Municipal Science & Technology Commission, Beijing 100029, China
c
Department of Molecular, Cellular and Developmental Biology, University of Colorado, Boulder, CO
80309, USA
Received 23 July 2019; received in revised form 15 October 2019; accepted 16 October 2019
Available online 21 October 2019
KEYWORDS
Menthol;
Molecular dynamics
simulations;
Permeability
Abstract Objective: Transdermal drug delivery systems represent a critical focus in the phar-
maceutics field; however, their use is limited by the fact that many drugs usually pass through
the skin with low permeability. Menthol is a common penetration enhancer because of its high
penetration-enhancing efficiency and safety. Our research aimed to reveal the penetration-
enhancing mechanisms of menthol via a multiscale study.
Methods: First, the interaction of menthol with the stratum corneum was studied using verti-
cal Franz diffusion cells obtained from the abdominal skin of rats as a model. Then, the skin
samples were observed via transmission electron microscopy. Finally, the interaction of
different concentrations of menthol with a mixed lipid model of the stratum corneum was
investigated via molecular dynamics simulation using the GROMOS 54A7 force field on a micro-
cosmic level.
Results: At concentrations of 3.5% or lower, menthol changed the original structure of the stra-
tum corneum to varying degrees, which increased its fluidity and facilitated the permeation
and storage of menthol. Menthol increased the fluidity of the stratum corneum mainly via
two mechanisms. First, menthol had strong hydrogen-bonding capability, and it could compete
for the lipidelipid hydrogen bonding sites, thereby weakening the stability of the hydrogen-
bonding network connecting the skin lipids. In addition, menthol had strong affinity for choles-
terol, probably due to their similar molecular structures, suggesting that the incorporation of
menthol would increase the fluidity of the lipid membrane similarly to cholesterol.
Conclusion: The penetration-enhancing mechanism of menthol was explained using in vitro
and molecular dynamics simulation methods. These findings may advance the basic research
* Corresponding author.
E-mail address: shixinyuan01@163.com (X. Shi).
Peer review under responsibility of Beijing University of Chinese Medicine.
https://doi.org/10.1016/j.jtcms.2019.10.001
2095-7548/ª2019 Beijing University of Chinese Medicine. Production and hosting by Elsevier B.V. This is an open access article under the CC
BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Available online at www.sciencedirect.com
ScienceDirect
journal homepage: http://www.elsevier.com/locate/jtcms
Journal of Traditional Chinese Medical Sciences (2019) 6, 347e354
of transdermal drug delivery systems and facilitate the discoveries of novel penetration en-
hancers.
ª2019 Beijing University of Chinese Medicine. Production and hosting by Elsevier B.V. This is
an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/
by-nc-nd/4.0/).
Introduction
At present, higher standards have been enacted in terms of
precision, compliance, and convenience regarding routes of
drug delivery.
1
In the pharmaceutics field, transdermal drug
delivery systems (TDDSs) have attracted increasing atten-
tion and recognition.
2,3
Compared with oral drug adminis-
tration, TDDSs can avoid the hepatic first-pass effect and
gastrointestinal inactivation. They can also maintain a
constant systemic drug level and prolong the curative ef-
fects of drugs. Despite these advantages, the use of TDDSs
is limited because most drugs usually pass the skin with low
permeability.
4
It has become clear that the stratum cor-
neum (SC) constitutes the first but crucial obstacle to the
penetration of exogenous substances into or via human
skin. Regarding the intact SC, the intercellular space is
filled with lamellar hydrated lipid bilayers of ceramides
(CERs), fatty acids, and cholesterol (CHOL), which take
continuous crystalline, semicrystalline, gel, and other
forms.
5,6
Drugs mostly penetrate through this intercellular
route in the SC; therefore, penetration enhancers are often
used to interact with skin lipids to disrupt these domains
and assist in the delivery of drugs across the SC.
7,8
Menthol, derived from Mentha haplocalyx BRIQ., is one
of the most commonly used skin penetration enhancers.
