A new method for the preparation of a purified glucosylceramide and ceramide from shiitake mushroom

Abstract

Ingestion of plant and fungal glucosylceramides is known to reduce colon carcinogenesis and skin barrier damage in mice and humans. However, such effects in animal experiments have not been revealed for plant and fungal ceramides because the content of ceramides contained in plants and fungi is so low that the large amount required for animal experiments is difficult to obtain. Noting that the fungus shiitake mushroom (Lentinus edodes) is rich in a glucosylceramide, (4E,8E)-N-d-2′-hydroxypalmitoyl-1-O-β-d-glucopyranosyl-9-methyl-4,8-sphingadienine [Glc-d19:2(4E,8E,9Me)-h16:0], a new method for purifying the fungal glucosylceramide was developed using ethanol precipitation and a high performance liquid chromatography. We also developed a new method to produce large amounts of a ceramide [d19:2(4E,8E,9Me)-h16:0] from this purified glucosylceramide using human glycoside hydrolase family 30 glucocerebrosidase (imiglucerase). These methods will be useful for elucidating the physiological function by ingestion of fungal ceramides in animal experiments.

Dietary Fungal Glucosylceramide and Ceramide Reduce the Formation of Aberrant Crypt Foci in 1,2-Dimethylhydrazine-Treated Mice: Differences in the Role of Glucosylceramide and Ceramide

“…GlcCer [Glc-d19:2(4E,8E,9Me)-h16:0] from shiitake mushroom was purified using ethanol precipitation and HPLC equipped with a reverse-phase column as described. 22 The purified fungal Cer [d19:2(4E,8E,9Me)-h16:0] was prepared by hydrolysis of this purified fungal GlcCer using human GH30 GCase expressed in Chinese hamster ovary cells (imiglucerase, Ieda Chemical, Tokyo, Japan). 22 Animals.…”

“…22 The purified fungal Cer [d19:2(4E,8E,9Me)-h16:0] was prepared by hydrolysis of this purified fungal GlcCer using human GH30 GCase expressed in Chinese hamster ovary cells (imiglucerase, Ieda Chemical, Tokyo, Japan). 22 Animals. Four-week-old female Bagg Albino (BALB)/c mice were obtained from Japan SLC, Inc. (Shizuoka, Japan) and housed in isolator cages at 22 °C under a 12 h light/dark cycle.…”

“…Fungal GlcCer and Cer were analyzed in positive ion mode with nitrogen as the collision gas and quantified by multiple reaction monitoring (MRM) as described previously. 22 The…”

“…22 Furthermore, because human glycoside hydrolase family (GH) 30 glucocerebrosidase (GCase) hydrolyzes plant GlcCer to Cer 23 and rice GH1 GCase hydrolyzes fungal GlcCer to Cer, 24 we also developed a new method to produce large amounts of a Cer species, [d19:2(4E,8E,9Me)-h16:0] (fungal Cer) from this purified fungal GlcCer using human GH 30 GCase. 22 In the present study, to clarify the roles of sphingolipids in intestinal impairment in vivo, we investigated the effects of these fungal GlcCer and Cer on ACF formation and colon inflammation. Mice were treated with DMH for ACF formation and chronic colon inflammation, and the effects of diets supplemented with the fungal GlcCer or Cer on their colon impairments were evaluated by the number of ACF and expression levels of inflammation-related cytokines and apoptosis-related proteins.…”

“…In a previous study, noting that shiitake mushroom (Lentinula edodes) is rich in a GlcCer species, (4E,8E)-N-D-2′-hydroxypalmitoyl-1-O-β-D-glucopyranosyl-9-methyl-4,8sphingadienine [Glc-d19:2(4E,8E,9Me)-h16:0] (fungal GlcCer), we used this fungus to develop a new method for purifying the GlcCer using ethanol precipitation and high performance liquid chromatography (HPLC). 22 Furthermore, because human glycoside hydrolase family (GH) 30 glucocerebrosidase (GCase) hydrolyzes plant GlcCer to Cer 23 and rice GH1 GCase hydrolyzes fungal GlcCer to Cer, 24 we also developed a new method to produce large amounts of a Cer species, [d19:2(4E,8E,9Me)-h16:0] (fungal Cer) from this purified fungal GlcCer using human GH 30 GCase. 22 In the present study, to clarify the roles of sphingolipids in intestinal impairment in vivo, we investigated the effects of these fungal GlcCer and Cer on ACF formation and colon inflammation.…”