Improvement of the pharmacological activity of menthol via enzymatic β-anomer-selective glycosylation

Article abstract of DOI:10.1186/s13568-017-0468-0

Menthol has a considerable cooling effect, but the use range of menthol is limited because of its extremely low solubility in water and inherent flavor. (?)-Menthol β-glucoside was determined to be more soluble in water (>27 times) than (?)-menthol α-glucoside; hence, β-anomer-selective glucosylation of menthol is necessary. The in vitro glycosylation of (?)-menthol by uridine diphosphate glycosyltransferase (BLC) from Bacillus licheniformis generated (?)-menthol β-glucoside and new (?)-menthol β-galactoside and (?)-menthol N-acetylglucosamine. The maximum conversion rate of menthol to (?)-menthol β-d-glucoside by BLC was found to be 58.9%. Importantly, (?)-menthol β-d-glucoside had a higher cooling effect and no flavor compared with menthol. In addition, (?)-menthol β-d-glucoside was determined to be a non-sensitizer in a skin allergy test in the human cell line activation test, whereas menthol was a sensitizer.

Full text of DOI:10.1186/s13568-017-0468-0

et al. AMB Expr (2017) 7:167
Choi
DOI 10.1186/s13568-017-0468-0
Open Access
ORIGINAL ARTICLE
Improvement of the pharmacological
activity of menthol via enzymatic
β-anomer-selective glycosylation
Ha‑Young Choi1,2, Bo‑Min Kim1, Abubaker M. A. Morgan1, Joong Su Kim3 and Won‑Gon Kim1,2*
Abstract
Menthol has a considerable cooling efect, but the use range of menthol is limited because of its extremely low
solubility in water and inherent favor. (−)‑Menthol β‑glucoside was determined to be more soluble in water (>27
times) than (−)‑menthol α‑glucoside; hence, β‑anomer‑selective glucosylation of menthol is necessary. The in vitro
glycosylation of (−)‑menthol by uridine diphosphate glycosyltransferase (BLC) from Bacillus licheniformis generated
(−)‑menthol β‑glucoside and new (−)‑menthol β‑galactoside and (−)‑menthol N‑acetylglucosamine. The maximum
conversion rate of menthol to (−)‑menthol β‑d‑glucoside by BLC was found to be 58.9%. Importantly, (−)‑menthol
β‑d‑glucoside had a higher cooling efect and no favor compared with menthol. In addition, (−)‑menthol β‑d‑
glucoside was determined to be a non‑sensitizer in a skin allergy test in the human cell line activation test, whereas
menthol was a sensitizer.
Keywords: Menthol, Menthol β‑glycosides, Glycosyltransferase, Solubility, Cooling
without the need for a protection and deprotection pro-
cesses. α-Anomer-selective glucosylation of menthol
by yeast α-glucosidase has been reported (Nakagawa
et al. 1998), but to the best of our knowledge, β-anomer-
selective glucosylation of menthol has not yet been
reported.
Introduction
Menthol is used in a wide variety of products as an addi-
tive to foods, medicines, cosmetics, and cigarettes owing
to its inherent mint favor and refreshing feelings. Te
use range of menthol, however, is limited because of its
extremely low solubility in water and favor (Kamatou
et al. 2013; Patel et al. 2007). Glycosylation is one method
than can improve both the water solubility and biologi-
cal activity of non-glycosylated compounds (Ahmed et al.
2006). Sugar moieties are known to improve the pharma-
cokinetic properties and/or afect the biological activity
of glycosides in important natural pharmaceutical prod-
ucts (Weymouth-Wilson 1997). Chemical synthesis of
menthyl glucosides was reported to produce an anomeric
mixture of α- and β-anomers (Sakata and Iwamura 1979).
As an eco-friendly process, enzymatic synthesis is
superior to chemical synthesis because the enzymatic
reactions proceed regioselectively and stereoselectively
Bacilli have been used as a source of glycosyltrans-
ferases for the modifcation of natural products. Uridine
diphosphate (UDP)-glycosyltransferases from Bacillus
sp. HH1500 (Rabausch et al. 2013) and B. cereus (Ahn
et al. 2009) were reported to glucosylate macrolide and
favonoid substrates. UDP-glycosyltransferase BLC
from B. licheniformis has been known to glycosylate a
variety of natural products such as geldanamycin ana-
logs, epothilone A, resveratrol, chalcone, and various
favonoids (Koirala et al. 2014; Parajuli et al. 2014; Wu
et al. 2012). Additionally, BLC was reported to catalyze
β-anomer-selective glycosylation of geldanamycin ana-
logs and epothilone A. Tese reports led us to study
β-anomer-selective glycosylation of menthol by BLC.
