The multifunctional role of ectoine as a natural cell protectant

Abstract

The protective properties of ectoine, formerly described for only extremophilic microorganisms, can be transferred to human skin. Our present data show that the compatible solute ectoine protects the cellular membrane from damage caused by surfactants. Transepidermal water loss measurements in vivo suggest that the barrier function of the skin is strengthened after the topical application of an oil in water emulsion containing ectoine. Ectoine functions as a superior moisturizer with long-term efficacy. These findings indicating that ectoine is a strong water structure-forming solute are explained in silico by means of molecular dynamic simulations. Spherical clusters containing (1) water, (2) water with ectoine, and (3) water with glycerol are created as model systems. The stronger the water-binding activity of the solute, the greater the quantity of water molecules remaining in the cluster at high temperatures. Water clusters around ectoine molecules remain stable for a long period of time, whereas mixtures of water and glycerol break down and water molecules diffuse out of the spheres. On the basis of these findings, we suggest that the hydrogen bond properties of solutes are not solely responsible for maintaining the water structure form. Moreover, the particular electrostatic potential of ectoine as an amphoteric molecule with zwitterionic character is the major cause for its strong affinity to water. Because of its outstanding water-binding activity, ectoine might be especially useful in preventing water loss in dry atopic skin and in recovering skin viability and preventing skin aging.

Full text

The multifunctional role of ectoine as a natural
cell protectant
Ruediger Graf, PhD
a,
⁎, Soheila Anzali, PhD
b
, Joachim Buenger, PhD
a
,
Frank Pfluecker, PhD
a
, Hansjuergen Driller, PhD
a
a
Department of Cosmetics and Food, Merck KGaA, 64293 Darmstadt, Germany
b
R&D New Technology Evaluation, Merck KGaA, 64293 Darmstadt, Germany
Abstract The protective properties of ectoine, formerly described for only extremophilic microorgan-
isms, can be transferred to human skin. Our present data show that the compatible solute ectoine protects
the cellular membrane from damage caused by surfactants. Transepidermal water loss measurements in
vivo suggest that the barrier function of the skin is strengthened after the topical application of an oil in
water emulsion containing ectoine. Ectoine functions as a superior moisturizer with long-term efficacy.
These findings indicating that ectoine is a strong water structure-forming solute are explained in silico
by means of molecular dynamic simulations. Spherical clusters containing (1) water, (2) water with
ectoine, and (3) water with glycerol are created as model systems. The stronger the water-binding
activity of the solute, the greater the quantity of water molecules remaining in the cluster at high
temperatures. Water clusters around ectoine molecules remain stable for a long period of time, whereas
mixtures of water and glycerol break down and water molecules diffuse out of the spheres. On the basis
of these findings, we suggest that the hydrogen bond properties of solutes are not solely responsible for
maintaining the water structure form. Moreover, the particular electrostatic potential of ectoine as an
amphoteric molecule with zwitterionic character is the major cause for its strong affinity to water.
Because of its outstanding water-binding activity, ectoine might be especially useful in preventing water
loss in dry atopic skin and in recovering skin viability and preventing skin aging.
© 2008 Elsevier Inc. All rights reserved.
Introduction
Ectoines, as small organic molecules, occur widely in
aerobic, chemoheterotrophic, and halophilic organisms that
enable them to survive under extreme conditions. These
organisms protect their biopolymers (biomembranes, pro-
teins, enzymes, and nucleic acids) against dehydration
caused by high temperature, salt concentration, and low
water activity by substantial ectoine synthesis and enrich-
ment within the cell.
The organic osmolyte ectoine (Fig. 1) and hydroxyectoine
are amphoteric, water-binding, organic molecules. They are
generally compatible with the cellular metabolism without
adversely affecting the biopolymers or physiologic processes
and are so-called compatible solutes.
1
The protective function of the compatible solutes in a low-
water environment may be explained by the “preferential
exclusion model”: The solutes are excluded from the
immediate hydration shell of, for example, a protein because
of an unfavorable interaction with the protein surface. The
⁎Corresponding author.
E-mail address: ruediger.graf@merck.de (R. Graf).
0738-081X/$ –see front matter © 2008 Elsevier Inc. All rights reserved.
doi:10.1016/j.clindermatol.2008.01.002
Clinics in Dermatology (2008) 26, 326–333

consequence is preferential hydration of the protein, thus
promoting its native conformation. Because compatible
solutes do not interact directly with the protein surface, the
catalytic activity remains unaffected.
