Materials
Gallic acid, deuterium oxide (D2O), 1,1-diphenyl-2-picrylhydrazyl (DPPH), 3-(3,4-dihydroxylphenyl)-l-alanine (l-DOPA), mushroom tyrosinase, and β-arbutin were obtained from Sigma-Aldrich (St. Louis, MO, USA). All chemical reagents were commercially available and of analytical reagent grade.
Enzyme preparation
Dextransucrase (EC 3.2.1.11) was obtained from L. mesenteroides B-512 FMCM (KCCM 11728P), which were cultured on LM medium with 2% (w/v) glucose, as previously described (Moon et al. 2007a). The fermented culture was harvested, centrifuged, and concentrated with 30 K hollow fibers (Millipore, Bedford, MA, USA). The enzyme activity was measured at 28 °C with 0.1 M sucrose in 20 mM sodium acetate (pH 5.2) for different reaction periods. The reactants were spotted on a thin-layer chromatography (TLC) silica gel 60 plate (Merck, Darmstadt, Germany) and developed twice in an acetonitrile–water (85:15, v/v) solution. The TLC plate was visualized by spraying with N-(1-naphthyl)-ethylenediamine-H2SO4 solution and heating at 121 °C for 10 min. The content of fructose released from sucrose was measured by the evaluation of its density using the NIH Image Program (http://rsb.info.nih.gov/nih-image) with a standard compound. One unit (U) was defined as the amount of enzyme that caused the release of 1 μmol of fructose per minute at 28 °C in 20 mM sodium acetate buffer (pH 5.2).
Synthesis, purification, and identification of gallic acid glucoside
The reactants (1 L), which consisted of 325 mM gallic acid, 355 mM sucrose, and B-512 FMCM dextransucrase (0.55 mU/mL), were incubated in 20 mM sodium acetate (pH 5.2) at 28 °C for 6 h and boiled for 10 min to stop the enzyme action. Glucosylated gallic acid was confirmed by using TLC plate analysis (Merck, Darmstadt, Germany) at 25 °C. The reaction mixtures were placed on TLC plates and developed twice in the following solvent systems: (1) nitromethane/1-propanol/water (2:5:1.5, v/v/v) or (2) ethyl acetate/acetic acid/water (3:1:1, v/v/v) with gallic acid, fructose, and sucrose as the standard materials. Subsequently, the developed plate was visualized by spraying with N-(1-naphthyl)-ethylenediamine-H2SO4 solution and heating at 121 °C (Moon et al. 2007a) or UV exposure, as previously described (Seo et al. 2005).
The reaction mixture (1 L) was partitioned with n-butanol to obtain the modified gallic acid products from the upper layer. The modified products were further concentrated under vacuum to 50 mL by using a rotary evaporator (EYELA, Tokyo, Japan) at 47 °C. The sample was applied to a 4.0 × 75 cm silica gel column. After the removal of the remaining sugars with distilled water (total, 2.5 L; flow rate, 1 mL/min), gallic acid glucoside was extracted with 85% (v/v) acetonitrile in water. The compound was purified by high-pressure liquid chromatography (HPLC) under the following conditions: column TSK-GEL amide-80, 5 μm (Waters, Milford, MA, USA); 80% (v/v) acetonitrile in water mobile phase; 1.0 mL/min flow rate; RID-10A RI detector (Shimadzu, Tokyo, Japan).
Purified gallic acid glucoside (2 mg/mL) was mixed with 2,5-dihydroxybenzoic acid (1 mg/mL) in a ratio of 1:1 (v/v), loaded, and dried on a stainless-steel plate at 25 °C. The molecular mass of the sample was measured by MALDI-TOF (Voyager DE-STR, Applied Biosystems, Poster, CA, USA) in a linear mode with delayed extraction (75 laser shots) and an acceleration voltage of 65 kV.
Optimization of gallic acid glucoside production
The influence of sucrose, enzyme, and gallic acid on the reaction was detected by using response surface methodology (RSM). The experimental data were applied via the response surface regression procedure with the following polynomial equation (Abe et al. 2000):
$$ \begin{aligned}Y &= \beta_{0} + \beta_{ 1} {\text{x}}_{ 1} + \beta_{ 2} {\text{x}}_{ 2} + \beta_{ 3} {\text{x}}_{ 3} \\ & \quad + \beta_{ 1 1} {{\text{x}}_{1}}^{ 2} + \beta_{ 2 2} {{\text{x}}_{ 2}}^{ 2} + \beta_{ 3 3} {{\text{x}}_{ 3}}^{ 2} \\ & \quad + \beta_{ 1 2} {\text{x}}_{ 1} {\text{x}}_{ 2} + \beta_{ 1 3} {\text{x}}_{ 1} {\text{x}}_{ 3} + \beta_{ 2 3} {\text{x}}_{ 2} {\text{x}}_{ 3} . \end{aligned}$$
The regression and graphical analysis of the data were computed by Design-Expert 7.0.0 central composite design (CCD) RSM software (State-Ease, Minneapolis, MN, USA). The effects of separate parameters and interactions were analyzed by analysis of variance and the equation and model terms were analyzed by Fisher’s test for model significance. The fit quality for the model equation was indicated by the coefficient of determination (R
2) and an adjusted R
2. Preliminary experiments led to the selection of three factors (dextransucrase unit, sucrose, and gallic acid concentration) for the optimization of the production conditions of gallic acid glucoside, with the following values: dextransucrase from L. mesenteroides, 61–1238 mU/mL; sucrose, 10–700 mM; and gallic acid, 30–619 mM.