Herein, we report that the water solubility of the
β-anomer of menthyl glucoside is superior to its
α-anomer, and Bacillus glycosyltransferase BLC catalyzes
*Correspondence: wgkim@kribb.re.kr
2 Department of Bio‑Molecular Science, KRIBB School of Bioscience, Korea
University of Science and Technology (UST), Yusong, Daejeon, Republic
of Korea
Full list of author information is available at the end of the article
© The Author(s) 2017. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License
(http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium,
provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license,
and indicate if changes were made.

et al. AMB Expr (2017) 7:167
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Quantitative analysis of menthol glucoside by LC–MS/MS
Te concentrations of menthol glucoside in the water
solubility test or the BLC reactions were analyzed by
LC–MS/MS in MRM mode. Te samples were subjected
to an HPLC system (Luna C18(2), 100 × 2.0 mm, 3 μm,
Phenomenex, Torrance, CA, USA) connected to a QTrap
3200 with a Turbolon Spray source (AB SCIEX, Singa-
pore). Te column was maintained at 20 °C with a fow
rate of 0.4 mL/min and a gradient of 0.1% (v/v) formic
acid in H2O (A) and 0.1% (v/v) formic acid in acetonitrile
(B) at 40% B to 90% B for 7 min. MRM was performed
by selecting the two mass ions set specifcally for the
selected analytes to detect the transition from parent
ion to product ion, i.e., m/z 319 > 139 for (−)-menthol
β-glucoside. For analysis of (−)-menthol β-d-glucoside,
the Turbolon Spray source-dependent parameters were
optimized to the following values: 10 psi curtain gas, high
collision gas, 5500 V ion spray voltage, 150 °C tempera-
ture, and 12 psi ion source gas. Te compound-depend-
ent parameters were optimized to the following values:
15 eV collision energy, 8.5 V entrance potential, 126 V
declustering potential, and 4 V collision cell exit poten-
tial. For analysis of (−)-menthol α-d-glucoside, the Tur-
bolon Spray source-dependent parameters were the same
as those for (−)-menthol α-d-glucoside. Te compound-
dependent parameters were optimized to the following
values: 15 eV collision energy, 4 V entrance potential,
41 V declustering potential, and 4 V collision cell exit
potential.
the β-anomer-selective glycosylation of (−)-menthol.
Additionally, the cooling ability and skin sensitization
properties of (−)-menthol β-d-glucoside were improved
compared with those of menthol.
Materials and methods
Chemicals
(−)-Menthol, UDP-glucose, UDP-galactose, UDP-
N-acetylglucosamine, and UDP-glucuronic acid were
purchased from Sigma-Aldrich (St. Louis, MO, USA).
Te chemical synthesis of (−)-menthol β-glucoside and
(−)-menthol α-glucoside was conducted by MediGen Inc
(Daejeon, Korea).
Cloning, expression, and purifcation of glycosyltransferase
Glycosyltransferase BLC was cloned, expressed, and
purifed from the genomic DNA of B. licheniformis
DSM13, as reported previously (Wu et al. 2012). Briefy,
the amplifed PCR products were purifed, digested
with BamHI and XhoI, and cloned into the same sites
of the pET28a vector. Te recombinant expression vec-
tors were transformed into E. coli BL21(DE3). Te E. coli
BL21(DE3) culture transformed with the recombinant
expression vector was induced with isopropyl-1-thio-
β-d-galactopyranoside (IPTG) (at a fnal concentration
of 0.5 mM). After centrifugation of the cell lysate, BLC
enzyme was purifed by nickel–nitriloriacetic acid (Ni–
NTA) column chromatography (Qiagen). Te fractions
eluted with 50 and 100 mM imidazole were pooled and
concentrated with an Amicon Ultra-15 instrument (Mil-
lipore). Te concentrated enzyme was dialyzed overnight
at 4 °C using 100 mM Tris–HCl (pH 8.0) containing 20%
(vol/vol) glycerol and stored at 4 °C until use.