2,3
Yu and Nagaoka
5
reported interesting results on mole-
cular dynamic simulations performed for water-ectoine
mixture models around chymotrypsin inhibitor 2. According
to their statement, ectoine maintains water at the surface by
slowing down the water diffusion around a protein, where it
is most needed, whereas it does not directly interact with
macromolecules themselves. Thus, ectoine plays an indirect
role in the alteration of the solvent properties and the
modification of the stability of proteins.
4
Ectoine minimizes the denaturation that occurs on the
removal of water molecules by making the unfolding less
favorable.
6
In addition, hydroxyectoine, with its OH group,
can at least partly replace those water molecules lost from the
hydrate shell (replacement hypothesis); in this way, the
native structure of the biopolymers can be further stabilized.
Compatible solutes are amphiphilic in nature and capable of
“wetting”hydrophobic proteins, thus improving their
hydration capability.
7
The structure-forming and breaking
properties of compatible solutes indirectly influence the
hydration shells and thus the activities of the proteins
involved.
8
In this way, halophilic organisms and other bacteria use
ectoine to protect their cytoplasmic biomolecules against
heat, freezing, dryness, and osmotic stress.
9
Ectoine and
hydroxyectoine can be isolated from halophilic bacteria on a
large scale and thus are available as active ingredients for
skin care.
10
The protective properties of ectoine, formerly described
only for microorganisms, could be transferred to human skin.
Human skin is situated at the interface of the organism and its
environment and therefore is exposed to a variety of
environmental assaults. The stratum corneum in particular
provides a barrier to the evaporation of water from the viable
epidermis. Many factors work to compromise this barrier and
increase the rate of water loss from the skin. Exposure to
extreme environmental conditions, including cold, dry
winter weather, frequent washing with soap and hot water,
or the exposure to surfactants, may cause skin dryness. In
addition to dryness, the cumulative effect of external factors,
such as radiation, wind, and temperature extremes, leads to
accelerated skin aging.
11,12
Various investigations underline the outstanding anti-
aging properties of ectoine. Epidermal dendritic Langerhans
cells are the single most important antigen-presenting cell
population in the skin. The number of Langerhans cells
decreases significantly in aged skin, whereas the decrease in
skin exposed to the sun is greater than that in skin protected
from the sun.
13-15
Topically applied ectoine shows an
immunoprotective potential on the sun-exposed skin of
healthy subjects. The ultraviolet-induced reduction of
Langerhans cells has been prevented by pretreatment with
ectoine before sun exposure.
16
The exposure of primary human keratinocytes to ultra-
violet A provokes the formation of ceramide by a singlet
oxygen-mediated mechanism. As a consequence of the
increased ceramide level, an intracellular signaling cascade is
activated, leading to expression of the proinflammatory
intercellular adhesion molecule-1. These negative effects are
effectively prevented by ectoine as a result of its singlet
oxygen-quenching properties.
17,18
Because the activity of
antioxidant enzymes and the levels of nonenzymatic
antioxidants decrease with age,
19,20
ectoine could prevent
such oxidative damage in skin.
Skin in particular, which is susceptible to water loss
because of the absence of an optimal skin barrier (eg, the skin
of the elderly, atopic skin, or after surfactant treatment),
shows increased transepidermal water loss (TEWL) and
diminished moisturization.
21
The goal of the present study was to investigate the effect
of ectoine on the moisturization status and barrier function of
the skin after topical application in vivo. Furthermore,
different molecular dynamic simulation systems were
created in silico to compare models of water, water-ectoine,
and water-glycerol. The outstanding activity of ectoine as a
strong water structure former was evaluated against glycerol
as a commonly used humectant in cosmetics.
Materials and methods
Membrane assay
The membrane assay is based on the photometric
quantification of free hemoglobin released from erythrocytes
with a partially damaged membrane provoked by surfactants.
For the different experiments, the erythrocytes are treated as
Fig. 1 Molecular structure of ectoine with the two tautomeric forms (A) and its hydrophilic surface colored according to the corresponding
atomic partial charges (B).
