Part 16

These experiments indicate that an acute form of poisoning may be induced by feeding concentrated aqueous extracts of Astragalus mollissimus and Aragallus lamberti from Hugo, Colo., and Imperial, Nebr., to rabbits, and that if the extract is given in smaller and repeated doses a more prolonged or chronic condition may follow.

The rabbits showing the chronic effects of these plants exhibit symptoms which have a marked parallelism with those reported as occurring in larger herbivora (horses and cattle) on the range when locoed; that is, the loss of appetite (Experiment No. 9), the emaciation and loss in weight (Experiment No. 9), the dullness and stupor, with more or less anesthesia (Experiment No. 7), the disturbance in the visual function (Experiment No. 9), and the mental symptoms (Experiment No. 6). The occasional abortion compares with what has been observed in larger animals. The dried Astragalus mollissimus and Aragallus lamberti still retained their poisonous properties, as we were able to kill with aqueous extracts of the dried plants made in the laboratory under the proper conditions.

=EXPERIMENTS ON SHEEP.=

Experiment No. 1.--On May 31, 1906, a sheep weighing 32.2 kilos was fed with a concentrated aqueous extract of 1,000 grams of the fresh Astragalus mollissimus preserved in chloroform water. The temperature at 11 o'clock, the time of feeding, was 103.4°F. At 11.45 a.m. this dose was repeated. At 12 o'clock the temperature was 104.1°F. At 12.45 the animal urinated. At 1.10 p.m. a similar extract of 2,000 grams was fed. The total liquid used was 1,500 c.c. On June 1 no symptoms were noted. On June 5 an extract of 3,000 grams of fresh Aragallus lamberti and 3,000 grams of Astragalus mollissimus was fed. After feeding this the animal could be easily turned over on its back and its ear pricked with impunity. The animal at this time weighed 30.8 kilos. On June 6, at 11 a.m., the temperature was 104°F. The sheep had numerous soft stools, and was very dull, and would not eat. On June 7 the temperature was 103.7°F. and the sheep still refused to eat. On the 8th the temperature was 103.2°F. at 10.40 a.m., and the stools were still numerous and soft.

There were then fed 640 c.c., representing the aqueous extract of 4,000 grams of the fresh Aragallus lamberti. The animal could be easily turned on its back. It weighed at this time 28.57 kilos. On June 9, at 10.47 a.m., the temperature was 103.4°F. The sheep still did not eat, but had no diarrhea. It now weighed 27.9 kilos, and the temperature was 103°F. at 10.45 a.m.

On June 13 the animal began to eat, and 1,700 c.c. of fluid, representing 5,500 grams of the fresh Aragallus lamberti, were fed. The temperature at 12.30 p.m. was 103°F. On June 14 the temperature was 103.4°F., the animal weighed 28.3 kilos, and refused food. On June 16 the weight was 28.3 kilos; the temperature at 2 p.m. was 103.5°F. There was no diarrhea.

On June 19 the aqueous extract of 1,000 grams of the dried Astragalus mollissimus was fed with 420 c.c. of water. The temperature was 102.6° F. On June 20 the temperature was 102.9°F. at 10.45 a.m.

On June 21 500 c.c., representing the aqueous extract of 1,000 grams of the dried Astragalus mollissimus, were again fed. The animal now weighed 26.9 kilos. On June 26 the animal weighed 26 kilos, and its gait was very uncertain. The temperature was 104.2°F. It was fed 300 c.c. of fluid, representing the extract of 400 grams of the dried Astragalus mollissimus. On June 29 the animal weighed 26.8 kilos and the temperature was 102.8°F. It was fed the extract of 1,000 grams of dried Astragalus mollissimus in 500 c.c. of water. On June 30, at 10.45 a. m., the temperature was 104.2°F. The animal was very dull and died at night.

At autopsy the intestines and stomach merely appeared pale. There were no worms, and the lungs and other organs appeared normal.