Isolation and structure determination of three
biosynthesized menthol glycosides
To obtain a large amount of menthol glycosides for NMR
study, the preparative-scale reaction containing UDP-
d-glucose (2 mM, 39.1 mg), menthol (1 mM, 5 mg), 3 μM
BLC in 32 mL was performed for 18 h, producing men-
thol glucoside (0.41 mM, 4.2 mg) representing 41.2%
conversion of menthol. Te preparative-scale reaction
containing UDP-d-galactose (2 mM, 78.2 mg), menthol
(8 mM, 80 mg), and 3 μM BLC in 64 mL was performed
for 18 h, producing of menthol galactoside (0.125 mM,
2.6 mg) representing 1.56% conversion of menthol. To
purify the menthol glycosides from the reaction mix-
ture, the reaction mixture was partitioned with water-
saturated butanol, and the butanol layer was evaporated
in vacuo. Te resulting residue was purifed by thin layer
chromatography (TLC) on silica gel 60 F254 plates (Merck
No 1.05715.0001: Darmstadt, Germany) developed with
chloroform:methanol (3:1) to produce menthol glucoside
and menthol galactoside with Rf values of 0.61 and 0.58,
respectively, as detected with I2 vapor. Teir structures
Glycosylation of menthol using BLC
Analytical glycosylation reactions were performed in
a total volume of 50 μL containing purifed His-tagged
recombinant BLC (3 μM), 1 mM menthol, 2 mM UDP-
glycosides (UDP-glucose, UDP-galactose, UDP-N-acetyl-
glucosamine, and UDP-glucuronic acid), 1 mM MgCl2,
and 50 mM Tris–HCl (pH 8.0) (Wu et al. 2012). Te
reaction mixtures were incubated at 30 °C for 18 h. For
determination of the kinetic parameters for menthol, the
reactions were performed for 10 min with varying con-
centrations of menthol from 0.25 to 4 mM. Triplicate
reactions were performed. All reactions were quenched
by the addition of 450 μL MeOH and then centrifuged.
Te supernatants were qualitatively detected by LC–ESI–
MS or quantitatively analyzed by LC–MS/MS operat-
ing in multiple reaction monitoring (MRM) mode, as
described below.

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were confrmed by 700 MHz Bruker, BioSpin nuclear USA), seeded at 0.5 × 106 cells/mL in a 24-well plate, and
magnetic resonance (NMR) analysis including one- cultured with chemicals for 24 h. When DMSO was used
dimensional 1H NMR, 13C NMR and two-dimensional as a solvent, its fnal concentration in the culture media
NMR-correlation spectroscopy (COSY), hetero-nuclear was less than 0.2%. Te cell viability was evaluated by MTT
single quantum coherence (HSQC), and heteronuclear assay. Te concentration resulting in 75% cell viability,
multiple bond connectivity (HMBC).
referred to as CV75, was calculated based on the analysis of
viable cells. Cells were incubated for 24 h with test chemi-
cals at three or four concentrations of a 1.2-fold serial dilu-
Water solubility determination
Te water solubilities of (−)-menthol β-d-glucoside and tion starting at 1.2 × CV75. Te cells were analyzed for
(−)-menthol α-d-glucoside were tested. Each compound CD54 (with anti-CD54-FITC antibodies; DAKO, Denmark)
was dissolved at 20 mg/mL in distilled water. Te solu- and CD86 expression (with anti-CD86-FITC antibodies;
tion was vortexed for 3 min, held overnight at 25 °C, and BD Pharmingen) by fow cytometry. FITC labeled-mouse
centrifuged at 6500g for 10 min. Te concentration of IgG1 was used as an isotype control. Te relative fuores-
each compound in the supernatants was quantitatively cence intensity (RFI) values of CD54 and CD86 were deter-
analyzed by LC–MS/MS analysis in MRM mode.
mined at >50% of cell viability and calculated as follows:
ꢀ
ꢁ
MFI of chemical-treated cells − MFI of chemical-treated isotype control cells
ꢁ
ꢀ
MFI of vehicle control cells − MFI of vehicle isotype control cells
Topical cooling test
where MFI is the mean fuorescence intensity. If the RFIs
of CD54 and CD86 were greater than 200 and 150%,
respectively, the test chemical was judged as a sensitizer,
and otherwise, it was considered a non-sensitizer.
Following approval of the study by the Public Insti-
tutional Review Board Designated by the Ministry of
Health and Welfare (P01-201705-13-002), Korea, 10
healthy males and females were recruited from the local
institute’s population.
Results
Te topical cooling test was performed as reported previ-
ously (Ottinger et al. 2001). Solutions of 1% (−)-menthol or
(−)-menthol β-d-glucoside were prepared by frst dissolving
100 mg of the product using 50 μL ethanol and diluting to
10 mL with water. Tus, the quantity of the alcoholic solu-
tion in the tested solutions was 0.5%. Twofold diluted solu-
tions were prepared with 0.5% ethanol. An aliquot (0.2 mL)
of solutions containing between 0.031 and 1.0% of the cool-
ant in water was applied to a circular area (~10 cm2) of the
skin surface on the inside of a forearm, midway between the
wrist and the elbow, and rubbed for 30 s. In parallel, an ali-
quot (0.2 mL) of 0.5% ethanol was applied as a blank onto
the skin of the other forearm. After 30 s, the skin was dried
with a towel. A panel of 10 subjects was asked to identify the
arm with a detectable “cooling” sensation and to rank the
perceived cooling intensity on a scale from 0 (no efect) to 5
(very strong). Te values evaluated in three diferent sessions
over 2 days were averaged. Te values between individuals
and separate sessions difered by no more than two scores.