327Multifunctional role of ectoine as a natural cell protectant

follows: (1) Human erythrocytes (2 ×10
8
cells/mL) are
treated for 1 hour with 0%, 0.1%, 0.5%, 1%, and 5% ectoine
to determine the effect of ectoine concentration; and (2) 2 ×
10
8
erythrocytes/mL are treated for 0 (control), 6, 18, and
24 hours with 1% (w/v) ectoine to determine the effect of the
incubation time. Both sets of cells are stressed for 10 minutes
with 0% to 0.04% sodium dodecyl sulfate (SDS) solution,
and the number of cells in lysis is determined spectro-
scopically via the content of free hemoglobin. With two
absorption peaks at 540 and 575 nm, hemoglobin can be
quantified by the determination of absorption at 575 nm,
with the molar absorbance coefficient of 0.125 mmol/L
oxyhemoglobin at A
575nm
= 2.0.
22
The results are shown as
the difference (%) of cells in lysis as a function of the
concentration of ectoine against an untreated control. The
experiment is repeated five times.
Determination of the transepidermal water loss
in vivo
The volar forearm of five volunteers is treated twice daily
for 1 week with an oil in water emulsion (2 mg/cm
2
) con-
taining 0% (placebo), 2%, and 5% ectoine. To achieve a
synthetic increase in TEWL by damaging the skin barrier, the
skin is occlusively treated with 80 μL SDS (2% in water) in
an aluminium chamber for 24 hours. The TEWL is deter-
mined in an acclimatized room at 22°C with an air humidity
of 60% using a TEWAmeter TM210 (Courage + Khazaka,
Koeln, Germany). The TEWL values are visualized before
and after treatment with ectoine-containing emulsion and
after damaging the skin barrier with SDS.
Determination of skin moisture by corneometry
Ectoine treatment and subsequent dehydration
with silica
The skin of the volar forearm of five volunteers is treated
twice daily for 1 week with a cosmetic formulation (2 mg/
cm
2
) containing 0% (placebo), 2%, and 5% ectoine. The
moisture content of the skin is determined with a
Corneometer before application and, after 1 week, 4 hours
after the final application. Silica gel 60 (0.2 g/cm
2
) is applied
under occlusion for 2 hours (dehydration step). On re-
moval of the silica gel, the skin moisture is determined
after 10 minutes, 2 hours, 4 hours, and 24 hours.
Ectoine treatment for long-term hydration
The skin of the volar forearm of five volunteers is treated
twice daily for 12 days with a cosmetic formulation (2 mg/
cm
2
) containing 0% (control), 0.5%, and 1% ectoine. The
skin hydration is determined by corneometry starting at day 8
until day 12. On day 12, the application is stopped for 7 days,
finalizing this experiment on day 19 with a last measurement
of hydration. The measurements are carried out in an
acclimatized room at 22°C with an air humidity of 60%.
Molecular dynamic simulations
The Schrödinger package Impact (Integrated Modelling
Program using Applied Chemical Theory
23
) is used for
molecular dynamic simulations (with OPLS-2005 force
field parameters and partial charges). The OPLS-2005
force field uses experimental data from the liquid state
and quantum mechanical calculations. It is calculated
from the sum of the intramolecular bond, angle, and
torsion motions to set the constituent parameters and the
nonbonded interaction as a van der Waals term together
with an electrostatic term.
Three spheres have been created for: (1) water only; (2) an
ectoine-water mixture; and (3) a glycerol-water mixture. The
creations of spheres are as follows: For each ingredient
(ectoine and glycerol), a 3 ×3×3 matrix is created. For this
purpose, 27 molecules of each ectoine or glycerol are
clustered per sphere. A minimization is performed using the
surface generalized born method with 500 steps of steepest
descent, followed by 500 steps of conjugated gradient.
Ectoine and glycerol are placed in a rectangular box, and
soaking of simple point charge water with a dimension of
70 ×70 ×70 Å is performed.
The spheres are cut out with a radius of 30 Å away from
the centroid atoms. The size of spheres of 30 Å in radius is
sufficient to cover more than one solvation shell for solutes
calculated in spheres. The reason for having so many water
molecules is to ensure that there are at least two shells of
water molecules around the solutes. In addition, we can
examine and compare the indirect effect of solutes on water
molecules on such a large scale.
