Notizen: Isolation of Amavadin, a Vanadium Compound Occuring in Amanita Muscaria

Abstract

The toadstool Amanita muscaria is relatively rich in vanadium and contains up to 120 ppm vanadium per g dry weight 1 . Therefore we investigated the possibility of isolating a vanadium chelate from it.The vanadium distribution does not show a high enrichment in any part of the mushroom, only the stems showing somewhat higher concentrations (Table 1) The vanadium compound, which we call Amavadin, can be extracted with water, methanol, and ethanol. Preliminary experiments with Sephadex A 25 showed that all vanadium occurs in the form of amavadin. Dialysis and gel chromatography indicate a molecular weight below 1000. Electrophoresis reveals the strongly acidic nature of amavadin. In the range of pH 1 to 10, it migrates as an anion. Pure amavadin is isolated by a combination of ion-exchange and gel chromatography. Isolation of amavadin:The mushrooms were collected in the Black Forest and the Schönbuch. They were stored at -30°. To extract amavadin, one 1 of methanol is added to each kg of frozen mushrooms. The thawed mixture is ground in a blender and filtered. The filtrate is acidified with acetic acid (to 0.1 N). DEAE-cellulose is added (Whatman DE 52, 40 ml cellulose per kg of mushrooms), and the slurry is stirred overnight. The next day the cellulose is placed in a chromatography column, washed with 0.5 N acetic acid, and the amavadin is eluted with 0.2 N phosphate buffer (pH 5.8, 200 ml per kg of mushrooms). The eluate is diluted with water (1 : 2) and absorbed on Sephadex A 25 (Pharmacia) in a chro-207 matography column. Amavadin is eluted with 0.4 N phosphate buffer (pH 5.8). The lyophilized amavadin fraction, detectable by its green color, is extracted with methanol. The combined extracts are further purified on Dowex 50 W X 8 (Baker, H® form) and Sephadex G 25. Yield: ca. 40 mg amavadin per kg (50%, based on the vanadium content of fresh amavadin per 50 kg (50%, based on the vanadium content of fresh mushrooms).Amavadin is a blue compound without a melting point. Above 170° the blue color of the crystals slowly disappears. The crystals turn irreversibly yellow at 185° and brown at 220°. Amavadin is stable in dilute acids and ammonia, but decomposes in IN sodium hydroxide.The IR spectrum of amavadin shows a very strong C = 0 band (1600-1650 cm" 1 ) and a V = 0 band (985 cm -1 ) 2 . The eletronic spectrum of amavadin is similar to those of other vanadium ( +4) compounds 3 . It has the following absorption peaks ( in water) :775 nm (e = 19.3) 270 nm (shoulder, E = 6800) 715 nm (e=18.9) 235 nm (£ = 12300) 565 nm (e = 23.5) 218 nm (shoulder, £ = 12600).The electron spin resonance spectrum indicates also the presence of V 4 ® in amavadin 4 .The molecular weight of amavadin, determined osmometrically, was found to be 415. Titration with sodium hydroxide gave an equivalent weight of 210, indicating two free acid groups per molecule. Requests for reprints should be sent to Prof.

Vanadium in biology—the case of theAmanitatoadstools

“…The ionisation constants of these protons were estimated by Anderegg, Koch and Bayer in a recent paper and the values reported are pK1 = -0.6 and pK2 = 0 ± 0.2, which agree with the fact that at pH > 1 amavadine migrates as an anion in electrophoresis experiments (Bayer and Kneifel, 1972). Such a strong di-carboxylic acid, isolated as a solid, is indeed very unusual and leads one to suspect that there might be some inconsistencies in the data, but the investigation of the thermodynamic stability of the vanadium complexes of HIDA and HIDPA reveals still further inconsistencies (Bemski eta/., 1986;Felcman eta/., 1984;Anderegg et al, 1987).…”

“…The EPR hyperfine structure of oxidised amavadine was also reported and interpreted assuming the formation of a nitroxyl radical. These facts were already mentioned in the first papers by the same authors (Bayer and Kneifel, 1972;Kneifel and Bayer, 1973), but no quantitative data was presented at that time.…”

“…Bertrand, 1943;Meish et al, 1979;Koch et al, 1987 and Frausto da Silva et al (unpublished). This was achieved in 1972 when Bayer and Kneifel isolated a vanadium containing compound from a German variety of Amanita muscaria (Black Forest and Schoenbuch) which they named "amavadine" (Bayer and Kneifel, 1972). About 40 mg of the compound were obtained per kg of the fresh mushroom by an elaborate procedure which included * Table 2 Some properties of amavadine.…”

“…The properties of "amavadine", as reported by these authors, are summarised in Table 2 * According to Bayer and Kneifel, 1972.…”

“…By necessity, the complex must have one (coordinated?) water molecule, two non-ionised hydroxyl groups and two readily ionisable protons which can be titrated (Bayer and Kneifel, 1972).…”