Orellanine is a nephrotoxin found in various Cortinaceae mushroom species. Unintentional consumption after these species were confused with edible mushrooms such as Cantharellus tubaeformis has caused several casualties. In this work, a quantitative HPLC-ESI-MS/MS method for total orellanine in Cortinarius rubellus, spiked blood plasma, and a mushroom stew prepared from C. tubaeformis with the addition of a single specimen of C. rubellus is presented. The existence of mono- and diglucosylated orellanine in C. rubellus was also proven, although quantitative analysis could not be obtained for the glucosides due to rapid hydrolyzation to orellanine in the extract. Extraction with 3 M HCl or water mainly yielded orellanine, while MeOH or acidified MeOH mainly extracted mono- and diglucosylated orellanine. The highest recovery of total orellanine was obtained with 3 M HCl, which was subsequently used for quantitative analysis. A C18 HPLC column and low pH in the eluents retained all these toxins. Orellanine could be detected at a 4.9 ng/mL level in all extracts, which is well below the threshold for acute toxic effects. Additionally, the fragmentation pattern of orellanine upon electrospray MS/MS was probed. The method described is useful for two important applications. First, it allows quantitative analysis of processed food products that may be contaminated by orellanine from Cortinaceae mushrooms. Second, orellanine is currently being evaluated as a potential cure of metastatic renal cancer, and this work provides a method for monitoring orellanine at low concentrations within the therapeutic interval in blood serum

orellanine, orellanine glucosides, nephrotoxin, Cortinaceae

There is only one chemically distinct structure:

SVGMWSMILES3D molIonize

 

[O-][n+]1ccc(O)c(O)c1-c1c(O)c(O[C@@H]2O[C@H](CO)[C@@H](O)[C@H](O)[C@H]2O)cc[n+]1[O-]

414.323 g/mol (C₁₆H₁₈N₂O₁₁)

Orellanine is a nephrotoxin found in various Cortinaceae mushroom species. Unintentional consumption after these species were confused with edible mushrooms such as Cantharellus tubaeformis has caused several casualties. In this work, a quantitative HPLC-ESI-MS/MS method for total orellanine in Cortinarius rubellus, spiked blood plasma, and a mushroom stew prepared from C. tubaeformis with the addition of a single specimen of C. rubellus is presented. The existence of mono- and diglucosylated orellanine in C. rubellus was also proven, although quantitative analysis could not be obtained for the glucosides due to rapid hydrolyzation to orellanine in the extract. Extraction with 3 M HCl or water mainly yielded orellanine, while MeOH or acidified MeOH mainly extracted mono- and diglucosylated orellanine. The highest recovery of total orellanine was obtained with 3 M HCl, which was subsequently used for quantitative analysis. A C18 HPLC column and low pH in the eluents retained all these toxins. Orellanine could be detected at a 4.9 ng/mL level in all extracts, which is well below the threshold for acute toxic effects. Additionally, the fragmentation pattern of orellanine upon electrospray MS/MS was probed. The method described is useful for two important applications. First, it allows quantitative analysis of processed food products that may be contaminated by orellanine from Cortinaceae mushrooms. Second, orellanine is currently being evaluated as a potential cure of metastatic renal cancer, and this work provides a method for monitoring orellanine at low concentrations within the therapeutic interval in blood serum

orellanine, orellanine glucosides, nephrotoxin, Cortinaceae

There is only one chemically distinct structure:

SVGMWSMILES3D molIonize

 

[O-][n+]1ccc(O[C@@H]2O[C@H](CO)[C@@H](O)[C@H](O)[C@H]2O)c(O)c1-c1c(O)c(O[C@@H]2O[C@H](CO)[C@@H](O)[C@H](O)[C@H]2O)cc[n+]1[O-]

576.464 g/mol (C₂₂H₂₈N₂O₁₆)

