*Corresponding author. Tel.:#1-217-333-3260; fax:#1-217-244-7246. 1Emeritus Professor of Botany.
E-mail address:d-seigler@uiuc.edu (D.S. Seigler)
Sambunigrin and cyanogenic variability in
populations of
Sambucus canadensis
L. (Caprifoliaceae)
Rex A. Buhrmester
!
, John E. Ebinger
!
,1
, David S. Seigler
"
,
*
!Eastern Illinois University, Charleston, IL 61920, USA
"Department of Plant Biology, University of Illinois, Urbana, IL 61801, USA
Received 7 April 1999; accepted 11 June 1999
Abstract
The presence or absence of cyanogenic glycosides was determined for individuals from nine populations of Sambucus canadensis L. (elderberry) of east-central Illinois. In most of the populations tested, all or most of the individuals did not produce these compounds, in one, all were cyanogenic, whereas in another population this trait was highly variable. Addition of the enzyme emulsin to negative tests did not result in any further release of cyanide. The glycoside responsible is (S)-sambunigrin. ( 2000 Elsevier Science Ltd. All rights reserved.
Keywords:Cyanogenesis; Sambunigrin;Sambucus canadensis; Elderberry; Variation; Caprifoliaceae
1. Introduction
Many plants synthesize compounds capable of liberating hydrogen cyanide upon hydrolysis (Seigler, 1976; Seigler and Brinker, 1993). This process usually results from the presence of cyanoglycosides or cyanolipids that are broken down byb-glucosides to either sugars or fatty acids, an aldehyde or ketone, and hydrogen cyanide (HCN). Cyanogenic glycosides have been found in at least 2650 plant species representing more than 130 families, having been reported from bacteria, fungi, lichens, ferns,
fern-allies, gymnosperms and angiosperms (Aikman et al., 1996; Gibbs, 1974; Seigler, 1976; Tjon Sie Fat, 1979). The speci"c cyanogenic compound responsible, however, has been isolated from only about 300 species (Brinker and Seigler, 1992; Seigler and Brinker, 1993).
Several species of the genus Sambucus have previously been shown to be cy-anogenic. Among these areS.glaucaNutt.,S.racemosaL. andS.sieboldianaBlume ex Miquel (seed), (Bohm and Glennie, 1971; Gibbs, 1974; Hegnauer, 1989). A species distributed widely in Europe and Middle Asia,S.nigraL., has been shown to contain the cyanogen (S)-sambunigrin (Bourquelot and Danjou, 1905a}e) as do the American speciesS.racemosa,S.callicarpaGreene andS.microbotrysRydberg (Hegnauer, 1989). In a subsequent study, both (R)-prunasin and (S)-sambunigrin, as well as the meta-substituted compounds (R)-holocalin and (S)-zierin, were reported (Jensen and Niel-sen, 1973) inS.nigra. Materials ofS.caeruleaRaf.,S.ebulisL.,S.gaudichaudianaDC.,
S.melanocarpaGray, andS.pubensMichx. formaxanthocarpagave negative tests for cyanide by the picrate method (Bohm and Glennie, 1971).
The common and widespread North American elderberry,Sambucus canadensis, has been reported to lack cyangenic capabilities (Bohm and Glennie, 1971). In contrast to the European species above, the presence and amount of the cyanogenic compounds varies widely among naturally occurring populations of S. canadensis
(Aikman et al., 1996). The compound responsible has not previously been identi"ed.
2. Methods and materials
2.1. Population studies
Nine populations ofSambucus canadensisfrom Coles County in east-central Illinois were selected for study. These were mostly roadside populations in disturbed sites that were growing in full-sun for most of the day. Within each population, 30 individuals were chosen randomly and each marked for multiple sampling. Leaf material of each marked individual was tested for HCN production with Feigl}Anger paper (Feigl and Anger, 1966; Tantisewie et al., 1969). A small amount of each lea#et (about 200 mg) was crushed, placed in a straight-sided glass vial, moistened with distilled water, a strip of Feigl}Anger paper was added to the vial without touching the sample, and held in place with a cork. The vials were examined at 1, 8, and 24 h for any color change; no changes were observed after 8 h, suggesting that bacterial or fungal release of cyanide was not involved. A positive test was indicated by a change in the Feigl}Anger paper from white to blue. The test was considered weak if only part of the paper turned a light blue (HCN#), moderate if most of the paper turned a light to medium blue (HCN##), and strong if the entire paper turned deep blue (HCN###). A weak reaction indicates that there was approximately 2}20 mg of HCN per kg fresh weight, a moderate reaction 21}50 mg of HCN per kg fresh weight, and a strong reaction more than 50 mg of HCN per kg fresh weight (Dickenmann, 1982). All individuals in each population were tested three to
"ve times during a three to eight day period. Six of the nine populations were resampled in early August 1999. Approximately the same numbers of cyanogenic plants were observed. Addition of phosphate bu!er (pH 6.8) and the enzyme emulsin to negative tests from these populations did not result in additional release of cyanide.
