Chemicals
(−)-Menthol, UDP-glucose, UDP-galactose, UDP-N-acetylglucosamine, and UDP-glucuronic acid were purchased from Sigma-Aldrich (St. Louis, MO, USA). The chemical synthesis of (−)-menthol β-glucoside and (−)-menthol α-glucoside was conducted by MediGen Inc (Daejeon, Korea).
Cloning, expression, and purification of glycosyltransferase
Glycosyltransferase BLC was cloned, expressed, and purified from the genomic DNA of B. licheniformis DSM13, as reported previously (Wu et al. 2012). Briefly, the amplified PCR products were purified, digested with BamHI and XhoI, and cloned into the same sites of the pET28a vector. The recombinant expression vectors were transformed into E. coli BL21(DE3). The E. coli BL21(DE3) culture transformed with the recombinant expression vector was induced with isopropyl-1-thio-β-d-galactopyranoside (IPTG) (at a final concentration of 0.5 mM). After centrifugation of the cell lysate, BLC enzyme was purified by nickel–nitriloriacetic acid (Ni–NTA) column chromatography (Qiagen). The fractions eluted with 50 and 100 mM imidazole were pooled and concentrated with an Amicon Ultra-15 instrument (Millipore). The 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.
Glycosylation of menthol using BLC
Analytical glycosylation reactions were performed in a total volume of 50 μL containing purified His-tagged recombinant BLC (3 μM), 1 mM menthol, 2 mM UDP-glycosides (UDP-glucose, UDP-galactose, UDP-N-acetylglucosamine, and UDP-glucuronic acid), 1 mM MgCl2, and 50 mM Tris–HCl (pH 8.0) (Wu et al. 2012). The 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 concentrations 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. The supernatants were qualitatively detected by LC–ESI–MS or quantitatively analyzed by LC–MS/MS operating in multiple reaction monitoring (MRM) mode, as described below.
Quantitative analysis of menthol glucoside by LC–MS/MS
The concentrations of menthol glucoside in the water solubility test or the BLC reactions were analyzed by LC–MS/MS in MRM mode. The 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, Singapore). The column was maintained at 20 °C with a flow 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 specifically 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 temperature, and 12 psi ion source gas. The compound-dependent 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 potential. For analysis of (−)-menthol α-d-glucoside, the Turbolon Spray source-dependent parameters were the same as those for (−)-menthol α-d-glucoside. The 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.
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 menthol glucoside (0.41 mM, 4.2 mg) representing 41.2% conversion of menthol. The 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 mixture, the reaction mixture was partitioned with water-saturated butanol, and the butanol layer was evaporated in vacuo. The resulting residue was purified 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 R
f
values of 0.61 and 0.58, respectively, as detected with I2 vapor. Their structures were confirmed by 700 MHz Bruker, BioSpin nuclear magnetic resonance (NMR) analysis including one-dimensional 1H NMR, 13C NMR and two-dimensional NMR-correlation spectroscopy (COSY), hetero-nuclear single quantum coherence (HSQC), and heteronuclear multiple bond connectivity (HMBC).
Water solubility determination
The water solubilities of (−)-menthol β-d-glucoside and (−)-menthol α-d-glucoside were tested. Each compound was dissolved at 20 mg/mL in distilled water. The solution was vortexed for 3 min, held overnight at 25 °C, and centrifuged at 6500g for 10 min. The concentration of each compound in the supernatants was quantitatively analyzed by LC–MS/MS analysis in MRM mode.
Topical cooling test
Following approval of the study by the Public Institutional 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.
The topical cooling test was performed as reported previously (Ottinger et al. 2001). Solutions of 1% (−)-menthol or (−)-menthol β-d-glucoside were prepared by first dissolving 100 mg of the product using 50 μL ethanol and diluting to 10 mL with water. Thus, the quantity of the alcoholic solution in the tested solutions was 0.5%. Twofold diluted solutions were prepared with 0.5% ethanol. An aliquot (0.2 mL) of solutions containing between 0.031 and 1.0% of the coolant 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 aliquot (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 effect) to 5 (very strong). The values evaluated in three different sessions over 2 days were averaged. The values between individuals and separate sessions differed by no more than two scores.
In vitro skin sensitization test
The 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% antibiotic–antimycotic mixture (Invitrogen Corp., Carlsbad, CA, USA), seeded at 0.5 × 106 cells/mL in a 24-well plate, and cultured with chemicals for 24 h. When DMSO was used as a solvent, its final concentration in the culture media was less than 0.2%. The cell viability was evaluated by MTT assay. The concentration resulting in 75% cell viability, referred to as CV75, was calculated based on the analysis of viable cells. Cells were incubated for 24 h with test chemicals at three or four concentrations of a 1.2-fold serial dilution starting at 1.2 × CV75. The cells were analyzed for CD54 (with anti-CD54-FITC antibodies; DAKO, Denmark) and CD86 expression (with anti-CD86-FITC antibodies; BD Pharmingen) by flow cytometry. FITC labeled-mouse IgG1 was used as an isotype control. The relative fluorescence intensity (RFI) values of CD54 and CD86 were determined at >50% of cell viability and calculated as follows:
$$\frac{{\left( {{\text{MFI of chemical-treated cells}} - {\text{MFI of chemical-treated isotype control cells}}} \right)}}{{\left( {{\text{MFI of vehicle control cells}} - {\text{MFI of vehicle isotype control cells}}} \right)}}$$
where MFI is the mean fluorescence 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.