Comparison of water solubility of (−)‑menthol β‑glucoside
and (−)‑menthol α‑glucoside
(−)-Menthol β-glucoside and (−)-menthol α-glucoside
were prepared by chemical synthesis according to the
a previously described method (Sakata and Iwamura
1979). Te chemical synthesis produced an anomeric
mixture, requiring tedious column chromatography
to purify (−)-menthol β-glucoside. Te water solubil-
ity values of (−)-menthol β-glucoside and (−)-menthol
α-glucoside were compared and found to be remark-
ably diferent (Table 1). Te solubility of (−)-men-
thol β-glucoside in water at 25 °C was 18.1 g/L, which
is 27 times higher than that (0.66 g/L) of (−)-menthol
α-glucoside. Tis result prompted us to conduct the
enzymatic β-anomer-selective glycosylation of menthol
(Fig. 1).
Enzymatic glucosylation of menthol
Te purifed BLC enzyme catalyzed the synthesis of
(−)-menthol glucoside using UDP-d-glucose as a sugar
donor (Fig. 2). Time-course production of (−)-menthol
glucoside was quantitatively determined using LC–MS/
MS in MRM mode (Fig. 3). BLC at 3 μM produced a max-
imum yield of nearly 58.9% in a molar ratio at 1 h. Te Km
and Kcat for (−)-menthol with BLC were determined to
be 1.74 0.6 mM and 94.5 2.33 min−1, respectively, in
our system (Additional fle 1: Figure S1).
In vitro skin sensitization test
Te human cell line activation test (H-CLAT) using THP-1
(human monocytic cell line) was performed as described by
Ashikaga et al. (2006). In brief, cells were cultured in RPMI
1640 medium (Invitrogen Corp., Carlsbad, CA, USA) with
10% FBS (v/v), 0.05 mM 2-mercaptoethanol, and 1% antibi-
otic–antimycotic mixture (Invitrogen Corp., Carlsbad, CA,

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Table 1 Summary of water solubility and pharmacological properties of (−)-menthol β-d-glucoside and (−)-menthol
Water
solubility (g/L)
Flavor
The threshold
concentration
for cooling (%)
Expression of activation
Irritating
sensation
to the skin
marker on THP‑1 cells
CD54
CD86
Menthol
NT
Yes
No
NT
0.125
0.06
NT
Positive
Negative
NT
Negative
Negative
NT
Yes
No
NT
Menthol β‑glucoside
Controla
18.1
0.66
NT not tested
a
Control for water solubility were menthol α-glucoside
Structure determination of menthol glycosides
After large-scale reactions followed by chromatographic
isolation, (−)-menthol glucoside and (−)-menthol galac-
toside were obtained as white powders, and their struc-
tures were confrmed by HR-ESIMS and NMR spectral
analysis (Additional fle 1: Figures S4–S6). Te anomeric
confguration of (−)-menthol glucoside and (−)-men-
thol galactoside was determined to be β based on the
large coupling constants of the anomeric protons at
δ 4.35 (J = 7.8 Hz) and δ 4.35 (J = 7.6 Hz), respectively
(Table 2). Because the yield of (−)-menthol N-acetyl-
glucosamine was too low, the structure of (−)-menthol
N-acetylglucosamine was determined using only MS/
MS and HR-ESI MS data (m/z 382.3 [M+Na]+, 360.3
[M+H]+, 204.2 [N-acetylglucosamine+H] +; HRESIMS:
m/z 360.2385 (M+H)+, calcd. 360.2381 for C18H34O6N).
To the best of our knowledge, the NMR data of men-
thol β-d-glucoside was reported for the frst time in this
study, and (−)-menthol β-d-galactoside and (−)-menthol
N-acetylglucosamine are novel compounds.
Fig. 1 Structure of (−)‑menthol β‑d‑glycosides synthesized by BLC, a
glycosyltransferase from B. licheniformis
Cooling efect
To determine whether glucosylation afects the cooling
sensation of menthol on the skin, topical cooling tests of
(−)-menthol β-d-glucoside were performed using men-
thol as the control. (−)-Menthol β-d-glucoside showed
a higher cooling efect than menthol (Fig. 4; Additional
fle 1: Figure S7) and was topically detectable at a con-
centration of 0.06%, whereas menthol imparted a cooling
sensation at a concentrations of >0.125%. Increasing the
concentration of (−)-menthol β-d-glucoside intensifed
the cooling efect on the skin, reaching the maximum
score of 5 at a concentration of 0.25%, whereas menthol
required a concentration of 1%.