9e11
However, the mechanism by which menthol increases skin
permeability at the molecular level remains unknown, and
uncovering this mechanism would meaningfully improve the
development of novel TDDSs.
In general, understanding biological mechanisms require
knowledge of both the detailed structural features of the
biological systems and the characteristics of their dynamic
behaviors, which cannot generally be ascertained using
conventional experimental methods. With the growth of
computer simulation techniques, molecular dynamics (MD)
has become a novel means for exploring biological mecha-
nisms, and considerable progress has been made in related
fields.
12,13
Therefore, MD simulations represent a good
strategy for studying the mechanisms by which menthol
enhances the permeability of drugs through the skin.
Recently, MD simulations have been used to study the
enhancement mechanism. For example, Wang et al used a
coarse-grained simulation to study the penetration mech-
anism of menthol.
14
This simulates a long time scale and a
large spatial scale at the mesoscopic level but blurs the
interaction between molecules compared with all-atom
simulations. Huang et al used an all-atom simulation to
calculate the potential of mean force between substances,
providing a mechanism by which menthol can promote
quercetin uptake.
15
However, those studies mainly focused
on simulation to explain the enhancement mechanism.
Thus, in this study, the effects of menthol on the SC were
observed using in vitro experiments and transmission
electron microscope (TEM, Japan Electron Optics Labora-
tory, Tokyo, Japan). Then, an all-atom simulation was used
to construct the SC model, and different concentrations of
menthol were added to study the mechanism by which
menthol affects the structure of the SC to explain the
enhancement mechanism of menthol.
Using MD simulations with the united atom GROMOS 54A7
force field,
16
we investigated the interaction of menthol
with a mixed skin lipid bilayer in this study. The lipid matrix
in the SC was modeled as a mixed lipid bilayer consisting of
CER 2, CHOL, and C24:0 free fatty acid (FFA) in a 2:2:1 M
ratio. In fact, many types of ceramide have been found in
the SC, but we could not represent all of them in our lipid
model because of computational overload when performing
atomistic simulations. To our knowledge, the most abun-
dant species among the ceramide family in the human SC is
CER 2, and the most common fatty acid chain length is
C24.
17
Therefore, we selected CER 2 as the representative
ceramide. Fig. 1 shows the 2-dimensional molecular struc-
ture and the united atom model of CER 2. Similarly, the
selected FFA was C24:0 based on its relative abundance in
the SC lipid layer.
18
In addition, we chose a mixed lipid
bilayer with a molar ratio composition of 2:2:1.
19,20
Excluding conventional experiments, we report MD simu-
lations of the mixed lipid bilayer using different concen-
trations of menthol in water in expectation of the discovery
of the transdermal penetration enhancement mechanisms
of menthol.
Materials and methods
Drugs and chemicals
Menthol (purity 98%) was obtained from the National
Institute for Food and Drug Control (Beijing, China).
Phosphate-buffered saline (PBS) was obtained from Beijing
Solarbio Science & Technology (Beijing, China). Tween-80,
1,2-propanediol (PG), and other solvents were of analytical
or high performance liquid chromatography grade, and all
Fig. 1 Two/three-dimensional structure of ceramide 2. (a)
Two-dimensional molecular structure; (b) United atom model
of ceramide 2.
348 L. Chen et al.
were obtained from Beijing Chemical Works (Beijing,
China).
Preparation of samples and skin
An appropriate weight of menthol was dissolved in a water/
PG (1:4 v/v) mixture and subsequently was diluted to five
concentrations (0 [control group], 1.0, 2.0, 3.5, and 5.0%
[m/m]).
Male SpragueeDawley rats (200 10 g, Sibeifu Labora-
tory Animals) were sacrificed after anesthesia. After
shaving off their abdominal hair, skin samples were excised
from each animal. Then, the subcutaneous tissue was
removed to make the surface smooth. Finally, samples
were obtained after washing in PBS.
