Synthesis of a Novel α-Glucosyl Ginsenoside F1 by Cyclodextrin Glucanotransferase and Its In Vitro Cosmetic Applications

Ginsenosides from Panax ginseng (Korean ginseng) are unique triterpenoidal saponins that are considered to be responsible for most of the pharmacological activities of P. ginseng. However, the various linkage positions cause different pharmacological activities. In this context, we aimed to synthesize new derivatives of ginsenosides with unusual linkages that show enhanced pharmacological activities. Novel α-glycosylated derivatives of ginsenoside F1 were synthesized from transglycosylation reactions of dextrin (sugar donor) and ginsenoside F1 (acceptor) by the successive actions of Toruzyme®3.0L, a cyclodextrin glucanotransferase. One of the resultant products was isolated and identified as (20S)-3β,6α,12β-trihydroxydammar-24ene-(20-O-β-D-glucopyranosyl-(1→2)-α-D-glucopyranoside) by various spectroscopic characterization techniques of fast atom bombardment-mass spectrometry (FAB-MS), infrared spectroscopy (IR), proton-nuclear magnetic resonance (1H-NMR), 13C-NMR, gradient heteronuclear single quantum coherence (gHSQC), and gradient heteronuclear multiple bond coherence (gHMBC). As expected, the novel α-glycosylated ginsenoside F1 (G1-F1) exhibited increased solubility, lower cytotoxicity toward human dermal fibroblast cells (HDF), and higher tyrosinase activity and ultraviolet A (UVA)-induced inhibitory activity against matrix metalloproteinase-1 (MMP-1) than ginsenoside F1. Since F1 has been reported as an antiaging and antioxidant agent, the enhanced efficacies of the novel α-glycosylated ginsenoside F1 suggest that it might be useful in cosmetic applications after screening.


Introduction
Ginseng saponins, referred to as ginsenosides, are one of the major bioactive substances of Panax ginseng Meyer, a commonly used traditional herbal medicine in Korea, China, and Japan. Ginsenosides have been reported to have antifatigue and antioxidant activities, improve brain function, enhance stamina, and regulate blood circulation with approval from the Korea Food and Drug 10-15 units of glucose) were dissolved in 20 mM sodium phosphate buffer (1 mL, pH 7.0). Next, 25 µL of Toruzyme ® 3.0L with initial activity of 3.0 KNU (kilo novo units)/g [17] was added to the reaction mixture and reacted at 50 • C for 2 h. and kept in boiling water for 5 min to inactivate the enzyme. The mixture was extracted three times with an equal volume of n-butanol, and the n-butanol layer was washed twice with distilled water to remove excess dextrin, dried in a rotary evaporator under vacuum [19], and dissolved in methanol for thin-layer chromatography (TLC).

Glycosylation of Ginsenoside F1
For further experimental analysis, F1 was used as a substrate. The effects of different concentrations of dextrin (0-7 mg) and Toruzyme ® (5-30 µL) and different reaction durations (0.5-3 h) on specificity of F1 glycosylation were examined using the procedure described above. For purification of glycosylated F1, F1 (500 mg, 1.56 mM, 1 eq) and dextrin (2g, 7.92 mM, 5 eq) were dissolved in 500 mL of 20 mM sodium phosphate buffer and then treated with 15 mL of Toruzyme ® 3.0 L.

Identification of Glycosylated Ginsenoside F1
Semiqualitative screening of the glycosylated products was carried out by TLC and high-performance liquid chromatography (HPLC) was carried out by Ramya et al., Quan et al., 2012 [13,23] with slight modifications. TLC was performed with silica gel plates (60 F254, Merck, Darmstadt, Germany) using the developing solvent CHCl 3 :CH 3 OH:H 2 O (65:35:10, v/v, lower phase). The TLC plates were dried, dipped in 10% H 2 SO 4 , and air dried with heating at 110 to 120 • C. The HPLC analysis was carried out on an Agilent 1260 series with a C 18 (250 × 4.6 mm, ID 5 µm) column using distilled water as solvent A and acetonitrile as solvent B mobile phases. The following gradient was used: A:B ratios of 80.