The shake algorithm is used to constrain the X-H bond,
which allows time steps of 2 fs. Elaborate equilibration runs
of 50 ps at 298.15 K are performed to allow for a careful
accommodation of water structure around the solutes
(ectoine and glycerol). Water oxygen atoms are fixed beyond
25 Å from the defined centroid atoms in each created sphere
in the equilibration. For the dynamic simulations, these
constraints are removed.
The dynamic simulations are performed for water and
water-glycerol for 200 ps and 500 ps at the temperature of
370 K with a temperature relaxation constant value of 0.01
ps. For the water-ectoine mixture, the simulation is
performed for 1 ns to demonstrate the effect of ectoine
with regard to water cluster formation in a long time frame.
The trajectories are recorded every 50 time steps.
Results and discussion
Barrier-improving effects
The membrane of the skin cell can become damaged, for
example, by exposure to surfactants present in washing and
skin-cleansing solutions. Thus, the use of active cleansing
328 R. Graf et al.

surfactants also leads to removal of fat from the skin,
increased TEWL, and dry skin.
For the evaluation of the membrane-protecting properties
of ectoine, the red blood cell (RBC) test was applied. This
assay is a biologic in vitro test for the rapid estimation of
membrane and protein-denaturing properties of surfactants.
The standard protocol uses erythrocytes, non-nucleated
blood cells containing hemoglobin. Because hemoglobin is
incapable of crossing the RBC membrane, it is not detectable
outside erythrocytes as long as the RBC membrane is intact.
The assay is based on the photometric quantification of the
hemoglobin released as a consequence of RBC plasma
membrane damage after exposure to surfactants, thus
providing a measure of surfactant aggressiveness.
The stabilization effect on cell membranes pretreated with
ectoine was evaluated. The erythrocytes were incubated for
10 minutes with SDS. SDS destabilizes the membranes of
untreated cells in such a way that lysis occurs in part and cell
components (eg, hemoglobin) are released. The hemoglobin
released serves as an indicator for the spectrophotometric
determination of the degree of cell membrane damage
provoked by SDS. Detecting the released hemoglobin
enabled the number of destroyed erythrocytes to be
determined in our experiments. A modified version of the
RBC test was used to determine the membrane stabilization
achieved by a test substance versus surfactant lysis. This
assay includes the RBC preincubation with a stabilizer
before the addition of surfactant as the lytic agent.
Fig. 2 shows that ectoine protects the cells from damage
caused by SDS treatment. The erythrocytes pretreated with
ectoine are shown to be more resistant to membrane damage
by SDS than those of untreated cells. No stabilizing effect
was observed in cells without ectoine, in which maximum
erythrocyte damage occurred (0% increase of membrane
stability). The higher the ectoine concentration, the greater
the protective effect against membrane damage (Fig. 2A).
Furthermore, the influence of prolonging the incubation
time was investigated. The membrane stability increased to
30% after 6 hours of pretreatment and to approximately 60%
after 24 hours. Thus, the longer the cells are pretreated with
ectoine, the greater the protective effect against membrane
damage by the surfactant SDS (Fig. 2B). The degree of cell
protection that has been linked with the degree of membrane
stabilization depends directly on the ectoine concentration
and the duration of ectoine pretreatment.
Ectoine thus protects the skin barrier against the
damaging effect (water loss) of SDS.
Fig. 3 In vivo determination of TEWL after damage of the skin
barrier by SDS. The forearm skin of the volunteers (n = 5) is treated
twice daily for 1 week with an oil in water emulsion (2 mg/cm
2
)
containing 0% (placebo), 2%, and 5% ectoine. To achieve a
synthetic increase in TEWL by damaging the skin barrier, the skin is
subsequently treated with 2% SDS in water for 24 hours and the
TEWL is determined. The diagram shows the TEWL before and
after treatment with emulsion containing ectoine and after damage
of the skin barrier with SDS.
Fig. 2 Evaluation of the membrane-stabilizing effect of ectoine in
surfactant-stressed cells. Human erythrocytes (2 ×10
8
cells/mL) are
treated (A) for 1 hour with 0%, 0.1%, 0.5%, 1%, and 5% ectoine
and (B) for 0 (control), 6, 18, and 24 hours with 1% ectoine. Both
sets of cells are stressed for 10 minutes with 0% to 0.04% SDS
solution, and the number of cells in lysis is determined spectro-
scopically via the content of free hemoglobin. The diagrams
illustrate the difference (%) of cells in lysis as a function of the
concentration of pretreated ectoine against an untreated control. The
experiment is repeated five times.