Skin permeation
Skin permeation was examined using vertical Franz diffu-
sion cells. Tween-80 (1.5% in PBS, pH 7.0e7.2, 0.01 M,
15 mL), maintained at 34 C, was used as the diffusion
medium. The magnetic stirring rate was set at 300 rpm, and
the effective diffusion area was 1.23 cm
2
. The donor
chambers were filled with samples of different concentra-
tions (2 mL each). At predetermined time intervals, sam-
ples (2 mL) were removed from each receptor chamber and
replaced with an equal volume of fresh receiver solution.
Samples were analyzed via gas chromatography (GC).
21
GC analysis
The concentration of menthol was determined by GC using
a HP-5 capillary column (30 m 0.32 mm 0.25 mm). The
flow rate of nitrogen (carrier gas) was 1 mL $min
-1
. The
column temperature of the GC oven was programmed as
follows: starting at an initial temperature of 100 C,
increased by 2 C$min
1
to 120 C, and then increased by
15 C$min
1
to 240 C (2 minutes). All samples (2 mL) were
injected in a split ratio (2:1). The temperature of the
injector and FID detector was 250 C.
TEM studies
After the permeation experiment, skin samples were stored in
2.5% glutaraldehyde. First, the skin samples were cut into
cubes (1 cm
3
) and washed three times in PBS to remove the
glutaraldehyde. Then, the skin samples were fixed in 1% osmic
acid. The samples were dehydrated in different concentra-
tions of acetone solutions. The tissues cubes were then sub-
jected to gradient infiltration in a mixture of different
concentrations of epoxy resin and acetone. The slices were
next embedded in the resinunder different temperatures, and
thin sections were obtained using an ultramicrotome. After
double staining with uranyl acetate and lead citrate, the
samples finally were observed via TEM at 80 kV.
22
Force field
The geometry of the three types of skin lipids and menthol
molecules were optimized using the Materials Studio 6.0
package, and then we submitted the optimized molecules
to the PRODRG server (http://davapc1.bioch.dundee.ac.
uk/cgi-bin/prodrg, access at November 20, 2018) and
obtained molecular topologies for use with GROMACS
(version 4.6.3).
23
The models for the three types of skin
lipids and the penetration enhancer were built with the
GROMOS 54A7 force field using the parameters of bonded
and nonbonded interactions derived from their molecular
topology files. The water model we chose was a simple
point charge (SPC).
24
Simulation system
The mixed skin lipid bilayer comprised 320 molecules, with
160 molecules in each leaflet. For the starting configura-
tion, the three different lipid molecules were randomly
arranged laterally in the bilayer. As the united atom MD of
such a complicated system is relatively slow, we restrained
the positions of both the head and tail atoms of each lipid
molecules to make the starting configuration of the bilayer
as similar to its equilibrated structure as possible using the
Packmol package.
25
The box type was defined as a cube. Surrounded by
water, the bilayer was packed in the middle, and the lipid/
water molecule ratio was 1:15. Menthol molecules were
added randomly to the aqueous region on both sides of the
bilayer in consideration of the periodic boundary condition
in simulations. An initial system is shown in Fig. 2.
This study involved five simulations for different con-
centrations of menthol. The mass fraction of menthol with
regard to the solvent water was set at 0, 3, 7, 10, or 15%.
The details are presented in Table 1. Note that MD simu-
lation underestimates the thermodynamic properties of the
reaction, making it difficult to match the quantitative
results.
Fig. 2 Initial configuration of the 7% menthol system. The
mixed lipid bilayer consisted of ceramide 2 (red), cholesterol
(yellow), and C24:0 free fatty acid (green). Water and menthol
are colored cyan and purple, respectively.
Penetration-enhancing mechanism of menthol 349
Simulation detail
In the research, all MD simulations were performed using
the GROMACS software package with single precision,
26
and
the molecular systems were displaying using the VMD pro-
gram (version 1.9.2).