Nuclear Magnetic Resonance Analysis
Structural elucidation of the new compound by NMR spectra ( 1 H NMR, 13 C NMR, gHSQC (heteronuclear single quantum correlation) and heteronuclear multiple bond correlation (gHMBC)) were performed using a Varian Unity INOVA AS 400 FT-NMR spectrometer (Varian, Palo Alto, CA, USA), and chemical shifts were expressed in δ (ppm), with tetramethylsilane (TMS) used as an internal standard. The dimethyl sulfoxide-d 6 (DMSO-d 6 ) was used as a solvent. Melting points were obtained using a Fisher-John's melting point apparatus. Optical rotations were measured on a JASCO P-1010 digital polarimeter. Infrared spectra were obtained on a Perkin Elmer Spectrum One FTIR spectrometer (Perkin-Elmer, Walthanm, MA, USA). High resolution fast-atom bombardment mass spectrometry (HR-FAB/MS) were recorded using a JEOL JMS-700 (JEOL, Tokyo, Japan) mass spectrometer.

Cell Lines and Cell Culture
Human dermal fibroblasts (HDF) were purchased from the Korean Cell Line Bank (Seoul, Korea). The cells were grown in Dulbecco's modified essential media (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin at 37 • C in a humidified atmosphere containing 95% air and 5% CO 2 .

Ultraviolet Irradiation and Sample Treatment
A high-pressure metal halide lamp (UVASUN 3000, Mutzhas, Munich, Germany) emitting wavelengths in the range of 340 to 450 nm was used as a UV source. Human dermal fibroblasts cells were seeded at 4 × 10 cell/dish in 60-mm culture dishes for 24 h. Prior to UV irradiation, cells were washed twice with phosphate buffer saline (PBS), and the medium was replaced with 1 mL of PBS. The incident dose at the surface of the cells was 66 mW/s. The spectral distribution of the UVASUN 3000 source was determined with a Beckman UV 5270 spectrophotometer (Beckman, Munich, Germany, FRG).

Cytotoxicity Assay
Human dermal fibroblasts cells were cultured at a density of 1 × 10 4 cells/well in 96-well flat-bottomed plates in a 5% CO 2 humidified atmosphere at 37 • C. After 24 h of culture, the medium was exchanged with medium containing different concentrations of ginsenoside F1 (F1) and α-glycosylated ginsenoside F1 (Glycosylated F1), and the cells were incubated for a further 24 h. Cell viability was determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay [24] with slight modification. Briefly, 10 µL of MTT solution (5 mg/mL) was added to each well and incubated for 4 h. After removal of MTT, the cells were lysed with 100 µL DMSO, and absorbance was measured at 570 nm using a microplate reader (Bio-Tek Instruments, Winooski, VT, USA).

In Vitro Tyrosinase Inhibition Activity
Tyrosinase from Agricus bisporus (mushroom) was purchased from Sigma Chemicals Co. (St Louis, MO, USA). Inhibition of tyrosinase activity was measured as previously described [22]. L-DOPA (3-(3,4-dihydroxyphenyl)-L-alanine, 0.83 or 3.3 mM) was used as the substrate, and 600 units of tyrosinase was added in the presence or absence of F1, glycosylated F1, or arbutin. The absorbance was measured at 475 nm in a microplate reader (Bio-Tek Instruments, Winooski, VT, USA).

Biotransformation of Minor Ginsenosides by Cyclodextrin glycosyltransferase (CGTase)
Among the major ginsenosides, Rb1, Rc, Re, and Rg1 have already been used as substrates for the synthesis of series of new α-glycosylginsenosides through transglycosylation [13,25,26]. However, after oral administration, the major ginsenosides were converted into minor ginsenosides by intestinal microflora. Therefore, we used minor ginsenosides CK, Rh2, F1, Rh1, aPPD, and aPPT as acceptors with dextrin as a sugar donor during CGTase enzyme transglycosylation. As a result, CK, Rh2, F1, and Rh1 yielded new transglycosylated compounds with different retention factor (R f ) values compared with known ginsenoside standards ( Figure S1). Among these, PPT type ginsenosides Rh1 and F1 showed more glycosylated products, possibly due to the glucose attached to α-OH at C-6 and another -OH at C-20 of the dammerendiol steroidal aglycone. We chose F1 for further studies because of the distinct separation of glycosylated products in addition to its previous reported application in cosmetics and skin care. PPD and PPT aglycone did not generate glycosylated products, indicating that sugar molecules are primarily involved in transglycosylation.