Antioxidant activity
The antioxidant activities of gallic acid and gallic acid glucoside were detected by using a DPPH scavenging assay (Abe et al. 2000). Samples at each concentration (0.01–2.0 mM) were dissolved in ethanol, reacted with a 0.1 M DPPH reagent for 10 min at 25 °C, and monitored at 517 nm by using a microplate reader (Molecular Devices, Sunnyvale, CA, USA). The radical scavenging activity was expressed as the percentage inhibition of DPPH radical concentration against the reference compound of ascorbic acid. The IC50 value was designated as the concentration of sample that resulted in a 50% reduction in DPPH radicals.
Anti-lipid peroxidation activity
The anti-lipid peroxidation effect was analyzed by ARA-L kit (ABCD GmbH, Berlin, Germany) with an HP-CLA chemiluminescence-measuring device (Tohoku Electronic Industrial, Tokyo, Japan). In accordance with the TIC (thermo-initiated chemiluminescence) method, the antioxidant species in the sample (gallic acid or gallic acid glucoside) were incubated with ample free radical-attached luminol to delay photon generation until the antioxidant species were consumed. The lag time(s) was proportional to the amount of antioxidant species in sample. Each sample (20 μL of 0.5, 1.0, and 5.0 mM) or α-tocopherol (20 μL of 10, 25, 50, and 100 μM/mL) was mixed with a reaction buffer (1.0 mL) at 37 °C, and the effects were measured (Sreejayan et al. 1997).
Tyrosinase inhibition
The incubation mixture consisted of l-DOPA (0–5 mM, l-β-3,4-dihydroxyphenyl alanine) and mushroom tyrosinase (10 U/mL), in the presence or absence of gallic acid or gallic acid glucoside (0–10 mM) as the inhibitor. Ten units of mushroom tyrosinase were used to find the Ki value. The nature of the inhibition was determined by a Dixon plot of the relationship between the reciprocal of the velocity of the reaction and the concentration of the inhibitor (gallic acid or gallic acid glucoside at 0–10 mM) at various substrate concentrations (0.1, 0.5, 1.0, and 5.0 mM l-DOPA). After the mixture was incubated at 37 °C for 10 min, the absorbance was measured at 475 nm by using a microplate reader (Molecular Devices, Sunnyvale, CA, USA), which allowed the calculation of the tyrosinase inhibition (Kim et al. 2010).
MMP-1 production and type 1 procollagen production by enzyme-linked immunosorbent assay (ELISA)
Human newborn foreskin fibroblast cells (HS68) were cultured in Dulbecco’s Modified Eagle’s Medium supplemented with 10% fetal bovine serum (Sigma-Aldrich, St. Louis, MO, USA) and 1% antibiotic–antimycotic (Sigma-Aldrich, St. Louis, MO, USA) in a humidified atmosphere with 5% CO2 at 37 °C. The HS68 cells (ATCC CRL 1635; Rockville, MD, USA) were subcultured in a 1:5 ratio for several passages until they reached 80–90% confluence (Ho et al. 2005; Watanabe et al. 2004). The serum-starved confluent cells were washed twice with phosphate-buffered saline (PBS), left in a thin layer of PBS, and exposed to UVB (100 mJ/cm2) from a UVB lamp (312 nm, Spectroline Model EB-160C, New York, NY). Immediately after irradiation, the cells were washed in a serum-free medium and the response was detected after incubation for 24 h. Prior to UVB irradiation, the cells were pretreated with arbutin (standard, Sigma-Aldrich, St. Louis, MO, USA), gallic acid, and gallic acid glucoside (10–100 μM/mL). The negative control (Control) comprised cells that did not receive UVB exposure and the positive control (UVB) comprised cells that received UVB exposure in the absence of an antioxidant compound (Ho et al. 2005; Watanabe et al. 2004). MMP-1 production was measured by using an ELISA kit (Merck & Co. Inc., Whitehouse Station, NJ, USA), as previously described (Ho et al. 2005). Type 1 procollagen content was measured by using a procollagen type I C-peptide ELISA kit (MK101, Takara, Tokyo, Japan), as previously described (Watanabe et al. 2004). Each sample was measured in triplicate.