Menthol glycosylation with other UDP sugars
To examine the selectivity of BLC enzyme for sugar
donors, menthol glycosylation was performed with
other sugar donors, including UDP-d-galactose, UDP-
N-acetylglucosamine, and UDP-d-glucuronic acid.
Identical reactions were performed by varying only the
UDP-sugar donor. Te incubation of menthol with UDP-
galactose and UDP-N-acetylglucosamine in the pres-
ence of BLC aforded new products corresponding to
(−)-menthol galactoside and (−)-menthol N-acetylglu-
cosamine, as confrmed by the LC–MS analysis (Addi-
tional fle 1: Figures S2, S3), whereas no new product was
detected with the use of UDP-glucuronic acid. Te con-
version of (−)-menthol to (−)-menthol galactoside was
1.56% in the large-scale preparation, as described in the
Materials and Methods section. Tis result showed that
BLC had a high selectivity for UDP-d-glucose as a sugar
donor.
Skin sensitizing potential
Te in vitro skin sensitization test was performed to deter-
mine the sensitizing potential by assessing the changes
in CD54 and CD86 expression in THP-1 cells using fow
cytometry after 24 h exposure to a test compound (Fig. 5).

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Fig. 2 LC–MS analysis of (−)‑menthol glycosylation reaction by BLC with UDP‑d‑glucose. a Total ion chromatogram of the reaction mixture at 0 h
incubation (control reaction); b total ion chromatogram of the reaction mixture at 18 h incubation; c ESI–MS spectrum of the new peak at 6.32 min
Menthol and (−)-menthol β-glucoside treatments were
Discussion
applied at three or four concentrations of a 1.2-fold serial
dilution starting at 250 and 125 μg/mL, respectively, which
were the concentrations that resulted in 75% cell viabil-
ity. As expected, menthol presented values >200% RFI
for CD54 activation marker, indicating the sensitizing
potential of menthol. However, (−)-menthol β-glucoside
presented values <150% RFI for both CD54 and CD86 acti-
vation markers, indicating that (−)-menthol β-glucoside is
a non-sensitizer. In fact, the sensory panel did not feel any
signifcant irritating sensation to the skin for (−)-menthol
β-d-glucoside during the topical cooling tests, whereas
menthol did produce an irritating sensation.
Menthol has well-known cooling characteristics and a
residual minty smell. Because of these attributes, men-
thol is used in a variety of consumer products, ranging
from confectioneries such as chocolate and chewing gum
to cosmetics, oral-care products such as toothpaste, in
over-the-counter medicinal products such as analge-
sics, and as an additive in cold compresses for its cool-
ing and biological efects (Eccles 1994; Patel et al. 2007).
Additionally, approximately one-quarter of the cigarettes
on the market contain menthol (Kamatou et al. 2013).
However, due to its poor solubility in water, the use of
menthol has been limited to direct mixing in the form

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Fig. 3 Time‑course production of (−)‑menthol β‑d‑glucoside using
UDP‑d‑glucose as a sugar donor with increasing concentration of
BLC (0.03–3 μM). The reaction mixtures were incubated for 10 min, 1,
6, and 18 h and quantitatively analyzed by LC–MS/MS in MRM mode
according to“Materials and methods”. The error bars represent the
standard deviation of three independent experiments
Fig. 4 Topical testing of (−)‑menthol β‑d‑glucoside and (−)‑menthol
on the inside of the forearm. The cooling efect on the skin was deter‑
mined using a topical test that scored the cooling intensity on a scale
from 0 (no efect) to 5 (very strong)
Table 2 1H- and 13C-NMR data (700 MHz, CD3OD) for bio-
synthesized (−)-menthol β-glucoside and (−)-menthol
β-galactoside
Position (−)‑Menthol β‑glucoside
H (multi., J in Hz) δc
(−)‑Menthol β‑galactoside
δH (multi., J in Hz) δc
δ
1
3.57 (1H, ddd, 10.6,
105, 4.2)
78.4 3.57 (1H, ddd, 10.8,
10.8, 4.4)
78.4
2
3
1.24 (1H, m)
49.5 1.22 (1H, m)
49.5
24.4
1.03 (1H, m); 1.63
(1H, m)
24.3 1.03 (1H, m); 1.65
(1H, m)
4
0.84 (1H, m); 1.68
(1H, m)
35.8 0.85 (1H, m); 1.67
(1H, m)
35.8
5
6
1.37 (1H, m)
32.9 1.38 (1H, m)
32.9
41.8
0.94 (1H, m); 2.12
(1H, m)