Specificity of Transglycosylation of Ginsenoside F1
Even though the effects of various factors on transglycosylation by Toruzyme were already reported [19,27], this should be validated for the effective synthesis of new compounds. Therefore, the effects of different concentrations of dextrin and CGTase (Toruzyme) on the degree of glycosylation were investigated by HPLC. As shown in Figure S2a, the 5:1 w/w ratio of dextrin: F1 showed the highest yield. There was no significant difference for greater than five volumes, and it was difficult to separate saponin after biotransformation due to the combined extraction of sugar with saponin in the recovery process. In addition, increasing the amount of enzyme rapidly increased the yield up to 20 µL of enzyme with 1 mg of F1 and 5 mg of dextrin, as determined by HPLC ( Figure S2b).

Characterization of Novel α-Glycosylated Ginsenoside F1
Water Solubility of Ginsenoside F1 and Novel α-Glycosylated Ginsenoside F1 Transglycosylation reactions catalyzed by CGTase are an efficient method to enhance the water solubility of various compounds [16,18,28]. Accordingly, the water solubility of α-glycosylated ginsenoside F1 was higher than that of F1 alone (data not shown). The soluble α-glycosylated ginsenoside F1 should not only facilitate investigation of the pharmacological activities of ginsenoside F1, but also may be useful as a cosmetics ingredient.

Comparison of Cell Viability of Ginsenoside F1 and Novel α-Glycosylated Ginsenoside F1 in Human Dermal Fibroblast Cells
To evaluate the effects of α-glycosylated ginsenoside F1 and ginsenoside F1 on the cell viability of HDFs, the cells were treated with different concentrations. Ginsenoside F1 reduced the cell viability of HDFs to a greater extent than α-glycosylated ginsenoside F1 (G1-F1) in a dose-dependent

Characterization of Novel α-Glycosylated Ginsenoside F1
Water Solubility of Ginsenoside F1 and Novel α-Glycosylated Ginsenoside F1 Transglycosylation reactions catalyzed by CGTase are an efficient method to enhance the water solubility of various compounds [16,18,28]. Accordingly, the water solubility of α-glycosylated ginsenoside F1 was higher than that of F1 alone (data not shown). The soluble α-glycosylated ginsenoside F1 should not only facilitate investigation of the pharmacological activities of ginsenoside F1, but also may be useful as a cosmetics ingredient.

Comparison of Cell Viability of Ginsenoside F1 and Novel α-Glycosylated Ginsenoside F1 in Human Dermal Fibroblast Cells
To evaluate the effects of α-glycosylated ginsenoside F1 and ginsenoside F1 on the cell viability of HDFs, the cells were treated with different concentrations. Ginsenoside F1 reduced the cell viability of HDFs to a greater extent than α-glycosylated ginsenoside F1 (G1-F1) in a dose-dependent manner ( Figure 3). The α-glycosylated ginsenoside F1 showed lower toxicity toward HDFs than ginsenoside F1 up to a concentration of 5 mg/mL. The cell viability was greater than 90% of that of the control cells up to 2 mg/mL. These results showed that ginsenoside F1 and α-glycosylated ginsenoside F1 have no significant cytotoxicity against skin cells. Thus, the inhibitory effect of these compounds on collagenase expression was not due to cytotoxicity of these compounds at concentrations up to 2 mg/mL.  (Figure 3). The α-glycosylated ginsenoside F1 showed lower toxicity toward HDFs than ginsenoside F1 up to a concentration of 5 mg/mL. The cell viability was greater than 90% of that of the control cells up to 2 mg/mL. These results showed that ginsenoside F1 and α-glycosylated ginsenoside F1 have no significant cytotoxicity against skin cells. Thus, the inhibitory effect of these compounds on collagenase expression was not due to cytotoxicity of these compounds at concentrations up to 2 mg/mL.

Inhibition of Tyrosinase Activity by Ginsenoside F1 and G1-F1
To investigate the tyrosinase inhibitory activity of G1-F1, the half maximal inhibitory concentration (IC50) values against mushroom tyrosinase were measured. The tyrosinase inhibitory activity of α-glycosylated ginsenoside F1 was higher than that of ginsenoside F1 but weaker than that of arbutin (Figure 4). It was previously reported that F1 can function as an anti-aging and antioxidant agent [11] and as a drug against skin cancer with antiproliferation and whitening functions [10]. Comparison of the

Inhibition of Tyrosinase Activity by Ginsenoside F1 and G1-F1
To investigate the tyrosinase inhibitory activity of G1-F1, the half maximal inhibitory concentration (IC50) values against mushroom tyrosinase were measured. The tyrosinase inhibitory activity of α-glycosylated ginsenoside F1 was higher than that of ginsenoside F1 but weaker than that of arbutin (Figure 4).  (Figure 3). The α-glycosylated ginsenoside F1 showed lower toxicity toward HDFs than ginsenoside F1 up to a concentration of 5 mg/mL. The cell viability was greater than 90% of that of the control cells up to 2 mg/mL. These results showed that ginsenoside F1 and α-glycosylated ginsenoside F1 have no significant cytotoxicity against skin cells. Thus, the inhibitory effect of these compounds on collagenase expression was not due to cytotoxicity of these compounds at concentrations up to 2 mg/mL.