We have isolated Hypoculoside, a new glycosidic amino alcohol lipid from the fungus Acremonium sp. F2434 belonging to the order Hypocreales and determined its structure by 2D-NMR (Nuclear Magnetic Resonance) spectroscopy. Hypoculoside has antifungal, antibacterial and cytotoxic activities. Homozygous profiling (HOP) of hypoculoside in Saccharomyces cerevisiae (budding yeast) revealed that several mutants defective in vesicular trafficking and vacuolar protein transport are sensitive to hypoculoside. Staining of budding yeast cells with the styryl dye FM4-64 indicated that hypoculoside damaged the vacuolar structure. Furthermore, the propidium iodide (PI) uptake assay showed that hypoculoside disrupted the plasma membrane integrity of budding yeast cells. Interestingly, the glycosidic moiety of hypoculoside is required for its deleterious effect on growth, vacuoles and plasma membrane of budding yeast cells.

glycosphingolipid, antifungal activities, Acremonium, antibacterial activities, hypoculoside, cytotoxic activities

13C NMR data:
Linkage	Residue	C1	C2	C3	C4	C5	C6	C7	C8	C9	C10	C11	C12	C13	C14	C15	C16	C17	C18	C19	C20	C21	C22	C23	C24
2	aDGlcp	96.7	73.5	74.0-75.2	74.0-75.2	71.9	62.7
	Subst	12.1	75.5	75.4	34.1	27.0	30.6-30.8	30.6-30.8	30.6-30.8	30.6-30.8	30.6-30.8	30.6-30.8	30.6-30.8	30.6-30.8	30.6-30.8	30.6-30.8	30.6-30.8	30.6-30.8	30.6-30.8	30.6-30.8	27.2	34.0	71.8	52.6	12.2


1H NMR data:
Linkage	Residue	H1	H2	H3	H4	H5	H6	H7	H8	H9	H10	H11	H12	H13	H14	H15	H16	H17	H18	H19	H20	H21	H22	H23	H24
2	aDGlcp	4.94	3.38	3.62	3.62	3.27	3.64-3.78
	Subst	1.12	3.74	3.69	1.44	1.31-1.49	1.26-1.34	1.26-1.34	1.26-1.34	1.26-1.34	1.26-1.34	1.26-1.34	1.26-1.34	1.26-1.34	1.26-1.34	1.26-1.34	1.26-1.34	1.26-1.34	1.26-1.34	1.26-1.34	1.32	1.43	3.68	3.25	1.20


1H/13C HSQC data:
Linkage	Residue	C1/H1	C2/H2	C3/H3	C4/H4	C5/H5	C6/H6	C7/H7	C8/H8	C9/H9	C10/H10	C11/H11	C12/H12	C13/H13	C14/H14	C15/H15	C16/H16	C17/H17	C18/H18	C19/H19	C20/H20	C21/H21	C22/H22	C23/H23	C24/H24
2	aDGlcp	96.7/4.94	73.5/3.38	74.0-75.2/3.62	74.0-75.2/3.62	71.9/3.27	62.7/3.64-3.78
	Subst	12.1/1.12	75.5/3.74	75.4/3.69	34.1/1.44	27.0/1.31-1.49	30.6-30.8/1.26-1.34	30.6-30.8/1.26-1.34	30.6-30.8/1.26-1.34	30.6-30.8/1.26-1.34	30.6-30.8/1.26-1.34	30.6-30.8/1.26-1.34	30.6-30.8/1.26-1.34	30.6-30.8/1.26-1.34	30.6-30.8/1.26-1.34	30.6-30.8/1.26-1.34	30.6-30.8/1.26-1.34	30.6-30.8/1.26-1.34	30.6-30.8/1.26-1.34	30.6-30.8/1.26-1.34	27.2/1.32	34.0/1.43	71.8/3.68	52.6/3.25	12.2/1.20

There is only one chemically distinct structure:

SVGMWSMILES3D molIonize

 

CC(N)C(O)CCCCCCCCCCCCCCCCCCC(O)C(C)O[C@H]1O[C@H](CO)[C@@H](O)[C@H](O)[C@H]1O

563.817 g/mol (C₃₀H₆₁NO₈)