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.

1. Addison, K. B., Rhodesia agric. J. 54, 521 (1957)

2. Addison, K. B., Rhodesia Farmer 28, 17 (1958)

3. Bell, E. A., Biochem. J. 70, 617 (1958)

4. Birdsong, B. A.; Alston, R.; Turner, B. L., Canad. J. Bot. 38, 499 (1960)

5. Bonner, J., Americ. J. Bot. 33, 1740 (1949)

6. Fearon, W. R.; Bell, E. A., Biochem. J. 59, 221 (1955)

7. Horowitz, N. H.; Srb, A. M., J. Biol. Chem. [Tokyo] 11, 265 (1929)

8. Jaffé, W. G., Arznei-mittel-Forsch. 10, 1012 (1960)

9. Kitagawa, M.; Tomiyama, T., J. Biochemistry [Tokyo] 11, 265 (1929)

10. Orru, A., Cesaris Demel, V., Quanderni Nutrizione. 7, 273 (1941); ref.: C. 1941, 631

11. Schwartz, H.; Maas, W. K., J. Bacteriol. 79, 794 (1960)

12. Shone, D. K., Rhodesia agric. J. 58, 18 (1961)

13. Steward, F. C., Pollard, J. K., Patchett, A. A., Wittkop, B., Biochim. Biophys. Acta [Amsterdam] 28, 308 (1958)

14. Tschiersch, B. Flora (Jena] 147, 405 (1959)

15. Tschiersch, B. Flora (Jena] 150, 87 (1961)

16. Volcani, B. E. and Snell, J. Biol. Chem. 174, 893 (1948)