329Multifunctional role of ectoine as a natural cell protectant

These data confirm our previous studies of further
cosmetically relevant surfactants in which ectoine showed
a stronger protective effect compared with the well-known
membrane stabilizer phosphatidylcholine.
24
These in vitro findings should also be approved in vivo.
Surfactants have also been used to cause dry skin.
25
For this
reason, after SDS treatment of the skin, the TEWL is
determined as a read-out parameter for the integrity of the
skin barrier. The barrier disruption can be expressed as a
change in TEWL, and the influence of ectoine can be
measured. The study is performed on the lower forearm of
healthy volunteers.
The application of a cosmetic emulsion containing
different amounts of ectoine leads to a remarkable
reduction of TEWL to 40% (Fig. 3). Fig. 3 shows that
skin pretreated with ectoine becomes less susceptible to
damage by the surfactant SDS. The ectoine emulsion thus
protects the skin against surfactant damage and the con-
sequent loss of water.
Protection against dehydration
One of the major goals of cosmetics is still the protection
of the skin against stress factors that lead to dehydration. Dry
air, particularly during periods of freezing or hot weather and
air conditioning, tends to dry out the skin considerably.
To demonstrate the protective effect of ectoine on skin
moisture, two cosmetic formulations with and without
ectoine were topically applied to the lower forearm of
volunteers twice daily for 1 week. The moisture content of
the skin was determined by corneometry, and the results are
shown in Fig. 4.
The diagram illustrates that ectoine in a cosmetic oil in
water emulsion protects the skin against dehydration. In
addition to this protection, ectoine also produces a higher
moisture content than the basic (placebo) formulation that
already contains 3% glycerol. The results also show that
ectoine, even after 24 hours, maintains a considerably greater
degree of skin moisture than untreated or placebo-treated
skin. Ectoine even protects skin against rapid dehydration
after direct application of hygroscopic silica gel. Skin
moisture can be maintained for a longer period of time by
topically applying ectoine.
Low humidity has been shown to stimulate epidermal
DNA synthesis and amplify the hyperproliferative response
to barrier disruption.
26
Stratum corneum morphology is also
Fig. 4 In vivo determination of skin moisture after treatment
with ectoine and subsequent dehydration with silica gel. The
forearm skin of the volunteers (n = 5) is treated twice daily for
1 week with an oil in water emulsion (2 mg/cm
2
) containing 0%
(placebo), 2%, and 5% ectoine. The moisture content of the skin
is determined before application and, after 1 week, 4 hours after
the final application. Silica gel 60 (0.2 g/cm
2
) is applied under
occlusion for 2 hours (dehydration). On removal of the silica
gel, the skin moisture is determined after 10 minutes, 2 hours, 4 hours,
and 24 hours.
Fig. 5 Long-term moisturizing effect with ectoine. The skin of the volar forearm of five volunteers is treated twice daily for 12 days with a
cosmetic formulation (2 mg/cm
2
) containing 0% (control), 0.5%, and 1% ectoine. The skin hydration is determined by corneometry starting at
day 8 until day 12 (A). On this day the application is stopped for 7 days, finalizing this experiment on day 19 with a last measurement of
hydration (B).
330 R. Graf et al.

influenced by a dry environment, and abnormal desquama-
tion is observed under low humidity.
27,28
With respect to our
findings in the “silica-dried skin model,”formulations
containing ectoine have a prophylactic effect against such
adverse processes in dry skin.
Moisture boost with long-term effect
In a further series of experiments, ectoine was evaluated
according to its long-term effect on skin moisture. The test
was carried out on the volar forearm of volunteers. Twice-
daily applications of 0.5% and 1% ectoine were applied for
12 days. The skin hydration was measured with a
Corneometer starting at day 8 until day 12. On day 12, the
application of ectoine was stopped for 7 days, detecting the
skin hydration finally at day 19. The results of this placebo-
controlled study underline the outstanding activity of
ectoine: After 8 days of application, the hydration increased
markedly up to 200% compared with the placebo-treated
skin and remained constant until the end of the testing period
(Fig. 5A). Although the topical application was stopped on
day 12, the actual hydration status was preserved for
approximately 7 days, underlining a significant long-term
moisturizing effect of ectoine (Fig. 5B).