Inhibition of Tyrosinase Activity by Ginsenoside F1 and G1-F1
To investigate the tyrosinase inhibitory activity of G1-F1, the half maximal inhibitory concentration (IC50) values against mushroom tyrosinase were measured. The tyrosinase inhibitory activity of α-glycosylated ginsenoside F1 was higher than that of ginsenoside F1 but weaker than that of arbutin (Figure 4).

Figure4
. Inhibitory effects of ginsenoside F1 and α-glycosylated ginsenoside F1 on Mushroon tyrosinase activity. Tyrosinase activity was measured using 3.3 mM L-DOPA as a substrate. Results are expressed as the percentage of inhibition by ginsenoside F1 and α-glycosylated compound. Arbutin was used as a positive control. Data represent the mean ± SD of triplicate experiments. *p < 0.05 compared with the control. F1: ginsenoside F1; G1-F1: α-glycosylated ginsenoside F1.
It was previously reported that F1 can function as an anti-aging and antioxidant agent [11] and as a drug against skin cancer with antiproliferation and whitening functions [10]. Comparison of the It was previously reported that F1 can function as an anti-aging and antioxidant agent [11] and as a drug against skin cancer with antiproliferation and whitening functions [10]. Comparison of the inhibition of tyrosinase activity showed that α-glycosylated ginsenoside F1 had a greater inhibitory effect on tyrosinase activity than ginsenoside F1, indicating that α-glycosylated ginsenoside F1 might be an efficacious anti-tyrosinase agent for use in cosmetics.
In addition to the number of sugars, their linkage positions and alpha vs. beta linkages affect pharmacological activities. For example, ginsenoside F1 and Rh1 have the same number of sugar moieties and the same molecular weight but different glucose attachment positions at C-20 and C-6, respectively. F1 showed significantly greater inhibition of viability than Rh1 in prostate cancer cell lines [30]. The glycosylation and nano formulations of ginseng saponins [13,25,26,31,32,33] and other steroidal saponins [19,27] has recently attracted increased interest.
The alpha isomers of glucose also exhibited significant activity, especially stronger inhibitory activity of α-arbutin on tyrosinase compared with β-arbutin [22]. Similarly, in comparison with the common beta isomers of glucose in ginsenosides, α-glycosyl ginsenoside was reported to have a reduced bitter taste [26], suggesting its potential as an additive in food products.

Conclusions
This study describes for the first time the glycosylation of ginsenoside F1 by CGTase and identification of a novel α-glucosylated F1 with an unusual α-D-glcp-(1→2)-β-D-glcp sugar chain (G1-F1). The novel compound G1-F1 showed lower cytotoxicity and stronger inhibitory activity against tyrosinase and collagenase (MMP-1) than ginsenoside F1. This novel G1-F1 may be a potential pharmacological active compound. A single α-glucosylated F1 was purified in this study, and other new glycosylated spots remain to be characterized.
The alpha isomers of glucose also exhibited significant activity, especially stronger inhibitory activity of α-arbutin on tyrosinase compared with β-arbutin [22]. Similarly, in comparison with the common beta isomers of glucose in ginsenosides, α-glycosyl ginsenoside was reported to have a reduced bitter taste [26], suggesting its potential as an additive in food products.

Conclusions
This study describes for the first time the glycosylation of ginsenoside F1 by CGTase and identification of a novel α-glucosylated F1 with an unusual α-D-glcp-(1→2)-β-D-glcp sugar chain (G1-F1). The novel compound G1-F1 showed lower cytotoxicity and stronger inhibitory activity against tyrosinase and collagenase (MMP-1) than ginsenoside F1. This novel G1-F1 may be a potential pharmacological active compound. A single α-glucosylated F1 was purified in this study, and other new glycosylated spots remain to be characterized.