41.8 0.93 (1H, m); 2.13
(1H, m)
7
2.31 (1H, m)
26.3 2.31 (1H, m)
16.4 0.79 (3H, d, 7.2)
21.6 0.89 (3H, d, 7.0)
22.8 0.96 (3H, d, 6.8)
101.4 4.35 (1H, d, 7.6)
75.2 3.14 (1H, m)
78.3 3.35 (1H, m)
72.0 3.30 (1H, m)
77.8 3.24 (1H, m)
26.3
16.4
21.6
22.8
101.5
75.2
78.3
72.1
77.8
8
0.81 (3H, d, 6.8)
0.87 (3H, d, 7.1)
0.93 (3H, d, 6.6)
4.35 (1H, d, 7.8)
3.14 (1H, m)
9
10
1′
2′
3′
4′
5′
6′
3.35 (1H, m)
Fig. 5 In vitro skin sensitization test of (−)‑menthol β‑d‑glucoside
and (−)‑menthol using human cell lines. The changes in CD54 (a)
and CD86 (b) expression on THP‑1 cells were measured after a 24‑h
exposure to a test compound. The RFI values were calculated and
compared
3.31 (1H, m)
3.24 (1H, m)
3.67 (1H, dd, 11.6, 5.4);
3.84 (1H, dd, 11.6, 2.3)
63.1 3.67 (1H, dd, 11.6, 5.2); 63.2
3.84 (1H, dd, 11.6, 2.4)
use of menthol in certain cooling products, such as cos-
metics. Tus, menthol derivatives with good solubility in
water and no favor that retain the cooling sensation have
been greatly desired. Glycosylation improves the water
of solid particles with the material to which the menthol
is to be added, making a suspension using an emulsifer,
or dissolving the menthol in an organic solvent such as
alcohol. Additionally, the inherent mint favor limits the

et al. AMB Expr (2017) 7:167
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solubility of drugs or natural products (Weymouth- concentration of menthol that maintains a cooling sensa-
Wilson 1997). In our study, (−)-menthol β-glucoside tion and minimizes irritation must be considered (Gillis
was shown to more soluble in water (>27 times) than et al. 2010). In this study, interestingly, (−)-menthol β-d-
(−)-menthol α-glucoside; hence, the β-anomer-selective glucoside exhibited higher cooling efects with no accom-
glucosylation of menthol is necessary. Limited glycosyla- panying irritation compared with menthol. We believe
tion of menthol has been reported. Water-soluble men- that the sugar moiety of (−)-menthol β-d-glucoside
thyl glycosides with monosaccharide or oligosaccharide might play an important role in its interaction with the
as a sugar unit have been synthesized by chemical meth- cold receptor and pain fbers.
ods, but the anomer-selective synthesis of methyl gly-
Many natural products currently used as therapeu-
coside is impossible due to a lack of regioselectivity and tics derive their biological functions or pharmacokinetic
stereoselectivity (Sakata and Iwamura 1979). α-Anomer- properties from the sugar components present in their
selective synthesis of menthyl glucoside was reported structures. For example, vancomycin loses its in vivo
using α-glucosidase from yeast (Nakagawa et al. 1998) or efcacy if the sugar moiety is removed, although in vitro
bacteria such as Xanthomonas (Nakagawa et al. 2000), activity remains (Nagarajan 1993). Doxorubicin has
but β-anomer-selective glucosylation of menthol has no antitumor activity without the sugar portion (Han
not yet been reported. We therefore undertook to syn- et al. 2011). Erythromycin contains sugar moieties that
thesize menthol β-glucoside by synthesizing the gluco- afect its molecular mechanism of action (Langenhan
syl analogs of menthol using UDP-glycosyltransferase et al. 2005). Diferent sugar moieties could also result in
BLC from B. licheniformis. Tree diferent glycoside alteration of biological activities, as observed in rebec-
derivatives with the β-confguration were successfully camycin (Animati et al. 2008). In addition to the cool-
produced in the reaction catalyzed by the BLC in the ing efect, menthol has diverse biological properties
presence of the corresponding NDP-sugars. Te maxi- such as analgesic, antifungal, antibacterial, anticancer,
mum glucoside conversion rate was determined from the anti-infammatory, antitussive, antiviral, and insecticidal
time-dependent study of menthol coupled with UDP- efects (Kamatou et al. 2013). In recent years, menthol
d-glucose. During the molar conversion, approximately has become one of the most efective terpenes used to
58.9% glucoside was produced after 1 h of incubation. enhance the dermal penetration of pharmaceuticals.