Ectoine retains the power of water
The protein-stabilizing effects of ectoine can be explained
by the preferential exclusion model as a consequence of
entropically favored surface minimization. The ability of
ectoine as a strong water structure-forming solute is further
processed in comparison with glycerol as a commonly used
humectant in cosmetics.
11
After the dynamic simulation time of 200 ps, as well as
1000 ps, the number of water molecules in the water-ectoine
complex remained unexpectedly constant. In contrast, the
performance of the water-glycerol complex: an extreme
corrosion was observed. The total number of water
molecules decreased significantly after 200 ps of dynamic
simulation, and only 2339 water molecules remained in the
sphere (Table 1).
To explain this phenomena, the total potential energy
(E
pot
) was calculated for the spheres containing water, water-
Fig. 6 Evaluation of the E
pot
-value of different water clusters.
During the dynamic simulation at 370 K, water molecules diffuse
out of the spheres and the total amount of water molecules
decreases. To explain this phenomenon, the total potential energy
has been calculated and plotted as the E
pot
-value. In this
experimental setup, the E
pot
-value can be adopted as the stored
energy or the energy of position of each system.
Table 1
t (ps) Water Water-glycerol Water-ectoine
0 3618 3429 3139
200 3026 2339 3138
500 NC 1288 3112
1000 NC NC 3103
The number of water molecules retained in spherical water clusters
during the dynamic simulation time. The simulation is carried out at 370
K, and the water molecules are counted after 0, 200, 500, and 1000 ps.
NC, Not calculated.
Fig. 7 Molecular dynamic simulation of different models
containing (A) water, (B) water and ectoine, and (C) water and
glycerol. The pictures are taken at the beginning of the simulation
(t = 0, A1, B1, C1) and after 200 ps (A2), 1000 ps (B2), and 500 ps
(C2) at a constant temperature of 370 K. Water clusters around
ectoine molecules remain stable for a long period of time, whereas
the cluster of water and glycerol breaks down and water molecules
diffuse out of the spheres. The pictures represent the number of
water molecules counted during the dynamic simulation as shown
in Table 1. The solutes are green.
331Multifunctional role of ectoine as a natural cell protectant

glycerol, and water-ectoine. In this experimental setup, the
E
pot
-value can be adopted as the stored energy or the energy
of position in such a system.
With regard to water and the water-glycerol complexes,
the E
pot
-values decreased dramatically during the simulation
time, whereas the E
pot
-value of the water-ectoine sphere
remained constant even throughout a longer simulation time
(Fig. 6). The E
pot
-value of the water-ectoine sphere remained
constant at the level indicated in the diagram (data not
shown). It is remarkable that the E
pot
-value of regular water
molecules per se was greater than that of the water-ectoine
mixture, indicating the strong organizing and complexing
properties of ectoine.
The dynamic simulation and animations, and the
statistical analysis, demonstrated that the water diffusion
out of the spheres was limited and decreased enormously by
adding ectoine molecules to the sphere (Fig. 7A and B; see
also the stick presentation of water and ectoine atoms in
Fig. 8). Even a 5-fold longer simulation time showed a stable
water structure form attributable to ectoine properties, which
is superior compared with water itself and outstanding
compared with a water-glycerol complex (Fig. 7).
We propose that the hydrogen bond properties of solutes
are not solely responsible for maintaining the water structure
form. Moreover, the particular electrostatic potential of a
compatible solute, such as ectoine, as an amphoteric
molecule with zwitterionic character is the major reason for
its affinity to water.
Conclusions
Our recent studies demonstrate the outstanding role of the
compatible osmolyte ectoine in preventing water loss caused
by surfactant-induced barrier damage. Ectoine functions as a
more potent moisturizer than glycerol and features long-term
moisturizing efficacy. These in vivo findings were explained
in silico by means of molecular dynamic simulations. Water
clusters around ectoine molecules remain stable for a long
period of time, whereas mixtures of water and glycerol are
disintegrated by the diffusion of water molecules out of the
spheres. Because of its strong water-binding activity, ectoine
may be especially useful in the prevention of dehydration in
dry atopic skin and the recovery of skin viability and
prevention of skin aging.
Acknowledgments
We thank Dr Jianxin Duan, of Schrödinger GmbH,
Mannheim, Germany, for the fruitful discussions and
technical support for the dynamic simulations.
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333Multifunctional role of ectoine as a natural cell protectant