Experiment No. 2.--A lamb weighing 15.4 kilos was fed on July 6, at 1.10 p.m., with 640 c.c. of fluid, representing the extract of 2,000 grams of Astragalus mollissimus. At 1.17 p.m. the animal could be turned on its back, and it regained its feet with difficulty. At 1.24 p. m. it urinated and had a stool. The lamb died during the night.

The autopsy the following morning showed the heart filled with clots; lungs normal save for hypostatic congestion. The cerebral and dural vessels were dilated. About 1-1/2 teaspoonfuls of bloody serum were found at the base of the brain. There was none in the lateral ventricles, and no clots. The kidneys exhibited no marked congestion. There was no fluid found in the peritoneal or the pleural or pericardial cavities. The first stomach, however, contained small hemorrhagic spots, and the second was black. There were small hemorrhages in the intestines.

Experiment No. 3.--July 13, 1906, a sheep weighing 19.5 kilos was fed with 640 c.c. of fluid, representing the extract of 2,000 grams of Aragallus lamberti. The temperature at the time of feeding, 1.10 p. m., was 105.3°F. At 1.49 p.m. the sheep could be easily turned on its back. At 2.23 p.m. the temperature was 103.6°F. At 3.42 p.m. the temperature was 103.5°F. At 4.20 p.m. the respiration was fairly rapid. On July 14, at 11.15 a.m., the temperature was 103.6°F. The sheep would run about but could easily be turned over. It had not eaten, but there was diarrhea present. July 15, at 3.30 p.m., the temperature was 104°F. The animal had eaten. On July 17 the temperature was 104°F. and the animal weighed 18.8 kilos. On the 27th it weighed 17.2 kilos; on August 29, 20.8 kilos.

Experiment No. 4.--A lamb weighing 19 kilos was fed August 21, 1906, with 740 c.c., representing the aqueous extract of 2,500 grams of the fresh Astragalus mollissimus, shipped to Washington in September, 1905. This animal ate at night, but the following day was dull. When seen on August 27 there was diarrhea present and the animal was still dull. On the 28th the animal died, weighing 16.7 kilos. There was no autopsy on account of decomposition.

Experiment No. 5.--A lamb weighing 15.6 kilos was fed on September 4, 1906, with an aqueous extract representing 3,500 grams of the dried Aragallus lamberti, 1,000 c.c. of water being used. The temperature at the time of feeding was 104.3°F. At 2.48 p.m. the animal on rising to its feet developed a slight tremor of the fore legs and showed marked disinclination to stand on its feet. The temperature was 104°F. The animal died at 4.25 p.m. The post-mortem was negative, save for some reddening of the second stomach.

These feeding experiments in sheep can not be considered quantitative, because, as is shown later, aqueous extracts of dried plants are often inactive, yet poisonous principles may be obtained from the plants by treatment with digestive fluids.

Extracts of dried loco plants vary much in their toxicity; with some the writer was unable to kill rabbits, even when an extract of 300 grams of the dried plant was used. It is interesting to note that when the field station was established at Hugo, Colo., in 1905, almost all the aqueous extracts of dried specimens sent to Washington would produce the acute symptoms of poisoning in rabbits, but during the third season of its existence many of the samples sent from the same area were much less active, if not inactive.

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FOOTNOTES:

There was a slight odor of chloroform noticed on opening the stomach, so that perhaps the imperfect removal of the chloroform due to a hurried evaporation of the extract should be taken into consideration in this case.

=LABORATORY EXPERIMENTS--CHEMICAL.=

The fact that the aqueous extract of 500 grams of the fresh Astragalus mollissimus, or of 200 grams (in some cases 100 grams) of the dried plant, when fed by mouth, would regularly kill a rabbit weighing about 907 grams, with certain definite clinical symptoms and pathological lesions, was at first arbitrarily selected as our test to aid in the isolation of the active principle. Later the production of chronic symptoms by the aqueous extract or digestion of 200 grams of these dried plants given in doses of 100 grams each on two successive days was considered essential. Carnivora, such as dogs and cats, vomit so easily as to render them unsuitable for these investigations. The aqueous extract was distilled with and without steam, also after acidifying with sulphuric acid, and likewise after the addition of magnesium oxid, but in all cases the distillate was inactive.

