• Journal of the Society of Cosmetic Scientists of Korea
• /
• v.44 no.3
• /
• pp.343-347
• /
• 2018

We synthesized 1, 2-hexanediol galactoside (HD-Gal) from HD using Escherichia coli (E. coli) ${\beta}-galactosidase$ (${\beta}-gal$), in which the reaction is generally called as transgalactosylation (reverse hydrolysis). In this study, we investigated how much HD-Gal and HD had a cytotoxic effect on HaCaT cell, in order to compare HD-Gal with HD in terms of the cytotoxicity of human skin cell. Cell proliferation assay and phase-contrast microscope observation were used for investigating the cytotoxicity. As a result, HD-Gal had not cytotoxic effect on HaCaT cell in the concentration range from 42.2 to 211 mM. In addition, when we observed the cells using microscopy, there was no change in the cell morphology. Meanwhile, when 42.2 mM and 84.4 mM HD were treated on HaCaT cell, we did not observe the cytotoxicity; however, when 168.8 mM and 211 mM HD were on HaCaT cell, HD had a higher cytotoxic effect on HaCaT cell. In addition, when HD was treated on the cells regardless of the concentration of HD, there were obvious changes in cell morphology and cell number. It was expected hopefully that HD-Gal would be applicable as a substitute for HD as a less toxic preservative in views of safety, health, and well-being.

• Journal of Life Science
• /
• v.26 no.5
• /
• pp.608-613
• /
• 2016

Recently, it has been reported that some preservatives used in cosmetics lead to skin problems. Among the many cosmetic ingredients, 1, 2-hexanediol (HD) is used as both a preservative and humectant. In order to develop safer ingredients, we studied the synthesis of 1, 2-hexanediol galactoside (HD-G) by a transgalactosylation reaction using β-galactosidase (β-gal)-containing recombinant Escherichia coli cells. The transgalactosylation reaction was carried out under high-lactose conditions for 24 hr. After 12 hr had elapsed, a new spot was identified by thin-layer chromatography (TLC) analysis, and we presumptively designated this new spot as HD-G. Then, we carried out the purification of the presumptive HD-G spot from the reaction mixture by using silica gel chromatography, and its mass was measured by electrospray ionization-mass spectrometry. The purified new spot on the chromatograph was identified a sodium adduct ion ([M+Na]⁺, m/z = 303.1423) of HD-G. In addition, when purified HD-G was hydrolyzed using commercially available E. coli β-gal, it was observed that a galactose molecule was released from HD-G. That is, it was demonstrated that HD-G is a galactoside derivative of HD. Finally, we confirmed that HD-G was enzymatically synthesized by E. coli β -gal as a new molecular entity. In the future, we plan to determine the minimum inhibitory concentrations of HD-G against different bacterial species. The cytotoxicity of HD-G against human skin cells will also be examined. It is expected hopefully that the galactosylation of HD would improve the functionality of HD-G.

• Journal of the Society of Cosmetic Scientists of Korea
• /
• v.43 no.4
• /
• pp.373-379
• /
• 2017

We carried out the enzymatic synthesis of 1, 2-hexanediol galactoside (HD-gal) by transgalactosylation reaction using recombinant Escherichia coli ${\beta}-galactosidase$ (${\beta}-gal$). The amounts of ${\beta}-gal$ and 1, 2-hexanediol (HD), pH, and temperature, respectively, were first optimized (${\beta}-Gal$, 4.8 U/mL; HD, 75 mM; pH, 7.0; temperature, $37^{\circ}C$). Under these optimal conditions, about 96% HD was converted to HD-gal. When we investigated the water holding capacities (WHCs) of HD and HD-gal using pig epidermis in the concentrations of 84.4, 126.6, 168.8, 211.0 mM, WHC of HD-gal was superior to HD. In particular, at 168.8 mM HD and HD-gal, WHC of HD-gal showed about 20% greater than that of HD. However, it was observed that MIC values against Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus of HD-gal were about three to ten times greater than those of HD, although MIC value of HD-gal against Enterococcus faecalis was almost the same as that of HD. Finally, it was concluded that the covalent bonding of a galactose molecule to HD (transgalactosylation) resulted in an increase in WHC of HD-gal and a decrease in anti-bacterial activity.

