Zur toxischen Wirkung der Jackbohne
B.Tschiersch (1962) Pharmazie 17, 621-623
About the toxic effects of the jack bean
The jack bean, Canavalia ensiformis (L.) D.C., belongs to the agriculturally utilized legumes of the tropics. Both, the pods and seeds, as well as the other plant parts are often used as stockfeed. In the literature, several reports indicate that the seeds and other parts of this plant lead to the development of toxic effects in animals.
Investigations about these manifestations of poisoning, primarily observed in agricultural practice, are to date only isolated. Not in all cases could a toxic effect of the plant be shown. Addison [1] observed no toxic effects after feeding young oxen with Canavalia meal that was fed together with silage and maize straw. The same result came from experiments with Jersey cows (Addison [2]). In contrast, Orru & Cesaris Demel [10] observed a toxic effect of Canavalia meal with rats.
More recently, for further clarification of this question, feeding trials with cattle were carried out by Shone [12]. A Canavalia meal suspension was given by stomach tube. The clear reaction of the animals showed that, indeed, a toxic substance must be contained in the seeds. According to the observations of Orru & Cesaris Demel, this substance is apparently thermolabile. The authors hold the view, that cooked seed flour is not toxic. Without further testing, this statement is accepted from other authors. Only this way can it be explained that one substance was totally overlooked in the search for the toxic principle: canavanine. This amino acid, in structure closely related to arginine, is present in high concentrations (up to 4%/dry weight) in the seeds of Canavalia [9].
Only recently was it shown that canavanine occurs also in a range
of other Papilioniaceae; in some of them in very
high concentrations [3, 4, 14, 15]. Canavanine plays an important
metabolic role in the storage of nitrogen in these plants [15].
Special attention was given to canavanine after its effect on
arginine metabolism, initially observed with microorganisms,
was noted [7, 11, 16]. An inhibitory effect of canavanine on
higher plants has also been established [5, 13]. No observations
about the effects of this compound on animal metabolism have yet
been reported.
In a series of feeding trials it was therefore tested whether Canavalia flour and equivalent amounts of canavanine sulfate allow the recogniton of comparable toxic effects in animal experiments. Seed flour of Canavalia ensiformis was used after the canavanine content was determined according to Fearon & Bell [6]. The canavanine sulfate batches used were of different origns (Light & Co., Ltd., Colnbrook; California Corp., Los Angeles).
The seed flour as well as the pure substance (Reinsubstanz) were
rubbed (gerieben) together with water and maize starch to give
a kneadable dough. This dough was dried and in that form offered
to the animals. As controls, maize-starch preparations were used;
one with added canavanine, the other only with water.
The result of the experiments was initially only judged by the behaviour of the animals. In later experiments it was also attempted, by qualitative and quantitative determinations of the symptoms observable in the organs of the animals, to gain some insight into the mechanism by which damage had occurred.
In the first trial 20mg canavanine/kg mouse were given. Within
the trial period of 48hrs no influence of the canavanine could
be observed, when compared to the arginine and water controls.
During the second trial, in which 200mg/kg mouse were given,
clear damages were already observable after a few hours. The
animals crouched (kümmerten) and reacted only very sluggish
to external stimuli. After conclusion of the trials, normal protein-rich
feed was given. The animals recovered quickly and no indication
of lasting damage was noticeable. When arginine(1g/kg) was fed
together with canavanine(200mg/kg), no differences in behaviour
compared to that of the two controls were observed.
In the third trial the concentration of canavanine was increased
to 2g/kg mouse. The arginine, as well as the water controls showed
no effect while all animals that had been fed with Canavalia
flour or with canavanine sulfate died within 24hrs.
It can therefore be said, that through the feeding of canavanine or canavanine-containing flour clear damages are caused in experimental animals. These damages can, especially with high doses, lead to animal death.
The same result was obtained from another series of experiments where we fractionated the Canavalia flour through exhaustive extraction by petroleum ether, followed by extraction with 70% ethanol. The petroleum ether soluble fraction, the 70% ethanol soluble fraction, as well as the residue were fed as maize-starch preparations.
Another group of animals was fed with seed flour that had been
boiled for 3hrs. To achieve clear effects, 2g Canavalia
flour/ animal which is equivalent to 3g/kg mouse of canavanine
were used.