27
First, the initial configurations were
relaxed through energy minimization to ensure that there
were no steric clashes or inappropriate geometries in the
simulation systems. To simulate real dynamics, we equili-
brated the lateral positions of the lipids and the solution
around the bilayers. An equilibration time of 5 ns was
chosen to bring the area per lipid (APL) of the bilayer to an
equilibrium value. In the equilibration phase, we arrived at
the correct temperature based on the kinetic energies of
these systems. The MD simulations were conducted in the
NPT ensemble using the methods of the velocity rescaling
thermostat
28
and Berendsen barostat.
29
The temperature
was set to 310 K. In the bilayer system, the pressure was
altered via semi-isotropic pressure coupling with a
compressibility of 4.5 10
5
bar
1
.
30
The time step was
1 fs. The cutoff for van der Waals and coulomb interactions
was 1.2 nm. We finally obtained 13.5-ns trajectory data for
the calculation of equilibrium properties.
Results
Skin permeation studies
Fig. 3 shows the cumulative amounts of different concen-
trations of menthol, from which Qn-t equations could be
obtained. Table 2 shows the equations’ corresponding
assessment parameters. The results illustrated that when
the concentration of menthol was 3.5% or lower, the
permeation rate and residence time of menthol itself
increased as the menthol concentration increased, indi-
cating that increasing the concentration was beneficial to
the transdermal penetration of menthol itself and the
ability of the skin to store menthol. When the concentra-
tion of menthol reached 5.0%, the transdermal rate of
menthol increased significantly, whereas the residence
time decreased. This indicated that the skin’s storage ca-
pacity for menthol was reduced at this concentration, and
menthol tended to penetrate the skin quickly.
TEM studies
As shown in Fig. 4, the ultrastructure of the SC is composed
of several unique lipid layers. In the untreated group
(Fig. 4a), the SC had a lamellar structure in which a plu-
rality of layers of lipid cells was arranged neatly and
closely. In the control group (Fig. 4b), the SC morphology
did not change, but it was slightly curved. When 1% menthol
was present (Fig. 4c), the structure of the SC remained
unaffected, but the number of wrinkles increased and the
gap between the layers became larger than that in the
untreated and control groups, leading to increases in
fluidity. When 2 or 3.5% menthol was present (Fig. 4e and
f), the SC displayed obvious peeling and shedding. There
was a cross between the layers, and thus, the SC became
disordered. Meanwhile, 5% menthol seriously damaged the
structure of the SC and induced lipid cells to form a single-
layer vacuolar structure. Worse, the complete SC
morphology could not be observed at 20 000
magnification.
APL analysis
The APL is a valuable parameter for examining the phase of
the membrane through the packing of the headgroups of
lipids. It is calculated by dividing the average box area by
half the amount of lipids in the system. The resulting APLs
are shown in Fig. 5, and we can see that APL increases as
the menthol concentration increased. It should be noted
that the number of menthol molecules added in our system
was low compared with the total number of lipids. In this
case, we hold that the presence of menthol enlarges the
gaps between lipid headgroups and induces loose packing of
lipids, especially at higher concentrations (mass fraction of
menthol, 10%).
Hydrogenebonded interaction
A lateral hydrogen bonding network between the lipid head-
groups often contributes to the cohesiveness of the skin
lipid.
31
To the best of our knowledge, menthol also has
hydrogen-bonding capability. Therefore, a hydrogenebonded
interaction exists between menthol and the skin lipid if
menthol molecules are sufficiently close to the lipid head-
groups. In GROMACS software, the hydrogen bonds were
calculated using the g_hbond. The default value of 0.35 nm
Table 1 Molecular composition of simulation systems.
Menthol concentration
(mass fraction, %)
CER* CHOL FFA SPC MEN
0 128 128 64 4800 0
3 128 128 64 4656 17
7 128 128 64 4464 39
10 128 128 64 4320 56
15 128 128 64 4080 83
Note: * The values present the number of molecules.
Fig. 3 Cumulative amounts of different concentrations of
menthol.
350 L. Chen et al.
was used for the cutoff radius, and we set the cutoff angle
to 60.