The concentrated aqueous extract was shaken by the Dragendorff method with petroleum ether, benzol, chloroform, ether, and amyl alcohol, both in alkaline and acid condition, but the shakings yielded no physiologically active body. Shakings by the Otto-Stas method also proved inactive. Lead acetate, lead subacetate, silver nitrate, mercuric chlorid, alcohol, phosphotungstic acid, trichloracetic acid, ammonium hydrate, sodium carbonate, sodium hydrate, Mayer's solution, uranyl acetate, silver oxid, and barium carbonate also failed to remove the active constituent. They gave heavy precipitates in all cases, but these proved inactive. Hydrocyanic acid was sought for with negative results. The pathological lesions in the very acute cases suggested in some respects oxalic acid, a saponin, a metal, or perhaps a toxalbumin as the active principle, but none of the precipitants for saponins, such as lead and copper, or the magnesium oxid method yielded a body which was active. Proteids were excluded by the fact that the various proteid precipitants--alcohol, trichloracetic acid, lead subacetate, mercuric sulphate or chlorid, and salting out with ammonium sulphate and sodium chlorid (complete saturation and half saturation)--failed to give an active precipitate. Glucosidal or alkaloidal bodies were also excluded. On dialysing for twenty-four hours, some of the active principle went into the dialysate and some remained in the dialyser. Ether yielded a precipitate from alcoholic solution which failed to kill. The possibility of the activity of the plants being due to its normal acidity was excluded by neutralizing the extract with sodium hydrate and precipitating the salts with alcohol. The filtrate proved active after removing the alcohol.

The negative results in looking for active alkaloidal, or glucosidal, or proteid bodies suggested that perhaps the action was due to some inorganic constituent. The writer then boiled the extract three minutes and as the filtrate was still found active and the proteid precipitate inactive became convinced of the inorganic nature of the active constituents, and finally incinerated the plant. The acid extract from this was also active, but death was delayed several hours. This was believed to be due to the insoluble form into which the compound was converted. In fact, the question of solubility and the avoidance of an acid reaction, which of itself may kill, are the main points to keep in mind.

These experiments indicated that the injurious action toward rabbits of the Astragalus mollissimus and Aragallus lamberti collected at Hugo, Colo., was due to one or more inorganic constituents, but it does not follow that all loco plants have the same poisonous principle nor that the same species occurring on all soils has the same poisonous action.

Of Astragalus mollissimus from Imperial, Nebr., collected in 1906, 200 grams were ashed in a platinum bowl and extracted with water. This aqueous extract when neutralized produced no marked symptoms in a rabbit and the weight of the animal remained about the same.

The ash undissolved after this extraction was then treated with acetic acid and water overnight, and after carefully evaporating off the acetic acid on the bath (tested by litmus paper) the residue was fed, partly in solution and partly suspended in water, to a rabbit weighing 1,800.2 grams. Next day the rabbit weighed 1,771.8 grams, showed paralysis of the limbs, and died during the morning. The stomach was intensely reddened and contracted.

An extract of a similar ash was made by boiling the same amount with a large quantity of 94 per cent alcohol. This was evaporated in vacuo and taken in water and fed to a rabbit weighing 1,459.9 grams. On the sixth day the animal died, having lost 70.9 grams in weight. The stomach showed reddening but no ulcers.

An acetic acid aqueous extract, made from the ash after the alcoholic extraction, proved inactive, showing that the alcohol had removed the active bodies. A 70 per cent alcohol extract of another ashed lot proved active, killing the rabbit overnight.