While all animals that had fed on canavanine-free fractions (petroleum
ether fraction and residue) survived the 48hr trial period without
any visible damage, all that had fed on the ethanol fraction
or the boiled seed flour died.
In later experiments, animals were killed after 12hrs or 24hrs; that is before the animals that fed on canavanine died [before they could die through canavanine effects](transl. comm.).
Investigations of the nitrogen-fractions of the liver showed
the changes which are presented in table 1
Table 1. Composition of N-fractions of liver after feeding
fractionated Canavalia flour(mg N/g liver [dryweight])
Feed contained
Total N/Prot. N/sol. N/Urine N
1g seed flour, boiled
157.05 142.3 414.71 1.26
Petrolether extract ex 2g flour
139.88 132.64 7.24 0.11
Ethanol extract ex 2g flour
161.87 144.64 17.23 2.63
Residue
150.98 137.08 13.9 0.26
The amount of urine-N shown for the liver is important in this context. After feeding canavanine-containing materials, a high increase in urine-N content of the livers can be seen.
Canavanine is obviously rapidly metabolized by arginase in the liver . For this assumption speaks the fact that no canavanine could be detected by paper chromatography in the soluble N-fraction of the liver.
Remarkable is also the change in the composition of free amino
acids. Besides a small increase in arginine concentration, an
amino acid can be found in the livers of animals that had fed
on canavanine, which could not be found in controls. This compound
takes the same position as citrulline on the paper-chromatogram.
The substance is ninhydrin and Ehrlich reagent positive and
is unchanged by hydrolysis with 6N HCl (Fig 1). Whether this
substance is citrulline, or possibly ureido-homoserine which
could have formed from canavanine, could not be decided.
The described changes of the amino-acid composition are probably the result of influences on the ornithine cycle. An explanation for the toxic effects of canavanine is probably more likely to be found in a disturbance of protein metabolism.
The liver because of its high arginase content is probably unsuitable
for such a study. Damages have been reported for other organs
[12], so that a much stronger effect must be assumed.
These findings are of special importance because Canavalia ensiformis is not only used as stockfeed but also for human consumption. They confirm the observations of other authors that Canavalia can have toxic effects. The scattered reports about poisoning by this plant probably stand in no relation to actual number of incidences that are caused by it in agricultural practice, because the cause is difficult to recognize.
Only in a few cases do poisonings end fatal, because the lethal dose (30 g seed flour/ kg body weight) is apparently only reached in exceptional cases. However, even small doses allow the recognition of clear effects. Shone [12] observed that milk production was markedly reduced after feeding Canavalia flour to dairy cows. The composition of the feed seems also to have had something to do with it. When Canavalia flour is given together with protein-rich fodder, then no or little effect is noted. Feed containing up to 30% Canavalia can, according to Shone[12], be given without danger. It is, however, to be expected that even thereby some detrimental effect on the animals occurs.
Likewise, Jaffé [8], with investigations about phytotoxins
of legumes, noted clear effects of Canavalia flour on
rats. A feed which contains only 20% of the flour leads to a
reduction in normal weight gain by 7%. Because he managed to
isolate a toxic protein fraction from Phaseolus seed, he
suspects a similar substance to act in Canavalia. No
fractionations of the flour, nor a control with cooked flour were
tested.
We could detect any difference in the level of toxicity between
boiled and raw flour during our investigations. In addition,
the protein fraction, meaning the flour extracted with petroleum
ether, followed by extraction with 70% ethanol, showed no toxicity.
the toxicity of Canavalia seeds can therefore not be explained
by the effect of phytotoxins (e.g. lectins). The toxic principle
is, moreover, present in the 70% ethanol fraction. The main component
of this so-called "soluble N" fraction is canavanine.
On the basis of the presented results it is very likely, that
this canavanine which is present in high concentration causes
the toxicity the seed material. No statements can be made as
yet about the mechanism of action in the animal organism. But
presumably, the activity is related to a dsitrubance of agrinine
functions, similar as in micro-organisms or plants.
Summary
The seeds of Canavalia ensiformis showed toxicity in a
feeding experiment with mice. By feeding fractionated seed material
and pure canavanine, it could be proven that the toxicity can
be traced back to canvanine, which is present in high concentration
in the seeds.
translated by
Dirk Enneking (1991)
Dep. Plant Science, University of Adeliade
Waite Agricultural Research Institute,
Glen Osmond 5064, South Australia,
ref.
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