32
The average numbers of lipidewater (LeW), lipid-
menthol (LeM), and lipidelipid (LeL) hydrogen bonds
per timeframe in bilayer systems containing different
concentrations of menthol are reported in Table 3.
Compared with the pure aqueous skin lipid bilayer sys-
tem, the LeW hydrogen bonds have been reduced in
systems containing menthol, similarly as the LeL
hydrogen bonds. We found that the sum of LeMand
LeL hydrogen bonds in each system containing a given
Fig. 4 Skin morphology after 22 hours of menthol treatment ( 20 000). (a) Untreated group; (b) control group; (c) 1% menthol;
(d) 2.0% menthol; (e) 3.5% menthol; (f) 5.0% menthol.
Table 2 Qnet equation assessment parameters.
Menthol
concentration/%
1.0 2.0 3.5 5.0
Linear
regression
equation
yZ7.340x þ8.199 y Z17.425x 25.211 y Z38.441x 115.570 y Z107.450x 201.520
R
2
0.974 0.928 0.958 0.965
J 7.340 17.425 38.441 107.450
T
lag
1.117 1.447 3.006 1.875
Fig. 5 Area per lipid for the mixed lipid bilayer systems.
Table 3 Average number of lipidewater (LeW), lipid-
menthol (LeM), and lipidelipid (LeL) hydrogen bonds per
timeframe in systems containing different concentrations of
menthol.
Menthol concentration
(mass fraction, %)
L*eWLeMLeL
0 379.4 e93.2
3 326.4 5.1 87.6
7 263.6 10.4 81.4
10 214.4 16.7 75.6
15 135.7 22.1 71.9
Note: * The lipid contained ceramide 2, cholesterol, and C24:0
fatty acid.
Penetration-enhancing mechanism of menthol 351
concentration of menthol was approximately equal to
the number of LeL hydrogen bonds in the pure aqueous
skin lipid bilayer system, suggesting that LeL hydrogen
bonds are partially replaced by LeMhydrogenbondsin
systems containing menthol. Thus, menthol has strong
hydrogen-bonded interactions with the skin lipid head-
groups, competing for hydrogen bond sites on the lipid
headgroups and thus disturbing the ordered lipid pack-
ing. It appears that menthol competes only the
hydrogen bond sites between the lipid headgroups but
not with the LeW hydrogen bond sites. Menthol accu-
mulates at the interface (Fig. 6) and drives some water
molecules out of the lipid headgroups, thereby breaking
the LeW hydrogen bonds. As presented in Table 3,the
quantity of LeW hydrogen bonds decreased as the
menthol concentration increased in systems.
The CER tail angle
When menthol interacts with the lipid bilayer, it is unclear
whether it affects the CER tail angle. As stated previously,
the APL becomes larger as the menthol content is
increased. We therefore analyzed the distribution of CER
tail angles in various skin lipid bilayer systems containing
different concentrations of menthol (Fig. 7). However, the
results revealed little differences in the CER tail angles
among different systems, with the values on average
distributing between 80 and 90. In view of this finding,
menthol may not disturb the bonded interaction of lipids.
The loose packing of lipid molecules is irrelevant to the CER
tail angle.
Menthol-lipid radial distribution functions (RDFs)
To describe the affinity between menthol and skin lipids, it
was of interest to analyze the menthol-lipid RDFs, modeling
the effective interactions between the molecule pairs. The
GROMACS program g_rdf can calculate RDFs, and in this
paper, RDFs were calculated around the center of mass of
molecules. In Fig. 8, we present the simulation results in
the form of RDFs of various skin lipids around menthol. At
all concentrations, the first peak of the RDFs of menthol-
CHOL is located at the shortest distance, reflecting that
menthol has the strongest affinity for CHOL among the
three types of skin lipids.
CHOL and menthol are both ringlike compounds. Thus,
we inferred that the strong intermolecular interaction be-
tween menthol and CHOL is related to the similarities of
their molecular structures, which is instructive for discov-
ering novel penetration enhancers. Furthermore, CHOL can
induce fluidity of the densely packed lipids in the skin lipid
bilayer.
