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

Date Published: August 29, 2017

Publisher: Springer Berlin Heidelberg

Author(s): Ha-Young Choi, Bo-Min Kim, Abubaker M. A. Morgan, Joong Su Kim, Won-Gon Kim.


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.

Partial Text

Menthol is used in a wide variety of products as an additive to foods, medicines, cosmetics, and cigarettes owing to its inherent mint flavor and refreshing feelings. The use range of menthol, however, is limited because of its extremely low solubility in water and flavor (Kamatou et al. 2013; Patel et al. 2007). Glycosylation is one method than can improve both the water solubility and biological activity of non-glycosylated compounds (Ahmed et al. 2006). Sugar moieties are known to improve the pharmacokinetic properties and/or affect the biological activity of glycosides in important natural pharmaceutical products (Weymouth-Wilson 1997). Chemical synthesis of menthyl glucosides was reported to produce an anomeric mixture of α- and β-anomers (Sakata and Iwamura 1979).

Menthol has well-known cooling characteristics and a residual minty smell. Because of these attributes, menthol 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 analgesics, and as an additive in cold compresses for its cooling and biological effects (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 of solid particles with the material to which the menthol is to be added, making a suspension using an emulsifier, or dissolving the menthol in an organic solvent such as alcohol. Additionally, the inherent mint flavor limits the use of menthol in certain cooling products, such as cosmetics. Thus, menthol derivatives with good solubility in water and no flavor that retain the cooling sensation have been greatly desired. Glycosylation improves the water solubility of drugs or natural products (Weymouth-Wilson 1997). In our study, (−)-menthol β-glucoside was shown to more soluble in water (>27 times) than (−)-menthol α-glucoside; hence, the β-anomer-selective glucosylation of menthol is necessary. Limited glycosylation of menthol has been reported. Water-soluble menthyl glycosides with monosaccharide or oligosaccharide as a sugar unit have been synthesized by chemical methods, but the anomer-selective synthesis of methyl glycoside is impossible due to a lack of regioselectivity and stereoselectivity (Sakata and Iwamura 1979). α-Anomer-selective synthesis of menthyl glucoside was reported using α-glucosidase from yeast (Nakagawa et al. 1998) or bacteria such as Xanthomonas (Nakagawa et al. 2000), but β-anomer-selective glucosylation of menthol has not yet been reported. We therefore undertook to synthesize menthol β-glucoside by synthesizing the glucosyl analogs of menthol using UDP-glycosyltransferase BLC from B. licheniformis. Three different glycoside derivatives with the β-configuration were successfully produced in the reaction catalyzed by the BLC in the presence of the corresponding NDP-sugars. The maximum glucoside conversion rate was determined from the time-dependent study of menthol coupled with UDP-d-glucose. During the molar conversion, approximately 58.9% glucoside was produced after 1 h of incubation. BLC from B. licheniformis DSM 13 is reported to glycosylate diverse substrates such as geldanamycin analogs (Wu et al. 2012), epothilone A (Parajuli et al. 2014), chalcone (Pandey et al. 2013), and various flavonoids (Koirala et al. 2014). The maximum conversion rates of epothilone A and 3-hydroxyflavone to its respective glucosides by BLC were reported to be approximately 26 and 90%, respectively, at 3 h incubation using UDP-d-glucose. In this study, BLC catalyzed the glycosylation of menthol (C10H20O; molecular weight, 156), a notably small cyclic monoterpene alcohol. This result proves the remarkable flexibility of an aglycone substrate of BLC.




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