BLC from B. licheniformis DSM 13 is reported to glyco- Tus, a study on the efects of menthol glycosides on
sylate diverse substrates such as geldanamycin analogs these biological activities is of interest.
(Wu et al. 2012), epothilone A (Parajuli et al. 2014), chal-
Te current study demonstrates a higher water solu-
cone (Pandey et al. 2013), and various favonoids (Koirala bility, an improvement in cooling efects and a lack
et al. 2014). Te maximum conversion rates of epothilone of sensitization of (−)-menthol β-glucoside, thereby
A and 3-hydroxyfavone to its respective glucosides by demonstrating its potential as a new cooling agent.
BLC were reported to be approximately 26 and 90%, β-Anomer-selective glucosylation of (−)-menthol was
respectively, at 3 h incubation using UDP-d-glucose. In achieved using the BLC glycosyltransferase from B.
this study, BLC catalyzed the glycosylation of menthol licheniformis. Because BLC uses the relatively expensive
(C10H20O; molecular weight, 156), a notably small cyclic UDP-glucose as a donor substrate, the in vivo production
monoterpene alcohol. Tis result proves the remarkable of (−)-menthol β-glucoside using a strain engineered
fexibility of an aglycone substrate of BLC.
with BLC could be considered for industrial production
Although improvement of the water solubility of men- of menthol β-glucoside.
thyl glycosides is known (Sakata and Iwamura 1979), no
reports exist regarding the enhancement of their pharma-
cological properties. Menthol acts on the highly sensitive
cold receptor TRPM8 located on the cell membrane of
sensory neurons (McKemy et al. 2002). Menthol contains
many active forms, but (−)-menthol produces the strong-
est cooling efects. Low concentrations of (−)-men-
thol elicit a cooling sensation (Schafer et al. 1986), but
increased concentrations result in irritation and local
anesthetic efects (Eccles 1994). Te feeling of irritation
might be due to the action of (−)-menthol on pain fb-
ers (Eccles 1994). A concentration of 0.2% (−)-menthol
causes both cooling and irritation. Tus, an optimal
Additional fle
Additional fle 1. Structural elucidation, LC–MS and HR‑ESIMS data of
(−)‑menthol β‑glucoside, (−)‑menthol β‑galactoside, and (−)‑menthol
N‑acetylglucosamine. Lineweaver–Burk plot of (−)‑menthol glucosyla‑
tion by BLC with respect to menthol (Figure S1). LC–MS analysis of
(−)‑menthol glycosylation reaction by BLC with UDP‑d‑galactose (Figure
S2) and UDP‑d‑N‑acetylglucosamine (Figure S3). 1H NMR spectra of syn‑
thesized (−)‑menthol α‑glucoside and (−)‑menthol β‑glucoside (Figure
S4). 1H and 13C NMR spectra of (−)‑menthol β‑glucoside (Figure S5) and
(−)‑menthol β‑galactoside (Figure S6) prepared by BLC‑catalyzed gly‑
cosylation reactions. HMBC data of the menthol glucoside and menthol
galactoside prepared by BLC‑catalyzed glycosylation reactions (Figure
S7). Topical cooling test (Table S1).

et al. AMB Expr (2017) 7:167
Choi
Page 8 of 8
Abbreviations
Animati F, Berettoni M, Bigioni M, Binaschi M, Felicetti P, Gontrani L, Incani
UDP: uridine diphosphate; MRM: multiple reaction monitoring; TLC: thin layer
chromatography; NMR: nuclear magnetic resonance; H‑CLAT: human cell line
activation test; THP‑1: human monocytic cell line; RFI: relative fuorescence
intensity.
O, Madami A, Monteagudo E, Olivieri L, Resta S, Rossi C, Cipollone A
(2008) Synthesis, biological evaluation, and molecular modeling stud‑
ies of rebeccamycin analogues modifed in the carbohydrate moiety.
ChemMedChem 3:266–279
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M, Ito Y, Suzuki H, Toyoda H (2006) Development of an in vitro skin
sensitization test using human cell lines: the human Cell Line Activation
Test (h‑CLAT). I. Optimization of the h‑CLAT protocol. Toxicol In Vitro
20:767–773
Authors’ contributions
WGK designed the research and wrote the paper; HYC and BMK performed
the research. HYC, BMK, JSK, and WGK analyzed the data. All authors read and
approved the fnal manuscript.