33
Therefore, we can speculate that the incorpora-
tion of menthol would increase the fluidity of the lipid
membrane because of its similarity to and affinity for CHOL.
Discussion
In this research, to understand the penetration-enhancing
mechanism of menthol, in vitro and molecular dynamics
simulation methods were used to study the action of
menthol on the SC.
First, the interaction between menthol and the SC was
studied using vertical Franz diffusion cells obtained from
abdominal skin as a model. Abdominal skin was subse-
quently observed via TEM. The results illustrated that at
concentrations of 3.5% or lower, menthol changed the
original structure of the SC to varying degrees, thereby
increasing its fluidity and facilitating the permeation and
storage of menthol. Moreover, 5.0% menthol caused func-
tional damage of the SC, thereby destroying its barrier
function and menthol storage capacity.
To clarify the mechanism by which menthol affects the
structure of the SC at a microcosmic level, we conducted a
series of simulations of the mixed lipid bilayer in the
presence of different concentrations of menthol (mass
fraction, 0%e15%). We first analyzed the APL of the bilayer,
which generally reflects the phase behavior of membranes.
We found that the APL of the bilayer increased as the
menthol concentration increased. In other words, the gaps
between lipid headgroups were enlarged, and the bilayer
was liquefied by the action of menthol. Then, the
hydrogen-bonded interactions between menthol and skin
Fig. 6 Snapshot of a 15% menthol-involved system. The
mixed bilayer consists of ceramide 2 (red), cholesterol (yel-
low), and C24:0 free fatty acid (green). Water and menthol are
colored cyan and purple, respectively.
Fig. 7 The distribution of ceramide tail angles under
different concentrations of menthol. The ceramide tail angle
“a” as shown on the left reflects the extension of the double
aliphatic chains of ceramide 2.
352 L. Chen et al.
lipids were examined, and the results suggested that
menthol had strong hydrogen-bonding capability and that it
could even compete for the LeL hydrogen bonding sites,
thereby weakening the stability of the hydrogen bonding
network connecting the skin lipids, which explained the
loose packing of lipids (the increase of the APL). In addi-
tion, we suspected that the increase of the APL of the
mixed skin lipid bilayer is probably attributable to the
enlargement of the CER tail angle. However, we observed
no significant differences in the CER tail angle distributions
among the various bilayer systems containing different
concentrations of menthol. Finally, we explored the
effective interactions between menthol and different skin
lipids using RDFs. The strongest affinity existed between
CHOL and menthol. Interestingly, menthol and CHOL are
extremely similar in molecular structure. Both are ringlike
and amphiphilic compounds. The molecular length of CHOL
is noticeably shorter than that of other lipids, and thus,
CHOL plays a role in modulating the spatial arrangement of
the tails of longer chainlike lipids, thereby endowing the
skin lipid bilayer with fluidity. Accordingly, we suggested
that the incorporation of menthol would also interfere with
the lateral dense packing of skin lipids, thereby increasing
the permeability of the membrane.
Conclusion
The penetration enhancement mechanism of menthol was
explained using in vitro and molecular dynamics simulation
methods. These findings may advance the basic research of
TDDSs, and they may be instructive for discovering novel
penetration enhancers.
Funding
This work was supported by the Municipal Natural Science
Foundation of Beijing (7162122).
Declaration of competing interest
The authors declared no conflicts of interest.
CRediT authorship contribution statement
Liping Chen: Data curation, formal analysis, and writing ‒
original draft. Lina Ma: Data curation, software, and proj-
ect administration. Shufang Yang: Software and formal
analysis. Xiaowen Wu: Methodology and data curation.
Xingxing Dai: Supervision. Shifeng Wang: Methodology.
Xinyuan Shi: Funding acquisition and writing ‒review &
editing.
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Fig. 8 Radial distribution functions of the skin lipid around menthol in systems with a range of menthol concentrations (3%e15%).
Abbreviations: MEN: menthol; CER: ceramides; FFA: free fatty acid; CHOL: cholesterol.
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