ISOLATION AND STRUCTURE ELUCIDATION OF ...

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ISOLATION AND STRUCTURE ELUCIDATION OF CYANOGENIC GLYCOSIDES ADOLF NAHRSTEDT

Institut für Pharmazeutisohe BioZogie der Teohnischen Universität, D-3300 Braunsohweig, West Germany

I. INTRODUCTION In the last ten to twenty years the increased interest in cyanogenic compounds has led to better analytical procedures. Among these sample preparation, conditions of extraction, methods in gas chromatography (GLC), high pressure liquid chromatography (HPLC) and proton magnetic resonance (lH-NMR) have been fully developed. Besides older summaries (Seifert, 1955; Dilleman, 1958) there are some excellent reviews in the last decade covering the cyanogenic glycosides (Eyj61fsson, 1970a; Seigler, 1975a,b) and cyanolipids (Mikolajczak, 1977) which contain much information on methods of isolation and structure elucidation. The present paper on cyanogenic glycosides reviews the literature through 1979 on the most useful of these methods together with our own experience in this field. 11. SAMPLE PREPARATION

AND EXTRACTION

Structure elucidation involves the isolation of substances which are normally extracted from crude plant or animal material. Cyanogenic glycosides require special attention as most of them are not particularly stable, and isomerization and decomposition must be avoided during isolation. The normal way is to extract material which has been air dried carefully. However, one may have to accept loss in glycoside content because hydrolyzing enzymes Cglucosidases) become active after damage to the plant material. In some plants (e.g. Heterodendron oZeaefoZium, Hübel, 1978) which contain high levels of tannins the activity of glucosidases is reduced. Some workers extract fresh plant material, which is sometimes frozen, and inactivate the hydrolyzing enzymes during extraction. In this case cold or hot methanol or ethanol Ce.g. Gondwe et aZ., 1978, for tetraphyllin; Gmelin et aZ., 1973, for holocalin; Jensen and Nielsen,

146

A. NAJ-IRSTEDT

1973, for sambunigrin; Butterfield et al., 1975, for acacipetalin) have been used as ~xtracting media. Sometimes formic or acetic acid (Reay, 1969, for dhurrin; Schwarzmaier, 1976, for taxiphyllin) have been combined with the alcohol. Aqueous perchloric acid (Bough and Gander, 1971, for dhurrin) and even hot water (e.g. Eyj6lfsson, 1970b, for triglochinin) have also been used (Fig. 1). Extraction of fresh plant material is possible only if the plants are available near the laboratory. Since .t h i s cannot be realized in every case a suitable alternative method is to collect plants and place them directly in liquid nitrogen or dry ice, grind them under liquid nitrogen or dry ice in the lab and dry the resulting powder in a frozen condition by lyophilization (e.g. Nahrstedt, 1970, 1976a, b; Hübel and Nahrstedt, 1979). The material prepared in this manner is ready for extraction or can be stored for a long time at -l8°C. ~

fresh !llant material ~

I

- 1\""",.00"""

air dried

I (somet;~

j,oz..,)

-9ti!lQiDg..Jn.J:J2_

1

freeze dried

---i

(..ady

fo< storog~ -tB"C)

1

Iextroction I

petrcl, "'.one (lipids)

direct extroction

c) MeOH, EtOH

c) boiling in R-OH b) cold R-OH (.AcOH)

b) MeOHI EIOAc Azeotrop

cl 0,3 M Heia,.

EIOAc d) EtOEI c)

lavoo

Fig. 1

~ l:> In H20

I

(sat. with

HtJ)

J

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Iexlractl

Different ways of extracting fresh or dried plant material in order to obtain an extract containing cyanogenic glycosides

Dry material should be extracted first with petroleum ether to remove lipophilic compounds. Then, depending on ttre gLycus i de mvo Ived, different salvents are po ssfbl.e . Since all cyanogenic glycosides are rather polar substances, methanol or ethanol are excellent solvents. An azeotropic mixture of methanol and ethylacetate reduces the extraction temperature and avoids extraction of large amounts of polar substances such as sugars, same amino acids and peptides

CYANOGENIC GLYCOSIDES: STRUCTURE AND ELUCIDATION

147

(Nahrstedt, 1971). Cardiospermin-5-0-(4-hydroxy)-benzoate, for instance, is extractable with pure ethylacetate (Nahrstedt, 1976a), and prunasin with diethylether saturated with water. This latter method also allows the separation of prunasin from amygdalin, which is not soluble in this solvent (Trim, 1955). Even at this stage isolation has to be performed carefully to avoid decomposition, isomerization or derivatization of some cyanogenic glycosides. O-Glc-Glc

)