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46:618–630
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Han AR, Park JW, Lee MK, Ban YH, Yoo YJ, Kim EJ, Kim E, Kim BG, Sohng JK, Yoon
YJ (2011) Development of a Streptomyces venezuelae‑based combinato‑
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doxorubicin and its biosynthetic intermediates. Appl Environ Microbiol
77:4912–4923
Author details
1 Superbacteria Research Center, Korea Research Institute of Bioscience
and Biotechnology, Yusong, Daejeon 305‑806, Republic of Korea. 2 Depart‑
ment of Bio‑Molecular Science, KRIBB School of Bioscience, Korea University
of Science and Technology (UST), Yusong, Daejeon, Republic of Korea. 3 Infec‑
tion Control Material Research Center, Korea Research Institute of Bioscience
and Biotechnology, Jeongeup‑si, Jeollabuk‑do 56212, Republic of Korea.
Acknowledgements
The authors thank the Korea Basic Science Institute for NMR measurements.
Kamatou GP, Vermaak I, Viljoen AM, Lawrence BM (2013) Menthol: a simple
monoterpene with remarkable biological properties. Phytochemistry
96:15–25
Competing interests
The authors declare that they have no competing interests.
Koirala N, Pandey RP, Parajuli P, Jung HJ, Sohng JK (2014) Methylation and sub‑
sequent glycosylation of 7,8‑dihydroxyfavone. J Biotechnol 184:128–137
Langenhan JM, Grifth BR, Thorson JS (2005) Neoglycorandomization and
chemoenzymatic glycorandomization: two complementary tools for
natural product diversifcation. J Nat Prod 68:1696–1711
McKemy DD, Neuhausser WM, Julius D (2002) Identifcation of a cold recep‑
tor reveals a general role for TRP channels in thermosensation. Nature
416:52–58
Availability of data and materials
The data supporting the conclusions of this article are included within the
article and Additional fle 1.
Consent for publication
Not applicable.
Nagarajan R (1993) Structure–activity relationships of vancomycin‑type glyco‑
peptide antibiotics. J Antibiot 46:1181–1195
Ethic approval and consent to participate
Topical cooling testing was approved by the Public Institutional Review Board
Designated by the Ministry of Health and Welfare (P01‑201705‑13‑002), Korea.
Written consent was received from all participants.
Nakagawa H, Yoshiyama M, Shimura S, Kirimura K, Usami S (1998) Anomer‑
selective glucosylation of l‑menthol by yeast α‑glucosidase. Biosci
Biotechnol Biochem 62:1332–1336
Nakagawa H, Dobashi Y, Sato T, Yoshida K, Tsugane T, Shimura S, Kirimura K,
Kino K, Usami S (2000) Alpha‑anomer‑selective glucosylation of menthol
with high yield through a crystal accumulation reaction using lyophilized
cells of Xanthomonas campestris WU‑9701. J Biosci Bioeng 89:138–144
Ottinger H, Soldo T, Hofmann T (2001) Systematic studies on structure and
physiological activity of cyclic alpha‑keto enamines, a novel class of“cool‑
ing”compounds. J Agric Food Chem 49:5383–5390
Pandey RP, Li TF, Kim EH, Yamaguchi T, Park YI, Kim JS, Sohng JK (2013) Enzy‑
matic synthesis of novel phloretin glucosides. Appl Environ Microbiol
79:3516–3521
Funding
This study was supported by the Intelligent Synthetic Biology Center of the
Global Frontier Project funded by the Ministry of Education, Science and
Technology (2011‑0031944), the Korea Institute of Planning and Evaluation
for Technology in Food, Agriculture, Forestry and Fisheries (IPET) through the
Agri‑Bio Industry Technology Development Program funded by the Ministry
of Agriculture, Food and Rural Afairs (MAFRA) (114145‑3), and the KRIBB
Research Initiative Program, Republic of Korea.
Parajuli P, Pandey RP, Koirala N, Yoon YJ, Kim BG, Sohng JK (2014) Enzymatic
synthesis of epothilone A glycosides. AMB Express 4:31
Patel T, Ishiuji Y, Yosipovitch G (2007) Menthol: a refreshing look at this ancient
compound. J Am Acad Dermatol 57:873–878
Publisher’s Note
Springer Nature remains neutral with regard to jurisdictional claims in pub‑
lished maps and institutional afliations.
Rabausch U, Juergensen J, Ilmberger N, Bohnke S, Fischer S, Schubach B,
Schulte M, Streit WR (2013) Functional screening of metagenome and
genome libraries for detection of novel favonoid‑modifying enzymes.
Appl Environ Microbiol 79:4551–4563
Received: 23 June 2017 Accepted: 18 August 2017
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Wu CZ, Jang JH, Woo M, Ahn JS, Kim JS, Hong YS (2012) Enzymatic glycosyla‑
tion of nonbenzoquinone geldanamycin analogs via Bacillus UDP‑glyco‑
syltransferase. Appl Environ Microbiol 78:7680–7686
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