• Journal of the Korean Applied Science and Technology
• /
• v.33 no.2
• /
• pp.286-292
• /
• 2016

1, 2-Hexanediol galactoside (HD-gal) has been synthesized from 1, 2-hexanediol (HD), a cosmetic preservative, using recombinant Escherichia coli ${\beta}$-galactosidase (${\beta}$-gal) at the high lactose concentration (300 g/l). To confirm the molecular structure of synthesized HD-gal, NMR ($^1H$- and $^{13}C$-) spectroscopy and mass spectrometry of HD-gal were conducted. $^1H$ NMR spectrum of HD-gal showed multiple peaks corresponding to the galactocyl group, which is an evidence of galactocylation on HD. Downfield proton peaks at ${\delta}_H$ 4.44 ppm and multiple peaks from ${\delta}_H$3.96~3.58 ppm were indicative of galactocylation on HD. Up field proton peaks at ${\delta}_H$ 1.60~1.35 ppm and 0.92 ppm showed the presence of $CH_2$ and $CH_3$ protons of HD. $^{13}C$ NMR spectrum revealed the presence of 21 carbons suggestive of ${\alpha}$- and ${\beta}$-anomers of HD-gal. Among 12 carbon peaks from each anomers, the 3 peaks at dC 68.6, 60.9 and 13.2 ppm were assigned to be overlapped showing only 21 peaks out of total 24 peaks. The mass value (protonated HD-gal, m/z = 281.1601) from mass spectrometry analysis of HD-gal, and $^1H$ and $^{13}C$ NMR spectral data were in well agreement with the expecting structure of HD-gal. For further study, the minimum inhibitory concentrations (MICs) of HD-gal against bacteria will be investigated, and, in addition, cytotoxicity to human skin cells of HD-gal will be examined. It is expected that it will eventually be able to develop a new cosmetic preservative, which have low cytotoxicity against human skin cell and maintains antimicrobial effect.

• Journal of the Korean Applied Science and Technology
• /
• v.33 no.3
• /
• pp.498-506
• /
• 2016

1, 2-Hexanediol galactoside (HD-gal) has been previously synthesized from 1, 2-hexanediol (HD), in which recombinant ${\beta}$-galactosidase (${\beta}$-gal) of Escherichia coli (E. coli) was used for transgalactosylation reaction. In this study, a method for HD-gal purification from the reaction mixture was particularly investigated. Using ${\beta}$-gal-containing E. coli, HD-gal was synthesized from 75 mM HD for 48 hr under 300 g/l lactose concentration. Then, HD-gal synthesis from HD was confirmed by TLC analysis, and the existence of E. coli ${\beta}$-gal during 48 hr-reaction was also confirmed by Western blotting, in which the conversion yield of HD to HD-gal reached about 94% during 48 hr. To establish an efficient method for HD-gal purification, we carried out the solvent extraction of the reaction mixture, followed by silica gel chromatography, particularly in order to remove the residual HD. Two water-immiscible solvents, such as methylene chloride and ethyl acetate, were investigated comparatively to find out appropriate solvent. Then, it was found that residual HD was almost removed when ethyl acetate extraction of water phase of reaction mixture was carried out four times. Subsequently, silica gel chromatography was carried out, and purified HD-gal could be finally obtained. The production yield for HD-gal from 75 mM HD was $8.9{\pm}0.6%$ (n=3) (mole basis) or $21.1{\pm}1.4%$ (n=3) (weight basis). For further study, using purified HD-gal, we will investigate the minimum inhibitory concentrations (MICs) of HD-gal against bacteria. In addition, cytotoxicity to human skin cells of HD-gal will be examined.