2.2. Cyanogenic glycoside determination
Dried leaf material ofSambucus canadensis(20.8 g) (J. E. Ebinger 25235, voucher deposited at EIU) was extracted with 80% aqueous methanol by heating to boiling for 10 min. The extract was "ltered through "lter paper and concentrated under vacuum to yield a solid residue (2.3 g). Based on determination of hydrogen cyanide liberated on hydrolysis by the sandwich-picrate method involving thin layer chromatography on Silica Gel 60 plates (ethyl acetate, methanol, water, 79 : 11 : 10), only one cyanogenic band was present. This band hadR
&0.58 and gave a gray color
when sprayed with anisaldehyde-sulfuric acid spray reagent (Wagner et al., 1984). A small portion of the sample from above (about 1 mg) was treated sequentially with trimethylchlorosilane (TMCS, 20 ml), pyridine (20ll) and bis(trimethylsilyl)
Table 1
Cyanide concentrations in the individuals of nine populations ofSambucus canadensisfrom east-central Illinois along with the date and time of each test. Thirty individuals were tested in each population
Population Date/time HCN! HCN# HCN## HCN###
acyanogenic 2}20 mg 20}50 mg '50 m
all individuals in four populations tested negative for cyanide production each time tested, and in three populations only an occasional individual tested weakly positive for cyanide. In one population (d5), however, approximately two-thirds of the individuals consistently gave a strong positive test each of the"ve times tested, other individuals gave a negative test, and a few consistently gave weak to moderately positive tests.
In most populations, each individual gave the same test results each time. Inconsist-ency did occur in some populations; however, usually one or a few individuals gave a weak test one day and a negative test on other days. There are many possible reasons for these inconsistent test results, including the time of day the sample was taken, the age of the sample material, the time of year the sample was taken, and the genetic or environmental e!ects on the plants causing them to start or stop the production of the cyanogenic glycoside or the enzyme that breaks down the glycoside. In most instances, the test paper started to change color within an hour, and for strong tests it was not uncommon for the results to be completed in 1 h; no positive reactions required more than 8 h, and most required much less. This largely precludes positive tests due to the presence of bacteria or fungi.
It is possible that some of the individuals that tested negative had produced HCN earlier in the year and were no longer cyanogenic when the present tests were performed. Horton and Ebinger (1989) found that individuals of some species varied in cyanide production at di!erent times during the growing season. Reversal in the cyanogenic reaction may also be linked to environmental change. When the rhizome of a strongly cyanogenic elderberry plant was transplanted to the greenhouse, the new growth tested negative for the presence of cyanide. Also, the fruits of some populations gave a strong positive reaction within 10 min, whereas the leaves of these same plants were negative for cyanide production.
Resampling of six of the nine populations in August 1999 indicated that approxim-ately the same percentage of cyanogenic plants were present in each case. Addition of emulsin to negative tests did not result in the release of additional cyanide, indicating that the materials sampled lacked the cyanogenic glycoside, and that negative tests were not simply due to lack of enzyme.
3.2. Cyanogenic glycoside
The cyanogenic glycoside was established to be (S)-sambunigrin by gas chromato-graphic separation of the TMS-derivative. This was done by comparison with stan-dards of (S)-sambunigrin and (R)-prunasin under conditions that separated both compounds.
3.3. Other American species
of this taxon in Franklin Co., NY, tested positive, although 10 of 10 collections in Franconia Notch State Park, Grafton Co., New Hampshire proved positive.
Acknowledgements
We wish to thank Elizabeth Bartlett for extraction of the sample and Alexander Schenck and Matthias Lechtenberg for assistance with determination of sambunigrin by gas chromatography.
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